Neuropharmacology and analgesia Neuropeptide Y is analgesic in rats after plantar incision Suraj M. Yalamuri, Timothy J. Brennan, Christina M. Spofford n Department of Anesthesia, University of Iowa, 200 Hawkins Drive, 6 JCP Iowa City, IA 52242, United States of America article info Article history: Received 11 June 2012 Received in revised form 16 October 2012 Accepted 23 October 2012 Available online 2 November 2012 Keywords: NPY (Neuropeptide Y) Incision Pain behavior Gene Protein expression abstract Previous work has demonstrated that neuropeptide tyrosine (NPY), Y 1 receptor and Y 2 receptor are critical in modulation of pain after nerve injury. We hypothesized that NPY was important for nociception after surgical incision. As a model of postoperative pain, rats underwent a plantar incision in one hindpaw. Western blots were used to quantify changes in protein expression of NPY, Y 1 receptor and Y 2 receptor after incision in skin, muscle, and dorsal root ganglion (DRG). Pain-related behaviors were tested after incision in rats treated with intrathecal NPY, Y 1 receptor antagonist (BIBO3304 – Chemical Name: N-[(1R)-1-[[[[4- [[(Aminocarbonyl)amino]methyl]phenyl]methyl]amino]carbonyl]-4-[(aminoiminomethyl)amino]butyl]-a- phenyl-benzeneacetamide ditrifluoroacetate), Y 2 receptor antagonist (BIIE0246 – Chemical Name: N-[(1S)- 4-[(Aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]- butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentane- acetamide), combined NPYþantagonists, morphine, or vehicle. Pain behaviors were tested after incision in rats treated with locally applied intraplantar injections of NPY, Y 1 receptor and Y 2 receptor antagonists or vehicle. NPY protein expression was significantly downregulated in muscle for two days after incision. In contrast, Y 1 receptor and Y 2 receptor protein expression was upregulated in both skin and muscle. A single intrathecal injection of NPY reduced cumulative guarding pain scores, as did morphine. The intrathecal administration of Y 2 receptor antagonist also reduced pain scores; findings that were not observed when drugs were administered locally. Intrathecal Y 2 receptor antagonists and NPY improved mechanical threshold and heat withdrawal latency 2 h after incision. Intrathecal administration of NPY and/or central blockade of Y 2 receptor attenuated pain behaviors early after incision (postoperative day (POD) 1–2 ). Y 1 receptor antagonist administration blocked the anti-hyperalgesic effect of NPY. Together these data suggest a role for spinal NPY in postoperative pain. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Pain is an important parameter in patient recovery and satisfac- tion after surgery. For many patients, pain due to surgical incision can be severe despite treatment with conventional use of opioids, non-steroidal anti-inflammatory drugs (NSAIDs) and local anes- thetics (Apfelbaum et al., 2003). Research into post-operative pain mechanisms has provided some insight into the potential factors involved in the initiation and maintenance of pain after surgery. One factor showing significant regulation in expression after plantar incision is neuropeptide Y (NPY) (Spofford and Brennan, 2012). NPY is a 36 amino acid peptide, which is widely distributed in the central and peripheral nervous systems. Peripherally, NPY is abundant in the sympathetic nervous system, where it is stored and released with norepinephrine. NPY exerts its biological action via five G-protein coupled receptors (Y 1 receptor–Y 5 receptor) that have been characterized based on their physiological effects. The physiological mechanisms affected by each of these receptors has been partially explored in the neurovascular system (Hodges et al., 2009; Lin et al., 2004) as well as pain from diabetes and intradermal capsaicin (Franco-Cereceda and Liska, 1998; Gibbs et al., 2007). However, there are no reports about the role of NPY in plantar incision. Previous work by others has suggested NPY may sensitize primary sensory afferents. The Y 1 receptor and Y 2 receptor are the likely receptors involved in the transmission and modulation of pain after spared nerve injury (Intondi et al., 2008). However, the role of each of these receptors is controversial with some reports suggest- ing these receptors can have a pronociceptive, antinociceptive, or mixed effects (Brumovsky et al., 2007; Smith et al., 2007). In order to elucidate the role of NPY in post-incision pain processing in the peripheral nervous system, we examined the role of NPY in a well-established plantar incision pain model (Brennan et al., 1996). We hypothesize that NPY and its receptors, Y 1 receptor and Y 2 receptor, are important for nociception after surgical incision. 2. Materials and methods 2.1. Animals This study was approved by the Institutional Animal Care and Use Committee at the University of Iowa and all experiments Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/ejphar European Journal of Pharmacology 0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.10.036 n Corresponding author. Tel.: þ1 319 356 2633; fax: þ1 319 356 2940. E-mail address: [email protected] (C.M. Spofford). European Journal of Pharmacology 698 (2013) 206–212
Transcript
European Journal of Pharmacology 698 (2013) 206–212
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology
0014-29
http://d
n Corr
E-m
journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Neuropeptide Y is analgesic in rats after plantar incision
Suraj M. Yalamuri, Timothy J. Brennan, Christina M. Spofford n
Department of Anesthesia, University of Iowa, 200 Hawkins Drive, 6 JCP Iowa City, IA 52242, United States of America
a r t i c l e i n f o
Article history:
Received 11 June 2012
Received in revised form
16 October 2012
Accepted 23 October 2012Available online 2 November 2012
acetamide), combined NPYþantagonists, morphine, or vehicle. Pain behaviors were tested after incision in
rats treated with locally applied intraplantar injections of NPY, Y1 receptor and Y2 receptor antagonists or
vehicle. NPY protein expression was significantly downregulated in muscle for two days after incision. In
contrast, Y1 receptor and Y2 receptor protein expression was upregulated in both skin and muscle. A single
intrathecal injection of NPY reduced cumulative guarding pain scores, as did morphine. The intrathecal
administration of Y2 receptor antagonist also reduced pain scores; findings that were not observed when
drugs were administered locally. Intrathecal Y2 receptor antagonists and NPY improved mechanical
threshold and heat withdrawal latency 2 h after incision. Intrathecal administration of NPY and/or central
blockade of Y2 receptor attenuated pain behaviors early after incision (postoperative day (POD) 1–2 ). Y1
receptor antagonist administration blocked the anti-hyperalgesic effect of NPY. Together these data suggest
a role for spinal NPY in postoperative pain.
& 2012 Elsevier B.V. All rights reserved.
1. Introduction
Pain is an important parameter in patient recovery and satisfac-tion after surgery. For many patients, pain due to surgical incisioncan be severe despite treatment with conventional use of opioids,non-steroidal anti-inflammatory drugs (NSAIDs) and local anes-thetics (Apfelbaum et al., 2003). Research into post-operative painmechanisms has provided some insight into the potential factorsinvolved in the initiation and maintenance of pain after surgery. Onefactor showing significant regulation in expression after plantarincision is neuropeptide Y (NPY) (Spofford and Brennan, 2012).
NPY is a 36 amino acid peptide, which is widely distributed inthe central and peripheral nervous systems. Peripherally, NPY isabundant in the sympathetic nervous system, where it is stored andreleased with norepinephrine. NPY exerts its biological action viafive G-protein coupled receptors (Y1 receptor–Y 5 receptor) thathave been characterized based on their physiological effects. Thephysiological mechanisms affected by each of these receptors hasbeen partially explored in the neurovascular system (Hodges et al.,
ll rights reserved.
: þ1 319 356 2940.
M. Spofford).
2009; Lin et al., 2004) as well as pain from diabetes and intradermalcapsaicin (Franco-Cereceda and Liska, 1998; Gibbs et al., 2007).However, there are no reports about the role of NPY in plantarincision. Previous work by others has suggested NPY may sensitizeprimary sensory afferents. The Y1 receptor and Y2 receptor are thelikely receptors involved in the transmission and modulation of painafter spared nerve injury (Intondi et al., 2008). However, the role ofeach of these receptors is controversial with some reports suggest-ing these receptors can have a pronociceptive, antinociceptive, ormixed effects (Brumovsky et al., 2007; Smith et al., 2007).
In order to elucidate the role of NPY in post-incision painprocessing in the peripheral nervous system, we examined the roleof NPY in a well-established plantar incision pain model (Brennanet al., 1996). We hypothesize that NPY and its receptors, Y1 receptorand Y2 receptor, are important for nociception after surgical incision.
2. Materials and methods
2.1. Animals
This study was approved by the Institutional Animal Care andUse Committee at the University of Iowa and all experiments
S.M. Yalamuri et al. / European Journal of Pharmacology 698 (2013) 206–212 207
adhered to the ethical guidelines for investigations of experi-mental pain in conscious animals. Adult male Sprague-Dawleyrats (Harlan, Indianapolis, IN, USA), 250 to 350 g, were used for allexperiments. Rats were housed in groups of 2 on a 12-h light and12-h dark schedule with food and water made available adlibitum.
2.2. Plantar incision
Plantar incision was made as previously described (Brennanet al., 1996). Briefly, rats were anesthetized with isoflurane (1.5–3%). The right hindpaw was prepared with Povidone iodine anddraped. A 1-cm longitudinal incision was made 0.5 cm from theheel on the right hindpaw through the skin, fascia, and the plantarflexor digitorum brevis muscle. The flexor digitorum brevismuscle was raised and stretched. The wound was closed with2 mattress sutures with 5-0 nylon on an FS-1 needle. Shamsurgeries consisted of all procedures except incision. All rats wereallowed to recover from anesthesia before being returned to anew, clean cage for testing.
2.3. Western blot analysis
Incised and sham-operated rat hindpaw plantar skin, andflexor digitorum brevis muscle were frozen in liquid nitrogenand stored at �80 1C. Frozen plantar muscle and skin tissue wascrushed in liquid nitrogen using a pestle and mortar and trans-ferred to ice-cold 0.2 M RIPA lysis buffer [100 mM Tris–HCl ,pH 7.4, 300 mM NaCl, 0.5% deoxycholic acid, 2% NP-40, 2 mMEDTA] (Millipore, Billerica, MA, USA) containing protease andphosphatase inhibitor cocktails (Sigma–Aldrich, St. Louis, MO,USA) for 45 min before being homogenized on ice. Lumbar level4 and 5 dorsal root ganglion (DRG) from incised and sham-operated rats were pooled and frozen in liquid nitrogen withsubsequent storage at �80 1C. DRG were processed in the samemanner as skin and muscle except they underwent disruptionwith a 60-sonic dismembrator (Fisher Scientific, Pittsburg, PA,USA) for 60 s. Lysates were centrifuged at 10,000 g for 20 min at4 1C and protein concentration of the supernatant was quantifiedusing the BCA protein assay (Pierce, Rockford, IL, USA). All proteinlysates were denatured by boiling in reducing sample buffer[1.25% 0.5 M Tris–HCl , pH 6.8, 2% sodium dodecyl sulfate (SDS),10% glycerol, 1.5% 2-b mercaptoethanol, 0.0025% bromophenolblue] for 5 min at 99 1C. Thirty mg of total protein for each samplewas electrophoresed (130 V for 1.5 h) on 4–20 % SDSPAGE gels(Bio-Rad, Hercules, CA, USA). Proteins were transferred ontopolyvinylidene fluoride (PVDF) membranes (0.45 mm Immobi-lon-P; Millipore) by semidry electrophoresis (15 V for 1 h). Forimmunodetection, membranes were incubated in blocking buffer3% (w/v) BSA and 0.01% (v/v) Tween 20 in 0.1 M Tris-bufferedsaline (TTBS) (20 mM Tris, 500 mM NaCl, pH 7.5) for 1 h at roomtemperature followed by washing with 0.1 M TTBS. Membraneswere then incubated overnight with rabbit anti-NPY (FL-97)(1:200) or goat anti-Y 1 receptor (A-17) (1:200) or goat anti-Y 2
receptor (L-17) (1:200) (Santa Cruz Biotechnologies, Santa Cruz,CA, USA) diluted in blocking buffer 4 1C while being gently rocked.Membranes were washed 3 times in 0.1 M TTBS and incubated inIR Dye 680CW conjugated donkey anti-goat or donkey anti-rabbitIgG secondary antibody (1:5000; LI-COR Biosciences, Lincoln, NE,USA) in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE,USA) for 2 h at room temperature. Where appropriate, afterincubating the membranes in RestoreTM stripping buffer (Pierce,Rockford, IL, USA), membranes were incubated in blocking bufferand reprobed for the housekeeping gene, actin, overnight at 4 1Cusing mouse anti-actin (1:1000; Millipore) diluted in blockingbuffer. Membranes were washed in 0.1 M TTBS and incubated in
IR Dye 800CW conjugated donkey anti-mouse IgG secondaryantibody (1:5000; LI-COR Biosciences) in Odyssey blocking bufferfor 2 h at room temperature. All membranes were imaged usingthe Odyssey infrared imaging system (LI-COR Biosciences) at scanintensity and resolution of 1 and 84 mm, respectively. NPY, Y1
receptor and Y2 receptor protein expression levels were deter-mined using integrated intensities quantified using Image J 1.41o(NIH, Bethesda, MD, USA). An unloaded gel lane integrateddensity value was subtracted from the integrated densities fromNPY, Y1 receptor, Y2 receptor, and actin bands. These values werethen used to normalize expression of NPY, Y1 receptor, and Y2
receptor to the internal loading control, actin. Each time pointstudied consisted of 4 rats that underwent incision and 2 rats thatunderwent sham surgery. Data from sham rats pooled afterpreliminary analysis showed no change in NPY, Y1 receptor, andY2 receptor protein bands or levels in the tissues across all timepoints.
2.4. ELISA
Incised and sham-operated rat right hindpaw L4 and L5 DRGwere pooled and frozen in liquid nitrogen and stored at �80 1C.Frozen DRG was pounded in liquid nitrogen using a pestle andmortar and transferred to ice-cold 1� DPBS (Invitrogen, GrandIsland, NY, USA) containing protease and phosphatase inhibitorcocktails (Roche, Indianapolis, IN, USA) for 45 min with occasionalvortexing before being homogenized on ice. Lysates were cen-trifuged at 10,000 g for 15 min at 4 1C and protein concentrationof the supernatant was quantified using the BCA protein assay(Pierce, Rockford, IL, USA). The NPY levels of DRG were quantifiedusing NPY EIA kit (Phoenix Pharmaceuticals, Belmont, CA, USA).The standards and protein lysates were pipetted into the wellsprecoated with NPY-specific rat antibody. An enzyme-linkedpolyclonal antibody specific for rat NPY was added to the wellsand incubated for 2 h. Any unbound antibody–enzyme reagentwas removed by washing. Following the wash HRP substratesolution was added to the wells. The enzyme reaction wasstopped using 2 N HCl. The microplates were read at 450 nm.
2.5. Behavioral studies
We measured withdrawal threshold to mechanical stimuli, with-drawal latency to radiant heat, and nonevoked guarding painbehavior. Following 3 days of acclimation to the testing environmentand personnel, baseline measurements for all 3 behavioral tests weremade. All personnel were blinded to the treatment group. Forguarding behavior, a cumulative pain score was used to assessnonevoked pain behavior as described previously (Brennan et al.,1996). Briefly, unrestrained rats were placed onto a plastic meshsurface (8�8-mm2 grid) under a plastic chamber (21�27�15 cm3)for 15 min before testing. Ipsilateral and contralateral hindpawswere assessed for each animal for 1 min repeating every 5 min for1 h (30 min for intrathecal drug administration). Following each1 min observation period of both hindpaws, a score was assignedto each paw; a 0, 1, or 2 score was given accordingly. A 0 score wasrecorded when the hindpaw touched the mesh causing skin toblanch, a score of 1 was recorded when the hindpaw was partiallyoff the mesh surface, and a score of 2 was recorded when thehindpaw was completely off the mesh surface. The sum of the 12scores (0–24) recorded for each paw was obtained. The differencebetween the scores from the incised paw and nonincised paw wasthe cumulative pain score.
For withdrawal threshold to mechanical stimuli, unrestrained ratswere individually placed onto a plastic mesh surface (12�12-mm2
grid) under a plastic chamber (21�27�15 cm3) for approximately30 min of acclimation. Using openings created by the mesh surface,
S.M. Yalamuri et al. / European Journal of Pharmacology 698 (2013) 206–212208
calibrated Semmes–Weinstein monofilaments (North Coast Medical,Morgan Hill, CA, USA) were applied from underneath the meshsurface to the glabrous skin of the hindpaw. Each filament wasapplied once until the filament bent. We started with the lowest forceof 13 mN and incrementally increased until an abrupt paw with-drawal from the mesh was observed or a force of 228 mN wasreached. If a rat failed to respond to the 228 mN filament, the nextfilament (522 mN) was recorded as the threshold. Each test wasrepeated after a 5-min test-free interval, for a total of 3 tests per rat.The lowest force from the 3 tests producing a response wasconsidered the withdrawal threshold. For withdrawal latency toradiant heat stimuli, unrestrained rats were individually placed ontoa 3-mm thick glass surface (40�40 cm) under a plastic chamber(21�27�15 cm3) for approximately 15 min of acclimation. Afocused radiant heat source was applied from underneath the glassfloor onto the center of incision site. The latency to evoke withdrawalwas determined with a cut-off value of 30 s to prevent radiant heatevoked injury to the hindpaw. Each rat was tested 3 times, with a5 min interval between tests. The mean paw withdrawal latency wasdetermined from 3 tests. Guarding pain was performed before with-drawal tests to avoid the potential influence of withdrawal responseson guarding pain. Pain behavior experiment time points were base-line, 2 h postoperative (PO 2 h) and postoperative days 1-5, (POD 1-5).
2.6. Intraplantar drug administration and behavior testing
In a separate series of experiments after hindpaw incision wasmade and the wound sutured, the skin glue, DermabondTM (Ethicon,Inc, Somerville, NJ, USA) was applied to seal the incision and toprevent drug leakage. When the glue had dried, 100 mL of either 0.9%saline, NPY (300 nM), Y1 receptor antagonist (BIBO3304) (0.2 mM),or Y2 receptor antagonist (BIIE0246) (1 mM) (all drugs from TocrisBiosciences, Ellisville, MO, USA) was injected subcutaneously into thehindpaw. These doses previously shown to be efficacious in a painmodel using hindpaw inflammation (Gibbs et al., 2006) or sparednerve injury (Intondi et al., 2008). All rats were allowed to recover for2 h before undergoing pain behavior testing. Only guarding and heatresponses were measured. Mechanical thresholds could not bemeasured because the DermabondTM seal reduced sensitivity tomechanical stimuli in the area where the filaments were applied(unpublished observation).
2.7. Intrathecal drug administration and behavior testing
In a separate group of rats, plantar incision was performed andthe rats were allowed to recover from anesthesia. After recovery,rats were lightly anesthetized with 1% isoflurane and theintrathecal space at the level of L4-L5 was accessed with a27 gauge 1.5 in. hypodermic needle. A tail flick or hindpaw twitchmotor response was used as confirmation for the needle beinglocated in the intrathecal space. After aspirating to confirm non-vascular access, either 0.9% saline, 10 mg of morphine sulfate(Baxter Healthcare Corporation, Deerfield, IL, USA), NPY Y1 recep-tor antagonist (BIBO3304), Y2 receptor antagonist (BIIE0246) (alldrugs from Tocris Biosciences, Ellisville MO, USA), NPYþY1
receptor antagonist, NPYþY2 receptor antagonist was injectedinto the intrathecal space in a volume of 10 ml followed by a 10 mlflush of saline. Guarding, heat responses, and mechanical thresh-olds were assessed 2 h after incision.
2.8. Statistical analysis
All data were analyzed by Prism 5.0 software (GraphPadSoftware, San Diego, CA, USA) and Po0.05 was consideredsignificant. For qRT–PCR data, analysis of significant change in
NPY, Y1 receptor, and Y2 receptor gene expression, t-tests withSAM analysis were performed on each gene at each time pointcomparing the mean delta CT (DCT) values from the incised groupto the sham-operated group (n¼4 rats per group). Data wereexpressed as mean7S.D. For western blot data, differencesbetween means were determined by 1-way analysis of variancefollowed by Dunnett’s post hoc tests versus sham. Data wereexpressed as mean7S.E.M. For behavioral data, guarding andwithdrawal latency to heat data was analyzed using 2-wayanalysis of variance (ANOVA), followed by 1-way ANOVA andpost hoc t-test with Bonferroni correction. Data were expressed asmean7S.E.M. Friedman’s 2-way ANOVA followed by Kruskal–Wallis with Dunns’ post hoc test was used for withdrawalthreshold to mechanical stimulation. Data are expressed asmedian and interquartile range.
3. Results
3.1. Expression of NPY, Y1 receptor, and Y2 receptor in skin following
plantar incision
We recently reported that compared to sham, NPY messengerRNA (mRNA) in skin was immediately upregulated at 1 and 4 hafter plantar incision and Y1 receptor mRNA expression wasdownregulated early after incision with a maximal response atPOD 1. Y2 receptor mRNA expression did not change in skin afterplantar incision (Spofford and Brennan, 2012).
Western blot analysis after incision showed that NPY proteinsignal in the skin was too low to be accurately analyzed andproduced unreliable fold change values (Fig. 1A). However, bothY1 receptor and Y2 receptor protein were elevated after incisionwith a maximal fold change occurring at POD 1 of 3.2271.50 forY1 receptor (Fig. 1B) and 7.3972.26 for Y2 receptor (Fig. 1C).Y2 receptor protein showed the greatest change and was elevatedat 12 h to 2 days after incision.
3.2. Expression of NPY, Y1 receptor, and Y2 receptor in muscle
following plantar incision
NPY mRNA expression was previously shown to be signifi-cantly upregulated 2 days after incision (Spofford and Brennan,2012). Y1 receptor mRNA expression was downregulated earlyafter incision with a maximal response of �6.0170.4 occurringon POD 1. While the Y2 receptor mRNA expression did not changein skin, it showed a significant bi-phasic response in muscle. Themaximal downregulation was at POD 1 at �48.1770.01 and itwas maximally upregulated at POD 2 with a fold change of51.76726.02 (Spofford and Brennan, 2012).
Western blot analysis revealed a trend of NPY protein down-regulation and receptor upregulation after incision. NPY proteinwas decreased at POD 1 (�3.2172.03) and POD 2 (�2.1970.77;Fig. 2A). Y1 receptor protein was elevated after incision with amaximal fold change of 1.8970.42 occurring at POD 5 for muscle(Fig. 2B). Once again, Y2 receptor protein showed the greatestchange and was elevated at 12 h to 2 days after incision. Themaximal response of 13.3675.41 occurred on POD 1 (Fig. 2C).
3.3. Expression of NPY, Y1 receptor, and Y2 receptor in dorsal root
ganglion following plantar incision
In our previous work, we have shown that NPY, Y1 receptor,and Y2 receptor mRNA were detected in L4-L5 DRG, but did notshow any changes following plantar incision, (Spofford andBrennan, 2012). We were unable to detect NPY, Y1 receptor, orY2 receptor protein in L4-L5 DRG via a western blot analysis (data
Fig. 1. Time course of protein expression of NPY (A), Y1 receptor (B), and Y2
receptor (C) protein in skin following plantar incision. Data are expressed as mean
fold change (incision/sham)7S.E.M. n¼10 in sham group and n¼4 in incised
group. *Po0.05; **Po0.01; ***Po0.001.
Fig. 2. Time course of protein expression of NPY (A), Y1 receptor (B), and Y2
receptor (C) protein in muscle following plantar incision. Data are expressed as
mean fold change (incision/sham)7S.E.M. n¼10 in sham group and n¼4 in
incised group. *Po0.05; **Po0.01; ***Po0.001.
S.M. Yalamuri et al. / European Journal of Pharmacology 698 (2013) 206–212 209
not shown). In order to increase sensitivity of detection, we used acommercially available NPY ELISA to detect and quantify NPYprotein expression. On POD 1, the concentration of NPY in theDRG was increased, but failed to reach statistical significance(0.2670.22 v. 0.0870.07; P¼0.093).
3.4. Behavioral studies
To determine whether NPY could be analgesic in the periphery,intraplantar administration of NPY, Y1 receptor antagonist, and Y2
receptor antagonist was performed. These drugs did not affectguarding pain scores or heat hyperalgesia (Fig. 3). Withdrawalthreshold to von Frey filaments was not tested after local drugadministration as noted in the methods.
In contrast to local administration of NPY, intrathecal admin-istration of NPY decreased cumulative guarding pain scores 2 hafter incision (1.871.0 v. 8.271.5); an effect that remainedthrough POD 1 (4.271.2 v. 8.571.4) compared to saline controls(Fig. 4A). The Y2 receptor antagonist also decreased cumulativeguarding pain scores 2 h after incision (4.871.2 v. 8.271.5) andPOD 1 (5.570.8 v. 8.571.4; Fig. 4A). Combined administration ofNPYþY2 receptor antagonist reduced guarding pain scores for alonger duration with a significant reduction seen through POD 2(3.871.0 v. 5.271.0; Fig. 4B). This decrease in pain scores was
Fig. 3. Effect of intraplantar administration of saline, NPY, Y1 receptor antagonist, or Y2 receptor antagonist following plantar incision on nonevoked guarding pain (A) and
withdrawal latency to heat (B). Data are expressed as mean7S.E.M. n¼6 per group. Base¼baseline. HRS¼hours.
Fig. 4. Effect of intrathecal administration of saline, morphine, NPY, Y1 receptor antagonist, Y2 receptor antagonist following plantar incision on nonevoked guarding pain
(A). Effect of intrathecal administration of saline, Y1 receptor antagonistþNPY, and Y2 receptor antagonistþNPY following plantar incision on nonevoked guarding pain (B).
Data are expressed as mean7S.E.M. n¼6 per group. *Po0.05; **Po0.01; ****Po0.0001. Significance compared against saline group at each time point.
Fig. 5. Effects of intrathecal administration of saline, morphine, NPY, Y1 receptor antagonist, Y2 receptor antagonist following plantar incision on heat withdrawal latency (A).
Effects of intrathecal administration of saline, Y1 receptor antagonistþNPY, and Y2 receptor antagonistþNPY following plantar incision on heat withdrawal latency (B). Data
are expressed as mean7S.E.M. n¼6 per group. *Po0.05. Significance compared against saline group at each time point.
S.M. Yalamuri et al. / European Journal of Pharmacology 698 (2013) 206–212210
greater to the time frame and effect seen with intrathecalmorphine (Fig. 4A). The Y1 receptor antagonist did not changepain behaviors compared with saline controls and the combined
administration of Y1 receptor antagonistþNPY reversed theanti-hyperalgesic effects of NPY at PO 2 h (6.871.8 v. 1.871.0)and POD 1 (8.070.6 v. 4.271.2; Fig. 4B).
Fig. 6. Effects of intrathecal administration of saline, morphine, NPY, Y1 receptor antagonist, Y2 receptor antagonist following plantar incision on mechanical withdrawal
threshold (A). Effects of intrathecal administration of saline, Y1 receptor antagonistþNPY, and Y2 receptor antagonistþNPY following plantar incision on mechanical
withdrawal threshold (B). Data are expressed as expressed as median and interquartile range. n¼6 per group. *Po0.05. Significance compared against saline group at each
time point.
S.M. Yalamuri et al. / European Journal of Pharmacology 698 (2013) 206–212 211
Intrathecal administration of NPY increased heat withdrawallatency 2 h after incision (6.770.7 s v. 2.670.2 s) an effect thatwas significant again on POD 3 (8.271.3 s v. 5.271.1 s; Fig. 5A)compared to saline controls. As seen in the guarding pain test, theadministration of NPY was similar to the effect seen withintrathecal morphine 2 h after incision (6.171.9 s; Fig. 5A). Y2
receptor antagonist (4.370.7 s) and combined Y2 receptor antag-onistþNPY (4.270.9 s) also increased heat withdrawal latency2 h after incision compared to saline controls (2.670.2 s; Fig. 5Aand B). Withdrawal latency did not differ from saline controlswith the administration of Y1 receptor antagonist (Fig. 5A) andthe combined administration of NPYþY1 receptor antagonistattenuated the analgesic effect of NPY (Fig. 5B).
Withdrawal threshold to von Frey filaments was increased 2 hpost-incision after intrathecal administration of NPY (2097197 mN),Y2 receptor antagonist (2347178 mN), and morphine (2467210mN) compared to saline controls (58715 mN; Fig. 6A). There was nodifference in the withdrawal threshold at other times and with otherexperimental groups (Fig. 6A and B).
4. Discussion
4.1. NPY expression after incision
NPY, Y1 receptor, and Y2 receptor are found in the periphery(Bjur et al., 2009; Brumovsky et al., 2007; Lin et al., 2004; Lundberget al., 1983) and NPY is increased in the DRG following neuropathicor inflammatory injury (Intondi et al., 2010; Landry et al., 2000). Inskin, mRNA expression of NPY is upregulated early after incisionand receptors are downregulated (Spofford and Brennan, 2012).After incision, NPY protein levels were decreased and both Y1
receptor and Y2 receptor protein levels were increased.The pattern of the trends observed in skin was exaggerated in
muscle: the level of NPY protein was decreased whereas theprotein level of both the receptors was markedly increased.Specifically, Y2 receptor protein was increased when pain beha-viors were the greatest (POD 1 and 2). mRNA expression showedregulation at earlier time points compared to that of protein.
DRG expression of mRNA for NPY, Y1 receptor, and Y2 receptorwas not regulated after incision (Spofford and Brennan, 2012). Wequantified NPY protein in the DRG using a sensitive ELISA andfound no changes after incision on POD 1. This measure quantifiesthe amount of protein, but does not address the release of NPY, alimitation of the study. Taken together, these data suggest thatNPY and its receptors are differentially regulated in skin andmuscle following incision. Given the many sites of NPY storageperipherally, these changes could be due to a response to wound
healing, sympathetic response following incision, and/or modula-tion of nociceptive signals.
4.2. Pain behaviors and NPY
Previous studies have suggested that peripheral Y1 receptorsattenuate capsaicin-evoked mechanical allodynia (Gibbs et al.,2006). This antinociceptive effect led us to hypothesize that localadministration of NPY and its antagonists after plantar incisioncould have an effect on spontaneous and evoked pain behaviors.In this study, intraplantar administration of NPY and receptorantagonists after plantar incision did not result in modulation ofpain behaviors as measured by guarding pain and heat hyper-algesia. This suggests that, peripherally, NPY and its receptors donot have a critical role in pain after incision. This may be due todifferences in the type and degree of injury amongst pain models(Honore et al., 2006).
Others have suggested that NPY exerts its analgesic effect atthe dorsal horn and/or the DRG as opposed to locally in the tissue(Smith et al., 2007; Thomsen et al., 2007). In this study we foundthat intrathecal administration of NPY was analgesic after inci-sion. Intrathecal administration of NPY has been shown to be bothpro and anti-nociceptive in models of inflammatory and neuro-pathic pain and that antagonism of Y1 receptor or Y2 receptor hasbeen shown to reverse behavioral signs attenuated by NPY inthese models (Intondi et al., 2008). We found that after plantarincision, selective blockade of Y2 receptor alone reduced painbehaviors. Additionally, blockade of Y1 receptor reversed the anti-nociceptive effects of NPY when co-administered . Taken togetherthese data suggest that exogenous NPY acts on the Y1 receptor toproduce an analgesic effect. This is in agreement with previousstudies in an inflammatory model of pain that have shown that Y1
receptor knockout increases nociception and that the antihyper-algesic effects of NPY are mediated by Y1 receptors (Kuphal et al.,2008; Naveilhan et al., 2001; Taiwo and Taylor, 2002).
We have shown that a single dose of intrathecally administeredNPY was sufficient to attenuate postoperative pain behaviors for48 h. Administration of Y2 receptor antagonist was also reducednociceptive behaviors for 24 h. While receptor antagonists havebeen shown to reverse the effects of exogenously administeredNPY, this is the first report demonstrating that the sole adminis-tration of Y2 receptor antagonist could have anti-hyperalgesiceffects for postoperative pain. A possible mechanism accountingfor the attenuation of early postoperative pain by Y2 receptorantagonist can be explained by the Y2 receptor-mediated controlof NPY release. In the rat hypothalamus, sole administration of Y2
receptor antagonist, BIIE0246, enhanced Kþ stimulated NPY release(King et al., 2000). Here we suggest a similar mechanism at the
S.M. Yalamuri et al. / European Journal of Pharmacology 698 (2013) 206–212212
spinal level where Y2 receptor antagonism leads to an increase inNPY release, which exerts its analgesic effect through the Y1
receptor subtype. A similar mechanism of action has been proposedfor the antidepressant and anti-anxiolytic behaviors observed inrats after sole administration of BIIE0246 (Morales-Medina et al.,2012; Bacchi et al., 2006).
5. Conclusions
After plantar incision, protein expression of NPY was down-regulated in skin and muscle. The receptors for NPY were upregu-lated in skin and muscle. The regulation of NPY and its receptorsoccurs when pain behaviors are the greatest (POD 1 & 2). A singledose of intrathecally administered NPY reduced pain behaviors for24–48 h and was as effective as morphine. Our data suggests thatNPY produced this analgesic effect through NPY’s action on theY1 receptor. Combined, these results support a role for NPY in acutepostoperative nociception at the spinal level.
Funding
Foundation for Anesthesia Education and Research to CMS.
References
Apfelbaum, J.L., Chen, C., Mehta, S.S., Gan, T.J., 2003. Postoperative pain experi-ence: results from a national survey suggest postoperative pain continues tobe undermanaged. Anesth. Analg. 97, 534–540.
Bacchi, F., Mathe, A.A., Jimenez, P., Stasi, L., Arban, R., Gerrard, P., Caberlotto, L.,2006. Anxiolytic-like effect of the selective Neuropeptide Y Y2 receptorantagonist BIIE0246 in the elevated plus-maze. Peptides 27, 3202–3207.
Bjur, D., Alfredson, H., Forsgren, S., 2009. Presence of the neuropeptide Y1 receptorin tenocytes and blood vessel walls in the human Achilles tendon. Br. J. SportsMed. 43, 1136–1142.
Brennan, T.J., Vandermeulen, E.P., Gebhart, G.F., 1996. Characterization of a ratmodel of incisional pain. Pain 64, 493–502.
Brumovsky, P., Shi, T.S., Landry, M., Villar, M.J., Hokfelt, T., 2007. Neuropeptidetyrosine and pain. Trends Pharmacol. Sci. 28, 93–102.
Franco-Cereceda, A., Liska, J., 1998. Neuropeptide Y Y1 receptors in vascularpharmacology. Eur. J. Pharmacol. 349, 1–14.
Gibbs, J.L., Diogenes, A., Hargreaves, K.M., 2007. Neuropeptide Y modulates effectsof bradykinin and prostaglandin E2 on trigeminal nociceptors via activation ofthe Y1 and Y2 receptors. Br. J. Pharmacol. 150, 72–79.
Gibbs, J.L., Flores, C.M., Hargreaves, K.M., 2006. Attenuation of capsaicin-evokedmechanical allodynia by peripheral neuropeptide Y Y1 receptors. Pain 124,167–174.
Hodges, G.J., Jackson, D.N., Mattar, L., Johnson, J.M., Shoemaker, J.K., 2009.Neuropeptide Y and neurovascular control in skeletal muscle and skin. Am.
tide Y reduces behavioral and molecular markers of inflammatory or neuro-pathic pain. Pain 137, 352–365.
Intondi, A.B., Zadina, J.E., Zhang, X., Taylor, B.K., 2010. Topography and time courseof changes in spinal neuropeptide Y immunoreactivity after spared nerveinjury. Neuroscience 165, 914–922.
King, P.J., Williams, G., Doods, H., Widdowson, P.S., 2000. Effect of a selectiveneuropeptide Y Y2 receptor antagonist, BIIE0246 on neuropeptide Y release.
increases nociception and prevents the anti-allodynic actions of NPY. Nutrition24, 885–891.
Landry, M., Holmberg, K., Zhang, X., Hokfelt, T., 2000. Effect of axotomy onexpression of NPY, galanin, and NPY Y1 and Y2 receptors in dorsal root gangliaand the superior cervical ganglion studied with double-labeling in situ
peripheral neuropeptide Y receptors in sympathetic modulation of acutecutaneous flare induced by intradermal capsaicin. Neuroscience 123, 337–347.
Lundberg, J.M., Terenius, L., Hokfelt, T., Goldstein, M., 1983. High levels ofneuropeptide Y in peripheral noradrenergic neurons in various mammalsincluding man. Neurosci. Lett. 42, 167–172.
Morales-Medina, J.C., Dumont, Y., Benoit, C.-E., Bastianetto, S., Flores, G., Fournier, A.,Quirion, R., 2012. Role of neuropeptide Y Y1 and Y2 receptors on behavioral
despair in a rat model of depression with co-morbid anxiety. Neuropharmacol-ogy 62, 200–208.
Naveilhan, P., Hassani, H., Lucas, G., Blakeman, K.H., Hao, J.-X., Xu, X.-J.,Wiesenfeld-Hallin, Z., Thoren, P., Ernfors, P., 2001. Reduced antinociceptionand plasma extravasation in mice lacking a neuropeptide Y receptor. Nature
mechanisms of NPY analgesia. Peptides 28, 464–474.Spofford, C., Brennan, T., 2012. Gene expression in skin, muscle and dorsal root
ganglion after plantar incision in the rat. Anesthesiology 117, 161–172.Taiwo, O.B., Taylor, B.K., 2002. Antihyperalgesic effects of intrathecal neuropeptide Y
during inflammation are mediated by Y1 receptors. Pain 96, 353–363.Thomsen, M., Wortwein, G., Olesen, M.V., Begtrup, M., Havez, S., Bolwig, T.G.,
Woldbye, D.P.D., 2007. Involvement of Y5 receptors in neuropeptide Y agonist-induced analgesic-like effect in the rat hot plate test. Brain Res. 1155, 49–55.
