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Genetic and Phenomic Determinants of Basal Mechano-sensitivity and Spread of Neuropathic Pain Following Transection of the Infraorbital Nerve in Mice
By
Daniel Froimovitch
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Physiology
University of Toronto
© Copyright by Daniel Froimovitch 2011
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Genetic and Phenomic Determinants of Basal Mechano-sensitivity and Spread of
Neuropathic Pain Following Transection of the Infraorbital Nerve in Mice
Daniel Froimovitch
Master of Science
Department of Physiology University of Toronto
2011
ABSTRACT
Craniofacial nerve injury occasionally causes spread of mechanical hypersensitivity in
humans. We modeled this abnormality by transecting the infraorbital nerve (IONX) in male and
female mice of the 23 AXB-BXA recombinant inbred lines and their progenitor strains,
comparing their responsivity to 7 applications of a 0.2 gram Von Frey filament to the ears, paws
and tail. When normalizing their mechano-responsivity on postoperative days 14 and 21 by the
preoperative values, subtracting data of sham-operated from IONX mice, highly contrasting
line/strain-specific differences were demonstrated. Similar line/strain-specific variability in the
spread of mechano-allodynia to the paws post-IONX was demonstrated in our novel 3 minute
place-avoidance paradigm, assessing parameters of mobility on a smooth surface versus a pro-
allodynic granular surface. These genetically-controlled, widespread changes in mechano-
sensitivity caused by IONX were minimally sexually dimorphic and mapped to intervals on
chromosomes 5, 9, and 13. Further analysis is needed to identify the causative genes.
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ACKNOWLEDGEMENTS
I would like to thank Professor Ze'ev Seltzer for his continual patience and guidance throughout
the duration of my work towards this Master’s degree. Professor Seltzer was my mentor, and
though I will miss being his student, I am happy to take with me all of the lessons that I learned
from him, both in science and in life. I would also like to thank Professors Barry Sessle and
Siew-Ging Gong for their kind help and advice along the way. I am also indebted to Mariam
Mashregi and Elaheh Soleimannejad for their substantial individual contributions to my study,
and to David Tichauer and Merav Yarkoni-Abitbul for their kind assistance. I’d like to thank the
lovely Anne Siu for her inimitable presence and for being such a wonderful person. I would like
to especially thank my extraordinary parents, Mark and Carol, for thoroughly supporting me
throughout this entire challenging experience, as well as my admirable brothers, Ira and Adam,
for reminding me every so often, that I am not really alone.
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TABLE OF CONTENTS
1.0 INTRODUCTION (p.1-19) 1.1 Definitions of Acute Pain and Chronic Pain (p.1) 1.2 Characteristics of Neuropathic Pain (p.1-2)
1.2.1 Trigeminal Neuropathic Pain (p.2-3) 1.2.2 Enhancement of Neuropathic Pain by Sympathetic Activity (p.3) 1.2.3 Mechanisms of Widespread Neuropathic Pain: Central Sensitization (p.3-6)
1.3 Animal Models of Pain (p.6-7) 1.3.1 Animal Models of Neuropathic Pain (p.7)
1.3.2 Animal Models of Trigeminal Neuropathic Pain (p.7-8) 1.3.3 IONX Model of Widespread Neuropathic Pain (p.8)
1.4 Heritability of Pain (p.8) 1.4.1 Heritability of Basal Nociception (p.9) 1.4.2 Heritability of Chronic Pain (p.10-11) 1.4.3 Sexual Dimorphism in Pain Proneness and Analgesia (p.11-12) 1.5 Syntenic Conservation between Mouse and Human (p.12) 1.6 Quantitative Trait Loci and Genetic Mapping (p.12-13) 1.6.1 Single Marker Analysis (p.13-14) 1.6.2 Interval Mapping (p.14-15) 1.6.3 Mating Systems in the Production of Panels for QTL Mapping (p.15-16) 1.7 Trait Correlograms (p.16) 1.8 Haplotype-based Genetic Mapping (p.16) 1.9 Preliminary Findings Regarding Genetic Propensity for Pain in Progenitor
Strains (p.16-17) 1.10 Hypotheses (p.17) 1.11 Aims and Rationale (p.17-19)
2. 0 METHODS (p.20-31) 2.1 Animals (p.20) 2.2 Surgical Procedures (p.20) 2.3 Von Frey Testing (p.21-24) 2.4 Mechanical Place Avoidance Test (M-PAT) (p.24-25) 2.5 Effect of Stimulus Repetition on Mechano-responsivity (p.25) 2.6 Side Differences (p.25) 2.7 Differences across Baselines and Surgery-induced Changes in Sensitivity (p.25-27) 2.8 Correlation of Responsivity across Body Loci (p.27) 2.9 Surgery-induced Changes in Mechanical Responsivity (p.27) 2.10 Spread of Mechanical Hyper-responsivity (p.27-29)
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2.11 QTL Mapping (p.29) 2.12 Pearson Product-moment Correlations (p.29) 2.13 Bootstrap Test (p.29) 2.14 Permutation Test (p.30) 2.15 Narrow-sense Heritability (p.30) 2.16 Additive Effect of a QTL (p.30-31) 2.17 Haplotype Analysis (p.31)
3.0 RESULTS (p.32-66)
3.1 Mechano-responsivity in Naïve mice (p.32-41) 3.1.1 Adaptation versus Sensitization to the Stimulus (p.32-34)
3.1.2 Side Differences (p.35) 3.1.3 Differences across Testing Periods (BL1 versus BL2) (p.35-36) 3.1.4 Correlation of Responsivity to Mechanical Stim. across Tested Body Loci (p.36-39) 3.1.5 Mechanical Place Avoidance Test (M-PAT) (p.39) 3.1.6 Correlation between M-PAT Parameters and Responsivity to VF Stimulation (p.39-41)
3.2 Changes in Mechano-responsivity following Infraorbital Nerve Injury (IONX) (p.41-51) 3.2.1 Side Differences (p.41-42) 3.2.2 Differences between PO1 and PO2 (p.42) 3.2.3 IONX-induced Effects on Mechanical Responsivity (p.42-43) 3.2.4 Effect of IONX on Behaviour in the M-PAT (p.43-49) 3.2.5 The effect of IONX on the Correlation between Tactile Sens. and M-PAT (p.50) 3.2.6 Pain Spread following IONX (p.50-51) 3.3 Gender Differences in Basal Mechano-responsivity and Changes Post-IONX (p.52-54) 3.3.1 Gender-specific Mechanical Responsivity (p.52-53) 3.3.2 Gender Differences in Sensitization to the Repeated Stimulation (p.53-54) 3.3.3 Gender Differences in the Mobility Test (M-PAT) (p.54) 3.4 Genetic Considerations in Baseline Mechano-responsivity and M-PAT Parameters and Effect of IONX (p.54-66 ) 3.4.1 Line Differences in Mechano-responsivity (p.54-56) 3.4.2 Heritability (h2) of Basal and Postoperative Mechano-responsivity (p.56) 3.4.3 Heritability (h2) of Basal and Postoperative M-PAT Parameters (p.57) 3.4.4 QTL maps of Basal Mechanical Sens., Effect of IONX, and M-PAT (p.58-61) 3.4.5 Assessment of Correlation between Mechanical Responsivity pre- and post-IONX and Spontaneous Neuropathic Pain (p.61-66)
4.0 DISCUSSION (p.67-91) 4.1 Changes in Responsivity of Naïve Animals within a Testing Session across 7 Trials (p.67-68) 4.2 Recategorization of Response Types and Interpretation of Withdrawal Responses and Methodological Pitfalls (p.68-69) 4.3 Changes in Responsivity of Naïve Animals across Testing Sessions (p.69-70)
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4.4 Sensitivity in Discreet Body Regions of Naïve Animals (p.70-71) 4.5 Behaviour of Naïve Mice in the Mechanical Place Avoidance Test (M-PAT) (p.72) 4.6 Correlation between M-PAT Behaviour and Mechano-responsivity in Naïve Mice (p.72-73) 4.7 Changes in Mechano-responsivity following IONX (p.73-74) 4.8 Effect of IONX on M-PAT Behaviour (p.74) 4.9 Correlation between Mechanical Responsivity and M-PAT Parameters Post-IONX (p.74-76) 4.10 Gender Differences (p.76) 4.11 Strain Distribution of Basal Mechanical Responsivity Levels (p.77-78) 4.12 Genetic Effects on the Correlation in Basal Mechano-responsivity across the Tested Body Loci, and Effect of IONX (p.78) 4.13 Estimation of Heritability (p.79) 4.14 Mapping QTLs for Mechano-responsivity and for the Effect of IONX (p.80-81) 4.15 Mapping QTLs for M-PAT Parameters and for the Effect of IONX (p.81-82) 4.16 Candidate Genes in the Mapped QTLs (p.82-85) 4.17 Correlation between Mechano-responsivity pre- and post-IONX and Spontaneous Neuropathic Pain (p.85-87) 4.18 Findings Corroborating the Hypotheses (p.87-88) 4.19 Summary of Other Significant Findings of the Study (p.88) 4.20 Future Directions (p.88-91)
5.0 APPENDIX (p.92-120) 6.0 REFERENCES (p.121-133)
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LIST OF ABBREVIATIONS
AXBn RI mouse lines derived from A/J female crossed with C57BL/6 male BDNF Brain-derived neurotrophic factor BL Baseline BXAn RI mouse lines derived from A/J male crossed with C57BL/6 female CCI Chronic Constriction Injury CM Centimorgan Chr. Chromosome CNS Central Nervous System Crfbp Corticotropin Releasing Factor Binding Protein DRG Dorsal Root Ganglion EMG Electromyography F1 Filial 1 (first generation offspring from a genetic cross) GABA Gaba-Aminobutyric Acid GEPR Genetically Epilepsy-Prone Rats h2 Narrow-sense Heritability HA High Autotomy HYPER Hyperalgesia HYPO Hypoalgesia IAN Infra-alveolar Nerve IASP International Association for the Study of Pain IC Inferior Colliculus ION Infraorbital Nerve IONX Infraorbital Nerve Transection JNK-3 (c-Jun N-terminal kinase)-3 L Left L1 Lamina 1 L2 Lamina 2 LA Low Autotomy LDP Line Distribution Pattern LEW Outbred Lewis Rats LRS Likelihood Ratio Statistic MAPK10 (Mitogen-Activated Protein Kinase)-10 Mb Mega-base pair M-PAT Mechanical Place Avoidance Test NMDA N-Methyl-D-aspartate pIONL Partial Infra-orbital Nerve Ligation PNI Peripheral Nerve Injury PNS Peripheral Nervous System PO Post-operative PSL Partial Sciatic Ligation QTL Quantitative Trait Locus R Right RF Recombination Frequency RI Recombinant Inbred
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SD Sprague Dawley Outbred Rat SG Substantia Gelatinosa SHR Spontaneously Hypertensive Rat SIA Stress-induced Analgesia SIH Stress-induced Hyperalgesia SMP Sympathetically-maintained pain SNL Spinal Nerve Ligation SNP Single Nucleotide Polymorphism SPARCl1 (Secreted Protein Acidic and Rich in Cysteine)-like Protein-1 TG Trigeminal Ganglion VF Von Frey VARenv Environmental Variance VARgen Genetic (Allelic) Variance WK Wistar-Kyoto Outbred Rat WS Weighted Score
LIST OF FIGURES Figure 1 (p.22) Figure 2 (p.22) Figure 3 (p.24) Figure 4 (p.33-34) Figure 5 (p.37) Figure 6 (p.37-38) Figure 7 (p.40) Figure 8 (p.45-47) Figure 9 (p.48) Figure 10 (p.49) Figure 11 (p.92-93) Figure 12 (p.52) Figure 13 (p.53) Figure 14 (p.56) Figure 15 (p.57) Figure 16 (p.59) Figure 17 (p. 61) Figure 18 (p.62-63) Figure 19 (p.65) Figure 20 (p.84) Figure 21 (p.86) Figure 22 (p.86)
LIST OF TABLES Table 1 (p.21) Table 2 (p.26) Table 3 (p.28) Table 4 (p.94) Table 5 (p.94) Table 6 (p.95)
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Table 7 (p.38) Table 8 (p.95) Table 9 (p.96) Table 10 (p.96) Table 11 (p.97-98) Table 12 (p.99) Table 13 (p.100) Table 14 (p.101) Table 15 (p.101) Table 16 (p.102) Table 17 (p.102-103) Table 18 (p.103) Table 19 (p.104-107) Table 20 (p.108) Table 21 (p.108) Table 22 (p.109) Table 23 (p.110) Table 24 (p.111) Table 25 (p.111) Table 26 (p.112) Table 27 (p.113) Table 28 (p.114) Table 29 (p.115) Table 30 (p.116-117) Table 31 (p.118-120) Table 32 (p.63) Table 33 (p.64) Table 34 (p.66)
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1.0 INTRODUCTION 1.1 Definitions of Acute Pain and Chronic Pain
The International Association for the Study of Pain (IASP) has defined pain as “an
unpleasant sensory and emotional experience associated with actual or potential tissue damage or
described in terms of such damage” (137). Acute pain is considered pain that is perceived in
response to a noxious stimulus, and generally lasts for the duration of the stimulus. This sensory
phenomenon has been proposed to have three adaptive functions: 1) to warn the individual of the
existence of real tissue damage, 2) to warn the individual of the probability that tissue damage is
about to occur by realizing that a stimulus has the potential to cause such damage, and 3) to warn
a social group of danger of injury to any one of its members as soon as it exists for one member
(66). Chronic pain occurs as a result of pathological processes affecting the sensory nervous
system. This type of pain outlasts the period of tissue damage and repair. Chronic pain can occur
without any apparent tissue damage, referred to as idiopathic. Injury to the nervous system can
result in a class of chronic pain referred to as neuropathic pain. The IASP has defined
neuropathic pain as: “Pain initiated or caused by a primary lesion or dysfunction in the nervous
system” (137). Chronic pain can cause extreme distress for the individual, family and economy,
and its adaptive value remains elusive (39).
1.2 Characteristics of Neuropathic Pain
Peripheral neuropathic pain is classified as pain resulting from injury to the peripheral
nervous system (PNS), the division of the nervous system consisting of primary afferent fibres of
the spinal nerves, cranial nerves, autonomic nerves, and all tributaries thereof (1). This type of
pain is commonly caused by mechanical trauma, metabolic diseases, neurotoxic chemicals,
infection, or tumor invasion (39). This condition involves plastic changes both within the PNS
and the Central Nervous System (CNS), the division of the nervous system consisting of the
brainstem, spinal cord, and brain. When primary afferent fibres are injured, they develop ectopic
sources of neural impulse firing and oscillatory membrane fluctuations. These impulses can
originate from the neuroma, a swelling of nervous tissue at the site of injury of the nerve, as well
as in the soma of the injured fibres, located in the Dorsal Root Ganglia (DRG) associated with
the damaged nerves (in the spinal system) and the trigeminal ganglion ((TG) in the trigeminal
system). This may result in spontaneous pain, perceived as arising from the original field of
innervation of the injured nerve (25). There is also hypersensitivity of nearby uninjured
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nociceptors (25) and signal amplification at the CNS, at second-order neurons in the dorsal horn,
which become hyperexcitable (105). In addition, the postsynaptic impact of intact nociceptive A-
delta and C-fibres that innervate nearby regions of the body may underlie spreading of stimulus-
evoked pain beyond the territory innervated by the injured nerve (‘extra-territorial’ pain). Central
collateral projections from such fibres may reach central nociceptive neurons, resulting in
widening of receptive fields within the CNS. As well, signaling of intact non-nociceptive
primary afferents, such as A-beta fibres, may become perceived as painful (1). Consequently,
after nerve injury, various regions of the body can become allodynic and/or hyperalgesic. The
former refers to a state in which a normally innocuous stimulus to a body region is perceived as
painful (1). The latter refers to an intensification of pain that is elicited by a normally painful
stimulus (1).
1.2.1 Trigeminal Neuropathic Pain
Damage to the TG or one of its tributary nerves can lead to persistent pain within and
outside the field innervated by the injured nerve, including spread outside the orofacial region.
This pain is a common symptom of trigeminal pathologies such as Trigeminal Neuralgia and
Anaesthesia Dolorosa. Trigeminal neuralgia is characterized by the IASP as “sudden, usually
unilateral, severe, brief, stabbing, recurrent pains in the distribution of one or more branches of
the fifth cranial nerve” (137). It is postulated to be the result of compression of the trigeminal
nerve root by blood vessels as in an aneurism or by an impending tumour at the site of pressure
along the nerve; there is a consequent degeneration of myelin, which leads to abnormal
depolarization of the nerve and ectopic impulses, which may be perceived as painful (61).
Anaesthesia Dolorosa is a related condition of neurogenic craniofacial pain that often results
from neurosurgical or traumatic lesions of the trigeminal nerve or its tributaries or the ganglion
or destructive nerve blocks carried out in an attempt to rid the patient of the pain arising from
trigeminal neuralgia (62). Current pharmacological treatment for such craniofacial pain
syndromes is partially effective at best.
Human pain syndromes, including those of the trigeminal region, exhibit a high degree of
inter-individual variability in pain levels (18). The same aetiology can trigger a maximal pain
response in one individual and none in another. Conversely, strikingly different aetiologies can
result in reportedly similar sensory manifestations. In some cases of idiopathic trigeminal
neuralgia, an intense pain and sensory abnormalities are present with no aetiological correlate of
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any sort to explain it. As well, there is high variability in the pain levels of animals subject to
model injuries, including those of the trigeminal system (8, 9, 21).
1.2.2 Enhancement of Neuropathic Pain by Sympathetic Activity
It has been shown that sensory symptoms of peripheral nerve injury can be reduced
following pharmacological block of sympathetic release or surgical destruction of sympathetic
ganglia (sympathectomy) (98). Such procedures have been shown to be clinically efficacious in
neuropathic complications, which are predominantly manifested in the spinal system, such as
Causalgia and Reflex Sympathetic Dystrophy (98). Evidence indicates that Peripheral Nerve
Injury (PNI) can result in increase in responsivity of sensory neurons after injury, rather than the
once widely assumed notion that the cause of hypersensitivity was a global increase in
sympathetic drive (99). With regards to changes in sympathetic efferents, however, there is often
collateral sprouting onto the DRG of injured nerves, and peripherally at the neuroma (109). This
allows for greater access of sympathetic output to the sensory fibres than in the non-injured state.
The alpha-2 adrenoreceptors and, in a minor way, the alpha-1 receptor have been shown to be
the key mediator in coupling sympathetic output to sensory changes. Noradrenaline and
adrenaline released into circulation and at the injured sites, bind to the alpha-2 receptor along
nerve cells and lead to generation of ectopic electrical signals (100). There is suggestion that
transcripts encoding adrenoreceptors, while constitutively expressed in the DRG in the uninjured
state, may be upregulated after axonal injury, accounting for increased and ectopic distribution
of adrenoreceptors along sensory nerve axons (97).
Following PNI, the increase in adrenosensitivity promotes a tonic barrage of electrical
signals toward the CNS, due to the exposure of nociceptive fibres to catecholamines (1). This
contributes to the sensitization of the CNS (see below), resulting in low-threshold afferents
sending nociceptive signals to the brain (i.e. allodynia). This state is known as Sympathetically-
Maintained Pain (SMP) and can be reduced by therapeutic measures to reduce activation of
alpha-adrenoreceptors along nociceptive fibres (1). It has also been shown that this influence of
sympathetic drive on neuropathic pain is a feature that varies between different genetic groups of
animals (101).
1.2.3 Mechanisms of Widespread Neuropathic Pain: Central Sensitization
Intense and ongoing nociceptive activity can trigger a state of heightened sensitivity in
the CNS, referred to as Central Sensitization. This can be evoked during inflammatory pain,
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from prolonged and intense signaling generated from nociceptors (1,39). Peripheral nerve injury
can also trigger such long-term use-dependent alterations in the functioning of the CNS via the
injury-induced persistent barrage of ectopic input from the site of injury or the ganglia of the
injured nerve (39). This can lead to hypersensitivity of sensory pathways, including those
involved in nociceptive transmission. This may partially serve as the pathological substrate for
widespread pain after peripheral nerve injury. As well, large-diameter, low-threshold A-beta
fibres, which normally subserve non-painful sensation from low-intensity stimulation, can
functionally shift to trigger pain sensations from these same stimuli (such as light touch).
After nerve injury, polysynaptic and monosynaptic inputs from A-beta fibres (which
normally terminate in the dorsal horn laminae 3-5) appear also in the more superficial laminae of
the dorsal horn in the spinal cord and medullary dorsal horn of the trigeminal system, an area
that usually receives and processes A-delta and C-fibre nociceptive signals. This has been
demonstrated, for example with C-Fos activation of neurons in laminae 1-2 from light tactile
stimulation of the hindpaw after sciatic nerve crush (41, 48). This phenomenon can also occur in
regions of the spinal cord which correspond to regions of the periphery that are outside those
corresponding to those normally processing primary inputs of the injured nerve (41). Second
order neurons in the spinal cord and brainstem change to having lower excitatory thresholds,
rendering them excitable by low threshold afferent input that normally does not activate them
(57, 105). Here, a normally nociceptive-specific neuron broadens the range of afferent input that
is capable of exciting it. As well, there may be a reduction in activity of inhibitory inter-neurons
in the spinal cord or brainstem (105). These neurons normally synapse onto the central terminals
of primary afferent fibres or onto the postsynaptic second order neurons in the spinal cord
conferring glycinergic or Gaba-Amino Butyric (GABA-ergic) inhibition (105).
After peripheral nerve injury this inhibition can be reduced resulting in more easily
excitable or spontaneously active neurons in nociceptive pathways (42). Normally there is a
tonically active descending inhibitory circuit projecting from various supra-spinal regions,
descending to the spinal cord and modulating nociceptive transmission therein (105). After PNI,
this inhibitory system can be weakened (105). Descending serotonergic input can switch from
being inhibitory to facilitatory (44). Descending noradrenergic inhibition can also be reduced
(43). As well, due to changes in ion gradients across nociceptive lamina 1 neurons, GABAergic
input can switch from being hyperpolarizatory to depolarizatory (45). This effect is commonly
thought to be ultimately the result of injury-induced activation of microglial cells in the spinal
cord or brainstem (45, 167). Microglia multiply and concentrate at the site of spinal cord entry
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segments of the injured nerve, likely as an innate immune response (45). They become activated,
partly function as phagocytes in clearing the region of debris from degenerated primary afferent
axons and secondary neurons. They are thought to become activated partly by way of the barrage
of electrical input to the CNS from injured nerves. This causes the release of a plethora of agents
from primary afferent terminals in the spinal or medullary dorsal horn that act as chemo-
atractants and activators of microglia (104). Concurrently, these leukocytes are induced into the
activated state by signals arising from interruption in axonal transport in primary afferents (104).
Among several cytokines, activated microglia cells release Brain-Derived Neurotrophic
Factor (BDNF) which acts on lamina 1 cells and downregulates expression of the gene encoding
potassium chloride cotransporter isoform-2 in neurons, leading to a reversal of the trans-
membrane chloride gradient, such that GABA-induced opening of chloride channels results in
cellular efflux of chloride and thus paradoxically facilitates nociceptive neurons in their
electrical transmission (45,46). The resulting depolarization of membrane potential can lead to
the protein-tyrosine kinase Src-mediated potentiation of dorsal horn N-Methyl-D-aspartate
(NMDA) receptors (the predominant receptor for the excitatory neurotransmitter, glutamate) via
its Src-mediated phosphorylation. This results in facilitation of transmission within the spinal
cord. It has also been demonstrated that the barrage of nociceptive input to the CNS resulting
from PNI induces excitotoxic effects on cells within the dorsal horn (105). This occurs primarily
by glutamate-mediated apoptosis of GABAergic inhibitory interneurons in the CNS (47, 150).
This results in reduced inhibitory currents in superficial laminae cells, rendering them more
excitable (47).
A proliferation of microglial cells in the spinal and medullary dorsal horns,
corresponding to the region of central termination of the injured nerve, has also been observed
following PNI (103, 167). As well, infiltration of circulating monocytes, which become activated
microglial cells, has been observed (103). It has been shown that PNI leads to two distinct glial
responses within the spinal cord and brainstem, which influence pain perception thereafter. The
first of these responses is microglial activation, contributing to initial sensitization of the
nociceptive pathway. This is followed by astrocyte activation, which is strongly suggested to be
a contributor of the maintenance of neuropathic pain after PNI (102, 58). It has been shown that
injury to a branch of the trigeminal nerve preceding injury to the sciatic nerve can prime the
latter nocifensive effects and cause them to occur at an accelerated rate (106). That is, injury to
the trigeminal system can alter the CNS response to a succeeding injury to a peripheral nerve
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that is located remotely along the rostral-caudal axis of the body. This effect of priming has also
been noted to be strain-dependent in rats (106).
It has also been shown that continual peripheral nociceptive input can allow for the
maintenance of chronic pain from a remote focus in the body (3). This has been referred to as
‘state-dependent central sensitization’ (3) and has been demonstrated by the alleviation of
mechano-allodynia at a discreet locus by the reduction of ongoing nociceptive input from a
remote locus of the body (3). Similarly, secondary hyperalgesia from a burn on the forearm
could be eliminated by application of topical anaesthetic to the zone of primary hyperalgesia, the
site of the burn (107). This suggests dynamic maintenance of extra-territorial allodynia by
continual nociceptive input (3, 107).
1.3 Animal Models of Pain
Zimmerman expanded the IASP definition of pain to apply it to animals. He defined it as:
“...an aversive sensory experience caused by actual or potential injury that elicits progressive
motor and vegetative reactions, results in learned avoidance behaviour, and may modify species
specific behaviour, including social behaviour” (53). The inability of animals to verbally
communicate presents a considerable obstacle in the development of pain models. Given this
problem, animal models of pain have been designed to allow for evaluation of pain based on
anthropomorphic interpretation of their reactive behaviours. Instead of through verbal
communication, the subject’s sensory experience is inferred on the basis of its behavioural
reactions to various controlled stimuli (in the case of stimulus-evoked pain) or its spontaneous
behaviour (in the case of spontaneous pain). Behavioural reactions to pain are referred to as
‘nocifensive’ and are the basis on which the animal subject’s pain is inferred and evaluated by the
experimenter. Nocifensive behaviours can be categorized into three main groups; simple
reflexes, organized unlearned behaviours, and organized learned behaviours (60). Such
nocifensive behaviours include flexion withdrawal reflexes ridding the body of contact with the
stimulus, licking, biting or rubbing the stimulated region, or learned aversion of the stimulus in
the form of classical or operant conditioning. The majority of the models eliciting stimulus-
evoked pain in animals use reflexive measures such as the latency to a withdrawal response, the
threshold intensity at which a withdrawal is elicited and the rate of such responses as indices of
pain. Many studies have shown a tight relationship between the nociceptive withdrawal reflex
threshold and response strength (as seen by Electromygraphy (EMG)-recorded muscle
activation) and reported pain in response to noxious mechanical (63, 64, 65) and thermal stimuli
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(67). However, several counter-arguments to the validity of these indices as a measure of pain
have been posited. There is the viewpoint that a decrease in withdrawal threshold to punctate
tactile stimuli (e.g., Von Frey (VF) filament application) may in many cases suggest
hyperesthesia rather than allodynia (4). That is, hypersensitivity of a tactile withdrawal reflex
may not necessarily indicate a concomitant alteration in pain perception. Some dissociation has
also been shown between reflex sensitivity and pain perception in humans (13).
1.3.1 Animal Models of Neuropathic Pain
The majority of models for neuropathic pain involve injury of a peripheral nerve with
ensuing sensory phenomena that are reminiscent of the clinical features of neuropathic pain. In
general, a leftward shift in the stimulus-response function is observed. That is, subjects exhibit a
reduction in the sensory threshold that is required to elicit a nocifensive response (allodynia). As
well, subjects exhibit intensified nocifensive responses to normally noxious stimuli
(hyperalgesia). In some cases, the occurrence of paraesthesias or dysaesthesias is inferred (4).
Usually behavioural abnormalities begin 1 to 5 days following the injury and peak 7-21 days
following it. At this point postoperatively, sensory abnormalities plateau and remain at a
constant heightened state for several weeks or decline to normalcy earlier, in some pain models,
depending on the species, genetic background and gender. Established models differ in the
region of and type of injury, including; transection of a tightly ligated sciatic nerve,
deafferentation or section of a dorsal nerve root or several roots (4, 96), loose chronic
constriction of the sciatic nerve (11), tight ligation of about 50% of the common sciatic nerve
(50) or a spinal segmental nerve (52), or lesion to two of the tributaries of the sciatic nerve (51).
Several of the latter models have demonstrated pain spread to regions remote from the injured
site, as with contralateral allodynia and hyperalgesia in rats reminiscent of mirror image pain in
humans with limb amputation (50). As well, remote rostro-caudal spread was suggested in rats
after dorsal rhizotomy in the cervical region exhibiting hypersensitivity of the tail withdrawal
reflex (96). There are other models that are used infrequently.
1.3.2 Animal Models of Trigeminal Neuropathic Pain
A number of models for trigeminal pain have been established with use of rats. Vos et al.
adapted the Bennett and Xie Sciatic Chronic Constriction Injury (CCI) model for the Infraorbital
nerve (ION) of rats. This model demonstrates behavioural signs reminiscent of clinical features
of trigeminal neuralgia, namely; spontaneous pain, mechanical allodynia, and heat hyperalgesia
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(12, 54). As well, there are studies in which complete transection or nerve crush is applied to a
portion of the ION, lingual, or alveolar tributaries of the trigeminal nerve that have been used to
elucidate post-injury neural changes (56, 57, 58, 59, 95). Pain spread to regions outside of those
innervated by the injured nerve has been demonstrated by allodynia in regions contralateral to
the ION lesion (12), or in the vibrissae of a rat with Infraorbital Alveolar Nerve (IAN) injury
only (95) (this region not being innervated by the respective injured nerve). The PSL model (50)
was adapted to the ION of the mouse (PIONL) in an attempt to simulate trigeminal neuralgia.
This model exhibited signs reminiscent of clinical symptoms of trigeminal injury such as
mechanical allodynia (55).
1.3.3 IONX Model of Widespread Neuropathic Pain
The trigeminal pain model used in this study entails transection of the ION in the mouse.
After nerve section, the corresponding field of innervation (including the upper lip, gums, upper
lateral vestibule, and the cheek up to the lower eyelid, whisker pad) is rendered totally
anaesthetic to external stimuli, yet widespread mechanical and thermal allodynia and
hyperalgesia may ensue in varying levels depending on the genetic background of the studied
strain (157), environmental variables such as diet (26) and social caging conditions (27), gender
(27), type of nerve injury (29) and stimulus modality (4). Nearby and remote spread of pain has
been shown to occur in the ears, forehead, hindpaws and tail (157). This model has been adapted
by members of our group to the mouse (157) from established models existing for the rat (12, 59,
95). For the purposes of the present study, which aimed at elucidating genetic and phenomic
determinants of neuropathic pain ensuing from trigeminal injury, this model was adapted for use
of the mouse due to the higher abundance of genetic assays as well as the high number of
recorded genetic markers available for this species, allowing for a higher resolution of genetic
mapping.
1.4 Heritability of Pain
Pain is generally classified as a quantitative trait, one that is normally expressed in a
continuous distribution throughout a population (112). This is distinguished from a Mendelian
trait, which is generally expressed in one of two categorical forms throughout a population (112).
9
1.4.1 Heritability of Basal Nociception
Basal nociceptive levels are genetically controlled to a large extent. It has been shown
that inbred Dark Agouti rats are significantly less sensitive to noxious thermal stimulation than
are the albino Wistar-Kyoto rat (68). As well, it has been indicated that inbred Fisher (F344) rats
are significantly more sensitive to heat stimulation than are Genetically Epilepsy-Prone (GEPR)
Rats (69). Levels of basal nociception have been demonstrated to vary across mice strains.
Likewise, specific sensory modalities vary across strains independently of one another. Thermal
and mechanical sensitivity are genetically controlled by two distinct mechanisms in mice (9, 73,
114). This was accomplished using 12 inbred strains of mice and rating them according to their
sensitivity to various pain assays including ones for thermal and mechanical sensation. A lack of
correlation was shown among strains between mechanical and thermal responsivity, implying
distinct mechanisms behind them (9, 114). Strain- and gender-dependent variability in response
to analgesics has also been demonstrated in rats (72) and mice (71). There are various evidence
in support of the notion that the endogenous opioid system, which serves as an inhibitory
regulator of nociception, underlies an important part of the genetic control of basal nociception
(82). The Spontaneously Hypersensitive Rat strain (SHR) shows a considerably low response
level to multiple tests of acute nociception (78), and it has been demonstrated that SHR rats
possess , on average, a higher than normal density of opioid receptors in various regions of the
CNS, many of which have been implicated in the pain system (80). As well, the hypoalgesic
responses observed in the above nociceptive assays can be reversed by administration of an
opioid antagonist such as Naloxone in these rats (80). Mogil et al. demonstrated evidence that a
candidate gene involved in the regulation of thermal nociception encodes the delta-opioid
reception for which the endogenous opioid, enkephalins, are the ligand (82).
Another potential system underlying the genetic control of basal sensitivity is
endogenous GABAergic and glycinergic inhibition in the CNS. It has been long known that
disinhibition of these inhibitory systems by exogenous agents results in considerable mechanical
allodynia (83). Correlation has been shown among various rat strains in GABAergic inhibition as
seen in density of hypothalamic GABA receptors (84), GABAergic neuron abundance in the
Inferior Colliculus (IC) (85) and basal tactile withdrawal thresholds (69). As well, in relation to
proneness to chronic pain, a link has been shown between rat strains selected for high proneness
to spontaneous neuropathic pain and seizure proneness, as induced by exogenous GABA-
blocking agents (87). It has been postulated that seizure proneness is linked to basal sensitivity
and neuropathic hypersensitivity due to their correlation to central inhibition (87).
10
1.4.2 Heritability of Chronic Pain
Human pain syndromes, including those of the trigeminal region, exhibit a high degree of
inter-individual variability in pain levels (18). The same aetiology can trigger a maximal pain
response in one individual and none in another. As well, there is high variability in the pain
levels of animals subject to model injuries, including those of the trigeminal system (8, 9, 21). A
strong intra-familial correlation with respect to susceptibility to trigeminal neuralgia has been
shown (2). The familial clustering occasionally observed may reflect a heritable proneness to the
potential triggers of the syndrome, such as vascular compression of part of the trigeminal nerve
root and resulting demyelination (2). This may reflect a genetic control for disease-susceptibility,
rather than for pain per se ensuing from a specific aetiology. Moreover a few genetic loci have
already been identified for neuropathic pain-related traits in mice (5, 110) and rats (14) such as
spontaneous pain after denervation of a limb. A Mendelian pattern of inheritance of
susceptibility to such spontaneous pain in the rat has been suggested (8, 22) and an oligogenic
control of the same pain trait was suggested in mice (8). It has been indicated that there is no
overlap between the genetic basis for spontaneous pain proneness and that for heightened
responsiveness to stimuli after nerve injury (69), yet contrary evidence has been found in support
of a unitary control for both such phenomena (70). It is plausible that the propensity to develop
trigeminal neuropathic pain after peripheral nerve injury is controlled by a unique set of genetic
factors. The identification of the genetic basis of this condition provides an opportunity for the
development of analgesics which target the respective genetic loci and their encoded
mechanisms, allowing individualized pain medicine as per the genotype an individual carries at
these loci (24, 112)
Calculating the heritability of propensity for various types of acute and chronic pain was
done using inbred mouse lines (9, 114). As well, heritability of sensitivity to analgesics was
calculated using this approach (113). This narrow-sense heritability is indicated by the ratio of
the variance between genetic groups over the total variance (9, 94). As well, one can estimate the
number of genetic loci that affect the trait of interest by the following formula (94):
(Highest strain mean – Lowest mean) / (4 * (Variance between strains)
It has been observed that several properties of self-mutilatory behaviour (‘autotomy’)
induced in the Neuroma Model are highly influenced by the strain of animal used. Three outbred
strains: Sprague Dawley (SD) rat, Wistar-Kyoto Outbred (WK) rat, and Spontaneously
Hypertensive Rat (SHR) were shown to differ from one another in the frequency, onset and
severity of autotomy following peripheral neurectomy (74) As well, Lewis Outbred (LEW) rats
11
were shown to autotomize much less severely and frequently than Sabra rats (75). This study
also showed that selective breeding from the same strain (Sabra) resulted in lines expressing high
or low levels of autotomy following the same denervation injury, as well as the penetrance of the
behavioural trait within a population (75). Selective inbreeding was conducted in order to
develop two populations of rats, which contrasted in the expressivity of autotomy, with complete
penetrance of the expected phenotype in each population. Crossing the two lines resulted in a
Filial 1st generation (F1) hybrid, which invariably showed low autotomy behaviour as in one of
the parental lines. Backcrossing these F1 animals onto the low autotomy (LA) parent resulted in
roughly 100% LA offspring. The same backcross onto the high autotomy (HA) parental line
resulted in roughly 50% HA offspring (76). This suggested that the propensity for autotomy after
nerve injury is inherited as a monogenic recessive trait. Reconciling this finding with the fact
that strains of mice and rats have been produced, which exhibit the expressivity of autotomy
along a continuous distribution, indicates that the trait is likely controlled by one or two major
genes with various modifying genes and with substantial environmental influence. Quantitative
trait mapping has been used to locate a region on murine chromosome 15, which controls for
much of the variance seen in expressivity of the trait in mice (5). The identity of the causative
gene or group of genes has been established recently by members of our group in collaboration
with the groups of Darvasi and Devor (110), but several other possible candidates exist which
are harboured within the same respective locus (158). It is commonly inferred that genes
controlling this trait are likely those that code for biophysical properties of ion channels, as this
relates to the electrogenicity of sensory neurons and the ectopic firing that is implicated in both
spontaneous and stimulus induced neuropathic pain (88). In addition, genes may encode for
synthesizing and catabolizing enzymes that catalyze the production or degradation of
neurotransmitters and neuropeptides, and receptors (82). Proneness to hypersensitivity to stimuli,
as measured using models of partial nerve injury, has also demonstrated strain-dependent control
(88,89).
1.4.3 Sexual Dimorphism in Pain Proneness and Analgesia
It has been a common observation that there is disparity between the genders with regards
to pain sensitivity (112). In the majority of such reported cases, females exhibit greater
sensitivity than males, as seen in animal models and studies on humans (112). As well, it has
been shown that woman have been more sensitive to various opioid analgesics, while the reverse
relation between the sexes has been apparent for nonhuman subjects (115). There has been
12
evidence of a strong interaction between genetic background and gender in controlling pain
propensity (112). Mogil demonstrated that male SD rats have lower thresholds to thermal
noxious stimuli than females of this strain, while the reverse gender relationship holds for Long
Evans rats (116). As well, Kest et al. demonstrated strain-specific gender differences in response
to morphine, with different strains showing positive, negative or no correlation between the
genders (117). Chesler et al. showed that in regards to basal sensitivity to mechanical stimulation
in a group of rat strains, it was the interaction of gender and strain that was a stronger
determinant than gender alone (118). It has also been demonstrated that several genes show a
sex-specific effect on sensitivity of the organism, including ones located on chromosomes 4 and
8 of the murine genome (119, 120). The former region encodes an opioid receptor, the sequence-
variance of which has been shown to be sex-specific in its effect on thermal pain sensitivity in
males only (119).
1.5 Syntenic Conservation between Mouse and Human
Homology between species refers to sequence similarity in genes between the species.
Synteny refers to the chromosomal location of these genes being the same between two or more
species (yet the order of the genes may not be the same) (90). Sequence conservation and
synteny serve as an index of the degree of evolutionary relatedness of two species, or an index of
the degree of evolutionary steps since the stage at which their common ancestor existed. Mural et
al. compared mouse chromosome 15 with the extensively studied human chromosome 21. These
chromosomes share roughly 25 Mb pairs in synteny. It was shown that out of 731 genes on the
mouse chromosome, a human homologous gene was found for all but 14 of the murine genes.
Conversely, it was shown that the human chromosome contained only 21 genes that were not
found in the respective murine chromosome (91). There is strong evidence that the murine and
human genomes have a high percentage of genes in common, thus justifying in part the use of a
pathological model of mice to be used to make inferences about mechanisms for corresponding
human pathologies and their genetic control (91).
1.6 Quantitative Trait Loci and Genetic Mapping
It is believed that complex traits, such as propensity for neuropathic pain are controlled
by multiple genes (112; but see Devor et al. 75,76, as described above). A common method of
identifying the areas of the genome, which contain such contributive genes (hereafter referred to
as ‘quantitative trait loci’ or QTL), is to make use of a panel of mice, which co-vary at known
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genetic loci and in the expression levels of the trait of interest. The current study makes use of a
particular type of panel classified as Recombinant Inbred (RI) mice lines. The following are the
steps in producing and using a panel of RI mice to identify a region of the genome that is linked
with a certain trait. Initially, two genetically distinct inbred strains are identified as contrasting in
expressivity of the trait of interest. These strains are then bred together. Since the progenitors are
homozygous at every locus, the resulting F1 are all identically heterozygous to one another at the
loci, which differed between the parents. That is, each F1 mouse possesses one set of
chromosomes inherited from one parent and another set that is inherited from the other parent.
During the production of gametes, some regions of each chromosome may exchange their
respective genetic material with that of the homologous chromosome. This process is referred to
as ‘recombination’. Consequently, the offspring of crossed F1s inherit unique combinations of
allelic variants (each of parental origin) on each chromosome. These F2s are then brother-sister
mated for approximately 20 generations. The resulting mice are now homozygous at almost each
loci (as the parents were), each have a unique array of allelic variants throughout the genome.
QTL mapping is performed under the assumption that there is no occurrence of meiotic
interference in the progenitor lines. That is, the recombination frequency between any two points
along a chromosome is consistently a function of both a random variable and the distance
between the two points.
1.6.1 Single Marker Analysis
Early QTL mapping was performed by use of single informative markers, each of which
being polymorphic (111). A t-statistic was calculated to screen for significant trait value
differences between allelic groups at each marker. If a significant difference was found, this
suggested that the respective marker is located near a causal sequence. This method does not
allow for a distinction between the phenotypic effect of a causative sequence and the proximity
of that sequence to the respective informative marker. As well, if the causative sequence is not
the same one as the marker sequence, there is high probability for a downward bias in the
estimation of the effect of the QTL (77). Moreover, all putative QTLs in proximity to a particular
marker are underestimated by this exact factor. This is highly limiting with regards to resolving
the spatial and functional parameters of a QTL within the marker-zone. For example, by
examining the effect of allelic substitution at the marker locus, it is impossible to discern
between a QTL whose effect is great yet is located far from the pertinent marker and a QTL
whose effect is minimal yet is positioned nearby the marker. To demonstrate this effect: Let RF
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be the Recombination Frequency between an informative marker, M1, and the putative QTL. A
QTL’s effect is measured by the difference between the phenotypic means of the two genotypic
groups corresponding to the informative marker nearest to that locus. If Z is the apparent effect
of an allelic substitution at the QTL (this being suggested by the difference between the
phenotypic means of the two genotypic groups of the nearest marker) and P is the actual effect of
substitution at a nearby QTL, then the true phenotypic effect of the QTL has been
underestimated by a factor of (1-2RF), that is P = Z(1-2RF).
Example (using real numbers): Let the Recombination Frequency (RF) between the nearest
marker and a QTL be 0.2. This means that there is a 20% probability in gametogenesis that these
two genetic loci will be separated and an 80% chance (1-RF) probability that they will segregate
together. Let Z be the actual phenotypic effect of allelic substitution at the QTL obtained from
the difference between the means corresponding to the two genotypic classes at the QTL. The Z
value would be 100 (i.e.,. the phenotypic effect of the QTL is 100). However, the two phenotypic
means suggested by marker substitution are 80 (since in 20% of cases an allelic substitution at a
marker is detached from the supposed allelic substitution at the QTL. That is, no substitution
actually occurs and so the apparent effect is zero (deceptively so). Since 20% of the cases show
effects diminished to zero, the net average of all marker substitutions will be lowered by 20%
(with all other factors being equal), hence the effect will be 100-20 = 80. Conversely, the
alternate genotypic class will show the true effect of zero in 80% of the cases, while 20% of the
cases will show an effect size of 100, thus the net average will be augmented by 20%. The actual
effect, P, of substitution at the QTL here is, therefore, 80-20 = 60, which is obtained by applying
the factor 0.6 (1-2(RF)) to the apparent effect, Z. Thus the formula P = Z(1-RF) holds true in this
specific case and the QTL effect is considerably underestimated (in this case by 40%).
1.6.2 Interval Mapping
Interval mapping is a QTL mapping strategy that allows for increased confidence in
delineating the region of the genome showing allelic variation, which associates with variation in
the phenotype of interest (10). This method involves comparing variance at every locus along the
genome with variance in phenotype (10). An estimate is made of the probability of inheritance of
a specific parental allele at each genomic locus of a line of mouse, based on that mouse’s allelic
inheritance at the two nearest flanking markers. To illustrate, take the example of a causative
locus bordered by two markers in a recombinant inbred line. Here, every genomic locus takes the
15
form of either one of the parental alleles on both chromosomes, the same allele being inherited
on each chromosome. Assume the markers are 10 cM apart from each other (1 cM being an
estimate of the genomic distance between two loci such that there is a 1% chance of
recombination between them during meiosis). Assuming that a putative QTL between the two
markers is 2cM from the left marker, if the left flanking marker is the allelic form of A and the
right marker is the form of B, then there is a 80% probability that the putative QTL is in the form
of the A allele and a 20% probability of it being in the form of the B allele. One can establish a
conditional phenotypic distribution pertaining to each locus (i.e., a mixture of distributions the
components of which include the expected populations inheriting the A allele and that of the B
allele (10)). As opposed to merely assessing variance at each of the informative markers along
the genome, this method assesses association between every locus in the genome and phenotype.
1.6.3 Mating Systems in the Production of Panels for QTL Mapping
QTL mapping firstly requires two populations of different genetic lineages who contrast
in the phenotype of interest. From this starting point, one can have offspring produced, which
combine the parental genomes in any number of ways. A backcross population is generated by
first mating two inbred progenitor strains, contrasting in phenotype, followed by mating an F1
with one of the parents. The resulting F2 offspring each possess a mosaic of alleles derived from
the two progenitors (a greater percentage of which is derived from the parent that was
backcrossed onto). The mosaic offspring can be inbred with one another for several generations
(typically 20) until each genomic locus is homozygous. At this point, any further breeding within
that strain cannot result in functional meiotic recombination, and all further offspring are almost
entirely genetically indistinguishable (i.e., isogenic). This allows for the genotyping of an
individual from that population, which can reliably apply to all others of that population and
descendants thereof, obviating the need for further genotyping down the line. A strain of this sort
is referred to as RI, as described above. A different type of RI strain can be produced by inter-
breeding the first generation of offspring population (F1) to generate a third generation
population (F2) of individuals with a mosaic of parental alleles. These individuals can be inbred
and formed into different lineages until complete homozygosity is achieved within each strain.
An F2 intercross and inbreeding is used in the current study. Within such a population, the
number of recombinations is quadrupled in comparison to the backcross population, providing
more informative markers and consequently higher resolution mapping (77, 78). Congenic
strains are generated by backcrossing repeatedly, each generation selecting for an individual,
16
with a particular variant at a specific genomic region. Eventually, the offspring will possess the
variant selected for, in addition to the rest of the genome being derived entirely from the parent
used for backcrossing. This allows for fine mapping, once a region has been detected which
contains a QTL, and for the original QTL to be dissected and resolved to discreet causative
sequences therein.
1.7 Trait Correlograms
WebQTL is a software package harbouring several genetic analytical tools, allowing
among others, the user to input data pertaining to the trait values of each relevant strain and draw
Pearson-moment correlations between traits (160). This software provides information about the
mechanistic correlations between traits. If traits are shown to lack entirely in correlation between
one another, this would warrant producing separate QTL maps for each trait, as they are likely
governed by discrete genes. Since panels of RI lines have been used for phenotyping hundreds of
traits, such correlograms facilitate identifying traits that are seemingly entirely different from a
trait under investigation, which apparently shares mechanisms and genetic control with the
studied trait.
1.8 Haplotype-based Genetic Mapping
An alternative approach to mapping genomic regions controlling a phenotype of interest
involves assessing association between specific groups of Single Nucleotide Polymorphisms
(SNPs) that are commonly transmitted as a single unit (‘haploblock’) with trait levels (115). In a
haploblock, this non-random association of SNPs on different loci is referred to as linkage
disequilibrium (‘LD’; 115) and can be quantified by dividing the number of SNPs within a
defined region of genome by the number of haplotypic variants of that region. A high LD value
would justify utilizing this region as if it were a unitary polymorphism, the variation of which
can be correlated with phenotypic variation.
1.9 Preliminary Findings Regarding Genetic Propensity for Pain in Progenitor Strains
A preliminary study by our group indicated a contrast in the level of mechanical pain
spread between C57BL/6J and A/J mice following IONX (157). For each body locus that was
tested, a greater level of mechano- and thermo-hypersensitivity was observed in the A/J mice
than in the C57BL/6J mice. Since this study was carried out under the same environmental
conditions, this difference suggested the presence of a genetic influence on neuropathic pain
17
development in various body regions following IONX. It was inferred from this finding that the
genetic background of A/J and C57BL/6J mice confers a predisposition to pain spread following
IONX or a protective property against this symptom, respectively for each strain. There is also
the possibility that both such genetic influences exist in the respective strains. This phenotypic
contrast between genetic groups provides an opportunity to shed light on specific genetic factors
mediating an influence over pain spread following IONX. By use of a panel of RI mice lines
(AXB-BXA) descendent of the two parental strains of the preliminary study, each line inheriting
a unique mosaic of parental genes, it is possible to delineate regions of the mouse genome which
may account for some of the variation in pain proneness observed. Moreover, identifying such
regions can be followed by assessment of the known functions of the genes therein, with the
ultimate interest of identifying candidate genes mediating this pain spread. Uncovering such
causative genes, the verification of which would warrant subsequent gene validation experiments
(e.g., use of congenic or knockout strains of mice and transcription profiling) can elucidate
mechanistic components underpinning the pathology of pain spread following IONX, the genetic
basis of which has not yet been studied as of yet. The pathological substrates for pain spread
following IONX revealed in this way may prove to be of clinical relevance to other forms of
chronic pain development, and/or to a better understanding of the mechanisms of pain in mice,
and likely also in humans (see 1.5).
1.10 HYPOTHESES
Hypothesis A: IONX in mice causes altered mechanical responsivity in extra-territorial body
loci.
Hypothesis B: Normal and altered mechanical responsivity in extra-territorial body loci
following IONX are gender-specific.
Hypothesis C: Normal and altered mechanical responsivity in extra-territorial body loci
following IONX are controlled by genetic determinants.
1.11 AIMS and RATIONALE
These Hypotheses will be addressed in the following way:
AIM-1:
I. To determine the inter-individual mechano-responsivity of naïve mice of the A and B strains
and their descendant AXB-BXA mice, by analyzing the following aspects:
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Adaptation versus sensitization to the stimulus: Basal mechano-responsivity will be tested by
recording nocifensive reflexes in response to 7 trials, each administering a punctate mechanical
stimulus in the ‘Von Frey method’ (see Methods) to a select number of body loci (i.e., ears,
hindpaws, and tail). Repeating the stimulus 7 times will be done in order to identify the typical
response to this stimulus. However, rather than a typical, stable response, mice could present an
unstable pattern manifesting in either adaptation or sensitization to the repetitive stimulation.
Side differences: While there is no expectation that such differences manifest in naïve mice,
there may be unintended procedural flaws that could have wrongly presented as side differences.
Moreover, not finding such differences could justify compiling the data for bilateral body sites,
thereby increasing the number of observations and improving the statistical power.
Differences across testing periods (BL1 versus BL2): Carrying out the testing session twice,
several days apart, will allow for: (i) a training session of the animals in the testing paradigm, (ii)
a better characterization of a typical response pattern to the mechanical stimulus that is not
biased by possible diurnal fluctuations, and (iii) test whether or not mice learn from their
experience in the first session and alter responsivity to the stimuli in the second.
Correlation of responsivity to mechanical stimulation across select body loci: The aim of this
analysis is to test whether or not responsivity of naïve mice to stimulation of the ears
(representing a craniofacial locus), is different from the hindpaws and tail (representing spinal
loci). It is anticipated that the ears are more sensitive than the hindpaws and tail, but also that
responsivity is a global trait that is mouse-specific across all body loci.
Mechanical Place Avoidance Test (M-PAT): While the responsivity to a punctate stimulus is
considered a nocifensive reflex that is largely controlled by segmental mechanisms (4), It is of
interest to assess ‘higher order’ processing of nociceptive stimuli, which will be tested by way of
an adapted place avoidance test, that presents mice with the option of avoiding a region having a
floor that has an aversive texture/roughness by choosing to stay in a nearby region with a floor of
a non-aversive quality. Correlating between M-PAT behaviour and responsivity to Von Frey
filament stimulation will allow for assessment of how much of the variance in the reflexive
behaviour can be explained by variance in ‘higher order’ behaviour, and the extent to which these
are independent behavioural entities.
II. To characterize the changes in mechanical responsivity following IONX, by analyzing the
following aspects: (i) Does the mechanical responsivity of mice post-IONX differ from sham-
operated mice in two postoperative sessions (days 14 and 21 PO), each group normalized in
19
relation to its basal mechanical responsivity (using the same parameters as in Aim-1 above) (ii)
The extent to which such injury alters the responsivity to mechanical stimuli in extra-territorial
body loci located further away caudally from the ION receptive field, will be assessed.
As for AIM-1(I), correlating between the M-PAT avoidance behaviour test and testing
the responsivity to Von Frey filament stimulation in IONX denervated and sham-operated mice
will allow for assessment of whether or not IONX affects segmental processing of mechanical
responsivity and/or ‘higher order’ behaviour. Note that in the somatosensory system the perioral
region is the most rostral in the body and ears, hindpaws, and tail are located further and further
caudally.
AIM-2 (gender specificity):
I. To compare the inter-individual extent of basal mechanical responsivity and extra-territorial
spread of mechanical responsivity post-IONX versus sham operation between males and
females.
Mechanical responsivity of naïve mice of the A and B strains and their descendant AXB-
BXA mice was compared between genders. This comparison will be repeated for IONX-injured
and sham-operated mice of the same lines-strains, by using the same parameters as done in
AIMs-1,2 above.
II. To compare the M-PAT avoidance behaviour of male and female mice and correlate between
this behaviour test and responsivity to mechanical responsivity.
AIM-3 (genetic considerations)
I. To characterize the inter-line/strain differences in the mechanical responsivity of naïve male
and female mice of the AXB-BXA RI lines and their parental strains, and repeat this analysis for
IONX-injured and sham-operated mice of the same lines/strains, by using the same parameters
as in AIMs-1,2 above.
II. To characterize the inter-line/strain differences in the M-PAT assay.
III. To map QTLs controlling the inter-lines/strains variability in basal mechanical responsivity
and the changes that IONX causes to this responsivity, including the extent of extra-territorial
pain spread post-IONX, in mice of the AXB-BXA RI lines and their parental strains.
IV. To identify candidate genes in these QTLs that control basal mechanical responsivity and
changes that IONX causes to this responsivity.
20
2.0 METHODS
2.1 Animals
Breeding nuclei were acquired from Jackson Laboratories (Bar Arbor, MI) and inflated
on demand at the vivarium of the Faculty of Dentistry. Data from both genders of the following
recombinant inbred lines/strains was obtained: AXB-1/, AXB-2/ AXB-4/, AXB-5/, AXB-6/
AXB-8/, AXB-10/, AXB-12/, AXB-13/, AXB-15/, AXB-19/, AXB-24/, BXA-1/, BXA-2/,
BXA-4/, BXA-7/, BXA-8/, BXA-11/, BXA-12/, BXA-13/, BXA-14/, BXA-24/, BXA-25/
(names of all lines/strains include the suffix PgnJ). As well, data was collected from the 2 strains
parental to the above lineage: A/J and C57BL/6J. Mice were between 8 to 12 weeks of age and
between 25 to 30 grams during the span of the experiment. Each tested group consisted of
between 3 to 10 subjects. Subjects were identified and coded with a permanent marker applied
to the proximal third of the tail. Food (Harlan Rodent Standard Brand) and tap water were
supplied ad libitum. Operated mice received the food in a jar softened with tap water. Mice were
operated on either the left or right side, at random.
2.2 Surgical Procedures
a) IONX: Mice were anaesthetized by inhalation of Halothane. The upper lip on one side was
retracted and kept for the duration of the operation at this position to expose the vestibulum. The
side operated randomly alternated between animals. A 3mm incision was made with a #11
scalpel underneath the upper lip, on the lateral aspect of the vestibulum. Separating the mucosal
flaps exposed the ION, which is situated underneath the levator labii muscle. The ION was
transected with iridectomy scissors. The wound was not closed with sutures due to its small size.
The upper lip was replaced to its natural position, it closed the wound flaps adequately. A small
pledget of the haemostatic agent Surgicell was applied to the wound to seal it and prevent food
particles from entering the wound, and promote healing. The anaesthesia was then discontinued
and the mouse was placed in a resuscitation chamber, heated to 32oC, until the mouse was on its
feet and resumed normal awake behaviour that included grooming, walking, social interaction,
exploration, etc. At this stage the mouse was placed back in its cage.
b) Sham surgery: The sham surgery entailed an identical procedure as above except for cutting
the ION.
21
2.3 VF Filament Testing
(i) VF monofilament: A single nylon monofilament was used
and produced from a Berkley 4-lb breaking-force fishing wire.
The filament was attached to a plastic rod with glue, then
trimmed to calibrate it to a bending force of 0.2g by applying
it perpendicularly on a digital scale until bending to a C-shape
and recording the bending force as the weight (in grams),
averaged across 10 trials (see Table 1, with SEM values).
Choosing this stimulus intensity involved a compromise
between our intention to show at each locus inter-individual
and inter-strain/line differences in threshold (see below) and
suprathreshold (see below) responses, and our preference to
use only one stimulus intensity that would enable high-
throughput phenotyping that was needed due to the large
number of tests. Using the same intensity also facilitated
direct comparisons of the responsivity in the three tested loci
(i.e., ears, hindpaws and tail).
Table 1: Weight measurements using digital scale for 10 trials of Von Frey Hair application
Trial # Force (g)
1 0.21 2 0.19 3 0.18 4 0.17 5 0.21 6 0.21 7 0.18 8 0.22 9 0.2 10 0.21 average 0.198 SEM 0.0053
A 0.2 g filament was selected as a stimulus for all body sites. When applied to the ears, a
filament having 0.2g bending force elicited, in all naïve mice, a threshold response (see
definition below), but only in a subset of these mice a threshold response in the hindpaws and
tail, as well as suprathreshold responses (see definition below) in some of these three tested loci.
(ii) Restraining Cages: Mice were placed in two types of restraining cages to limit their ability
to avoid stimulation by running around, but spacious enough to stand, sit, and turn around with
some difficulty. For testing the ears, the mice were placed in a small cage (4X7cm, 4cm high)
produced from mesh metal, fixed by screws to a block of wood, and varnished by a protective
lacquer (Figure 1). For testing the paws and base of the tail the mice were placed in another cage
(8.5cm in diameter, 9.5cm high) produced from an opaque plastic cylinder with mesh metal as its
floor (Figure 2). This cage enabled the hindpaw plantar surface to be accessible to Von
Frey filaments applied from underneath without the mouse having visual cues about impending
stimuli.
22
Figure 1a,b:
a b
Figure 2a,b:
a b
(iii) Testing racks: Testing the ears was done concurrently on 25 mice, one of each line-strain,
gender and surgery group (i.e., IONX, sham-operated or unoperated naïve), each placed in an
individual cage (as in Fig. 1a) on a stairwell-like rack, in 3 rows separated by an opaque wall so
as to visually isolate each mouse from its peers. Testing the paws and tail was also done
concurrently on 25 mice in individual cylindrical cages, suspended from 2 racks, each holding up
to 14 cages (Figure 2b) enabling access of the experimenter to the tested loci from underneath,
through the mesh metal floor.
(iv) Response scale: It should be noted that the terms, “responsivity” and “sensitivity”, although
commonly have two distinct meanings, will heretofore be used interchangeably in this study. As
well, the terms “hyperresponsivity” and “hypersensitivity” will be used interchangeably. A
threshold response was considered a "uni- or oligo-segmental response" typically involving
muscles associated with moving the stimulated locus only, responding in a reflex, ballistic
manner, whereas a suprathreshold response entailed a "multi-segmental response" of the whole
body, necessitating considerably more complex coordination of many muscle groups including
Figure 1a,b: Side view (a), and front view (b), of a mouse in the restraining cage used for Von Frey testing of ears.
Figure 2: a. Underneath view of a mouse in a cylindrical testing chamber used for Von Frey testing of the hindpaws and tail. b. View of the testing rack used to hold multiple testing chambers for high throughput Von Frey testing of tail and hindpaws.
23
those moving the stimulated locus as well as those of the torso and extremities, and likely
implementing complex supra-spinal neural processing.
Facial responses: The stimulus intensity elicited in the ears a threshold response comprising a
slight withdrawal reflex, which entailed a twitch of the ear and head away from the stimulus. In a
variable percentage of applications, this stimulus elicited a suprathreshold response that
comprised any response supplementary to the threshold reflex, including a robust head jerk,
whole body turning, facial long washing strokes with the forepaws (4), or a rapid head shaking
episode that could be interpreted as an attempt by the mouse to rid itself of a stimulus that may
have been perceived as painful, or potentially painful, that occasionally lingers on as an “after-
sensation” (4). The ears were tested as a site innervated by a separate major branch of the
trigeminal nerve, as well as other cranial nerves and those of the cervical spinal cord.
Hindpaw responses: A threshold response manifested in a slight twitch or paw withdrawal, and
a suprathreshold response included a robust paw flick occasionally accompanied by licking,
extended paw elevation, and whole body turning. The forepaws were not tested due to difficulty
in applying a stimulus to them, as this region is frequently in motion as the animal grooms itself
and probes its surroundings.
Tail responses: A threshold response was counted as a faint tail flick and suprathreshold
responses were expressed as a robust flick accompanied occasionally by head turning to the tail
and whole body turning. The presence of a threshold or suprathreshold response in any tested
locus was recorded to a PC computer Excel spreadsheet. A handheld small numeric keypad
facilitated rapid data entry of the values: 0=for no response, 1=for a threshold response, and
2=for a suprathreshold response, and a high throughput phenotyping.
(v) Procedure: Behavioural tests were conducted from 9am to 6pm. Mice were transferred from
their habitat to the testing room, and individually placed into the restraining cages and left to
habituate for 20 min. This study was planned to phenotypically screen mice of 25 different
lines/strains, in a statistically powered number of mice per surgery group (IONX, sham-operated
and naïve animals), and gender, in a number of stimulus repetitions that would enable a faithful
determination of the typical response type per body locus, per group, repeated in two testing
sessions. Thus, the developed sensory testing protocol enabled to collect response data in a high-
24
throughput fashion that involved testing each locus in a rapid succession for all 25 mice on one
side, then for all mice in the subsequent round on the other side, and so on until completing 7
trials/side/animal/25 mice. The stimulated side order was random. The stimulus presentation
order for the hindpaws and tail was different than in the ears, beginning with one hindpaw tested
for all 25 mice in rapid succession, followed by the other hindpaw for all 25 mice, and then the
tail for all mice. Thus, the interval between two successive stimuli of the same locus was 5-6
minutes, long enough to prevent peripheral receptor sensitization. If on one testing day the right
ear was stimulated first, the other ear was the first to be stimulated on the next testing session.
This sequence was repeated 7 times/locus/session, in two testing sessions 2 days apart, in the
week preceding surgery. These testing sessions are referred to as Baseline 1 (BL1) and Baseline
2 (BL2), respectively. Since BL1 was the first instance the mice were exposed to the stimuli, this
testing period was regarded as a training session. All mice were also tested on days 14 and 21,
following surgery. Preliminary work showed that by PO day 14, the model had reached its peak
effect of pain spread and this was sustained for several more weeks. These sessions are referred
to as Postoperative day 1 (PO1) and Postoperative day 2 (PO2), respectively. These testing
periods were chosen based on preliminary results showing that this pain model manifests peak
symptoms both in A/J and C57BL/6J by day 21 following surgery (157).
2.4 Mechanical Place Avoidance Test (M-PAT)
Mice were placed in a chamber (16cmX8cm, 20 cm high) with opaque plastic walls (Figure
3a), equally divided into two zones: an abrasive/rough region and a smooth one (Figure 3a).
Figure 3a-c
Activity of the mouse was recorded by a Microsoft webcam camera (640X400 pixels),
placed over an arena comprising 8 testing chambers (Figure 3b). AnyMAZE software (Version
4.3; Stoelting, Inc.) was used to record locomotor activity and quantify various aspects of the
a b c
Figure 3a-c: a: View from above of a mouse in the M-PAT testing chamber showing the two zones of different granularity. b: View from above of 8 mice being tested in their individual chambers. c: View of a mouse in its testing chamber as monitored by the ANY-Maze software.
25
activity per zone, including the distance walked and time spent in each region (see the
photograph of the PC computer monitor in Figure 3c). The AnyMAZE software computes for
every mouse the time, distance and speed of walking in the two regions. Group averages and
SEM values were plotted in histograms. Significance of the differences in the groups’ averages,
when comparing these parameters for walking in the two regions in the BL1 versus BL2 and
BL2 versus PO1 and PO2 sessions, was evaluated using a paired t-test, separately for the two
genders, adjusting for multiple comparisons using the Bonferroni factor, and the results
presented in a tabulated form and in histograms. Next, the responsivity in the tested body loci to
VF stimulation and the M-PAT parameters was correlated between, by calculating the Spearman
rank correlation coefficient (rho) and the p values associated with these coefficients, adjusted by
the Bonferroni factor. This was done for BL1 vs. BL2 and BL2 vs. PO1 and PO2 sessions,
separately for males and females. BL2 was used to compare with postoperative scores due to its
closer proximity in time to such sessions.
2.5 Effect of Stimulus Repetition on Mechano-responsivity
For every tested body locus, the frequency (in %) of No Responses, Threshold Responses
and Suprathreshold Responses was calculated for mice of all 25 lines-strains, separately for 192
males and 190 females, separately for stimuli numbers 1, 4 and 7, and for each testing session.
The distribution of these response types is shown graphically (Figure 4). This was followed by
computing, for each group, the significance of the differences in the distribution of the 3
response types in trials 1, 4 and 7 by the χ2-test. P≤0.05 was considered significant (for all tests).
Results are shown in a tabulated form. Since multiple comparisons were made, potentially
inflating the alpha level, a conservative approach was taken and the alpha level was adjusted by
applying a Bonferroni correction.
2.6 Side Differences
The distribution of response types to stimulation of the two ears and of the two hindpaws
was compared in each group for trials 1-7 using the χ2-test adjusted by the Bonferroni factor. The
results are charted using histograms.
2.7 Differences across Baselines and Surgery-induced Changes in Sensitivity
Since no side differences were found, data of the two sides was combined (as follows) for
subsequent data analysis. For each of the 7 trials the responses were recategorized as 0 if the
26
stimuli elicited No Responses in both ears, 1 if there were two occurrences of “Threshold
Responses” (one in each ear), or one occurrence of “No Response” in one ear and a
“Suprathreshold Response” in the other, or one occurrence of “No Response” and one
“Threshold Response”, and 2 if there were two occurrences of “Suprathreshold Responses” (one
in each ear), or a “Suprathreshold Response” in one ear and a “Threshold Response” in the other.
These new response categories reflected three levels of overall responsivity to the stimulus in the
two sides: “None”, “Weak”, and “Strong” responsivity (Table 2). The distributions of these
categories are presented graphically.
Table 2: Recategorization of the response types of “No Response”, “Threshold Response” and
“Suprathreshold Response” (in brackets – their values in the Excel and SPSS datasheets) based
on their occurrence in the right and left sides of the body. If a certain type occurred in response
to a stimulus in the left side and the same or a different type in the right side – a new Score was
assigned as shown below. This Score was then categorized into None (for a Score=0), Weak (for
a Score=1), and Strong (for a Score=2).
Right Left Score Response Category
No Response (0) No Response (0) 0 None
No Response (0) Threshold Response (1) 1 Weak
No Response (0) Suprathreshold Response (2) 1 Weak
Threshold Response (1) Threshold Response (1) 1 Weak
Threshold Response (1) Suprathreshold Response (2) 2 Strong
Suprathreshold Response (2) Suprathreshold Response (2) 2 Strong
These new response categories, which lumped the responses to stimulation in the right
and left body loci, were compared between the two baseline testing periods (BL1 and BL2) for
each trial, separately for each locus and gender (Table 6). For the tail, the original response types
were used (since there was no need for lumping over the two sides of the body).
The distribution of None, Weak and Strong response categories in the 4 testing sessions
(BL1, BL2, PO1, and PO2) was compared using the χ2-test, adjusted by the Bonferroni factor.
The results for trials 1-7 were charted using histograms. I also calculated for every mouse a
Cumulative Difference Score by combining the scores of the 7 trials, and then subtracted the
27
Cumulative Difference Score of BL2 from the cumulative difference Score of PO1 and PO2, and
then separately averaged the resulting values for sham-operated and IONX, separately for males
and females.
Thus, BL2 data, collected one to two days prior to surgery, were selected to represent the
baseline condition. Comparing PO1 - BL2 and PO2 – BL2 average group (±SEM) data was done
by paired t tests.
2.8 Correlation of Responsivity across Body Loci
The distribution of ‘None’, ‘Weak’ and ‘Strong response’ categories in each locus was
correlated to every other locus separately for trials 1-7 for each testing session and gender,
computing the Spearman rank correlation coefficient and P values, adjusted by the Bonferroni
factor. The results were presented graphically using pie charts.
2.9 Surgery-induced Changes in Mechanical Responsivity
A Chi Square-test was used to compare the frequency of ‘No response’, ‘Threshold’ and
‘Suprathreshold response’ types, for each trial, comparing for each surgery type and gender
between PO1 and BL2, and PO2 and BL2, and presented the χ2 and P values in a tabulated form,
highlighting the significant values, and the distributions in histograms that included the
following graphic representation of the significant values: * =0.05 ≤ P > 0.01; ** =0.01 ≤ P >
0.005; *** = P ≤ 0.005; a trend of significance was designated by # = 0.05 < P ≤ 0.1. In bold font
are highlight P values that remained significant after a Bonferroni adjustment of the alpha level.
2.10 Spread of Mechanical Hyperresponsivity
Traditionally, mechanical allodynia is defined as a reduced pain threshold, demonstrated
by the appearance of a nocifensive response to a stimulus that is normally innocuous. Reduced
pain thresholds can be demonstrated by producing a stimulus-response curve, using a set of Von
Frey filaments having ascending bending forces (20). Each stimulus intensity is repeated a
number of times to determine the “typical response” for that intensity. Repeated stimulation with
a subthreshold intensity results in 100% of the trials not eliciting a response. Increasing the
intensity, still within the innocuous intensity range, is expected to elicit a few nocifensive
responses out of, say 10 trials. Allodynia can then be defined in a different way, i.e., the intensity
that elicited a statistically increased incidence of limb/head withdrawals out of N trials,
compared to a baseline incidence that is <50% (20). An intensity that elicited in the baseline an
28
incidence >50% is referred to as noxious stimulus, and on this basis, mechanical hyperalgesia
can be defined as a significant increase in the response incidence compared to the baseline rate.
A slightly different approach was used, combining allodynia and hyperalgesia into “hyper
responsivity”. For every trial a recategorized response type was computed that combined the two
sides of the ears and separately of the two sides of the hindpaws, and calculated a Cumulative
Score for the 7 repeated trials, for every testing session. Next, the Cumulative Score of the
second baseline session (BL2; see rationale above) was subtracted from the second postoperative
testing session (PO2). Positive difference scores indicated an increased responsivity following
surgery (compared to the BL2) and were categorized as ‘HYPER’. Difference scores not meeting
this criterion (i.e., negative or 0) were categorized as ‘NOT’. This was done separately for the 3
tested body loci (ears, hindpaws, and tail) and separately for each surgery group (sham and
IONX). Next, every mouse was assigned a ‘Spread Type’ based on the combination of these
categories (i.e., HYPER and NOT) across the 3 body loci, using the 8 possible permutations
(Table 3). The incidence of the various Spread Types was then counted for the whole cohort, and
the % incidence of the two most-widespread hyperresponsivity Spread Types (i.e., ‘Hyper-
responsivity spread to all tested loci’, and ‘Hyper-responsivity to only the hindpaws and tail’) was
then calculated for the mice of each strain-line, separately for the sham operated and IONX
mice, and the subtracted values of IONX(%) – Sham(%) were defined as the IONX-produced
most-widespread hyperresponsivity types for QTL mapping.
Table 3: Catagories of pain spread patterns observed in mice following IONX as measured by
VF filament stimulation. “NOT” denotes a lack of change in responsivity in the corresponsing
body locus when comparing levels prior to, and following surgery. “HYPER” denotes
hyperalgesia observed at that locus.
29
2.11 QTL Mapping
For interval mapping purposes, since there was no significant side differences found, the
recategorized response types were used, for which each of the 7 trials combined the responsivity
of the two ears or paws. Next, a single weighted (WS) score was computed for every mouse,
combining the recategorized response types of all 7 trials in BL1 and the 7 trials of BL2, into a
single value that also assigned twice the weight for the occurrences of Strong responses
compared to Weak ones, and no value to None, as shown in the following equation:
WS = (NWEAK +2NSTRONG ) / (NNONE+WEAK+ STRONG)
BL 1 and BL 2 were combined in order to boost the sample sizes. This WS was
calculated for BL1+BL2, PO1 and PO2, for every mouse, and then averaged for each strain-line,
gender, surgery group, and testing period. Since several lines did not have enough mice for a
specific gender, the data of the two genders was combined for each strain-line in the population.
A difference score was calculated for each strain-line, subtracting the combined BL value from
PO1 and PO2 values, separately for sham and IONX. Finally, the average sham group was
subtracted from the IONX group/strain-lines, resulting in 25 values, one per strain-line denoting
the net-IONX effect/strain-line.
The 23 unique RI lines and 2 parental strains that were used in this study were all
genotyped using the Welcome-CTC-Illumina microarray for the 8,514 informative
markers (microsatellites and SNPs), which were used in QTL mapping in this study (160).
Web2QTL, an online genomic mapping software (160), was implemented by inputting all
phenotypic values as calculated per strain-line, per gender, per surgery group or combined into a
net-IONX effect. For interval mapping the software computes the significance of linkage
between each genetic locus and trait variance.
2.12 Pearson Product-moment Correlations
The linear correlation between various traits measures in this study was determined by
calculating the Pearson product-moment coefficient and P values. This was done using the
biostatistics software SPSS (version 18.0).
2.13 Bootstrap Test
The Bootstrap Test repeats the interval mapping process 1000 times, each time randomly
eliminating the value of one strain-line from the population of 25 values, and replacing its value
with that of another strain-line in the panel, which therefore, is represented twice. This way, the
30
software conducts an interval mapping 1,000 times and in each reiteration it records the genetic
location of the peak of the QTL having the highest Likelihood Ratio Statistic (LRS) value (see
below), stacking these locations and plotting the percentage of times out of 1,000 that a peak
was found at a certain locus. A value below 10% at a putative QTL indicates the presence of
outliers in the trait values for the population.
2.14 Permutation Test
This test outputs a P value that indicates how confident one can be that the reported QTL
is not a result of mere chance. The trait values of the strains-lines are reassigned randomly,
followed by remapping. This is done 1000 times, each for a different reassignment of values. If
the original QTL is shown to have a significance of 0.005, this indicates that it achieved higher
LRS values than were obtained in 95% of the random sampling maps. This suggests that there is
a relatively low probability that putative QTL had been identified out of chance alone, and a
much higher probability that it was detected due to conferring an actual causal link to the
phenotype of interest.
2.15 Narrow-sense heritability This is defined as the degree to which additive genetic variance controls the total variance of the
trait in the population. The narrow-sense heritability (h2) is computed by calculating the trait
variance between strains-lines ('allelic variance', VARgen) divided by the total trait variance that
comprises the sum of the allelic variance (VARgen) and the average variances within the lines-
strains (VARenv). The formula for computing narrow-sense heritability is as shown below:
h2 = VARgen / (VARgen + VARenv)
2.16 Additive Effect of a QTL
This can be viewed as a measure of the expected effect on the phenotype of substituting
one allele at each marker locus along the genome with its alternate allelic form. Since each
strain-line possesses pairs of identical chromosomes, comparing the observed effect of allelic
substitution is always superimposed by a second identical allelic substitution. Therefore, to
estimate the additive effect of a single allelic substitution, there was computation of the
difference between the average phenotypic score of individuals with a particular allele at the
marker locus and the average score for those with the alternate allele at this marker locus. This
31
difference was then divided by two, to estimate the effect on phenotype of a monoallelic
substitution at the putative QTL.
2.17 Haplotype Analysis
This feature of the Web2QTL software provides a graphic representation of the haplotype
sequence possessed by each strain-line in the study, throughout the entire genome, stacked by the
ascending trait values order, one on top of the others. Haplotypes inherited from parents A and B
are coloured differentially, thereby allowing the user to note which strain-line inherited which
parental allele at specific loci and identify ‘outlying’ strains-lines in the local haplotypic
structure of the QTL for which the inherited haplotype does not fit with the trait value as
expected from inheriting the genotype and phenotype of the fitting parental strain. For such
outliers, the misfitting trait values must be controlled by other genetic loci located elsewhere on
the genome, identified as additional QTLs.
32
3.0 RESULTS
The main findings/conclusions are summarized above each section, followed by a
detailed description of the results (including statistics and graphs) from which these conclusions
were drawn. To facilitate the readability of this chapter most Tables and some Figures appear in
the Appendix.
3.1 Mechanical Sensitivity in Naïve Mice
3.1.1 Adaptation versus Sensitization to the Stimulus
Main findings: No instances of adaptation to the repeated mechanical stimuli were seen.
Transient and progressive sensitization types were seen in the hindpaws and tail, respectively.
NO response to repeated stimulation was observed with respect to the ears.
Detailed analysis: Despite the sufficiently long inter-stimulus interval (~5-6 min), it is possible
that stress caused by single caging in a restrictive cubicle, and the repetitive stimulation, may
have caused analgesia that could manifest in adaptation to the stimulus along the 7 trials of the
testing session. Alternatively, these conditions may have produced sensitization to the stimuli.
Either way, if these processes were prominent, it would be difficult to characterize a typical
response type for all 7 repetitions as a basis for further data analysis. To test these two
possibilities, the data were taken from all mice regardless of their genetic background, and
analyzed separately for each gender. This was achieved by comparing for every tested body
locus the rate of various responses (i.e., No Response, Threshold Response, and Suprathreshold
Response) in trials 1, 4, and 7 (Figures 4a-f for males and Figures 4g-l for females).
Repeating the stimuli in the same tested locus 7 times over the course of ~1.5 h caused
two types of changes across trials 1, 4 and 7 (Figure 4): (1) ‘Progressive hyperresponsivity’
(suggesting progressive sensitization). This type was seen in the left hindpaw, both in BL1 and
BL2, as an increase in the combined response rates for the Threshold and Supra-threshold types
(green arrows), both for males and females, (2) ‘Transient hyperresponsivity’ which was
manifested in a more complex change in responsivity that resembled an inverted U-shape
distribution, where the response rate increased from the 1st to the 4th stimulus but was normalized
by the 7th trial (Table 4 in Appendix).
33
0%20%40%60%80%100%
Response type frequency EarsSup
rathresh
old
Thresho
ld
NO_Res
ponse
LeftRigh
t
0%20%40%60%80%100%
Response type frequency Paws
Suprath
reshold
Thresho
ld
NO_Res
ponse
LeftRigh
t
**
0%20%40%60%80%100%
Response type frequency TailSup
rathresh
old
Thresho
ld
NO_Res
ponse
***
0%20%40%60%80%100%
Response type frequency EarsSup
rathresh
old
Thresho
ld
NO_Res
ponse
LeftRigh
t
0%20%40%60%80%100%
Response type frequency Paws
Suprath
reshold
Thresho
ld
NO_Res
ponse
LeftRigh
t ****
0%20%40%60%80%100%
Response type frequency TailSup
rathresh
old
Thresho
ld
NO_Res
ponse
******
BL1 BL2
M A L E S
Figure 4a-l: The proportion of response types to stimulation of the VF filament for trial numbers
1,4, and 7, separately for BL1 and BL2. ‘No Response’ is shown in blue bars, ‘Threshold
Response’ in black bars, and ‘Suprathreshold Response’ in red bars; stimulation of the tail in
males (a,b) and in females (c,d), stimulation of the hindpaws in males (g,h) and in females (i,j),
stimulation of the ears in males (e,f) and same in females (k,l). For these Figures, and
heretofore, * designates 0.01<p≤0.05; ** 0.005<p≤0.01 and *** denotes p≤0.005. Yellow and
green arrows indicate transient and progressive changes in responsivity respectively.
Figure 4a-f:
Figure 4g-l:
a. b.
c. d.
f. e.
BL1
BL2
Tail Tail
Paws Paws
Ears Ears
34
A transient hyperresponsivity was mainly seen in the tail, both in BL1 and BL2, both in
males and females, but also in the right hindpaw of both genders and left paws for females
(orange arrows). Repeated stimulation of the ears did not show a change in responsivity
throughout the testing session, neither in BL1, nor in BL2, in both genders. In the appendix,
Cells in Table 4, marked by a bold frame, show values that survived a Bonferroni adjustment,
were found for both genders, mainly for 7 stimuli in the tail and in the left hindpaw during BL2.
Thus, this analysis shows that both for males and females, in BL1 and BL2, there was a
difference in responsivity across repeated trials (comparing trials 1,4, and 7) in the two tested
spinal loci (i.e., paws and tail) that manifested in spinal loci presenting a progressive or transient
hyperresponsivity, but the ears, representing a craniofacial region, showed no such pattern.
0%20%40%60%80%100%
Response type frequency Ears
Suprath
reshol
d
Thresh
old
NO_Re
sponse
Left
Right
0%20%40%60%80%100%
Response type frequency Paws
Suprath
reshol
d
Thresh
old
NO_Re
sponse
Left
Right *
#
0%20%40%60%80%100%
Response type frequency TailSup
rathres
hold
Thresh
old
NO_Re
sponse
***
0%20%40%60%80%100%
Response type frequency Ears
Suprath
reshol
d
Thresh
old
NO_Re
sponse
Left
Right
0%20%40%60%80%100%
Response type frequency Paws
Suprath
reshol
d
Thresh
old
NO_Re
sponse
Left
Right #
***
0%20%40%60%80%100%
Response type frequency TailSup
rathres
hold
Thresh
old
NO_Re
sponse
******
F E M A L E S
BL1 BL2
g. h.
i. j.
l. k.
Tail Tail
Paws Paws
Ears Ears
35
3.1.2 Side Differences
Main finding: Generally no side differences were seen in the responsivity of the paws and ears.
Detailed analysis: Genetic mapping of QTLs is done by inputting a single trait value for each
tested inbred mouse strain-line, therefore, the more mice tested per strain-line – the more
representative this single value is for that strain-line. Combining the values of the right and left
tested sides of the body could be one way of achieving the goal of increasing the data points per
strain-line. While we are not aware of side differences reported about the responsivity to
nocifensive tests anywhere in the body of rodents, the following analysis was done to
substantiate this claim, justifying pooling the data across body sides in subsequent analyses.
Having noted that repeated mechanical stimulation caused hyperresponsivity in the paws and tail
(see 3.1.1 above), data of all 7 trials cannot be lumped into a single value per mouse and then per
strain-line. However, it is possible to lump the data of the two sides for each trial by itself. For
each trial, a comparison was made for the distribution of response types across the right and left
sides, for each tested locus, separately for BL1 and BL2, and separately in males and females. As
shown in Table 5 (Appendix), only 4 out of 56 right-to-left comparisons showed significant side
differences between the distribution of response types to the same stimulus in the same locus and
trial number. Two of the four were marginally significant, and after adjustment of the alpha level
due to multiple comparisons, none of these 4 comparisons remained significant. Therefore, the
conclusion drawn from this analysis is that there are no side differences in mechanical
responsivity, justifying pooling data of both sides for subsequent analysis.
3.1.3 Differences across Testing Periods (BL1 versus BL2)
Main finding: Mechanical responsivity did not differ across the 2 baseline testing sessions at
any tested loci.
Detailed analysis: Since no side differences were found, occurrences of the “No Response”,
“Threshold Response” and “Suprathreshold Response” types were combined for both ears and
both paws, separately for each of the 7 trials, by recategorizing these response types. As shown
in Table 2 (Methods), if there were “No Responses” for both ears, or both paws, these trials were
scored a value of 0. A value of 1 was scored for trials having an occurrence of two “Threshold
Responses”, or one occurrence of “No Response” and one “Suprathreshold Response”, or one
occurrence of “No Response” and one “Threshold Response”. A value of 3 was scored for trials
36
having occurrences of one or two “Suprathreshold Responses” or one “Suprathreshold
Response” and one “Threshold Response” type. These new response categories reflected three
levels of overall responsivity to the stimulus: “None”, “Weak”, and “Strong” responsivity
(Methods, Table 2).
It was found that both for males and for females, there was no significant difference
between BL1 and BL2 in the response profile of stimulation in the ears. In the paws, for males it
was found that the first 4 trials showed significant differences in BL1 versus BL2. Figure 6a,b
and Table 6 (Appendix) show that for the paws and tail, both males and females had trials
showing significant increases from BL1 to BL2 in the “Weak” responses (for the paws) or
Strong responses (for the tail). However, following a Bonferroni correction, none of these
comparisons remained significant anywhere tested, suggesting that repeating the session twice
had no effect on the animal learning to avoid the stimuli or increasing the responsivity to these
stimuli. Thus, based on these results, it was concluded that data can be pooled across BL1 and
BL2 into a single value per site (i.e., ears, paws, and tail) for both for males and females and
used for further analyses.
3.1.4 Correlation of Mechanical Stimulation Responsivity across Tested Body Loci
Main findings: A gradient in mechanical responsivity was seen across the tested loci in the
order of: ears>paws>tail. Significant correlations were seen between the responsivity in ears
and the 2 other loci, and only moderate correlations between the paws and tail.
Detailed analysis: In this analysis, a comparison was made for the mechanical responsivity of
each locus to every other locus, separately for each trial and for each baseline period, as well as
for each gender. Table 7 (Appendix) shows the results of a Chi Square test that compared the
instances of None, Weak and Strong responses in the ears and paws and the No response,
Threshold, and Suprathreshold responses in the tail, for BL1, BL2, males and females.
Table 7 on page 38 shows that the distribution of response types is significantly different
across the body sites. Figure 6 shows a schematic representation of the body of the mouse
including the three loci tested in this study. The pie charts in Figure 6 show the average
distribution of response types across the 7 trials for each locus, separately for BL1 and BL2,
males and females. The charts for the ears show that there were no “No responses”, whereas such
responses were seen in the paws as well as in the tail.
37
Figure 5a,b: The proportion of bilateral response types in the hindpaws, ears and tail to the
stimulation with the VF filament in each of the 7 trials, in each baseline period, for males (a) and
females (b). Bilateral response types in the ears and paws were recategorized as described in the
text and methods. Those of the tail were retained in their original settings.
Trial number Trial number Trial number
Interestingly, the distribution in the paws and tail were also significantly different from
each other (Table 7). These graphs show that there is a gradient in responsivity: ears showed the
highest proportion of Suprathreshold/Strong responses, followed by the tail and finally the paws.
For Threshold/Weak responses, the order in decreasing responsivity was ears>paws>tail. The
order of No response/None was tail>paws>ears.
Figure 6: Schematic representation of the mouse’s body indicating the tested body loci (ears,
hindpaws, and tail), for which mechano-responsivity was tested. Pie charts near each locus
indicate the proportions of the bilaterally recategorized response types elicited from stimulation
at that locus, counted across the 7 trials, for BL1 and BL2, and for males and females. For each
BL1
BL2
BL1
BL2
a
b
38
Males Females BL1 BL2 BL1 BL2
Males Females BL1 BL2 BL1 BL2
BL1 BL2 BL1 BL2 Males Females
None / No response
Weak/ Threshold
Strong / Suprathreshold
of the trials, bilateral response type in the ears and paws were recategorized as “None”, “Weak”
or “Strong”, whereas responsivity in the tail used the original scoring values: “No Response”,
“Threshold Response”, and “Suprathreshold Response”.
Table 7: Chi Square tests comparing the proportion of response types across each body locus
(ears, hindpaws, and tail) in the baseline testing periods (BL1 and BL2) for males and females.
Testing period
Statistic
Gender Males Females
EARS vs. PAWS vs. TAIL PAWS vs. TAIL EARS vs. PAWS vs. TAIL PAWS vs. TAIL BL1 Chi 988.3 146.2 971.6 194.7
p <0.000 <0.000 <0.000 <0.000 BL2 Chi 832.9 208.6 738.1 184.4
p <0.000 <0.000 <0.000 <0.000
Table 8 (Appendix) shows the Spearman correlation coefficients and the p-values
associated with responsivity in the tested loci. P-values in red font yielded a value of p≤0.05, and
those highlighted in grey even passed a Bonferroni correction for multiple comparisons. In
males, comparing paws and tails in BL2, but not BL1, for trials 4, 5, and 7 showed significant
positive correlations with weak-to-moderate r values ranging from 0.24-0.28. In females, such
39
correlations were seen for trials 3 and 4 in both baseline sessions (with r values ranging from
0.23-0.27, respectively). However, no significant correlations were found when comparing the
ears versus paws (in both genders) or ears versus tail (in males). In females, the mechanical
responsivity of the ears significantly correlated with that of the tail in trial 4 (for BL1) and trials
3 and 5 (for BL2). Generally, most significant correlations in the responsivity between tested
body loci were noted for trials 3-5.
3.1.5 Mechanical Place Avoidance Test (M-PAT)
Main findings: Mice learned to prefer being in the rough zone in the M-PAT and mice walked
less distance in general across baseline sessions.
Detailed analysis: Figure 7 and Table 10 (Appendix) show parameters measured for males and
females comparing the mobility in each zone between baseline periods. In both genders, the time
spent in each zone was not significantly different between baseline sessions. However, the
travelled distance was significantly higher in BL1 than in BL2 for both zones (Table 10,
Appendix, Figures 7a-c). Table 9 (page 92) shows the P values of the paired t-tests that
compared the Time, Distance and Speed of mobility in the two zones. In BL1, neither males nor
females significantly preferred one zone over the other (p> 0.05). In BL2, in the rough zone,
females spent significantly more time and walked a significantly longer distance at a slower
speed. Males showed similar significant results but only for distance and speed in the rough
zone. Thus, this assay shows that from BL1 to BL2 mice learned to slightly prefer the Rough
zone that perhaps felt more similar to their cage bedding than did the Smooth zone. Note,
however, that while this preference was significant, the effect size was very minor.
3.1.6 Correlation between M-PAT Parameters and Responsivity to Von Frey Stimulation
Main findings: Mechanical responsivity in the hindpaws did not correlate with M-PAT
parameters, yet in the ears and tail showed some instances of correlation between the two
assays.
Detailed analysis: Arguably, applying a VF filament to the paw of a rodent elicits a sensation
that is not similar to the sensation perceived when standing or walking on a granular surface,
since the 0.2 gram VF filament exerts a punctate singular pressure whereas a granular surface
exerts its effect on all 4 paws against gravity, exerting ~25% of the body weight (~6 gram) per
40
paw. Finding a significant and positive correlation between mechanical responsivity to the VF
stimulation and M-PAT parameters in intact rodents could elucidate whether these sensations
share common mechanisms, justifying further analysis of whether this correlation changes post-
IONX versus sham operation.
Figure 7: Graphs comparing various parameters of the place preference assay; a. time spent in
each zone over a 3 min testing period; b. distance travelled in each zone over 3 min in the two
baseline periods for males and females; c. average speed travelled by the mice in each zone over
a 3 min period, by males (M) and females (F).
If corroborated, one may be able to use M-PAT parameters as predictors of hyper- or
hypo-responsivity following denervation procedures that are extra-territorial to the nerves of the
extremities.
0102030405060708090
100
M F
Time (
sec)
Gender
Time
BL1_smooth
BL2_smooth
BL1_rough
BL2_rough
0
0.2
0.4
0.6
0.8
1
1.2
1.4
M F
Distan
ce (m
)
Gender
Distance
0
0.005
0.01
0.015
0.02
0.025
M F
Spee
d (m/
sec)
Gender
Speed
*** *** ******
b
C
a
41
Spearman rank correlation coefficients were used to correlate in intact mice the data for
each of the 7 VF stimulation trials in the paws, tail, and ears against the time and distance
travelled in the M-PAT test, separately for males and females, and for BL1 and BL2. As shown
in Table 11a (Appendix), no significant correlations were found between the M-PAT parameters
of males and mechanical responsivity in the paws, for any trial of BL1 or BL2, and a similar
trend was found in females Table 11b (Appendix), with only a weak correlation coefficient for
trial 4 that did not reach significance following the Bonferroni correction.
Surprisingly, however, both for intact males and females, and both in BL1 and BL2,
highly significant correlations were found between several VF stimulation trials in the ears and
tail and the distance travelled on the smooth and rough zones of the M-PAT test, which survived
the Bonferroni correction (Table 11, page 93). Generally, both genders displayed the same trait
of a correlation in the distance travelled in both zones with increased responsivity to VF
stimulation in the ears and tail.
3.2 Changes in Mechanical Sensitivity following IONX
The presence of extra-territorial mechanical hyperresponsivity following IONX was
determined by comparing the responses of mice of the AXB-BXA RI lines and their parental
strains to VF stimulation in a select number of body regions not innervated by the ION (i.e., ears,
hindpaws, and tail), at two PO time points in mice post-IONX or sham operation, to those of the
same mice when they were naive (as determined in 3.1 above). In addition, parameters of their
mobility in the M-PAT place preference test that assessed avoidance of ambulation on a pro-
allodynic surface, was compared between baseline and post-IONX or sham operation. Since the
pro-allodynic attributes of the granularity of this surface are perceived via the sensory apparatus
of the paws, changes in place preference post-IONX versus sham operation arguably represents
extra-territorial hyperresponsivity of this region of the body or fear/anxiety caused by the IONX.
3.2.1 Side Differences
Main findings: No significant side difference was found postoperatively in the distribution of
response types between the ipsilateral and contralateral sides to the IONX or sham surgery.
Detailed analysis: The distribution of response types (No Response, Threshold Response and
Suprathreshold Response) were compared between the ipsilateral and contralateral sides, for
each of the 7 trials, for the ears and paws sham-operated mice and mice post-IONX, and
42
separately in males and females, and for the two PO testing periods, day 14 (PO1) and 21 (PO2)
postoperatively). As in the comparable analysis of the naïve mice (see 3.1 above), Chi Square
tests were used for this analysis. In none of these comparisons was there a significant side
difference in the response distribution between the ears or paws following surgery (Table 12,
Appendix), justifying pooling data bilaterally for the ears and paws for the subsequent analyses.
3.2.2 Differences between PO1 and PO2
Main findings: Only the tail in females showed a consistent difference between PO1 and PO2.
Detailed analysis: There was no significant difference between the scores measured for
mechanical responsivity in the ears and paws between PO1 and PO2, for IONX- and sham-
operated mice, males or females (Table 13, Appendix, p.96). However, for females but not
males, both the sham-operated and IONX mice showed a significant difference in the distribution
of response types of the tail that manifested as an increased proportion of the Suprathreshold
Responses from PO1 to PO2. The mechanical responsivity of the tail in females showed a
consistent difference between PO periods for both the sham-operated and IONX groups, which
for two of the trials remained significant after a Bonferroni correction. Therefore, it was decided
to not pool data for both PO time periods.
3.2.3 IONX-induced Effects on Mechanical Responsivity
Main findings: IONX induced an increase in responsivity to mechanical stimuli in the ears (in
males but not in females), and in the paws and tail of both genders.
Detailed analysis: The second baseline period (BL2) was used in the analysis (Tables 14,15;
Figures 8a-f), since it was the closest in time to the PO testing sessions. Moreover, the
confounding effects of training were minimized by using the second baseline session as a basis
for PO comparisons. Therefore, it was thought to be a more accurate reflection of a typical
response prior to surgery.
Figures 8a-f show distributions of response types for BL2, PO1, and PO2, for 7 trials,
separately for males and females, and tested body locus, as well as results of Chi Square tests
comparing the distributions of BL2 versus PO1 and BL2 versus PO2, showing many instances of
significant differences both for IONX and sham-operated groups of the two genders, generally,
more so in the middle and late trials (Figures 8a-f, Table 14,15). Moreover, there were
43
considerably more trials of significant difference between BL2 versus PO1 and BL2 versus PO2
in the IONX group compared to the sham group. Table 15 shows that when subtracting the
counts of such significantly different trials in the sham-operated from the IONX groups, the net-
IONX effect amounts to 2/21 stimulation trials in the ears, paws and tail of male mice versus
0/21 trials in females (for the comparison of PO1 to BL2). More significantly different trials
were seen in BL2 versus PO2 than BL2 versus PO1: 5/21 for males and 3/21 for females. This
suggests that IONX caused increasing hyperresponsivity from PO1 to PO2.
In a different analysis, the sum of bilateral recategorized scores of the 7 trials in BL2 was
subtracted from the respective sum of scores in PO1 and separately also from those of PO2,
separately for the sham-operated and IONX groups, per body locus, separately for males and
females (Table 16). Next, these difference scores were averaged per group, SEM values were
calculated, and the results shown in Figures 9a,b. Group averages of the two surgery types were
compared with t tests. The results for both PO1 and PO2 indicate that the group averages for
responsivity of the ears did not change postoperatively as much as those of the paws and tail. In
the paws, both for males and females, the difference scores where significantly higher for the
IONX groups than the sham-operated groups. A similar result was seen in the tail of male, but
not female mice, such that both in PO2 and PO1, IONX resulted in considerably more
reponsivity than in BL2.
3.2.4 Effect of IONX on Behaviour in the M-PAT
Main findings: Males, but not females, spent more time immobile in the rough zone following
IONX.
Detailed analysis: Figures 10a-c present the group averages, SEM values for the time, distance
and speed travelled by 247 and 250 male and female naïve mice (respectively) and for 71-93
sham-operated and post-IONX male and female mice (respectively) in the Smooth and Rough
zones of the M-PAT. These Figures show the baseline values on the right side panels and PO1
and PO2 data to their left. In BL1, both males and females did not prefer one zone over the other,
as shown by the lack of significant difference in the time, distance and speed of mobility in these
zones. In BL2, both males and females were able to differentiate between the Smooth and Rough
zones by spending significantly more time in the Rough zone (for females) and walking a longer
distance, but at a slower speed on the Rough zone compared to the Smooth zone. This supports
our choice of regarding BL1 as a training session and using BL2 for comparisons with the
44
operated groups. Following surgery, regardless of its type, in PO1 this surface discriminability
disappeared, as no significant differences between the time, distance, and speed across the two
zones were evident both for males and for females.
In PO2 females post-IONX (and not males nor in sham-operated female mice), there was
a partial recovery in place preference, manifesting in significantly more time spent in the Rough
zone compared to the Smooth zone. Since such a significant difference was not seen in the
Distance travelled in the Rough zone, this suggests that in PO2, but not PO1, the females froze
more in the Rough zone.
Table 17a (in the Appendix) shows the P values associated with a comparison of these
parameters for the Smooth and Rough zones in the PO1 or PO2 testing periods, against
respective data in BL2. The statistical results post-Bonferroni correction show that females, but
not males, walked in PO1 and PO2 a shorter distance postoperatively both in the Smooth and
Rough zones, whether sham-operated or post-IONX (see Figure 11, Appendix). Table 17b shows
that compared to BL2, females post-IONX, but not sham-operated mice slowed down their speed
on both the Smooth and Rough zones in PO1. In PO2, compared to BL2, females of both surgery
types significantly slowed down their speed in both zones. Male mice of both surgery types also
slowed their speed but only in the Rough zone. IONX-related slowing (that was not seen in
sham-operated mice) was only seen in females in PO1, but note that this slowing was seen when
walking in both zones.
Figures 11a-c show difference scores, resulting from subtracting the M-PAT BL2
baseline values from the respective PO1 and PO2 values, for each mouse, averaged for all mice
per gender and surgery group. Figure 11a shows that the average Distance in both M-PAT zones
were negative, indicating that the mice generally walked a shorter distance in PO1 and PO2 than
in BL2, and that this was significant for females, but not males, and regardless of surgery type.
But, as shown in Figure 11b, the Time in the Smooth or Rough zones did not significantly
change in PO1 or PO2, regardless of genders or surgery type.
Figure 11c shows that females post-IONX significantly slowed down their Speed when
walking in the Smooth and Rough zones in PO1 compared to BL2. This was not seen in females
post-sham and also in none of the male surgery groups. In PO2 the males of both surgery groups
slowed down in the Rough but not the Smooth zone. Thus, in PO2 males of both surgery groups
were able to discriminate between the two zones. In PO2 females of both surgery groups slowed
down in the Smooth and Rough zones.
45
Figure 8a-f: Comparison of the distribution of response types to VF stimulation between BL2 and the PO1
and PO2 testing periods for males and females. The horizontal axis displays the stimulus trial number. The
veritcal axis displays the percentage of occurence of each reponse type: None (white columns), Weak (grey
columns) and Strong (black colums) for the ears and paws; and for the tail: No response (white columns),
Threshold responses (grey columns) and Suprathreshold responses (black colums).
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
*
1 2 3 4 5 6 7
#*
Sham IONX
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
Sham IONXb. Ears (females)
a. Ears (males)
BL2 PO1 PO2 BL2 PO1 PO2
46
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 2 3 4 5 6 7 1 2 3 4 5 6 71 2 3 4 5 6 7
*
1 2 3 4 5 6 7
*
1 2 3 4 5 6 7
* #
1 2 3 4 5 6 7
* # *
*** *Sham IONX
1 2 3 4 5 6 7 1 2 3 4 5 6 7
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 2 3 4 5 6 7 1 2 3 4 5 6 7
***
*** **
***
1 2 3 4 5 6 7
* *#
***
1 2 3 4 5 6 7
** *
************
SHAM IONX#
** ****
***
c. Paws (males)
d. Paws (females)
BL2 PO1 PO2 BL2 PO1 PO2
47
1 2 3 4 5 6 7 1 2 3 4 5 6 7
* *#
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
**** * *# #
1 2 3 4 5 6 7
** **
***
***Sham IONX
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
** *
***
***
1 2 3 4 5 6 7
** **
* #
1 2 3 4 5 6 7
*
**** *##
1 2 3 4 5 6 7
******** ******
Sham IONX
e. Tail (males)
f. Tail (females)
BL2 PO1 PO2 BL2 PO1 PO2
48
Figures 9a,b: Results of comparisons between sham-induced and IONX-induced changes in
mechanical responsivity, expressed in the group averaged difference sum scores for the 7 trials
per session, normalized by BL2 sum scores, for each surgery group. An independent-samples t-
test was conducted to assess for significance of the difference between surgery groups separately
by gender, for PO1 in Figure 9a and PO2 in Figure 9b.
Finally, Table 18 (Appendix) shows the results of independent samples t-test comparing
parameters of the M-PAT assay for PO1 and PO2 data (normalized against naïve scores), in the
sham-operated groups to those in the IONX groups, noting that both in PO1 and PO2, males but
not females, spent a significantly shorter time in the Smooth zone after IONX than after sham
operation (see also Figure 12a below). No significant difference was found between the IONX
and sham groups in the distance (Figure 12b) or speed in the Smooth or Rough zones (Figure
12c).
Aver
age
diffe
renc
e Sc
ores
(P
O-B
L2)
PO1-BL2
Males_ShamMales-IONXFemales_ShamFemales_IONX
Aver
age
diffe
renc
e Sc
ores
(P
O-B
L2)
PO2-BL2
Males_Sham
Males-IONX
Females_Sham
Females_IONX
a
b
* #
#
* * *
* *
49
Figure 10a-c: Mobility parameters of the M-PAT assay, showing in a. the average time spent, b. the
distance traveled, and in c. the speed of walking in the Rough and Smooth zones by mice of each gender
and surgery group of. T-tests were done to compare group averages between Rough and Smooth zones.
70
75
80
85
90
95
100
105
110
115
120
Smooth Rough Smooth Rough Smooth Rough Smooth Rough
Tim
e (s
ec)
Sham IONX Sham IONX
PO1 PO2
***
Smooth Rough Smooth Rough
BL1 BL2
m f m f m f m f m f m f m f m f m f m f m f m f
***
Smooth Rough Smooth Rough
BL1 BL2***
***
0
0.005
0.01
0.015
0.02
0.025
Smooth Rough Smooth Rough Smooth Rough Smooth Rough
Spee
d (m
/sec
)
Sham IONX Sham IONX PO1 PO2
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
Smooth Rough Smooth Rough Smooth Rough Smooth Rough
Dist
ance
(m)
Sham IONX Sham IONX PO1 PO2
Smooth Rough Smooth Rough
BL1 BL2
***
***
m f m f m f m f m f m f m f m f m f m f m f m f
m f m f m f m f m f m f m f m f m f m f m f m f
c.
b.
a.
50
3.2.5 IONX Effect on the Correlation between Mechanical Sensitivity and M-PAT
Parameters
Main findings: IONX-induced changes in responsivity to VF filament stimulation in the ears and
tail correlated with some M-PAT parameters.
Detailed analysis: For each postoperative testing session a correlation was computed between
tactile responsivity to VF filament stimulation (separately for each of the 7 trials in the three
tested body loci) and the M-PAT parameters, separately for sham- and IONX-operated male and
female mice (Tables 19a-d, Appendix). The number of trials showing a significant correlation in
Table 19a-d is summarized in Table 20 (Appendix) and shown below in Figure 13. No
significant correlations were found between mechanical responsivity (in any tested body locus)
and Time in the Smooth zone, in any tested period, both for males and females. For females
(right panel), no significant correlations were found between any M-PAT parameter and
mechanical responsivity in PO1 or PO2 compared to BL2 (sham-operated or IONX mice). For
males (Left panel) there were no significant correlations between any M-PAT parameter and
mechanical responsivity in PO1 compared to BL2. In PO2 fewer correlations were found than in
BL1 and BL2. To summarize these findings: surgery, regardless of its type, was associated with
reduced correlations between mechanical responsivity and mobility on the M-PAT assay (as
compared to the naïve state).
It cannot be excluded that in females this reflected the effect of repeated stimulation, as
shown by the reduction in the number of significant correlations from BL1 to BL2. But this
cannot be applied to males for which the opposite effect of repeated testing on such correlations
was observed.
3.2.6 Pain Spread following IONX
Main findings: Data were analysed by mouse strain/line and spread of pain following sham
surgery and IONX surgery. Spread patterns were categorized based on body sites, which showed
an increase in mechanical responsivity following surgery. There was a variable range of spread
types in each strain/line, but most abundant were those showing spread to the paws and tail or to
the paws/tail/ears, seen in 46.5% of the sham group and 44.0% of the IONX group.
Detailed analysis: This analysis was performed on data of the PO2 testing period which showed
the peak postoperative local changes. Since there was no significant side and minor gender
51
differences the following analysis was perfomed on the joint dataset, combining bilateral values
for both genders. For every mouse a cumulative score was produced for the 7 repeated trials, per
body locus, for every testing session. The cumulative score of the second baseline session (BL2)
was subtracted from the second postoperative testing session (PO2). Positive difference scores
indicated an increased responsitivity to mechanical stimulation following surgery (compared to
the baseline) and were categorized as ‘HYPER’, denoting mechanical hyperresponsivity.
Difference scores not meeting this criterion (i.e., negative or 0), were categorized as ‘NOT’, to
denote the lack of hyperresponsivity (as per Table 3, Methods). This was done for the 3 tested
body loci (ears, hindpaws and tail) and separately for each surgery group (sham and IONX).
Based on Table 3, showing the 8 possible permutations of HYPER or NOT, every mouse
of the 378 mice was assigned a ‘Spread Type’. Table 21 (Appendix) shows the counts and
relative abundance (in %) of the various Spread Types, separately for each surgery group and
their relative proportion. When lumping all mice into two groups by surgery type a striking effect
of IONX compared to sham can not be seen. In bold type are the details for Spread Types 5 (i.e.,
spread of hyperresponsivity to all three tested body loci) and 7 (i.e., hyperresponsivity in the
distal tested loci – hindpaws and tail) that were most abundant in this population, accounting for
46.5% and 44.0% of all 8 possible types (for the sham and IONX groups, respectively). The
common denominator of these 2 Spread Types is the development of mechanical hyperalgesia in
the hindpaws and tail. As shown below, however, when dissecting the data of these 2 Spread
Types by strain-line there were strong indications of a genetic effect that is lost when averaging
regardless of the genetic background.
3.3 Gender Differences in Basal Mechano-responsivity and Changes Post-IONX
Most of the comparisons between the genders have already been addressed throughout
the Results chapter and details can be found above. In brief:
No laterality differences were found in either gender, justifying pooling data across sides into
a single value per body locus, testing period and gender.
No differences were found between BL1 and BL2, justifying pooling data across testing
periods into a single value per body locus and gender.
A weak (yet significant) correlation was found in both genders in the responsivity to
mechanical stimulation in the tail and paws. In females, but not in males, there was a weak
(yet significant) correlation between the responsivity in the ears and tail. No significant
corelations were found between the responsivity in the paws and ears, in either gender.
52
Figure 12a-c: Results of comparison of normalized scores following surgery for IONX and
sham surgery groups. a,b, and c show comparisons for scores in time spent in each zone, distance
walked on each zone, and average walking speed for each zone, respectively. Males and Females
are considered separately.
The following items highlight a few gender-based comparisons that were not addressed prior.
3.3.1 Gender-specific Mechanical Responsivity
Main findings: There was generally no effect of gender on responsivity.
Detailed analysis: Table 22 shows the results of a statistical comparison of the distribution in
males versus females of response categories for trials 1,4 and 7, for each testing period, using
Chi-square tests. A gender difference was only noted in trial 4 of PO1 in the paws of sham-
-20
-15
-10
-5
0
5
10
PO1
PO2
IONX-S
ham (PO
-BL2)
(sec)
PO1 PO2
Males
Females
***
-0.3
-0.25
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
IONX-S
ham (PO
-BL2)
(cm)
PO1 PO2
Males
Females
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
Smoot
h
Rough
Smoot
h
Rough
IONX-S
ham (PO
-BL2)
(cm/se
c)
PO1 PO2
a.
b.
c.
Tim
e Di
stan
ce
Spee
d
53
operated mice and in trial 1 of PO1 in the same group. Thus, it is concluded that generally there
were very few gender-related differences in sham-operated mice and none in naïve (BL1, BL2)
or IONX mice.
Figure 13: The number of stimulation trials applied to the three tested loci (7 in the ears, 7 in the
hindpaws and 7 in the tail, in total 21 trials) that showed a significant correlation between
mechanical responsivity (as measured by the VF filament) and M-PAT parameters, lumping data
for all three tested body sites, for BL1, BL2, PO1 and PO2, separately for males and females and
by surgery type.
3.3.2 Gender Differences in Sensitization to Repeated Stimulation
Main findings: No difference was observed in sensitization between genders.
Detailed analysis: Table 4 (Appendix) and Figure 4 show that for both genders there was a
transient or progressive sensitization in the responses to repeated stimulation along the testing
sessions (i.e., from trial 1 through 7) of the paws and tail. But this result did not specify whether
there was a gender difference in this process. This question was addressed here by categorizing
the change in the response pattern during each session throughout trials 1 to 7, as to whether they
increased or decreased in response magnitude from trial 1 to 4. The same was done for a change
from trial 1 to 7. Table 23 (Appendix) shows schematically all possible combinations of response
types to stimulation in trials 1,4 and 7, as well as the Response Change Category, defined as two
levels of Adaptation (numerically coded as ‘-2’ and ‘-1’), one level of No Change (coded as ‘0’),
0
1
2
3
4
5
6
7
8
BL1
BL2
PO1
PO2
PO1
PO2
BL1
BL2
PO1
PO2
PO1
PO2
N/2
1 tr
ials Time in Smooth
Distance in Smooth
Distance in Rough
Males Females
Intact Sham IONX Intact Sham IONX
54
and two levels of Sensitization (numerically coded as ‘+1’ and ‘+2’), and finally, one level of
Mixed Changes (coded as ‘M’; only a few such instances were observed and not included in the
following analysis). The frequency of the 5 change types (excluding ‘M’) was then calculated for
each study group (i.e., naïve, sham-operated and IONX) and compared across the genders. This
was done using Chi-square test (Table 24).
Only in the tail, for just one testing period (PO1), in only one of the groups (sham-
operated mice) was there a significant gender difference, but it did not ‘survive’ a Bonferroni
correction. It is concluded that there was no significant difference across the genders in the type
of change in responsivity during each testing session.
3.3.3 Gender Differences in the Mobility Test (M-PAT)
Main findings: Table 25 shows that there were no gender effects on the behaviour in this test
prior to, and following surgery in either surgery group.
3.4 Genetic Considerations in Baseline Mechanical Sensitivity and M-PAT Parameters and
Effect of IONX
The analyses described above did not take into consideration the genetic background of
the mice, and pooled all mice regardless of their line or parental strain. The following is concise
list of findings with respect to genetic considerations in mechanical responsivity and M-PAT
parameters and effect of IONX.
3.4.1 Genetic Differences in Mechanical Sensitivity
Main findings: The Strain/Line Distribution Patterns (LDPs) of mechanical responsivity levels
per tested locus, show a continuous increment in levels across the 25 strains-lines, with the ears
showing the lowest values. A significant correlation was found in the baseline mechanical
responsivity of the three body loci across the 25 strains/lines. The signiciant correlation between
the responsivity in the paws and tail was still present post-IONX, but those between the ears and
paws or tail disappeared postoperatively, suggesting commonly shared genetic control of the
changes in the tail and paws post-IONX, different from the genetic control of the changes post-
IONX in the ears.
Detailed analysis: The following results delineate the contribution of genetic variability to the
overall variability seen in the tested traits. Since there was a very minimal gender effect on the
55
tested traits, the following genetic data analysis was done regardless of the gender, pooling males
and females into one group per line-strain. As described in the Methods, for every mouse the
data of the two tested sides were combined, assigning a double value for the occurrence of
Suprathreshold Responses, combining the data of all 14 trials per animal, per testing session,
then averaging per line/strain. Line/strain average (±SEM) responsivity to mechanical
stimulation in the three tested loci, of naïve animals in the BL2 testing period, are shown in
Figure 14, arranged in an ascending order based on the lines/strains responsivity in the tails, a
trait that showed the highest range of values across these lines/strains.
Table 26 shows, for each tested trait, the range across lines/strains, calculated as the
difference between the line/strain having the maximal and minimal values, normalized by the
value of the line/strain having the minimal value. This range was higher for the paws and tail
than for the ears.
For Figures 15a-c, the trait levels across all 25 lines/strains were rank ordered from the
least sensitive (ranked number 1 out of 25) to the most sensitive line/strain (ranked number 25
out of 25). Using these values, Figures 15a-c show correlograms of the responsivity across body
loci, with p=0.023, p=0.000082, and p=0.0061 for the positive correlations between ears and
paws, ears and tail, and paws and tail, respectively. Thus, when considering the genetic
background in this analysis, sensitivities across body loci are significantly correlated, i.e., a line
that is sensitive in one body locus is also sensitive in the other two loci, and vice versa, justifying
pooling the responsivity weighted scores for each line across the three tested body loci into one
value for QTL mapping.
Figures 16a,b show the respective LDPs for PO1 and PO2. These histograms are based
on differential scores, which subtracted for each line/strain the average sham-normalized value
(i.e., each normalized by its BL2 values) from the IONX-normalized value. Figures 15d-f show
the corresponding correlograms for the same data as in Figures 16a,b for PO1 and PO2, with
non-significant correlations between ears and tail or between ears and paws, while the correlation
between paws and tail is positive and significant.
Thus, compared to sham-operated mice, IONX produced (both in PO1 and PO2) a net
IONX effect that is variable across the lines/strains, such that for some the IONX caused a
relative hyperresponsivity manifested by positive difference scores (as shown in Figures 16a,b),
in other lines/strains the opposite effect was observed, i.e., hyporesponsivity, and in yet other
lines/strains no net-IONX effect was observed.
56
The conclusion drawn from the correlogram in Figure 15e is that IONX had a similar net
effect on the paws and tail, presumably via a shared genetic control, whereas the lack of
significant correlations in Figures 15d,f between the ears and paws and ears and tail points to the
possibility that IONX had a different net effect on the mechanical responsivity of the ears than of
the paws and tail, suggesting that the phenotypic plasticity in the ears is likely under a different
genetic control than the two more caudal body loci tested.
Figure 14: Line/strain distribution plots of the mechanical responsivity combined for males and
females, showing the average score of each recombinant inbred line and their parental strains for
responsivity at each tested body loci. The average line/strain of naïve animals in the BL2 testing
session arranged in an ascending order based on responsivity in the tail, a trait that shows the
highest variability.
3.4.2 Narrow sense heritability (h2) of Basal and Postoperative Mechanical Sensitivity
Main findings: Heritability values ranged from 0.71 to 0.83 for the various traits phenotyped in
the study.
Detailed analysis: Narrow sense heritability h2 scores for basal responsivity ranged from 0.71 to
0.83, indicating a surprisingly high level of genetic control (Table 27 in Appendix, Figure 17a).
The h2 scores were higher for traits of naive mechano-responsivity than for the same traits
measured following surgery. Generally, heritability scores tended to decline as a function of time
since a similar decline was seen in IONX and sham groups across time (Table 27, Figure 17a).
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
A13 B2 A6 A2 B7 A19 B B25 B4 B13 A10 A15 A1 B12 A12 A5 B1 B14 B8 A8 A4 B24 B11 A24 A
Aver
age L
ine Re
spon
sivity
Strain / Line
Naive (Baseline; m+f)Tail Paws Ears
Aver
age
Mec
hani
cal
Resp
onsi
vity
57
3.4.3 Narrow sense heritability (h2) of Basal and Postoperative M-PAT Parameters
Main findings: Heritability values ranged from 0.49 to 073 for the various M-PAT traits
phenotyped in the study.
Detailed analysis: The same analysis was performed on the M-PAT data, showing in Figure 17b
similar estimates of heritability as for the mechanical responsivity, including the effect of
repeated testing that reduced the h2 estimates in PO1 and PO2.
Figure 15a-c: Correlations between the baseline mechanical responsivity of the 25 lines/strains
in the three body loci. Each correlogram shows the regression line, its equation and the values of
R2 and the p value associated with this line, for: a. Tails vs. ears, b. Paws vs. tail, and c. Paws vs.
ears. d-f. Correlations between the net-IONX effect on mechanical responsivity of the 25
lines/strains in the three tested body sites, in the two postoperative testing sessions PO1 and PO2,
normalised for each strain/line by its baseline and sham values. d. Tails vs. ears, e. Paws vs. tail,
and f. Paws vs. ears.
a b c
d e f
58
3.4.4 QTL Maps of Basal Mechanical Sensitivity, Effect of IONX, and M-PAT Parameters
Main findings: A number of QTLs were mapped for all traits studied in this project. A QTL of
significant LRS was found on chromosome 5 associated with spread of mechanical
hyperresponsivity following IONX. Another QTL of significant LRS was found on chromosome
13 for two traits - mechanical responsivity in the Tail in PO1 and Time_in_Smooth in BL1. A
QTL on Chromsome 9 was mapped independently for several traits such as responsivity in the
Tail in PO1 and Time_in_Smooth in BL1. Finally, no correlation was seen between traits
measured in this study that may relate to stimulus-evoked mechanical responsivity and those of
spontaneous neuropathic nocifensive behaviour measured in an earlier study by this lab.
Detailed analysis: QTL mapping of these traits was based on a single representative value per
line or parental strain. By statistically assessing the linkage between these values for the basal
and postoperative mechanical responsivity and an available genetic map for these lines/strains,
QTLs were mapped harbouring genetic loci having a major effect on the variance in these traits.
Since very minimal gender effects were found in the analyses (see 3.3), the QTL were mapped
for a joint dataset including males and females.
Table 28 (Appendix) shows the mechanical hyperresponsivity Spread Types for each
animal of all 20 lines/strains for which such calculation could be done, having at least 5
individuals per line/strain for both surgery types, and the total counts and relative abundance (in
%) of the various Spread Types per lines/strain, for each surgery group, and the difference scores
in these values between IONX and sham, are shown in Table 29 (Appendix). Calculating for
each line/strain the % of mice having Spread Types 5 or 7, for each surgery type and the
difference scores in these values between IONX and sham (Table 29, Appendix) shows that for
some lines the values are positive, indicating a higher prevalence of such Spread Types in the
IONX than sham groups for that line and vice versa in other lines/strains, or no difference in
other lines/strains.
The data fed into the Web2QTL software included BL2 data for the naïve mice, the net
IONX effect for the operated mice [i.e., The Difference Scores (IONX-BL2)-(sham-BL2)]
(Table 30a,b, Appendix). In addition, QTLs were mapped for extraterritorial spread of
hyperresponsivity. As an example of a genetic map that is produced by the Web2QTL software,
Figure 18 shows a genome-wide map of all 19 murine autosomal chromosomes and the X
chromosome for the trait: “% of mice/line showing a net-IONX extra-territorial spread of
mechanical hyperresponsivity that included paws and tail or ears, paws and tail (i.e., Spread
59
Types 5 or 7)”, resulting from subtracting the PO2 sham-operated group averages from the
IONX group averages, both normalized by their respective BL2 values. Marked by the blue line,
3 QTLs cross the suggestive threshold, and one QTL even crosses the significance threshold.
Figure 16a,b: Line/strain distribution patterns of the net-IONX effect of the mechanical
responsivity, combined for males and females of each line/strains, for the three tested loci,
arranged in an ascending order based on the responsivity in the tail. Line/strain average values
are of IONX-sham difference weighted scores of PO1 and PO2, each normalized by BL2 values.
Note that lines/strains with negative values in these histograms denote lines presenting with
hyporesponsivity (for which the normalized sham responsivity was higher than the normalized
IONX group.
The significant QTL in chromosome 5 is shown in Figure 19. The results of the bootstrap
test show a single peak at 49.4%, indicating that when reitrerating the linkage 1,000 times, each
time omitting data of one line/strain and substituting it with the data of another randomly
-1.8
-1.3
-0.8
-0.3
0.2
0.7
1.2
A24 A4 B1 A19 A6 A2 A15 A1 A5 B14 A13 B2 B A10 B13 B12 A8 A12 B11 B7 B25 A B4 B24 B8
Aver
age
Line
Res
pons
ivity
Strain / Line
PO1 (m+f)Tail Paws Ears
-1.8
-1.3
-0.8
-0.3
0.2
0.7
1.2
A24 A4 A19 A1 A15 B2 B25 B13 B A6 A12 B12 A13 A A2 B7 B4 B24 A8 B1 A5 A10 B14 B11 B8
Aver
age
Line
Res
pons
ivity
Strain / Line
PO2 (m+f)Tail Paws Ears
Mec
hani
cal R
espo
nsiv
ity (B
asel
ine-
norm
aliz
ed IO
NX-
Sham
diff
eren
ce)
b
c
60
selected line/strain, 494 of the QTL peaks which appear out of the 1000 trials are localized to a
marker loci in this region of 102-107Mb, in full agreement with the peak as determined by the
LRS test. In alternating red/green lines, Figure 19 shows that the additive effect of 20 units on
the trait level from a single allelic substitution in this QTL region is conferred by the B
(C57BL/6J) genotype.
Figure 19 shows a map only of chromosome 5, including the haplotype structure for this
chromosome. As seen across the peak region of this QTL, for most lines it is “paved” by
intervals originating from the green B parent, except ‘outliers’ (i.e., AXB-1, AXB-4 and BXA-
13) whose phenotype may not represent the true level for these lines since the data included too
few animals or their trait levels are determined by other genetic loci elsewhere in the genome, or
affected by an interaction with the non-genetic ‘environmental’ effects.
The confidence length of the QTLs was determined as the length of interval whos
corresponding LRS value is above the significant LRS level for that analysis. Browsing the list
of 176 candidate genes in the confidence length of the QTL in chromosome 5 (see Table 31,
Appendix), based on biological relevance, enabled to shortlist 3 candidate genes (Table 32).
Their relevance to the mapped trait with which this QTL was linked is discussed below in 4.15.
By using the same QTL mapping process described above for the trait, “Spread of
mechanical hyperresponsivity post-IONX”, QTLs were mapped for the following traits: Baseline
mechanical responsivity of naïve mice in the three tested body loci, the changes observed to this
trait post-IONX in these loci in PO1 and PO2, as well as the M-PAT parameters for BL1, BL2,
PO1, and PO2 (Table 33). This Table shows that all of these traits showed QTLs, however, the
QTL mapped on chromosome 5 for the trait “Spread of mechanical hyperresponsivity post-
IONX” and that on chromosome 13 for “Hyperresponsivity in the tail” were the only significant
QTLs, all other 18 were mapped at a suggestive statistical significance.
These suggestive QTLs were included in Table 33 since their presence was indicated by
two different mapping methods – the Permutation Test and the Bootstrap Test. Of note, there are
two QTLs that encode both a trait related to mechanical responsivity or its response to IONX and
M-PAT parameters. The first is located on chromosome 9, spanning a confidence interval from
~100-115Mb and associated with mechanical responsivity in BLs in the Paws and Tail and in
PO1 and PO2, as well as for Time_in_Smooth in BL1. The second QTL is on chromosome 13,
mapped from 94.0-96.0Mb, and is linked with the same traits as the one on chromosome 9: i.e.,
mechanical responsivity in the Tail in PO1 and Time_in_Smooth in BL1.
61
Figure 17a,b: Estimated values of the ‘narrow-sense heritability’ (h2) of responsivity to (a)
mechanical stimulation in the ears, paws, and tail, and for (b) M-PAT parameters, for each
surgery group (IONX and sham), prior to basline testing (BL) and following surgery (PO1 and
PO2).
3.4.5 Assessment of Correlation between Mechanical Responsivity Pre- and Post-IONX
and Spontaneous Neuropathic Pain
Main Findings: No significant correlation was seen between mechano-responsivity in this study
and spontaneous pain behaviour observed in a previous study on the same panel of mice.
Detailed Analysis: This analysis tested whether spontaneous neuropathic pain in the Neuroma
Model (using archival data from a previous study (158)) is correlated with the traits quantified in
the present study (i.e., mechanical responsivity in the three tested body loci, in intact mice and
Mechano-responsivity a
b M-PAT
62
post-IONX, as well as “Spread types 5,7” in sham-operated, IONX and net-IONX). The
parameters for the self-mutilation behaviour following total hindpaw denervation by sciatic and
saphenous neurectomy included: (i) the incidence in each RI line of scores ≥3 on day 36
postoperatively, the last day of behavioural follow up; (ii) the average line score on day 36; and
(iii) the line average of the first day that self-mutilation had been observed. Finding a significant
correlation could indicate a common genetic control of the correlated traits. This analysis used
trait values for all 23 AXB-BXA lines and the parental lines A, B.
Figure 18: Genome-wide QTL plot for spread of mechanical hyperresponsivity post-IONX. The
X-axis shows the physical distance along the 19+X chromosomes, in Megabases (Mb). The Y-
axis on the left side is in LRS units, which stands for the Likelihood Ratio Statistic, indicating
the confidence with which the software’s algorithm can propose that at the marker locus there is
a linkage between the variability in the trait levels across the lines/strains and the allelic phases
of these lines/strains at that locus. The area of the LRS plot shows two thresholds of significance,
one in grey, delineating LRS values that are associated with a “suggestive significance” level,
and in pink, a genome-wide “significance” level (p<0.005). This composite graph also shows a
histogram with the ordinate on the right side showing, in yellow, the results of the bootstrap test
(in % confidence units), indicating how confident is the algorithm in assessing the linkage at a
given locus, calculated differently than the LRS (see above). Another plot on this graph also has
an ordinate on the right side and shows (in green) units in which the trait was measured. This
plot is shown throughout the genome in alternating green and red lines, indicating the identity of
the parental strain (A/J in green, B [C57BL/6J] in red) whose allele at the marker locus
contributes to an increse in the trait values (units of increase are shown in green on the right side
ordinate). Their contribution at each marker locus is mutually exclusive (i.e., only one can have
the additive effect per locus).
63
Table 34 shows the resulting p values and correlation coefficients. Highlighted in red are
correlations yielding significant p values between all three hindpaw self-mutilation parameters
and mechanical responsivity of intact mice in the ears and on PO1 post-IONX. However, none of
these remained significant after a Bonferroni correction.
Table 32: List of 3 select known genes and their encoded proteins in mouse chromosome 5,
located within the confidence interval of the QTL linked with spread of mechanical
hyperresponsivity post-IONX. N = serial number of known genes in chromosome 5; Start (Mb) =
position (in Mb) where the gene sequence begins; SNP count = the number of known
polymorphic nucleotides comprising the respective gene.
N Gene symbol
Mb Start
(Mb)
Gene
Length
(Kb)
SNP
Count Encoded protein
766 Nkx6-1 102.0882 5.515 0 NK6 transcription factor r...
773 Mapk10 / JNK3 103.337 303.387 4 mitogen-activated protein ...
787 Sparcl1 104.5081 34.978 125 SPARC-like 1 (mast9, hevin...
64
Table 33: List of Quantitative Trail Loci (QTLs) identified in various chromosomes (chr.) for
various sensory traits in naïve and denervated conditions. Testing assays and parameters
included Von Frey testing (VF) at the Ears, Paws and Tail, and M-PAT test parameters for each
testing session (including Baseline 1 and 2 (B1 and B2) and Postoperative 1 and 2 (P1 and P2).
The parental allele that effected the trait level (a vs. b), and the interval location and span
(including start, peak and end of the QTL; in Mb; for the two mapping algorithms – Permutation
test and Bootstrap test). A Likelihood Ratio Statistic (LRS) was computed as the output of the
former algorithm, to indicate the presence or absence of a QTL at a genomic locus, and whether
the QTL was associated with a significant (Sig.) or suggestive (Sug.) level. The Bootstrap test
resulted in identification of the peak location (in Mb) and the number of reiterative mapping runs
in which a peak was identified in that location (5 of 1,000 runs).
Start Peak End Peak %
1 Naïve B1,2 Ears VF 1 a 92 92 92 92-95 45.2 10 *2 Denervated P2 Mobility Time_Smooth 1 b 23 23 23 20-23 33.9 12 *
Naïve B2 Mobility Distance_Smooth 2 b 168 169-170 170 168-175 62.8 12.3 *Naïve B2 Mobility Distance_Rough 2 b 168 169-170 170 168-175 58.4 11 *
4 Naïve B2 Mobility Time_Smooth 2 b 108 108-110 110 106-110 50.8 11 *5 Denervated P1 Mobility Distance_Smooth 3 b 8 8-16.0 16 8-16.0 58 12.3 *6 Naïve P2 Tail VF 3 b 47 47.5 48 47-48 40.5 10.5 *
Naïve B1,2 Mobility Time_Smooth 3 b 108 108-112 112 109-114 49.2 12.5 *Denervated P1 Mobility Distance_Smooth 3 b 102 102-104 104 102-104 19.8 12.1 *Denervated P2 Mobility Distance_Rough 3 b 98 98-101 101 97-101 30 10 *
8 Naïve P2 Ears VF 4 a 118 188-119.5 119.5 118-119 61.7 11.5 *9 Denervated P1 Mobility Time_Smooth 5 a 76 77 78 73-78 60.8 13 *
ConditionTesting period
Trait
Chr.Body region
Tested parameter
Interval location and confidence length (Mbp)Parental
allelePermutation test Bootstrap test LRS
score
Statistics
Sug. Sig.QTL
Number
3
7
Denervated P1 Paws VF 6 a 84 84-87.5 88 84-85.5 33.9 15 *Denervated P2 Paws VF 6 a 82 84-86 85 84-86 42.6 15 *
11 Denervated P2 Paws VF 9 b 114.5 114.6 114.7 114.6-115 50.1 12.5 *Naïve B1,2 Paws VF 9 b 100 103.5-106 111.5 103.5-106 35.9 15.5 *Naïve B1,2 Tail VF 9 b 102.8 103-104 104 103-104 38.1 15 *
Denervated P2 Tail VF 9 b 99.5 103.5-106 111.5 103.5-106 32.7 16 *Denervated P1 Paws VF 9 b 102 102-102.5 103 101-103 61.5 15 *
Naïve B1 Mobility Time_Smooth 9 a 113.5 113.6 114.5 104-108 33.7 12.5 *13 Naïve B1 Mobility Distance_Rough 10 a 6.5 6.5 6.5 6.0-7.0 56.8 11 *
Naïve B1 Mobility Distance_Rough 10 a 80 80-82 82 80-82 56.1 10.1 *Naïve B1 Mobility Distance_Smooth 10 a 80 80-82 82 80-82 54.6 11 *
15 Naïve B2 Mobility Distance_Rough 13 b 7 7-25.0 25 9-12.0 35 12 *Denervated P1 Tail VF 13 b 95 95.5 96 94-96 81 16.2 *
Naïve B1 Mobility Time_Smooth 13 b 94 95 98 94-96 62.9 12.4 *17 Naïve B1,2 Ears VF 16 a 22.8 22.9-23.5 23.6 22.9-23.5 38.8 11 *18 Denervated P2 Mobility Time_Smooth 17 b 27 29 30 27-32 54 13 *19 Denervated P2 Paws VF 18 b 64 66 66.5 55-57 55.1 17.5 *
14
16
10
12
9b Spread of Sensivity P2 vs. B2 Types
5,7 VF 5 b 102 104-105 107 104-107 72.0 14.9 *
65
Figure 19: Map of chromosome 5. The horizontal bars comprising red/green/blue/grey intervals
indicate the haplotypes inherited from the parental lines throughout the chromosome (green=B
parent, red=A parent, blue=regions of heterozygosity, grey= regions of undetermined genotype).
The lines are stacked in increasing levels of the trait from lowest below and upwards. This map
was based on phenotypes contributed by all 25 lines, including those that had few animals/line.
66
Table 34: P values and Pearson correlation coefficients for the correlations between mechanical
responsivity and self mutilation in the 23 AXB-BXA RI lines and their two parental lines, A and
B.
67
4.0 DISCUSSION
The main findings of this study include the demonstration of significant genetically
determined differences in mechano-responsivity to VF stimulation in various body loci, prior to,
and following IONX (vs. sham), in the AXB/BXA panel of RI mice and their progenitor strains.
A novel place-avoidance assay was used with the purpose of shedding further light on
mechanical allodynia in the paws of the animals following IONX. A number of QTLs were
identified pertaining to the genetic control of the nocifensive traits assessed in naïve mice and
mice following trigeminal nerve injury.
4.1 Sensitization to Repeated Mechanical Stimulation in Naïve Mice
This analysis was done on all mice, separately for each gender and testing session. No
adaptation was seen across the 7 trials in BL1 or BL2. The 2 craniofacial sites that were tested in
this study (i.e., R, L ears) showed no sensitization in responsivity across repeated trials (i.e., from
trial 1 to 4 and 7). However, the spinal regions that were tested (i.e., R, L hindpaws and tail) did
exhibit such changes as a function of trial number. The progressive hyperresponsivity seen in the
hindpaws may have been due to the animal becoming progressively more attentive to the
anticipated stimulus. In this way, the animal becomes more stressed and fearful with each
succeeding stimuli. The transient hyperresponsivity which was predominantly seen in the tail but
also in the hindpaws may have been a combination of 2 phases, the first manifests as
progressively increasing responsivity due to stress and fear to the stimuli in the early trials. The
resumption toward normalcy can be explained in several ways. In the tail, in many occasions the
animal repositioned it by slightly raising it above the mesh surface, thus, the spasticity of the tail
muscles (to keep the tail raised against gravity) may have mechanically stretched and tightened
the tail skin, rendering it less sensitive to succeeding mechanical stimuli by the Von Frey
filament.
This may have resulted in progressively weaker stimuli in the latter trials. Alternatively,
repeated testing may first have caused stress-induced hyperalgesia (SIH) that was later
substituted by stress-induced analgesia (SIA) in the animal (79). Usually, SIH and SIA depend
on the type of stressor. Thus, it is possible that two different attributes of the stimulation setup
may have produced each phenomenon in its due course, for example, SIH (say, due to the
alarming quality of the stimulus) may have been first to manifest, followed by SIA (say, due to
the painfulness or the perceived alarming qualities of the stimulus), a condition known to often
promote pituitary release of endogenous opioids (such as enkephalin and β-endorphin) into the
68
blood circulation, that jointly suppress nociceptive transmission (121). As well, anxiety, fear and
stress can activate antinociceptive descending circuits, such as the raphe-spinal noradrenergic
and/or serotonergic, and other pathways (121,122). Thus, while these mechanisms of SIH may
have delayed the effect of SIA that manifest in the return of the responsivity of the tail to the
initial levels, it is not clear why the repeated testing in the ears did not manifest in
hyperresponsivity. Perhaps the stimulus intensity selected for the ears was not appropriate (i.e.,
too strong or presented in a way that precluded the possibility of provoking hyperresponsivity.
Moreover, regional differences in SIH and SIA have been documented before (140).
The sensitization process is most likely under genetic control and could be studied by
analyzing the data per each strain/line rather than all mice in one group. If the data were variable
across strains/lines, one could map QTLs that would then lead to identifying candidate genes
related to SIA and/or SIH, fear and anxiety, and perhaps also of learning avoidance behaviour.
4.2 Recategorization of Response Types, Interpretation of Withdrawal Responses, and
Methodological Pitfalls
Since there were no side differences, occurrences of the “No Response”, “Threshold
Response” and “Suprathreshold Response” types were combined for both ears and both paws,
separately for each of the 7 trials, by recategorizing these response types into None, Weak and
Strong responses, respectively. In case the response in one side was a No Response and on the
other side a Suprathreshold Response, such instances were recategorized as the middle level - a
Weak response. Thus, some instances of Suprathreshold Response were “lost” in favour of Weak
Responses. Likewise, such instances falsely up scaled a No Response to the Weak category,
giving the impression that the stimulus was met by a response, albeit a Weak one. These re-
categorizations, therefore, resulted in skewing the distribution of the original response types
when categorizing instances where the response type in the two sides of the body were not the
same. Another example of such skewing is instances of Threshold Responses and Suprathreshold
Responses, recategorized as Strong Responses. Thus, rather than recategorizing such instances as
Weak responses they were placed in the Strong category, thereby compensating for their “loss”
in the previous example (above). Out of a total of 1722 and 1743 trials (for BL1 and BL2 in
males and females, respectively) that were recategorized in this way, only 43-54 of the trials
were of the first example (~2.5-3.1%) and 33-53 were of the second example (1.9-3.0%). Thus,
the accuracy of the re-categorization was very satisfactory, such that the recategorized response
types faithfully reflected the original responses per side.
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It cannot be excluded that in some cases, responses interpreted as a withdrawal threshold
may have actually been missed for instances when the animal was about to move anyways,
regardless of the stimulus. Likewise, there might be an error on the experimenter’s side to
explain a withdrawal in terms of response to pain, which could rather be a response to itch that
the mechanical stimulus produced, or a startle response.
Another potential shortcoming of the stimuli used in this study is that when approaching
the ears with the Von Frey filament it was impossible to prevent the animal from seeing the
ballistically approaching stimulus, occurring immediately before its ears and eyes. These
additional sensory cues might have affected the response repertoire of the mouse by priming the
animal for the impending ear stimulation. This may explain why there was invariably a
Threshold Response when stimulated in this locus. Because of the conspicuous presentation of
this stimulus, the animal may have perceived the stimulus as alarming and responded in a startle
reflex rather than a nocifensive response. If this characteristic is what the assay reports, it is
plausible that any sensory changes resulting from injury to the ION may have gone undetected.
This, however, becomes less of an issue when testing the caudal sites (i.e., tail and hindpaws)
where the impending stimuli were mostly invisible to the animal.
4.3 Changes in Responsivity of Naïve Animals across Testing Sessions
A general increase in responsivity was observed in naïve animals from the first to the
second preoperative baseline testing sessions in the spinal body regions tested. This may have
been due to a heightened expectation of stimulation that the animals gained from a repeated
session. Repeated testing may have played a stronger role in responsivity of the caudal sites since
testing these sites is not immediately apparent to the animal (since it is from below the animal,
likely beyond its field of vision and more difficult to hear). Since the testing of the ears is
conducted directly before the animal’s eyes and ears, the animal may already be highly primed
for stimulation, moments before it occurs.
Judging by the responses of individual strains/lines to the surgery, especially when
subtracting the sham values from the IONX, each normalized by its own naïve values in BL2, it
was clear that some strains/lines responded to IONX by hyperresponsivity while others by hypo-
responsivity, and the rest did not change their responsivity post-IONX. Therefore, post hoc,
lumping of all mice into one big group based on surgery type must have masked an important
contribution of the genetic variation. Therefore, it may not be correct to infer from the minor
overall effects, found when comparing all mice lumped into surgery groups, regarding the effect
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of repeated stimulation. Perhaps a better way to assess whether there was time dependency
independent of the surgery type when considering the genetic background as a covariate in an
ANOVA for Repeated Measures. Nevertheless, using the BL2 values to represent the naïve
animal was the best that could be done in this study, as these values were determined closer in
time to the postoperative testing sessions, to partially offset the effect of time and repeated
measurements. However, perhaps an even better way would have been to repeat the baseline
sessions until the effect of repetition had saturated in all lines/strains, and then introduce the
surgery. The saturation process is likely also under genetic control that could be studied by
mapping QTLs and perhaps identifying candidate genes related to fear and anxiety, learning
avoidance behaviour, and more.
4.4 Sensitivity in Discreet Body Regions of Naïve Animals
Using the same stimulus intensity for all tested loci (0.2g) allowed for a direct
comparison of the response types across body loci. Mice exhibited a rostro-caudal gradient in
responsivity to the same stimulus at the different loci, as measured by Suprathreshold/Strong
response frequency. The rostral sites (ears) showed the most number of Strong responses,
followed by the tail, and then the hindpaws. Moreover, the ears showed the highest frequency of
any response type. The tail showed a higher proportion of exaggerated (i.e., Suprathreshold)
responses than Strong responses in the hindpaws. For reasons presented in 4.2, it is believed that
such comparisons are permissible since both category scales reflect the same incremental
response entities that were in the vast number of cases faithful to the original categories without
compromise. Thus, for No response/None categories the order was tail>paws>ears, for
Threshold/Weak responses it was ears>paws>tail, and for Suprathreshold/Strong responses the
order was ears>tail>paws.
Of note, the site that exhibited the lowest percentage of suprathreshold responses was the
hindpaws. This may be related to the constant low threshold mechanical input triggered from
sustained weight-bearing forces applied uniquely to the plantar surface of the paws, which floods
the dorsal horn of the spinal cord with such inputs. Based on the Gate Control Theory of Pain (1)
this constant input may lead to an ongoing partial suppression of nociceptive inputs from the
hindpaw, manifesting in a reduction in the percentage of exaggerated nocifensive responses to
stimulation. This argument assumes that the application of the Von Frey filament serves as a
nociceptive stimulus, based on the observed fact that the animal uses the same response to such
stimulus as it does to more overtly noxious stimuli, i.e., limb withdrawal, which is also used by
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humans who can additionally rate the aversive aspects of such stimuli as painful using verbal
mode of communication.
When the data was analyzed regardless of the genetic background, i.e., studying all mice
in the same group, a very weak but significant correlation in responsivity was found between
most body sites (except for the paws vs. ears). All correlations observed were positive. This
indicates that an individual with relatively high responsivity in one region will likely have a
relatively high responsivity in other regions and vice versa. The correlations reached significance
generally within the window of trials 3-5. This indicates that earlier and later trials have become
more site-specific. Furthermore, the trials in the middle of the session occurred at a time when
the progressive and/or transient hyperresponsivity in various body loci were observed. Thus, it is
possible that the increased correlation in mechanical responsivity in the middle trials reflects a
‘Whole-body Entraining Mechanism’ whereby the CNS controls the responsivity to mechanical
stimuli in a way that overcomes the individual responsivity profile of each body locus by itself,
so that the whole body responds in the same manner. The brain is known to control the input to
pain ascending pathways by way of inhibitory and facilitatory descending controls from cortex
and other subcortical nuclei in the brainstem (1, 39), and these may be the means by way the
mechanisms of ‘Whole-body Entraining Mechanism’ may take place. To the best of our
knowledge – such a mechanism has not been identified in the somatosensory system before. If
validated by future research, this mechanism could shed light on the way the CNS processes
sensory inputs and maintains a ‘body schema’, integrating inputs in the frontal and back midline
structures, and rostro-caudal inputs into one contiguous map, and the way this perhaps undergo
disruptions during the development and maintenance of neuropathic pain following denervation
and diseases. One might assume that the Entraining mechanism is also controlled genetically and
could be analyzed using the data collected in this study, as well.
Toward the end of the testing session, there was a reduction in the number of significant
inter-regional correlations. This may suggest that when SIA sets in to restrain the
hyperresponsivity in body loci showing transient hyperresponsivity, the correlations between
these sites weakened. Not in all sites there was a transient hyperresponsivity while in some there
was hyperresponsivity, thus correlations disappeared toward the end of the session. The data of
this study are compatible with the possibility that the facilitation in responsivity in the middle
trials may result from a consorted action that is brought about under stressful conditions, and
manifests before SIA operates. More work is needed to substantiate this hypothesis.
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4.5 Behaviour of Naïve Mice in the Mechanical Place Avoidance Test (M-PAT)
The M-PAT assay is a new test that was developed with the original purpose of testing
the efficiency of candidate analgesic drugs in rats following hindpaw nerve injuries, as well as
for the purposes of the present study in mice (144). This test is a modification of similar assays
that have used place preference (142, 143), which for the purposes of the present study was
shortened to 3 minutes per run. Furthermore, mice were confined to a considerably smaller
testing chamber than that typically used in the other place preference assays, with the assumption
that this would enable them to realize rapidly the presence of 2 zones having contrasting
granularity, Rough and Smooth and make a rapid decision that would enable high throughput
testing. The expectation was that animals having mechanical allodynia in the paws will travel
shorter distance, spend less time and walk at a slower speed on the Rough zone, to avoid pain.
Since naïve animals do not have allodynia, the expectation was that such animals should not
prefer the Smooth zone, to provide sufficient dynamic range for them to distinguish their lack of
preference as naïve animals from avoiding the Rough zone postoperatively. In fact, in BL1, there
was no place preference for males or females, but in BL2, some minor preference was noted by
spending slightly more time and longer distance in the Rough zone, accompanied by a moderate
inter-zonal difference in speed, manifested in males and females walking on the Rough zone at a
slower speed than the Smooth zone. This in itself may have not reflected antalgic gait, avoiding
walking rapidly on a noxious surface, but perhaps more in consideration with the motor aspects
that relate to the physical ‘unevenness’ of the surface while walking.
4.6 Correlation between M-PAT Behaviour and Mechanical Sensitivity in Naïve Mice
The M-PAT assay was originally designed with the expectation that some of its place
preference-related mobility parameters would at least partially predict mechanical responsivity to
VF stimulation in the hindpaws. In fact, no significant correlation was seen between the data of
these two assays. This hypothesis was based on the fact that since it is mainly the paws of the
animal that makes direct contact with the surface of the floor, it is the mechanical responsivity of
this locus that is assessed, and thus parameters measured in the M-PAT assay would at least
partially correlate to the VF filament responsivity data in the hindpaws. It should be noted that
the VF filament responsivity was not evaluated for forepaws, because of technical difficulties.
There is a possibility that there exists a better correlation between the responsivity of the
forepaws and the M-PAT output parameters, since compared to the hindpaws, it is more the
forepaws that are presumably used to survey a foreign terrain which lies ahead of the animal.
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In light of the lack of correlation with the paw mechanical responsivity, surprisingly, both
in males and females, and both in BL1 and BL2, it was the tail the ears, whose tactile
responsivity was found to correlate with the distance travelled in the M-PAT assay in both zones.
These correlations were weak-to-moderate and negative, such that the less sensitive mice were in
the tail and ears to mechanical stimulation the longer distance they travelled in both the smooth
and rough zones. Because the correlation was not zone-specific the correlation is probably with
general mobility, as might be recorded with an Open Field Test. It could have been argued that
animals exhibiting a higher mechanical responsivity would be more impulsive, hence walk more.
However, this is not compatible with the data that showed that the more sensitive mice were to
mechanical stimuli in the tail and ears the less distance they walked. Thus, these results are more
compatible with the suggestion that the more sensitive a mouse was to stimulation in those body
loci the more they froze as an expression of nervousness, fearfulness of novel stimuli and in fact
displayed more guarding behaviour. Nevertheless, if this was the case, why was such a
correlation not observed for the hindpaws? Ears and tail, in their protruding from the body core,
may be more vulnerable body parts to insults and predators, and therefore, their mechanical
responsivity is used as a nocifensive alarming sense that is correlated with emotive protective
traits such as fear of bodily injury. Thus, when ignoring the place preference aspect of this assay,
the mobility in this test seems to be indicative of these traits. More work is needed to substantiate
this explanation.
4.7 Changes in Mechanical Sensitivity following IONX
It is surprising that in the ears, compared to the baseline responsivity (i.e., BL2), no
significant differences were found following IONX or sham operation in PO1 or PO2, since sites
at more caudal regions that are further away from the surgery site did show changes in
responsivity following surgery. The lack of surgery-dependent changes in responsivity in the
ears may reflect limitations in the testing method for this locus. By using a stimulus-intensity too
strong, no dynamic range may have been left to showing a net-IONX effect (manifested in an
increased incidence of Suprathreshold responses postoperatively in the IONX mice compared to
the sham-operated ones). Contrary to my findings, Iwata had observed spread of mechanical
hyperresponsivity within the trigeminal system, following transection of the IAN to the whisker
pad, which is innervated by the ION. Iwata’s study, however employed a VF testing method
allowing for finer scaling of responsivity. This may have provided more opportunity to observe
small changes in responsivity levels (59).
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A novel finding of the study was the occurrence of spread of hypersensitivity to a spinal
locus from injury to a trigeminal locus. With respect to the caudal sites, both surgery groups
exhibited significantly increased responsivity between the baseline and postoperative periods
(Figs. 8,9). It is possible that these changes were time-dependent, showing an increase as a
function of time. The lack of time-dependent change in responsivity from BL1 to BL2 in any site
(see sections 4.1-4.4 and Table 6 on basal sensitivity), may be due to the short time lapse
between BL1 and BL2. But since PO1 was more than 14 days later, one cannot exclude the
possibility that the significant differences between BL2 and PO1, and BL2 and PO2 in the sham
group reflected a time effect. Alternatively, it is possible that the sham operation in itself
produced this effect. Despite the significant effect of sham operation on mechanical responsivity
in the distal sites, IONX produced in these remote sites (but not ears), an even larger increase in
responsivity, both in PO1 and PO2, mainly in males. This indicates that nerve injury caused a
significant net increased responsivity in extraterritorial sites remotely from the surgical field but
not nearby fields. It is known that nerve injury causes increased afferent inputs from the injured
nerve (1, 39), central sensitization of the neurons whose primary afferent source is the injured
afferents (1, 34, 58) and decreased or imbalanced segmental and descending inhibitions and
facilitations (39). Thus, the expectation is that areas innervated by other branches of the
trigeminal nerve may exhibit increased responsivity, which has been documented before
following inferior alveolar nerve section (95). Therefore, the lack of effect in the ears in the
present study may reflect another mechanism that “balanced out” or offset the expected
hyperexcitation. The spread of hypersensitivity to the caudal sites may have been attributed to
changes in the balance of descending facilitative and inhibitory input to such sites. More work is
needed to identify the mechanisms by which this offsetting effect on the spreading
hyperexcitability is mediated.
There were individuals in both the sham and IONX groups, and more interestingly whole
lines/strains, that showed hyporesponsivity, rather than hyperresponsivity. This phenomenon is
seen clinically as well (135,136). The mechanisms underlying this dichotomous effect of nerve
injury is likely controlled genetically as well. Moreover, different mechanisms may be involved
in each behavioural outcome, each controlled by a different pool of genes (145). Therefore,
pooling all mice into one analytical process may mask such differential genetic contributions and
in follow up analyses of the present dataset this should be probed.
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4.8 Effect of IONX on M-PAT Behaviour
As was done for the mechanical sensitivity, data - the BL2 session was used as
representing the naïve state, relating to the first baseline period as a training session, in which the
animal learned that there are two zones and its preference about staying in one with respect to the
other, and the test duration. Thus, the differences between zones manifested only in the second
session due to the animals learned ability to avoid the aversive features. While intact animals
learned from BL1 to BL2 to distinguish between the zones, postoperatively, in PO1 this
preference was lost both in sham-operated and IONX mice of both genders. This result was
associated with shorter walking distance and slowing of the speed in all groups regardless of
surgery type and gender. In PO2, IONX-operated males (and a trend for females) again
discriminated between the zones, now by increased freezing in the Rough zone, since they spent
there more time without walking a longer distance. This freezing could be interpreted as
manifesting a fear of walking in this zone. The preference to stand still, rather than move about,
may be related to there being distinct sensations evoked by each behaviour. The stationary
mouse in the rough zone is presumably receiving a continual stimulus of mechanical pressure
applied to the plantar surface of the paws, one that is static, topically and temporally. The
walking animal experiences a more varied range of mechanical stimuli, as various regions of the
paws may brush against the surface of the floor, and contact between each paw and the floor
occurs sporadically. This latter scenario is reminiscent of cases in which a subject describes
allodynia induced by an object brushing against the skin. This symptom has been classified as
“dynamic allodynia”, and has been shown to be subserved by different sets of physiological
structures than those of allodynia induced by persistent/static pressure to the skin (166).
4.9 Correlation between Mechanical Responsivity and M-PAT Parameters Post-IONX
The distribution of None, Weak and Strong response types of all mice to each mechanical
stimulus in the tested loci was correlated with the main M-PAT parameters (Time_in_Smooth,
Distance_in_Smooth, and Distance_in_Rough), separately for males and females of each surgery
group. As the original proposal for the M-PAT was that it might provide an assessment of higher
order processing of aspects of allodynia (rather than information limited to segmental
brainstem/trigeminal or spinal reflex activity), it is not unreasonable that the two assays may not
be correlated to each other. An inference made in this study, however, was that the Von Frey
stimuli which induced exaggerated, complex responses (denoted as “suprathreshold”) would
necessarily involve supraspinal (when stimulating spinal body loci) or rostral to the trigeminal
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brain stem nuclei (when stimulating craniofacial loci) CNS structures required for coordination
and execution of such motor behaviours when moving the whole body or at least part of the
trunk, forepaws and head (when responding vigorously to craniofacial loci), or hindpaws, tail
and lower trunk (when responding to tail or hindpaw stimulation). A future analysis of the
available dataset could involve correlating only the % suprathreshold responses to M-PAT
parameters, rather a weighted score that lumped the Suprathreshold-, Threshold- and No-
Responses into an overall single value of responsivity. Given the supposition that both
behaviours involve supraspinal activity, there may be a greater chance that these two sets of data
would correlate. Surprisingly, significant correlations were seen in naïve mice between
responsivity in the ears and tail but not the hindpaws and distance travelled in males receiving
sham surgery. Fewer correlations were noted for other body loci. However, postoperatively no
such correlations were seen; instead, sham-operated male, but not female mice and also not
IONX male and female mice, showed significant correlations between the distance walked in
both zones and the mechanical responsivity in the paws. Thus, it seems that IONX disrupted such
a correlation. More work is needed to identify the mechanisms underlying this effect of IONX.
4.10 Gender Differences
Several past studies have indicated disparity between the genders in pain sensitivity, the
majority of which demonstrated a greater sensitivity to pain in female subjects, both in animal
models and in humans (112, 115). A strain-by-gender interaction has also been documented,
showing that the disparity between genders may be nonexistent or varied, depending on genetic
background (112). In the present study, response patterns to mechanical stimulation were
compared for each test between genders, and with the following exceptions they failed to show
sexual dimorphism. The exceptions were: (i) greater net-IONX effect in males than in females in
mechanical responsivity in the paws and tail both in PO1 and PO2; (ii) males discriminated more
between the M-PAT zones by freezing more in the Rough zone. Thus, in the cohort of mice
tested in this study, all descending from A and B parents, there was a general/consistent lack of
gender difference in most of the comparisons made. Regrettably, this cohort did not include
sufficient males and females for each studied strain/line, therefore, it cannot be ruled out that
there was an interaction between genome and gender in this RI panel as well. It is possible that
this specific panel of mice represented genotypes for which there were inherently minimal
differences between the genders.
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4.11 Strain Distribution of Basal Mechanical Responsivity Levels
In most studied traits, it was found that the values for the parental strains contrasted
enough to be positioned in or near the flanks of the line distribution patterns that included the RI
daughter AXB/BXA lines. This result lends more support to using this panel to study the
phenomic and genomic underpinning of the traits phenotyped in this study. On this basis, it is
expected that the majority of the progeny of these two parental strains would rank somewhere in
an intermediate position between that of the two parental strains. However, it is also expected
that some of the progeny will inherit specific allelic patterns that lead to trait expression that
outrank beyond the range delineated by the parental strains, either higher or lower than the latter.
This is referred to as ‘transgressive segregation’ (138) and has been described in a number of
studies on the inheritance pattern of various traits (139) including neuropathic pain in the
Neuroma Model, documented by our lab (5). This phenomenon may be explained by the
potential masking of trait effects of an allele in the homozygous parental, being unmasked in the
recombinant inbred offspring. Thus, the epistatic interaction of parental alleles may manifest in
offspring who surpass or fall short of range of trait levels delineated by the progenitor strains.
Other than a few exceptions, the line distribution patterns for all tested traits showed a
generally continuous distribution of values among the progeny lines and parental strains. This
pattern of gradual trait levels within a recombinant inbred population indicates a polygenic
pattern of inheritance (i.e., a higher number of effective genetic loci controlling a trait under
study is expected to manifest in a graded distribution pattern where trait levels are the sum of
small effects in many genes (94, 138)).
Sensitivity in the ears was shown to be the least variable trait among lines and parental
strains. This would be expected, given the minimal difference between the expression of this trait
in the parental strains. Sensitivity in the caudal regions, however, showed a much higher level of
contrast between the parental strains, and a much greater variability among the RI panel. It is
possible that the low variability in ear responsivity among lines was due to an overlap in relevant
genetic determinants in the parental strains, leading to a narrow spectrum of variability in trait
levels in their progeny. Alternatively, the apparent lack of variability in this trait may be related
to our methods of stimulation and scaling the responses. Stimulation in the ears invariably
elicited some form of response - either threshold or exaggerated (Suprathreshold- or Strong-
responses). Whereas, stimulation in the hindpaws and tail would occasionally not be followed by
any response, allowing for an extra level of categorization in these body loci, if the change post-
IONX manifested as hyperresponsivity. Due to the lack of opportunities for 'zero responses' in
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the ears, and thus a lower resolution scale of categorizing responses when measuring ear
responsivity, variability in this trait's levels may have appeared to be artificially diminished. As
discussed above, it is also possible that because stimuli delivered in the ears where in full view of
the animals, this promoted a response more affected by these cues, as well as anticipation and
stress, masking the true tactile responsivity than elsewhere in the body. The spinal sites being
tested by the monofilament applied from below the animal, when it is not expecting and/or
seeing it approaching the target, were bound to reflect a more accurate measure of true tactile
responsivity. Thus, the genetic mapping that was performed in this study based on these traits
may have unraveled QTLs that, for the mechanical responsivity of the ears, actually related to
variability in stress response rather than to mechanical responsivity per se.
4.12 Genetic Effects on Basal and Post-IONX Mechanical Sensitivity
Baseline mechanical responsivity was rank ordered across the lines and parental strains
by increasing mechanical responsivity. Despite the weak correlation in the mechanical
responsivity across tested body loci, found when pooling all mice into one group, separately by
gender and surgery type, when repeating this correlations in the ranked strain/line data, a strong
correlation was found between the responsivity in all three body loci, indicating that a strain with
a given degree of responsivity in one body locus will exhibit similar responsivity elsewhere in
the body. This suggests a shared genetic control of mechanical responsivity across the body,
including interestingly, between spinal and craniofacial body sites. This suggests that the “pain
types” and the genetic control patterns that have been documented by the group of Mogil et al.,
and other investigators, cataloguing pain traits in naïve mice of inbred strains using assays for
spinal system body loci, may be applicable to the craniofacial region as well (9,73,112).
A similar analysis was done for the PO data, rank ordering mechanical responsivity data
in the 25 lines and parental strains, pooled for males and females, based on the “net-IONX
effect” in each tested body locus. The rank orders per loci were then correlated for every two of
the three loci. It was found that the significant correlations, observed in naïve animals between
the rank orders for the ears and paws and for the ears and tail, did not reoccur postoperatively.
The most reasonable explanation for the net-IONX-associated ‘disappearance’ of the inter-locus
correlations seen in naïve mice is the lack of net-IONX effect in the ears while changing the
mechanical responsivity in the paws and tail. This may suggest that different genetic factors
control baseline mechanical responsivity and the changes to this responsivity after IONX
(adjusted for the effect of sham-operation) in the craniofacial and spinal regions (112).
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4.13 Estimation of Heritability
The ‘narrow-sense heritability’ (h2) reflects contributions from genetic and non-genetic
(‘environmental’) determinants, which each explains the variability in the ‘typical’ line-specific
trait values by calculating the ratio of the genetic variance over the total variance:
h2 = VARgen / (VARgen + VARenv)
The values of h2 calculated in the present study for naïve mice were somewhat higher
than those computed for mechanical responsivity by Mogil et al. (9), suggesting that the relative
contribution of the genetic control over the traits studied in the present project was higher were
the estimates of Mogil et al. This may be due to the following reasons: (i) while both studies
used the VF filament assay to determine the mechanical responsivity at the paws, the method
used by them to determine the response magnitude of the animal to the stimulus (the “up-down”
method) was different from the method which was used in this study (i.e., by response
magnitude), resulting in my data showing less VARenv (e.g., experimenter-related variability).
Likewise, it is possible that other environmental influences differed between the studies, for
example the diet which is known to affect pain traits in naïve and denervated rodent models of
neuropathy (26); (ii) this difference was due to a higher genetic variance (i.e., VARgen) in the
strains/lines tested, compared to theirs. Since Mogil et al. used 11 inbred lines of generally
diverse genetic backgrounds, whereas this work studied 23 RI daughter lines and their parental
lines (who were included in the Mogil line panel), one might expect to observe a higher genetic
diversity (i.e., higher VARgen) in their panel than in this study, hence a higher h2. However, the
results were, in fact opposite, undermining the plausibility of the explanation offered by this
argument. Alternatively, Mogil’s panel may have included lines that were not as genetically
diverse. Moreover, by using a panel that is less than half the number of lines than the one used in
this study, it is possible that VARgen was in fact smaller in their panel.
An interesting trend was observed in the present study for the h2 scores to
diminish following surgery. But since this decline in heritability occurred both in sham and
IONX groups, it is proposed that this was the result of a time effect per se. The surgical
procedure may have introduced new environmental variables (related to the method of surgery)
that would likely have augmented the non-genetic VARenv contribution. As well, more time
had passed before taking postoperative measurements, and this too may have allowed for more
opportunity for environmental confounds (such as caging conditions, etc.) to ‘dilute’ the
heritability estimates (94, 141).
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4.14 Mapping QTLs for Mechanical Sensitivity and Effect of IONX
The confounding effects of environmental variance on QTL detection should be stressed.
The greater the degree of environmental variance, the more limited is one’s capacity to
distinguish the effects of a single gene amongst phenotypic variation due to background noise. It
should be noted that the limited amount of QTLs detected in this study may not reflect the
complete list of QTLs governing expression of these traits. Some relevant QTLs may exist, yet
confer an effect that is too minimal to be detected through the means used in this study. It this
sense, this study may only permit detection of QTLs with major effects on the phenotype.
The candidate genes found in this study are known to have SNPs in located within their
coding region or within regulatory regions. The WebQTL software did not provide information
regarding where along the genome each informative SNP was situated. It also should be noted
that, since the phenotypes assessed in this study incorporated both the motor and sensory
systems, genes appearing to be pain-related may actually pertain more to motor function.
The first QTL that was identified for a pain related behaviour in mice was named, Pain1, and
localized to the central region of chromosome 15 (5). This QTL was mapped for spontaneous
pain behaviour in the same AXB/BXA panel of RI mice as the one used here, following
unilateral transection of the sciatic and saphenous nerves (5). A subsequent study corroborated
this finding using a separate backcross panel of mice showing a QTL in the same genomic region
(92). Subsequent other studies using various panels of mice and various pain models were used
to identify putative QTLs for pain in other regions of the murine genome. Such findings include;
Nociq1 and Nociq2, on chromosome 9 and 10 for responsivity to inflammatory pain from
formalin injection (164), and Tpnr1 on chromosome 4 for responsivity to a heat stimulus in the
paws (165). Wilson et al. found Nociq1 on chr. 9 with a peak region at approximately 110Mb,
arguably overlapping with the a QTL found in several maps for a number of different pain traits
in the present study, which mapped 12 different QTLs for various aspects of mechanical
responsivity in intact mice, as well as changes to this responsivity post-IONX vs. sham
operation. Four of these QTLs were mapped for mechanical responsivity of intact animals in the
ears (on chrs. 1,16), paws (on chr. 9) and tail (on chr. 9). Nine QTLs were mapped for the net-
IONX effect, of these one was mapped for the change in mechanical responsivity in the ears (on
chr. 4; for PO2), paws [one on chr. 6, both for PO1 and PO2, 2 on chr. 9, one for PO1 and an
adjacent one for PO2 (these might be the same QTL), and one on chr. 18, for PO2], and tail (one
QTL on chr. 13, for PO2).
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Being able to map two traits in the same QTL supports the validity of the finding. Indeed,
the QTL on chr. 6 mapped a net-IONX effect in the paws both for PO1 and PO2. Likewise, the
chr. 9 QTL is shared amongst 4 sensory traits including responsivity of intact animals in the tail
and paws, as well as a net-IONX effect in these two body loci (i.e., the change in mechanical
responsivity in the paws - for PO1, and in the tail - for PO2). Three QTLs were of special
interest:
(i) The QTL on chr. 9, while reaching only suggestive significance levels, is unique as it was
linked to responsivity in the paws and tail, both in the intact state and post-IONX. Since it was
found that the responsivity in the two body loci correlated significantly both in intact mice and
post-IONX, the locus mapped to chr. 9 may control both of these traits.
(ii) The QTL on chr. 13 reached actual significance in its linkage to the net-IONX effect on the
tail mechanical responsivity in PO1. The Permutation mapping test identified the peak LRS of
this QTL at ~95.5 Mb, and validated this peak in the Bootstrap test as located in an interval
between ~94 and ~96 Mb.
(iii) The QTL on chr. 5, also reaching significance, is unique since it mapped the extent of spread
of mechanical hyperresponsivity post-IONX. Identifying the gene controlling this trait may have
clinical implications as it can be used as a target for developing novel painkillers that operate in
the CNS. Moreover, labeling the protein product it encodes can be used to study the mechanisms
involved in the spreading of pain to extra-territorial regions.
4.15 Mapping QTLs for M-PAT Parameters and Effect of IONX
Twelve different QTLs were mapped for various aspects of mobility in the M-PAT assay
in intact mice, as well as the changes to this behaviour following IONX compared to sham
operations. Seven QTLs were mapped for intact animals, Distance_Smooth was mapped to QTLs
on chrs. 2 and 10; Distance_Rough on chrs. 2, two QTLs on chr. 10 and one on 13;
Time_Smooth on chrs. 2,3,9 (note that Time_Rough complements Time_Smooth to 3 min hence
there was no logic in duplicating the analysis for this tautologic data). Changed mobility in the
M-PAT arena post-IONX was mapped for Time_Smooth on chrs. 1 (for PO2), 5 (for PO1), and
17 (for PO2); Distance_Smooth was mapped to chrs. 3 (two QTLs, both for PO1);
Distance_Rough was mapped to chr. 3 (for PO2). QTLs on chrs. 2 and 10 were each mapped for
two correlated M-PAT traits and it is not surprising that they were both mapped to the same
QTL. Three QTLs were of special interest:
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(i) The QTL on chr. 9, while reaching only suggestive significance levels, is unique as it was
linked to responsivity in the paws post-IONX (in PO2) as well as to Time_Smooth (in BL1).
This is not the same QTL on chr. 9 that was mentioned above (see 4.13).
(ii) The QTL on chr. 13, reached actual significance in its linkage to the net-IONX effect on the
tail mechano-responsivity in PO1, as well as Time_Smooth (in PO2).
iii) The QTL on chr. 5, reached significance in its linkage to the trait for propensity for
exhibiting spread-type 5 or 7 following IONX (in PO-2).
4.16 Candidate Genes in the Mapped QTLs
For several of the mapped QTLs, lists of genes were browsed which were harboured
within the QTLs interval length, but for brevity, in the Results, this process was only described
for one of these QTLs, located on chr. 5, spanning an interval of 10Mb (from 100Mb – 110Mb),
and linked to the spread of mechanical hyperresponsivity in PO2 to the tail/paws or
tail/paws/ears (Spread types 5 and 7). Out of the genes located in this interval, 3 candidate genes
were selected based on their documented relevance to neural functions or specifically to pain and
showed them in Table 32. Nkx6-1 encodes for a transcription factor that is needed for neural
development and could be relevant to plasticity in the CNS post-IONX and other neuropathies.
However, since it has no known SNPs in it, it is less likely a candidate to explain the variance in
trait levels across the RI panel. It may still have SNPs that are currently unknown.
Browsing the two other genes in this Table using the Allen Brain Atlas
(http://www.brain-map.org/) allowed for assessment of whether a candidate pain gene in Table
32 is expresssed in the CNS of a naïve 56 days old adult male mouse of the C57BL/6J (B)
parental strain, and a 4 days old newborn of the same strain. This atlas used the in situ
hybridization method to label brain and spinal cord slices using radiocatively labelled riboprobes
that complement unique sequence intervals in 20,000 murine genes. It was found that the 2 other
genes in the Table are strongly expressed in neurons: Mapk10 (aka Jnk3) encodes for MAPK10 -
mitogen activated protein kinase-10, and Sparcl1 (aka Hevin and Mast9), encoding SPARCl-1
(secreted protein acidic cysteine-rich)-like 1 protein. Figure 20 shows in the top right
photomicrograph that Mapk10 is expressed most likely only in neurons, including in the dorsal
horn (see upper left micrograph). The photomicrographs below map such neurons in the spinal
cord (using a false colour palette that shows very low expression in black, and increasing levels
of expression by 'hotter' colours), showing (in the left panel) low expression levels of Mapk10 in
laminae I and II (substantia gelatinosa, SG) that process nociceptive inputs. Unfortunately, no
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expression maps were available for this gene in the brain. Figure 20b shows corresponding maps
for Sparcl1, which is more abundantly expressed in the spinal cord (including upper dorsal horn
layers) than Mapk10. The false colour panel on the right shows that there is relatively stronger
expression of Sparcl1 in LI, but like Mapk10, there is a relatively low expression in SG. The
histological slide in c taken from The Alan Brain Atlas shows that Sparcl1 is robustly expressed
throughout the brain, including the cortex, hippocampus, cerebellum and elsewhere. This
suggests that the gene plays a role in neural functioning.
MAPK-10 (Mitogen-activated Protein Kinase-10) which is also referred to as JNK-3 (c-
Jun N-terminal kinase-3), an enzyme that is heavily expressed in the nervous system of mice and
can be activated by noxious peripheral stimuli (148,149). This enzyme has been shown to play a
role in mediating neuronal cell death from excitotoxic synaptic input as can occur in the nervous
system following nerve injury (150). In fact, this enzyme has been shown to partly mediate
neural apoptosis, specifically following axotomy of a cranial or spinal nerve (151). Nerve cell
death can be induced by high levels of input of excitatory neurotransmitters such as glutamate, a
barrage of which is received by the CNS by primary afferents during and following peripheral
axotomy (39,152). The death of central inhibitory neurons through this process can lead to a
diminished inhibitory tone in the sensory pathways. Heightened nociceptive input to the CNS
can ensue from disinhibition, and may partially account for pain spread as seen in this study
(39,152). Since the JNK-3 enzyme has been shown to mediate stressed-induced neural death, it is
plausible that variation in its gene sequence (and likely its amino acid composition) in our panel
of RI mice may account for the phenotypic variation of pain spread observed following IONX.
The other gene that was found in the QTL identified on chromosome 5, linked to the
phenotype of pain-spread was sparcl1. This gene codes for the protein SPARCl-1 ((secreted
protein acidic and rich in cysteine)-like protein-1) of the SPARC family, known to localize in the
pericellular matrix and link extracellular signals to cellular processes (153, 154). Little is known
about the specific function of SPARCl-1 in the CNS, though it has been suggested to play a
critical role in neural migration in the cortex during embryonic development in the mouse (155).
The enzyme’s dense expression in neurons suggested a role in brain development. The
polymorphic gene sequence renders it a potential factor in mediating pain-spread following
IONX. Using a similar process, a few other QTLs were studied, and it was found that some
include a number of interesting candidate pain genes. More work is needed to validate their
candidacy. On chr. 13, the Crfbp gene was found.
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Figure 20a-c: Photomicrographs of lumbar spinal cord and brain transverse sections of an intact B
mouse, labelling the expression levels of Mapk10 (in a) and Sparcl1 (in b). A transverse section of the
adult mouse brain labeled Sparcl1 mRNA is shown in c.
a.
b.
c.
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This gene encodes for Corticotropin-releasing factor binding protein, which modulates
the affinity of the CRF peptide hormone for its respective receptor (125). CRF is a central
contributor in stress-induced analgesia, shown to reduce nociceptive thresholds in a dose-
dependent administration (126). It has been shown that this hormone may potentiate
glutamatergic NMDA currents in dopaminergic neurons via CRF binding to CRFbp, ligated to
CRF receptor 2, activating intra-neuronal signal transduction pathways that involve PLC and
PKC, which in turn activate the NMDAr (125).
Sv2c is a gene located within the above QTL on chromosome 13, encoding the synaptic
vesicle glycoprotein 2c, a member of a family of proteins (SV2) that, as the name implies, plays
a role in synaptic release in several brain regions, as well as in endocrine cells (128, 127).
The gene Cacna2d2 that encodes the voltage-gated calcium channel α2δ2 subunit is
harboured in the chr. 9 QTL between 107.2Mb – 107.4Mb, slightly upstream this QTL’s peak.
This QTL has been linked to a number of traits related to mechanical responsivity in intact mice
and to hyperresponsivity post-IONX (see above 4.14i). The strong relevance to neural function
and to pain supports its candidacy. This subunit is one of three auxiliary proteins that comprise
high-voltage-activated (HVA) calcium channels (131) (Figure 21). The α2δ subunits are encoded
by at least three distinct genes (132), each of which is differentially expressed throughout the
mouse’s body.
The α2δ2 gene, which has at least one splice-variant, is expressed ubiquitously
throughout the nervous system (129) and plays a role in modulating calcium current influx
through the channel (130). As well, expression levels of the subunit have been shown to correlate
to the voltage-dependence of the channels activation and inactivation (129). Of special interest,
this subunit is the ligand-binding site of two commonly used analgesics for neuropathic pain
(Pregabalin and Gabapentin) (134), which make this gene a prime candidate for controlling pain
traits studied here. Figure 22 shows that Cacna2d2 mRNA is expressed in neurons throughout
the mouse spinal cord.
4.17 Correlation between Mechanical Sensitivity Pre- and Post-IONX and Spontaneous
Neuropathic Pain
The AXB-BXA RI panel has been used by several investigators to map QTLs for various
traits, most of which have no direct relationship to pain. Correlating the data for these traits could
unravel shared genetic control between traits. The only trait documenting QTLs for chronic pain
in the AXB-BXA RI panel was based on the Neuroma Model, a preparation used to elucidate
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pathophysiological mechanisms mediating spontaneous neuropathic pain and the response to
analgesic agents. Seltzer et al. used this model to identify a QTL on chr. 15 that they named
Pain1 (5). Recently they identified a gene encoding another subunit of the Ca++ channel, Cacng2,
that plays a role in spontaneous neuropathic pain behaviour in these mice, other inbred strains of
mice, and even in women postmastectomy (110).
Figure 21. Schematic illustration of the three subunits comprising the voltage-gated calcium
channel. The α2δ subunits are an auxiliary subunit and are involved in regulation of the Ca++
influx across the transmembrane pore, which is formed by the α1 subunit.
Figure 22. Distribution of mRNA expression of Cacna2d2 in the lumbosacral region of the
spinal cord dorsal horn.
Elahipanah et al. in our lab has used archival data from the study of Seltzer et al., that
were acquired using the same RI panel, and mapped additional QTLs on other chromosomes for
the self-mutilation behaviour (156). These same trait values were used for all 23 AXB-BXA
lines and the parental strains A, B, and correlated them with the traits quantified in the present
study on mechanical responsivity and spread of hyperresponsivity post-IONX (Table 34). This
analysis allowed for a direct comparison of basal tactile responsivity and changes post-IONX (vs.
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sham operation) and spontaneous neuropathic pain behaviour in the Neuroma Model. While an
interesting correlation was found between mechanical responsivity in the ears (in BL and PO1)
and all 3 parameters of spontaneous neuropathic pain, none remained significant post-Bonferroni
correction.
These generally negative results are compatible with those of previous studies showing a
lack of correlation between the same spontaneous neuropathic pain trait of self-mutilation,
baseline mechanical responsivity in the paw, and changes following partial hindpaw denervation
both in the SNL and PSL models, both in rats and mice (9,69,73,146,147). Thus these results
support the conclusions drawn by those studies that separate physiological mechanisms, and thus
genetic controls, are involved in the mediation of each of these pain phenotypes (69,112).
It should be noted that the nerve injury in studies on the Neuroma Model on spontaneous
neuropathic pain was that of peripheral nerves projecting from the spinal system (5), whereas the
current study used a peripheral nerve injury in the trigeminal system. A large body of literature
proposes that these two areas of the sensory nervous system have fundamental differences in
their normal physiology, as well as in their pathophysiology following nerve trauma
(1,23,29,36,38,61). With this knowledge, it is not surprising that there would be a lack of
correlation found between the expression patterns of pain triggered by trauma to these partially
dissimilar neural systems, each having unique features in their underlying neural mechanics.
While there are common histological structures involved in mediating basal sensory input and
input following nerve trauma, such as the nerve fibres involved, a number of substantial
functional changes in the sensory pathways following nerve injury have been described and
reviewed in the Introduction (1,39). These functional changes reflect an altered neural state that
is likely regulated by separate genetic determinants than those regulating sensory transmission
and processing in the naïve state (1,69).
4.18 Findings Corroborating or refuting the Hypotheses
In summary, the following findings corroborate or refuted the Hypotheses:
Hypothesis A: IONX causes altered mechanical responsivity in extra-territorial body loci.
Main Results:
IONX was associated with an increased responsivity to mechanical stimuli in the ears (in
males but not in females), and in the paws and tail in both genders.
In the M-PAT assay IONX was associated in males with freezing in the Rough zone,
thereby likely exhibiting avoidance of walking on this pro-allodynic surface.
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Hypothesis B: Normal mechanical responsivity and altered mechanical responsivity in extra-
territorial body loci following IONX are gender-specific.
Main Results:
There were minimal differences observed between genders in the traits phenotyped.
In males but not females IONX was associated with an increased responsivity to
mechanical stimuli in the ears.
In females, but not in males, there was a weak (yet significant) correlation between the
responsivity in the ears and tail
In the M-PAT assay males, but not females, exhibited Rough zone freezing.
Hypothesis C: Normal mechanical responsivity and altered mechanical responsivity in extra-
territorial body loci following IONX is controlled by genetic determinants.
Main Results:
Striking differences were shown across the 25 strains/lines in the levels of mechanical
responsivity of naïve mice and in the response to IONX. Since all mice were tested by the
same unbiased investigator who was not aware of the strains/lines tested, and under
identical testing conditions, these differences strongly indicate a genetic control of the
inter-strain/line variability.
LDPs of all traits showed a gradual difference in trait levels across the strains/lines,
indicating a polygenic heritability mode.
These differences enabled:
- Estimating the heritability and determine that these traits are under strong
genetic control, explaining ~75-83% of the variability in the levels of mechanical
responsivity of naïve mice and ~73-75% of their behaviour in the M-PAT assay,
and that ~55-71% of the change in mechanical responsivity and ~50-73% of the
change in M-PAT parameters post-IONX are also controlled by genetic
determinants.
- Mapping QTLs for these traits throughout the genome, some of which were
shared between mechanical responsivity in certain body loci, and certain
parameters of the M-PAT assay, both in naïve mice and post-IONX.
QTLs of significant LRS were found on:
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o Chr. 5, associated with spread of mechanical hyperresponsivity
following IONX.
o Chr. 13, for mechanical responsivity in the Tail in PO1 and
Time_in_Smooth in BL1.
o Chr. 9, for several traits such as responsivity in the Tail in PO1
and Time_in_Smooth in BL1.
4.19 Summary of Other Significant Findings of the Study
When averaging the data for all naïve mice (separately by gender but regardless of the
genetic background) no indications were found that repeating the stimulation with a Von
Frey filament in any tested locus for 7 times during a testing session was associated with
response adaptation.
Instead, a transient and progressive sensitization was seen in the hindpaws and tail,
respectively, but not the ears.
No side differences were seen in the mechanical responsivity of the paws and ears.
Mechanical responsivity of any tested locus did not differ across the 2 baseline testing
sessions.
In the M-PAT assay, naïve mice learned from BL1 to BL2 to walk a longer distance in the
Rough zone but do it at a slower speed than in the Smooth zone.
Responsivity in the hindpaws to mechanical stimuli in naïve mice did not correlate with
any M-PAT parameter, yet the ears and tail showed some instances of correlation
between the two assays.
A gradient in mechanical responsivity was seen across the tested loci in the order of:
ears>paws>tail.
A few instances of correlation were seen between the ears and the other loci; moderate
correlation was seen between the responsivity of paws and that of the tail.
No significant correlation was found between traits measured in this study and
spontaneous neuropathic pain-like behaviour from another study by this lab.
4.20 Future Directions
Due to the large span of the genomic intervals identified in this study as putative QTLs,
further genetic assays are required in order to narrow down the causative regions, with the
ultimate aim of isolating the contribution of a specific gene (or genes) involved in controlling the
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phenotypic variation of the studied traits. The use of congenic strains can be helpful to this end.
The production of a congenic strain involves the substitution of a segment of DNA (containing a
putative QTL) from one strain onto the genomic background of another strain. If the former
strain has been shown to contrast with the latter strain in expression of a trait of interest, and the
substituted region contains a putative QTL, then the congenic strain can be used to validate the
effect of this specific genomic region on the phenotype (22). This method has been used to
validate the presence of a previously identified QTL controlling proneness to behaviour
suggestive of spontaneous pain in mice (22). As well, the congenic approach can be used to
reduce the confidence interval that is known to contain the causative locus, by producing
congenic strains with incrementally inserted segments (131). Mogil et al. used this congenic
approach to narrow down the interval of a previously identified QTL, Tpnr3, on chromosome 17
in mice for thermal nociception, as measured by hindpaw withdrawal thresholds to a heat
stimulus. The production of congenic and sub-congenic strains allowed this investigator to
narrow down the size of the putative QTL to a region harboring as few as 17 known genes (161).
Another method of refining or narrowing the confidence interval of the putative QTLs, is to use
an advanced intercrossed panel of lines of mice (162). This involves randomly inter-breeding the
F2 generation (as discussed in 1.6.3) and the resultant generations (as opposed to several
generations of brother-sister mating, as is done in the RI approach). This breeding system allows
for a resultant panel of mice representing a more densely distributed mosaic of recombinant
sequences, facilitating finer mapping of QTL (161). Another powerful mapping assay is the
computational haplotyping mapping method of Peltz (168) that is based on phenotyping a panel
of 15-20 inbred strains of mice for which a highly dense genetic map exists, that is based on
millions of SNPs. Peltz et al. used this information to create short haplotypes shared among some
strains of the panel and not others. Using this computational method, our collaborators Zhang et
al. (University of Zhejiang, Hangzhou, China) currently phenotype male and female mice of this
panel using identical methods and IONX model like those used in the present study to map the
same traits using the Peltz method. When available, comparing the present results to theirs will
facilitate identifying short haplotypes within the QTLs identified here, as a method to refine the
positional mapping of candidate genes controlling the spread of mechanical hyperresponsivity
post-IONX and basal mechanical responsivity in the mouse.
To further validate the candidacy of the genes identified in this study, a common
approach involves profiling the expression of the putative genes in a trait-relevant tissue in naïve
mice and mice following IONX or sham operation (112). This approach may provide further
91
evidence for the supposed involvement of a gene in pain spread based on its up- or down-
regulation following nerve injury (112). This approach was used by Persson et al. to strengthen
the hypothesis of involvement of specific voltage-gated sodium channel subunits in controlling
levels of spontaneous pain after axotomy of a peripheral nerve section in five strains of mice
varying in proneness to this trait (162). Persson et al. showed that genes encoding such subunits
were upregulated in the DRG following axotomy of a peripheral nerve. It was hypothesized that
the stereotyped hyperexcitable state of the primary afferent neuron following axotomy was
controlled in part by changes in expression levels of various genes within the cell body in the
DRG. Moreover, it was hypothesized that changes in the electrical properties of the nerve fibres
may be related to changes in production of voltage-gated sodium channels (known to be directly
related to electrogenicity of primary afferent neurons) and their insertion into the cell membrane.
Such genes are differentially regulated within the DRG following nerve injury and correlated
with the level of neuropathic pain observed in mice strains (162).
It would be of interest to phenotype this panel of RI mice for responsivity levels
regarding sensory modalities other than mechano-responsivity, such as thermal responsivity. In
fact, experiments in this vein are ongoing in our lab. In this way, questions can be addressed
concerning whether or not there exists an overlap between genetic underpinnings of different
sensory modalities. To corroborate or refute the present findings, the use of other nerve injury
models can be used, such as the transection of the IAN, shown to result in pain spread to a
neighbouring craniofacial region (59). As well, models of chronic inflammatory pain in mice can
be employed for the purpose of addressing whether or not the genetic influences of pain spread
are specific to the experimental conditions and the model used in the present study. Other
methods of VF scaling can be employed, such as the Chaplan method (22) to test whether pain
spread to the ears is detectible via another mechanical stimulation approach. As well,
electrophysiological recordings from the CNS can be taken in order to further elucidate the
physiological mechanisms underlying spread of hyperresponsivity to mechanical stimulation.
Furthermore, immunohistochemistry can be employed in order to assess cellular changes within
the CNS, which may additionally reveal underlying mechanisms governing the phenomics of
such traits.
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5.0 Appendix
Figure 11a-c: Distance travelled in the Smooth and Rough zones separately for males and
females in the PO1 and PO2 testing periods. b. Time spent in the Smooth zone. Note that Time
in the Rough zone is not shown since it is complementary to 180 seconds for all mice in all
groups. c. Speed of mobility in the Smooth and Rough zones. Data for the Time in the Smooth
zone and the Distance travelled in the Smooth and Rough zones were then normalized for each
mouse in PO1 and PO2 by subtracting the respective baseline (BL2) values, and then these
difference scores were averaged per group. The group difference scores in Figure 9a show that
the distance travelled by all groups was shorter in PO1 and PO2 than BL2, but to a variable
extent depending on the type of surgery and gender.
-0.5
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Females_Sham Females_IONX
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a. Distance
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b. Time (sec) in Smooth zone
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Table 4: Statistical analysis comparing the distribution of response types across trials number 1,
4 and 7 for BL1 and BL2, males and females, using X2 test. Highlighted in bold font are
comparisons yielding a p≤0.05; comparisons still significant after a Bonferroni adjustment of the
alpha level are highlighted in a bold frame.
Tested Locus
Side Testing period
Males Females X2 p-value X2 p-value
Ears R
BL1 3.253 0.51 3.228 0.52 BL2 1.002 0.90 0.292 0.99
L BL1 2.920 0.57 1.018 0.90 BL2 1.947 0.74 3.812 0.43
Paws R
BL1 1.569 0.81 10.652 0.031 BL2 9.909 0.042 8.659 0.07
L BL1 14.183 0.007 7.832 0.097 BL2 15.950 0.003 14.960 0.005
Tail BL1 15.172 0.004 24.774 0.00006 BL2 18.343 0.001 32.418 0.000002
Table 5: Results of statistical analysis comparing the distribution of response types between left
and right sides for tested body locus, and each trial (numbers 1-7) for BL1 and BL2, males and
females, using the X2 test. Highlighted in red font are comparisons yielding a p≤0.05; in green
font are comparisons showing a trend toward significance, with 0.1<p>0.05.
Trial number Tesing period Statistics BL1 BL2 BL1 BL2 BL1 BL2 BL1 BL2
Chi square 0.387 0.259 0.00 0.016 9.812 6.174 0.381 5.947P-value 0.82 0.87 1.00 0.99 0.007 0.045 0.82 0.051
Chi square 1.616 0.523 2.453 1.039 1.725 0.854 2.771 1.465P-value 0.44 0.77 0.29 0.59 0.42 0.65 0.25 0.48
Chi square 0.517 4.010 0.805 0.332 5.017 0.369 0.311 3.116P-value 0.77 0.13 0.66 0.84 0.081 0.83 0.85 0.21
Chi square 6.462 0.843 3.696 0.629 1.847 1.921 1.157 2.666P-value 0.040 0.65 0.15 0.73 0.39 0.38 0.56 0.26
Chi square 0.578 3.226 2.199 9.883 0.941 0.167 1.252 4.948P-value 0.74 0.19 0.33 0.0071 0.62 0.91 0.53 0.084
Chi square 3.817 0.568 0.703 0.197 4.403 1.861 2.708 3.173P-value 0.14 0.75 0.70 0.90 0.11 0.39 0.25 0.20
Chi square 0.980 1.044 0.454 2.077 3.628 7.172 4.971 4.767P-value 0.61 0.59 0.79 0.35 0.16 0.027 0.083 0.092
Males Females FemalesBody locus Ears Paws
6
7
Gender
1
2
3
4
5
Males
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Table 6: Chi Square and p-values for comparisons of responsivity in each body locus between
the two baseline periods for each stimulus trial. P-values highlighted in red are those that reached
significance (p≤0.05). Values highlighted in green have reached a suggestive level.
Table 8: Spearman rank correlation coefficients (r) with respective p-Values for the camparisons
of responsivity of select body loci (ears, hindpaws, and tail) to one another for each of the 7
stimulus trials, for baseline 1 (BL1) and 2 (BL2), for males (M) and females (F). P-Values
highlighted in red are those that reached significance in the absence of Bonferroni correction.
Those highlighted in gray have reached significance after Bonferroni correction.
Body locus Ears Paws Tail Ears Paws TailStatistics Gender
Chi square 0.726 6.113 2.863 0.045 5.745 5.437p-value 0.70 0.047 0.24 0.98 0.057 0.066
Chi square 2.921 13.037 0.972 0.000 10.245 6.096p-value 0.23 0.0015 0.62 1.00 0.006 0.047
Chi square 0.179 11.605 12.310 2.287 1.544 7.791p-value 0.91 0.003 0.002 0.32 0.46 0.020
Chi square 0.799 9.900 3.613 0.011 5.495 10.723p-value 0.67 0.007 0.16 0.99 0.06 0.005
Chi square 0.110 2.437 3.023 0.109 11.472 12.079p-value 0.95 0.30 0.22 0.95 0.003 0.002
Chi square 2.875 4.930 4.162 0.331 5.046 10.214p-value 0.24 0.09 0.12 0.85 0.08 0.006
Chi square 0.119 4.258 2.195 0.315 12.629 6.390p-value 0.94 0.12 0.33 0.85 0.0018 0.041
Trial number
1
FemalesMales
7
2
3
4
5
6
r p-Value r p-Value r p-Value r p-Value r p-Value r p-Value r p-ValueBL1 Paws_Tail 0.14 0.026 0.12 0.063 0.14 0.023 0.12 0.062 0.021 0.001 0.16 0.012 0.14 0.03BL2 Paws_Tail 0.15 0.019 0.01 0.11 0.06 0.31 0.28 0.0000056 0.24 0.0001 0.06 0.35 0.24 0.00011BL1 Ears_Paws 0.026 0.69 0.95 0.13 0.02 0.72 0.13 0.035 0.19 0.002 0.093 0.14 0.07 0.3BL2 Ears_Paws 0.38 0.55 0.073 0.25 0.06 0.37 0.03 0.62 0.082 0..19 0.058 0.36 0.02 0.79BL1 Ears_Tail 0.16 0.78 0.2 0.001 0.1 0.11 0.12 0.066 0.16 0.11 0.17 0.006 0.19 0.002BL2 Ears_Tail 0.079 0.21 0.022 0.73 0.1 0.12 0.11 0.09 0.16 0.009 0.17 0.002 0.01 0.87BL1 Paws_Tail 0.14 0.031 0.18 0.004 0.1 0.12 0.23 0.00022 0.16 0.013 0.2 0.001 0.06 0.37BL2 Paws_Tail 0.21 0.001 0.21 0.001 0.27 0.000019 0.17 0.006 0.12 0.069 0.2 0.001 0.17 0.008BL1 Ears_Paws 0.047 0.46 0.004 0.95 0.07 0.31 0.15 0.019 0.14 0.026 0.09 0.16 0.07 0.31BL2 Ears_Paws 0.063 0.32 0.047 0.46 0.04 0.54 0.05 0.39 0.88 0.17 0.037 0.56 0.01 0.83BL1 Ears_Tail 0.15 0.019 0.16 0.01 0.21 0.001 0.24 0.00014 0.12 0.094 0.075 0.24 0.18 0.004BL2 Ears_Tail 0.04 0.45 0.16 0.014 0.25 0.000058 0.03 0.67 0.28 0.00001 0.13 0.35 0.07 0.25
SexTested period
Loci correlated
M
F
7Trial_N
1 2 3 4 5 6
96
Table 9: Results of paired t-tests comparing behavioural parameters between the two different
zones in the M-PAT during each baseline testing session. Highlighted: p≤0.05; bold = values
significant past a Bonferroni correction.
p value
Gender Surgery type
Testing period Time Distance Speed
Males
Naïve BL1
0.05 0.86 0.98 Females 0.85 0.20 0.20
Males BL2
0.14 0.0012 <0.000 Females 0.0024 <0.000 <0.000
Males
Sham PO1 0.28 1.00 0.20 IONX 0.85 0.27 0.35 Sham
PO2 0.98 0.96 0.46
IONX 0.0051 0.13 0.024
Females
Sham PO1
0.54 0.73 0.52 IONX 0.87 0.46 0.65 Sham
PO2 0.16 0.0047 0.13
IONX 0.12 0.20 0.03
Table 10: Results of paired t-tests comparing behavioural parameters in specific zones of the
M-PAT between the two baseline testing sessions. Highlighted: p≤0.05; bold = values significant
past a Bonferroni correction.
Gender Zone Parameter t df p-Value
Males Smooth
Time -1.25 241 0.21 Distance 4.46 241 0.000013
Rough Time 1.24 241 0.21 Distance 3.58 241 0.00041
Females Smooth
Time 1.23 246 0.22 Distance 5.64 246 0.000000046
Rough Time -1.23 246 0.22 Distance 3.45 245 0.00066
97
Table 11a,b: Spearman rank correlation coefficients with respective p-values for responsivity of
tested body loci in naïve mice as measured via Von Frey stimulation for males (a) and females
(b) versus parameters measured with the Mecahnical Place Avoidance test (M-PAT; i.e., time
spent and distance travelled on each surface type). P-values highlighted in red cells reached
significance, and those with bold larger font designate values still significant after a Bonferroni
correction of p≤0.0001.
a. Males
Time in Smooth
Zone
Distance in Smooth Zone
Distance in Rough Zone
Time in Smooth
Zone
Distance in Smooth
Zone
Distance in Rough Zone
r2 -.31 -.34p value 0.000020 0.0000030
r2 -.21 -.21 -.17 -.19p value 0.0037 0.0037 0.019 0.010
r2 -.24 -.23p value 0.0011 0.0019
r2 -.21 -.22 -.23 -.23p value 0.0034 0.0022 0.0018 0.0015
r2 -.24 -.25 -.32 -.33p value 0.0010 0.00055 0.000012 0.0000037
r2 -.25 -.30p value 0.00067 0.000034
r2 -.17 -.16 -.32 -.34p value 0.020 0.030 0.000012 0.0000031
r2
p valuer2
p valuer2
p valuer2
p valuer2
p valuer2
p valuer2
p valuer2 -.15 -.16
p value 0.045 0.033r2 -.18 -.19 .19
p value 0.016 0.0095 0.010r2 -.33 -.32 .25 -.29 -.32
p value 0.0000056 0.0000090 0.00060 0.000084 0.000010r2 -.23 -.29 -.32 -.30
p value 0.0016 0.000077 0.000010 0.000040r2 -.30 -.34 .21 -.27 -.27
p value 0.000025 0.0000019 0.0036 0.00023 0.00017r2 -.18 -.28 -.31
p value 0.014 0.00010 0.000017r2 -.15 -.21 -.19 -.19
p value 0.042 0.0033 0.010 0.0092
1
2
3
4
5
6
7
Ears
Paws
Tail
1
2
3
4
4
5
6
7
Tested Period
StatisticTrial number
Tested Body SiteGender
5
6
7
1
2
3
BL1 BL2
Males
98
b. Females
Time in Smooth
Zone
Distance in Smooth Zone
Distance in Rough Zone
Time in Smooth
Zone
Distance in Smooth
Zone
Distance in Rough Zone
r2 -.26 -.25 -.29 -.27p value 0.00052 0.0010 0.00012 0.00033
r2 .17 -.28 -.27 -.19 -.18p value 0.025 0.00020 0.00028 0.01169406 0.017
r2 .18 -.28 -.30 -.20 -.21p value 0.022 0.00016 0.000082 0.0088 0.0064
r2 -.24 -.25 -.21 -.19p value 0.0016 0.0010 0.0050 0.014
r2 .18 -.21 -.21p value 0.020 0.0049 0.0052
r2 -.20 -.18p value 0.0096 0.016
r2 -.18 -.16 -.18 -.19p value 0.021 0.032 0.017 0.015
r2
p valuer2
p valuer2
p valuer2 -.17 -.16
p value 0.023 0.039r2
p valuer2
p valuer2
p valuer2
p valuer2 -.25 -.29 -.21 -.18
p value 0.0010 0.00015 0.0058 0.019r2 -.40 -.40 -.26 -.29
p value 0.000000043 0.000000043 0.00060 0.000096r2 -.38 -.37 -.39 -.45
p value 0.00000019 0.00000061 0.00000013 0.0000000011r2 -.30 -.30 -.28 -.28
p value 0.000074 0.000064 0.00017 0.00021r2 -.21 -.25 -.22 -.22
p value 0.0059 0.00077 0.0042 0.0045r2 -.20 -.23
p value 0.0087 0.0030
Ears
Paws
Tail
6
7
3
4
5
6
7
1
1
2
Gender Tested Body Site
Trial number Statistic
Tested Period
BL1 BL2
Females
7
1
2
3
4
5
6
2
3
4
5
99
Table 12: Results of statistical analysis comparing the distribution of response types between
the sides ipsi- and contralateral to the surgery, for each trial (numbers 1-7) for PO1 and PO2,
males and females, using X2 test.
Trial number
Statistics Tesing period
X2 P X2 P X2 P X2 P X2 P X2 P X2 P
Sham 0.673 0.95 0.168 1.00 0.191 1.00 1.722 0.79 0.921 0.92 0.046 1.00 0.528 0.97IONX 0.168 1.00 2.261 0.69 1.235 0.87 0.224 0.99 0.050 1.00 0.246 0.99 0.389 0.98Sham 0.409 0.98 2.189 0.70 0.176 1.00 0.165 1.00 0.040 1.00 0.043 1.00 0.188 1.00IONX 0.145 1.00 0.040 1.00 0.807 0.94 1.050 0.90 0.805 0.94 0.378 0.98 0.000 1.00Sham 0.159 1.00 0.144 1.00 0.168 1.00 0.039 1.00 0.059 1.00 0.054 1.00 0.896 0.93IONX 1.356 0.85 0.527 0.97 3.295 0.51 2.427 0.66 1.617 0.81 0.482 0.98 0.246 0.99Sham 0.531 0.97 2.972 0.56 1.157 0.89 6.012 0.20 0.246 0.99 0.391 0.98 2.876 0.58IONX 0.046 1.00 1.594 0.81 0.443 0.98 0.049 1.00 5.114 0.28 0.596 0.96 1.841 0.76Sham 2.117 0.71 1.690 0.79 1.182 0.88 1.072 0.90 0.536 0.97 0.131 1.00 4.415 0.35IONX 0.000 1.00 0.814 0.94 0.653 0.96 0.093 1.00 0.154 1.00 2.682 0.61 0.116 1.00Sham 6.234 0.18 5.725 0.22 0.542 0.97 1.989 0.74 0.185 1.00 3.062 0.55 0.390 0.98IONX 0.915 0.92 0.223 0.99 2.897 0.58 2.272 0.69 0.461 0.98 1.603 0.81 1.166 0.88Sham 1.106 0.89 0.460 0.98 2.328 0.68 1.100 0.89 1.757 0.78 1.633 0.80 3.213 0.52IONX 4.887 0.30 0.336 0.99 2.262 0.69 0.521 0.97 0.945 0.92 0.485 0.97 3.332 0.50Sham 0.580 0.97 1.089 0.90 2.219 0.70 0.904 0.92 1.107 0.89 4.781 0.31 0.095 1.00IONX 0.582 0.97 2.055 0.73 1.675 0.80 0.184 1.00 3.348 0.50 0.277 0.99 5.603 0.23
MalesPO1
PO2
FemalesPO1
PO2
Ears
MalesPO1
PO2
FemalesPO1
PO2
Paws
71 2 3 4 5 6Body locus Gender
100
Table 13: Results of statistical analysis comparing the distribution of response types in PO1 versus PO2 for each surgery type (sham operated and IONX), for each trial (numbers 1-7) for males and females, using X2 test. Highlighted in blue are P values showing a trend toward significance (0.1<p>0.05); in red font are comparisons having a value of p≤0.05 and in purple are those that remained significant after a Bonferroni correction.
Tested
period
Tr
ials nu
mber T
ested
site
12
34
56
7
Sham
ChiEar
s0.11
2
2.137
1.040
0.733
2.88
8
0.51
0
0.138
P value
Ears
0.998
0.71
1
0.90
4
0.94
7
0.577
0.973
0.99
8
ION
XChi
Ears
0.690
0.80
9
1.72
0
0.03
4
0.303
-
1.18
7
P va
lueEar
s0.95
3
0.937
0.787
1.000
0.99
0
1.00
0
0.880
Sham
ChiPaw
s0.92
6
1.075
1.607
2.745
2.14
0
1.83
1
0.359
P value
Paws
0.921
0.89
8
0.80
8
0.60
1
0.710
0.767
0.98
6
ION
XChi
Paws
2.282
0.31
7
0.56
3
4.62
7
1.854
0.106
0.51
8
P va
luePaw
s0.68
4
0.989
0.967
0.328
0.76
3
0.99
9
0.972
Sham
ChiTail
6.237
1.36
3
1.56
0
2.37
5
1.756
1.731
0.47
8
P va
lueTail
0.182
0.85
1
0.81
6
0.66
7
0.780
0.785
0.97
6
ION
XChi
Tail1.08
4
1.089
7.982
5.743
0.40
5
0.79
9
0.473
P value
Tail0.89
7
0.896
0.092
0.219
0.98
2
0.93
9
0.976
Sham
ChiEar
s2.73
2
2.807
0.011
0.009
0.69
1
0.24
7
0.500
P value
Ears
0.604
0.59
1
1.00
0
1.00
0
0.952
0.993
0.97
4
ION
XChi
Ears
0.015
0.07
4
2.06
2
0.14
3
0.118
1.528
0.10
5
P va
lueEar
s1.00
0
0.999
0.724
0.998
0.99
8
0.82
2
0.999
Sham
ChiPaw
s3.52
4
3.602
3.369
4.762
1.81
1
4.28
5
2.446
P value
Paws
0.474
0.46
3
0.49
8
0.31
3
0.770
0.369
0.65
4
ION
XChi
Paws
2.831
0.09
4
3.35
7
3.39
7
4.130
5.953
0.11
1
P va
luePaw
s0.58
6
0.999
0.500
0.494
0.38
9
0.20
3
0.999
Sham
ChiTail
25.119
4.997
17.629
9.663
10.5
70
19.236
10.8
98
P value
Tail0.00
005
0.28
8
0.00
1
0.04
7
0.032
0.001
0.02
8
ION
XChi
Tail6.37
7
17.656
15.1
51
25.4
72
11.519
3.83
0
18.166
P va
lueTail
0.173
0.00
1
0.004
0.00004
0.021
0.429
0.00
1
Females
Gender
Surger
ySta
tistic
Postop
erative
ly
Males
101
Table 14: Results of statistical analysis comparing the distribution of response types in BL2
versus PO1 or PO2 for each surgery type (sham operated and IONX), for each trial (numbers 1-
7) for, males and females, using X2 test. Highlighted in green are P values showing a trend
toward significance (0.1<p>0.05); in red are comparisons having a value of p≤0.05 and in bold
frame are those that remained significant after a Bonferroni correction.
Table 15: Counts of the number of trials (out of 7) showing a significantly different distribution
in the response types to VF stimulation, when comparing PO1 versus BL2 and PO2 versus BL2,
for the sham-operated group, IONX group and the difference between these groups (IONX-
sham).
Locus Gender Sham IONX IONX-Sham
PO1 PO2 PO1 PO2 PO1 PO2
Ears Males 0 0 1 0 1 0
Females 0 0 0 0 0 0
Paws Males 1 1 1 4 0 3
Females 5 3 6 7 1 4
Tail Males 1 4 2 6 1 2
Females 4 5 3 4 -1 -1
Total Males 2 5 4 10 2 5
Females 9 8 9 11 0 3
102
Table 16: The group total difference mechanical responsivity scores between the postoperative
periods and baseline levels of BL2 for the sham-operated and IONX groups at each tested body
site, per gender. This data was used to calculate group averages and SEM shown in Figures 9a,b,
and for the t-tests, the results of which are also shown in those Figures.
Table 17a-c: a. P values associated with comparisons of the two postoperative (PO1 and PO2)
testing periods against the second baseline testing session (BL2) for Time in Smooth and
Distance in Smooth and Rough, for males and females. b. Group averages (AVG), SEM and
number (N) of male and female mice sham-operated or post-IONX. Comparison of BL2 data to
those of the PO1 and PO2 testing periods for Time and Distance on the Smooth and Rough zones
of the M-PAT assay. p Values associated with comparisons of the two postoperative (PO1 and
PO2) testing periods against the second baseline testing session (BL2) for Time in Smooth and
Distance in Smooth and Rough, for males and females. c. The same comparisons but for Speed
in Smooth and Rough. In red are comparisons yielding significantly different group averages,
and in bold font are p values that remained significnat following a Bonferroni correction.
Gender Surgery
type
Tested
periods PO1 - BL2 PO2 - BL2
n mice Body locus
EARS PAWS TAIL EARS PAWS TAIL
Males Sham 93 -23 28 19 -13 37 99
IONX 92 4 112 153 2 112 179
Females Sham 94 30 57 115 29 90 177
IONX 99 12 122 90 0 169 190
Gender Males Females a. Time and Distance Surgery type Sham IONX Sham IONX
Zone M-PAT Parameter Compared groups p-Values
BL2 vs. PO1 0.051 0.47 0.31 0.62 Time
BL2 vs. PO2 0.25 0.0051 0.44 0.58 BL2 vs. PO1 0.48 0.37 0.01 0.0002
Smooth
BL2 vs. PO2 0.29 0.059 8.40E-06 0.00026 BL2 vs. PO1 0.13 0.6 0.0006 0.0002
Rough Distance
BL2 vs. PO2 0.099 0.23 0.0005 1.14E-05
103
Table 18: Results of independent samples t-test comparing parameters of the M-PAT assay for
PO1 and PO2 data normalized against naïve scores, in the sham-operated to the IONX groups.
Gender
P-Values when comparing normalized sham-operated vs. normalized IONX PO1 PO2 PO1 PO2
Smooth Rough Smooth Rough Smooth Rough Smooth Rough Time Distance Distance Time Distance Distance Speed
Males 0.027 0.92 0.73 0.0027 0.97 0.27 0.63 0.99 0.15 0.57 Females 0.28 0.43 0.83 0.86 0.89 0.74 0.25 0.62 0.39 0.72
b. Speed: BL2 PO1 PO2 PO1 vs. BL2 PO2 vs. BL2 Gender Surgery
type Stats Smooth Rough Smooth Rough Smooth Rough Smooth Rough Smooth Rough
N 94 94 94 85 93 93 AVG 1.13 1.15 0.99 0.98 0.94 0.96 SEM 0.11 0.13 0.10 0.11 0.09 0.11
Sham
P 0.10 0.01 0.85 0.0076 N 90 90 92 79 92 92
AVG 1.14 1.22 1.06 1.12 1.10 0.95 SEM 0.12 0.13 0.11 0.14 0.12 0.10
Males
IONX
P 0.47 0.79 0.84 0.0035 N 82 81 83 83 71 71
AVG 1.14 1.20 1.02 0.97 1.09 0.94 SEM 0.13 0.14 0.11 0.11 0.12 0.10
Sham
P 0.09 0.06 0.0073 0.000073 N 89 89 89 89 77 77
AVG 1.17 1.16 0.93 0.90 1.01 0.87 SEM 0.13 0.13 0.09 0.10 0.11 0.10
Females
IONX
P 0.0058 7E-04 8E-05 8.50E-06
104
Table 19a-d: Correlation of tactile responsivity and M-PAT parameters. Spearman rank correlation
coefficients and respective p-values for responsivity of tested body loci postoperatively as measured via
Von Frey stimulation for males and females versus behavioural parameters measured with the Place
Preference Assay (M-PAT; i.e., time spent and distance travelled on each surface type) (a-d). P-values
highlighted in red cells reached significance, and those with bold larger font designate values still
significant after a Bonferroni correction of p<0.0008.
a. Sham-operated male mice: M-PAT parameters vs. Von frey responsivity
105
b. Sham-operated female mice: M-PAT parameters vs. Von frey responsivity
106
c. IONX-injured male mice: M-PAT parameters vs. Von frey responsivity
107
d. IONX-injured female mice: M-PAT parameters vs. Von frey responsivity
108
Table 20: Table showing the count of significant correlations found between any body site and
parameters of the M-PAT for males and females separately.
Table 21: Possible permutations of hyperalgesia (‘HYPER’) or its lack thereof (‘NOT’) in
tested regions (ears, hindpaws, and tail). Each unique repetoire of changes in regional
responsivity was catagorized as a particular ‘Spread Type’. The number of occurences of each
spread type in each surgery group is shown, as well as the percentage of each surgery group that
showed the particular spread types of 5 (hyperalgesia in all tested regions) and 7 (hyperalgesia in
the two most distal regions).
109
Table 22: Results for Chi Square test comparing genders in response type distribution at each
testing trial, tested body locus, surgery group (IONX and sham) and testing session: baseline 1
(BL1), baseline 2 (BL2), Postoperative 1 (PO1), and Postoperative 2 (PO2). Cells highlighted in
red show significant comparisons; in green font are values showing a trend for significance and
in bold larger font – those surviving the Bonferroni correction.
Chi p Chi p Chi p1 0.121 0.94 1 0.194 0.91 0.029 0.994 0.071 0.97 4 0.076 0.96 0.053 0.977 0.042 0.98 7 0.122 0.94 0.083 0.961 0.013 0.99 1 0.726 0.70 0.759 0.684 1.265 0.53 4 0.407 0.82 0.118 0.947 0.167 0.92 7 0.525 0.77 0.323 0.851 0.106 0.95 1 2.272 0.32 0.615 0.744 2.972 0.23 4 15.656 0.00040 2.365 0.317 6.570 0.037 7 0.745 0.69 0.842 0.661 1.670 0.43 1 2.777 0.25 0.656 0.724 0.883 0.64 4 4.059 0.13 0.501 0.787 6.398 0.041 7 1.856 0.40 1.789 0.411 0.557 0.76 1 20.469 0.000036 0.053 0.974 2.572 0.28 4 5.538 0.063 3.826 0.157 1.303 0.52 7 6.754 0.034 4.222 0.121 1.567 0.46 1 2.912 0.23 0.537 0.764 1.930 0.38 4 7.505 0.023 2.624 0.277 0.363 0.83 7 0.866 0.65 6.536 0.038
NaïvePostoperatively
Testing session
Body locusTesting session
Trials Trials
PO1
PO2
PO1
PO2
PO1
PO2
EARS
PAWS
TAIL
BL1
BL2
BL1
BL2
Sham IONX
BL1
BL2
110
Table 23: The change in the response pattern from trial 1 to 4 and thence to 7 was scored on a
scale from -2 to +2, according to the following formula.
‘-2’ = Changes where both response types in trials 4 and. 7 were weaker than that of trial 1.
‘-1’ = Changes where just one of the response types in trials 4 and 7 were weaker than that of
trial 1.
‘0’ = No change in trials 4 and 7 compared to trial 1.
‘+1’ = Changes where just one of the response types in trials 4 and 7 were stronger than that of
trial 1.
‘’+2’ = Changes where both response types in trials 4 and 7 were stronger than that of trial 1.
‘M’ = Cases where the response increased or decreased from trials 1 to 4 from Weak to Strong or
to None (respectively), but then switched direction from trial 4 to trial 7, changing from Strong
to None or from None to Strong.
Response type
Type of change Response Change
Category Score
Trials 1 4 7
Trials 1 4 7
Trials 1 4 7
Strong response
Weak response
None
Adaptation -2
Strong response
Weak response None
Adaptation -1
Strong response
Weak response None
No change 0
Strong response Weak response
None
Sensitization +1
Strong response Weak response
None
Sensitization +2
Strong response Weak response
None
Mixed M
Score
111
Table 24: Results of Chi Square test comparing the counts of Response Change Categories
observed for each gender. Comparisons were made between genders for each surgery group
(sham and IONX), baseline testing session (BL1 and BL2), and postoperative testing session
(PO1 and PO2).
Table 25: Results of independent groups t-tests comparing parameters of mobility measured in
the M-PAT test between genders for each surgery group. Parameters measured were Time-spent-
in-smooth zones (in sec; T_S), Distance-traveled-in-smooth and -rough zones (D_S, D_R), in
both baseline sessions (BL1 and BL2) and postoperative sessions (PO1 and PO2).
BL1 BL2 PO1 PO2 PO1 PO2Chi square 6.775 1.269 1.370 0.709 2.074 2.687
P value 0.15 0.87 0.85 0.95 0.72 0.61Chi square 4.201 4.772 1.089 2.497 2.396 3.360
P value 0.38 0.31 0.90 0.65 0.66 0.50Chi square 3.314 3.316 5.291 1.814 11.933 6.557
P value 0.51 0.51 0.26 0.77 0.018 0.16Sham
Testing session
StatisticsEars Paws Tail
Naïve
IONX
112
Table 26: Range of observed trait values among the RI panel of mice and the parental strains,
calculated as the difference between the lines/strains having the maximal values normalized by
that of the line/strain having the minimal value.
Ears 0.5Paws 2.6Tail 3.8Ears 0.6 0.6Paws 2.2 1.4Tail 2.1 2.2Ears 0.8 0.6Paws 2.2 1.2Tail 2.7 1.6
Smooth-Time 1.2Smooth Distance 25.2
30.7Smooth-Time 2.6 1.5Smooth Distance 22.2 38.1
38.1 22.2Smooth-Time 1.9 5.2Smooth Distance 15.3 56.2
29.2 43.2
Period
Naïve Sham IONX
Rough Distance
Weighted score
Test ParameterTested locus
Tactile sensitivity
M-PAT
Rough Distance
Rough Distance
BL
PO1
PO2
BL2
PO1
PO2
113
Table 27: Computed values of narrow-sense heritability (h2) of responsivity of each tested body
region to Von Frey stimulation and respective scores for mobillity traits of mice as assessed in
the M-PAT, for each surgery group (IONX and sham), prior to and following surgery on two
separate postoperative testing sessions.
Test Tested locus / M-PAT parameter Testing period
Heritability (h2) SHAM IONX
Tactile responsivity
Ears
BL 0.75
PO1 0.65 0.69
PO2 0.62 0.68
Paws
BL 0.83
PO1 0.65 0.70
PO2 0.60 0.72
Tail
BL 0.75
PO1 0.56 0.64
PO2 0.64 0.54
M-PAT
Time in Smooth BL1 0.74 BL2 0.73
Distance in Smooth BL1 0.74 BL2 0.71
Distance in Rough BL1 0.74 BL2 0.72
Time in Smooth PO1 0.68 0.55 PO2 0.64 0.74
Distance in Smooth PO1 0.64 0.64 PO2 0.53 0.62
Distance in Rough PO1 0.68 0.49 PO2 0.59 0.58
114
Table 28: Computed and categorized Mechanical Hyperresponsivity Spread Types for each
animal of all 20 line/strain for which such data could be calculated, seperately by surgery type
(See Table 3 for an index of categories Spread Types).
SHAM IONX SHAM IONX SHAM IONX SHAM IONX SHAM IONX6 5 5 1 7 6 7 7 5 37 5 1 8 7 5 5 1 7 55 5 8 8 2 5 7 7 5 67 8 7 5 4 8 8 7 1 71 4 5 2 8 1 1 7 2 52 2 7 7 7 5 1 2 5 7
4 7 7 7 4 1 6 7 71 8 1 4 8 6 15 5 1 8 6 7 4 6 8
3 5 5 5 3 5 7 7 7 57 6 1 5 7 6 6 5 32 4 8 8 6 3 1 4 15 7 8 4 7 7 7 5 1 22 8 5 5 6 7 6 1 5 38 2 7 7 1 7 7 1 8 37 7 6 5 8 7 7 1 2 77 7 3 6 1 1 6 5 5 75 8 7 6 5 5 7 7 77 6 7 4 5 7 5 78 8 2 3 7 8 8 6 27 7 2 3 5 5 7 1 56 5 3 7 4 6 3 1 87 1 3 6 8 4 3 6 56 4 7 6 8 8 3 4 5 78 7 6 6 4 1 1 4 5 15 8 1 7 7 4 5 8 4 53 7 5 5 5 7 2 3 7 85 5 7 8 1 2 7 1 5 46 2 1 5 8 7 5 7 3 73 1 6 4 1 6 4 1 2 26 6 1 5 7 4 7 15 2 7 1 2 7 8 8 88 8 5 1 5 5 8 5 76 5 6 3 8 5 7
3 5 5 1 76 5 3 8 72 7 5 6 37 1 7 57 6 5 5
5 7 75 5 53 577
Spread typeLine/
strain
Spread type
A15
Line/ strain
Line/ strain
Spread type Line/
strain
Spread type Line/
strain
A13 A4
B14
B4
A5B
B2
B7
B11
A10A19
A6
B12
B24
A8B25
A12
A2
B13
Spread type
Spread type
Spread type
Spread type
Spread type
Spread type
115
Table 29: The percentage of mice of 20 strains/lines having Spread Types 5 or 7, for each
surgery type, and the difference scores in these values between IONX and sham.
Line/strain
% mice per strain/line having
Spread Types 5 or 7
Sham
(%)
IONX
(%)
IONX-Sham
(%)
BXA13 71.4 50.0 -21.4
AXB4 40.0 20.0 -20.0
AXB6 57.1 37.5 -19.6
AXB19 63.6 45.5 -18.2
BXA2 57.1 40.0 -17.1
BXA25 50.0 33.3 -16.7
BXA14 25.0 12.5 -12.5
BXA24 71.4 62.5 -8.9
BXA12 50.0 41.7 -8.3
AXB10 50.0 44.4 -5.6
AXB5 45.5 40.0 -5.5
C57BL/6J 30.0 25.0 -5.0
AXB12 42.9 42.9 0.0
AXB15 27.3 30.0 2.7
AXB13 42.9 50.0 7.1
BXA4 36.4 50.0 13.6
AXB8 41.7 57.1 15.5
AXB2 33.3 50.0 16.7
BXA7 60.0 81.8 21.8
BXA11 42.9 66.7 23.8
116
Table 30a,b: Average trait values per line/strain inputted into the WEBQTL software. In (a),
trait values correspond to mechanical responsivity as measured by Von Frey stimulation in BL2
and the ‘net-IONX effect’ for the operated mice [i.e., The Difference Scores (IONX-BL2)-
(sham-BL2)] in each tested body site (ears, hindpaws, and tail) for each postoperative session
(PO1 and PO2) . In (b), trait values correspond to tested parameters of the M-PAT in Naïve mice
in BL1 and BL2 and the ‘net-IONX effect’ for mice at each postoperative session.
a.
PO1 PO2 PO1 PO2 PO1 PO2ABF1 X X X X X X X X XBAF1 X X X X X X X X X
B X X X -0.071 -0.089 0.021 0.113 0.041 0.024A 1.725 0.850 1.275 X X X X X X
AXB1 1.175 0.613 0.650 X X X X X XAXB2 1.264 0.555 0.545 0.257 0.300 -0.093 0.033 -0.203 0.103AXB3 X X X X X X X X XAXB4 1.322 0.656 0.844 -0.105 0.005 -0.150 -0.165 -0.705 -0.335AXB5 1.195 0.810 0.750 0.090 -0.010 0.230 0.140 -0.100 0.200AXB6 X X X 0.071 0.025 -0.113 -0.063 -0.204 0.050AXB7 X X X X X X X X XAXB8 1.192 0.820 0.844 0.126 -0.066 -0.018 0.099 0.104 0.165AXB9 X X X X X X X X X
AXB10 X X X 0.142 0.246 0.179 0.121 0.042 0.200AXB11 X X X X X X X X XAXB12 1.177 0.685 0.677 -0.148 -0.060 0.086 0.102 0.117 0.052AXB13 X X X 0.123 0.111 -0.029 -0.160 -0.040 0.086AXB15 1.165 0.700 0.635 0.040 0.040 -0.070 -0.060 -0.190 -0.110AXB17 X X X X X X X X XAXB19 1.171 0.743 0.567 -0.062 0.003 -0.172 -0.042 -0.215 -0.243AXB21 X X X X X X X X XAXB23 X X X X X X X X XAXB24 1.633 0.933 1.033 X X X X X XBXA1 1.400 0.980 0.780 X X X X X XBXA2 X X X -0.070 -0.037 -0.057 -0.173 0.037 -0.067BXA4 1.265 0.710 0.610 0.043 0.051 0.206 0.061 0.282 0.157BXA7 1.230 0.685 0.565 0.180 -0.010 0.050 0.190 0.190 0.140BXA8 1.620 0.780 0.820 X X X X X X
BXA11 1.250 0.833 0.961 -0.225 -0.033 0.192 0.267 0.167 0.450BXA12 X X X -0.038 -0.012 0.117 0.287 0.072 0.070BXA13 1.350 0.683 0.617 0.200 -0.066 -0.023 0.120 0.051 0.011BXA14 1.233 0.800 0.787 0.163 0.100 0.234 0.229 -0.052 0.232BXA16 X X X X X X X X XBXA18 X X X X X X X X XBXA20 X X X X X X X X XBXA22 X X X X X X X X XBXA23 X X X X X X X X XBXA24 1.293 0.857 0.907 0.057 0.029 0.143 0.257 0.400 0.157BXA25 1.176 0.686 0.590 0.061 0.189 -0.100 0.078 0.261 0.003BXA26 X X X X X X X X X
Lines / Strains
Ears Paws TailPaws Tail
BL2Naïve
EarsDiff score (IONX-BL2)-(Sh-BL2)
Operated
117
b.
BL1 BL2 BL1 BL2 BL1 BL2 PO1 PO2 PO1 PO2 PO1 PO2
ABF1 X X X X X X X X X X X XBAF1 X X X X X X X X X X X X
B 1.15 1.13 1.20 1.05 86.92 89.22 0.06 -0.25 0.04 -0.32 -1.87 0.00A 0.14 0.10 0.14 0.06 73.70 52.58 X X X X X X
AXB1 1.23 1.09 0.99 0.94 76.83 90.96 X X X X X XAXB2 1.98 1.36 1.73 1.36 75.61 91.01 0.17 -0.26 0.21 -0.25 -5.89 -25.06AXB3 X X X X X X X X X X X XAXB4 0.73 0.59 0.71 0.42 65.24 82.66 0.48 0.29 0.38 0.33 7.01 1.90AXB5 2.01 1.60 1.89 1.47 85.10 84.00 -0.64 -0.44 -0.55 -0.11 3.04 -2.34AXB6 1.21 1.31 1.07 1.15 83.69 81.17 -0.21 -0.18 -0.38 -0.36 -20.23 -30.58AXB7 X X X X X X X X X X X XAXB8 1.22 0.81 1.31 0.79 97.07 94.22 0.48 0.19 0.18 0.24 -12.02 20.81AXB9 X X X X X X X X X X X X
AXB10 1.10 0.83 1.30 0.89 106.49 100.39 0.13 0.52 0.08 0.25 -4.04 -22.78AXB11 X X X X X X X X X X X XAXB12 1.10 0.75 0.81 0.70 73.97 79.40 0.30 0.01 0.23 -0.01 -4.89 -2.85AXB13 0.81 1.10 0.73 0.81 79.00 79.15 0.16 -0.08 0.11 0.00 -16.89 -18.63AXB15 0.98 0.81 1.08 0.70 77.68 76.02 -0.07 -0.07 0.00 -0.08 18.33 19.47AXB17 X X X X X X X X X X X XAXB19 2.11 1.96 1.86 1.57 87.68 87.15 -0.72 -0.14 -0.37 0.15 36.32 12.22AXB21 X X X X X X X X X X X XAXB23 X X X X X X X X X X X XAXB24 0.17 0.23 0.24 0.24 118.40 76.80 X X X X X XBXA1 0.15 0.06 0.16 0.11 96.44 105.72 X X X X X XBXA2 1.41 1.11 1.29 0.83 79.38 62.73 0.22 0.04 0.11 0.02 17.15 8.37BXA4 0.72 0.66 0.64 0.73 99.08 96.05 0.48 0.15 0.46 -0.10 12.52 -26.31BXA7 1.70 1.59 1.82 1.57 89.96 88.13 0.37 0.94 0.35 0.82 -0.89 4.75BXA8 0.22 0.13 0.23 0.13 84.86 77.04 X X X X X X
BXA11 0.73 0.62 0.88 0.64 98.13 101.84 0.18 0.04 -0.06 -0.12 -0.46 -10.35BXA12 1.67 1.29 1.74 1.29 96.75 106.06 -0.59 -0.40 -0.74 -0.34 4.53 -21.26BXA13 0.94 1.14 0.82 1.07 90.90 84.04 1.10 -0.01 0.83 0.20 12.04 -17.16BXA14 0.70 0.51 0.85 0.66 91.71 114.14 -0.17 -0.05 -0.14 -0.10 -19.18 -30.98BXA16 X X X X X X X X X X X XBXA18 X X X X X X X X X X X XBXA20 X X X X X X X X X X X XBXA22 X X X X X X X X X X X XBXA23 X X X X X X X X X X X XBXA24 0.91 0.97 1.21 1.25 106.39 106.80 0.35 0.44 0.34 0.25 -14.10 -30.97BXA25 1.10 0.83 0.88 0.59 84.44 69.80 -0.10 -0.36 -0.24 -0.17 -13.57 13.10BXA26 X X X X X X X X X X X X
OperatedNaïve
DistanceRough
TimeSmooth Rough Smooth
Distance TimeLines / Strains
114
Table 31: List of all known genes in mouse chromosome 5 mapped to the confidence interval of
the QTL associated with spread of mechanical hyperresponsivity post-IONX. N = serial number
of known genes in chromosome 5; Start (Mb) = position (in Mb) where the gene sequence
begins; SNP count = the number of known polymorphic nucleotides within each gene sequence.
Note that gene symbols are “clickable” fields, enabling the reader to browse genetic databases
for additional information documented about a gene of interest.
N Gene symbol Start (Mb) Gene Length (Kb) SNP
Count Encoded protein
741 4930405H06Rik 100.054854 1.227 0 RIKEN cDNA 4930405H06 gene
742 4930522N08Rik 100.112017 2.036 0 RIKEN cDNA 4930522N08 gene
743 A830083F22 100.159872 23.120 3 hypothetical protein A8300...
744 4930535B17Rik 100.376332 0.913 0 RIKEN cDNA 4930535B17 gene
745 Hnrpd 100.384954 23.003 81 heterogeneous nuclear ribo...
746 4930524J08Rik 100.408079 0.603 2 RIKEN cDNA 4930524J08 gene
747 Hnrpdl 100.462597 5.644 8 heterogeneous nuclear ribo...
748 Enoph1 100.469032 28.749 105 enolase-phosphatase 1
749 BC062109 100.506891 81.936 39 cDNA sequence BC062109
750 5830403M04Rik 100.506980 17.188 31 RIKEN cDNA 5830403M04 gene
751 Sec31a 100.602275 0.050 0 SEC31 homolog A (S. cerevi...
752 2310034O05Rik 100.639719 7.349 2 RIKEN cDNA 2310034O05 gene
753 LOC545785 100.731388 5.989 5 hypothetical protein LOC54...
754 5430416N02Rik 100.849860 8.666 30 RIKEN cDNA 5430416N02 gene
755 Lin54 100.871057 56.535 246 lin-54 homolog (C. elegans...
756 Cops4 100.947477 29.317 2 COP9 (constitutive photomo...
757 Plac8 100.982751 18.473 4 placenta-specific 8
115
758 Coq2 101.083744 19.531 85 coenzyme Q2 homolog, preny...
759 Hpse 101.108504 40.198 102 heparanase
760 Hel308 101.191166 36.453 5 helicase, mus308-like (Dro...
761 Mrps18c 101.227777 5.709 0 mitochondrial ribosomal pr...
762 Ccdc98 101.233820 16.134 1 coiled-coil domain contain...
763 4930458D05Rik 101.269772 5.357 0 RIKEN cDNA 4930458D05 gene
764 Agpat9 101.275247 52.874 10 1-acylglycerol-3-phosphate...
765 4930447E19Rik 101.461569 0.702 0 RIKEN cDNA 4930447E19 gene
766 Nkx6-1 102.088215 5.515 0 NK6 transcription factor r...
767 9430085M18Rik 102.132625 2.244 0 RIKEN cDNA 9430085M18 gene
768 Cds1 102.194148 58.723 2 CDP-diacylglycerol synthas...
769 Wdfy3 102.264603 234.337 7 WD repeat vs. FYVE domain ...
770 1700013M08Rik 102.908226 0.787 0 RIKEN cDNA 1700013M08 gene
771 Arhgap24 102.910409 416.547 14 Rho GTPase activating prot...
772 2900056B19Rik 103.154150 0.702 0 RIKEN cDNA 2900056B19 gene
773 Mapk10 103.336966 303.387 4 mitogen-activated protein ...
774 2900063K03Rik 103.631772 0.795 0 RIKEN cDNA 2900063K03 gene
775 4930429D17Rik 103.659393 68.655 1 RIKEN cDNA 4930429D17 gene
776 4921523P09Rik 103.772992 1.134 0 RIKEN cDNA 4921523P09 gene
777 1700021F02Rik 103.775616 1.702 0 RIKEN cDNA 1700021F02 gene
778 5430427N15Rik 103.783323 0.481 0 RIKEN cDNA 5430427N15 gene
779 Ptpn13 103.854210 173.170 7 protein tyrosine phosphata...
780 Slc10a6 104.034729 23.693 1 solute carrier family 10 (...
116
781 1700016H13Rik 104.077611 7.132 1 RIKEN cDNA 1700016H13 gene
782 Aff1 104.183180 101.161 2 AF4/FMR2 family, member 1
783 Klhl8 104.291068 49.180 39 kelch-like 8 (Drosophila)
784 Hsd17b13 104.384460 21.923 46 hydroxysteroid (17-beta) d...
785 Hsd17b11 104.418783 32.032 104 hydroxysteroid (17-beta) d...
786 Nudt9 104.476029 18.357 99 nudix (nucleoside diphosph...
787 Sparcl1 104.508129 34.978 125 SPARC-like 1 (mast9, hevin...
788 A830049A06 104.538235 6.807 3 hypothetical protein A8300...
789 Dspp 104.599730 9.416 10 dentin sialophosphoprotein
790 Dmp1 104.631635 11.486 27 dentin matrix protein 1
791 Ibsp 104.728305 12.183 40 integrin binding sialoprot...
792 Mepe 104.754347 13.283 77 matrix extracellular phosp...
793 D930016D06Rik 104.793112 0.050 0 RIKEN cDNA D930016D06 gene
794 Spp1 104.864136 5.933 2 secreted phosphoprotein 1
795 Pkd2 104.888475 46.363 8 polycystic kidney disease ...
796 BC005561 104.946651 4.753 0 cDNA sequence BC005561
797 Zfp33b 105.012008 1.630 2 zinc finger protein 33B
798 C230055K05Rik 105.242186 46.901 23 RIKEN cDNA C230055K05 gene
799 Abcg3 105.364075 47.661 79 ATP-binding cassette, sub-...
800 5830443L24Rik 105.443171 39.409 24 RIKEN cDNA 5830443L24 gene
801 BC057170 105.508316 30.981 39 cDNA sequence BC057170
802 Gbp4 105.544785 23.786 46 guanylate nucleotide bindi...
803 LOC626578 105.644717 23.835 62 macrophage activation 2 li...
117
804 Mpa2l 105.699720 22.998 51 macrophage activation 2 li...
805 EG634650 105.752045 23.446 70 predicted gene, EG634650
806 B230204H03Rik 105.827924 1.251 0 RIKEN cDNA B230204H03 gene
807 4930542N06Rik 105.842306 2.383 0 RIKEN cDNA 4930542N06 gene
808 Lrrc8b 105.844793 70.415 118 leucine rich repeat contai...
809 Lrrc8c 105.948489 89.484 312 leucine rich repeat contai...
810 C230066G23Rik 106.009674 1.117 1 RIKEN cDNA C230066G23 gene
811 Lrrc8d 106.129828 114.400 181 leucine rich repeat contai...
812 2810473G09Rik 106.159692 0.797 3 RIKEN cDNA 2810473G09 gene
813 D830014E11Rik 106.260704 0.752 0 RIKEN cDNA D830014E11 gene
814 1700061D13Rik 106.295391 0.406 0 RIKEN cDNA 1700061D13 gene
815 Zfp326 106.305620 39.211 78 zinc finger protein 326
816 4632409D06Rik 106.387686 2.765 6 RIKEN cDNA 4632409D06 gene
817 4930458A03Rik 106.780553 12.715 22 RIKEN cDNA 4930458A03 gene
818 4930432H08Rik 106.793579 2.289 4 RIKEN cDNA 4930432H08 gene
819 Barhl2 106.881541 5.644 1 BarH-like 2 (Drosophila)
820 EG666892 106.986757 16.980 18 predicted gene, EG666892
821 Zfp644 107.045759 80.090 250 zinc finger protein 644
822 Hfm1 107.330907 24.002 0 HFM1, ATP-dependent DNA he...
823 Cdc7 107.393340 20.110 1 cell division cycle 7 (S. ...
824 Tgfbr3 107.535590 183.024 19 transforming growth factor...
825 A830010M20Rik 107.748991 0.050 0 RIKEN cDNA A830010M20 gene
826 Brdt 107.760212 19.548 0 bromodomain, testis-specif...
118
827 Abhd7 107.832531 26.519 0 abhydrolase domain contain...
828 A830011I04 107.833841 27.088 1 hypothetical protein A8300...
829 Aytl1b 107.860567 3.491 0 acyltransferase like 1B
830 1700028K03Rik 107.963729 13.667 0 RIKEN cDNA 1700028K03 gene
831 Glmn 107.978042 48.552 1 glomulin, FKBP associated ...
832 Rpap2 108.026753 64.102 2 RNA polymerase II associat...
833 A930041C12Rik 108.059268 3.601 1 RIKEN cDNA A930041C12 gene
834 1700013N18Rik 108.071519 0.050 0 RIKEN cDNA 1700013N18 gene
835 5830490A12Rik 108.085028 1.426 0 RIKEN cDNA 5830490A12 gene
836 5830411K02Rik 108.117189 0.997 0 RIKEN cDNA 5830411K02 gene
837 4930428O21Rik 108.127230 5.269 0 RIKEN cDNA 4930428O21 gene
838 Gfi1 108.145673 7.690 0 growth factor independent ...
839 A430072P03Rik 108.155417 2.404 0 RIKEN cDNA A430072P03 gene
840 Evi5 108.173813 130.313 25 ecotropic viral integratio...
841 Rpl5 108.329608 7.461 11 ribosomal protein L5
842 2900024C23Rik 108.337356 78.696 131 RIKEN cDNA 2900024C23 gene
843 Gm621 108.379485 0.050 2 gene model 621, (NCBI)
844 Mtf2 108.494760 43.261 50 metal response element bin...
845 Tmed5 108.550665 10.945 5 transmembrane emp24 protei...
846 Ccdc18 108.561932 100.036 22 coiled-coil domain contain...
847 Dr1 108.697915 11.625 1 down-regulator of transcri...
848 Pigg 108.741943 35.855 14 phosphatidylinositol glyca...
849 9330198I05Rik 108.789205 3.697 5 RIKEN cDNA 9330198I05 gene
119
850 Pde6b 108.817391 43.370 168 phosphodiesterase 6B, cGMP...
851 Atp5k 108.862271 1.126 2 ATP synthase, H+ transport...
852 D830035I06 108.863389 1.643 1 hypothetical protein D8300...
853 Mfsd7 108.870072 7.838 7 major facilitator superfam...
854 Pcgf3 108.890350 41.768 141 polycomb group ring finger...
855 Cplx1 108.947572 31.474 87 complexin 1
856 LOC100042571 108.979099 3.274 10 hypothetical protein LOC10...
857 Gak 108.998432 60.326 111 cyclin G associated kinase
858 Tmem175 109.059020 17.262 23 transmembrane protein 175
859 0710007G10Rik 109.073841 0.274 0 RIKEN cDNA 0710007G10 gene
860 Dgkq 109.076062 13.726 82 diacylglycerol kinase, the...
861 Idua 109.098124 15.453 23 iduronidase, alpha-L-
862 Slc26a1 109.098902 5.686 4 solute carrier family 26 (...
863 Fgfrl1 109.123520 12.397 5 fibroblast growth factor r...
864 Rnf212 109.158326 21.648 13 ring finger protein 212
865 1700047L14Rik 109.192860 5.073 6 RIKEN cDNA 1700047L14 gene
866 1810008K16Rik 109.206253 18.129 23 RIKEN cDNA 1810008K16 gene
867 Vmn2r8 109.226211 11.562 26 vomeronasal 2, receptor 8
868 Vmn2r9 109.271965 9.564 12 vomeronasal 2, receptor 9
869 Vmn2r10 109.424557 10.898 43 vomeronasal 2, receptor 10
870 Vmn2r11 109.475891 12.580 33 vomeronasal 2, receptor 11
871 Vmn2r12 109.514867 12.016 21 vomeronasal 2, receptor 12
872 Vmn2r13 109.585086 36.040 35 vomeronasal 2, receptor 13
120
873 Vmn2r14 109.644520 9.121 2 vomeronasal 2, receptor 14
874 Vmn2r15 109.715287 11.288 22 vomeronasal 2, receptor 15
875 Vmn2r16 109.759399 34.101 139 vomeronasal 2, receptor 16
876 Vmn2r17 109.849031 33.375 74 vomeronasal 2, receptor 17
877 Crlf2 109.983727 4.237 4 cytokine receptor-like fac...
121
6.0 REFERENCES
1) Devor, M., Seltzer, Z. Pathophysiology of damaged nerves in relation to chronic pain. In: Wall, P and Melzack R, Textbook of Pain, 4th edition. Churchill Livingstone, London 1994, pp. 751-796.
2) Kirkpatrick, D.B. 1989. Familial trigeminal neuralgia: case report. Neurosurgery, 24;758-761.
3) Gracely, R.H., Lynch, S.A. and Bennett, G.J. 1992. Painful neuropathy: altered central processing maintained dynamically by peripheral input. Pain, 51;175- 194.
4) Zeltzer, R. and Seltzer, Z. A practical guide for use of animal models for neuropathic pain. In: J. Boivie (Ed.): Touch Temperature and Pain, Seattle 1994, pp. 337-379.
5) Seltzer, Z., Wu, T., Max, M.B. and Diehl, S.R. 2001. Mapping a gene for neuropathic pain-related behavior following peripheral neurectomy in the mouse. Pain, 93;101-106.
6) Seltzer, Z., Beilin, B.Z., Ginzburg, R., Paran, Y. and Shimko, T. 1991. The role of injury
discharge in the induction of neuropathic pain behavior in rats. Pain, 46;327-336.
7) Nomura, H., Ogawa, A., Tashiro, A., Morimoto, T., Hu, J.W. and Iwata, K. 2002. Induction of Fos protein-like immunoreactivity in the trigeminal spinal nucleus caudalis and upper cervical cord following noxious and non-noxious mechanical stimulation of the whisker pad of the rat with an inferior alveolar nerve transection. Pain, 95;225-238.
8) Devor, M. and Raber, P. 1990, Heritability of symptoms in an experimental model of
neuropathic pain. Pain, 42;51-67. 9) Mogil, J.S., Wilson, S.G., Bon, K., Lee, S.E., Chung, K., Raber, P., Pieper, J.O., Hain, H.S.,
Belknap, J.K., Hubert, L., Elmer, G.I., Chung, J.M. and Devor, M. 1999. Heritability of nociception I: responses of 11 inbred mouse strains on 12 measures of nociception. Pain, 80,67-82.
10) Broman, K.W. 2001. Review of statistical methods for QTL mapping in experimental
crosses. Lab animal, 30;44-52.
11) Bennett, G.J. and Xie, Y.K. 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33;87-107.
12) Vos, B.P., Strassman, A.M. and Maciewicz, R.J. 1994. Behavioral evidence of trigeminal
neuropathic pain following chronic constriction injury to the rat's infraorbital nerve. Neuroscience, 14;2708-2723.
13) Willer, J.C., Boureau, F. and Albe-Fessard, D. 1979. Supraspinal influences on nociceptive
flexion reflex and pain sensation in man. Brain Research, 179;61-68.
122
14) Devor, M., Gilad, A., Arbilly, M., Yakir, B., Raber, P., Pisante, A. and Darvasi, A. 2005. pain1: a neuropathic pain QTL on mouse chromosome 15 in a C3HxC58 backcross. Pain, 116;289-293.
15) Complex Trait Consortium 2003. The nature and identification of quantitative trait loci: a
community's view. Nature Reviews Genetics, 4;911-916. 16) Wilson, S.G. and Mogil, J.S. 2001. Measuring pain in the (knockout) mouse: big challenges
in a small mammal. Brain research, 125;65-73. 17) Arbilly, M., Pisante, A., Devor, M. and Darvasi, A. 2006. An integrative approach for the
identification of quantitative trait loci. Animal Genetics, 37(Suppl. 1),7-9. 18) Diatchenko, L., Slade, G.D., Nackley, A.G., Bhalang, K., Sigurdsson, A., Belfer, I.,
Goldman, D., Xu, K., Shabalina, S.A., Shagin, D., Max, M.B., Makarov, S.S. and Maixner, W. 2005. Genetic basis for individual variations in pain perception and the development of a chronic pain condition. Human Molecular Genetics, 14;135-143.
19) Lan, H., Chen, M., Flowers, J.B., Yandell, B.S., Stapleton, D.S., Mata, C.M., Mui, E.T.,
Flowers, M.T., Schueler, K.L., Manly, K.F., Williams, R.W., Kendziorski, C. and Attie, A.D. 2006. Combined expression trait correlations and expression quantitative trait locus mapping. PLoS Genetics, 2;e6.
21) Shir, Y., Raber, P., Devor, M. and Seltzer, Z. 1991. Mechano- and thermo-sensitivity in rats
genetically prone to developing neuropathic pain. Neuroreport, 2;313-316. 20) Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M. and Yaksh, T.L. 1994. Quantitative
assessment of tactile allodynia in the rat paw. Neuroscience Methods, 53;55-63. 22) Raber, P., Del Canho, S., Darvasi, A. and Devor, M. 2006. Mice congenic for a locus that
determines phenotype in the neuroma model of neuropathic pain. Experimental Neurology, 202;200-206.
23) Sessle, B., Levigne, G., Lund, J., and Dubner, R., Peripheral Mechanisms of Orofacial Pain.
In: Mathews, B. and Sessle, B. (Eds.): Orofacial Pain, 2nd edition, Quintessence Publishing Chicago, 2008, pp. 27-34.
24) Kim, H. and Dionne, R.A. 2009. Individualized pain medicine. Drug Discovery Today.
Therapeutic Strategies, 6;83-87. 25) Wall, P and Melzack R: Peripheral Mechanisms of Cutaneous Nociception. In: Wall, P and
Melzack R (Eds.), Textbook of Pain, 3rd edition, Churchill Livingstone, London 1994, pp. 11-45.
26) Shir, Y., Sheth, R., Campbell, J.N., Raja, S.N. and Seltzer, Z. 2001. Soy-containing diet
suppresses chronic neuropathic sensory disorders in rats. Anesthesia and Analgesia, 92;1029-1034.
123
27) Devor, M., Gilad, A., Arbilly, M., Nissenbaum, J., Yakir, B., Raber, P., Minert, A., Pisante, A. and Darvasi, A. 2007. Sex-specific variability and a 'cage effect' independently mask a neuropathic pain quantitative trait locus detected in a whole genome scan. European Journal of Neuroscience, 26;681-688.
28) Zeltser, R., Beilin, B., Zaslansky, R. and Seltzer, Z. 2000. Comparison of autotomy behavior
induced in rats by various clinically-used neurectomy methods. Pain, 89,19-24. 29) Sessle, B., Iwata, K., Dubner, R.: Central Mechanisms of Orofacial. in: Mathews, B. and
Sessle, B. (Eds.): Orofacial Pain, 2nd edition, Quintessence Publishing Chicago, 2008, pp. 35-43.
30) Seltzer, Z. and Mogil, J. Pain and Genetics. in: Sessle, B., Lavigne, G, Lund, J., Dubner, R.
(Eds.), Orofacial Pain, 2nd edition, Quintessence Publishing, Chicago 2008, pp. 69-78.
31) Wall, P.D. and Gutnick, M. 1974. Ongoing activity in peripheral nerves: the physiology and pharmacology of impulses originating from a neuroma. Experimental Neurology, 43;580-593.
32) Coderre, T.J., Grimes, R.W. and Melzack, R. 1986. Deafferentation and chronic pain in
animals: an evaluation of evidence suggesting autotomy is related to pain. Pain, 26;61-84. 33) Kim, S.H. and Chung, J.M. 1992. An experimental model for peripheral neuropathy
produced by segmental spinal nerve ligation in the rat. Pain, 50;355-363. 34) Wall, P.D., Devor, M., Inbal, R., Scadding, J.W., Schonfeld, D., Seltzer, Z. and Tomkiewicz,
M.M. 1979. Autotomy following peripheral nerve lesions: experimental anaesthesia dolorosa. Pain, 7;103-111.
35) Suzuki, R. and Dickenson, A. 2005. Spinal and supraspinal contributions to central
sensitization in peripheral neuropathy. Neuro-Signals, 14;175-181. 36) Sessle, B.J. 1986. Recent developments in pain research: central mechanisms of orofacial
pain and its control. Endodontics, 12;435-444. 37) Chiang, C., Y., Park, S., J., Kwan, C., L., Hu, J., W., Sessle, B., J. 1998. NMDA receptor
mechanisms contribute to neuroplasticity induced in caudalis nociceptive neurons by tooth pulp stimulation. Neurophysiology, 80;2621-2631.
38) Sessle, B.J. 2000. Acute and chronic craniofacial pain: brainstem mechanisms of nociceptive
transmission and neuroplasticity, and their clinical correlates. Critical Reviews in Oral Biology and Medicine, 11;57-91.
39) Costigan, M., Scholz, J. and Woolf, C.J. 2009. Neuropathic pain: a maladaptive response of
the nervous system to damage. Annual Review of Neuroscience, 32;1-32.
124
40) Devor, M. 2006. Centralization, central sensitization and neuropathic pain. Focus on sciatic chronic constriction injury produces cell-type-specific changes in the electrophysiological properties of rat substantia gelatinosa neurons. Neurophysiology, 96;522-523.
41) Bester, H., Beggs, S. and Woolf, C.J. 2000. Changes in tactile stimuli-induced behavior and
c-Fos expression in the superficial dorsal horn and in parabrachial nuclei after sciatic nerve crush. Comparative Neurology, 428;45-61.
42) Thompson, S.W., Woolf, C.J. and Sivilotti, L.G. 1993. Small-caliber afferent inputs produce
a heterosynaptic facilitation of the synaptic responses evoked by primary afferent A-fibres in the neonatal rat spinal cord in vitro. Neurophysiology, 69;2116-2128.
43) Rahman, W., D'Mello, R. and Dickenson, A.H. 2008. Peripheral nerve injury-induced
changes in spinal alpha(2)-adrenoceptor-mediated modulation of mechanically evoked dorsal horn neuronal responses. Pain, 9;350-359.
44) Bee, L.A. and Dickenson, A.H. 2008. Descending facilitation from the brainstem determines
behavioural and neuronal hypersensitivity following nerve injury and efficacy of pregabalin. Pain, 140;209-223.
45) Coull, J.A., Beggs, S., Boudreau, D., Boivin, D., Tsuda, M., Inoue, K., Gravel, C., Salter,
M.W. and De Koninck, Y. 2005. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature, 438;1017-1021.
46) Keller, A.F., Beggs, S., Salter, M.W. and De Koninck, Y. 2007. Transformation of the output
of spinal lamina I neurons after nerve injury and microglia stimulation underlying neuropathic pain. Molecular Pain, 3;27.
47) Scholz, J., Broom, D.C., Youn, D.H., Mills, C.D., Kohno, T., Suter, M.R., Moore, K.A.,
Decosterd, I., Coggeshall, R.E. and Woolf, C.J. 2005. Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury. Neuroscience, 25;7317-7323.
48) Soares, S., von Boxberg, Y., Lombard, M.C., Ravaille-Veron, M., Fischer, I., Eyer, J. and
Nothias, F. 2002. Phosphorylated MAP1B is induced in central sprouting of primary afferents in response to peripheral injury but not in response to rhizotomy. The European Journal of Neuroscience, 16;593-606.
49) Lombard, M.C., Nashold, B.S.,Jr, Albe-Fessard, D., Salman, N. and Sakr, C. 1979.
Deafferentation hypersensitivity in the rat after dorsal rhizotomy: a possible animal model of chronic pain. Pain, 6;163-174.
50) Seltzer, Z., Dubner, R. and Shir, Y. 1990. A novel behavioral model of neuropathic pain
disorders produced in rats by partial sciatic nerve injury. Pain, 43;205-218. 51) Decosterd, I. and Woolf, C.J. 2000. Spared nerve injury: an animal model of persistent
peripheral neuropathic pain. Pain, 87;149-158.
125
52) Kim, S.H. and Chung, J.M. 1992. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain, 50,355-363.
53) Zimmermann, M. Behavioural investigations of pain in animals. In: Assessing Pain in Farm
Animals, Duncan, I.J.H., Molony Y. (Eds.), 4th edition, Publications of the European Communities, Bruxelles 1986, pp. 16–29.
54) Imamura, Y., Kawamoto, H. and Nakanishi, O. 1997. Characterization of heat-hyperalgesia
in an experimental trigeminal neuropathy in rats. Brain Research, 116;97-103. 55) Xu, M., Aita, M. and Chavkin, C. 2008. Partial infraorbital nerve ligation as a model of
trigeminal nerve injury in the mouse: behavioral, neural, and glial reactions. Pain, 9;1036-1048.
56) Bongenhielm, U., Boissonade, F.M., Westermark, A., Robinson, P.P. and Fried, K. 1999.
Sympathetic nerve sprouting fails to occur in the trigeminal ganglion after peripheral nerve injury in the rat. Pain, 82;283-288.
57) Henry, M.A., Freking, A.R., Johnson, L.R. and Levinson, S.R. 2006. Increased sodium
channel immunofluorescence at myelinated and demyelinated sites following an inflammatory and partial axotomy lesion of the rat infraorbital nerve. Pain, 124;222-233.
58) Okada-Ogawa, A., Suzuki, I., Sessle, B.J., Chiang, C.Y., Salter, M.W., Dostrovsky, J.O.,
Tsuboi, Y., Kondo, M., Kitagawa, J., Kobayashi, A., Noma, N., Imamura, Y., Iwata, K. 2009. Astroglia in medullary dorsal horn (trigeminal spinal subnucleus caudalis) are involved in trigeminal neuropathic pain mechanisms. Neuroscience, 29;1161-1171.
59) Iwata, K., Imai, T., Tsuboi, Y., Tashiro, A., Ogawa, A., Morimoto, T., Masuda, Y.,
Tachibana, Y. and Hu, J. 2001. Alteration of medullary dorsal horn neuronal activity following inferior alveolar nerve transection in rats. Neurophysiology, 86;2868-2877.
60) Dubner, R. and Ren, K., Assessing transient and persistent pain in animals. In: Textbook of
Pain: Wall P.D., and Melzack R. (Eds.), 4th edition, New York 1999, pp. 1082-1099. 61) Zakrzewska, J. Trigeminal, eye and ear pain. In: The Textbook of Pain, Wall P.D. and
Melzack, R. (Eds.), 4th edition, Churchill Livingstone, New York 1999, pp. 739-750. 62) Boivie, J. Central Pain. In: Textbook of Pain: Wall, P.D. and Melzack, R. (Eds.), 4th edition,
Churchill Livingstone, New York 1999, pp. 371-394.
63) Willer, J.C. 1977. Comparative study of perceived pain and nociceptive flexion reflex in man. Pain, 3;69-80.
64) Dowman, R. 1991. Spinal and supraspinal correlates of nociception in man. Pain, 45;269-
281. 65) Chan, C.W. and Dallaire, M.. Subjective pain sensation is linearly correlated with the
flexion reflex in man. Brain Research, 479;145-150.
126
66) Dennis, S. and Melzack, R. Perspective on phylogenetic evolution of pain expression. In: Animal Pain: Perception and Alleviation, (Eds.), 1st edition, American Physiological Society, Maryland 1983, pp. 156-160.
67) Hardy, J.D. 1953. Thresolds of pain andreflex contraction as related to noxious stimulation. Applied Phsyiology, 5;725-739. 68) Xu, X.J., Plesan, A., Yu, W., Hao, J.X. and Wiesenfeld-Hallin, Z. 2001. Possible impact of
genetic differences on the development of neuropathic pain-like behaviors after unilateral sciatic nerve ischemic injury in rats. Pain, 893;135-145.
69) Shir, Y., Zeltser, R., Vatine, J.J., Carmi, G., Belfer, I., Zangen, A., Overstreet, D., Raber, P.
and Seltzer, Z. 2001, Correlation of intact sensibility and neuropathic pain-related behaviors in eight inbred and outbred rat strains and selection lines. Pain, 90;75-82.
70) Ziv-Sefer, S., Raber, P., Barbash, S. and Devor, M. 2009. Unity and diversity of neuropathic
pain mechanisms: Allodynia and hyperalgesia in rats selected for heritable predisposition to spontaneous pain. Pain, 146;148-157.
71) Belknap, J.K., Mogil, J.S., Helms, M.L., Richards, S.P., O'Toole, L.A., Bergeson, S.E. and
Buck, K.J. 1995. Localization to chromosome 10 of a locus influencing morphine analgesia in crosses derived from C57BL/6 and DBA/2 strains. Life Sciences, 57;117-124.
72) Kasson, B.G. and George, R. 1984. Endocrine influences on the actions of morphine: IV.
Effects of sex and strain. Life Sciences, 34;1627-1634. 73) Mogil, J.S., Wilson, S.G., Bon, K., Lee, S.E., Chung, K., Raber, P., Pieper, J.O., Hain, H.S.,
Belknap, J.K., Hubert, L., Elmer, G.I., Chung, J.M. and Devor, M. 1999. Heritability of nociception II. 'Types' of nociception revealed by genetic correlation analysis. Pain, 80;83-93.
74) Wiesenfeld, Z. and Hallin, R.G. 1981. Influence of nerve lesions, strain differences and
continuous cold stress on chronic pain behavior in rats. Physiology and Behavior, 27;735-740.
75) Inbal, R., Devor, M., Tuchendler, O. and Lieblich, I. 1980. Autotomy following nerve injury:
genetic factors in the development of chronic pain. Pain, 9;327-337. 76) Devor, M. and Raber, P. 1990. Heritability of symptoms in an experimental model of
neuropathic pain. Pain, 42;51-67. 77) Lander, E.S. and Botstein, D. 1989. Mapping Mendelian factors underlying quantitative traits
using RFLP linkage maps. Genetics, 121;185-199. 78) Haldane, J.B. and Waddington, C.H. 1931. Inbreeding and Linkage. Genetics, 16;357-374. 79) Zamir, N., Simantov, R. and Segal, M. 1980. Pain sensitivity and opioid activity in
genetically and experimentally hypertensive rats. Brain Research, 184;299-310.
127
80) Kujirai, K., Fahn, S. and Cadet, J.L. 1991. Receptor autoradiography of mu and delta opioid peptide receptors in spontaneously hypertensive rats. Peptides, 12;779-785.
81) Saavedra, J.M. 1981, Naloxone reversible decrease in pain sensitivity in young and adult
spontaneously hypertensive rats. Brain Research, 209;245-249. 82) Mogil, J.S. 1999. The genetic mediation of individual differences in sensitivity to pain and
its inhibition. Proceedings of the National Academy of Sciences, 96;7744-7751. 83) Yaksh, T.L. 1989. Behavioral and autonomic correlates of the tactile evoked allodynia
produced by spinal glycine inhibition: effects of modulatory receptor systems and excitatory amino acid antagonists. Pain, 37;111-123.
84) Smith, C.C., Hauser, E., Renaud, N.K., Leff, A., Aksentijevich, S., Chrousos, G.P., Wilder,
R.L., Gold, P.W. and Sternberg, E.M. 1992. Increased hypothalamic [3H]flunitrazepam binding in hypothalamic-pituitary-adrenal axis hyporesponsive Lewis rats. Brain Research, . 569;295-299.
85) Ribak, C.E. and Morin, C.L. 1995. The role of the inferior colliculus in a genetic model of
audiogenic seizures. Anatomy and Embryology, 191;279-295. 86) Seltzer, Z., Attal, U., Devor, M., Levi, Z., Neumann, S. and Shavit, Y., 1993. Neuropathic
pain related behavior and epileptogenesis are co-inherited in rats. Pain, 6(Suppl.); S186. 87) Seltzer, Z., Levi, Z. and Devor, M., 1992. Epileptogenesis in rats selected genetically for
high and low propensity for neuropathic pain behavior. J. Basic Clin. Physiol. Pharmacol. 23(Suppl.),39.
88) Liu, C.N., Raber, P., Ziv-Sefer, S. and Devor, M. 2001. Hyperexcitability in sensory neurons
of rats selected for high versus low neuropathic pain phenotype, Neuroscience. 105;265-275.
89) Xu, X.J., Plesan, A., Yu, W., Hao, J.X. and Wiesenfeld-Hallin, Z. 2001. Possible impact of
genetic differences on the development of neuropathic pain-like behaviors after unilateral sciatic nerve ischemic injury in rats. Pain, 89;135-145.
90) Gregory, S.G, Sekhon M, Schein J, Zhao S, Osoegawa K, Scott CE, Evans RS, Burridge PW,
Cox TV, Fox CA, Hutton RD, Mullenger IR, Phillips KJ, Smith J, Stalker J, Threadgold GJ, Birney E, Wylie K, Chinwalla A, Wallis J, Hillier L, Carter J, Gaige T, Jaeger S, Kremitzki C, Layman D, Maas J, McGrane R, Mead K, Walker R, Jones S, Smith M, Asano J, Bosdet I, Chan S, Chittaranjan S, Chiu R, Fjell C, Fuhrmann D, Girn N, Gray C, Guin R, Hsiao L, Krzywinski M, Kutsche R, Lee SS, Mathewson C, McLeavy C, Messervier S, Ness S, Pandoh P, Prabhu AL, Saeedi P, Smailus D, Spence L, Stott J, Taylor S, Terpstra W, Tsai M, Vardy J, Wye N, Yang G, Shatsman S, Ayodeji B, Geer K, Tsegaye G, Shvartsbeyn A, Gebregeorgis E, Krol M, Russell D, Overton L, Malek JA, Holmes M, Heaney M, Shetty J, Feldblyum T, Nierman WC, Catanese JJ, Hubbard T, Waterston RH, Rogers J, de Jong PJ, Fraser CM, Marra M, McPherson JD, Bentley DR. 2002. A physical map of the mouse genome. Nature, 418;743-750.
128
91) Mural, R.J., Schein, J., Scott, CE., Hutton, RD. 2002. A comparison of whole-genome shotgun-derived mouse chromosome 16 and the human genome. Science, 296;1661-1671.
92) Devor, M., Gilad, A., Arbilly, M., Yakir, B., Raber, P., Pisante, A. and Darvasi, A. 2005.
pain1: a neuropathic pain QTL on mouse chromosome 15 in a C3HxC58 backcross. Pain, 116;289-293.
93) Devor, M. and Raber, P. 1990. Heritability of symptoms in an experimental model of
neuropathic pain. Pain, 42;51-67. 94) Falconer, D. S., Mackay, T.F.C. Introduction to Quantitative Genetics. 4th edition,
Longmans Green, Harlow, Essex, UK, 1996, pp. 164-180. 95) Tsuboi, Y., Takeda, M., Tanimoto, T., Ikeda, M., Matsumoto, S., Kitagawa, J., Teramoto, K.,
Simizu, K., Yamazaki, Y., Shima, A., Ren, K. and Iwata, K. 2004. Alteration of the second branch of the trigeminal nerve activity following inferior alveolar nerve transection in rats. Pain, 111; 323-334.
96) Kriz, N., Yamamotova, A., Tobias, J. and Rokyta, R. 2006. Tail-flick latency and self-
mutilation following unilateral deafferentation in rats. Physiological Research, 55;213-220.
97) Perl, E.R. 1994. A reevaluation of mechanisms leading to sympathetically related pain. In:
Fields, H.I., Liebeskind, J.C. (Eds). Pharmacological Approaches to the Treatment of Chronic Pain: New Concepts and Critical Issues. IASP Press, Seattle, pp. 129-150.
98) Viel, E., Ripart, J., Pelissier, J. and Eledjam, J.J. 1999. Management of reflex sympathetic
dystrophy. Annales de Medecine Interne, 150;205-210. 99) Wall, P.D. and Gutnick, M. 1974. Ongoing activity in peripheral nerves: the physiology and
pharmacology of impulses originating from a neuroma. Experimental Neurology, 43;580-593.
100) Devor, M. 1983. Nerve pathophysiology and mechanisms of pain in causalgia. Autonomic
Nervous System, 7;371-384. 101) Chung, K. 1997. Strain differences in sympathetic dependency of neuropathic pain in a rat model. Neurosci. Abstr. 23;2356. 102) Tanga, F.Y., Raghavendra, V. and DeLeo, J.A. 2004. Quantitative real-time RT-PCR
assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. Neurochemistry, 45;397-407.
103) Beggs, S. and Salter, M.W. 2010. Microglia-neuronal signalling in neuropathic pain
hypersensitivity. Current Opinion in Neurobiology, 20;474-480.
129
104) Hathway, G.J., Vega-Avelaira, D., Moss, A., Ingram, R. and Fitzgerald, M. 2009. Brief, low frequency stimulation of rat peripheral C-fibres evokes prolonged microglial-induced central sensitization in adults but not in neonates. Pain, 144,110-118.
105) Latremoliere, A. and Woolf, C.J. 2009. Central sensitization: a generator of pain
hypersensitivity by central neural plasticity. Pain, 10;895-926. 106) Benoliel, R., Eliav, E. and Tal, M. 2002. Strain-dependent modification of neuropathic pain
behaviour in the rat hindpaw by a priming painful trigeminal nerve injury. Pain, 97;203-212.
107) Hardy, H.G. Wolff and H. Goodell. Methods for the study of pain thresholds. In Pain Sensations and Reactions: Hardy, H.G. Wolff and H. Goodell. (Eds.), 1st. edition; Williams and Wilkins, New York, 1952, pp. 34-60.
108) Staud, R., Robinson, M.E., Vierck, C.J.,Jr and Price, D.D. 2003. Diffuse noxious inhibitory
controls (DNIC) attenuate temporal summation of second pain in normal males but not in normal females or fibromyalgia patients. Pain, 101;167-174.
109) Small, J.R., Scadding, J.W. and Landon, D.N. 1996. Ultrastructural localization of
sympathetic axons in experimental rat sciatic nerve neuromas. Neurocytology, 25;573-582.
110) Nissenbaum, J., Devor, M., Seltzer, Z., Gebauer, M., Michaelis, M., Tal, M., Dorfman, R.,
Abitbul-Yarkoni, M., Lu, Y., Elahipanah, T., delCanho, S., Minert, A., Fried, K., Persson, A.K., Shpigler, H., Shabo, E., Yakir, B., Pisante, A. and Darvasi, A. 2010. Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2. Genome Research, 20;1180-1190.
111) Sax, K. 1923. The Association of Size Differences with Seed-Coat Pattern and
Pigmentation in PHASEOLUS VULGARIS. Genetics, 8;552-560. 112) Mogil, J.S. 2003. The Genetics of Pain. IASP Press, Seattle. pp. 1-15.
113) Wilson, S.G., Smith, S.B., Chesler, E.J., Melton, K.A., Haas, J.J., Mitton, B., Strasburg, K., Hubert, L., Rodriguez-Zas, S.L. and Mogil, J.S. 2003. The heritability of antinociception: common pharmacogenetic mediation of five neurochemically distinct analgesics. Pharmacology and Experimental Therapeutics, 304;547-559.
114) Lariviere, W.R., Wilson, S.G., Laughlin, T.M., Kokayeff, A., West, E.E., Adhikari, S.M.,
Wan, Y. and Mogil, J.S. 2002. Heritability of nociception. III. Genetic relationships among commonly used assays of nociception and hypersensitivity. Pain, 97;75-86.
115) Craft, R.M. 2003. Sex differences in opioid analgesia: from mouse to man, Clinical Journal
of Pain.19;175-186. 116) Mogil, J.S., Chesler, E.J., Wilson, S.G., Juraska, J.M. and Sternberg, W.F. 2000. Sex
differences in thermal nociception and morphine antinociception in rodents depend on genotype. Neuroscience and Biobehavioral Reviews. 24;375-389.
130
117) Kest, B., Wilson, S.G. and Mogil, J.S. 1999. Sex differences in supraspinal morphine
analgesia are dependent on genotype. Pharmacology and Experimental Therapeutics, 289; 1370-1375.
118) Chesler, E.J., Wilson, S.G., Lariviere, W.R., Rodriguez-Zas, S.L. and Mogil, J.S. 2002.
Identification and ranking of genetic and laboratory environment factors influencing a behavioral trait, thermal nociception, via computational analysis of a large data archive. Neuroscience and Biobehavioral Reviews, 26;907-923.
119) Mogil, J.S., Richards, S.P., O'Toole, L.A., Helms, M.L., Mitchell, S.R. and Belknap, J.K.
1997. Genetic sensitivity to hot-plate nociception in DBA/2J and C57BL/6J inbred mouse strains: possible sex-specific mediation by delta2-opioid receptors. Pain, 70;267-277.
120) Mogil, J.S., Richards, S.P., O'Toole, L.A., Helms, M.L., Mitchell, S.R., Kest, B. and
Belknap, J.K. 1997, Identification of a sex-specific quantitative trait locus mediating nonopioid stress-induced analgesia in female mice. Neuroscience, 17;7995-8002.
121) Bodnar, R.J., Kelly, D.D., Brutus, M. and Glusman, M. 1980. Stress-induced analgesia:
neural and hormonal determinants. Neuroscience and Biobehavioral Reviews, 4;87-100. 122) Review of Medical Physiology: Ganong, W.F. 19th edition, 2006. p. 139.
123) Marenholz, I., Heizmann, C.W. and Fritz, G. 2004. S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochemical and Biophysical Research Communications, 322;1111-1122.
124) Nikitin, V.P., Kozyrev, S.A., Shevelkin, A.V. and Sherstnev, V.V. 2002. The effects of
antibodies against proteins of the s100 group on neuron plasticity in sensitized and non-sensitized snails. Neuroscience and Behavioral Physiology, 32;25-31.
125) De Biasi, M. and Dani, J.A. 2003. Stress hormone enhances synaptic NMDA
response on dopamine neurons. Neuron, 39;387-388. 126) Vit, J.P., Clauw, D.J., Moallem, T., Boudah, A., Ohara, P.T. and Jasmin, L. 2006.
Analgesia and hyperalgesia from CRF receptor modulation in the central nervous system of Fischer and Lewis rats. Pain, 121;241-260.
127) Janz, R. and Sudhof, T.C. 1999. SV2C is a synaptic vesicle protein with an unusually
restricted localization: anatomy of a synaptic vesicle protein family. Neuroscience, 94;1279-1290.
128) Bajjalieh, S.M., Peterson, K., Shinghal, R. and Scheller, R.H. 1992, SV2, a brain synaptic
vesicle protein homologous to bacterial transporters. Science, 257;1271-1273. 129) Hobom, M., Dai, S., Marais, E., Lacinova, L., Hofmann, F. and Klugbauer, N. 2000.
Neuronal distribution and functional characterization of the calcium channel alpha2delta-2 subunit. The European Journal of Neuroscience, 12;1217-1226.
131
130) Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F. and Dascal, N. 1991. The roles of the subunits in the function of the calcium channel. Science, 253;1553-1557.
131) Hofmann, F., Lacinova, L. and Klugbauer, N. 1999. Voltage-dependent calcium channels:
from structure to function. Reviews of Physiology, Biochemistry and Pharmacology, 139;33-87.
132) Klugbauer, N., Lacinova, L., Marais, E., Hobom, M. and Hofmann, F. 1999, Molecular diversity of the calcium channel alpha2delta subunit. Neuroscience, 19;684-691.
133) Field, M.J., Li, Z. and Schwarz, J.B. 2007. Ca2+ channel alpha2-delta ligands for the
treatment of neuropathic pain. Medicinal Chemistry, 50;2569-2575. 134) Taylor, C.P. 2009. Mechanisms of analgesia by gabapentin and pregabalin-calcium channel
alpha2-delta [Cavalpha2-delta] ligands. Pain, 142;13-16. 135) Chen, J. and Lariviere, W.R. 2010. The nociceptive and anti-nociceptive effects of bee
venom injection and therapy: a double-edged sword. Progress in Neurobiology, 92;151-183.
136) Baumgartner, U., Magerl, W., Klein, T., Hopf, H.C. and Treede, R.D. 2002. Neurogenic
hyperalgesia versus painful hypoalgesia: two distinct mechanisms of neuropathic pain. Pain, 96;141-151.
137) Merskey, H. 1979. Pain terms: A list with definition and notes on usage: Recommendations
by the IASP Subcommittee on Taxonomy. Pain, 6;249-252. 138) Lynch, M. and Walsh, B. Genetics and Analysis of Quantitative Traits. Sinauer Associates,
Sunderland, 1998, pp.73-96. 139) Rieseberg, L.H., Archer, M.A. and Wayne, R.K. 1999. Transgressive segregation,
adaptation and speciation. Heredity, 83;363-372. 140) Martenson, M.E., Cetas, J.S. and Heinricher, M.M. 2009. A possible neural basis for stress-
induced hyperalgesia. Pain, 142;236-244. 141) Bergen, S.E., Gardner, C.O. and Kendler, K.S. 2007. Age-related change inheritability of
behavioral phenotypes over adolescence and young adulthood: a meta-analysis. Twin Research and Human Genetics, 10;423-433.
142) Davoody, L., Quiton, R.L., Lucas, J.M., Ji, Y., Keller, A. and Masri, Conditioned place preference reveals tonic pain in an animal model of central pain. Pain, In Press.
143) LaBuda, C.J. and Fuchs, P.N. 2000. A Behavioral Test Paradigm to Measure the Aversive
Quality of Inflammatory and Neuropathic Pain in Rats. Experimental Neurology, 163;490-494.
144) Soleimannejad, E., Mashregi, M., Froimovitch, D., Biton, S., Alter, G., Seltzer, Z. 2010.
Novel high-throughput screening for testing avoidance of pro-allodynic thermal and mechanical stimuli in rodents. IASP World Congress on Pain, abstract.
132
145) Lariviere, W. R., Nair, H. K., Peng, X., Spence, J. S., Chesler, E. J. 2010. Quantitative Trait Locus (QTL) mapping and genetic correlation analysis of inflammatory hypersensitivity. IASP World Congress on Pain, abstract.
146) Kauppila, T. 1998. Correlation between autotomy-behavior and current theories of
neuropathic pain. Neuroscience and Biobehavioral Reviews, 23;111-129. 147) Decosterd, I., Allchorne, A. and Woolf, C.J. 2004. Differential analgesic sensitivity of two
distinct neuropathic pain models. Anesthesia and Analgesia, 99;457-463. 148) Martin, J.H., Mohit, A.A. and Miller, C.A. 1996. Developmental expression in the mouse
nervous system of the p493F12 SAP kinase. Brain research, 35;47-57. 149) Yang, D.D., Kuan, C.Y., Whitmarsh, A.J., Rincon, M., Zheng, T.S., Davis, R.J., Rakic, P.
and Flavell, R.A. 1997. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature, 389;865-870.
150) Choi, D.W. 1988. Glutamate neurotoxicity and diseases of the nervous system. Neuron,
1;623-634. 151) Keramaris, E., Vanderluit, J.L., Bahadori, M., Mousavi, K., Davis, R.J., Flavell, R., Slack,
R.S. and Park, D.S. 2005, c-Jun N-terminal kinase 3 deficiency protects neurons from axotomy-induced death in vivo through mechanisms independent of c-Jun phosphorylation. Biological Chemistry, 280;1132-1141.
152) Zimmermann, M. 2001. Pathobiology of neuropathic pain. European journal of
Pharmacology, 429;23-37. 153) Scalabrini, D., Fenoglio, C., Scarpini, E., De Riz, M., Comi, C., Venturelli, E., Cortini, F.,
Piola, M., Villa, C., Naldi, P., Monaco, F., Bresolin, N. and Galimberti, D. 2007. Candidate gene analysis of SPARCL1 gene in patients with multiple sclerosis. Neuroscience Letters, 425;173-176.
154) Bornstein, P. and Sage, E.H. 2002. Matricellular proteins: extracellular modulators of cell
function. Current Opinion in Cell Biology, 14;608-616. 155) Gongidi, V., Ring, C., Moody, M., Brekken, R., Sage, E.H., Rakic, P. and Anton, E.S.
2004. SPARC-like 1 regulates the terminal phase of radial glia-guided migration in the cerebral cortex. Neuron, 41;57-69.
156) Elahipanah, T. 2010. Genetic Determinants of Spontaneous Neuropathic Pain following
Denervation of Sciatic and Saphenous Nerves in Mice. Thesis for Masters of Science, The Department of Physiology, University of Toronto.
157) Zhang, S., Deniz, B., Patel, U., Lu, Y., Sessle, B., HU, J., and Seltzer, Z. 2006. Wide-spread
ipsi- and contralateral abnormal pain behaviour in mice following unilateral infraorbital neurectomy is determined genetically and is sexually dimorphic. Society for Neuroscience. Abstract.
133
158) Yarkoni, M., Zhang, S., Tichauer, D., Elahipanah, T., Dorfman, R., Seltzer, Z. 2009, Genome-wide expression analysis in the mouse and candidate gene approach in humans reveal novel conserved neuropathic pain genes. University of Toronto Faculty of Dentistry Annual Research Day. Abstract.
159) Gau, X., Zhang, S., Zhu, B., Zhang, H. 2007. Investigation of specificity of auricular
acupuncture points in regulation of autonomic function in anesthetized rats. Autonomic Neuroscience, 138;50-56.
160) Chesler, E.J., Lu, L., Wang, J., Williams, R.W. and Manly, K.F. 2004. WebQTL: rapid
exploratory analysis of gene expression and genetic networks for brain and behavior. Nature Neuroscience, 7;485-486.
161) Mogil, J.S., Ritchie, J., Sotocinal, S.G., Smith, S.B., Croteau, S., Levitin, D.J. and
Naumova, A.K. 2006. Screening for pain phenotypes: analysis of three congenic mouse strains on a battery of nine nociceptive assays. Pain, 126;24-34.
162) Darvasi, A. and Soller, M. 1995. Advanced intercross lines, an experimental population for
fine genetic mapping. Genetics, 141;1199-1207. 163) Persson, A.K., Gebauer, M., Jordan, S., Metz-Weidmann, C., Schulte, A.M., Schneider,
H.C., Ding-Pfennigdorff, D., Thun, J., Xu, X.J., Wiesenfeld-Hallin, Z., Darvasi, A., Fried, K. and Devor, M. 2009. Correlational analysis for identifying genes whose regulation contributes to chronic neuropathic pain. Molecular Pain, 5;7.
164) Wilson, S.G., Chesler, E.J., Hain, H., Rankin, A.J., Schwarz, J.Z., Call, S.B., Murray, M.R.,
West, E.E., Teuscher, C., Rodriguez-Zas, S., Belknap, J.K. and Mogil, J.S. 2002. Identification of quantitative trait loci for chemical/inflammatory nociception in mice. Pain, 96;385-391.
165) Mogil, J.S., Richards, S.P., O'Toole, L.A., Helms, M.L., Mitchell, S.R. and Belknap, J.K.
1997. Genetic sensitivity to hot-plate nociception in DBA/2J and C57BL/6J inbred mouse strains: possible sex-specific mediation by delta2-opioid receptors. Pain, 70;267-277.
166) Field, M.J., Bramwell, S., Hughes, J. and Singh, L. 1999. Detection of static and dynamic
components of mechanical allodynia in rat models of neuropathic pain: are they signalled by distinct primary sensory neurons? Pain, 83;303-311.
167) Xie, Y.F., Zhang, S., Chiang, C.Y., Hu, J.W., Dostrovsky, J.O. and Sessle, B.J. 2007.
Involvement of glia in central sensitization in trigeminal subnucleus caudalis (medullary dorsal horn). Brain, Behavior and Immunity, 21;634-641.
168) Wang, J., Liao, G., Usuka, J., Peltz, G. 2005. Computational genetics: from mouse to
humans. Trends in Genetics, 9;526-532.
