The effects of shaking on the eye and central nervous system of
mice and Barbados Green Monkeys
by
Jin Han (Patrick) Kim
A thesis submitted in conformity with the requirements for the degree of
Master of Science
Graduate Department of Laboratory Medicine and Pathobiology
University of Toronto
Copyright by Patrick J.H. Kim 2009
ii
Abstract
The effects of shaking on the eye and central nervous system of
mice and Barbados Green Monkeys
Patrick J.H. Kim
Master of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
2009
Shaken baby syndrome is a clinicopathologic syndrome characterized by a triad of findings:
subdural hemorrhage, retinal hemorrhage and axonal injury. Although shaking is widely
believed to cause the triad, it is not yet entirely clear if shaking without head impact can produce
the triad. Initial attempts to test the effect of shaking in mouse pups were unsuccessful as neither
controlled continuous vibration nor pulse acceleration caused any of the components of the
triad. With no other convenient modeling system available, a pilot study with three adult
subhuman primates was conducted. Although a conclusive statement cannot be made, manual
shaking did not immediately cause hemorrhagic injuries to the primates’ brains and eyes. Future
studies should test for delayed development of axonal injury. In addition, a comparative
anatomical study should also be conducted to test the validity of the adult primate as a model
system for human infant injuries.
iii
Foundations
It is those who know little, and not those who know much, who so positively assert that this
or that problem will never be solved by science.
- Charles Darwin (1809 ~ 1882), Introduction to The descent of man and selection in relation to
sex (1871)
iv
Acknowledgements
I thank the members of my advisory committee for their support and guidance: Drs. C.
Bergeron (chair), M. Johnston, and M.S. Pollanen (supervisor). I also thank Dr. T.H. Rose, Ms.
E. Bidgoli and Ms. A. Mainiero for reading and editing the earlier drafts of this thesis. Special
thanks go to Drs. K. Cunningham, I. Kitulwatte and D.N. McAuliffe for valuable discussions
regarding general issues in forensic pathology.
I acknowledge the Department of Laboratory Medicine and Pathobiology for the visiting
trainee graduate award. I was fortunate to have the help of Mr. D. Kang and Ms. M. Currie,
histotechnologists, who answered countless questions I had about processing animal tissues. I
would like to thank Ms. B. Anders and Ms. R. Perri for their support in procurements. I also
thank all other members at the Forensic Pathology Unit, Office of the Chief Coroner for Ontario
for their daily support.
Special appreciation goes to Ms. J. Manias for her technical assistance and Dr. S. Nag for
graciously providing laboratory space and equipments for immunostaining.
I would like to thank Ms. T. McCook and L. Penny at Division of Comparative Medicine for
their assistance in animal handling. Also, Dr. K. Banks at DCM provided invaluable help in
establishing animal protocols. I would also like to thank everyone at BPRC for their hospitality
and assistance. Mr. D. Clutterbuck provided expert assistance in autopsy and photography. All
gross photographs for the Barbados Green Monkey study were taken by Mr. Clutterbuck.
Finally, my very special gratitude goes to my supervisor, Dr. M.S. Pollanen for his constant
support of this thesis through challenges and unexpected delays. This project is dedicated to my
family.
v
Part of the materials in this thesis was presented at the Canadian Association of
Neuropathologists annual meeting in 2007 in a talk “Non-Impact Head Injury in Infants –
Mouse Model of Shaken Baby Syndrome”, which won the Morrison H. Finlayson award.
vi
Table of contents
Abstract ii
Foundation iii
Acknowledgements iv
Table of contents vi
List of tables xi
List of figures xii
Abbreviations xiv
Chapter 1. Introduction and literature review 1
1.1. Introduction 1
1.2 Infant head and neck 3
1.2.1 Anatomical considerations 3
1.2.2. Pattern of head and neck injuries in child abuse 3
1.2.2.1. Injuries by blunt impact 3
1.2.2.2. Injuries by sudden accelerations of the head 4
1.2.2.3. Typical head and neck injuries seen in child abuse 4
1.3. Rise of Shaken Baby Syndrome 6
1.3.1. Historical considerations 6
1.3.2. Caffey’s Lecture in 1972 7
1.3.3. Retrospective case series vs. Anecdotal case reports 9
1.4. Study of Shaken Baby Syndrome 12
1.4.1. Animal models of Shaken Baby Syndrome 12
1.4.2. Mechanical models of Shaken Baby Syndrome 13
1.4.3. Evidence-based reviews of the retrospective case series 14
1.5. Current points of controversy 18
1.5.1. Pathologic nature of ‘pure shaking’ 18
1.5.1.1. Immediate traumatic injuries due to shearing and traction 18
vii
1.5.1.2. Hypoxic and ischemic encephalopathy due to brainstem injury 19
1.5.2. Specificity of the triad 19
1.5.2.1. Short fall debate 22
1.5.2.2. Confounding medical conditions 23
1.5.3. Nature of subdural hemorrhages 24
1.6. SBS in criminal justice system 26
1.6.1. Harris appeal and Shaken Baby Syndrome cases review in Britain 26
1.6.2. Goudge inquiry in Ontario 27
Chapter 2. Experimental design 28
2.1. Hypothesis 28
2.2. Research Objective 28
2.2.1. Overall 28
2.2.2. Specific aims 29
2.3. Rationale 30
2.3.1. Advantages of animal model 30
2.3.2. Indication from previous studies 30
2.3.3. American Academy of Pediatrics technical report (2001) 31
2.4. Material and methods 33
2.4.1. High frequency vibration of postnatal mice 33
2.4.1.1. Mice 33
2.4.1.2. Shaking apparatus 33
2.4.1.3. Displacement measurement 36
2.4.1.4. Anesthesia and euthanasia 36
2.4.1.5. Tissue processing and histology 36
2.4.2. Manual shaking of Barbados Green Monkeys 40
2.4.2.1. Barbados Green Monkeys 40
2.4.2.2. Accelerometer 41
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2.4.2.3. Anesthesia and euthanasia 41
2.4.2.4. Postmortem examination 47
2.4.2.5. Tissue processing and histology 47
2.4.2.6. Immunohistochemistry 48
2.5. Experimental protocol 49
2.5.1. Mice 49
2.5.1.1. Constant vibration 49
2.5.1.2. Pulse acceleration 50
2.5.1.3. Hinge-point 50
2.5.1.4. Displacement measurement 51
2.5.2. Barbados Green Monkeys 52
2.5.2.1. Sham (Negative control) 52
2.5.2.2. Anterior-posterior shaking 52
2.5.2.3. Lateral shaking 53
2.6. Ethics of animal use 56
2.6.1. Mice 56
2.6.2. Barbados Green Monkeys 56
Chapter 3. Results: Mice 57
3.1. Summary of findings 57
3.2. Constant vibration study 57
3.3. Pulse acceleration study 58
3.4. Hinge-point study 58
3.5. Vibration frequency and displacement 63
Chapter 4. Results: Barbados Green Monkey 66
4.1. Summary of findings 66
4.2. Physiological responses after shaking 67
ix
4.3. Anatomical and histologic findings 73
4.3.1 Brain and cervical spinal cord 73
4.3.2. Eyes and retina 102
4.3.3. Neck 102
4.3.4. Fingertip bruising 102
4.4. Accelerometry data 111
4.4.1. Sham animal 111
4.4.2. AP animal 111
4.4.3. Lat animal 111
Chapter 5. Discussion 119
5.1. Mice 119
5.1.1. General discussion 119
5.1.2. Negative findings: What do they mean? 120
5.1.3. Role of mouse model in future investigations of Shaken Baby Syndrome 122
5.2. Barbados Green Monkeys 124
5.2.1. General discussion 124
5.2.2. Negative findings: What do they mean? 125
5.2.3. Role of Barbados Green Monkey model in future investigations of Shaken Baby Syndrome 128
5.3. Back to Shaken Baby Syndrome debate: significance of experiments performed 130
5.3.1. True non-impact head injury protocol 130
5.3.2. Mechanical properties of Barbados Green Monkey manual shaking 130
5.3.3. Empirical data for Shaken Baby Syndrome discussion 131
Chapter 6. Future directions 133
6.1. Primate model of Shaken Baby Syndrome 133
6.1.1. Axonal injury 133
6.1.2. Describing head and neck movement during shaking 134
x
6.1.3. Ethical consideration 135
6.1.4. Anthropomorphic model 135
6.1.5. Mechanical properties of tissues and finite element modeling 136
6.2. Hypoxia as cause of subdural hemorrhages 137
6.2.1. Perinatal and neonatal intradural hemorrhages 137
6.2.2. Implication for Shaken Baby Syndrome ‘unified hypothesis’ 137
6.3. Medical investigations of Shaken Baby Syndrome 139
6.3.1. Retrospective and prospective studies using APP immunostaining 139
6.3.2. Cerebrospinal fluid analysis 139
6.4. Possible mechanism of Shaken Baby Syndrome triad development 140
Chapter 7. References 141
Appendix 150
A. Experimental protocols 150
A-1. Routine H&E staining protocol 150
A-2. Routine LFB/H&E staining protocol 152
B. Ethics 154
B-1. Revised Barbados Green Monkey manual shaking research proposal 154
B-2. Response to reviewers’ comments 165
xi
List of tables
Table 1-1. Summary of cases used by Caffey in 1972 article
Table 2-1. Selection of Barbados Green Monkey for the study
Table 2-2. Barbados Green Monkey Anesthesia/Euthanasia records
Table 4-1. Barbados Green Monkey morphometric dimensions
Table 6-1. Proposed experimental groups for the study of axonal damage from manual shaking
of Barbados Green Monkey
xii
List of figures
Figure 1-1. Photomicrographs of traumatic axonal injury (low/high power, APP)
Figure 1-2. Flow chart of two possible mechanisms of shaking injuries
Figure 2-1. Experimental setup of high frequency vibration of postnatal mice
Figure 2-2. Whole mount of postnatal mice head and neck
Figure 2-3. Overall photograph of an Barbados Green Monkey
Figure 2-4. Mounting of accelerometer
Figure 2-5. Experimental setup of manual shaking of Barbados Green Monkeys
Figure 3-1. Photomicrographs of A. Dural space, B. Retina, C. Cerebral cortex
Figure 3-2. Focal subarachnoid hemorrhage
Figure 3-3. Displacement over time plot of the shaking apparatus
Figure 4-1. Anterior-posterior shaking
Figure 4-2. Lateral shaking
Figure 4-3. Brain and cervical spinal cord of Sham animal. Gross View.
Figure 4-4. Brain and cervical spinal cord of Anterior-posterior animal. Gross View.
Figure 4-5. Brain and cervical spinal cord of Lateral animal. Gross View.
Figure 4-6. Coronal sections of Sham animal brain after formalin fixation
Figure 4-7. Horizontal sections of Sham animal brainstem after formalin fixation
xiii
Figure 4-8. Coronal sections of Anterior-posterior animal brain after formalin fixation
Figure 4-9. Horizontal sections of Anterior-posterior animal brainstem after formalin fixation
Figure 4-10. Coronal sections of Lateral animal brain after formalin fixation
Figure 4-11. Horizontal sections of Lateral animal brainstem after formalin fixation
Figure 4-12. Photomicrographs of cervical spinal cord of Barbados Green Monkey
Figure 4-13. Photomicrographs of pons of Barbados Green Monkey
Figure 4-14. Photomicrographs of medulla of Barbados Green Monkey
Figure 4-15. Photomicrographs of cerebral cortex of Barbados Green Monkey
Figure 4-16. Photomicrographs of posterior corpus callosum of Barbados Green Monkey
Figure 4-17. Retina after formalin fixation.
Figure 4-18. Photomicrographs of retina.
Figure 4-19. Posterior neck dissection.
Figure 4-20. Fingertip bruising found of body surfaces.
Figure 4-21. Accelerometry tracing from Sham treatment
Figure 4-22. Accelerometry tracing from Anterior-posterior animal shaking
Figure 4-23. Accelerometry tracing from Lateral animal shaking
xiv
Abbreviations
APP -amyloid precursor protein
AAP American Academy of Pediatrics
AP Anterior-posterior
BGM Barbados Green Monkeys
BPRC Barbados Primate Research Center
CSF Cerebrospinal fluid
CVP Central venous pressure
DAB 3,3’-Diaminobenzidine
G Gravitational acceleration unit (1G = 9.80665 m/s2)
H&E Hematoxylin-eosin stain
ICP Intracranial pressure
IDH Intradural hemorrhages
IM Intramuscular
IV Intravenous
LACC Local Animal Care Committee
Lat Lateral
LFB Luxol-fast-blue stain
PBS Phosphate buffered saline
RBC Red blood cell
RH Retinal hemorrhages
SBS Shaken baby syndrome
xv
SDH Subdural hemorrhages
TAI Traumatic axonal injury
tDAI Traumatic diffuse axonal injury
1. Introduction and literature review
1.1 Introduction
When a person dies, it is in the public interest to understand how the person came to die. This
is especially true in the case of deceased babies where public interest may be highly emotional,
and legal action may follow if any unlawful actions contributed to death. During death
investigations, forensic pathologists conduct autopsies and provide key evidence that may
influence the direction of the investigations. By performing a postmortem examination, a
pathologist seeks to determine the cause, and mechanism of death as well as facilitate a
determination of the manner of death. A postmortem examination consists of a series of steps
starting from the scene investigation and collection of the history, external and internal
examination of the body, ancillary testing on samples collected from the examination, and
interpretation of the findings to create a knowledgeable and balanced opinion.
In the past ten years, a considerable degree of controversy in the field of pediatric forensic
pathology in Toronto has come to public exposure by a series of wrongful conviction claims. As
Commissioner Goudge stated in his report for the Inquiry into Pediatric Forensic Pathology in
Ontario, the problematic issues that arose did not solely represent “a simple failure of any
individual, but in addition it represents systemic issues” 1. The bases of the controversy are
intricate and fundamental differences in the methods of gaining and interpreting knowledge in
the fields of medicine, science and the criminal justice system.
During the past three years at the University of Toronto and the Provincial Forensic
Pathology Unit, Ontario, I have focused on the topic of shaken baby syndrome (SBS) which
illustrates Commissioner Goudge’s statement very clearly. SBS is a combination of a clinical
and pathological findings in infants that are believed to be violently shaken by their caregivers 2.
1
In my effort to understand the SBS controversy, I came to appreciate that each group that
receives and interprets such findings made by forensic pathologists has a specific knowledge
base and unique requirements that influence how that information will be utilized. From the
scientists’ perspective, the goal is to discover the truths about the natural world by objectively
asking questions, and testing thoughfully developed hypotheses through experimentation. Due
to the very nature of this approach, existing frameworks to understand the truth (theories) are
questioned when new observations do not conform to the accepted perspective and the cycle of
inquiry begins anew. In medicine, the goal is to save lives, and the knowledge gained through
repetitive experience is just as important as scientific knowledge. When a new scientific
paradigm does not fit the previously accepted dogma, medicine reacts in a conservative manner
until a new body of experience confirms or refutes the scientific knowledge. In contrast, the
justice system uses expert opinions as a foundation of understanding that will be evaluated along
with other factual evidence from the death investigation. However, the justice system as a whole
or the expert witnesses themselves may fail to recognize that opinion evidence is subject to
change with advances in science and medicine. Failure to recognize the ever changing base of
scientific and medical knowledge could lead to devastating consequences as illustrated by
wrongful convictions associated with SBS.
This dissertation represents my effort to address the SBS controversy in an objective and
scientific manner. In chapter one, I have reviewed the literature to present the contentious issues
at hand and how they have affected the criminal justice system. In the subsequent chapters, I
have detailed my approach to the investigation of the issues surrounding the effect of shaking on
human infants with murine and Barbados Green Monkeys (BGM) models. A discussion of the
experimental results is found in chapter five which forms the basis of the future directions in
chapter six.
2
1.2 Infant head and neck
1.2.1 Anatomical considerations
The temporally disproportionate development of the anatomy of the head and neck in human
infants leads to its distinct morphology and susceptibility to external mechanical forces, such as
through shaking. The initially large head-to-trunk ratio of the neonate decreases until it reaches
that of the adults. In addition, the neck musculature continues to develop during infancy to
support the large cranium in bipedal locomotion. Other anatomic differences between the infants
and the adults include greater tensile strength of the scalp due to its higher collagen content,
patency of cranial sutures that gradually ossify over time, continued development of the
meningeal layers as the dura is not yet firmly attached to the inner table. Furthermore, the
relatively high water content of the brain decreases during the early years of life as a result of
continuous myelination of the axons. These developmental patterns lead to distinct
physiological properties during infancy that are most prominent immediately after birth and
gradually diminish with time as the child physically matures into an adult. The mechanically
important characteristics of an immature head and neck anatomy are: 1. A large head supported
by undeveloped neck structures, 2. A greater range of movement for the brain within the
cranium, and 3. A scalp which can stretch greatly without being restricted by the cranium.
1.2.2. Pattern of head and neck injuries in child abuse
1.2.2.1. Injuries by blunt impact
Blunt impact can be defined as a transfer of energy by a (typically) non-penetrating contact
event. The impact energy is transmitted to the different layers underneath the impact site. The
extent of tissue damage is determined by the force transmitted by the impact event and by the
injury threshold of the tissue. The extent of injury caused by the same impact varies for different
3
tissues. In addition, anatomical and morphological features must be considered to correlate with
the type of force that caused the observed injuries. The aforementioned anatomical properties of
infants have been thought to alter the extent of the blunt impact injuries from what would be
seen in adults after similar impact events. These differences pose a great challenge in
ascertaining the impact event from the pattern of injuries. Some of these factors include: 1. A
large head supported by a weak neck, which results in a higher susceptibility to the acceleration
after the impact, 2. A pliable cranium allows for more space for the brain to move independently
of its underlying structures following impact, potentially resulting in further damage, and 3.
Finally, as a consequence of the scalp’s increased elasticity and tensile strength, the extent of the
damage to the scalp could be inconsistent with the injuries seen on the structures underneath.
1.2.2.2. Injuries by sudden acceleration of the head
Most investigations of injuries caused by sudden acceleration of the head have been
conducted regarding motor vehicle crashes. It is now understood that, in adults, both linear and
rotational acceleration can cause damage to the head and neck without direct impact.
Acceleration injuries include: 1. Stretch injuries to the brainstem, 2. Traumatic diffuse axonal
injury, and 3. Fractures of vertebrae in severe accelerations. These injuries can occur in
combination with impact (impact-acceleration) and when they do, it is hard to determine the
contribution of each mechanism to the injuries.
1.2.2.3. Typical head and neck injuries seen in child abuse
Child abuse may be defined as “a large, complex group of human behaviours characterized
by traumatic interactions between parents or other caretakers and the infants and children of all
ages under their care, as well as between strangers and children during casual contact” 3. It is
not limited to physical trauma; other forms of maltreatment such as emotional as well as sexual
4
abuse, and various forms of neglect could also be part of child abuse. The American Academy
of Pediatrics (AAP) recognizes failure to thrive as another sign of child abuse 4. In chronic child
abuse, it is not uncommon to find at postmortem examination multiple bruises of different ages
on the scalp and face, skull fractures, retinal hemorrhage (RH), chronic and acute subdural
hemorrhages, generalized brain swelling and cerebral contusions of varying ages. Other injuries
to the body include long bone and rib fractures, metaphyseal fractures, visceral injuries and
burns. Although many of these injuries are primary consequences of specific physical
mechanisms, it is important to note that the broad timeframe of child abuse could lead to chronic
and secondary manifestations of the primary injuries.
5
1.3. Rise of the Shaken Baby Syndrome (SBS)
1.3.1. Historical considerations
Although the famous French forensic pathologist Ambroise Tardieu first described the
patterns of child abuse in 1860 5, it was not until early in the twentieth century that physicians
started to recognize child abuse as a separate entity. While most injury patterns were similar to
those of adults and thus generally reflected well understood mechanisms of infliction, some
unique injuries were seen in these infants that did not follow the pattern of direct impact
inflicted injuries. The term ‘whiplash injuries’ were used to describe the set of clinical findings
in abused babies that was thought to be caused by a whiplash motion of the head due to sudden
acceleration or deceleration. These clinical findings included gross hemorrhages and contusions
over the surface of the brain and upper cervical cord, cerebral concussion, and subdural
effusions 6.
From 1964 to 1969 Ommaya et al. published a set of studies in which the heads of primates
were subjected to sudden acceleration, causing a constellation of injuries similar to that of
‘battered babies’ 7-12. In 1971, Guthkelch published a case report in which he found subdural
hemorrhages in babies who had been shaken and noted a similarity to Ommaya’s experimental
findings 13. This, he felt, strongly correlated with a shaking mechanism. However, since the
findings that were supposedly caused by a whiplash mechanism were reported in the context of
battered babies, other cranial findings such as skull fractures and scalp bruising were present
and were often used as an indicator of an abusive event. It should be mentioned that other causes
of such intracranial injury in infants such as birth trauma were already known at this time.
6
1.3.2. Caffey’s Lecture in 1972
In 1972, Dr. J. Caffey gave an Abraham Jacobi lifetime achievement award lecture entitled
“On the theory and practice of shaking infants: Its potential residual effects of permanent brain
damage and mental retardation” 14. In this lecture, Caffey first described the metaphyseal bone
lesions in infants that were then believed to be pathologic features of shaking. Caffey then
reviewed the clinical and pathological findings of abusive trauma from multiple previous reports
along with three cases of his own. Of 29 cases of ‘whiplash-shaking’ (which Caffey mistakenly
reported as a total of 27 cases), bone lesions were found in six cases, surgically treatable
subdural hemorrhage (SDH) in three cases, and retinal hemorrhage (RH) in three cases. Results
from only one postmortem examination were mentioned in these cases of traumatic brain injury
(Table 1-1). Reporting on these seemingly uncommon incidents, Caffey primarily drew from his
experience and the Ommaya study 10 to conclude that in ‘whiplash-shaking’ causes 1. Bilateral
subdural hematoma caused by ‘indirect acceleration traction stresses’ are frequently
undiagnosed 2. Bilateral retinal lesions are indicative of subclinical chronic subdural hematoma
and 3. Early cerebral edema could lead to diffuse gliosis and cause infantile obstructive
hydrocephalus. Caffey also argued that the bilaterality of SDH and RH and the lack of external
signs of impact were suggestive of a non-impact origin such as ‘whiplash-shaking’. Caffey
described at length examples of child abuse and stated that infant shaking ‘appears to be
practiced widely’ and the number of incidents ‘cannot be even estimated satisfactorily’. He
concluded that ‘whiplash-shaking is always potentially pathogenic to some degree’, and when
repeated, ‘even relatively mild shaking…are probably more pathogenic than … more violent
and conspicuous shakings during wilful assault’.
7
Table 1-1. Summary of 29 cases mentioned in Caffey’s 1972 article
Case history Findings
Example 1 (15 cases)
An infant-nurse admitted killing three infants and injuring 12 more by shaking. Shaking and pounding was done to help infants burp
“Traumatic brain injury” in one fatal case
Example 2 (4 cases)
Two cases of young children being shaken, a case of infant being shaken and banged against the crib, and a case of an infant being shaken and beaten to death
No mention of pathologic findings
Example 3 (3 cases)
a) A case of an infant who died after being shaken to stop paroxymal coughing b) A case of an infant who was found convulsing and vomiting; parents admitted to shaking c) A case of an infant with broken femur; parents admitted to shaking
a) SDH
b) SDH, RH, bruising on forearms that fit adult hands
c) No other pathologic findings mentioned
Example 4 (1 case)
A case of young infant who was gripped by legs and shaken upside down
Multiple massive involucra and metaphyseal avulsions in femurs and tibias
Example 5 (1 case)
“Believed to be whiplash-shaken” Compression fractures of vertebral bodies
Example 6 (3 cases)
a) Two cases of infants who were yanked upwards to prevent from falling b) A case of an infant who was shaken and swung onto the bed by 8 year old sibling
a) Massive involucra of the radius and ulna in one, and avulsion metaphyseal fracture and traumatic involucra of tibia in another b) Traumatic involucra of both femurs
Example 7 (2 cases)
a) A case of an infant who was grabbed by the legs and swung around b) A case of an infant who was gripped by the thorax and shaken violently
a) SDH and RH b) RH
Total (29 cases)
Heterogeneous history with many with concurrent impact events
1 Traumatic brain injury 3 SDH 3 RH Remainder with bone lesions
8
1.3.3. Retrospective case series vs. Anecdotal case reports
In his 1972 lecture, Caffey stated that the findings of subdural hemorrhage, retinal
hemorrhage, and diffuse gliosis are due to the mechanical shearing or traction force generated
from shaking. However, none of his 29 case examples could qualify as pure shaking incidents
where the possibility of an impact event could be ruled out. Most cases had a history of
concurrent impact whether or not evidence of an impact could be found on examination.
Subsequent case reports also claimed that the reported findings are due to inertial force applied
during shaking. However, it is now understood that these cases might have had evidence of
impact that was missed or disregarded as a contributory factor at the original examination.
Although inadequate by today’s standards, the series of articles by Ommaya et al. 7-12 and
Caffey 14 was enough to raise awareness of SBS among physicians. The newly created clinical
entity of SBS was now considered to be a ‘previously ignored’ clinical presentation of child
abuse. Many retrospective studies using a group of known physical abuse cases were published
confirming the presence of SDH and RH along with axonal injuries which were detected by
silver stain on microscopic slides of the brain 15-20. From these studies, traumatic Diffuse Axonal
Injury (tDAI) was proposed as a histological correlate of the shaking injuries, and the swelling
of the brain originally described was then considered as a secondary response to such injuries.
The triad was now redefined to be 1. SDH, 2. RH and 3. tDAI. This had very significant legal
implications since infants with tDAI could not be expected to be alert for any period of time
after the initial injury (lucid interval). tDAI by definition involves disruption of the reticular
activating system that would render the infants immediately unconscious. As result, the finding
of tDAI at postmortem examination implies that the last caregiver who witnessed the infant
alive is the one to blame for the demise. The finding of tDAI was regarded as independent
9
pathologic evidence of shaking and many convictions were made without confessions, based on
the finding of tDAI at postmortem examination.
There are two major shortcomings of these retrospective studies which need to be
understood. First is the inherent fundamental limitation of retrospective studies. Retrospective
studies rely on the indirect correlation between the majority of findings present in a group of
cases and the criteria used to select such a group. It is a very powerful approach to defining a
clinical entity if the selection criteria have been designed to confer specificity. However, the
methods by which the findings were observed cannot be ascertained with this approach, and
even with well-devised selection criteria, it can only illustrate a strong correlation at best. As
mentioned previously, retrospective studies in the 1980s and 1990s did not have proper selection
criteria to correlate the shaking mechanism with the observed injuries. In the first group of
reports, the study criteria included all suspected abuse cases and simply attributed all cases
without other significant findings to shaking 21, 22. The other group of studies analyzed fatal
infant head trauma and attributed those without evidence of impact to the result of shaking 23, 24.
With such broad criteria, it is not inconceivable that the findings are heterogeneous and do very
little to isolate the possible mechanisms for the injuries present. Furthermore, both of these
approaches are inadequate in ascertaining the mechanism of injury, since the ‘evidence’ of
shaking does not derive from the findings themselves but rather from the assumption that these
injuries are caused by shaking. These studies should serve as the starting point in the search for
mechanisms responsible for injuries that cannot be explained by known mechanisms such as
impact to the head.
Another approach in medicine to define or refute a clinical entity is by the accumulation of
anecdotal reports. By examining cases that do not fit the existing paradigm, anecdotal case
reports may help in refining clinical entities. If enough anecdotal reports are accumulated, the
10
existing entity may cease to be accepted altogether and a newer entity could be defined. When
infant head injuries in the context of child abuse were beginning to be recognized as a separate
entity, numerous anecdotal reports of birth trauma presenting in suspicious circumstances were
also published. Once SBS was defined and the triad was being used as pathognomonic of SBS,
many anecdotal reports were soon published and formed the basis for the controversies which
are still being debated now. These anecdotal reports may be grouped into following categories;
1. Cases of infant head trauma caused by an impact-related mechanism presenting with the triad
but with no sign of impact 25, 2. Cases presenting with the triad of SBS where alternate causes
are known (such as disease conditions or resuscitation efforts) 26-38, or 3. Cases demonstrating
the potential misdiagnosis of the triad in other disease processes 39-44. Anecdotal case reports
constantly evaluate the validity of a medical entity where the mechanism is not known.
Unfortunately, similar to the process of elimination, it is an indirect approach and cannot be
used to directly confirm whether shaking could cause the triad.
11
1.4. Study of SBS
1.4.1. Animal models of SBS
The first animal models of inertial head injury were published in 1964 7. Ommaya
demonstrated an experimental concussion caused by occipital impact acceleration in monkeys.
The study presented a series of linear acceleration thresholds for experimental cerebral
concussion, and proposed that these curves could be used as a baseline for non-impacting
impulsive loads. Throughout the late 1960s and early 1970s, Ommaya et al. reported a series of
experiments where whiplash motion was modelled as a single episode of acceleration delivered
by a mechanical actuator 7, 9, 45-48. Gennarelli et al. 18 and others 16, 49-53 further characterized this
model. It showed that all putative markers of SBS (except retinal hemorrhages which were
unexamined) were produced by a single rotational acceleration to the head on the coronal plane
(side-to-side on lateral axis) to the head. It is interesting that the rotational acceleration on the
sagittal plane (which is commonly believed to be a type of acceleration an infants’ head would
experience in an abusive shaking event) only produced a concussion while the lateral rotational
acceleration produced more severe effects including SDH and coma. Histological methods using
silver impregnation correlated diffuse axonal injury with the acceleration injuries. It should be
noted that in 1981, skull fracture, SDH and hypoxic encephalopathy were described as part of
the main findings of acceleration injuries, whereas in an article published in 1982 using the
same model system, none of the animals had such findings. This set of studies has been cited as
the strongest scientific evidence of the pathogenic nature of shaking to date. However, such an
important discrepancy in the results from this model system has not been properly tested by
other researchers due to the difficulties in conducting primate experiments. Since 1998 a few
studies have used the swine model to reproduce Gennarelli’s primate experiments with variable
12
results 54-56. The need for a conclusive animal model is well recognized by all participants in the
SBS controversy 57-61.
1.4.2. Mechanical models of SBS
Most anthropomorphic simulations have been done using scale models of adults, and the data
has been extrapolated to give an injury threshold for infants. In 1987, Duhaime et al. published a
seminal study which showed that the acceleration injury threshold established by Gennarelli’s
primate experiments could not be reached by shaking an anthropomorphic model irrespective of
the range of neck characteristics used 62. On the other hand, impact of the head of the
anthropomorphic model generated accelerations that were in a similar range to the Gennarelli
primate model injury thresholds. In the same paper, postmortem examination findings of 13
fatal infant head injuries that were diagnosed as SBS were reviewed. All 13 cases had findings
that were suggestive of impacts, such as scalp bruising or a skull fracture. The findings of this
study were reaffirmed by another anthropomorphic study in 2003 63, where the authors
concluded that:
These findings suggest that inflicted impacts against hard surface may be more frequently associated with
clinically significant inertial brain injuries than vigorous shaking or falls from less than 1.5m. In addition, there are
no data showing that maximum change in angular velocity and peak angular acceleration of the head experienced
during shaking and inflicted impact against unencased foam is sufficient to cause SDHs or primary TAIs in an
infant.
The main criticism against such studies is the issue of scaling. The argument centers on the
premise that the mechanical properties of the infants are not a simple linear scale reduction to
that of adults. As discussed earlier, infants have very distinct anatomical and physiological
properties but there is very scant data available that demonstrates the contribution of such
distinct characteristics to altering the mechanical scaling. Because of this, most
13
anthropomorphic studies to date have employed an approach that included theoretical extremes
of a given mechanical property, resulting in data that could not be conclusively correlated to
human infant shaking events.
1.4.3. Evidence-based reviews of the retrospective case series
Another shortcoming of the retrospective studies from the 1980s and 1990s is not due to an
imperfect scientific basis but rather a technical one. In these studies, silver-based stains of brain
tissue were used in neuropathology to detect axonal lesions along with H&E to show other
changes within the brain. More recently, immunostaining of the brain for β-amyloid precursor
protein (βAPP) expression has been accepted as the new norm for brain injuries (Figure 1-1) 64-
68. βAPP is a neuronal protein with various native functions including synaptic formation and
repair 69. It is mostly known as the precursor form of the β-amyloid protein (transmembrane
domain of βAPP) that forms characteristic plaques in Alzheimer’s disease. βAPP is normally
transported from neuronal cell bodies to the synapses by axoplasmic flow 70. Disruption of the
axons produces an accumulation of βAPP, histologically characterized by an ‘axonal retraction
ball’ formation. It is also reported that βAPP expression increases after axonal injury 66. βAPP
immunostaining is much more specific than silver stains. In 2001, Geddes et al. published a
two-part retrospective study of 53 non-accidental head injuries in infants and children which
showed that there is no significant difference in pathology between the shaken only group and
the impact group (These papers are sometimes called Geddes 1 and 2) 71, 72. Also, βAPP
immunostaining of the cases confirmed only two cases of tDAI present in the shaking group. In
2003, vascular changes following hypoxic/ischemic encephalopathy were proposed as a possible
alternate causal mechanism to the ‘shaking triad’. In these studies, it was discovered that the
expression of βAPP in the brain is not limited to traumatically damaged axons but can also be
expressed by the axons in response to direct and/or indirect effects of hypoxia (This paper is
14
sometimes called Geddes 3) 73. Following Geddes’ paper, the evidentiary nature of the previous
retrospective reports was questioned, and more evidence-based reviews of the previously
reported SBS cases were published 59, 74, 75. In these reviews, at times more opinionated than
objective, the conclusions varied from “cause of death in the SBS victims was a global cerebral
ischemia induced by a multifactorial process” in one review 74 to “whiplash shaking without
impact is the cause of death of this subset of infant homicides” in another review 59. The
opinions varied greatly on most of the critical points of the SBS diagnosis and they are
discussed in the following section.
15
Figure 1-1. Photomicrographs of traumatic axonal injury in human brain. A. Numerous axonal retractions balls (brown) are seen. (100x magnification) B. High power view of the axonal retraction balls. Slight positive staining of the neuronal bodies is also seen. (400x). βAPP immunostaining using DAB method.
16
17
1.5. Current points of controversy
1.5.1. Pathogenesis of the triad - Pathologic nature of ‘pure shaking’
Currently there are two hypotheses which may account for the development of the triad after
shaking without an impact component (i.e. pure shaking). First is the traditional hypothesis
which focuses on the inertial load to the various anatomical interfaces from repeated
acceleration and deceleration. The second hypothesis attributes the triad to secondary
permeability changes due to hypoxia/ischemia that is caused by initial brainstem damage
(Figure 1-2) or another inciting cause of hypoxia. No direct experimental evidence that supports
either of these two hypotheses has been reported, while many retrospective studies have been
aimed at confirming one over the other.
1.5.1.1. Immediate traumatic injuries due to shearing and traction
In this hypothesis, the supposed fragility of the infant structures is key when they are exposed
to a great inertial loading through repeated acceleration/decelerations generated by shaking.
According to this hypothesis, the bridging veins which cross the dura-arachnoid interface would
break under the inertial load to cause SDH, while the traction generated by the vitreous fluid
would cause retinal hemorrhages. Also, undermyelinated infant axons are more susceptible to
damage under an inertial load. It is postulated that the injuries to the axon would be diffuse due
to the generalized inertial load. This hypothesis explains the frequent bilaterality of
hemorrhagic lesions in suspected SBS cases as well as the presence of the diffuse axonal
injuries.
The critical weakness of this hypothesis is that the injury threshold of any of the structures
involved in such a mechanism is not known, or studied in the context of each other. Only a few
mechanical parameters of infant tissue are available. None of the studies have measured such
18
parameters or considered the contribution from connected structures. This hypothesis cannot
account for the frequent absence of neck injuries in suspected SBS cases where some studies
have suggested that the injury threshold for neck or vertebral injuries is much lower than for
intracranial injuries.
1.5.1.2. Hypoxic ischemic encephalopathy due to brainstem injury
In this hypothesis, violent shaking of the infant head causes damage to the brainstem and
upper cervical spinal cord via stretching or hinge trauma (pinching) within a confined space. As
a result, apnea due to damage to the brainstem then triggers a hypoxic response. This in turn
leads to changes in vessel permeability that could allow leakage of blood within the dura, and
then into the subdural space or retina. This hypothesis was first suggested in 1999 76. Recent
retrospective studies using βAPP have shown that the axonal lesions that were previously
thought to be traumatic were hypoxic in nature 32, 73, 77.
This alternative hypothesis (also called the ‘unified hypothesis’) fits well with the typical
presenting history of supposed SBS infants who are usually found in a state of apnea or “vital
signs absent” by the caregiver. However, an exhaustive resuscitation effort usually follows such
a discovery, and hypoxic/ischemic encephalopathy is a frequent finding in post-resuscitation
infants. In such cases, the assessment of the relative contribution to injury by the traumatic
mechanism versus the hypoxic remains highly controversial 78, 79.
1.5.2. Specificity of the triad
Putting the pathogenic nature of pure shaking aside, the next biggest question is the
specificity of the triad. This question was brought to light by the accumulation of anecdotal
reports where mechanisms other than shaking alone resulted in the triad of findings. In addition,
a retrospective review of previously diagnosed SBS cases revealed that many such cases have
19
Figure 1-2. Comparison of traditional and unified hypothesis. Green arrows: Traditional hypothesis. Red arrows: Unified hypothesis. (Modified from Geddes 3, 2003)
20
21
some evidence of impact to the head that was originally undetected or described as a co-finding
of ‘shaken-impact syndrome’. Part of this controversy can be explained because SBS was first
defined in the context of general physical child abuse where an impact to the head was not an
exclusionary criterion. For any professional involved in the assessment of the triad, it is
important to remain aware of other known mechanisms that could result in the triad.
1.5.2.1. Short fall debate
In 2001, Plunkett published a study where 18 fatal head and neck injuries in infants and
children involving playground equipment were collected from the Consumer Product Safety
Commission database 34. Of these18 cases, 12 were witnessed by a non-caregiver to fall from
equipment at various heights between 0.6 and 3 metres. The results showed that SDH and RH
are not specific indicators of the shaking mechanism since 13 of 18 cases had SDH, whereas 4
of 6 cases where the retina was examined had RH. Also, 12 of 18 infants or children had a lucid
interval which indicated that the tDAI might occur at much higher forces than SDH.
The study faced an immediate backlash where many of the study’s limitations were pointed
out. Spivack argued that injuries from a contact force of a fall are fundamentally different than
injuries from inertial forces of shaking or impact following shaking 80. Levin argued that RH in
accidental falls is extremely rare as the author “had to search literally tens of thousands of
records” to identify the four cases 81. These two comments clearly demonstrated the fundamental
division in a field where some professionals would contest the facts, methodology or limitations
stated in an article and dismiss the findings of any study that did not conform to their paradigm.
In the author’s reply to these comments, Plunkett pointed out that the physics of a fall or ‘slam’
is identical and the number of cases examined for RH was 6 out of 18 cases where a
22
funduscopic retinal examination was performed. In his word of caution, Plunkett ended a letter
to the editor with the following comments 82:
I hope that my study encourages us to re-examine our concepts regarding traumatic brain injury (TBI) and the
relative importance of inertial or impulsive loading (whiplash) and contact. Dr. Caffey’s “theory,” accepted for
almost 30 years, taught in medical schools, approved as an ICDA-9 “codable disease” and testified to as “truth” in
court, is based on a misinterpretation of early pioneering experiments performed for the automotive and space
industry. Ommaya published a landmark study in 1968 showing that TBI could be produced in rhesus monkeys by
acceleration of the head alone (with the midneck as a fulcrum) and no contact. However, the level of acceleration
he used to cause these injuries was 10,000–100,000 r/s2, with the lower limit being the concussion threshold. (Ten
thousand r/s2 at a radius of 6 inches is 5,000 f/s2 or 156 G’s). Caffey called Ommaya after his (Caffey’s) 1972
article was published and discussed it with him. Ommaya told him that he (Caffey) was misinterpreting his
(Ommaya’s) studies, but Caffey either didn’t understand or forgot to tell us. This misinterpretation is repeated in
Caffey’s 1974 article. And here we are today. A WWII paratrooper aphorism concerning chute-deployment failure
says it best: “It is not the fall that kills you. It’s when you hit the ground.”
This undoubtedly led to more studies which either re-examined the injury database 83, 84 or
used anthropomorphic models to confirm the forces involved in short falls 63, 85, 86.
Unfortunately, a fundamental misunderstanding of Ommaya’s experimental results still forms
the basis of many of the articles published on SBS which have claimed to be authenticated by
the vast clinical experience of physicians.
1.5.2.2. Confounding medical conditions
There are many anecdotal clinical reports where at least one of the triad has been described in
other natural and more benign medical conditions. SDH has been described in a variety of
circumstances including birth trauma 39, 87, impact trauma that does not have any other external
findings, and as a complication of a shunting procedure for hydrocephalus 27. Although reports
are rare, cerebrovascular thrombosis has been described as a delayed injury presentation of head
23
and neck trauma 34, 88-91. Also, SDH has been described as a complication of non-traumatic
conditions such as glutaric aciduria type-1 33, 92, hemodialysis 93, rhinocerebral mucormycosis 30,
Menkes disease 35 and more. Also, the possibility of misinterpretation of subdural effusion or
mass as SDH has been well reported.
Some physicians now believe that RH may occur in any circumstance where the intracranial
pressure (ICP) is increased 94, 95. This also includes circumstances where venous outflow is
obstructed leading to secondary increase in ICP. Accidental head injuries resulting in increased
ICP could show RH and in rare circumstances, RH has been documented in cases where
resuscitation efforts involved chest compression 96.
1.5.3. Nature of subdural hemorrhages
Although SDH has been described for many types of cranial trauma, the very nature of the
subdural space is still not uniformly agreed upon. Initially believed to be a cavity between the
dura and arachnoid, newer ultrastructural studies showed that it is a loosely filled interface
between the dura and arachnoid that is prone to cavitation 97-99. Fluid channels within the dura
itself have also been demonstrated 100. Whether it is tearing of bridging veins or leakage of blood
after hypoxia, there are important questions regarding SDH that need to be addressed to
accurately correlate it to the chain of events that might cause this bleeding.
SDH can be diagnosed directly (at surgery or at postmortem examination) or indirectly (by
imaging). Once developed, an acute SDH can either be reabsorbed or persist to become a
chronic SDH. In classical medical teaching, acute SDH is described as a collection of freshly
clotted blood along the contour of the brain surface, without extension into the depths of sulci
101. Once the original bleeding is limited, SDH may organize in a sequence where the clot is
24
lysed followed by highly vascularised granulation tissue formation and fibroblast growth into
the hematoma. The SDH could then retract leaving a thin layer of reactive connective tissue 102.
The age of the SDH is highly important in legal proceedings where multiple, temporally
distint causes of SDH are involved. Since the development and resolution of SDH are both slow
processes, the aging of multiple SDHs of different ages can pose a great challenge. Hemosiderin
and fibrin detection methods can aid in determining the age of SDH 102, but the resolution of the
temporal sequence is still in the range of few days to weeks. Some physicians suggest that the
aging of SDH can also be complicated by a phenomenon called ‘re-bleeding’ 42, 103, 104. This
suggests that the vascularised granulation tissue is prone to bleed even with minor trauma that
would not normally result in SDH formation. It could then be mistaken for an acute SDH where
greater forces are thought to be required.
25
1.6. SBS in criminal justice system
SBS has been hotly debated in the criminal justice system in many countries since the
controversy surrounding its specificity has been raised. Since many SBS convictions were based
on the pathologic evidence (presence of the triad), the possibility of potential miscarriages of
justice triggered large scale reviews in Britain and Canada. The British review was conducted
between 2004 and 2006 while the review of SBS in Ontario, Canada is still in process as of June
2009. Below are the summaries of both reviews to date.
1.6.1. Harris appeal and SBS cases review in Britain
Following two contentious infant homicide cases, a large scale review of infant homicides
was conducted. Following this review, a group of 88 cases was identified as SBS and were
further reviewed by AG Lord Goldsmith 105. At the same time, the Court of Appeal heard four
cases involving SBS, the most famous of which was the case of Lorraine Harris. Harris was
accused of killing her four month old son by shaking and was later convicted and sentenced to
three years imprisonment. The baby had been found having difficulty breathing, and Harris
admitted shaking the baby in an attempt to help. A home visit was made by a general
practitioner who did not find RH or any signs of abuse. However, the baby was found vital signs
absent about an hour later and resuscitated by the emergency crew. Upon arrival at the hospital,
the baby was noted to have an extensive bilateral RH and later died in hospital.
The appeals court examined the extensive areas of controversy in the SBS debate. The court
heard from Dr. Geddes about the ‘unified hypothesis’, and examined the fine points of the
controversy in detail. All of the current topics were discussed including the degree of force
required to cause injuries such as SDH, short falls, and the significance of RH. In the end, the
court quashed the conviction of Harris on the ground that fresh evidence heard in the appeal
26
might reasonably have affected the jury’s decision to convict, therefore rendering the conviction
unsafe.
With regard to the triad and the degree of force required to cause the triad, the appeals court
stated that “in cases where the triad alone is present, triad alone cannot automatically or
necessarily lead to a conclusion that the infant has been shaken”. However, the court also stated
that the triad remains a strong pointer to SBS. The court also acknowledged that although the
force required to cause the triad in the vast majority of the cases is more than rough handling,
there are rare or very rare cases where such injuries could be caused by little force.
1.6.2. Goudge inquiry in Ontario
The Inquiry into Pediatric Forensic Pathology was established to conduct a systemic review
and assessment of pediatric forensic pathology in Ontario from 1981 to 2001. The inquiry was
in part triggered by a series of cases in which the possibility of wrongful conviction was raised
due to the errors made by a pathologist who conducted several postmortem examinations. Many
challenging cases of pathologic diagnosis in forensic settings were reviewed. Among his
recommendations, the commissioner noted that there is a “significant evolution in pediatric
forensic pathology relating to shaken baby syndrome and pediatric head injuries warrants a
review... because of the concern that there may have been convictions that should now be seen
as miscarriages of justice”. After the release of the commissioner’s report 1, a review was
announced of previously diagnosed SBS cases in the province of Ontario. This review is
currently in progress.
27
2. Experimental design
2.1. Hypothesis
a. High frequency, low amplitude angular acceleration of the postnatal mouse head can
produce the triad (SDH, RH and axonal injury) and be a model for SBS
b. Manual shaking of adult BGM can produce the triad and be a model for SBS
2.2. Research objective
2.2.1. Overall
As discussed above, the four main approaches to the fundamental question of whether
shaking can cause injuries are: retrospective review, anecdotal case report, biophysical modeling
and experimental animal models. Both retrospective review of cases and anecdotal case series
provide valuable input to the debate but cannot ultimately answer the question due to their
inherent limitations. Retrospective studies rely on the history of a putative shaking event to
make a causal connection between shaking and injuries. Indisputably, anecdotal case evidence
does support a linkage between the triad and historical evidence of shaking. However, recent
experimental approaches using anthropomorphic models have not provided corroborative data in
support of SBS. In fact, one major drawback to anthropomorphic models is the inability to
produce the triad, thus limiting its value in answering the fundamental question. A well-
designed animal model could describe the mechanical parameters while providing the
pathophysiological mechanisms of injuries. Past attempts at modeling SBS have produced
inconclusive data due to several factors. The models have failed to reproduce the precise
shaking motion of the head and have utilized impact-related methods such as impact-
28
acceleration 9, 106, 107 and fluid percussion 108-114. The aforementioned baboon-macaque model
also fails to meet this criterion as only a single large acceleration was applied to the animals.
In the first part of the study, experiments were designed to test the feasibility of using mouse
pups as a model of SBS. If feasible, the model will represented a convenient method to
scientifically address the controversies regarding the specificity and characteristics of shaking
injuries in human infants. In the second part of the study, a small number of adult BGM will be
manually shaken to experimentally test the effect of inertial forces on the head and neck of an
animal that is anatomically and physiologically similar to humans.
2.2.2. Specific aims
Specific aims of the experiments were
I. To determine if high frequency, low amplitude vibration of mouse pups under general
anesthesia can cause subdural hemorrhage, retinal hemorrhage and diffuse axonal
injury/hypoxic encephalopathy as are seen in human infant head injury without evidence of
impact.
II. To determine if manual shaking of Barbados green monkeys (Cercopithecus aethiops) under
general anesthesia can cause subdural hemorrhage, retinal hemorrhage and diffuse axonal
injury/hypoxic encephalopathy as are seen in human infant head injury without evidence of
impact.
and
III. To describe physical parameters such as frequency and acceleration of both anterior-
posterior and lateral manual shaking of BGMs.
29
2.3. Rationale
2.3.1. Advantages of animal model
Animals have a distinct advantage in modeling human diseases, as they are a true living
system where complex physiological responses to experimental variables can be measured.
Responses may be evaluated temporally (progression), which allows for further studies on how
the responses could be interrupted (treatment) or prevented. Once the experimental protocol
establishes in an animal a condition that resembles the human disease, this can be used as a
model to study various aspects of the human disease by adjusting different variables in a
controlled manner. However, a fundamental assumption underlies animal models of human
disease: that the differences between the human and the animal can be ignored. Also, another
common assumption in animal models of human injury is that the mechanism of injury itself is
irrelevant as long as the presentation of the injury in the animal is similar to the human injury. In
establishing animal models for SBS, the potential effect of these common assumptions must be
carefully evaluated, as the mechanism of injury itself is in question. Species differences in
craniocervical anatomy result in vastly different ranges of head and neck movements in
response to external forces. As well, most currently available animal models of head injury
could not be used to study SBS because the mechanisms of injury infliction in these models
include an impact component.
2.3.2. Indication from previous studies
There is very limited literature available on animal models of SBS. In rodent models used by
Bonnier et al., linear shaking of postnatal pups reportedly gave rise to some RH and focal white
matter lesions including some atrophy, although the changes were not clear from the
photomicrographs published 115, 116. If these results could be replicated, it would represent a
30
convenient model to test the effect of inertial force injuries in a large sampling group. However,
this model did use mechanical means (linear rotating shaker) rather than manual shaking to
generate the inertial force, which is not a true representation of SBS where the force is applied
manually.
On the other hand, the baboon-macaque model of Gennarelli 16, 49, 52, 53 showed that either an
indirect impact angular acceleration or a single pneumatic rotational acceleration of a primate’s
head could cause SDH, concussion and axonal injuries. The experiments showed that the lateral
rotational acceleration of the head (side-to-side acceleration/deceleration of the head) with the
neck being the fulcrum caused more damage than the anterior-posterior rotational acceleration
(front-to-back acceleration/deceleration of the head). This primate model holds an advantage
over the rodent model, as the animals used are more anatomically similar to humans. The larger
size of the animals also permits some direct comparison of the shaking parameters without
scaling of the data.
Anthropomorphic studies also generated some very valuable data that were considered in
designing my experiments. First, anthropomorphic studies showed that the maximum
acceleration that could be generated manually is around 12 G which is well below the injury
threshold reported in Gennarelli’s model 62, 63. Secondly, they demonstrated that the key
difference between shaking and impact was the duration of the peak force applied to the head.
The difference in acceleration between shaking and impact was explained almost exclusively by
the difference in the peak force duration.
2.3.3. American Academy of Pediatrics technical report (2001)
In this technical report regarding SBS 2, the AAP states that a shaking event that generates
SBS ‘must be so violent that individuals observing it would recognize it as dangerous and likely
31
to kill the child’. This also sets the guideline for what is considered to be shaking in a modeling
system. Any model of SBS should address all aspects of this statement. For the purpose of
establishing an animal model for SBS, the AAP statement could be rewritten as ‘the model for
SBS should involve manual shaking that applies repeated oscillation with great enough force
that onlookers would see it as dangerous and likely to kill the animal’.
32
2.4. Material and methods
2.4.1. High frequency vibration of postnatal mice
2.4.1.1. Mice
CD-1 mice are outbred laboratory mice that are frequently used in experiments where no
specific genetic characteristics are required. General weight and size for male and female adults
are 25 to 30 g and 20 to 25 g respectively. Mature female CD-1 mice give birth to a litter of 8 to
12 pups after 21 days of gestation. Postnatal day 8 pups are considered to be developmentally
similar to full term human newborn infants 117. Pups open their eyes and start foraging for food
around postnatal day 15 and can be weaned by day 21. In this experiment, eight untimed
pregnant mice were purchased from Charles River laboratory and housed at the Division of
Comparative Medicine at University of Toronto. Animals had free access to food and water, and
nest material was provided. The birthdates of the pups were recorded by monitoring the cage
each morning.
2.4.1.2. Shaking apparatus
A standard analog vortex mixer capable of speeds up to 3000 rpm was used as shaking
apparatus in this experiment (Fisher Scientific, Catalog #02-215-365). Cotton fitted 50 ml
conical bottom tubes were used as a restraint that permitted a sitting position of the pups while
free head movement was allowed (Figure 2-1). In the article by Bonnier et al. 115, a horizontally
rotating shaker was used to ‘shake’ mice at 900 rpm in a way that did not limit head movement
or cause chest compression. However, the precise information regarding the apparatus and
parameters used in the Bonnier experiments could not be obtained and thus, my experiments
should not be considered as replication of the Bonnier study.
33
Figure 2-1. Experimental setup of high frequency vibration of postnatal mice. A. Vortex mixer with cotton-fitted tube on top. B. Cotton-fitted tube with P15 mouse in situ.
34
35
2.4.1.3. Displacement measurement
The vibration frequency, displacement of the head of the analog vortex mixer and top of the
tube were measured using a laser-optical electronic system at one-thousandth second intervals
(Micro-Epsilon, Model #. optoNCDT 1401). Data output was then saved to a comma-separated
values file format and calculations were made using a Microsoft Excel program.
2.4.1.4. Anesthesia and euthanasia
Animals were anesthetized prior to shaking by the inhalation of vapourized Isoflurane (5%
v/v) until no reflex retraction was observed upon pinching of a distal hind limb. In cases where
animals were observed to regain consciousness during the procedure, shaking was immediately
terminated and more isoflurane was promptly given. All animals were euthanized by overdose
of Isoflurane. Once euthanized, all animals were immediately submerged in 10% buffered
formalin for fixation. For larger animals of advanced age (postnatal day 21 (P21) and up), an
incision was made in the abdomen to facilitate fixation.
2.4.1.5. Tissue processing and histology
After fixation, serial whole mount sections of the head and neck were prepared by overnight
decalcification in 10% formic acid followed by coronal serial sections in five millimetre slices.
Sections were then processed for paraffin embedding in a Shandon Excelsior tissue processor™
(Thermo Electron Corporation). The overnight processing cycle started with two 30 minute
changes of 10% buffered formalin, then six one hour changes of alcohol in sucessively higher
concentrations (starting at 60%, last two changes in absolute ethanol) to dehydrate. Three one
hour changes of Clearene® were followed by three 80 minute immersions in 61°C paraffin.
Tissues were left immersed in 61°C paraffin until embedding.
36
Blocks were cooled to room temperature and sectioned at 5 μm, floated on a warm water
bath, mounted on glass slides coated with egg white albumin to prevent loss during staining, and
baked at 60 degrees overnight. Both eyes were included in the coronal head sections and
examined histologically (Figure 2-2). Slides were then stained with routine hematoxylin-eosin
(H&E) (Appendix A-1).
37
Figure 2-2. Whole mount sections (5 μm thickness) of mouse head and neck regions. Left-to-right: Eyes to cervical spine. H&E stain.
38
39
2.4.2. Manual shaking of Barbados green monkeys
2.4.2.1. Barbados green monkeys
Although Barbados green monkeys (BGM, C.aethiops) are not native to the Caribbean island
of Barbados, they were brought to the island as pets on slavery ships during the 17th century.
BGM have since naturalized and are considered an agricultural pest with an established wild
population of over 14,000. C.aethiops, old-world monkeys that are phylogenetically closely
related to vervets, have been extensively used for medical research including polio vaccine
production. The Barbados Primate Research Centre (BPRC) was originally established to
control the wild population. It is an accredited primate research facility with veterinary and
technical support. In this study, four male adult BGM were initially screened and medical
records of each animal were retrieved. Three animals were then chosen based on their health
status and weights: B4830 for sham treatment, B0098 for anterior-posterior (AP) shaking, and
B2038 for lateral (Lat) shaking (Table 2-1).
Table 2-1. Selection of BGM for the study
Cage Number Animal ID Weight* (kg) Height** (mm)
2A1 B2038 5.90 469.9
2A3 B4830 5.50 508.0
2A6 B0098 5.68 482.6
* Weight of the animals at initial health screening. Differ slightly from the weight recorded at the time of experiment.
** Height is measured from crown-to-rump.
40
The weights of the animals resemble the 50th percentile of two month old human infants. A
day before the experiments, vital signs of the animals were recorded and the head, wrist and
ankle of the animals were shaved under ketamine sedation to facilitate mounting of
accelerometers and the placement of an intravenous (IV) catheter (Figure 2-3) by the facility
veterinarian.
2.4.2.2. Accelerometer
In the previous anthropomorphic experiments, acceleration of the manual shaking was
reported to be in the range between 8 to 9 Gs with the maximum reaching 12 G 63. Two triaxial
accelerometers were used in this study to measure acceleration during shaking (Microstrain Inc.
Model# G-Link-10G). These accelerometers record up to 10 G and designed to withstand up to
the maximum of 500 G. Thus, the integrity of the measurements is preserved even after peaks
over the maximum recording range. The maximum range of these accelerometers was slightly
below the maximum peak acceleration reported in the anthropomorphic studies. However, these
were the only directly mountable wireless accelerometers available (small form factor, wireless)
on the market. The accelerometers were directly mounted onto the top of the animal’s head and
the wrist using medical tape and gauze (Figure 2-4). Data output was originally recorded by
proprietary software from the manufacturer, then exported as a comma-separated values file
format. The calculations and accelerometry traces were made using a Microsoft Excel program.
2.4.2.3. Anesthesia and euthanasia
Animals were given an intramuscular injection of ketamine by the facility veterinarian while
their movements were restricted using a pull out mechanism of the cage. After a complete loss
of motor control was observed, the animal was transferred to the surgical suite where vital signs
were recorded. An IV catheter was placed in the great saphenous vein and a valium/ketamine
41
cocktail was administered intramuscularly as an anesthetic agent. The state of anesthesia was
confirmed by examining jaw tone. Anesthetic agent was administered intramuscularly as needed
throughout the procedure (Table 2-2). The animals were euthanized by injection of pentobarbital
through the IV catheter at the end of the experiment by the facility veterinarian.
Table 2-2. Anesthesia and euthanasia records of BGM
Animal ID Drug Dose Route Time
Sham
Ketamine 0.6 ml IM 9:15 AM
Ketamine/Valium 2.0 ml IM 9:28 AM
Ketamine/Valium 0.5 ml IM 9:38 AM
Ketamine/Valium 1.0 ml IM 9:53 AM
Pentobarbital Sodium 2.5 ml IV 10:56 AM
AP
Ketamine 0.6 ml IM 1:56 PM
Ketamine/Valium 2.5 ml IM 2:02 PM
Ketamine/Valium 1.0 ml IM 2:42 PM
Ketamine/Valium 1.5 ml IM 3:24 PM
Ketamine/Valium 1.0 ml IM 4:12 PM
Pentobarbital Sodium 2.5 ml IV 4:29 PM
Lat
Ketamine 0.6 ml IM 8:45 AM
Ketamine/Valium 3.0 ml IM 9:03 AM
Ketamine/Valium 1.0 ml IM 9:53 AM
Ketamine/Valium 1.0 ml IM 10:51 AM
Ketamine/Valium 0.6 ml IM 11:03 AM
Pentobarbital Sodium 2.5 ml IV 11:18 AM
42
Figure 2-3. Preanesthetized BGM with IV catheter placement. Animal was sedated with IM injection of ketamine in cage. Once sedated, head, wrist and ankle were shaved to facilitate the mounting of the accelerometers and IV catheter placement.
43
44
Figure 2-4. Mounting of accelerometer on BGM. Wireless accelerometer unit was secured onto BGM head by gauze and medical tapes. Tapes were carefully placed to avoid limiting jaw movement.
45
46
2.4.2.4. Postmortem examination
A postmortem examination was performed on all animals. External photographs were taken
at the beginning of each postmortem examination to document any visible injuries, and gross
dissections and findings were photographed throughout. A layered dissection of the posterior
neck was performed to detect neck injuries. The brain was removed in continuity with the
cervical spinal cord using a posterior approach. The eyes were removed in continuity with the
retrobulbar optic nerves after orbital roof removal. The eyes, brain, spinal cord and clivus were
fixed in 10% buffered formalin before being transported to the University of Toronto in a semi-
wet state according to international regulations on shipping of biological specimens (not
immersed in formalin but wrapped in damp cloths in a liquid tight container).
2.4.2.5. Tissue processing and histology
The brain and the spinal cord were serially sectioned in the coronal plane. All regions of the
brain and cord were sampled, with larger sections bisected or trisected to fit within blocking
cassettes. The globes of the eyes were bisected to reveal the retina, floated on 10% buffered
formalin and photographed. Eyes were embedded in toto in a direction where the longitudinal
cross section profile of the eyes (semi-disk with optic nerve entry in middle sections) could be
seen. The clivus was decalcified in 1% formic acid solution for one week before serial
sectioning along the parasagittal plane. The sections were then processed for paraffin
embedding in the manner described above. Blocks were sectioned at 5 μm, floated on a warm
water bath, mounted on glass slides coated with egg white albumin to prevent loss during
staining, and baked at 60 degrees overnight. Mollifex® solution was used for on spot
decalcification of the clivus blocks. All slides were histologically examined after routine H&E
staining and all neural tissues were also examined with Luxol fast blue (LFB)/H&E staining.
47
2.4.2.6. Immunohistochemistry
Formalin fixed, paraffin embedded brain and spinal cord tissues were sectioned 5μm and
mounted on Fisher FrostedPlus© slides. Sections were deparaffinized in two five minute
changes of xylene, rehydrated in a series of absolute and a successively lower concentration of
ethanol (two three minute changes in absolute alcohol, 3 minutes in 90% alcohol, 2 minutes
each in 70% and 50% alcohol), then brought to distilled water. Slides were submerged
completely in a beaker containing citrate buffer adjusted to pH 6.0 and boiled for 10 minutes for
the antigen retrieval. The beaker was then removed from the heat and allowed to cool on the
benchtop for 20 minutes. Sections were further cooled by washing in a change of phosphate
buffered saline (PBS) on a shaker for three minutes. Endogenous peroxidase activity was
blocked by treatment with 0.3% methanolic peroxide (0.5 ml of 30% H2O2 in 50ml methanol)
immersion for 20 minutes and washed with distilled water for three minutes on a shaker. Slides
were then rinsed in two three minute changes of PBS (pH 8.0). Excess buffer was removed by
careful blotting and a circle was drawn around the section with a hydrophobic pen to localize the
antibody solutions. Non-specific antibody binding sites were blocked by incubating sections
with 250 μl of normal goat serum (Jackson #005-000-121, 1/20, 15 minutes) in a wet chamber.
The block solution was then rinsed away by careful blotting with a Kimwipe and the section
incubated for 2 hrs at a room temperature with 250 μl per slide of diluted antibody (Zymed #13-
0200, 1/180). After primary antibody incubation, slides were washed three times in PBS (3 mins
each on shaker). Antibody binding was detected using a biotin-streptavidin detection system
(Biotinylated Goat-antiMouse, Jackson #115-135-003, 1/850, 30 minutes; Strepavidin
conjugated with horseradish peroxidase, Jackson #016-030-084, 1/650, 30 minutes) with 3,3’-
diaminobenzidine as the chromogenic substrate.
48
2.5. Experimental protocol
2.5.1. Mice
2.5.1.1. Constant vibration
In this study, two groups of mouse pups at different postnatal ages (P8 and P15, n=20 and 19
respectively) were subjected to varying degrees of shaking under anesthesia to examine the
effect of the shaking frequency. Standard sterile technique was used to handle the mice and a
circulating warm water blanket was used to help maintain body temperature. Mice were
anesthetized, then their weights were recorded using a top-loading balance. An identification
number was marked on their backs using a permanent marker. Mice were then placed in a
cotton-fitted 50ml conical bottom tube in a sitting position and the chest movements were
restricted by the careful placement of cotton balls. Mice were then shaken using an analog
vortex mixer for 30 seconds at various speeds between 1000 rpm to 3000 rpm facing forward.
Animals were then removed from the tube and allowed to regain consciousness before being
returned to the cage. Two control animals of the same age were subjected to a sham treatment
where they were anesthetized and returned to the cage after regaining consciousness. Mice were
immersed in soiled bedding before being returned to the cage to reduce the chance of maternal
rejection. Any animals that died during the procedure were fixed in 10% buffered formalin
immediately. All surviving animals and the control animals were then euthanized by an
isoflurane inhalation overdose 48 hours after the procedure. Once euthanized, all animals were
immediately submerged in 10% buffered formalin for fixation. The same set of experiments was
repeated at a 90° clockwise rotated position (n=15 at each time point) to examine the effect of
the rotational plane.
49
2.5.1.2. Pulse acceleration
In this experiment, a single angular rotational acceleration experiment as described in the
literature (baboon-macaque model of SBS) was combined with vibrational shaking. This
modification was designed to capture the shaking motion more accurately by allowing multiple
angular accelerations to be applied to the brain. Anesthetised pups were divided into three
groups by age (P8, P15 and P21, n=5 for each time point) and then subjected to pulsating
vibrations (continuous pulsation between 20 to 50 Hz, one minute in total) using the shaking
apparatus described above. Two control animals of the same age were subjected to a sham
treatment where they were anesthetized and returned to the cage after regaining consciousness.
The surviving and the control pups were euthanized 48 hours after the procedure. For P21 mice,
an incision was made in the abdomen after euthanasia to facilitate satisfactory fixation. The
same set of experiments was repeated with the position rotated 90° clockwise (n=5, 5 and 4
respectively) to examine the effect of the rotational plane.
2.5.1.3. Hinge-point
In this experiment, both constant vibration and pulse acceleration experiments were repeated
with modification of restraint to change the location of the hinge-point of the head movement.
P22 pups (n=11) were restrained in a manner that allowed no head movement, simulating a
shaking event where a baby is grabbed by the head. This was achieved by allowing the mice to
sit deeper into the tube with the neck extended and carefully placing cotton balls to restrict
movements within the tube. Two control animals of the same age were subjected to a sham
treatment where they were anesthetized and returned to the cage after regaining consciousness.
50
2.5.1.4. Displacement measurement
Direct measurement of mouse head acceleration could not be made as continuous contact of
the laser beam with the mouse head could not be established during the vibration. Instead, the
displacement measurements were made by aligning a laser beam with the top of the conical
bottom tube. The rotational acceleration was then calculated by approximating the movement of
the vortex head as a circular motion (acircular = 4π2rf2, where acircular is uniform circular
acceleration, r is radius of the orbit and f is frequency of the circular motion).
51
2.5.2. Manual shaking of Barbados green monkeys
2.5.2.1. Sham (Negative control)
The animal was preanesthetized using a ketamine injection and the state of general anesthesia
was induced with an intramuscular injection of valium/ketamine cocktail. Once anesthesia was
confirmed, the initial vital signs and morphometric parameters were recorded. Small, wireless
accelerometers were mounted directly onto the forehead and wrist using gauze and medical
tapes. The animal was firmly held by the chest just below the scapulae in the back and below the
ribcage in the front with the surrounding skin pulled tight (Figure 2-5). The animal was gently
moved in a rocking motion for a minute to simulate parental cradling of an infant. The rocking
motion was first made in AP axis and repeated in lateral axis (sagittal and coronal plane,
respectively). General anesthesia was maintained for an hour after rocking with monitoring of
vital signs. The animal was the euthanized by an intravenous pentobarbital injection.
Funduscopic examinations were also made during the survival interval. The animal underwent
postmortem examination as described above.
2.5.2.2. Anterior-posterior shaking
This animal was used to establish a baseline for the AP shaking (front-to-back,
acceleration/deceleration of the head on sagittal plane) which is commonly believed to be the
type of shaking involved in SBS. After induction of anesthesia and mounting of the
accelerometers, the animal was firmly held by the chest in the manner described above and
manually shaken in an anterior-posterior fashion for a minute continuously at the maximum
speed, producing maximum head displacement at each flexion and extension. General
anesthesia was maintained for an hour while vital signs were continuously monitored.
Funduscopic examinations were also made during the survival interval. The animal was then
52
euthanized by intravenous injection of pentobarbital. The animal underwent postmortem
examination.
2.5.2.3. Lateral shaking
The same procedure as the AP shaking described above was repeated on the third animal
where the only difference was that the animal was shaken in the lateral axis (side-to-side,
acceleration/deceleration of the head on coronal plane). Lateral shaking produced more damage
in the Gennarelli baboon-macaque model when compared to AP shaking.
53
Figure 2-5. Experimental setup of manual shaking of BGM. The animal is held at chest level with scaled background.
54
55
2.6. Ethics of animal use
2.6.1. Mice
The experimental protocol was approved by University of Toronto Local Animal Care
Committee (LACC, Protocol #2000-6688). All standards set out in the Care and Use of
Experimental Animals guideline by the Canadian Council on Animal Care were followed. A
facility veterinarian was available for consultation throughout the experiment.
2.6.2. Barbados green monkeys
Application for animal use was made to the University of Toronto LACC, which was
externally reviewed on the applicant’s request (Appendix B-1). Revisions were made along with
the submission of the response to the reviewers (Appendix B-2). The protocol was approved by
LACC in a revised form (Protocol #2000-7466). The protocol was then reviewed by the
institutional animal care and use committee at BPRC and a joint experimental protocol (Protocol
#281008A) was established. The facility veterinarian was in attendance throughout the
experiments to assist with all animal handling.
It is understood that the use of primates in medical research is very contentious. All
alternative means of testing should be considered. It is further accepted that the ethics of animal
rights are a legitimate and important barrier to the use of primates in many experimental studies.
However, the application was made in the belief that the potential knowledge gained from the
proposed experiment outweighs the ethical arguments against it.
56
3. Results: Mice
3.1. Summary of findings
By using several different experimental settings for shaking the heads of mice, we have been
unable to produce meningeal, brain or spinal cord injuries of any type. Mouse pups were
subjected to a high frequency vibration (20 to 50 Hz) under anesthesia while restrained in a
manner that minimized chest compression but allowed free head movement. In both continuous
high frequency vibrational shaking and pulse acceleration of the head, no central nervous system
injury was detected in the whole mount section of the head (Figure 3-1). In contrast to previous
reports, change in rotational plane did not result in more severe trauma. When attempts were
made to deliver vibration directly to the head by restricting both head and neck movement, no
injuries were seen.
3.2. Constant vibration study
Two groups of mouse pups at different postnatal ages (P8 and P15, n=20 and 19 respectively)
were subjected to shaking for 30 seconds under anesthesia at a constant frequency. When
subjected to 20 to 50 Hz of continuous high frequency vibration, neither group of mouse pups
showed any signs of injuries. No SDH, RH, hypoxic injuries or evidence of traumatic axonal
injury were seen with routine microscopic examination. The same set of experiments was
repeated at a 90° clockwise rotated position (N=15 for each time point) and also produced no
injuries. There were three intra-procedural deaths. One pup had a focal microscopic
subarachnoid hemorrhage (Figure 3-2) without SDH, RH or brain swelling. No abnormalities
were found in the other two animals. No signs of functional neurological damage was seen
immediately after the shaking as all animals that survived the shaking procedure had locomotion
57
comparable to the control animals. No maternal rejection was seen and no sign of
developmental delay or malnutrition was observed.
3.3. Pulse acceleration study
Three groups of anesthetized pups (P8, P15 and P21, n=5 for each time point) were subjected
to continuous pulsating vibrations between 20 to 50 Hz for 1 minute using the shaking apparatus
and repeated five times. The same set of experiments was repeated at a 90° clockwise rotated
position (n=5, 5 and 4 respectively) to examine the effect of the rotational plane. No SDH, RH,
hypoxic injuries or evidence of traumatic axonal injury were seen on routine microscopic
examination. There were 10 intra-procedural deaths in which no injuries were found upon
histological examination. No signs of functional neurological damage were seen immediately
after the shaking as all animals that survived the shaking procedure had locomotion comparable
to the control animals. No maternal rejection was seen and no sign of developmental delay or
malnutrition was observed.
3.4. Hinge-point study
The P22 pups (n=11) were shaken while restrained in a manner that restricted the head and
neck movements. Histological examination revealed no evidence of injuries or hypoxia.
However, this procedure resulted in a higher mortality rate than the previous procedures as 5 out
of 11 pups were killed during the vibration. This is likely due to the higher level of chest
compression needed to restrain the pups during the procedure. No signs of functional
neurological damage were seen immediately after the shaking as all animals that survived the
shaking procedure had locomotion comparable to the control animals. No maternal rejection
was seen and no sign of developmental delay or malnutrition was observed.
58
Figure 3-1. Photomicrographs of postnatal mouse. A. Meninges with no SDH (50x). B. Retina showing no hemorrhages (200x). C. cerebral white matter with no axonal damages (400x). H&E stain.
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Figure 3-2. Photomicrograph of a local SAH found in intra-operative death (200x). Hemorrhage was limited to subarachnoid space and thought to be non-lethal. H&E stain.
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3.5. Vibration frequency and displacement
Vibrational frequency closely matched the frequency range provided by the manufacturer
(25-50Hz). There was a general agreement between the experimental acceleration calculated
from the displacement data (~20 G at maximum) and the circular approximated theoretical
acceleration (6-25 G). Figure 3-3 shows an initial one second plot of displacement measured
with the vortex machine dial set on 6. The vibration frequency measured was 40Hz, with
consistent amplitude of 5 millimeters.
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Figure 3-3. Displacement over time plot of the shaking apparatus
64
-2.0
-1.0
0.0
1.0
2.0
3.0
4.0
5.0
Displacement (millimeters)
Tim
e (s
econ
ds)
Dis
pla
cem
ent
of
vort
exer
hea
d o
ver
tim
e
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4. Results: Monkey
4.1. Summary of findings
Overall, violent, manual shaking in a manner that conforms to the American Academy of
Pediatrics description of the shaking event in SBS did not cause any immediately hemorrhagic
lesions in eyes, dura, cerebrum, brainstem or spinal cord of the animals. Also, there were no
injuries to the posterior neck musculature or the clivus. Fingertip bruises were noted on the torso
of both animals that were subjected to shaking. The animals used in this study were comparable
in weight to 2-month-old human infants (Table 4-1).
Table 4-1. BGM morphometric dimensions at time of autopsy
Animal ID
Head circumference*
(mm)
Neck circumference
(mm)
Chest circumference**
(mm)
Shoulder to elbow
(mm)
Elbow to
wrist (mm)
Hip to
knee (mm)
Knee to
ankle (mm)
Sham 280 225 340 170 140 170 170
AP 270 180 350 170 160 170 170
Lat 285 210 340 140 120 170 170
*Head circumference measured over supraorbital ridge-ear-occiput after shaving of the head.
**Chest circumference measured directly over nipples with the fur firmly pressed against the skin.
Anterior-posterior shaking lasted 43 seconds with 125 shakes while lateral shaking lasted 53
seconds with 178 shakes. The maximum frequency reached for AP shaking was 3.2 Hz with an
average frequency of 2.9 Hz. The maximum frequency reached for Lat shaking was 3.7 Hz with
an average frequency of 3.4 Hz. Both types of shaking achieved full extension/flexion and
saturated the accelerometer recordings along the axis of the shaking motion (Figure 4-1). The
video recording clearly showed that the movement of head during lateral shaking was not
limited to the coronal plane. The head followed ‘figure 8’ pattern in its motion where the neck
66
was repeatedly hyperextended or hyperflexed on top of being extended in the coronal plane
(Figure 4-2). The accelerometer data confirmed this secondary movement axis of the lateral
shakes as the acceleration peaks on the sagittal (AP) axis also reached the recording range limit
of the accelerometer (~10 G). Incidental findings at postmortem examination include an old
amputated digit and a 7 cm retroauricular, suboccipital scalp scar on the sham animal. Also, a
shaving abrasion of 3 cm in diameter was found on the suboccipital scalp of both the sham and
Lat animal. The Lat animal had an old scar on the left knee.
4.2. Physiological responses after shaking
Heart rate and temperature were monitored throughout the experiment including one hour
survival interval after shaking under general anesthesia. A slight drop in body temperature was
observed in all animals including the sham animal. However, it should be noted that the room
temperature was maintained at 18°C during the procedure and it is unclear if the slight
hypothermia was due to the procedure (anesthesia and/or shaking) or the environment. There
was a slight elevation in the heart rate immediately after shaking for both the AP and Lat
animals. Breathing was also shallower immediately after shaking, but both the heart rate and
breathing rate was within the normal range and returned to the baseline within few minutes.
At 38 minutes after shaking, a slight twitching of the left ear was observed in the AP animal
along with fasciculation of the left masseter muscle. A loud snoring was also observed with a
small amount of clear, foamy discharge from nose and mouth. The twitching and snoring
continued for approximately one minute, then disappeared. A slight shivering was observed at
43 minutes after shaking. No RH was seen upon funduscopic examination at any time point
during the survival interval although the examination became difficult due to the presence of
tears.
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The Lat animal had a similar course during the survival interval. Shivering was observed at
26 minutes after shaking with non-specific movements at 33 minutes after shaking. These
movements lasted about one minute, then disappeared. Possible deviation of the eyes was seen
at 41 minutes after shaking and snoring was observed at 42 minutes after shaking.
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Figure 4-1. Still frames from AP animal manual shaking video. Frames show various head and neck positions at A. full extension to B. full flexion. Atypical extension was also observed (C) where head was seen slightly rotated to the left.
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Figure 4-2. Still frames from Lat animal manual shaking video. Head and neck positions during lateral shaking showed much more complex movement where the exception of E, all head positions had a component of sagittal extension/flexion, rotation and lateral flexion.
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4.3. Anatomic and histological findings
4.3.1 Brain and cervical spinal cord
At postmortem examination, no scalp contusions or subgaleal or meningeal hemorrhages
were seen in any of the shaken animals. No SDH, subarachnoid hemorrhage or extradural
hemorrhage were seen on the surface of the exposed spinal cord (Figure 4-3 to 4-5). After
fixation in 10% buffered formalin, horizontal sections of brainstems and coronal sections of
brains were made and were free of visible macroscopic hemorrhages (Figure 4-6 to 4-11).
Histologically, the brain and the spinal cord were examined with both H&E and LFB/H&E
stains (Figure 4-12 to 4-16). No microscopic SDH or subarachnoid hemorrhages were seen in
any of the animals. As expected in highly mobile animals, axons of the corticospinal tracts in all
animals including the sham animal appear to be enlarged compared to that of the humans. βAPP
immunostaining revealed no visible accumulation of βAPP in axons in all of the sections
examined. Some weak neuronal staining of βAPP was seen in all sections. Sections examined
with βAPP immunostaining from each animal were: upper cervical spinal cord, pons, medulla,
superior cerebral hemisphere at the level of anterior commisure, and posterior corpus callosum.
A known βAPP positive human brain tissue sample was used as a positive control for the
immunostaining procedure (Figure 1-1), while incubation with PBS buffer solution without the
primary antibody was used as a negative control for each animal. Minimal non-specific
background staining was seen in all sections (Figure 4-12, inlay).
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Figure 4-3. Brain and cervical spinal cord of Sham animal. A. Posterior aspects of brain, cerebellum and cervical spinal cord after removal of spinous processes. Dura has been reflected to expose the spinal cord. B. Brain and cervical spinal cord after fixation. Superior view. C. Brain and cervical spinal cord after fixation. Inferior view.
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Figure 4-4. Brain and cervical spinal cord of AP animal. A. Posterior aspects of brain, cerebellum and cervical spinal cord after removal of spinous processes. Dura has been reflected to expose the spinal cord. B. Brain and cervical spinal cord after fixation. Superior view. C Brain and cervical spinal cord after fixation. Inferior view.
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Figure 4-5. Brain and cervical spinal cord of Lat animal. A. Posterior aspects of brain, cerebellum and cervical spinal cord after removal of spinous processes. Dura has been reflected to expose the spinal cord. B. Brain and cervical spinal cord after fixation. Superior view. C Brain and cervical spinal cord after fixation Inferior view.
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Figure 4-6. Coronal sections of sham brain after formalin fixation.
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Figure 4-7. Horizontal sections of sham brainstem after formalin fixation.
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83
Figure 4-8. Coronal sections of AP brain after formalin fixation.
84
85
Figure 4-9. Horizontal sections of AP brainstem after formalin fixation.
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Figure 4-10. Coronal sections of Lat brain after formalin fixation.
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89
Figure 4-11. Horizontal sections of Lat brainstem after formalin fixation.
90
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Figure 4-12. Photomicrographs of cervical spinal cord of BGM. A. to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. Inlay is negative control of immunostaining procedure where the section was incubated with PBS buffer without βAPP antibody. D. to F. AP animal. G. to I. Lat Animal. (All 400x.). No hemorrhagic or axonal injuries were seen.
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Figure 4-13. Photomicrographs of pons. A. to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. D. to F. AP animal. G. to I. Lat Animal. (All 400x.). No hemorrhagic or axonal injuries were seen.
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95
Figure 4-14. Photomicrographs of medulla. A to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. D to F. AP animal. G to I. Lat Animal. (All 400x.). No hemorrhagic or axonal injuries were seen.
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Figure 4-15. Photomicrographs of frontal cerebral cortex. A to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. D to F. AP animal. G to I. Lat Animal. (All 200x.). No hemorrhagic or axonal injuries were seen.
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99
Figure 4-16.Photomicrographs of posterior corpus callosum. A to C. Sham animal. H&E stain, LFB/H&E stain, and βAPP immuno stain, respectively. D to F. AP animal. G to I. Lat Animal. (All 400x). No hemorrhagic or axonal injuries were seen.
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4.3.2. Eyes and retina
No RH or optic nerve sheath hemorrhages were seen after the removal of the orbital roof and
enucleation. Globe of the eyes were bisected to reveal retina, and the examination while floating
in 10% buffered formalin revealed no RH (Figure 4-17).
Histologically, no microscopic RH or perioptic nerve sheath hemorrhages were seen in any
of the sections examined using the H&E stain (Figure 4-18).
4.3.3. Neck
Layered posterior neck dissection revealed no gross structural damage (Figure 4-19). Serial,
parasagittal sections of clivus from all three animals showed no hemorrhages or structural
damage upon microscopic examination.
4.3.4. Fingertip bruising
Subcutaneous bruises extending to the myofacial plane were observed on both shaken animals
(Figure 4-20). These bruises were located over bony prominences at the points where the
animals were held during shaking such as the scapula, thoracic spinous processes and the ventral
torso, inferior to the nipples. The shapes of the bruises ranged from circular to irregular and the
sizes measured from 0.5 cm in diameter to 4 cm by 1 cm.
Hemorrhages representing the subcutaneous bruises extended into the fibroadipose tissue but
not into muscles (Figure 4-20 C).
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Figure 4-17. Retina after formalin fixation. Globe of the eye was bisected to reveal retina on posterior hemisphere. Photographs were taken while posterior globe was floated on 10% buffered formalin. A. and B. Sham animal (left (A) and right eyes (B)). C. and D. AP animal. E and F. Lat animal. No grossly visible RH was seen.
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Figure 4-18. Photomicrographs of retina. No microscopic RH was seen. A and B. Sham animal. C and D. AP animal. E and F. Lat animal. (All 200x).
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Figure 4-19. Posterior neck dissection. Layered dissection of posterior neck muscles revealed no structural damage or bleeding. A and B. Sham animal. C and D. AP animal. E and F. Lat animal.
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Figure 4-20. Fingertip bruising found on body surfaces. A. Skin bruising over ribcage just below nipples. B. Bilateral bruising over scapulae found after skin reflection. C. Photomicrograph of myofacial hemorrhage associated with skin bruising. H&E stain, 100x.
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4.4. Accelerometry Data
4.4.1. Sham animal
Both front-to-back and lateral rocking of the sham animal generated a maximum tangential
acceleration of 1 to 2 G with mean frequency of ~1 Hz. Figure 4-21 shows a plot of the
tangential accelerations measured while being gently rocked in AP direction. In this plot, blue
line denotes a tangential acceleration in front-to-back direction and red line denotes a tangential
acceleration in side-to-side direction. The sweep number corresponds to number of times the
measurements are taken by the accelerometer.
4.4.2. AP animal
In AP shaking, most of the acceleration peaks measured in front-to-back direction reached
the upper recording limit of the accelerometer. Figure 4-22 shows a plot of the tangential
accelerations measured while being violently shaken in AP direction. In this plot, blue line
denotes a tangential acceleration in front-to-back direction and red line denotes a tangential
acceleration in side-to-side direction. Accelerations on side-to-side direction reached the
maximum of ~5 G. Overall, the animal was shaken front to back 125 times in 43 seconds of
manual shaking (mean frequency: ~2.9 Hz). Maximum frequency reached was ~3.2 Hz.
4.4.3. Lat animal
In Lat shaking, most of the acceleration peaks measured in the side-to-side direction reached
the upper recording limit of the accelerometer. Figure 4-23 shows a plot of the tangential
accelerations measured while being violently shaken in lateral direction. In this plot, the blue
line denotes a tangential acceleration in front-to-back direction and the red line denotes a
tangential acceleration in side-to-side direction. Interestingly, some of the peak accelerations in
111
the front-to-back direction also reached the limit while the rest of the peaks in this direction had
a mean value of ~8 G. Overall, the animal was shaken side-to-side 178 times in 53 seconds of
manual shaking (mean frequency: ~3.4 Hz). Maximum frequency reached was ~3.7 Hz.
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Figure 4-21. Accelerometry tracing from Sham treatment.
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-2
-1.5
-1
-0.5
0
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Figure 4-22. Accelerometry tracing from Anterior-posterior animal shaking.
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Figure 4-23. Accelerometry tracing from Lateral animal shaking.
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5. Discussion
5.1. Mice
5.1.1. General discussion
Rodents have been used for modeling human head injuries. Relative ease of handling and
lower operational cost have contributed to their wide use in various medical research. Previous
rodent models of head injuries focused primarily on impact trauma or impact-induced angular
acceleration trauma. In these models, a wide range of brain injuries from mild concussion to
diffuse axonal injury has been documented. However, the utility of these models in describing
repeated inertial injuries of shaking is low, since neither the mechanism (non-impact induced
rotational acceleration) nor the manner (repeated oscillatory motion) is reliably replicated in
these models.
There are only a few rodent studies that evaluated a non-impact mechanism of brain injury.
Non-impact induced angular acceleration combined with hypoxia has been reported to cause
brain lesions in adult rats 118. Bonnier’s articles in 2002 and 2004 represent one of the very few
rodent head injury studies that combined the mechanism and manner (through the use of s linear
rotating shaker at 900 rpm for 15 seconds) to be addressed in modeling SBS. In this model, RH,
focal axonal injuries and focal white matter hemorrhages were reported, but the assessment of
the figures provided showed the general overstaining for most immunostains. It is also puzzling
that no astrocytic reaction was seen despite the injuries mentioned.
There was a total of 18 intra-procedural deaths in the three studies.16.5% mortality rate in
our study is lower than the 27% mortality rate reported in Bonnier’s study. Without any positive
intracranial findings, there are two main causes that could account for the intra-procedural
deaths. The first is the possibility of accidental anesthetic overdose. Since the anesthetic agent
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(isoflurane) was vaporized and administered via inhalation, the amount of the agent that entered
the animal largely depended on the animal’s pulmonary function. The lungs of younger pups are
not yet fully developed and individual variation may have been great. The second possible cause
of intra-procedure death is mechanical asphyxia due to the restraint used to immobilize the
animal within the tube. This may have prevented chest expansion during inhalation. The higher
mortality rate in the pulse acceleration (10 of 29) and hinge-point study (5 of 11) supports this
speculation as more adjustment of the fitting was required for the pulse acceleration study (5
repetitions) and a larger amount of cotton was used in the hinge-point study to limit both chest
and head movements.
5.1.2. Negative findings: What do they mean?
In this experiment, high frequency vibration of postnatal mice did not result in any injuries
commonly thought to be caused by shaking. There are three potential explanations why this
experimental setup did not result in injuries.
First is the difference in craniocervical anatomy between human infants and mouse pups.
During vibration, the head of the mouse generally followed the movement of the tube which
closely approximates a circular motion. The mice’s heads were observed to ‘jerk’ out of the
circular path from time to time, particularly when the vortex machine was changing speeds
(start/end and pulse acceleration). Since these pups were anesthetized, any voluntary movement
could be ruled out. These jerking motions could potentially be explained by the craniocervical
anatomy of the mice. The foramen magnum in quadrupedal animals is positioned in the occiput,
oriented vertical to the ground 119. The position of the foramen magnum gradually moves
towards the inferior surface of the occipital bone in higher order animals. The vertical position
of the foramen magnum and atlanto-occipital joint of rodents limits their range of neck
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extension. If greater range is required the trunk has to move together with the head. Also, in
younger pups, head and neck development are incomplete and the neck circumference is larger
than the head circumference. This also does not permit true shaking of the head where the neck
needs to act as a fulcrum. The jerking movements observed during high frequency vibration
could be interpreted as movement of trunk when the sudden change of acceleration exceeded the
extension limit of the neck.
Second, the force applied to the mouse pups may not have been high enough to cause head
and neck injuries. It is believed that brain weight is a critical factor in determining the force
required to create injuries by acceleration/deceleration in an inverse relationship where greater
non-impact acceleration would be required to cause injuries to a smaller brain. The brain weight
of the postnatal mice used in this study was not recorded since whole mount sections of the head
and neck regions were prepared without removal of the brain. However, with the overall weight
of the pups ranged from 6 g to 20 g, the brain weight would not exceed a few grams at the most.
In other animal studies of non-impact head injury, at least 350 krad/sec2 of angular acceleration
was required to cause a mild traumatic brain injury such as concussion in a rat model 106 (brain
weight = ~5 g), whereas in a piglet model 55 (brain weight = ~35 g), 110 krad/sec2 was sufficient
to cause SDH and axonal injuries. When scaled for the brain weight of 500 g, concussion, SDH
and tDAI threshold from Gennarelli’s baboon-macaque model 62 were 100 krad/sec2, 350
krad/sec2 and 400 krad/sec2 respectively. Applying the proposed inverse relationship, it is
expected that the amount of angular acceleration required to cause some type of injury in
postnatal mice model would exceed these figures. Although the angular acceleration was not
directly measured in this study, it is reasonable to project from the calculated tangential
acceleration that the high frequency vibration used did not apply enough force to cause any head
and neck injuries.
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Last, the lack of findings in mice pups might be a true indication that shaking does not cause
head and neck injuries. With the exception of Bonnier studies, all other animal models of non-
impact human head injury that resulted in positive findings employed forces that are orders of
magnitude greater than the forces generated by manual shaking in anthropomorphic studies.
With no experimental evidence showing that damage could result from such lesser force, the
negative findings in this study add to a body of evidence that the accelerations that are
comparable in magnitude to the accelerations from manual shaking does not cause head injuries.
However, due to limitations of the model discussed above, the results of this study should not be
considered definitive evidence that manual shaking does not cause head and neck injuries in
human infants.
5.1.3. Role of mouse model in future investigations of shaken baby syndrome
During the development of the experiment, it was a challenge to come up with a shaking
apparatus that would not cause chest compression or other trauma to the body while delivering
sufficient non-impact force to the head of the mouse pups. We could not think of any alternate
solution that could achieve this goal. Other means of accelerating the head of the animals
available in literature, such as impact-acceleration 106, 107 or fluid percussion 108-114 cannot be
applied to mouse pups as they would cause catastrophic damages to the pups overall at the level
of force projected to cause internal head and neck injuries. This limitation effectively eliminates
the possibility of using postnatal mice in replicating mechanism of manual shaking.
Despite the inability of replicating the shaking mechanism itself, the convenience of using
small animals such as mice should be utilized in studying the pathobiology of injury. For
example, the specific molecular mechanism for vessel permeability changes in the setting of
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hypoxia or ischemia is yet to be fully understood and could be studied by designing experiments
where hypoxia or ischemia is induced by means other than shaking.
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5.2. Barbados green monkeys
5.2.1. General discussion
Manual shaking of BGM in this study was designed to detect immediate hemorrhagic injuries
and possible physiologic responses suggestive of axonal damage. Several non-specific
physiologic responses that could be suggestive of axonal damage was observed during one hour
survival including hypothermia, twitching and deviation of eyes. Unfortunately, βAPP
immunostaining did not show any signs of axonal injury (no axonal positivity). This does not
rule out the possibility of such injury as the survival interval might not have been sufficient for
broad development of detectable βAPP accumulation in injured axons. Although it is widely
recognized that the time interval between cerebral injury and expression of βAPP is at least two
hours, there is growing evidence in the literature that it may be detected at earlier time intervals
after the injury 120. Longer time intervals allow broad development of βAPP-stainable axonal
retraction balls. However, they also allow secondary changes to the brain via generalized
swelling after initial traumatic injuries, leading to non-perfusion anoxia and florid hypoxic
ischemic encephalopathy. Therefore, allowing sufficient time for βAPP development could give
rise to confounding results that overshadow true injuries produced by shaking. This is indeed the
case for the most of the head-injured babies where ascertaining the mechanism of the initial
injury can be quite challenging without clear findings of an impact event.
In Gennarelli’s baboon-macaque model, lateral rotational acceleration generated significantly
more severe damage than did anterior-posterior (sagittal) rotational acceleration of the same
magnitude. However, Gennarelli did not offer any possible explanation why lateral acceleration
on the coronal plane resulted in more severe injuries. In manual shaking of BGM, although there
were no detectable injuries, lateral shaking generated more complex head movement than did
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AP shaking. As shown in Figure 4-23, tangential acceleration on both the sagittal and coronal
axes reached over 10 G, and where combined, tangential acceleration could be significantly
more. This secondary movement axis in lateral shaking may be due to the adaptation of
craniocervical junction in bipedal animals. The range of lateral flexion movement in higher
order animals, especially bipedal animals, is all but lost and this may create a secondary axis of
motion 121. The exact magnitude of combined tangential acceleration cannot be calculated since
the maximum peak values could not be recorded . This should be addressed in future
experiments using an accelerometer with a higher recording range.
Findings of fingertip bruising on the skin surface where the monkeys were held for shaking
shows that the force required to hold the animal during shaking is significant. Although findings
of non-specific punctate bruising is relatively common in chronically abused babies, recognizing
their significance as a possible sign of manual shaking could provide additional corroborative
evidence for the pathologist in providing a balanced opinion. Thus, a layered dissection of both
the anterior and posterior torso and arms (in case the baby was held by the arms) should be
performed in all unexpected deaths of infants as subcutaneous bruises on such locations could
be a possible sign of the shaking event.
5.2.2. Negative findings: What do they mean?
Dissimilarity due to developmental parameters
A frequent argument against using adult animals in modeling SBS is the belief that the
immature brain and meninges have different mechanical properties than those of adults. During
the external revision of the BGM experimental protocol, this question was raised by one of the
reviewers.
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“Scientifically, what this model does not address is the difference between the immature brain and its
coverings in the two-month-old human infant and that of the adult non-human primate brain. The infant
brain and its vessels would be expected to be more mobile and more fragile than an adult brain.”
As described in the introduction, human infants have distinct developmental parameters that
could potentially affect the response to external forces. A tensile scalp, pliable cranium,
underdeveloped meninges and blood vessels, and lack of the myelinations all could have
mechanical ramifications. These differences are most pronounced in the perinatal and neonatal
periods and are considered to be a contributing factor to the predominance of suspected SBS
cases in these age groups.
Currently available mechanical studies of infant vessel properties do not conclusively answer
this difficulty. These studies are limited in their inability to reproduce the complex mechanical
relationships that the infant brain and its vessels are exposed to including brain dimensions, the
anatomical location of vessels and axis of angular displacement. One of the aims of the BGM
study was to describe mechanical displacement parameters of manual shaking. For this purpose,
having dimensionally (size and weight) similar animals offered more relevant information that
could be compared to anthropomorphic studies in the literature.
Weight of the brain and the acceleration
In an anthropomorphic study using a scaled model of 1-month-old human infants (brain
weight = assumed to be 500 g), manual shaking generated a maximum of 2.64 krad/sec2, a
simulated fall from 0.9 metre onto concrete surface generated 89.4 krad/sec2, while inflicted
impact against a benchtop surface generated 173 krad/sec2. An older anthropomorphic study
using a similar scaled model showed that manual shaking could generate peak tangential
acceleration of 9.29 G with mean angular acceleration of 1.14 krad/sec2 while impact against a
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metal bar generated 428 G and 52.5 krad/sec2 respectively. When combined, these figures
suggest that manual shaking simply could not generate enough angular acceleration to cause any
injuries 62, 63.
The weights of the brains of the BGM used in this study were 76.0 g (AP animal) and 61.0 g
(Lat animal). From the injury thresholds above, it is not hard to project that manual shaking
cannot generate the acceleration thought to produce injuries. Unfortunately, the peak tangential
acceleration during manual shaking of BGM could not be determined due to the saturation of
the accelerometer’s upper recording limit. Without a measured maximum peak tangential
acceleration value, interpretation of the acceleration data obtained in this study is limited.
However, the accelerometry trace showed that the some peak individual oscillations were not
saturated and the tangential accelerations of those peaks were clustered around 10 G. Thus, it is
likely that peak tangential acceleration during manual shaking of BGM did not reach the
magnitude of the reported injury thresholds.
Craniocervical anatomy of Barbados green monkeys
Phylogenetically, monkeys are closely related to humans. It is generally accepted that their
anatomy resembles that of humans more closely than does the anatomy of non-primates. BGM
are essentially bipedal in their modality, thus the position of the foramen magnum is near the
base of the skull similar to humans. Also, the anatomy of the craniocervical junction permits full
extension and flexion of the neck, similar in the range that is observed in human. This permits a
true whiplash type of shaking motion to be replicated in BGM where, without support of neck
musculature after general anesthesia, the movement of the head is isolated from the trunk. This
craniocervical anatomic similarity is one of the most important benefits of using primates for the
initial experiments of SBS where several important mechanical parameters and physical
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responses could be studied. However, a thorough anthropological comparison study of BGM to
human must be carried out to document the subtle differences that would be required in
interpreting the data gathered from BGM studies. The general aims of such study are outlined in
the next chapter.
Fundamental question: Can shaking cause head and neck injuries?
The very fundamental question in the SBS debate still remains unanswered. Both traditional
and unified hypotheses hinge on the assumption that the shaking can cause some type of injury
(primary injury) that initiates cascade of responses (secondary injury). In this experiment, a
violent manual shaking of animals that are similar in dimensions to human infants did not cause
any acutely detectable hemorrhagic injuries. The pertinent negative results from this
experiments are: 1. Shaking did not cause shearing of bridging veins that is thought to cause
SDH in SBS, 2. Shaking did not cause immediate RH from vitreous-retinal traction, 3. No apnea
was observed immediately after shaking, and 4. No evidence of axonal injury was detected after
one hour survival interval under general anesthesia after shaking. On the other hand, the
limitations of this study were: 1. Mature animals were used instead of infantile/juvenile animals,
2. Brain weights were significantly less than those of human infants, 3. Accelerometer could not
capture the maximum peak acceleration, and 4. Insufficient time was allowed for the delayed
marker of injuries. Some of these limitations could be directly or indirectly addressed by the
approaches discussed in the following chapter.
5.2.3. Role of BGM model in future investigations of SBS
As discussed above, primates hold a distinct advantage over other animal models in studying
SBS. There are many physiological and mechanical aspects of the SBS debate that still need to
be described. First of all, the development of βAPP after a longer survival interval must be
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addressed using the same protocol established in this study. Along with the axonal injury study,
many of other studies that require experimental data input from primate studies are discussed in
the following chapter.
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5.3. Back to the shaken baby syndrome debate: Significance of experiments performed
5.3.1. True non-impact head injury protocol
This BGM study represents one of the first to use a non-impact mechanism of applying
rotational acceleration in animals. No previous animal studies have employed true shaking,
where the head undergoes fast repetitive oscillatory motion with the neck being the fulcrum.
Baboon-macaque studies by Gennarelli 16, 18, 50, 52, 53 and piglet studies by Margulies 54-56 both
failed in this regard, where only a single (or multiple, separate single accelerations over 15
minute time interval) large scale acceleration was applied to create the head injuries. Also, in
this BGM study, the force was delivered manually which also is a novel approach in animal
studies of SBS. By incorporating these two factors, this study meets the criteria of the violent
shaking event described by AAP 2 and what pediatricians would believe to cause the triad. This
is important because previous animal models that were able to generate part of the triad were
rejected since they failed to meet the description of a ‘shaking event’ in the context of child
abuse.
5.3.2. Mechanical properties of Barbados green monkey manual shaking
The frequency and tangential accelerations recorded in this study generally validate the
anthropomorphic data from Duhaime and Prange studies 62, 63. At full strength, an adult male
experimenter was able to generate frequency less than 4 Hz with the projected maximum
tangential accelerations in the realm of anthropomorphic data. However, without properly
measured maximum acceleration, further investigations using accelerometers with higher
calibrated recording range are required for meaningful comparison between BGM model and
anthropomorphic devices. The BGM study performed here still represents an improvement from
the anthropomorphic approach where no ‘worst case scenario’ assumptions on head and neck
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mechanical properties were needed. In addition, our BGM model not only allowed physical
recording of the mechanical parameters, but it also allowed correlation of such data with the
actual injuries created (had there been some type of detectable injuries). This is a unique aspect
of manual shaking protocol where precise control of forces applied is nearly impossible, yet
with the proper instrumentation, similar type of mechanical data recorded from the baboon-
macaque or piglet studies could be recorded.
5.3.3. Empirical data for shaken baby syndrome discussion
In the introduction, an example of rhetorical exchange in the current SBS debate was given.
The debate has reached a point where studies of SBS may not be analyzed rationally for their
methodology but simply criticized or applauded based on conclusions reached by the study and
on which side of the debate the reader stands. In one such exchange, opposite groups in the
debate have accused each other of ‘living in a glass house’ or doing ‘junk science’ 122, 123. This is
a serious subject, the consequences of which are as serious as infanticide and potential
miscarriage of justice, and opinions of ‘junk scientists’ or medical professionals ‘living in
glasshouses’ are not sufficient. There are multiple urgent steps that need to be taken to refocus
this highly charged debate into a rational, scientific one. A critical evaluation of retrospective
and anecdotal medical evidence for their study methodology such as selection criteria,
postmortem examination protocols and analytical methods is required. At the same time,
carefully designed scientific studies must be conducted to test the various hypotheses that could
arise from such re-evaluations.
This BGM study represents the first step in that direction. Although inconclusive due to the
limitations stated above, the model incorporated many aspects that are discussed in the field. It
is one of the first producing experimental data that can be critically evaluated, not based on the
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‘vast experience’ of the professionals who have worked in this field for many years. I recognize
that their contribution to the field has been invaluable and their input is still very much required
in both designing and evaluating the experimental studies. The BGM protocol used in this study
could be adapted and modified to gain more experimental data that will only help the
understanding of SBS. The body of experimental data can then be used to validate other studies
of SBS or to modify study designs to be more relevant to the issues being evaluated.
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6. Future directions
6.1. Primate model of SBS
6.1.1. Axonal injury
The experiments performed did not produce any detectable injuries. Thus, the question of
whether or not manual shaking can cause head injuries is still unanswered. The BGM study in
this thesis has established an experimental protocol that could be adapted with two different
survival intervals, allowing sufficient time for βAPP development should there be axonal
injuries (Table 6-1).
Table 6-1. Proposed experimental groups for the study of axonal damage from manual shaking of BGM
Animal ID
Axis of displacement
Survival interval between shaking and euthanasia (Hrs) Other
Sham 1 n/a One Anesthesia followed by euthanasia only Sham 2 n/a Six
AP 1 Anterior-Posterior One
AP 2 Anterior-Posterior Six
Lat 1 Lateral One
Lat 2 Lateral Six
The presence of widespread axonal injuries after a longer survival interval without immediate
hemorrhagic injuries in BGM could imply two possibilities. First, if the axonal injuries are truly
traumatic in origin, it could suggest that the shearing of the axon is the mechanism of injury and
since the threshold of vessel injuries are higher in adult BGM than in human infants, only
axonal injuries are detected. Secondly, if the axonal injuries are secondary consequences of
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hypoxic ischemic event, the mechanism of which these axonal injuries and other components of
the triad develop after shaking must be further studied. Both traditional and unified hypotheses
could explain the development of hypoxia (generalized brain swelling after diffuse axonal injury
or hypoxia/ischemia following apnea from brainstem injury) but the mechanism of which SDH
or RH could develop is not yet known.
6.1.2. Comparative anatomy and head and neck movement during shaking
One of the critical factors in determining behaviour of the head and neck movement during
application of the force is the shape of the craniocervical junction. A study by Penning
compared the craniocervical junctions of humans and other animals and their impact on various
neck movements 121. Careful measurements of the locations and joint surface angles in humans
and other animals were taken from different components of the craniocervical junction including
the occipital condyles, foramen magnum, atlas and axis vertebrae. The study then measured the
range of motions allowed in these animals and found important differences between
quadrupedal mammals and human. The study also included lateral flexion data from a primate
(Orangutan) that showed the most similarity to the human compared to other animals. Compared
to quadrupedal animals, humans have largely lost this lateral flexion. Also in human, this
motion is coupled with the atlanto-axial rotation.
A similar type of analysis is needed to confirm the anatomical similarities between human
infants and BGM. The study should include thorough measurements of the location and angles
of the components of the craniocervical joint. The range of motions including sagittal extension,
flexion and translation (occipito-axial translation and head-trunk translation), rotation about the
atlanto-occipital and atlanto-axial joints, and lateral flexion should be evaluated with or without
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the supporting neck musculature. This study could be done in conjunction with the axonal injury
study proposed above to minimize the number of animals required.
6.1.3. Ethical consideration
The proposed study represents a minimum number of animals required to confirm the
original findings and to determine if manual shaking could generate axonal damage. It also
gives an opportunity to use a higher-range accelerometer to measure maximum peaks of
tangential accelerations. Once baseline mechanical displacement parameters are established,
attempts of scaling could be made for future studies of smaller but developmentally and
physiologically similar animals.
6.1.4. Anthropomorphic model
The general lack of knowledge about properties of the neck of human infants has led to
problems in designing biofidelic anthropomorphic devices. In the study previously mentioned,
Prange et al. had to design a neck with negligible resistance to err on the side of a worst case
scenario 63. A hinge was used as the centre of rotation and provided no resistance to either
extension or flexion in sagittal plane. However, the hinge did not provide any range of motion in
other directions and the location of the centre of rotation had to be estimated. This resulted in no
information being provided regarding movements in coronal plane and likely overestimated the
rotational acceleration.
The design of biofidelic devices could benefit greatly if the craniocervical anatomy of
primates is confirmed to be similar to humans. In that case, the resistance of the neck in all
directions could be measured in primates either under anesthesia or during postmortem
examination to reduce the effect of developed neck musculature of adult primates. Also, such
study could determine the precise location of the centre of rotation which would provide more
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realistic estimation of the rotational acceleration. Combined with the information from the
comparative anatomy study above, a more complex hinge mechanism could be designed that
could account for the movements in both the sagittal and coronal planes.
6.1.5. Mechanical properties of tissues and finite element modeling
A reductionist approach in building in silico model of SBS in the past has been limited by the
lack of experimental data about mechanical properties of the tissues and the interface involved.
Most studies of mechanical properties of tissues have been limited to testing of selective tissue
types (vessels, scalp, skull, etc.) with no regards to the properties of the modular systems these
components build together 124. Also, the finite element modeling of SBS has been hampered by
the lack of experimental data the model should aim to emulate 125-129.
The development of an animal model of SBS could provide key information required in such
a reductionist approach. While an animal model could provide physiologic and pathologic
information, the finite element models could be used to complement the inherent shortcomings
of the animal models. Using in silico models, the subtle species differences identified in
comparative anatomic studies and the age specific differences in tissue properties could be
tested for their effect on shaking.
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6.2. Hypoxia as cause of subdural hemorrhages
6.2.1. Perinatal and neonatal intradural hemorrhages
In recent months, a series of papers were published that suggested a link between age related
meningeal development and the propensity of SDH in hypoxia. In a retrospective, observational
study, Cohen and Scheimberg analyzed postmortem examination findings of 25 fetuses of 24
weeks of gestation or older that died in utero and 30 infants of one month of age or younger,
selected for the presence of intradural hemorrhage (IDH) 130. Both falco-tentorial and parietal
IDH were examined grossly and histologically. A strong association was found between the
presence and degree of hypoxia, the amount of IDH and the presence of SDH especially in
neonates. The paper also suggested that the IDH and SDH are mechanistically linked by
eventual weakening of the dural border cell layer next to the arachnoid membrane upon
accumulation of IDH. Interestingly, Squire et al. demonstrated the lack of intradural space
(dural channels) in neonates which is thought to contribute to the occurrence and severity of
IDH in this age group. These spaces could function as a reserve cavity for reflux from various
venous sinuses present in dura 100, 104, 131. With these two studies combined, a correlation could
be made for the propensity of hypoxia related IDH and SDH in younger infants.
6.2.2. Implication for the shaken baby syndrome ‘unified hypothesis’
In 2003, Geddes 3 has proposed the ‘unified hypothesis’ which proposed that hypoxia could
be the underlying cause of SDH seen in previously diagnosed SBS cases 73. However, Geddes
failed to demonstrate a mechanistic link between hypoxia and SDH other than the typical
presenting history of apnea in these SBS cases and the presence of brainstem injuries in some of
the cases reviewed. The ‘unified hypothesis’ has since been challenged in court (discussed in
Chapter 1) and in the literature. However, the ‘unified hypothesis’ can now gain some support
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with the newly proposed link between hypoxia and IDH/SDH in neonates if it can be
demonstrated that manual shaking can cause enough damage to the brainstem to cause apnea
leading to hypoxia.
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6.3. Medical investigations of SBS
6.3.1. Retrospective and prospective studies using βAPP immunostaining
Geddes 2 72 and a study by Oehmichen 74 represent the retrospective efforts being made to
evaluate the incidence of tDAI in previously diagnosed SBS cases using βAPP. These studies
are limited in their scope as number of cases is limited in each study location and therefore
could be difficult to generalize. Continuing efforts should be made with this approach as it can
provide data to compare to results of the experimental studies. Both retrospective and
prospective analysis of cases should be carried out using standards such as case selection
criteria, types of investigations performed in each case and standard grading of the positive
findings. Ideally, such studies should involve multiple centres from different regions to
maximize the sample size and increase the consistency of the analysis.
6.3.2. Cerebrospinal fluid analysis
In clinical settings where an infant is in serious medical condition without a known cause,
sampling of the cerebrospinal fluid (CSF) is quite common. CSF is sterile in healthy individuals
and is isolated from the blood circulation by blood brain barrier. Blood tinge in a CSF sample is
an indication of cerebral bleeding where the presence of bacteria in CSF is an indication of
meningitis or generalized sepsis. Advanced techniques are available to study the components of
fluids and their relationships; those techniques could be applied to the study of CSF. In general,
even slight change in components of CSF could be measured against the normal state using
molecular techniques. CSF collected in clinical settings could be analyzed to identify candidate
markers for each type of injury (hypoxia or axonal injury) at different time points and could be
utilized as an sensitive analytical test of head injury.
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6.4. Possible mechanisms for Shaken Baby Syndrome triad development
1. Although our experimental findings must be interpreted in the context of the limitations
discussed, our results did not support the traditional hypotheses proposed to explain subdural
hemorrhages and retinal hemorrhages following infliction of injury.
2. The presence of axonal injury could not be confirmed histologically, as the survival
interval required for the broad development of the injury marker was insufficient. The
experimental protocols should be repeated with a longer survival interval after shaking.
3. Although the maximum acceleration of the monkey’s head could not be directly measured,
it is likely that manual shaking cannot generate enough force to cause the pattern of injuries
identified in previous animal studies. However, movement of the head during lateral shaking is
complex and needs to be studied further.
4. There is growing evidence that IDH and SDH can develop in the setting of hypoxia in
neonates. This premise might represent the mechanistic link between hypoxia and SDH in the
‘unified hypothesis’.
5. It is still inconclusive as to whether manual shaking can cause the triad in human infants.
Although manual shaking does not appear to generate sufficient force to cause immediate
diffuse injuries, it is still unclear if focal brainstem injury could be produced to account for the
development of the triad pattern of injury.
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7. References
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Appendix A-1. Routine Hematoxylin and Eosin staining protocol
Procedure
1. Deparaffinize in Clearene® – 5 changes, 5 minutes in total
2. Remove Clearene® with absolute alcohol – 5 changes, 10 dips each with thorough
draining between changes
3. Wash in running tap water until alcohol is removed – 10 dips
4. Put in Harris’ Hematoxylin – 5 minutes with occasional agitation, Harris’ Hematoxylin
should be filtered before each use
5. Wash off excess Hematoxylin in running tap water – 10 dips
6. Differentiate in acid alcohol – 5 dips
7. Wash well in running tap water – 10 dips
8. Blue in lithium carbonate – 2 minutes
9. Wash well in running tap water – 5 minutes, check nuclear staining under microscope.
If overdifferentiated, repeat from step 4. If underdifferentiated, repeat from step 6.
10. Stain in eosin – 2 minutes with frequent agitation
11. Rinse off excess eosin in running tap water – 10 dips
12. Dehydrate in absolute alcohol – 5 changes, 10 dips each
13. Clear in Clearene® – 5 changes, 10 dips each
14. Mount with Permount®
150
Solutions
1. Harris’ Hematoxylin
Hematoxylin 14 gram
95% Ethanol 120 ml
Aluminum ammonium sulphate 240 gram
Distilled water 2,400 ml
Mercuric oxide 6 gram
Glacial acetic acid 48 ml
Mix the alum and water and bring to a boil. Remove from heat and add the hematoxylin
dissolved in alcohol. Bring to a rapid boil again. Remove from heat and let it settle for
one minute. Add mercuric oxide very slowly at first. Bring it to boil again, the cool
quickly in cold water. Add glacial acetic acid.
2. Acid alcohol – 1% hydrochloric acid in 70% ethanol
3. Saturated Lithium Carbonate
Lithium carbonate 12 gram
Distilled water 1000 ml
4. Eosin – 1% Eosin Y in distilled water and add a few crystals of thymol (2-isopropyl-5-
methylphenol)
Eosin 30 gram
Distilled water 3000 ml
151
Appendix A-2. Routine Luxol Fast Blue/H&E staining protocol
Procedure
1. Deparaffinize in Clearene® – 5 changes, 5 minutes in total
2. Remove Clearene® with absolute alcohol – 5 changes, 10 dips each with thorough
draining between changes
3. Wash in running tap water until alcohol is removed – 10 dips
4. Stain in Luxol Fast Blue solution for 4 hrs at 60°C. Use Paraflim to cover the solution.
5. Rinse in 70% ethanol and wash in distilled water until alcohol is removed – 10 dips
6. Differentiate in lithium carbonate – 30 seconds
7. Wash well in distilled water – 10 dips
8. Differentiate in 70% ethanol – 30 seconds
9. Wash well in distilled water – 10 dips
10. Check staining with the microscope – Myelin and RBC should be blue, the rest of the
tissues should be colourless. If overstained, repeat from step 6.
11. Follow routine H&E protocol with following modifications – Harris hematoxylin 3
minutes, lithium carbonate 1 minute, and eosin 1 minute.
12. Rinse off excess eosin in running tap water – 10 dips
13. Dehydrate in absolute alcohol – 5 changes, 10 dips each
14. Clear in Clearene® – 5 changes, 10 dips each
15. Mount with Permount®
152
Solutions
1. Luxol fast blue
Luxol fast blue 0.1 gram
95% ethanol 100 ml
10% acetic acid 0.5 ml
2. Lithium carbonate (for step 6)
0.05% lithium carbonate
153
ANIMAL USE PROTOCOL AND RESEARCH ETHICS BOARD REVISED PROPOSAL FOR PEER REVIEW
TITLE A PRIMATE MODEL FOR THE SHAKEN BABY SYNDROME
INVESTIGATOR Michael S. Pollanen MD PhD FRCPath DMJ(Path) FRCPC
INSTITUTION Department of Laboratory Medicine and Pathobiology
University of Toronto SUMMARY OF REVISIONS MADE
1. The total number of proposed animals remains UNCHANGED 2. The episodic shaking experimental group has been ELIMINATED 3. Eliminated animals are REALLOCATED for a survival interval
experimental group. 4. There is NO LUCID INTERVAL in any of the animals. All animals will be
under general anesthesia for all experimental manipulations with no conscious survival interval (i.e., all animals with be euthanized under general anesthesia).
SUMMARY OF RESEARCH PROPOSAL A controversy has been developing in pediatric and forensic medicine over the past 10-15 years – whether shaking an infant can cause fatal head injury. It has traditionally been held that shaking causes fatal and serious cerebral injury – the shaken baby syndrome (SBS). Initial opposition to the SBS concept was not generally viewed as a serious or credible challenge to the received view that SBS is a specific form of abusive head trauma. Currently, however, there is growing scepticism in the forensic pathology community, and some believe that the SBS concept may not so firmly evidence-based as once held. This growing scepticism has found empirical support in the peer-reviewed literature, mostly from descriptive retrospective neuropathologic studies on putative cases of SBS and some biomechanical modeling experiments. However, there has been no definitive experimental data to provide key data to inform the underlying issue: does shaking cause injury? To date SBS has not been reproduced in an animal model. At this time some researchers believe that we are at an impasse – until SBS can be reproduced in a relevant animal model we will be suspended in the current state of oppositional scientific debate with little empirical evidence that can provide data-driven conclusions. In essence, we are currently at a point where there is a complete block to further scientific development in our understanding of SBS. The medical and scientific community needs a decisive experiment to provide new data to inform the debate.
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One clearly defined way forward is determining if shaking can cause the putative markers of SBS in an animal model that closely resembles an infant. The putative markers are the so-called SBS ‘triad’: subdural hemorrhage, retinal hemorrhage, and brain swelling. On this basis, I propose a ‘proof-in-principle’ experiment to determine if shaking can induce the triad in a small number of primates. This primate experiment would provide significant new knowledge to inform the discourse around SBS. RESEARCH PROPOSAL Background General Background Physical abuse of children is the leading cause of serious pediatric head injury. The shaken baby syndrome (SBS, or abusive head trauma), is widely viewed as a common and frequently fatal form of child abuse usually seen in children younger than two years of age, but occasionally observed in children up to five years of age1-3. The early descriptions of SBS in the 1970s suggested that the mechanism of head injury was a ‘whiplash’ motion of the head caused by rapid back and forth displacement of the infant’s head while the perpetrator was shaking the infant by grasping its trunk4, 5. In the American Academy of Pediatrics Technical Report on SBS, it is stated that the act of shaking is “so violent that individuals observing it would recognize it as dangerous and likely to kill the child”6. Despite authoritative claims of professional organizations such as the American Academy of Pediatrics and the National Association of Medical Examiners6, 7, a controversy has been developing in forensic medicine over the past 10-15 years – whether shaking on infant can cause fatal head injuries in infants. This debate has polarized the medical community into two camps: (i) Reputable physicians who strongly view the current medical evidence as
definitively supportive of shaking as a mechanism of injury that can be fatal or lead to permanent neurological impairment7-21.
(ii) Reputable physicians and non-medical scientists who view the current
medical and biomechanical evidence as either entirely non-supportive of shaking as a mechanism; or, at least, have doubt that the current evidence strongly supports the conclusion that shaking causes injury1, 22-34.
It is clearly and widely held in the medical community that shaking causes fatal and serious cerebral injury. It is undisputed that the concept of SBS would be identified by most pediatricians as a mainstream concept. In fact, the initial opposition to the SBS concept was not generally viewed as a serious or credible
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challenge to the established view that SBS was a coherent concept. However, there is growing scepticism about SBS in the forensic pathology community. Some forensic pathologists now question if the SBS concept is as firmly established on an adequate evidence-based as is widely believed. This scepticism has found some empirical support in the peer-reviewed literature, mostly from descriptive retrospective neuropathologic studies on putative cases of SBS21, 24, 25, 35; and some biomechanical modeling paradigms36-38. However, there has been no definitive experimental data to provide key data to inform the underlying issue: does shaking cause injury? At this time many physicians and scientists believe that we are at an impasse – until a relevant experimental model can be developed we will be suspended in the current state of scientific oppositional debate with little evidence that can provide data-driven conclusions. In essence, we are currently at a point where there is a complete block to any further development in our understanding of SBS. The medical and scientific community needs a decisive experiment to provide new data to inform the debate. It is generally accepted by the medical and scientific community that all currently available experimental animal models (e.g., rodent models) and biomechanical paradigms (e.g., biofidelic models) cannot provide a decisive experiment to resolve the SBS debate. All attempts to address this issue experimentally have failed to reproduce definitive results. All animal models have failed to produce the combination of lesions that are thought by many to represent markers of SBS. The biomechanical models have produced promising results, but the wider medical community has not accepted the data and continues to view that approach as only marginally relevant. Although retrospective clinicopathological and clinicoradiological studies have produced a range of important results, this approach will never be capable of providing definitive results on the key issue, since the mechanism of injury will always be scientifically disputable in any particular case (i.e., we will always ultimately rely on an assumption of validity of the history of the event, or a confession to ‘shaking’). Thus, one of the clearly defined ways forward in this debate is the development of a relevant animal model to determine if shaking can cause the putative markers of SBS as derived from autopsy studies of putative SBS cases. These putative markers comprise the so-called SBS triad: subdural hemorrhage, retinal hemorrhage, and brain swelling (i.e., cerebral edema and/or hypoxic encephalopathy with [or without] axonal lesions). On this basis, I am interested in exploring the use of a small number of primates to develop an initial experiment to determine if shaking can induce the triad. This primate experiment would provide significant new knowledge to inform the debate and may provide a ‘proof-in-principle’ answer to the SBS controversy.
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Preliminary Results We have studied putative shaking injury in a developing mouse pup model. Using several different specific experimental designs for shaking the heads of mice, we have been unable to produce meningeal, brain or spinal cord injuries of any type. Mouse pups (n = 40) were subjected to a high frequency vibration (20-50 Hz) under anesthesia while restrained in a manner that minimized chest compression but allowed free head movement. In both continuous high frequency vibrational shaking and pulse acceleration of the head, no central nervous system injury was detected in the whole mount section of the head. In contrast to previous reports, change in rotational plane did not result in more severe trauma. Attempts were made to deliver vibration directly to the head by restricting both head and neck movement, but again, no injuries were seen in this setting. The manuscript describing the details of the study is in preparation for publication. Rationale The failure to recapitulate the SBS triad in experimental animal models may relate to three important factors that all current animals models have failed to possess: (i) a relatively massive head providing significant inertia during motion; (ii) the precise craniocervical anatomy that permits a ‘shake’ to cause a rapid internal displacement of the brain and eyes, rather than simply vibrating the head on the neck; and (iii) any experimental mechanism of shaking that causes loading of head that an on-looker would unequivocally identify as shaking. The latter is not to be underestimated as an important variable since the American Academy of Pediatrics has established that this is a pivotal indicator of the lethality of shaking. On this basis, the most scientifically valid approach to studying SBS using an animal model is to use subhuman primates to determine if shaking can induce the triad. This primate experiment would provide significant new knowledge to inform the debate and may provide a ‘proof-in-principle’ answer to the SBS controversy. The animal of choice is the African green monkey. African green monkeys are among the most commonly used primates in biomedical research39, 40. These animals are small, easily handled, non-endangered, evolutionarily closely related to humans, and easily bred in captivity. Adult African green monkeys range in size from 4.1-5.5 kg and have top of the head to the base of the tail length of 426-490 cm41, 42. Based on these dimensional considerations adult African green monkeys are similar in weight to ~2 month infants. Most importantly, subhuman primates share with man important craniocervical anatomical similarities, which are not found in lower animals43.
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Research Plan Objective and Specific Aim The overall objective is to determine if SBS is a valid concept by reproducing the injuries in an animal model. Our specific aim is to determine if manual shaking of African green monkeys (C. aethiops) can cause head injuries including, subdural hemorrhage, retinal hemorrhage and diffuse axonal injury/hypoxic encephalopathy. Experimental Design In this experiment, healthy adult African green monkeys that were bred in captivity at Barbados Primate Research Centre (BPRC) will be screened for general health status and evidence of previous impact head injuries prior to the experiment. Animals with any scalp bruise or scars will be excluded from the experiment as will animals in generally poor health. General vital information of selected animals such as sex, height, weight, pulse, blood pressure and morphometric parameters including dimensions of the head will be recorded. A total of six animals will be used for this experiment: two will be used as controls and four will be subjected to manual shaking followed by euthanasia after a two hour survival interval under general anesthesia. All animals will be subjected to pre-anesthetic induction by intravenous propofol injection 30 minutes prior to the experiment. The state of general anesthesia will be maintained by isoflurane inhalation until eventual euthanasia by intravenous injection of barbiturates. The following parameters will be monitored from the initial anesthesia to euthanasia for all the animals: heart rate, temperature, oxygen saturation and breathing frequency. Any sign of deterioration will terminate the anesthesia and the animal will be promptly euthanized. Three small accelerometers will be attached to the head after induction of anesthesia and will be used to measure the acceleration experienced by the animal. Also, the shaking procedure will be recorded with a high-speed video camera to visualize the motion of the head and neck and calculate the maximal angle of the neck extension and flexion. In the experimental group, four animals will be subjected to shaking under anesthesia using the following two variables: axis of acceleration (sagittal vs. lateral) and survival interval after shaking (immediate euthanasia vs. 6 hours post-procedure survival under general anesthesia). The first animal will be shaken to establish a baseline for anterior-posterior (AP) shaking (sagittal acceleration-deceleration of the head). The animal will be firmly held by the chest and continuously shaken 20 times in anterior-posterior plane at the maximum head displacement generated manually. The animal will be euthanized by intravenous injection of barbiturates immediately after shaking. The second animal will be shaken in the anterior AP plane in the same manner described above. The animal will be kept under anesthesia for a further 6 hours after
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shaking and then euthanized. The same set of experiments will be repeated while shaking is applied in lateral plane, given the results of high-fidelity reproduction of axonal lesions in the Gennarelli-Thibault baboon-macaque model44-49. The control group will consist of a single negative control and a positive head injury control animal. The negative control animal will be subjected to pre-anesthetic induction, anesthesia and euthanasia without shaking. The positive control animal will be subjected to anesthesia in the same manner, and then the posterior aspect of the head will be subjected to controlled impact onto a flat, unyielding surface. The animal will be kept under anesthesia for 6 hours post-impact and then euthanized. After euthanasia, all animals will undergo postmortem examination using the standard autopsy protocol applied to cases of putative child abuse in the Province of Ontario. All gross findings will be documented and photographed. The brain, meninges, spinal cord, and eyes50 will be fixed in 10% buffered formalin (pH 7.4) prior to dissection. In addition to the latter specimens, the ribs and metaphyses of long bones will be studied macroscopically and microscopically. Tissue blocks will be processed for paraffin embedding. Exhaustive histologic examinations of the central nervous system, meninges, and eyes will be performed as in previous studies21, 24, 25, 51. Histologic sections from the central nervous system will be studied using conventional histologic preparations and immunohistochemistry using a variety of antibodies including: βAPP (marker for axonal injury), myeloperoxidase (neutrophils), CD-68 (macrophages), ubiquitin (extra-lysosomal proteolysis), GFAP (astroglia), and MAP-2 (dendrites). Macroscopic and microscopic data will be correlated with the data collected from the accelerometers. The average and maximal head acceleration achieved during shaking will be compared to the magnitude of acceleration and force described in biofidelic models of shaking. Expected Results The experiment will either result in the creation of some combination of subdural and retinal hemorrhages and diffuse axonal injury/hypoxic encephalopathy in monkeys, or it will not. If the results are positive, then this will be the first scientific-experimental evidence to support the existence of SBS. Thus, a positive result will provide powerful non-clinical validation of the prevailing or received view about SBS. If the results are negative, the issue will not be definitively resolved.
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Difficulties Anticipated There are no scientific barriers to the proposed research other than the possibility of a negative result that will not refute the hypothesis that SBS can be reproduced in primates. However, there are legitimate ethical considerations. There has been a justifiable movement away from the use of primates in medical research. This has been the case in head injury research since PETA animal activists brought attention to the NIH-sponsored Head Injury research program at the University of Pennsylvania in the laboratory of neurosurgeon, Dr. T. Gennarelli. It is accepted in the medical community by many researchers and by the applicant that animal rights ethics are a legitimate and important barrier to the development of many experimental models with primates. However, the considerable knowledge that can potentially be gained by this experiment is felt to balance the ethical arguments against it. Timeline The experiments will be performed at the Barbados Primate Research Centre (BPRC) over three days. Tissue removed from the animals will be analyzed at the University of Toronto over a period of several months. The study will be completed in less than one year leading to submission for publication and presentation at relevant scientific meetings.
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References 1. Whitwell HL: Head injury in the child. Edited by London, Arnold, 2006, p. 2. Gerber P, Coffman K: Nonaccidental head trauma in infants, Childs Nervous System 2007, 23:499-507 3. Trenchs V, Curcoy AI, Navarro R, Pou J: Subdural Haematomas and Physical Abuse in the First Two Years of Life, Pediatric Neurosurgery 2007, 43:352-357 4. Caffey J: On the theory and practice of shaking infants. Its potential residual effects of permanent brain damage and mental retardation, American Journal of Diseases of Children 1972, 124:161-169 5. Gennarelli TA, Thibault LE, Ommaya AK: Pathophysiologic Responses to Rotational and Translational Accelerations of the Head. Edited by 1972, p. pp. 296-308 6. American Academy of Pediatrics Committee on Child Abuse and Neglect: Shaken Baby Syndrome: Rotational Cranial Injuries - Technical Report, Pediatrics 2001, 108:206-210 7. Case ME, Graham MA, Handy TC, Jentzen JM, Monteleone JA: Position Paper on Fatal Abusive Head Injuries in Infants and Young Children, The American Journal of Forensic Medicine and Pathology 2001, 22:112-122 8. Guthkelch AN: Serious effects of shaking were described in 1971, British Medical Journal 1995, 310:1600 9. Margulies SS, Thibault KL: Infant skull and suture properties: Measurements and Implications for mechanisms of pediatric brain injury, Journal of Biomechanics 2000, 122:364-371 10. Graham DI: Paediatric head injury, Brain 2001, 124:1261-1262 11. Hymel KP, Jenny C, Block RW: Intracranial Hemorrhage and Rebleeding in Suspected Victims of Abusive Head Trauma: Addressing the Forensic Controversies, Child Maltreatment 2002, 7:329-348 12. Levin AV: For Debate: Shaken Baby syndrome, British Journal of Neurosurgery 2003, 17:15-16 13. Bonnier C, Mesples B, Gressens P: Animal models of shaken baby syndrome: revisiting the pathophysiology of this devastating injury, Pediatric Rehabilitation 2004, 7:165-171 14. Punt J, Bonshek RE, Jaspan T, McConachie NS, Punt N, Ratcliffe JM: The 'unified hypothesis' of Geddes et al. is not supported by data, Pediatric Rehabilitation 2004, 7:173-184 15. Reece RM: The evidence base for shaken baby syndrome: Response to editorial from 106 doctors, British Medical Journal 2004, 328:1316-1317 16. Healey K, Schrading W: A case of shaken baby syndrome with unilateral retinal hemorrhage with no associated intracranial hemorrhage, The American Journal of Emergency Medicine 2006, 24:616-639 17. Jenny C, Neglect CoCAa: Evaluating Infants and Young Children with Multiple Fractures, Pediatrics 2006, 118:1299-1303
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18. Margulies SS, Prange M, Myers BS, Maltese MR, Ji S, Ning X, Fisher J, Arbogast K, Christian CW: Shaken baby syndrome: A flawed biomechanical analysis, Forensic Science International 2006, 164:278-279 19. Wygnanski-Jaffe T, Levin AV, Shafiq A, Smith C, Enzenauer RW, Elder JE, Morin FD, Stephens D, Atenafu E: Postmortem Orbital Findings in Shaken Baby Syndrome, American Journal of Ophthalmology 2006, 142:233-240 20. Case ME: Abusive head injuries in infants and young children, Legal Medicine 2007, 9:83-87 21. Oechmichen M, Schleiss D, Pedal I, Saternus K-S, Gerling I, Meissner C: Shaken baby syndrome: re-examination of diffuse axonal injury as cause of death, Acta Neuropathologica 2008, 22. Jayawant S, Rawlinson A, Gibbon F, Price J, Schulte J, Sharples P, Sibert JR, Kemp AM: Subdural haemorrhages in infants: population based study, British Medical Journal 1998, 317:1558-1561 23. Geddes JF, Whitwell HL, Graham DI: Traumatic axonal injury: practical issues for diagnosis in medicolegal cases, Neuropathology and Applied Neurobiology 2000, 26:105-116 24. Geddes JF, Hackshaw AK, Vowles GH, Nickols CD, Whitwell HL: Neuropathology of inflicted head injury in children - I. Patterns of brain damage, Brain 2001, 124:1290-1298 25. Geddes JF, Vowles GH, Hackshaw AK, Nickols CD, Scott IS, Whitwell HL: Neuropathology of inflicted head injury in children - II. Microscopic brain injury in infants, Brain 2001, 124:1299-1306 26. Goldsmith W: Fatal pediatric head injuries caused by short-distance falls, American Journal of Forensic Medicine and Pathology 2001, 22:334-336 27. Uscinski R: Shaken baby syndrome: fundamental questions, British Journal of Neurosurgery 2002, 16:217-219 28. Donohoe M: Evidence-Based Medicine and Shaken Baby Syndrome. Part I: Literature Review, 1966-1998, The American Journal of Forensic Medicine and Pathology 2003, 24:239-242 29. Geddes JF, Tasker RC, Hackshaw AK, Nickols CD, Adams GGW, Whitwell HL, Scheimberg I: Dural haemorrhage in non-traumatic infant deaths: does it explain the bleeding in 'shaken baby syndrome'?, Neuropathology and Applied Neurobiology 2003, 29:14-22 30. Geddes JF, Plunkett J: The evidence base for shaken baby syndrome, British Medical Journal 2004, 328:719-720 31. Lantz PE: Response to Reece et al from 41 physicians and scientists, British Medical Journal 2004, 329:741-742 32. Lantz PE, Block RW: Junk Science and Glass Houses, Pediatrics 2004, 114:330 33. LeFanu J, Edwards-Brown R: Subdural and retinal haemorrhages are not necessarily signs of abuse, British Medical Journal 2004, 328:767 34. Miller M, Leestma JE, Barnes P, Carlstrom T, Gardner H, Plunkett J, Stephenson J, Thibault K, Uscinski R, Neidermier J, Galaznik J: A Sojourn in the Abyss: Hypothesis, Theory, and Established Truth in Infant Head Injury, Pediatrics 2004, 114:326
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35. Leestma JE: Case analysis of brain-injured admittedly shaken infants: 54 Cases, 1969-2001, The American Journal of Forensic Medicine and Pathology 2005, 26:199-212 36. Ommaya AK, Goldsmith W, Thibault LE: Biomechanics and neuropathology of adult and paediatric head injury, Journal of Neurosurgery 2002, 16:220-242 37. Goldsmith W, Plunkett J: A biomechanical analysis of the causes of traumatic brain injury in infants and children, The American Journal of Forensic Medicine and Pathology 2004, 25:89-100 38. Roth S, Raul J-S, Ludes B, Willinger R: Finite element analysis of impact and shaking inflicted to a child, International Journal of Legal Medicine 2007, 121:223-228 39. Ervin F, Palmour R: Primates for 21st century biomedicine: The St. Kitts vervet (Chlorocebus aethiops, SK). Edited by Washington DC, National Research Council, 2003, p. pp. 49-53 40. Carlsson H-E, Schapiro SJ, Farah I, Hau J: The use of primates in research: A global overview, Am J Primatol 2004, 63:225-237 41. Napier PH: Catalogue of Primates in the British Museum (Natural History) and Elsewhere in the British Isles. Part II: Family Cercopithecidae, Sub-family Cercopithecinae. Edited by London, British Museum (Natural History), 1981, p 42. Skinner JD, Smithers RHN: The mammals of the Southern African Sub region. Edited by South Africa, University of Pretoria, 1990, p 43. Penning L: Craniovertebral Kinematics in Man and Some Quadrupedal Mammals, Neuro-orthopedics 1995, 17/18:3-20 44. Adams JH, Gennarelli TA, Graham DI: Brain damage in non-missile head injury: observations in man and subhuman primates. Edited by 1982, p. pp. 165-190 45. Adams JH, Graham DI, Gennarelli TA: Neuropathology of Acceleration-Induced Head Injury in the Subhuman Primate. Edited by Grossman RG, Gildenberg PL. New York, Raven Press, 1982, p. pp. 141-150 46. Gennarelli TA, Segawa H, Wald U, Czernicki Z, Marsh K, Thompson C: Physiological Response to Angular Acceleration of the Head. Edited by Grossman RG, Gildenberg PL. New York, Raven Press, 1982, p. 47. Gennarelli TA, Thibault LE, Adams JH, Graham DI, Thompson CJ, Marcincin RP: Diffuse Axonal Injury and Traumatic Coma in the Primate, Annals of Neurology 1982, 12:564-574 48. Gennarelli TA: Head Injury in Man and Experimental Animals: Clinical Aspects, Acta Neurochirurgica 1983, Suppl. 32:1-13 49. Adams JH, Graham DI: Diffuse brain damage in non-missile head injury, Recent Advances in Histopathology 1984, 241-257 50. Gilliland MGF, Levin AV, Enzenauer RW, Smith C, Parsons MA, Rorke-Adams LB, Lauridson JR, La Roche GR, Christmann LM, Mian M, Jentzen JM, Simons KB, Morad Y, Alexander R, Jenny C, Wygnanski-Jaffe T: Guidelines for Postmortem Protocol for Ocular Investigation of Sudden Unexplained Infant Death and Suspected Physical Child Abuse, Am J Forensic Med Pathol 2007, 28:323-329
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51. Shannon P, Smith CR, Deck J, Ang LC, Ho M, Becker L: Axonal injury and the neuropathology of shaken baby syndrome, Acta Neuropathologica 1998, 95:625-631
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Response to reviewers’ comments on a proposal “A primate model for the shaken baby syndrome”
Patrick Jin Han Kim HBSc (MSc candidate) Michael S. Pollanen MD PhD FRCPath DMJ (Path) FRCPC
Department of Laboratory Medicine and Pathobiology, University of Toronto
________________________________________________________________ Overview
In recent ruling on a habeas corpus petition regarding disputed Shaken Baby Syndrome (SBS), an appeal court of the United States overturned a conviction for murder by accepting the defence expert opinion that the SBS was a flawed concept1. Also in the US, a new trial was ordered in a SBS case on the basis of new doubts about the reliability of the SBS concept. In the ruling, the court stated “that a significant and legitimate debate in the medical community has developed in the past ten years over whether infants can be fatally injured through shaking alone, whether an infant may suffer head trauma and yet experience a significant lucid interval prior to death, and whether other causes may mimic the symptoms traditionally viewed as indicating shaken baby or shaken impact syndrome.”2.
Almost every day, a news story could be found where a caregiver is charged with murder of a baby from shaking3-10. However, as one of the reviewers of the proposal pointed out, “in different parts of the world, the justice system proceeds on different bases concerning what is regarded as mainstream thinking in the area”. The origin of this confusion is from the very public disagreement between even the scientists and the experts in the field without empirical scientific evidence to support their positions11-18. Based on the evidence that is available at this moment, it is generally agreed that there are two fundamental questions regarding the shaking of the infants. First is whether or not shaking can cause subdural hemorrhage, retinal hemorrhage and diffuse brain damage, otherwise known as the ‘triad’. The question of specificity of these findings to shaking alone, which is essential in many legal proceedings, is the other fundamental question that still needs to be answered. Many important controversies such as lucid interval, the existence of traumatic diffuse axonal injury, the importance of hypoxic ischemic encephalopathy (as it could be secondary to traumatic injuries to the head or represents an alternate mechanism), and rebleeding of an old subdural hemorrhage. In this proposed pilot study, the investigators do not intend to resolve all of the issues described above. Instead, the investigators intend to establish a reproducible and relevant model of SBS to lay the foundations for the future studies that could resolve many of the issues that could not be achieved by other approaches. On this basis, we have chosen the most critical experimental variables to study in a relevant animal model: (i) axis of displacement; and (ii)
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time interval between shaking and euthanasia. The experimental approach captures essence of shaking motion described by American Academy of Pediatrics19. Although this means that the motion is not controlled rigorously (such as electronically-controlled motion devices), the pilot study aims to describe the mechanical parameters of the manual shaking action which there are great discrepancies in present literatures. In the following discussions, we address the specific issues raised by the reviewers.
Reviewer 1
The first reviewer shared the our view on the state of knowledge in this field and recognized the need of the proposed experiment. The reviewer raised a concern regarding a very important parameter of the proposed experiment: the time interval between shaking event and euthanasia. We have decided to modify the experimental protocol to address this issue. Reviewer 1 indicated: “From a purely technical standpoint, I wonder whether post-shaking anaesthesia for two hours is sufficient time to allow B-APP stainable axon retraction balls to form? They are regarded as taking at least two hours to do so in humans (Whitwell H., Forensic Neuropathology. Hodder Arnold. P97). It would seem wise to ensure there is sufficient time for this part of the pathology to form by perhaps extending this anaesthesia.” Although it is widely recognized that the time interval between cerebral injury and expression of βAPP is at least two hours20-22, there is growing evidence in the literature that it could be detected at earlier time intervals after the injury23. Although longer time intervals would allow broad development of β-APP stainable axonal retraction balls, it also allows secondary changes to the brain due to initial traumatic injuries via generalized swelling leading to non-perfusion anoxia and florid hypoxic ischemic encephalopathy24. Therefore, allowing sufficient time for βAPP development could give rise to confounding results that overshadows true injuries produced by shaking. Furthermore, tDAI related expression of βAPP has been disputed recently (as retrospective studies have shown that diffuse axonal injuries are not present in majority of the presumed shaken cases and those with positive βAPP immunostaining are either local axonal injuries in the brainstem known as stretch injuries or vascular (implying hypoxic) in nature25-27.
To resolve this issue, we have modified the experimental groups into two distinct time points: (i) immediate euthanasia (no survival) and (ii) 6 hour survival under general anesthesia. Immediate euthanasia will be used to access hemorrhagic injuries (RH, SDH) that do not require survival interval, and the 6 hour survival interval will be used to access the development of axonal findings as detected by immunohistochemical staining of βAPP.
Using these two distinct time points, any traumatically-induced hemorrhages detected at immediate euthanasia will lack any confounding effect from generalized hypoxic changes or alterations in vascular permeability that might
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occur in the 6 hour interval of survival. Therefore, any SDH or RH can be definitely linked to trauma alone.
On this basis, we have decided to postpone one of the research questions from the original protocol. We no longer propose to study the effect of episodic shaking. The number of animals to be used in the proposed experiments remains at six. We believe this is the minimum number of the animals required to address the experimental questions we have asked (Table 1).
Table 1. Summary of animal allotment including control animals.
Animal Number Axis of displacement
Time interval between shaking and
euthanasia (Hrs) Other
Negative control n/a n/a Anesthesia followed by
euthanasia only
Positive control n/a Six Traumatic blunt impact head and
brain injury
1 Anterior-Posterior Immediate
2 Anterior-Posterior Six
3 Lateral Immediate
4 Lateral Six
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Reviewer 2
The second reviewer raised concerns with regards to the appropriateness of the model and validity of shaking mechanism. The reviewer suggests an alternative model: a piglet model that has been used in vehicular trauma research. Although the piglet model has its advantages in modelling infant trauma, it has to be balanced with other anatomical and mechanical factors before being employed for this particular research. In general, following factors should be considered in selecting an appropriate animal model of human traumatic neuropathology28.
1. Ability to reproduce hypothetical mechanism
“In the experimental design, there is lack of detail on the frequency of the shaking “the animal will be firmly held by the chest and continue shaking 20 times in the anterior posterior plane at maximum head displacement generated manually.”” In a technical report on SBS, American Academy of Pediatrics stated that “the act of shaking leading to shaken baby syndrome is so violent that individuals observing it would recognize it as dangerous and likely to kill the child. Shaken baby syndrome injuries are the result of violent trauma.”19. At this time, the authors cannot determine the time needed to conduct 20 maximal head displacements (which will subsequently allow the rough estimation of the frequency). Part of the goals of this proposed pilot study is to establish these primary parameters that will be used for the subsequent studies. 2. Mechanical parameters/skull base geometry
“The objective and specific aim is to use non-human primates, specifically the African green monkey to reproduce the shaking injury in a model which reproduces the size and special anatomy of the human head and neck at two months. The experimental work proposed utilizes the African green monkey because “based on these dimensional considerations, adult African green monkeys are similar in weight to two months infants. Most importantly, subhuman primates share with man important cranial cervical anatomical similarities which are not found in lower animals.” This commentary indicates that the primate head would be approximately the same size as a two-month-old human infant and the relationship of the neck to the head would be similar to humans. This requires confirmation.” This comment raises two of the factors that the investigators considered in choosing subhuman primates for this proposed study. First is the dimensional similarity of subhuman primate head to that of the infants of the relevant age. Since one of the goals of this pilot study is to describe mechanical parameters of manual shaking such as frequency, maximum acceleration and trajectory, it is important to have the size and dimension closely matched. The dimensional similarity can be verified by comparing the relevant values from the growth charts of the infants29, 30 and that of the animals31, 32. More importantly, it is the geometrical consideration of the skull base, specifically the position of the
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foramen magnum and its orientation and the anatomy of the craniovertebral junction. We believe these anatomical factors shared by the African Green Monkeys are critical to success of the proposed experiment33, 34. Paleontological evidence shows that the position and orientation of foramen magnum in higher order species is linked to the evolutionary transition to the bipedalism where more ventral and horizontal which allows the position of the head to be more orthogonal to the ground35-37. This has important implication on the human head movement when compared to obligatory quadrupedal mammals (such as the piglet) as extension motion is reduced by 50 degrees, sagittal head-trunk translation is largely lost, and lateral flexion of the craniovertebral junction is also greatly reduced33. Due to these differences in mechanics of head movement, it is critical to employ anatomically similar animal in modeling shaking head injuries. Therefore, the use of African Green Monkeys in mechanical modelling of head injury is justified as they possess a clear anatomical similarity to human than the piglets. 3. Age matching/developmental stage “An alternate animal model which may have similar anatomic characteristics and be similar in the state of the development of the brain may be piglets. I understand that piglets have been used to study traumatic injury by the Insurance Institute of Highway Safety, Charlottesville, Virginia, USA. I do not know whether the information from these studies is publicly available.” There are studies published by a group in University of Pennsylvania using the swine model in this subject but generally are not accepted as true model of shaking. In these studies, the piglets are immobilized while their heads are subjected to a single, rapid (~40ms) angular acceleration controlled by actuator38-
40. It is ironic that this is the same group that initially explored baboon-macaque model with similar experimental set up and their study terminated due to ethical concerns raised by activist groups. Indeed, the subsequent swine studies essentially continued the research established the baboon-macaque model. However, the anatomical differences between human infants and piglets (described above) have resulted in the general belief that there is no valid model of shaking injuries in human infants, with exception of 11, 41-44. “Scientifically, what this model does not address is the difference between the immature brain and its coverings in the two-month-old human infant and that of the adult non-human primate brain. The infant brain and its vessels would be expected to be more mobile and more fragile than an adult brain. Our societal norm is to reduce invasive studies utilizing non-human primates, except when absolutely required. A key question is whether the brain anatomy and physiology of adult non-human primates is sufficiently similar to infant humans. The infant pig should be considered as an alternative animal model for these experiments.”
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The argument for the supposed fragility of the vessels in infants during shaking comes from the original lecture by Caffey as he proposed the mechanism of subdural hemorrhage during shaking is the tearing of the veins that transverses subdural space45. However, currently available mechanical studies of infant vessel properties do not give conclusive answer to this question as these studies are limited due to their inability to reproduce complex mechanical relationships that these vessels are exposed to such as brain dimensions, anatomical location of vessels and axis of angular displacement46. In this pilot study, one of the aims is to describe the mechanical displacement parameters of manual shaking. For this purpose, having dimensionally (size and weight of the head) similar animal, such as subhuman primates, will offer more relevant information for future studies in the field. Once baseline mechanical displacement parameters are established, attempts of scaling could be made for future studies of smaller but developmentally/physiologically similar animals. Overall, although piglet model could offer some advantages in modelling developmentally immature structures of the infant head, it does not possess anatomical (geometrical consideration of the skull base) and mechanical advantages of the primate model (ability to be shaken in a manner described by American Academy of Pediatrics and reproduce head motion of infants). As for pilot study, the study’s focus is to test plausibility of the model by limiting number of variables. Although these questions are important in the field, they could be studied better once the repeatable model is established. Conclusion The past attempts in modelling shaking head injuries in animals are limited in value due to the inappropriateness of the mechanisms that are used to inflict the injury. Although some models succeeded in reproducing the head injury38-40, 47-50, they failed to answer the fundamental question of whether or not shaking could cause these injuries. While this proposed study cannot address all questions, it serves as a starting point. The proposed experiments will help establish a direction for future research and shift the focus of the debate to empirical-experimental data. Once again, we believe the need for a primate model is justified. Much of the confusion in the field stems from the lack of agreement as to what SBS actually entails because there is no conclusive data that supports that shaking alone can produce lethal injuries. The closely related anatomical relationship between human and the subhuman primates will allow important new data to be gained from the proposed pilot study. These data can be the preliminary results for a more programmatically-focussed investigation of SBS using an experimental approach.
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