Pharmacological attenuation of chronic alcoholic pancreatitis induced hypersensitivity in rats

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1 Submit Mnuscript: Help Desk: DOI: 1.37/wjg.v1.i3.3 World J Gstroenterol Jnury 1; 1(3): 3-3 ISSN (print) ISSN 19- (online) Bishideng Publishing Group Inc. All rights reserved. Bsic Study Phrmcologicl ttenution of chronic lcoholic pncretitis induced hypersensitivity in rts ORIGINAL ARTICLE Sbrin L McIlwrth, Krin N Westlund Sbrin L McIlwrth, Krin N Westlund, Deprtment of Physiology, University of Kentucky, Lexington, KY 3-9, United Sttes Author contributions: Westlund KN designed the reserch nd edited the pper; McIlwrth SL performed the reserch, nlyzed the dt, nd wrote the pper. Supported by Ntionl Institutes of Helth, No. NINDS R1 NS391. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC.) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/./ Correspondence to: Krin N Westlund, PhD, Professor, Deprtment of Physiology, University of Kentucky, MS- Medicl Center, Rose St., Lexington, KY 3-9, United Sttes. kwhigh@uky.edu Telephone: Fx: Received: June, 1 Peer-review strted: June, 1 First decision: August, 1 Revised: August 1, 1 Accepted: September 9, 1 Article in press: September 3, 1 Published online: Jnury 1, Abstrct AIM: To chrcterize n lcohol nd high ft diet induced chronic pncretitis rt model tht mimics poor humn dietry choices. METHODS: Experimentl rts were fed modified Lieber-DeCrli lcohol (%) nd high-ft (%) diet (AHF) for 1 wk while control nimls received regulr rodent chow diet. Weekly behviorl tests determined mechnicl nd het sensitivity. In week 1 fsting glucose tolernce test ws performed, mesuring blood glucose levels before nd fter g/kg bodyweight intrperitonel (i.p.) injection of glucose. Post mortem histologicl nlysis ws performed by stining pncres nd liver tissue sections with hemtoxylin nd eosin. Pncres sections were lso stined with Sirius red nd fst green to quntify collgen content. Insulinexpressing cells were identified immunohistochemiclly in seprte sections. Tissue stining density ws quntified using Imge J softwre. After mechnicl nd het sensitivity becme stble (weeks -1) in the AHF-fed nimls, three different drugs were tested for their efficcy in ttenuting pncretitis ssocited hypersensitivity: Group Ⅱ metbotropic glutmte receptor specific gonist (R,R)--Aminopyrrolidine-,-dicrboxylte (APDC, 3 mg/kg, ip; Tocris, Bristol, United Kingdom), nociceptin (,, nmol/kg, ip; Tocris), nd morphine sulfte (3 mg/kg, µ-opioid receptor gonist; Bxter Helthcre, Deerfield, IL, United Sttes). RESULTS: Histologicl nlysis of pncres nd liver determined tht unlike control rts, AHF fed nimls hd pncretic fibrosis, cinr nd bet cell trophy, with stetosis in both orgns. Ft vcuoliztion ws significntly incresed in AHF fed rts (.% ± 1.1% in controls vs 3.% ±.%, P <.). Rts fed the AHF diet hd reduced fsting glucose tolernce in week 1 when pek blood glucose levels reched significntly higher concentrtions thn controls (17. ± 9. mg/dl in controls vs 11. ±. mg/dl, P <.). This concurred with 3. fold higher incidence of single nd smll -1 cell insulin-positive cell clusters (P <.). Insulin expressing islet of Lngerhns cells ppered hypertrophied while islet number nd re mesurements were not different from controls. Weekly behviorl tests determined tht mechnicl nd het sensitivities were significntly incresed by wk on AHF diet compred to controls. Hypersensitivity 3 Jnury 1, Volume 1 Issue 3

2 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity ws ttenuted with efficcy similr to morphine with single dose tretment of either metbotropic glutmte receptor /3 gonist APDC, or nociceptin, the endogenous lignd for opioid-receptor-like 1 receptor. CONCLUSION: The AHF diet induces chronic lcoholic pncretitis in rts with mesurble fetures resembling clinicl ptients with chronic pncretitis nd type 3c dibetes mellitus. Key words: Lieber-DeCrli diet; Orphnin FQ receptor; Metbotropic glutmte receptor; Liver stetosis; Pin; Behvior; Glucose tolernce; Type 3c dibetes mellitus The Author(s). Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: Chronic pncretitis is progressive nd potentilly ftl disese cused by persistent unresolved inflmmtion nd pncretic fibrosis. It cn be ccompnied by intrctble bdominl pin nd progress to type 3c dibetes mellitus (T3cDM) nd pncretic cncer. Animl models of cute pncretitis re typiclly chemiclly induced, invsive, of short durtion, nd hve high mortlity rte. This study chrcterizes diet-induced chronic rt model closely mimicking poor humn dietry choices to investigte therpies for lcoholic chronic pncretitis with developing T3cDM. The efficcy of cute opioid nd non-opioid phrmcologicl interventions re compred to morphine in pin-relted behvior tests. McIlwrth SL, Westlund KN. Phrmcologicl ttenution of chronic lcoholic pncretitis induced hypersensitivity in rts. World J Gstroenterol ; 1(3): 3-3 Avilble from: URL: DOI: INTRODUCTION Chronic pncretitis is progressive nd potentilly ftl disese cused by persistent unresolved inflmmtion nd pncretic fibrosis typiclly ccompnied by intrctble bdominl pin, prticulrly during the erly phse [1,]. Other symptoms include weight loss due to insufficiency of the exocrine pncres, type 3c dibetes mellitus (T3cDM), nd n incresed risk of developing pncretic cncer [3-13]. Fctors tht increse susceptibility to the diverse etiology of chronic pncretitis include lifestyle choices such s regulr lcohol consumption, prticulrly binge drinking, hyperlipidemi/hypertriglyceridemi cused by diet high in sturted ft, nd smoking, s well s heritble fctors [1,1,1-1]. Although %-7% of ptients with chronic pncretitis regulrly consume lcohol, only round 1% of ptients with lcohol use disorders develop this disese, indicting other contributing fctors [1,-]. Worldwide the incidence of chronic pncretitis is higher in the mle popultion, prtilly explined by the recently identified X chromosome linked genetic polymorphism in the cludin- gene [19]. Chief clinicl complint of fflicted ptients is intrctble pin nd currently recommended pin mngement therpies rnge from non-nrcotic nlgesics incresing to strong opites depending on the ptient s complints [-7]. Concurrent nlysis of mino cids in plsm nd serum of ptients with chronic pncretitis identified doubling of the glutmte concentrtion, the min excittory neurotrnsmitter of the nervous system nd known fcilittor of pin, while other mino cid levels re unffected or decresed [,9]. Knowledge of the underlying pthophysiology of chronic pncretitis resulting in this severe bdominl pin is still limited nd is needed for the development of better phrmcologicl tretments. Presently employed niml models poorly reproduce the clinicl etiology of chronic pncretitis s they re highly invsive, requiring lprotomy surgery nd/or utilize repetitive dosing with custic chemicls. The bdominl hypersensitivity induced with these experimentl procedures is better suited for modeling cute pncretitis [3,31]. Chronic pncretitis is multifctoril disese. Clinicl observtions indicte tht diets high in ft fcilitte the progression of lcoholic chronic pncretitis [1,1,1,]. Animl studies modeling chronic pncretitis hve reported limited or no success when ethnol is used lone [3-3]. The combintion of ftty cids nd ethnol is required to induce noticeble cell dmge to cultured pncretic cells while the ppliction of individul compounds lone is unble to injure these cells [37-39]. Recently, we estblished rt model of chronic pncretitis using modified Lieber-DeCrli diet with incresed dietry sturted ft content (%) [,1]. In the present study, the lcoholic chronic pncretitis rt model ws modified by further incresing the dietry sturted ft content to %. Unique to the current model is developing T3cDM, recognized by the Americn Dibetes Assocition nd the Ntionl Institutes of Helth, is n underdignosed secondry disese ssocited with exocrine pncretic dmge [,,1,13]. Cliniclly, between %-1% of ptients with dibetes re dignosed with T3cDM [,13]. This is the first niml model of chronic pncretitis tht develops T3cDM. The model is utilized here to quntify chronic pncretitis induced pin relted behviors tht persist t lest 1 wk for nlgesic testing. Using the lcohol nd high ft diet (AHF) induced rt chronic pncretitis model, the efficcy of cute phrmcologicl ctivtion of three inhibitory metbotropic Gi protein-coupled receptors (GPCR) ws tested. The gonists ctivte either Group Ⅱ metbotropic glutmte receptor nd receptor 3 (mglur/3), µ-opioid receptor (MOR), or the opioid receptor like-1 receptor (ORL-1). Signling through mglur/3 receptors ws initited by glutmte binding [,3], while the opioid receptors ORL-1 nd µ-opioid receptor MOR re ctivted by the endogenous neuropeptides nociceptin (lso clled orphnin FQ [,] ), enkephlins nd bet- 37 Jnury 1, Volume 1 Issue 3

3 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity endorphins, respectively [,7]. Their ctivtion results in downstrem signling events tht ctivte voltge-gted potssium chnnels to inhibit voltge-gted clcium chnnels. Thus, ctivting these metbotropic receptors on peripherl nociceptors ctivted by noxious stimuli inhibits the relese of neuropeptide modultors nd glutmte, the min excittory neurotrnsmitter in the nervous system [-]. Agonists for mglur/3 hve been shown to be nlgesic in niml models of somtic inflmmtory nd neuropthic pin [1-]. Informtion bout ORL-1 ctivtion indictes tht intrthecl nd peripherl pplictions of nociceptin ttenute hypersensitivity in inflmmtory nd neuropthic rodent pin models [-9], while intrcerebroventriculr injections produce hyperlgesi []. The efficcy of their ctivtion in reducing hypersensitivity in the chronic lcoholic pncretitis model in the present study is compred to morphine, the customry opite utilized for experimentl comprisons. Using the AHF rt chronic pncretitis model with developing T3cDM, we provide the first evidence tht peripherl ctivtion of inhibitory GPCR-medited signling cscdes through mglur/3 or ORL-1 re similrly efficcious to morphine in reversing hypersensitivity induced in this chronic viscerl pin model. MATERIALS AND METHODS Ethics sttement All niml procedures were conducted ccording to the guidelines for the ethicl tretment of experimentl nimls published by the Internl Assocition for the Study of Pin nd pproved by the University of Kentucky Institutionl Animl Cre nd Use Committee (IACUC#7-113). Induction of lcohol pncretitis Experiments were performed using totl of 1 mle Fischer rts weighing 3- g (Hrln Lbortories, Indinpolis, IN). Animls were housed individully on 1/1 h reverse light cycle so tht behviorl ssys were conducted during their ctive night phse. Food nd wter were given d libitum nd nimls were divided into two groups: (1) controls (n = 7) received regulr low soy rodent chow (Teklb #, Hrln, Indinpolis, IN) nd () lcohol nd high ft (AHF) diet fed nimls (n = 9) received modified Lieber-DeCrli diet. Along with the % ethnol liquid diet, dily portion of g lrd ws provided. The liquid diet (1 g) contined 1% LD11A (TestDiet, Richmond, IN), 9% mltodextrin, 1% pple juice, % ethnol, nd 3.3% corn oil. The AHF liquid diet ws initilly introduced without ethnol nd corn oil. On weekly bsis the ethnol content of the food ws incresed from %-% nd finlly corn oil ws lso dded, resulting in liquid mintennce diet contining % ethnol nd 3% corn oil in AHF fed nimls. Throughout this time AHF fed nimls lso received the g lrd supplement dily. Consumed liquid diet nd lrd were mesured dily nd verged throughout the experimentl time period to clculte the percentge of consumed dietry ft. Averge dily liquid diet consumption ws g, of which 7.% ws nutritionl vlue nd 7.% ws wter. Of this. g with nutritionl vlue, % ws ft (1. g). Animls consumed n verge of g of lrd dily, resulting in 7. g totl ft consumption per dy out of the totl 11. g of nutritionl diet. Thus, totl dily dietry ft consumption ws bout %. Behviorl testing Weekly behviorl ssys to determine mechnicl nd het sensitivity were performed on ll nimls. The bdomen ws shved t lest h prior to testing. Alcohol ws removed h prior to behviorl testing nd drug ppliction to minimize potentil interference of lcohol on motor control nd interctions with the compound of interest. Previous studies hd shown tht lcohol withdrwl in this experimentl setting did not interfere with outcomes of these behviorl ssys [1]. Animls were plced in individul Plexigls boxes on n elevted metl screen mesh (3 mm holes). Mechnicl sensitivity of the bdomen nd hindpws ws tested by probing the niml through the mesh from below using two different experimentl methods. Abdominl referred sensitivity ws chrcterized s previously reported by Ver-Portocrrero et l [1] (3). Briefly, the upper right bdominl qudrnt overlying the pncres ws probed 1 times ech in scending order with different von Frey filments eliciting 1. or. g bending force equivlent to 9. or. mn. This ssessed behvior in response to both sub- nd supr-threshold mechnicl stimuli, i.e., touch nd poke, to identify the time course for development of chronic mechnicl hypersensitivity. Stimuli were pplied t 1 s intervls nd the number of bdominl withdrwls counted. A single tril to determine the number of responses to ppliction of 1 stimuli with single von Frey filment ws performed per niml per time point. Mechnicl hypersensitivity thresholds of the hindpw footpds were subsequently determined while the nimls were still on the elevted mesh screen tble. For this mesure, the up-down method ws used incorporting series of von Frey filments exerting clibrted bending forces (.,., 1.,.,.,.,.,. g or 3.9,.9, 9., 19., 39.,., 9. mn [] ). Het sensitivity ws determined using hot plte nlgesiometer pprtus set t (Columbus Instruments, Columbus, OH). Animls were plced individully on the hotplte ( mm mm heted surfce) surrounded by 3 cm high Plexigls brrier nd the ltency until nocifensive response occurred, such s shking or licking the pw, jumping, or running ws recorded. Animls were immeditely removed fter the initil response nd cut-off time of s prevented burn injury. The het test ws performed 3 times t min 3 Jnury 1, Volume 1 Issue 3

4 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity intervls nd the ltencies verged. Drug tretments After mechnicl nd het sensitivity developed in the AHF-fed nimls, the efficcy of three different drugs in ttenution of pncretitis ssocited hypersensitivity ws determined to identify contributing signling pthwys: Group Ⅱ metbotropic glutmte receptor specific gonist, (R,R)--Aminopyrrolidine-,-dicrboxylte (APDC, 3 mg/kg, ip; Tocris, Bristol, United Kingdom), nociceptin (,, nmol/kg, ip; Tocris), nd morphine sulfte (3 mg/kg, subcutneous (s.c.); Bxter Helthcre, Deerfield, IL, United Sttes). All drugs were diluted in sterile sline nd tested fter hypersensitivity becme stble (weeks -1). Mechnicl nd het sensitivity were determined h before tretment nd 1,, nd h fter intrperitonel (ip) injection of single dose of drug or vehicle (sline) ws dministered. Efficcy of drug tretments ws tested once week in the sme nimls since no long-lsting chnges for cute use of these drugs is reported nor noted in the present study. Glucose tolernce test Prior to euthnsi, glucose tolernce test ws performed with ll nimls [3]. Rts were fsted for h nd then given glucose ( g/kg body weight, ip) in distilled sterile wter (% w/v solution). Glucose levels in til vein blood smples were mesured prior to nd t severl timepoints fter giving glucose (, 3,, 1, 1 min) using blood glucose meter nd pproprite test strips (FreeStyle, Abbott Dibetes Cre, Almed, CA). Histology Liver nd pncres smples were collected prior to perfusion of the niml nd immerse fixed overnight in % prformldehyde in phosphte buffered sline, trnsferred into 7% ethnol, nd prffin embedded. Sections were cut ( µm), rehydrted, stined for collgen fibers with Sirius red (Electron Microscopy Sciences, #37-), nd counterstined with fst green. Other sections were stined with Sirius red nd hemtoxylin nd eosin (HE) using routine histologicl protocols. Liver nd pncres smples were nlyzed fter cquiring five rndom imges per slide using ACT softwre nd Nikon microscope for nlysis using Imge J. A red color threshold mcro ws used to mesure the imge res with fibrous collgen stined red by Sirius red in rndom fst green stined sections per group. The re quntified is presented s rtio of the totl tissue re. Islets of Lngerhns were identified opticlly in HE nd Sirius red stined sections. Imges were tken of ll Islets, their sizes mesured, nd their rtio normlized to totl tissue re. Quntifiction of white spce mong the lobules of condensed pncres tissue ws performed tking rndom imges in HE nd Sirius red stined tissue sections, quntifying the rtio of unstined to stined tissue re. A threshold mcro determining white res ws used to ssess the ft content leched out in prffin processing in HE nd Sirius red stined tissue, nd the rtio of unstined white re to totl tissue re per imge ws determined. Immunofluorescent tissue stining Deprffinized nd rehydrted pncres tissue on slides ws incubted overnight in primry rbbit ntibody ginst insulin (H-, sc-91; Snt Cruz Biotechnology, Snt Cruz, CA) 1: t room temperture. Tissue ws wshed, incubted in got nti-rbbit secondry ntibody conjugted to Alex Fluor (Life Technologies, Grnd Islnd, NY) 1:1 for h t room temperture, wshed nd coverslipped in VectShield hrd set mounting medium with DAPI (Vectorlbs, Burlingme, CA). Fluorescent stining of ll proliferting insulin-positive cells in imges of whole pncres sectioned hed to til ws nlyzed using Nikon microscope with Metmorph softwre. Post-processing nd quntittive nlyses were conducted using Photoshop nd Imge J. Colocliztion of DAPI nd insulin ws used to count the number of cells in smll clusters of under 1 cells nd the counts normlized to totl tissue re in ech pncretic section. Grph Pd Prism nd Excel Microsoft softwre were used for sttisticl nlysis nd grphing of the dt. Sttisticl nlysis Dt re expressed s men ± SE. Sttisticl significnce of behviorl nd immunohistochemicl dt ws determined using nlysis of vrince tests nd Student t test with the significnce level set t P <.. Post hoc comprison included Student t tests to test sttisticl differences between control nd experimentl groups before nd fter drug tretments. RESULTS Chronic consumption of AHF diet induces histopthologicl chnges in pncres nd liver indictive of lcoholic pncretitis Adult mle Fischer rts were fed the lcohol nd high ft diet (AHF) d libitum for 1 wk. The liquid diet contins % (wt/vol) ethnol nd 3.3% (wt/vol) corn oil supplemented with dily dose of g sturted ft resulting in the dily consumed diet to contin % ft, modified from Lieber-DeCrli nd others [3,1,,]. Control nimls received stndrd rt chow pellets (Hrln Tekld ). Post mortem histopthologicl nlysis of the pncres using Sirius red, hemtoxylin, nd eosin stining reveled cinr trophy, fibrosis, nd necrosis in ll of the tissue smples from AHF diet fed rts (Figure 1A nd B). Pncret of AHF fed rts developed enlrged interlobulr spces, nd cellulr trophy. Pncretic interlobulr spces, mesured s the percent white re to stined tissue, were significntly incresed in AHF fed rts (Figure 1C; control =.9% ± 1.%, n = ; AHF = 39 Jnury 1, Volume 1 Issue 3

5 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity Control AHF A B C L Liver Pncres Pncres L L L D E F L L G H I L % Intrlobulr re % Collgen/totl protein % Ft vcuoliztion Control Control AHF AHF Control AHF Figure 1 Histopthologicl nlysis of the effects of lcohol nd high ft diet ft diet on pncres nd liver. A: Microgrph of pncres from control regulr chow fed rt stined with hemtoxylin nd eosin (HE); B: Microgrph of pncres from n lcohol nd high ft diet (AHF) fed rt stined with HE; C: Quntifiction of intrlobulr spces (re percentge) determined there ws significnt increse in pncret from AHF fed rts; D: Microgrph of pncres from control rt fed regulr chow with Sirius red stined collgen fibrosis nd fst green counterstin; E: Microgrph of pncres from n AHF fed rt with Sirius red stined collgen fibrosis nd fst green counterstin; F: Quntifiction of extrcellulr collgen deposits (stined red) s percent of totl tissue re. A significnt increse in collgen stining ws detected in pncret of AHF fed rts; G: Microgrph of liver from control rt fed regulr chow stined with HE; H: Microgrph of liver from n AHF fed rt stined with HE; I: Quntifiction of intrcellulr ft vcuoliztion (re percentge). Unstined res re ft vcuoles tht were significntly incresed in liver smples from AHF fed rts. P <., AHF vs control. L: Islet of Lngerhns; Solid rrow hed: Pncretic duct; Smll rrow: Blood vessel. A, B: Sclebr mm; D, E: Sclebr 1 mm; G, H: Sclebr 1 mm..7% ±.%, n = ; P <.) indicting cell shrinkge nd/or necrosis. Prenchyml fibrosis ws determined histologiclly s the rtio of the re of pncretic tissue collgen proteins stined with Sirius red to stining of non-collgenous proteins with fst green [-]. Smll mounts of interstitil collgen were detected in control tissue smples while the rtio of collgenous nd noncollgenous protein stining ws significntly incresed in AHF fed nimls (Figure 1D, F; control = 3.% ±.%; AHF =.% ± 1.7%; P <.). Collgen bundles were incresed in prticulr nd clerly visible surrounding pncretic ducts, lobes, nd blood vessels in tissue smples from the AHF experimentl group, while only diffuse stining loclized within nd mong the cinr cells ws observed in control nimls. In the liver, chronic AHF diet consumption produced clerly visible stetosis when tissue ws stined with Sirius red, hemtoxylin, nd eosin. Alcoholic ftty liver ws quntified by mesuring the unstined intrcellulr re in liver tissue smples remining of the severe ft vcuoliztion fter grded lcohol removl of the prffin. Ft vcuoliztion ws significntly incresed in AHF fed nimls compred to controls (Figure 1G-I; control =.% ± 1.1%; AHF = 3.% ±.%; P <.). Jnury 1, Volume 1 Issue 3

6 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity A Control B AHF Insulin DAPI C No. smll insulin clusters/mm pncretic tissue D Glucose (mg/dl) Control (n = ) AHF (n = ). Control AHF t /min Figure Chronic lcohol nd high ft diet induced insulin cell prolifertion nd decresed glucose tolernce. A: Microgrph of insulin immunorectivity in pncres from control rt fed regulr chow; B: Microgrph of insulin immunorectivity in pncres from rt fed lcohol nd high ft (AHF). In tissue smples from AHF fed rts insulin producing cells in the islets re trophied nd n increse of spurious proliferting insulin expressing cells (rrows) is detected in the pncres; C: Quntifiction of the number of single cells or smll insulin cell clusters per mm pncres tissue detected significnt increse in AHF fed rts; D: Intrperitonel glucose tolernce test t 1 wk determined tht glucose tolernce ws reduced in fsted AHF fed nimls. Blood glucose levels of AHF fed nimls peked to significntly higher levels nd 3 min post injection. P <., AHF vs control. A-B: Sclebr 1 mm. Immunohistochemicl nlysis of insulin expression in pncres tissue stined from nimls fed (A) regulr chow or (B) AHF diet determined there ws prominent prolifertion of insulin immunorective cells evident in AHF fed nimls (Figure A-C). After chronic AHF diet for 1 wk the pncres hd trophy of insulin producing cells in the islets nd n increse of spurious proliferting insulin expressing cells. The number of single to smll cell clusters of 1 or less ws 3. fold higher in AHF fed nimls (1.9 ±.19 per mm ) compred to controls (.33 ±.9 per mm, P <.) (Figure C). Chronic AHF diet consumption decreses glucose tolernce Weekly monitoring of blood glucose levels in nïve control nd AHF fed rts did not detect significnt differences throughout the experiment. Unfsted blood glucose concentrtions remined constnt t.1 ± 3.1 mg/dl in controls nd 3. ±. mg/dl in AHF fed rts. Glucose tolernce testing following ip injection of g glucose per kg bodyweight ws performed in week 1 fter fsting nimls for h to mesure metbolic efficiency of glucose clernce from the blood strem (Figure D). Animls fed AHF hd significntly higher pek blood glucose levels nd 3 min fter glucose injection (control min: 17. ± 9. mg/dl, 3 min: 11. ± 3.9 mg/dl; AHF min: 11. ±. mg/dl, 3 min: 13. ±. mg/dl, P <.), indicting reduced glucose tolernce. Chronic AHF diet consumption cuses mechnicl nd het hypersensitivity A chief complint of clinicl ptients with chronic pncretitis is intrctble pin. The pin is commonly focused in the upper bdomen but cn rdite to the bck with some ptients reporting overll sensitivity. In the niml model, mechnicl nd het sensitivity were monitored weekly (Figure 3). Abdominl sensitivity ws ssyed by testing the reflexive responses to low intensity (1 g = 9. mn force; Figure 3A) nd higher intensity ( g =. mn force; Figure 3B) mechnicl stimuli pplied with von Frey filments [1]. The Y-xis indictes the verge number of bdominl withdrwls in response to the bending force stimultions (1 ). Low intensity (1 g) mechnicl stimultion pplied on the bdomen elicited n verge of 3 of 1 responses from control rts fed stndrd chow diet (Figure 3A, open squres). Bseline responses to low intensity nd 1 Jnury 1, Volume 1 Issue 3

7 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity A No. of responses/1 1 1 g force bdomen B No. of responses/1 1 g force bdomen Control (n = 7) AHF (n = 9) t /wk t /wk C Hindpw D Hotplte Mechnicl threshold (g) 1 Ltency (s) t /wk t /wk Figure 3 Time course of nociceptive behviors in rts with chronic pncretitis evoked by mechnicl nd het stimuli. A, B: Abdominl sensitivity is determined by the number of bdominl withdrwls in response to von Frey filment bending force stimultions ( 1). Responses to (A) low intensity (1 g) nd (B) high intensity ( g) mechnicl stimultion elicited significntly more responses in lcohol nd high ft (AHF) fed rts strting in week ; C: Mechnicl thresholds of the hindpws were determined with von Frey filments using the up-down method. In week mechnicl sensitivity thresholds of AHF fed rts were significntly reduced nd did not recover while on the AHF diet; D: Het sensitivity ws determined by mesuring the response ltency (s) in the hotplte test ( ). In week, AHF fed rts demonstrted reduced response ltencies in the hotplte test which did not recover during the 1 wk experimentl time course. Open squres: control nimls. Solid circles: AHF fed nimls. P <., AHF vs control. higher intensity stimultion on the bdomen skin were not different between groups [1 g: control =.3 ±.; AHF = (1. ±. responses)/1; N.S.; g: control =. ±.; AHF = (. ±. responses)/1; N.S]. Responses incresed significntly during the experiment s durtion in the AHF group (1 g: P <.1; g: P <.1). In week, AHF fed rts responded with lmost twice s mny bdominl withdrwls to low intensity mechnicl stimultion thn controls [control = 3. ±.; AHF = (. ±.7 responses)/1; P <.; Figure 3A]. Similrly, the responses to g mechnicl stimultion of the bdomen ws significntly incresed in AHF nimls in week [control =. ±.; AHF = (9. ±.3 responses)/1; P <.]. Higher intensity ( g) mechnicl stimultion of the bdomen consistently elicited n verge of of 1 responses from control rts while AHF fed nimls responded every time fter week. Mechnicl nd het sensitivity of control nimls did not chnge during the study. Similrly, mechnicl thresholds for eliciting hindpw withdrwl in AHF fed rts decresed significntly in week from 1.7 g force to 9.3 ± 1. g force (P <.; Figure 3C). This referred hypersensitivity of the hindpw plntr surfce ws chrcterized by determining the mechnicl withdrwl threshold using the up-down method []. The mechnicl thresholds of the hindpws of control nimls did not chnge throughout the experiment (17. ±. g force). Hindpw mechnicl thresholds for AHF fed rts remined decresed t 7. ±.9 g force (P <.1) through the experiment end t week 1. Het sensitivity ws determined indirectly by mesuring the ltency (s) to n escpe response in the hotplte test ( ) (Figure 3D). Response ltencies on the hotplte, i.e., the time until nimls displyed signs of nociception such s shking the pw, licking, gittion, were not different between groups t bseline (control = 1. ±.1 s; AHF =.9 ±. s; N.S). In week hotplte sensitivity ws significntly decresed in the AHF fed group (control =. ±. s; AHF = 13.3 ± 1. s; P <.; Figure 3D). The reduced ltencies did not recover during the 1 wk experimentl time course. At the end of the experiment (1 wk), control nimls spend 1.3 ±. s on the hotplte before showing signs of distress while AHF nimls rected within 1. ±.3 s (P <.). Jnury 1, Volume 1 Issue 3

8 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity A No. of responses/1 APDC (3 mg/kg) - time course 1 g force, bdomen 1 B No. of responses/1 1 g force, bdomen Control (n = ) AHF (n = ) C Hindpw D Hotplte Mechnicl threshold (g) 1 Ltency (s) 1 Figure Time course of nociceptive responses to mechnicl nd het stimuli fter single systemic tretment with (R,R)--Aminopyrrolidine-,- dicrboxylte. A, B: Animls were treted with single ip dose of (R,R)--Aminopyrrolidine-,-dicrboxylte (APDC) (3 mg/kg). APDC reversed the increse in nociception responses 1 h post tretment evoked by (A) low intensity (1 g) bdominl stimultion nd (B) high intensity ( g) bdominl stimultion; C: Alcohol nd high ft (AHF) fed rts hd incresed hindpw mechnicl withdrwl thresholds; D: Response ltencies of AHF fed rts incresed 1 h fter APDC tretment in the hotplte test, but sensitivity of the controls ws not ltered. P <., AHF vs control. Anlgesic effects of APDC on AHF diet induced hypersensitivity Glutmte is widely expressed neuropeptide within the nervous system. When binding to group Ⅱ metbotropic glutmte receptors, mglur nd mglur3, Gi-protein coupled receptors, their ctivtion inhibits denylyl cyclse. This initites inhibitory signling cscdes tht result in reduced neuronl ctivity nd decrese the relese of further glutmte [9,]. The mglur/3 gonist APDC hs been shown to reduce inflmmtion induced mechnicl nd het hypersensitivity in mouse models of somtic pin [1,]. Here we used single dose of 3 mg/kg APDC (ip) to decrese mechnicl nd het hypersensitivity induced by chronic pncretitis, concentrtion previously shown to optimlly reduce hypersensitivity in cute somtic inflmmtory niml models. Within 1 h of APDC tretment, reversl ws noted of the incresed nociceptive response evoked by low intensity bdominl stimultion in AHF fed rts (Figure A). Responses to low intensity mechnicl stimultion of the bdomen were 3. ± 1., insted of. ±.9 responses to 1 stimuli observed t bseline. Responses of the controls 1 h fter APDC tretment (1 h: 3. ±.3 responses) remined the sme (- h: 3. ±. responses; Figure A). Responses of AHF fed rts to higher intensity mechnicl stimultion of the bdomen were similrly reduced t the 1 h timepoint [- h: (1 ± responses)/1; 1 h: (7. ±.7 responses)/1; P <.; Figure B]. Mechnicl thresholds of the hindpws (- h:. ± 1. g force; 1h: 17. ± 1.3 g force; AHF n =, P <.; Figure C) nd ltencies on the hotplte (- h: 1. ±. s; 1 h: 17.3 ± 1. s; AHF n =, P <.) improved significntly to vlues indistinguishble from control nimls (Figure D). The effect of APDC ws bolished h fter tretment. Nociceptin tretment reverses AHF diet induced hypersensitivity Nociceptin, n endogenous opioid-relted neuropeptide, binds to the opioid receptor like-1 receptor (ORL-1) whose peripherl ctivtion nd ctivtion in the spinl cord hs been shown to be nti-nociceptive [7,,9]. Both control nd AHF fed nimls received single ip injections of vehicle only,,, or nmol/kg nociceptin. Nociceptive responses to mechnicl nd het stimultion 1 h fter tretment with single ip dose of nociceptin in nimls with AHF induced pncretitis were compred 3 Jnury 1, Volume 1 Issue 3

9 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity to controls. Dose-dependent improvement ws observed fter the single dose of nociceptin for responses to low intensity mechnicl stimultion of the bdomen (Figure A), responses to higher intensity mechnicl stimultion of the bdomen (Figure B), nd mechnicl thresholds on the hindpws (Figure C). In AHF fed nimls nmol/kg nociceptin decresed bdominl hypersensitivity to low intensity (1 g) mechnicl stimultion [vehicle: control =. ±.; AHF = (.3 ±.7 responses)/1; p <.; nmol nociceptin: control = 3. ±.3; AHF = (.7 ±. responses)/1; n.s.; Figure A]. Abdominl withdrwl of AHF nimls to higher intensity ( g) mechnicl stimultion decresed from 9. ±.3 to. ±. responses fter tretment with the highest dose of nociceptin. This ws not different from vehicle treted control nimls [(. ±. responses)/1; Figure B]. Tretment of AHF fed nimls with nmol/kg nociceptin incresed mechnicl thresholds on the hindpws so tht they were no longer different from controls while not ltering the sensitivity in control nimls (vehicle: control = 1. ±.; AHF =.3 ±.7 g force; P <.; nmol: control = 1.7 ±.; AHF =.7 ± 1. g force; P <.; Figure C). Thus, dose of nmol nociceptin per kg body weight ws determined to be the most effective in reducing sensitized nociceptive responses in ll four behviorl ssys in AHF fed rts (Figure E-H). With this dose, bdominl withdrwls of AHF fed rts in response to low intensity mechnicl stimultion decresed to control levels t the 1 nd h timepoints. Responses of AHF fed nimls to mechnicl stimultion of the bdomen t 1 h were significntly decresed [1 g: - h =. ±.; 1 h = (3. ± 1.1 responses)/1; P <.; g: - h = 9. ±.; 1 h = (. ±. responses)/1; P <.; Figure E, F] nd returned to pre-tretment levels h fter the injection [1 g: h = (. ±.9 responses)/1; g: h = (9. ±.3 responses)/1]. Nociceptive responses of AHF fed rts to higher intensity bdominl mechnicl stimultion were decresed to control vlues only t the 1 h time point fter nociceptin tretment (Figure F). Mechnicl withdrwl thresholds of the hindpws of AHF fed nimls lso incresed significntly from. ± 1. g force prior to tretment to 17. ± 1.3 g force 1 h fter tretment (Figure G). Four hours fter nociceptin tretment, the mechnicl thresholds of AHF fed nimls with chronic pncretitis decresed gin, returning to their previous hypersensitive level within h ( h = 13.3 ±.; h = 7. ± 1. g force; Figure G). Nociceptin tretments hd no effect on mechnicl sensitivity in control nimls. In control nimls the nmol/kg dose incresed hypersensitivity but ws not significnt [(7. ±.3 responses)/1]. Vehicle tretments did not lter nociceptive responses of nïve or AHF fed nimls. Hotplte response ltencies of AHF nimls were incresed dose dependently from 1. ±. s (vehicle) to.1 ±. s ( nmol/kg nociceptin), but the improvement ws significnt only t the highest dose of nociceptin (Figure D). After systemic injection of nmol/kg nociceptin, for AFH nimls, the hotplte response ltency incresed from 1.1 ±.7 s prior to tretment to.1 ±. s 1 h fterwrds (P <.; Figure H). This improvement remined significntly lower thn in untreted control (. ±.9 s; P <.). Het responses of control nimls slightly decresed 1 h fter injection of nmol/kg nociceptin (vehicle: 19. ±.3 s; nmol: 1.7 ± 1. s; Figure H). The bility of nociceptin to reverse hypersensitivity of AHF fed nimls ws optiml t 1 h fter which it decresed nd ws completely bsent h fter injection. Comprison to effects of morphine on AHF diet induced hypersensitivity Mny studies hve demonstrted the efficcy of morphine in reversl of cute nd chronic pncretitis ssocited hypersensitivity in niml models s well s its use for pin mngement of chronic pncretitis ptients with pin. Single dose s.c. tretment with 3 mg/kg morphine sulfte solution resulted in significnt reduction of mechnicl nd het hypersensitivity in AHF fed rts (Figure ). Responses of AHF fed rts to low intensity mechnicl stimultion of the bdomen [- h: (. ±.3 responses)/1; 1 h: 1. ±. responses /1; h: (. ±. responses)/1; AHF n = ; P <.; Figure A] ws significntly reduced for up to h compred to responses before tretment. Similrly, responses of AHF fed rts to higher intensity mechnicl stimultion of the bdomen [- h: (9. ±. responses)/1; 1 h: (.3 ±. responses)/1; h: (. ±. responses)/1; AHF n = ; P <.; Figure B] ws significntly reduced for up to h compred to responses before tretment. Nïve nimls responded to higher intensity mechnicl stimultion of the bdomen with. ±. responses/1 which ws significntly reduced 1 h fter morphine tretment to 3.3 ±. responses/1 (P <.; Figure B). Mechnicl thresholds of the hindpws in the AHF diet group improved fter morphine tretment for up to h (- h: 7. ± 1. g force; 1h: 1.7 ± g force; h: 1. ±. g force; AHF n =, P <.; Figure C). No chnges in nïve nimls were mesured fter morphine tretment for either on the hindpw nociceptive withdrwl thresholds for low intensity mechnicl stimultion or the responses to low threshold (1 g) bdominl stimultion. Morphine tretment reversed het sensitivity of AHF fed nimls bck to response levels recorded in nïve nimls for up to h (- h: 1.7 ±.1 s; 1h:. ±.7 s; h: 1. ±.7 s; AHF n =, P <.; Figure D). Responses of nïve rts on the hotplte were not ffected by the 3 mg/kg morphine. Animls were tested during their ctive period nd did not pper somnolent for ny of the testing. After h no effects of morphine were detected in ny of the tests. DISCUSSION Here we present non-invsive dietry niml model of lcoholic chronic pncretitis induced in 3 wk but Jnury 1, Volume 1 Issue 3

10 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity Nociceptin - dose-response curve A 1 No. of responses/1 C Mechnicl threshold (g) 1 1 g force, bdomen Nociceptin (nmol/kg) B 1 No. of responses/1 Hindpw D Ltency (s) 1 g force, bdomen Control (n = ) AHF (n = ) Nociceptin (nmol/kg) Hotplte Nociceptin (nmol/kg) Nociceptin (nmol/kg) Nociceptin ( nmol/kg) - time course E 1 No. of responses/1 G Mechnicl threshold (g) 1 1 g force, bdomen F 1 No. of responses/1 Hindpw H Ltency (s) 1 g force, bdomen Hotplte Control + veh AHF + veh Control + nociceptin AHF + nociceptin Figure Dose-response curves nd time course of nociceptive responses to mechnicl nd het stimuli fter single systemic tretment with nociceptin. A, B: Comprisons of the number of responses to mechnicl stimultion on the bdomen skin 1 h fter tretment with nociceptin (ip) for control nd lcohol nd high ft (AHF) fed rts. Dose-dependent responses were reduced to (A) low intensity (1 g) nd (B) high intensity ( g) mechnicl stimultion of the bdomen; C: Nociceptin incresed hindpw mechnicl withdrwl threshold in AHF fed rts; D: Hotplte response ltencies of AHF fed nimls improved with the highest dose of nociception. In control nimls nociceptin did not lter mechnicl nd het sensitivity. Vehicle tretments hd no effect on mechnicl or het sensitivity of nimls from either group; E-H: Mechnicl nd het responses in nimls treted with either single systemic injection of nociceptin ( nmol/kg in sline) or with the sline vehicle; E: Abdominl withdrwls in response to low intensity (1 g) mechnicl stimultion decresed to control levels t the 1 nd h post injection timepoints of AHF fed rts. Mechnicl hypersensivity of vehicle treted AHF diet fed rts did not improve; F: Responses of AHF fed rts to high intensity ( g) bdominl mechnicl stimultion were decresed to control vlues only 1 h fter injection of nociception; G: Hindpws mechnicl withdrwl thresholds of AHF fed rts recovered to control vlues for up to h post injection; H: Hotplte response ltencies of AHF fed rts improved only t the 1 h time point fter nociceptin tretment. P <., AHF vs control. Jnury 1, Volume 1 Issue 3

11 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity A Morphine (3 mg/kg) - time course 1 1 g force, bdomen B 1 g force, bdomen Control (n = ) AHF (n = ) No. of responses/1 No. of responses/1 C Hindpw D Hotplte Mechnicl threshold (g) 1 Ltency (s) 1 Figure Time course of nociceptive responses to mechnicl nd het stimuli fter single systemic tretment with morphine. Animls were injected with morphine (3 mg/kg, subcutneous) nd responses to mechnicl nd het stimuli mesured. Morphine tretment of lcohol nd high ft (AHF) fed rts decresed the number of responses to (A) low (1 g) nd (B) high intensity ( g) mechnicl stimultion of the bdominl skin for up to h. Abdominl withdrwl responses of controls evoked by high intensity ( g) mechnicl stimultion were reduced 1 h fter tretment compred to before morphine. Hindpw (C) mechnicl withdrwl thresholds nd (D) response ltencies on the hot plte test of AHF fed rts were improved t 1 nd h fter morphine tretment. Controls were unffected by the low dose morphine. P <., AHF vs control. persisting for t lest 1 wk. The model replictes poor dietry choices of clinicl ptients dignosed with lcoholic chronic pncretitis. Recent estimtes reported tht 1%-7% of Americns consume 3- lcoholic beverges per dy which concurs with decrese in diet qulity nd n increse of dietry ft intke [7-7]. Concurrently, the Food nd Agriculture Orgniztion (FAO) of the United Ntions reported stedy increse of the verge dily dietry ft consumption per cpit in the United Sttes from 1 g in 199/9 to 11 g in /7 while it verged 131 g ft in the developed world [73]. Comprison of nutritionl profiles identified tht ptients with chronic lcoholic pncretitis consumed significntly more sturted ft nd niml protein compred to ptients suffering from lcohol consumption induced cirrhosis of the liver [1,7,7]. Other studies hve shown tht drinkers consume significntly more dietry ft nd clories on drinking dys [71,7,7]. Exctly this combintion is described s inducing chronic lcoholic pncretitis, with lcohol being the leding nd diet the secondry cuse [77,7]. Animl models of lcoholic chronic pncretitis nd ftty liver disese hve shown tht incresed dietry ft consumption results in higher heptic triglycerides nd ssocited ft vcuoliztion [,,79]. Hence, the AHF rt model of chronic pncretitis described here closely mimics highrisk humn dietry choices tht re prt of the etiology of chronic lcoholic pncretitis with liver stetosis. Presently fvored rodent models of pncretitis re highly invsive nd poorly reproduce disese progression seen in clinicl ptients. They often require lprotomy surgery to obstruct the pncretic duct, or direct instill chemicl irritnts such s the romtic nitro compound trinitrobenzene sulfonic cid or ethnol into the pncretic duct. Other models utilize the repeted suprphysiologicl ip injections of cerulein, n nlog of the gstrointestinl peptide hormone cholecystokinin, in combintion with n ip lipopolyscchride injection or single dose of the highly toxic orgnotin dibutyltin chloride into the til vein [3,31,]. All of these models induce rpid immune response tht is focused primrily on the pncres. By chemiclly modifying cell surfce proteins n inflmmtory cell rection is induced which ultimtely results in cinr cell deth with or without prior dysregulted digestive enzyme relese. Thus, other current models of pncretitis re better suited for study of cute Jnury 1, Volume 1 Issue 3

12 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity pncretitis [31,1,]. Animls cn recover in these cute models within -7 h s in most cses of cute pncretitis. However, in the AHF model the pthologicl nd functionl symptomtology of chronic lcoholic pncretitis persists. Regenertion of the dmged tissue is impired by the continued lcohol consumption, fcilitting the estblishment of chronic pncretitis [3]. Chronic pncretitis is multifctoril disese cused by combintion of regulr lcohol consumption, low qulity diet high with high ft content producing hyperlipidemi, smoking, nd hereditry fctors [1,19,1,3,7,]. The AHF model of chronic pncretitis focuses on the combined effects of lcohol nd diet high in ft on the pncretitis nd ssocited comorbidities. Chronic consumption of lcohol results in its metbolism not only in the liver, but lso in the pncres. Alcohol is typiclly metbolized vi cetldehyde into cetyl which is used for energy production. In the results presented here, liver stetosis induced by lcohol consumption [,] ws visible not only in the centrilobulr region of the liver where lipids re predominntly stored in ftty vcuoles [], but ws detected throughout the lrge re of smpled tissue in the AHF fed nimls indicting severity of disese progression. Metbolism of dietry lcohol nd ft converges on the sme enzymes, so tht when high mounts of lcohol nd ftty cids re consumed, cellulr signling molecules nndmide nd rchidonic cid, nd cytotoxic cetldehydes ccumulte within liver nd pncres tht re ble to directly ctivte ion chnnels such s Trnsient Receptor Potentil Ction Chnnel, Subfmily A, Member 1 (TRPA1); Trnsient Receptor Potentil Ction Chnnel, Subfmily V, Member 1 (TRPV1); nd Trnsient Receptor Potentil Ction Chnnel, Subfmily V, Member (TRPV) loclized in the cell membrnes of nociceptive sensory neurons innervting the pncres s well s on locl cells to induce cellulr dmge [,7-9]. In the pncres cetldehydes nd ftty cid ethyl esters cn further induce frgility of pncretic zymogen grnules nd cuse cellulr dmge to pncretic cells [7,,9,91]. Resulting cellulr trophy nd necrosis induce seprtion of pncretic lobules s well s peri- nd intrlobulr fibrosis, indictors of severe dmge to prenchyml tissue, were visible throughout the exmined pncretic tissue of AHF fed nimls. Cell dmge nd necrosis directly nd indirectly ctivte the pncretic locl immune stellte cells, nd ctivte nociceptors innervting the pncres, mplifying pin senstion nd pncretic tissue dmge through neurogenic inflmmtion [9,9,93]. Alcoholic chronic pncretitis not only results in dmge to the exocrine pncres but cn lso impir nd dysregulte insulin metbolism in the endocrine pncres which in ptients results in the development of dibetes mellitus type 3c [1,13]. In the present study, 1 wk of AHF diet resulted in decresed glucose tolernce in rts. While no chnges in islet of Lngerhns number nd size distribution were detected (dt not shown), fsting glucose tolernce ws impired, cellulr trophy of insulin cells ws evident histologiclly, nd 3. fold increse of smll clusters of insulin producing cells were detected in AHF fed rts. This suggested compenstory rection to bet cell trophy nd decresed effectiveness. In Koren study incresed lcohol consumption coincided with incresed dietry ft which concurred with decresed pncretic bet cell function [7]. Low-density lipoproteins, metbolic products of sturted fts, hve been shown to reduce bet-cell function, decresing glucose-stimulted insulin secretion nd cusing bet-cell prolifertion in primry cell culture experiments [9]. Exposure of cultured bet cells to combined ftty cids nd ethnol hs been shown to decrese their mitochondril ctivity nd denosine triphosphte (ATP) production nd increse relesed rective oxygen species, indictors of their decresed function [3]. Plteletderived growth fctor s well s interleukin-1β, two proinflmmtory cytokines nd signling molecules tht re incresed in pncretitis, hve been reported to induce bet-cell prolifertion [9]. The prolifertion of smll bet-cell groups observed in AHF fed pncres tissue suggest regenertive mechnism is ctive in the rts to compenste for ethnol nd ftty cid induced cell dmge. As in our previous study using similr protocol to induce chronic pncretitis, nimls fed AHF diet developed mechnicl hypersensitivity of the bdomen nd hindpws s well s incresed het sensitivity. Alcohol metbolism in chronic lcoholism is not confined to viscerl orgns but occurs in every tissue. As result of cellulr dmge produced by chronic lcoholism, resulting oxidtive stress cn lso be mesured in skeletl muscle [9]. Cellulr dmge releses molecules such s ATP nd incompletely metbolized lcohol products ctivte nd sensitize nociceptors throughout the body, resulting in hypersensitivity tht is not confined to the bdominl region. Activity induced centrl sensitiztion further excerbtes hypersensitivity to noxious nd normlly innocuous stimuli. Cliniclly, % of chronic pncretitis ptients require tretment for pin t some point during disese progression nd re treted depending on severity of the pin with non-nrcotic nlgesics nd s pin increses, ever incresing doses of opites s well s pncretic enzyme therpy. In extreme cses, nerve blocks nd eventully surgicl intervention my be required [,7]. While opioids re efficcious in ptients s well s in niml models of chronic pncretitis, even cute doses hve severe side effects nd my led to overdose. Long term use of morphine cn cuse ddiction nd constiption, its efficcy to reduce pin decreses with durtion of use, while ongoing pncretic inflmmtion is not improved. Yet, despite the side effects, prescription rtes for opioids hve incresed lmost exponentilly in the lst decde nd non-ddictive lterntives re very much needed [97,9]. We therefore investigted the hypothesis tht ctivtion of other Gicoupled GPCRs would decrese nociceptive signling. Initil focus ws on the inhibitory mglur/3 signling pthwy. Glutmte is the min excittory 7 Jnury 1, Volume 1 Issue 3

13 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity neurotrnsmitter tht depolrizes neurons by binding to its ionotropic nd metbotropic receptors. However, glutmte initites inhibitory signling cscdes when bound to the group Ⅱ metbotropic glutmte receptors, mglur nd mglur3 to dmpen cellulr excitbility [99,1]. Activtion of these Gi-coupled GPCRs negtively modulte denylte cyclse, lter synptic connectivity, nd decrese relese of glutmte [11]. Group Ⅱ mglurs re expressed throughout the nervous system, in primry sensory neurons, predominntly presumed nociceptors, in the dorsl horn of the spinl cord, s well s in viscerl orgns such s bet-cells of the pncres [1-]. After cute single dose tretment with the mglur/3 gonist APDC in the present study, the hypersensitized responses to mechnicl nd het stimuli of AHF nimls were no longer significntly different from those of controls. This ttenution ws of short durtion (1 h) suggesting tht systemic dministrtion of APDC my hve ctivted mglur/3 receptors on peripherl nociceptive sensory neurons, thus reducing nociception. Previous studies hve reported efficcious use of group Ⅱ gonists s nlgesics without ny pprent side effects in niml models of neuropthic nd cute inflmmtory pin without ltering nociceptive responses of control nimls [1,,,1-1]. Systemic use of mglur/3 gonists is ble to ctivte receptors on peripherl sensory neurons to decrese their ctivity nd centrl relese of glutmte, thus interrupting neurogenic inflmmtion nd fcilitting tissue regenertion. Due to their expression within the limbic system, mglur/3 receptors hve been studied in connection with schizophreni, lcohol nd drug ddiction, nd nxiety [19-111]. The potentil bility of single drug to be used not only for pin mngement in lcoholic chronic pncretitis, but lso to support ptients lcohol cesstion nd dietry mngement would be of immense benefit, simplifying dosing nd regulr use of phrmcologicl therpy. Prolonged use of different mglur/3 gonist, LY379, for pin mngement in somtic pin models showed tht repeted dily systemic dosing quickly resulted in desensitiztion nd lost bility to reverse hypersensitivity [3,]. Long-term experiments utilizing lower doses or novel mglur/3 gonists for repeted tretments re needed to investigte its efficcy in reversing lcoholic chronic pncretitis ssocited hypersensitivity. Nociceptin, lso clled orphnin FQ, is n endogenous opioid-like peptide tht does not bind to the clssicl opioid receptors but to ORL-1. Like mglur/3, ctivtion of this Gi-coupled GPCR lso inhibits denylte cyclse nd voltge-gted C + -chnnels while ctivting voltge-gted K + -chnnels which results in decresed neuronl ctivity nd decresed glutmte relese [,,11]. In the AHF diet induced chronic pncretitis niml group, cute systemic tretments with nociceptin successfully reversed the induced mechnicl nd het hypersensitivity in AHF fed nimls. Unlike morphine, these tretments did not lter nociceptive responses of control nimls. Morphine decreses mechnicl nd het sensitivity concentrtion-dependently in control nimls. In the present experiments morphine decresed mechnicl sensitivity in control rts while response ltencies in the hotplte test were not ffected. The Fischer F3 rt strin is known for hving higher nxiety compred to other rt strins [113]. Animls were cclimted to the experimentl set-up for mechnicl sensitivity ssys wheres the hotplte test posed novel environment which my hve ltered the effects of morphine in control Fischer F3 rts. Nociceptin nd downstrem signling pthwys initited by binding to the ORL-1 receptor hve been enigmtic. Initil reports described tht intrcerebroventriculr injections induced hyperlgesi nd high doses inhibited locomotion [,]. Subsequent studies using intrthecl injections demonstrted nlgesic properties of ORL-1 ctivtion by reducing cute inflmmtory nd nerve injury induced neuropthic hypersensitivity [,7,9,11]. Intrthecl nociceptin reduced peripherl relese of vsodiltory neuropeptides co-expressed with ORL-1, thus decresing edem fter cute inflmmtory insult by decresing dorsl root potentils nd glutmtergic trnsmission [1-117]. Due to the bsence of relible ntibodies for ORL-1, its locliztion still relies on in situ hybridiztion studies. Its mrna hs been loclized throughout nociceptive signling pthwys, including smll dimeter, presumbly nociceptive sensory neurons, within the superficil dorsl horn s well s round the centrl cnl, nd in multiple regions throughout the brin [11,11]. Reduction of hypersensitivity in AHF nimls fter systemic tretment with nociceptin ws most likely chieved by ORL-1 ctivtion within the peripherl nervous system nd resulting decrese in neuronl ctivity nd glutmte relese. While ctivtion of ORL-1 within the spinl cord is predicted to lso be ble to ttenute chronic pncretitis induced hypersensitivity, repeted use of systemic nociceptin my result in incresed crossing of the blood brin brrier which cn be compromised in cses of severe peripherl inflmmtion thus promoting pro-nociceptive effects similr to intrcerebroventriculr injections [119,1]. Combined tretment using morphine nd nociceptin provided synergistic nlgesi thus diminishing the mount of morphine needed to ttenute pin senstion nd reducing the risk of sensitiztion to morphine in previous study []. This opens the possible therpeutic option of employing the nociceptin-nop system for more efficient pin mngement of chronic pncretitis-ssocited pin while reducing side effects. In summry, the Gi-coupled GPCR signling pthwys downstrem from mglur/3 nd ORL-1 receptors investigted here, presented good lterntives to presently employed opioid pin therpies for the tretment of chronic pncretitis induced pin. Likewise, these studies suggest these gents hve potentil s djuvnt therpy to reduce the dosge of morphine derivtives nd relted side effects. Jnury 1, Volume 1 Issue 3

14 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity COMMENTS Bckground Chronic pncretitis is progressive nd potentilly ftl disese cused by persistent unresolved inflmmtion nd pncretic fibrosis. Pncretitis cn be ccompnied by severe intrctble bdominl pin. Currently recommended pin mngement therpies rnge from non-nrcotic nlgesics to strong opites with their ccompnying side-effects, but re not effective s long term tretments. Presently employed niml models poorly reproduce the clinicl etiology of chronic pncretitis s they re highly invsive, requiring lprotomy surgery nd/or utilize repetitive dosing with custic chemicls. The bdominl hypersensitivity induced with these experimentl procedures is better suited for modeling cute pncretitis. Reserch frontiers To dte no stisfctory pin therpy is vilble for tretment of chronic pncretitis nor is there suitble experimentl model for the study of chronic pncretitis resembling the clinicl syndrome with which to test potentil therpeutics. Knowledge of the underlying pthophysiology of chronic pncretitis resulting in this severe bdominl pin is needed for the development of better phrmcologicl interventions. Innovtions nd brekthroughs The niml model of chronic pncretitis is induced with % lcohol nd high ft diet similr to poor humn dietry choices. It produces hypersensitivity to touch with fine filments on the bdomen nd hs chrcteristics of type 3c dibetes mellitus (T3cDM), i.e., bnorml insulin histology nd intolernce to glucose. Reduction of hypersensitivity induced by the model ws demonstrted with the phrmcologicl ctivtion of three inhibitory metbotropic Gi proteincoupled receptors. Anlgesi ws demonstrted with single doses of drugs ctivting inhibitory glutmte nd opite-like receptors, mglur/3 nd ORL-1, respectively, in comprisons to morphine. Applictions These therpies for the tretment of chronic pncretitis ssocited pin re suggested s good lterntives to presently employed opioid pin therpies. Used s djuvnt therpy with morphine derivtives for more effective relief of chronic pncretitis pin, these gents would hve potentil to reduce the opite dosge nd relted side effects. Terminology Intrctble pncretic pin cnnot be llevited with current therpies. Using the % lcohol nd high ft diet (AHF) induced rt chronic pncretitis model, two therpies were tested, including nociceptin, n endogenous lignd for the opioid-receptor-like 1 receptor, nd (R,R)--Aminopyrrolidine-,- dicrboxylte, n ctivtor of inhibitory mglur/3 glutmte receptors. These therpies reduced the hypersensitivity tht develops on the bdominl skin of the rts with chronic pncretitis s effectively s morphine. Peer review This study demonstrted tht the AHF diet replicting high risk humn dietry choices induced chronic lcoholic pncretitis in rts with histologicl fetures nd developing glucose intolernce resembling clinicl ptients with chronic pncretitis nd T3cDM. So, this pper hs novelty nd innovtion. REFERENCES 1 Coté GA, Ydv D, Slivk A, Hwes RH, Anderson MA, Burton FR, Brnd RE, Bnks PA, Lewis MD, Disrio JA, Grdner TB, Gelrud A, Amnn ST, Billie J, Money ME, O Connell M, Whitcomb DC, Shermn S. Alcohol nd smoking s risk fctors in n epidemiology study of ptients with chronic pncretitis. Clin Gstroenterol Heptol 11; 9: -73; quiz e7 [PMID: 1977 DOI: 1.11/ j.cgh.1.1.] Nøjgrd C, Becker U, Mtzen P, Andersen JR, Holst C, Bendtsen F. Progression from cute to chronic pncretitis: prognostic fctors, mortlity, nd nturl course. Pncres 11; : [PMID: 1993 DOI: 1.197/ MPA.b13e311f9] 3 Mlk D, Hmmel P, Mire F, Ruft P, Mdeir I, Pessione F, Lévy P, Ruszniewski P. Risk of pncretic denocrcinom in chronic pncretitis. 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18 McIlwrth SL et l. Phrmcologicl ttenution of pncretitis induced hypersensitivity 799 DOI: 1.1/jphert.1.] 119 Ronldson PT, Dvis TP. Trgeting blood-brin brrier chnges during inflmmtory pin: n opportunity for optimizing CNS drug delivery. Ther Deliv 11; : 1-11 [PMID: 1 DOI: 1./tde.11.7] 1 Tin JH, Xu W, Fng Y, Mogil JS, Grisel JE, Grndy DK, Hn JS. Bidirectionl modultory effect of orphnin FQ on morphine-induced nlgesi: ntgonism in brin nd potentition in spinl cord of the rt. Br J Phrmcol 1997; 1: 7- [PMID: 9137 DOI: 1.13/sj.bjp.79] P- Reviewer: Zhng ZM S- Editor: M YJ L- Editor: A E- Editor: Wng CH 3 Jnury 1, Volume 1 Issue 3

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