Z. WARZECHA, A. DEMBIÑSKI, P. CERANOWICZ, J. STACHURA*,R. TOMASZEWSKA*, S. J. KONTUREK

EFFECT OF SENSORY NERVES AND CGRP ON THEDEVELOPMENT OF CAERULEIN-INDUCED PANCREATITIS AND

PANCREATIC RECOVERY

Department of Physiology and *Department of Clinical and Experimental Pathomorphology,

Jagiellonian University School of Medicine, Cracow, Poland

The function of primary sensory neurons is to receive and transmit information fromexternal environment and these neurons are able to release neuromediators from theactivated peripheral endings. The aim of this study was to determine the influence ofsensory nerves and administration of their mediator — calcitonin gene related peptide(CGRP) on the course of acute pancreatitis (AP). Ablation of sensory nerves wasperformed by neurotoxic dose of capsaicin (100 mg/kg). Single or repeated episodesof AP were induced by caerulein infusion (10 �g/kg/h for 5 h). Five repeated AP wereperformed once a week. Capsaicin at the dose which stimulates sensory nerves (0.5mg/kg/dose) or CGRP (10 �g/kg/dose) was administrated before and during or aftersingle induction of AP, as well as, after each induction of repeated AP. Rats werekilled at the time 0, 3 or 9 h after single induction of AP or two weeks after lastinduction of repeated AP. Ablation of sensory nerves aggravated pancreatic damage incaerulein-induced AP. Treatment with stimulatory doses of capsaicin or CGRP beforeand during single induction of AP attenuated the pancreatic damage in morphologicalexamination. This effect was also manifested by partial reversion of AP evoked dropin DNA synthesis and pancreatic blood flow (PBF). Administration of CGRP aftersingle AP induction aggravated histologically manifested pancreatic damage. Thefurther decrease in PBF and DNA synthesis was also observed. Animals with fiveepisodes of AP showed almost full pancreatic recovery two weeks after last inductionof AP concerning all parameters tested. In stimulatory doses of capsaicin treated rats,we observed the decrease in pancreatic amylase and fecal chymotrypsin activity, aswell as, the drop in DNA synthesis. Similar but less pronounced effects were observedafter treatment with CGRP. We conclude that effect of sensory nerves and CGRP onAP is two-phase and time dependent. Stimulation of sensory nerves or theadministration of CGRP during development of AP exhibits protective effects againstpancreatic damage induced by caerulein overstimulation. After induction of AP,persistent activity of sensory nerves and presence of CGRP aggravate pancreaticdamage and lead to functional insufficiency typical for chronic pancreatitis.

K e y w o r d s: capsaicin, acute pancreatitis, CGRP, pancreatic regeneration, pancreatic

functional insufficiency

INTRODUCTION

Primary sensory neurons serve for conduction of nociceptive information tothe central nervous system, but also activation of these neurons causes the

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2001, 52, 4, 679–704

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release of neuromediators from peripheral endings (1). Sensory fibers have aspecial sensitivity to capsaicin (2). Low doses of capsaicin result in thestimulation of sensory nerves accompanied with the release of CGRP and otherneuromediators (3, 4), whereas high doses of capsaicin lead to ablation ofsensory nerves with the decrease in plasma and tissue level of CGRP (5, 6).CGRP is widely distributed throughout the central, peripheral and entericnervous systems (7, 8). Within the enteric nervous system, CGRP-containingnerves have been found in large numbers among others in the stomach (9, 10),the intestine (8, 10) and the pancreas (9—12). CGRP is identified as a majormediator of thin, unmyelinated capsaicin-sensitive sensory neurons (3, 13).Capsaicin-sensitive primary afferent neurons and CGRP are involved indifferent aspects of the stomach pathology. The stimulation of sensory fibers,as well as, administration of exogenous CGRP was found to exert a protectiveeffect in different experimental models of gastric ulcers (14—16), whereas theablation of sensory nerves aggravates gastric mucosal lesions induced byvarious ulcerogenic factors (17, 18), inhibits the gastric mucosal growth (19)and prolongs gastric ulcer healing (20).

Beneficial effect of afferent nerve stimulation and CGRP administrationbefore ulcer induction has been attributed, at least in part, to improvement ofmucosal circulation (21, 22). In the pancreas, vascular mechanism was shown toplay an important role in exocrine secretion (23) and organ integrity. Thedevelopment of acute pancreatitis is associated with the decrease in pancreaticblood flow. Clinical and experimental studies have shown that pancreaticischemia may initiate acute pancreatitis and always aggravates pancreaticdamage (24—26). The severity of such experimental pancreatitis is closelycorrelated with tissue ischemia (27). Moderate and severe pancreatitis was foundto be accompanied with progressive decrease in pancreatic blood perfusion (27).

On the other hand, there is a growing number of evidence suggesting thatCGRP released from unmyelinated, afferent capsaicin-sensitive sensory nervesmay contribute to the chronic inflammatory response (28). Alterations ofintrapancreatic nerves including edema of nerve bundles and increased densityof peptidergic nerves with an intensification of immunostaining for CGRP havebeen seen in surgical specimens from patients and animals with chronicpancreatitis (29—31). Moreover, activation of these nerves may produce theneurogenic inflammation described as the local vasodilatation and plasmaextravasation (32).

The aim of this study was to determine: (a) the effect of sensory nerves andpretreatment with CGRP on development of acute caerulein-inducedpancreatitis, (b) the influence of CGRP administration after induction of acutepancreatitis on development of pancreatic damage, (c) the effect of prolongedstimulation of sensory nerves with low doses of capsaicin or CGRPadministration on pancreatic repair after repeated episodes of acute pancreatitis.

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MATERIALS AND METHODS

Animals and treatment

Studies were performed on male Wistar rats weighing 160—190 g (singleinduction of pancreatitis) or 120—140 g (repeated induction of pancreatitis) atthe start of experiments. The animals were housed in cages with wire meshbottoms, with normal room temperature and a 12-hour light-dark cycle. Drinkingof water and food were available ad libitum. The study was conductedfollowing the experimental protocol approved by the Committee for Researchand Animal Ethics of the Jagiellonian University.

Experiments were carried out in three separate series.First series of experiments were performed to determine the effect of sensory

nerves and pretreatment with CGRP on the development of single episode ofacute pancreatitis. The following groups of animals were used in this series ofthe experiment: (1) rats infused with saline (0.9% NaCl) s.c. to serve as thecontrol group; (2) rats infused with caerulein (caerulein-induced pancreatitis);(3) rats with stimulation of sensory nerves (capsaicin 2 � 0.5 mg/kg s.c., firstinjection 30 min before the start of saline infusion, second 3 h later) and infusedwith saline; (4) rats with ablation of sensory nerves (capsaicin 100 mg/kg s.c.)and infused with saline; (5) rats treated with CGRP (2 � 10 �g/kg s.c., firstinjection 30 min before the start of saline infusion, second 3 h later) andinfused with saline; (6) rats with ablation of sensory nerves, treated with CGRP(2 � 10 �g/kg s.c., first injection 30 min before the start of saline infusion,second 3 h later) and infused with saline; (7) rats with stimulation of sensorynerves (capsaicin 2 � 0.5 mg/kg s.c., first injection 30 min before the start ofcaerulein infusion, second 3 h later) and infused with caerulein; (8) rats withablation of sensory nerves and infused with caerulein; (9) rats treated with CGRP(2 � 10 �g/kg s.c., first injection 30 min before the start of caerulein infusion,second 3 h later) and infused with caerulein; (10) rats with ablation of sensorynerves treated with CGRP (2 � 10 �g/kg s.c., first injection 30 min before thestart of caerulein infusion, second 3 h later) and infused with caerulein.

Ablation of afferent sensory nerves was induced by pretreatment with capsaicin(Fluka, Buchs, Switzerland) in a total dose of 100 mg/kg, which was given in sixinjections (2.5 + 10 + 12.5 + 25 + 25 + 25 mg/kg s.c.) over 3 consecutive days.Two injections per day were performed in rats under ether anesthesia and arecovery period of 10 days was allowed before further experiments. To assess theeffectiveness of sensory denervation, the day before the induction of pancreatitis, adrop of capsaicin (0.33 mM) was instilled into rat eye, and animals showing anywiping movements were excluded from the study.

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Acute edematous pancreatitis was induced by caerulein infusion in rats keptin individual cages. Caerulein (Takus, Pharmacia & Upjohn GmbH, Erlangen,Germany) was diluted in saline and infused s.c. for 5 h at a dose 10 �g/kg/h andat a rate of 1.0 ml/h.

Rat, synthetic CGRP-I was obtained from Sigma Chemical Co, St. Louis,MO, USA. Animals from the first series of experiments were sacrificed after5 h of infusion with saline or caerulein.

Second series of experiments were performed to evaluate the influence ofCGRP administration after single induction of acute pancreatitis on developmentof pancreatic damage. The following groups of animals were used in this seriesof experiments: (1) control (intact rats infused with saline only); (2) caeruleininduced pancreatitis; (3) caerulein induced pancreatitis + CGRP (20 �g /kg ofCGRP given s.c. in two doses: first 10 �g/kg 30 min prior to caerulein infusionand second 10 �g /kg 3 h later, during caerulein infusion); (4) caerulein inducedpancreatitis + CGRP given at the time 1 h, 4 h, and 7 h after the end of caeruleininfusion (10 �g of CGRP per dose).

Acute edematous pancreatitis was induced by caerulein as in the first seriesof experiments. Rats were sacrificed at the time 0 h, 3 h or 9 h after cessationof caerulein infusion.

Third series of experiments was performed to determine the effect ofprolonged activity of sensory nerves or CGRP administration on the pancreaticrepair after repeated episodes of acute pancreatitis. The following groups ofanimals were used in this series of experiment: (1) rats infused with saline s.c.to serve as the control group; (2) rats with repeated induction of acutepancreatitis and treated with saline s.c. after each induction of acute pancreatitis;(3) rats with repeated induction of acute pancreatitis and stimulation of sensorynerves by capsaicin after each induction of pancreatitis; (4) rats with repeatedinduction of acute pancreatitis and treated with CGRP after each induction ofpancreatitis.

Five episodes of acute pancreatitis were performed by caerulein infusion atweekly intervals. After each induction of acute pancreatitis, saline, capsaicin orCGRP were administrated s.c. three times daily starting 1 h after termination ofcaerulein infusion. Capsaicin was used at the sensory nerve stimulatory dose:0.5 mg/kg/dose. CGRP was administrated at the dose 10 �g/kg/injection. Twoweeks after last induction of acute pancreatitis rats were sacrificed.

Determination of pancreatic blood flow

At the end of each series of experiments, animals were anesthetized withether, weighed and the abdominal cavity was opened. The pancreas was exposedfor measurement of blood flow in the pancreatic tissue by laser Doppler

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flowmeter using PeriFlux 4001 Master monitor (Perimed AB, Jrflla, Sweden).Blood flow was measured in five different portions of the pancreas. The pancreaticblood flow was presented as percent change from control value obtained in ratsinfused with saline.

Determination of plasma amylase activity and plasma interleukin 1-�

concentration

Immediately after measurement of pancreatic blood flow the abdominal aortawas exposed and blood was taken for plasma amylase and interleukin-1� (IL-1�)determination. Plasma amylase activity was determined by an enzymatic method[Amylase reagent set (kinetic), Alpha Diagnostic sp. z o.o., Warsaw, Poland].The values were expressed as units/liter (U/L) or units/pancreas. Plasma IL-1�

was measured in duplicate using the BioSource Cytoscreen rat IL-1� kit basedon a solid phase sandwich Enzyme Linked Immuno Sorbent Assay (ELISA)(BioSource International, Camarillo, California, USA). Concentration of plasmaIL-1� was expressed as pg/ml.

Determination of DNA synthesis and amylase activity in the pancreas

After the blood withdrawal, the pancreas was carefully dissected out from itsattachment to the stomach, the duodenum, and the spleen. Fat and excess tissuewere trimmed away. Samples of pancreatic tissue from each series of experimentwere taken for study of pancreatic DNA synthesis and morphologicalexamination. Additionally, samples of pancreatic tissue from the third series ofexperiment were taken for study of pancreatic amylase activity. The rate of DNAsynthesis in the portion of minced pancreatic tissue was determined byincubating the tissue at 37oC for 45 min in 2 ml of medium containing 8 �Ci/mlof [3H]thymidine ([6-3H]-thymidine, 20—30 Ci/mmol; Institute for Research,Production and Application of Radioisotopes, Prague, Czech Republic). Thereaction was stopped with 0.4 N perchloric acid containing carrier thymidine (5mM). Tissue samples were centrifuged and the precipitate washed twice in cold0.2 N perchloric acid and recentrifuged. RNA was hydrolyzed in 0.3 M KOHincubated for 90 min at 37oC. DNA and protein were reprecipitated with 10%perchloric acid. After standing for 10 min on ice, the tubes were centrifuged andthe supernatant was discarded. DNA in the residual pellets was solubilized in10% perchloric acid by heating at 70oC for 20 min. Denaturated protein wasremoved by centrifugation for 20 min. Using calf thymus as a standard, the DNAconcentration was determined by Giles and Myers procedure (33). Theincorporation of [3H]thymidine into DNA was determined by counting 0.5 mlDNA-containing supernatant in a liquid scintillation system. DNA synthesis was

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expressed as [3H]thymidine disintegrations per minute per microgram DNA(dpm/�g DNA).

The pancreatic amylase activity was determined in the portion of pancreatictissue which was weighed and placed in 2 ml pH 7.4 sodium — potassiumphosphate buffer containing 0.2 mg of trypsin inhibitor (Type I-S, Sigma, St.Louis, USA). Pancreatic tissues were homogenized, sonicated and centrifugedad 30,000 g for 10 min. Amylase activity in aliquot from the supernatant wasdetermined by the same enzymatic method as in plasma samples. Thepancreatic amylase activity was expressed as units per pancreas.

Determination of fecal chymotrypsin

Fecal chymotrypsin activity in the third series of experiment was measured bycolorimetric method with a test kit Chymo (Boehringer, Mannheim, Germany).Values were expressed as U/g of stool.

Histological examination

Samples of pancreatic tissue were excised, fixed in 10% formalin,embedded in paraffin and sections were stained with hematoxylin and eosin.The slides were examined histologically by two experienced pathologistswithout the knowledge of treatment given. The histological grading of edemawas made using a scale ranging from 0 to 3; 0 = no edema, 1 = interlobularedema, 2 = interlobular and moderate intralobular edema, and 3 = severeinterlobular and intralobular edema. Leukocyte infiltration was graded from 0(absent) to 3 for maximal alterations (diffuse infiltration in the entire pancreaticgland) Grading of vacuolization was based on the percentage of cells involved: 0= absent, 1 = less than 25%, 2 = 25—50% and 3 = more than 50%.

Additionally, samples from the third series of experiments were examined forhistological signs of chronic pancreatitis such as acinar cell necrosis, atrophy,fibrosis and tubular complexes. Findings of acinar necrosis, atrophy and fibrosiswere evaluated as lesion size score: 0 = absent, 1 = a lesion slightly shown in thelobule or intralobular region (less than one-half), 2 = a lesion widely shown inthe intralobular region (more than one-half), and 3 = a lesion shown acrosslobules and intralobular regions or with destruction of lobular architecture.Tubular complexes were expressed as: 0 = lack or 1 = presence of these lesions.

Statistical analysis

Results are expressed as means � S.E.M. and were analyzed by analysis ofvariance and t-Student test for unpaired values, with p < 0.05 consideredsignificant.

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RESULTS

First series of experiments

Subcutaneous administration of caerulein at a dose 10 �g/kg/h for 5 hinduced the acute edematous pancreatitis in all tested rats. Pancreata appearedgrossly swollen and enlarged with visible collection of edematous fluid.Pancreatic DNA synthesis was deeply decreased to 52% of control value (Fig.

1), the pancreatic blood flow was reduced by about 50% (Fig. 2). The plasmaamylase activity (Fig. 3) and the plasma IL-1� level (Fig. 4) were increasedeight and twofold, respectively. Histological changes were closely correlatedwith the biochemical findings (Table 1). There was moderate interlobular andintralobular edema, and in about half of rats severe intralobular edema. Theedema was accompanied by perivascular infiltration by neutrophils and thepresence of vacuolization in about half of the acinar cells.

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Fig. 1. Effect of saline (control), stimulatory (0.5 mg/kg) and neurotoxic (100 mg/kg) doses of capsaicin andCGRP given alone or in their combination with single saline or caerulein infusion on pancreatic DNAsynthesis. Mean � S.E.M. of 8—14 observations. aP < 0.05 compared with control, bP < 0.05 compared withcaerulein given alone, cP < 0.05 compared with the neurotoxic dose of capsaicin given in combination with

caerulein.

Stimulatory doses of capsaicin (0.5 mg/kg) given alone were without anysignificant effect on the pancreatic DNA synthesis (Fig. 1), plasma amylaseactivity (Fig. 3), plasma IL-1 (Fig. 4) or pancreatic histology. Pancreatic bloodflow was increased by 29% (Fig. 2).

Ablation of sensory nerves by high dose of capsaicin (100 mg/kg) caused asignificant decrease in pancreatic DNA synthesis and pancreatic blood flow by18 and 30%, respectively (Fig. 1 and 2). Plasma amylase activity showed asmall but significant increase (Fig. 3). Plasma IL-1� concentration wasincreased by 58% (Fig. 4). Histological examination has shown that ablation ofsensory nerves produced slight leukocyte infiltration and lack or slightinterlobular edema without vacuolization of acinar cells (Table 1).

CGRP given alone caused a significant increase in pancreatic blood flow by20% over the control value (Fig. 2), whereas other parameters were notsignificantly affected. Treatment with CGRP in combination with ablatorydoses of capsaicin (100 mg/kg) partly reversed the capsaicin-induced reduction

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Fig. 2. Effect of saline (control), stimulatory (0.5 mg/kg) and neurotoxic (100 mg/kg) doses of capsaicin andCGRP given alone or in their combination with single caerulein infusion on pancreatic blood flow. Mean ±S.E.M. of 8—14 observations. aP < 0.05 compared with control, bP < 0.05 compared with caerulein givenalone, cP < 0.05 compared with the neurotoxic dose of capsaicin given in combination with caerulein, dP < 0.05

compared with the neurotoxic dose of capsaicin given alone.

in pancreatic DNA synthesis (Fig. 1) and pancreatic blood flow (Fig. 2). In thisgroup of animals, plasma amylase activity was above the control value or valueobserved after CGRP or neurotoxic dose of capsaicin given separately (Fig 3).CGRP partly reduced the plasma IL-1� concentration in animals with ablationof sensory nerves (Fig. 4). Morphological features (Table 1) have shown thatCGRP prevented against perivascular leukocyte infiltration induced byneurotoxic dose of capsaicin.

Administration of the stimulatory dose of capsaicin with caerulein infusionreduced the severity of caerulein-induced pancreatitis. Pancreatic DNAsynthesis (Fig. 1) and pancreatic blood flow (Fig. 2) were higher, whereas anincrease in plasma amylase activity (Fig. 3) was smaller compared to caeruleininfusion alone. Also histological examination showed a reduction in pancreaticdamage (Table 1). Edema was limited to interlobular space in most cases, onlyin a few cases moderate intralobular edema was observed. Leukocyteinfiltration and acinar cells vacuolization were decreased. Moreover, an

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Fig. 3. Effect of saline (control), stimulatory (0.5 mg/kg) and neurotoxic (100 mg/kg) doses of capsaicin andCGRP given alone or in their combination with single caerulein infusion on plasma amylase activity. Mean �

S.E.M. of 8—14 observations. aP < 0.05 compared with control, bP < 0.05 compared with caerulein givenalone, cP < 0.05 compared with the neurotoxic dose of capsaicin given in combination with caerulein, dP < 0.05

compared with the neurotoxic dose of capsaicin given alone.

increase in plasma IL-1� concentration (Fig. 4) caused by caerulein waspartially reversed by stimulatory doses of capsaicin, but this effect wasstatistically insignificant.

Capsaicin-induced deactivation of sensory nerves (100 mg/kg) prior tocaerulein infusion aggravated pancreatic damage created by caerulein, whichwas manifested by an additional decrease in pancreatic DNA synthesis (Fig. 1)and a increase in plasma IL-1� concentration (Fig. 4). Alterations of pancreaticblood flow and plasma amylase activity were not statistically significant (Fig. 2

and 3). Histological examination revealed severe interlobular and intralobularedema in all animals. Leukocytic infiltration and vacuolization of acinar cellswere more pronounced than after caerulein given alone.

Treatment with CGRP during caerulein infusion attenuated the severity ofacute pancreatitis. Caerulein-induced a reduction in pancreatic DNA synthesis(Fig. 1) and pancreatic blood flow (Fig. 2) was partly, but significantlyreversed. Also morphological features showed improvement of pancreatichistology, edema was limited to the interlobular space in most cases, leukocyticinfiltration and acinar cells vacuolization were strongly reduced. Plasmaamylase activity (Fig. 3) and plasma IL-1� concentration (Fig. 4) were notaltered significantly.

Deleterious effect of the ablation of sensory nerves on caerulein inducedpancreatitis was completely reversed by CGRP administration. Pancreatic DNA

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Table 1. Effect of saline (control), stimulatory (0.5 mg/kg) and neurotoxic (100 mg/kg) doses ofcapsaicin and CGRP given alone or in their combination with single caerulein infusion onhistological signs of pancreatic damage.

EDEMA(0—3)

INFILTRATION(0—3)

VACUOLIZATION(0—3)

Saline (control) 0 0 0

Caerulein 2/3 1/2 2

Capsaicin 0.5 mg/kg 0 0 0

Capsaicin 100 mg/kg 0/1 1 0

CGRP 0 0 0

Capsaicin 100 mg/kg + CGRP 0/1 0 0

Capsaicin 0.5 mg/kg + Caerulein 1/2 0/1 1

Capsaicin 100 mg/kg + Caerulein 3 2 3

CGRP + Caerulein 1/2 0/1 1/2

Capsaicin 100 mg/kg + CGRP + Caerulein 2 1 2

Numbers represent the predominant histological grading in each group.

synthesis (Fig. 1) and pancreatic blood flow (Fig. 2) were increased, whereasplasma amylase activity (Fig. 3) and plasma IL-1� concentration (Fig. 4) weredecreased. These changes were statistically significant. Histologically, pancreaticdamage was reduced. Pancreatic edema and leukocytic infiltration were evensmaller than after caerulein given alone (Table 1).

Second series of experiments

Like in first series of experiments, subcutaneous infusion of caeruleinresulted in the formation of acute pancreatitis in all tested rats. Pancreatic DNAsynthesis was decreased by 42% at the time 0 h after the end of caeruleininfusion (Fig. 5) and an additional inhibition of DNA synthesis was observedafter 3 and 9 h. At the time 0, 3 and 9 h after the end of caerulein infusion,pancreatic blood flow was reduced by 30, 31 and 51%, respectively (Fig. 6).At the time 0 h after caerulein infusion, plasma amylase activity (Fig. 7) and

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Fig. 4. Effect of saline (control), stimulatory (0.5 mg/kg) and neurotoxic (100 mg/kg) doses of capsaicin andCGRP given alone or in their combination with single caerulein infusion on plasma interleukin-1�

concentration. Mean � S.E.M. of 8—14 observations. aP < 0.05 compared with control, bP < 0.05 comparedwith caerulein given alone, cP.05 compared with the neurotoxic dose of capsaicin given in combination with

caerulein, dP < 0.05 compared with the neurotoxic of capsaicin dose given alone.

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Fig. 5. Effect of CGRP given before and during or after single caerulein infusion on pancreatic DNAsynthesis. Mean � S.E.M. of 8—10 observations. aP < 0.05 compared with control, bP < 0.05 compared withcaerulein given alone at the same time of observation, cP<0.05 compared with animals treated with CGRP

before and during caerulein infusion at the same time of observation.

Table 2. Effect of CGRP administration before and during or after single caerulein infusion onhistological signs of pancreatic damage.

HISTOLOGY

EDEMA

(0—3)

INFILTRATION

(0—3)

VACUOLIZATION

(0—3)

0 h after caerulein infusion

Saline infusion (control)Caerulein aloneCGRP before and during caerulein

02/31/2

01/20

02

1/2

3 h after caerulein infusion

Caerulein aloneCGRP before and during caeruleinCGRP after caerulein

2/313

1/21

1/2

211

9 h after caerulein infusion

Caerulein aloneCGRP before and during caeruleinCGRP after caerulein

1/212

10

1/2

101

Numbers represent the predominant histological grading in each group.

plasma IL-1� concentration (Fig. 8) were increased about nine- and threefold,respectively and these parameters trended to an additional increase at the time3 and 9 h. Morphological examination of pancreata at the time 0 h revealed theinterlobular and moderate intralobular edema in half of the animals treated withcaerulein, the rest of animals treated with caerulein, revealed severe interlobularand severe intralobular edema (Table 2). The same feature of edema wasobserved at the time 3 h after cessation of caerulein infusion, whereas at thetime 9 h the edema was in most cases limited to the interlobular space. Edemawas accompanied by perivascular infiltration by leukocytes and the presence ofacinar cells vacuolization. The maximum infiltration by leukocytes andvacuolization were observed at the time 0 and 3 h, whereas at the time 9 hwere less pronounced (Table 2).

Treatment with CGRP before and during caerulein infusion attenuated theseverity of pancreatitis. The pancreatic edema was permanently reduced at thetime 0, 3 and 9 h after caerulein infusion (Table 2). Caerulein-induced reductionin pancreatic DNA synthesis was partly reversed by pretreatment with CGRP(Fig. 5) and this effect was statistically significant at the time 0, 3 and 9 h after

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Fig. 6. Effect of CGRP given before and during or after single caerulein infusion on pancreatic blood flow.Mean � S.E.M. of 8—10 observations. aP < 0.05 compared with control, bP < 0.05 compared with animals

treated with CGRP before and during caerulein infusion at the same time of observation.

cessation of caerulein-infusion. Caerulein evoked fall in pancreatic blood flow(Fig. 6), it was partly, but permanently and significantly reversed bypretreatment with CGRP. Administration of CGRP before and during caeruleininfusion was without significant effect on caerulein evoked increase in plasmaamylase activity at the time 0 and 3 h after the end of caerulein infusion (Fig.

7), whereas at the time 9 h, a significant reduction in this parameter wasobserved. Plasma IL-1� concentration in the group treated with CGRP beforeand during caerulein infusion obtained the same value as in the caerulein alonetreated group (Fig. 8) at the time 0 h after cessation of caerulein infusion, butlater, plasma IL-1� concentration decreased reaching at the time 9 h asignificanty lower level, than in the caerulein treated group. Alsomorphological features showed improvement of pancreatic histology whenCGRP was administered before and during caerulein infusion (Table 2).Pancreatic edema, leukocytic infiltration and acinar cells vacuolization werestrongly reduced at the time 0, 3 and 9 h after the end of caerulein infusion.

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Fig. 7. Effect of CGRP given before and during or after single caerulein infusion on plasma amylaseactivity. Mean � S.E.M. of 8—10 observations. aP < 0.05 compared with control, bP < 0.05 compared withcaerulein given alone at the same time of observation, cP < 0.05 compared with animals treated with CGRP

before and during caerulein infusion at the same time of observation.

Treatment with CGRP after caerulein infusion aggravated pancreaticdamage created by caerulein. Pancreatic DNA synthesis (Fig. 5) and pancreaticblood flow (Fig. 6) were decreased additionally and significantly reaching thelowest values at the time 9 h after cessation of caerulein infusion. Plasmaamylase activity (Fig. 7) was higher than in the caerulein alone treated group,but this difference was not statistically significant. A significant difference inplasma amylase activity was observed between animals treated with CGRPbefore and during caerulein infusion and animals treated with CGRP aftercaerulein infusion at the time 9 h after cessation of caerulein infusion.Administration of CGRP after caerulein infusion strongly increased the plasmaIL-1� concentration (Fig. 8). At the time 3 h after cessation of caeruleininfusion, histological examination revealed that CGRP given after caerulein,leads to severe inter-and intralobular edema in all cases (Table 2). Also, at thetime 9 h after caerulein infusion, pancreatic edema was strongly expressed inthis group of animals. In the group of animals treated with CGRP aftercaerulein infusion, leukocytic infiltration and vacuolization of acinar cells were

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Fig. 8. Effect of CGRP given before and during or after single caerulein infusion on plasma interleukin 1�

concentration. Mean � S.E.M. of 8—10 observations. aP < 0.05 compared with control, bP < 0.05 comparedwith caerulein given alone at the same time of observation, cP < 0.05 compared with animals treated with

CGRP before and during caerulein infusion at the same time of observation.

more pronounced than in caerulein alone treated group or in the group ofanimals treated with CGRP before and during caerulein infusion.

Third series of experiments

Two weeks after last episode of repeated acute pancreatitis, pancreata ofanimals treated with placebo (saline) showed almost full recovery. PancreaticDNA synthesis was not significantly different when compared with valuesobtained from intact rats without induction of pancreatitis (Fig. 9), whereaspancreatic blood flow was significantly increased by 26% (Fig. 9). Pancreaticamylase activity and fecal chymotrypsin activity reached 90 and 88% ofcontrol value, respectively (Fig. 10). Plasma amylase activity and plasma IL-1�

concentration were similar to control values (Fig. 11). Morphologicalexamination of pancreata obtained from spontaneously healed animals revealedslight interlobular edema and slight or lack of leukocytic infiltration and acinarcells vacuolization (Table 3). Acinar cell necrosis, atrophy, fibrosis or tubularcomplexes were not observed in pancreata obtained from animals with repeatedinduction of acute pancreatitis and treated with saline.

Treatment with capsaicin after each repeated episodes of acute pancreatitiscaused a delay in pancreata healing. In this group of animals, pancreatic DNAsynthesis (Fig. 9), pancreatic amylase activity (Fig. 10) and fecal chymotrypsinactivity (Fig. 10) were significantly lower than in the control group, as well as,in animals treated with saline after each induction of acute pancreatitis.Contrary to these findings, pancreatic blood flow was increased by 72% abovecontrol value (Fig. 9) and by 36% when compared with animals treated withsaline after each induction of acute pancreatitis. Also, administration ofcapsaicin in animals with repeated induction of pancreatitis caused an increasein plasma IL-1� concentration above control value and value observed foranimals treated with saline after each induction of acute pancreatitis (Fig. 11).Reduction in plasma amylase activity was insignificant (Fig. 11).Morphological features showed interlobular and moderate intralobular edemaand a mild stage of leukocytic infiltration, and acinar cells vacuolization inanimals treated with capsaicin (Table 3), but in most cases the acinar cellnecrosis, atrophy, fibrosis or tubular complexes were not observed.

Treatment with CGRP after each induction of pancreatitis produced markedreduction in pancreatic amylase activity and fecal chymotrypsin activity to thelevel as observed in animals treated with capsaicin (Fig. 10). Influence ofCGRP on pancreatic DNA synthesis (Fig. 9), pancreatic blood flow (Fig. 9),plasma IL-1� concentration and plasma amylase activity (Fig. 11) was similarto that produced by capsaicin, but changes evoked by CGRP were lesspronounced. Histological examination of pancreata obtained from rats treated

694

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Fig. 9. Effect of five episodes of caerulein-induced pancreatitis combined with administration of capsaicin(stimulatory doses) or CGRP on the pancreatic DNA synthesis and pancreatic blood flow. Mean � S.E.M. of8—12 observations. aP < 0.05 compared with control, bP < 0.05 compared with repeated caerulein-induced

pancreatitis + saline.

Table 3. Effect of five episodes of caerulein-induced pancreatitis combined with administrationof capsaicin or CGRP on histological signs of pancreatic damage.

CONTROL

(without

pancreatitis)

REPEATED

PANCREATITS

+

SALINE

REPEATED

PANCREATITIS

+

CAPSAICIN

REPEATED

PANCREATITIS

+

CGRP

edema 0 1 2 1/2

leukocyte infiltration 0 0/1 1 1

vacuolization 0 0/1 1 1

acinar cell necrosis 0 0 0 0

acinar cell atrophy 0 0 0 0

fibrosis 0 0 0 0

tubular complexes 0 0 0 0

Numbers represent the predominant histological grading in each group.

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Fig. 10. Effect of five episodes of caerulein-induced pancreatitis combined with administration of capsaicin(stimulatory doses) or CGRP on the pancreatic amylase activity and fecal chymotrypsin activity. Mean +S.E.M. of 8—12 observations. aP < 0.05 compared with control, bP < 0.05 compared with repeated

caerulein-induced pancreatitis + saline.

Fig. 11. Effect of five episodes of caerulein-induced pancreatitis combined with administration of capsaicin(stimulatory doses) or CGRP on the plasma amylase activity and interleukin-1� concentration. Mean +S.E.M. of 8—12 observations. aP < 0.05 compared with control, bP < 0.05 compared with repeated

caerulein-induced pancreatitis + saline.

with CGRP showed only interlobular edema or interlobular and moderateintralobular edema (Table 3). Leukocytic infiltration was slight and thevacuolization was found in less than 25% of acinar cells. Acinar cell necrosis,atrophy, fibrosis or tubular complexes were not found in any cases of animalstreated with CGRP after repeated induction of acute pancreatitis.

DISCUSSION

The present study confirms and extends our previous findings, thatstimulation of sensory nerves (34, 35) and administration of CGRP (36) beforeand during induction of pancreatitis reduces pancreatic damage evoked by theoverdose of caerulein. Morphological features and the increase in pancreaticblood flow and pancreatic DNA synthesis have shown an improvement ofpancreatic tissue condition. Stimulation of sensory nerves or CGRPadministration reduced leukocyte infiltration of pancreatic tissue but bothfactors were without significant effect on plasma IL-1� concentration. Also,treatment with CGRP did not affect plasma amylase activity significantly.These results suggest that protective effect of stimulation of sensory nerves andadministration of CGRP is mainly dependent on the improvement of pancreaticblood flow. Vasodilatation evoked by CGRP and an increase in pancreaticblood flow before vascular damage, allow for removal of active digestiveenzymes and mediators of inflammation from pancreatic tissue and attenuatethe pancreatic damage in pancreatitis. For the same reason plasma amylaseactivity and plasma IL-1� concentration remained increased.

Another beneficial mechanism of CGRP action can be dependent on theinhibition of exocrine pancreatic secretion (37, 38). Debas et al. (39) suggestedthat CGRP evoked inhibition of exocrine pancreatic secretion is indirect,neurally mediated and may be explained by the release of somatostatin. Theprotective effect of sensory nerve stimulation and administration of CGRPagainst caerulein-induced pancreatitis can be also dependent on the release ofnitric oxide (NO). Interaction between release and action of CGRP and NO isunclear. Some previous reports have suggested that release of CGRP is NOdependent (39, 40), others have suggested that CGRP acts by NO release (41,42). Moreover, it has been shown that CGRP stimulates NO liberation byacinar cells in the course of acute pancreatitis (43) and increases the activity ofNO synthase (NOS) (44). Stimulation of afferent sensory nerves results in therelease of endogenous CGRP (3) and NO (45), where both factors: CGRP andNO are strong vasodilators (23, 46). Furthermore, a reduction in NO synthesisby an inhibition of NOS aggravates the damage of the pancreas created bycaerulein (47) and the degree of this injury is almost the same as after sensory

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nerve ablation in combination with caerulein. Addition of L-arginine, asubstrate for NOS, reverses deleterious effect of NOS inhibition (47).

On the other hand, excessive amounts of NO cause the oxidative injury andcontribute to multiorgan oxidative stress in pancreatitis (48), and NO may alsoreduce the antioxidant capacity of injured organs by binding the SH group (49).Moreover, NO can induce pancreatitis by itself (50). These findings have shownthat the level of NO should to be within an appropriate range. Either excess orlack of NO can exhibit deleterious effect on pancreatic tissue. As initiallyreported for constitutive NOS, also inducible NOS activity may be associated toreduced leukocyte-endothelium interaction and platelet aggregation, as well as,protection of microcirculation (51). It is possible, that the stimulation of NOrelease by CGRP allows maintenance of a physiological amount of NO.

Protective effect of sensory nerve stimulation or pretreatment with CGRPagainst development of pancreatic damage in caerulein-induced pancreatitiswas seen immediately after the termination of caerulein infusion, as well as, 3and 9 h later. This observation indicates that it is a permanent protective effect.

The present study has shown that ablation of sensory nerves increases theseverity of caerulein-induced pancreatitis and this effect has been completelyreversed by exogenous CGRP. This observation and the information that lowdoses of capsaicin stimulate the release of CGRP (5) demonstrate thatprotective effects of sensory nerve stimulation in the pancreas could beattributed to CGRP release. Additional support for this hypothesis is thefinding that administration of a neurotoxic dose of capsaicin causes thepersistent decrease in tissue CGRP-like immunoreactivity (1, 52).

Another finding of our present study is observation that administration ofCGRP after induction of pancreatitis increases the pancreatic damage. Thisdeleterious effect of CGRP administration is in agreement with our previousreport (52) and with results obtained during induction of gastric lesions byLopez-Belmonte and Whittle (53). They showed that CGRP can exert bothanti- and pro-inflammatory action leading, respectively to reduction andaugmentation of gastric mucosal injury. This effect has been dependent on thetime of CGRP administration. Pretreatment with CGRP before induction ofgastric lesions protected gastric mucosa against injury, whereas administrationof CGRP after application of endothelin-1 exacerbated the mucosal damage.This effect of CGRP on the stomach, as well as, on the pancreas seems to bedependent on the degree of tissue and vascular damage. Induction of gastriculcer or induction of pancreatitis led to tissue and vascular damage and for thisreason addition of a vasodilator, such as CGRP increased the plasma proteinleakage from injured vessels to pancreatic tissue leading to tissue edema and amaximal decrease in pancreatic blood flow. This hypothesis is in agreementwith studies performed by Cambridge et al. (54) and Newbold et al. (55) whofound that treatment with CGRP promotes increasing vascular permeability

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leading to the production of edema. Moreover, there is a growing number ofevidence suggesting that CGRP released from unmyelinated, afferentcapsaicin-sensitive sensory nerves may contribute to the chronic inflammatoryresponse (28). Activation of these nerves may produce the neurogenicinflammation described as the local vasodilatation and plasma extravasation(32). Furthermore, CGRP and substance P may promote neutrophil adherenceto endothelium (57). It is well known that adhesion of leukocytes to microvascularendothelium is an early and rate limiting step in the inflammatory response(57) leading, among others, to induction of cytokines production. In our presentstudy, we found that treatment with CGRP after induction of pancreatitis,increases the plasma IL-1� concentration to a higher extent than in the grouptreated with caerulein alone and aggravates the pancreatic damage. Thisobservation is consistent with findings that IL-1� plays the crucial role in theinduction of cytokine cascade and development of pancreatitis (58). The use ofIL-1 naturally occurring receptor antagonist, almost completely attenuates arise in serum IL-6 and TNF-�, as well as, decreases the severity of acutepancreatitis as was shown in the study performed by Norman et al. (58).

The mechanisms that induce chronic pancreatitis remain unclear (59) andthe pathogenesis of this disease is under much debate as to whether it is a newprocess or a result of single attack or recurrent episodes of severe acutepancreatitis (60, 61). In our present study repeated induction of acuteedematous pancreatitis by caerulein failed to induce chronic pancreatitis. Twoweeks after the last episode of acute pancreatitis, pancreata from animalstreated with saline showed almost full functional and morphological recovery.This result is in agreement with our previous observations (62, 63) and dataobtained by others (64—66) that repeated induction of acute pancreatitis aloneis insufficient to induce chronic pancreatitis.

Sensory neurons play a role in regulation of the inflammatory and immuneresponses in peripherial tissue. The peripherial localized inflammation inducesincrease in the synthesis and transport of neuropeptides in sensory nervesinnervating inflamed tissue (67), as well as, the activation of these nerves mayproduce neurogenic inflammation described as the local vasodilatation andplasma extravasation (32). On the other hand, clinical (29, 30) and experimental(31) studies in chronic pancreatitis have shown an increase in the number anddiameter of intralobular and interlobular nerve bundles and the intensificationof immunostaining for sensory nerve mediators. In our present study, treatmentwith capsaicin was performed after each induction of acute pancreatitis leadingto continuous stimulation of sensory nerves. Long lasting activity of sensorynerves led to reduction in digestive enzyme amount in the pancreas causing thedecrease in pancreatic and fecal digestive enzyme activity. The treatment withcapsaicin inhibited the pancreatic regeneration as proven by the decrease in

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pancreatic nucleic acid content (63) and DNA synthesis. This pancreaticfunctional insufficiency is typical for chronic pancreatitis, however, thehistological examination did not reveal signs of chronic pancreatitis, such asacinar cell necrosis, atrophy, fibrosis or tubular complexes. In rats treated withCGRP pancreatic and fecal activity of digestive enzymes, as well as,morphological features were similar to noticed in the capsaicin treated groupbut less pronounced.

Finally, our present data demonstrate that the physiological role ofcapsaicin-sensitive sensory nerves and their mediator — CGRP is dependent onlocal protection against damage if noxious agent acts on intact tissue. Theprolonged activity of sensory neurons induced by multiple injection ofstimulatory dose of capsaicin, as well as, the prolonged administration ofCGRP on damaged tissue aggravate tissue lesions and lead to enzymaticinsufficiency typical for chronic pancreatitis.

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R e c e i v e d: October 5, 2001

A c c e p t e d: October 18, 2001

Author’s address: Artur Dembiñski, Department of Physiology, Jagiellonian UniversitySchool of Medicine, ul. Grzegórzecka 16, 31-531 Kraków, Poland, phone +48-12-421-10-06, fax+48-12-421-15-72.

E-mail mpdembin@cyf-kr.edu.pl

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