Gut 1995; 37: 427-433

Biochemical and morphological changes thatcharacterise recovery from necrotising biliarypancreatitis in the opossum

M Runzi, A Saluja, A Kaiser, D Gerdes, A Sengupta, M L Steer

AbstractThe events that characterise recoveryfrom severe biliary pancreatitis have notbeen defined. This study used a reversiblemodel ofnecrotising pancreatitis, inducedby obstructing the opossum common bilepancreatic duct (CBPD), to evaluate thisphenomenon. The CBPD of opossumswas obstructed with a balloon tippedcatheter for five days and then decom-pressed by removal of the catheter.Recovery was evaluated 0-90 days afterrelief of obstruction. Serum bilirubin andamylase values rapidly declined, reachingcontrol values 7-14 days after removal ofthe obstructing catheter. Pancreaticprotein and amylase values were tran-siently increased shortly after relief ofobstruction but returned to control values21 days after decompression. Pancreaticornithine decarboxylase activity and in-corporation of [3HJ-thymidine into DNAwere transiently increased 14 days afterduct decompression suggesting that re-generation occurs at approximately thattime. Foci ofpancreatic necrosis involvedroughly 400/0 of the gland at the time ofdecompression but these foci graduallydisappeared and the gland resembled thatof control animals 60 days after decom-pression. Evidence of fibrosis or collagendeposition in the pancreas was not notedat any time. These studies show thatrecovery after necrotising biliary pan-creatitis occurs comparatively rapidlyand the restitution ad integrum occurs.Recovery from necrotising acute pan-creatitis in this model is not associatedwith the development of chronic pan-creatitis.(Gut 1995; 37: 427-433)

Keywords: biliary pancreatitis, opossum.

The events that characterise structuralrecovery of the pancreas after a severe attack ofnecrotising pancreatitis have not been clearlydefined. Restitutio ad integrum is believed bymany to occur in humans.1 On the other hand,some recent reports have suggested that severenecrotising acute pancreatitis in humans maybe followed by fibrosis and evolve into chronicpancreatitis.24 This so called necrosis-fibrosishypothesis, however, remains controversial.Detailed morphological studies characterisingrecovery from acute necrotising pancreatitis in

humans have not been possible because, forthe most part, specimens of pancreatic tissuefrom patients recovering from pancreatitishave not been available. Animal studies havebeen of limited value as, for the most part, themethods of inducing pancreatitis result in onlymild glandular injury (duct ligation or secreta-gogue hyperstimulation in rodents) or causeextensive yet poorly controlled degrees ofdestruction (retrograde duct injection).5We have recently undertaken a series of

studies characterising a model of biliary pan-creatitis in the opossum. Obstruction of thecommon bile pancreatic duct (CBPD) in thatanimal with a balloon catheter results in acutenecrotising pancreatitis, which has a severitythat is dependent upon the duration of ductobstruction. With obstruction for five days,roughly 40% of acinar cells become necrotic.Relief of obstruction, by removal of the ballooncatheter, prevents further progression of thepancreatitis and, as noted previously, theanimals can recover.8 We reasoned that thismodel might permit further studies that couldcharacterise the events that occur duringrecovery from severe pancreatitis and that thecontroversial issue of whether or not severeacute pancreatitis evolves into chronic pancre-atitis could be examined in such studies. In thiscommunication, we report the results ofexperiments designed to examine these issues.We note that complete recovery occurscomparatively rapidly after relief of obstructionand that chronic pancreatitis does not develop.

Methods

Preparation of animalsRandomly trapped, apparently healthy,opossums (Didelphis Virginiana) of either sexweighing 1 1-5.8 kg were obtained fromR-Zoo (Neshkoro, WI), Northeastern Wild-life (South Plymouth, NY) or R Singleton(Arcachia, FL). Conditioned animals werehoused in individual steel cages in a climatecontrolled room with ambient temperature of23±3°C and with a 12 hour light/dark cycle.Animals were fed an opossum laboratory dietand given water ad libitum. Because trappedopossums commonly harbour a variety ofparasites, all animals were extensively de-wormed (Ivemectin, 1% solution, 0.2 ml/kgintramuscularly, Merck-Agvet, Brassway, NJ)before starting the experiments. In addition, allanimals were pre-treated with a five day courseof penicillin G (50 000 U/kg intramuscularly

Department ofSurgery, Beth IsraelHospital, HarvardMedical School,Harvard DigestiveDiseases Center,Boston, USAM RunziA SalujaA KaiserD GerdesM L Steer

Division ofPediatricGastroenterology andNutrition, BostonUniversity School ofMedicine, Boston,USAA Sengupta

Correspondence to:Dr M Steer, Department ofSurgery, Beth IsraelHospital, 330 BrooklineAvenue, Boston, MA 02215USA.Accepted for publication31 December 1994

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on alternate days). This prophylactic treatmentwith penicillin G was continued for 10 daysafter operation and induction of pancreatitis.This regimen of prophylactic penicillin treat-ment was chosen because opossums are verysusceptible to infection, especially withstreptococcus D, and fatal acute bacterialendocarditis is comparatively common amongthese animals if they are left untreated.9The experimental protocol was approved by

the Institutional Animal Care and UseCommittee. Animals were anaesthetised byintraperitoneal injection of pentobarbital (50mg/kg). Using sterile technique, an upper mid-line laparotomy was performed and the cysticduct was ligated to prevent the gall bladderfrom serving as a bile reservoir. Control shamoperated animals underwent duodenotomyand closure of the duodenotomy but no furthersurgical manipulation. In contrast, the experi-mental animals underwent placement of aballoon tipped catheter (size 4F, Shiley Inc,Irvine, CA) in the CBPD as previously des-cribed.8 For this purpose, the duodenum wasopened on its antimesenteric surface and thecatheter was passed into the ampulla of Vaterwithout performance of a sphincterotomy.After inflation of the balloon and closure of theduodenotomy, the catheter was secured to asubcutaneous site and the abdomen wasclosed. The control sham operated group ofanimals (n=3), which had only cystic ductligated, underwent sham re-operation (lapar-otomy) five days later and they were then killed36 days after that procedure. In the experi-mental animals, ductal obstruction wasrelieved by removing the balloon tippedcatheter, after a laparotomy under pentobarbi-tal anaesthesia, five days after catheter place-ment. At that time, those experimental animalswere randomly assigned to seven differentgroups based on the length of time that wasallowed to elapse between relief of obstructionand death. That time was as follows: group A(n=4), zero days; group B (n=3), one day;group C (n=4), two days; group D (n=4),seven days; group E (n=4), 14 days; group F(n=3), 21 days; group G (n=7), 65 days; andgroup H (n=3), 90 days. At those times,animals were killed by the intravenous admin-istration of a lethal dose of pentobarbital, theabdomen was opened, and blood as well aspancreas was harvested for various assays (seelater). In addition to the sham operated andexperimental groups of animals described, anon-operated control group of animal (n=4)were also studied. The results obtained in thatgroup were, in all aspects studied, not signifi-cantly different from those noted for the shamoperated control group.We chose to study recovery from acute

necrotising pancreatitis induced by a five dayperiod of duct obstruction after performingpreliminary studies to evaluate the relationbetween animal survival and the duration ofduct obstruction. Those studies (not shown)showed that all animals survived when theductal obstruction was relieved after five days.On the other hand, when ductal obstructionwas present for seven or more days, animal

death was common (approximately 80%) and,under those circumstances, most of theanimals died within 10-14 days of the time ofinitial ductal obstruction.

Assessment of acinar cell necrosis by morphometryComplete cross-sections of the head, body,and tail of the pancreas were fixed in 10 percent neutral phosphate buffered formaldehyde.Paraffin wax embedded sections (5 Km) werestained with haematoxylin and eosin, periodicacid Schiff, and Sudan black. They wereexamined by an experienced morphologist whowas not aware of the identity of the sample.Acinar cell necrosis was defined as destructionof acinar histo-architecture with the remainingtissue being composed of amorphous debris,cellular ghosts, and inflammatory cells. Theextent of necrosis was morphometrically quan-tified using a computerised image analysisvideo unit (model CCD-72, Dage-MTI,Michigan City, IN) equipped with the NIH-1200 image analysis software. As previouslyreported,7-9 foci of necrosis in this model ofacute pancreatitis are equally distributedbetween the head, body, and tail of thepancreas. Thus, morphometric data from thevarious sections obtained from each animalwere pooled for data analysis. Approximately10 randomly chosen high power microscopefields were examined for each tissue sampleand three tissue samples were evaluated foreach experimental animal.

Thymidine incorporationThe incorporation of [3H]-thymidine intotissue samples was used to evaluate the rate ofDNA synthesis. For these studies, animalswere given a bolus of [3H] -thymidine (100,uCi/kg, 24-5 mCi/mmol, Amersham, Arling-ton Heights, IL) two hours before death. [3H] -thymidine incorporation was evaluated aspreviously described.10 After tissue homogen-isation, [3H]-labelled DNA was precipitated byaddition of 20% trichloroacetic acid. Theprecipitates were washed twice in 10%trichloroacetic acid and radioactivity quanti-fied in a Packard Tricarb Scintillation counter(Packard, Sterling, VA). Results wereexpressed as counts per minute per mg DNA.

Immunocytochemistry for collagenFragments ofpancreas were fixed by immersionin neutral buffered picroformaldehyde for 18hours at 4°C. They were then washed exten-sively overnight with cold phosphate bufferedsaline (pH 7.4) and cryoprotected with 15%sucrose enriched ice cold phosphate bufferedsaline (pH 7.4). The samples were rapidlyfreeze blocked in Tissue-Tek OCT compound(Miles, Inc, Elkhart, IN) and stored at -80Cuntil sectioning. Cryosections of 10 Rum wereprepared and placed in gelatin subbed slides,which were allowed to dry overnight. They wererinsed in phosphate buffered saline and sites ofnon-specific protein binding were blocked byincubation with goat serum (10 minutes, room

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temperature). After washing they were exposedfor 18 hours at 4°C to the primary polyclonalantiserum (rabbit anti-collagen intravenously,#PS46, Monosan, Rotterdam, the Nether-lands), which had been diluted 1:40 before use.After thorough rinsing, they were exposed forone hour at room temperature to the secondaryantibody (goat antirabbit IgG conjugated withfluorescein isothiocyanate (ICN Immuno-biologicals, Lisle, IL), which had been diluted1:33 before use. Antigen was identified using anappropriate excitation filter and epi-illumina-tion with an Olympus BHT-2 micro-scope. Immunocytochemistry controls includedsamples in which the primary antibody wasomitted or in which the primary antiserum hadbeen previously exposed to purified collagen(100 ,ug/ml) for 24 hours at 4°C. Samples ofopossum achilles tendon and plantaris longustendon were examined as positive controls whilesamples of rat brain were examined as negativecontrols.

AssaysBlood was harvested from the right ventricleand serum prepared for measurement ofamylase activity as described by Pierre et all"as well as bilirubin content as describedby Winsten and Cehelyk. 12 Fragments ofpancreas were rapidly removed and weighed todetermine pancreatic wet weight. These frag-ments were then desiccated by incubation at160°C for 48 hours and re-weighed to deter-mine dry weight. Water content was calculatedas the difference between wet and dry weightand expressed as a per cent of the pancreaticwet weight. Other fragments of pancreas wereharvested for measurement of DNA, protein,amylase, ornithine decarboxylase (ODC), andhydroxyproline. The fragments were mincedwith scissors on ice and homogenised threetimes (20 seconds each) in ice cold buffer(50 mM phosphate, pH 7.4) using a Polytronhomogeniser (Brinkman, Westbury, NY).DNA was quantified fluorometrically usingHoechst dye 33258 and calf thymus DNA asthe standard.13 Protein was quantified accord-ing to Lowry et all 4 using bovine serumalbumin as the standard. Amylase was quanti-fied as described by Pierre et al.1 Ornithinedecarboxylase (ODC) activity was measured asdescribed by Langlois and Morrisset15 and cal-culated as [14C]-CO2 released per mg DNA.Hydroxyproline content was measured accord-ing to Ho and Pang'6 with slight modifications.In addition to hydroxyproline measurement,collagen formation was further evaluated usingthe chromatographic technique of Lopez deLeon and Rojkind.17 For these measurements,paraffin wax embedded cross sections of thehead, body, and tail regions of the pancreas(see Table) were deparaffinised, stained with0.1% Sirius red (Miles, Inc, Elkhart, IN), and0.1% Fast Green FCF (Sigma Chemicals,St Louis, MI) in saturated picric acid. Siriusred labels collagenous protein while FastGreen FCF labels non-collagenous protein.After incubation for 30 minutes at roomtemperature the stains were eluted with 0.1 N

NaOH in absolute methanol and the eluteddye was quantified in a spectrophotometer(Danbury, CT). The amount of collagenousprotein was expressed as a per cent oftotal pro-tein in the cross sections.

Analysis of dataStatistical analysis of the data was accom-plished using analysis of variance (ANOVA)and the Newman-Keul's test as a post hoc test.The data shown in the Figures represent mean(SEM) values. The absence of error bars indi-cates that the SEM was too small to illustrate.Significant differences in results were definedas those with a p value <0 05.

ResultsAll animals recovered quickly from anaesthesiaand displayed appropriate behaviour until thetime of death. Food and water consumptionfor control and experimental animals weresimilar. In accord with our previous finding,serum bilirubin concentration and amylaseactivity were increased five days after ductobstruction but then rapidly declined to thecontrol value after relief of ductal obstruction(data not shown). Pancreatic water contentwas also increased five days after duct obstruc-tion with a decline to control values notedshortly after relief of obstruction (data notshown). During the period of ductal obstruc-tion, pancreatic protein and amylase contentdeclined. Both pancreatic protein and amylasecontent returned to control values within oneweek after relief of obstruction, increased tovalues above the control level over the sub-sequent week (165 (10)% of control value),and then gradually declined.

Acinar cell necrosisMorphometry - five days after CBPD

obstruction, morphometric analysis at the lightmicroscope level showed that 42.5 (4.5)% ofacinar cells were necrotic. With relief ofCBPDobstruction, evidence of acinar cell necrosisrapidly diminished reaching a value of 17-3(2.5)% of acinar cells 14 days later and return-ing to the value noted in control sham operatedanimals by 60 days after relief of CBPDobstruction (Fig 1).

Microscopy - Figure 2(A)-(D) shows lightmicrographs from representative animals. Thenormal pancreas morphology is shown in (A)while that noted after CBPD obstruction forfive days is shown in (B). Figure 2(B) showsvarious features of acinar cell injury andnecrosis including single foci of necrotic cells(arrowheads in (B)) as well as the presence ofcellular ghosts. At this time, numerous inflam-matory cells as well as spindle cells, which maybe fibroblasts (asterisks in (B)) were seen in theinterstitial tissue spaces. Nine days after reliefof ductal obstruction, only infrequent necroticacinar cells were seen although inflammatorycells as well as spindle shaped cells were stillnoted in the interstitial space (asterisks in (C)).Ninety days after relief of CBPD obstruction,

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Time after decompression (days)Figure 1: Effect of bile pancreatic duct decompression onacinar cell necrosis. Opossums underwent obstruction of thebile pancreatic duct as described in the text anddecompression by removal of the balloon tipped catheterfivedays later. They were killed 0-90 days after decompression.Acinar cell necrosis was determined by morphometry, asdescribed in the text, and expressed as the per cent of totalsurface area that was occupied by necrotic cells. Asterisksshow values significantly different (p<0 05) from control.

no evidence of inflammation, acinar cellnecrosis, or fibrosis was seen (D) and thepancreatic morphology was similar to thatnoted for samples taken from control animals(A).

ODC activity andDNA synthesisDuct obstruction was associated with anincrease in ODC activity but no change in therate of incorporation of [3H]-thymidine intopancreatic DNA (Figs 3(A) and (B)). Withrelief of ductal obstruction, ODC activityrapidly returned to the pre-obstruction valuewhile the rate of [3H]-thymidine incorporationinto DNA fell below the control value.Subsequently, both ODC activity and the

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Figure 2: Effect of bile pancreatic duct decompression on pancreatic morphology.Representative light microscopic images for animals from thefollowing groups are shown:(A) Sham operated control; (B) CBPD obstruction forfive days with zero daysdecompression; (C) CBPD obstruction forfive days with 90 days decompression.Arrowheads in (B) point to areas ofacinar ceUl damage indicated by loss ofpolarity andarchitecture, loss ofcellular borders, and the presence ofamorphous debris as well as cellghosts. Asterisks in (B) and (C) indicate presence of inflammatory cells as well as spindleshaped cels.

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Time after decompression (days)Figure 3: Effects of bile pancreatic duct decompression onpancreatic ornithine decarboxylase (ODC) activity andincorporation ofpHI-thymidine into pancreatic DNA.Conditions are as described in Figure 1 legend. ODCactivity (A) was measured as described in the text andexpressed as cpm of['4C-CO2 released per mg DNA.[3HI-thymidine incorporation into DNA (B) was measuredas described in the text. Results represent mean (SEM)values for three to seven animals in each group with eachmeasurement being performed in duplicate. Asterisks showvalues significantly different (p<0 05) from control.

incorporation of [3H]-thymidine into pan-creatic DNA were transiently increased. Therise in DNA synthesis was first noted sevendays after relief of obstruction, reached a peakvalue on the 14th day after relief of obstruc-tion, and thereafter slowly declined over thesubsequent two months. The rise and fall ofODC activity after relief of obstruction weremore abrupt but ODC activity was also foundto reach a peak value 14 days after relief ofobstruction.

Collagen metabolismFigure 4 shows the effects of CBPD obstruc-tion as well as the relief of CBPD obstructionon pancreatic content of hydroxyproline andcollagen. Hydroxyproline values were approxi-mately fivefold higher than the control valueafter five days of CBPD obstruction and thosevalues remained increased during the initialseven days after relief of obstruction (A).Thereafter, pancreatic hydroxyproline valuesreturned to that found in samples taken from

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Time after decomFigure 4: Pancreatic hydroxyprolineafter relief ofCBPD obstruction. Pawhydroxyproline (A) was measured a.and expressed as ng hydroxyproline Iprotein. Pancreatic content of collageusing the Sirius Red-Fast Green FC.technique as described in the text anccollagen per mg non-collagenous protdescribed in the Figure 1 legend. Res(SEM) values for three to seven aniiwith each measurement being perfoonAsterisks show values significantly d,that notedfor control sham operated

sham operated control animslagen content was also increEductal obstruction and ren

increased during the initialrelief of ductal obstructionpancreatic collagen contenthat value noted for contranimals. Pancreatic contentgen was also evaluated usingchemical fluorescence micr(As Figure 5 shows, no increatic content of collagen duiduct obstruction or after reliotion could be shown using

DiscussionMost patients recover from a

pancreatitis, even if it is asso

sis of significant portions ofintrapancreatic events thatrecovery are poorly understoied this issue using an anim

pancreatitis, which closely resembles gall stoneinduced pancreatitis in humans. As previouslynoted, obstruction of the opossum CBPDresults in haemorrhagic necrosis of the glandand extensive peripancreatic fat necrosis. Theextent of parenchymal necrosis is dependenton the duration of duct obstruction and affectsroughly 40% of acinar cells after five days ofobstruction. During those five days of obstruc-tion, serum amylase activities and pancreaticwater content initially rise, then begin todecline, but remain above pre-obstructionvalues. Pancreatic protein and amylase con-tent, on the other hand, fall during the periodof duct obstruction. These various changes

I0 I10 I0 J2 probably reflect the fact that extensive intra-60 70 80 90pancreatic injury and intraparenchymalinflammation accompany obstruction of theopossum CBPD. We have also noted thatCBPD obstruction in the opossum is associ-ated with an increase in pancreatic ODC activ-ity but no change in the rate ofincorporation of[3H]-thymidine into pancreatic DNA. Thecells responsible for the rise in ODC activityhave not been identified. We would favour thehypothesis that it reflects infiltration andpossible replication of inflammatory cellsduring the early stages of pancreatitis but manyother potential mechanisms may underlie thesephenomena.The CBPD obstruction in this model can be

easily relieved by removing the balloon tippedcatheter from the CBPD. The fact that serum

60 70 80 9o bilirubin concentrations rapidly decline afterpression (days) removal of the catheter is consistent with theand collagen content conclusion that obstruction is relieved at thencreatic content of time of catheter removal. The progressives described in the text increase in the extent of acinar cell necrosis isper mg pancreatic abruptly halted by relief of obstruction and,n (B) was measured'F spectrophotometric over the following three weeks, the extent ofd expressed as jig necrosis rapidly falls. Other indicators of pan-win. Groups are as creatic injury also are favourably changed byults represent meanmals in each group relief of ductal obstruction. For example,ned in duplicate. serum amylase activities and pancreatic water!ifferent (p<0 05) from content, as well as the pancreatic content ofanimals. protein and amylase gradually return to their

pre-obstruction values. Pancreatic ODC activ-als. Pancreatic col- ity returns to the pre-obstruction value shortlyased five days after after relief of obstruction but then both ODCnained transiently activity and the rate of [3H]-thymidine incor-seven days after poration into pancreatic DNA, two indicators(B). Thereafter, of cell replication, rise approximately 14 days

It declined below after the relief of obstruction. A transientol sham operated increase in pancreatic protein and amylaseof types I-V colla- content is also noted at roughly the same time.g an immunocyto- It is probable that these changes reflect acinaroscopy technique. cell replication. Morphological evaluation ofase in the pancre- samples obtained 14 days after relief ofring the period of obstruction suggest the appearance of so calledef of duct obstruc- tubular complexes, which are believed tohis approach. reflect pancreatic regeneration'8-21 but identi-

fication of tubular complexes in this model ismade difficult by the fact that ductal obstruc-tion causes the lumena of many of the acini to

n episode of acute dilate and the cells lining the acinar lumena tosciated with necro- assume a more cuboidal shape. Conclusive evi-the pancreas. The dence that acinar cell replication occurs at thisaccompany that time during recovery would, therefore, require

ood. We have stud- performance of autoradiographic nuclearLal model of acute labelling studies, which were, unfortunately,

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Figure 5: Immunohistochemical demonstration of collagen. (A) shows representativesample taken from unoperated control animal. Arrowheads identify areas ofperiacinarcollagen in the basement membrane area at the basalateral surface of cells. (B) showsrepresentative section taken from opossum afterfive days ofCBPD obstruction while (C)shows representative section taken from animal subjected to five days ofCBPD obstructionfollowed by 90 days of ductal decompression. Arrowheads in (B) and (C) show areas ofperiacinar collagen which, although less fine in its appearance, does not seem to beincreased in amount when compared with that noted in the non-operated control groups(A). (D) shows section ofplantarus longus tendon (positive control). Controls were

negative (not shown). Calibration bar in (A) applies to all panels and is 100 gm long with10 ,um gradations.

not possible because of the very large doses ofradioactive tracer that would be needed forsuch studies in opossums. It would also beinteresting to find out if the increase in ODCactivity precedes the rise in [3H]-thymidineincorporation and to evaluate the temporalrelation between these events and the transientchanges in tissue protein as well as amylasecontent that we have seen. Unfortunately, use

of wild animals such as the opossum is associ-ated with considerable interanimal variation,which makes the temporal resolution of theseevents difficult if not impossible.We were particularly interested in noting

whether recovery from acute necrotisingpancreatitis was associated with changes con-sistent with the development of chronic pan-creatitis. On light microscopic examination ofthe tissue, no evidence of chronic inflamma-tion or fibrosis was noted. Deposition of col-lagen in interstitial areas of the pancreas is,however, the essential feature of chronic pan-creatitis and such changes might have beenoverlooked by light microscopic examination.To further explore this issue, we have used

three different but complementary techniquesto discover if relief of the ductal obstructionand recovery from acute necrotising pancrea-titis is associated with formation of collagen inthe pancreas. The first method entails directquantifications of pancreatic hydroxyprolinevalues. Those studies (Fig 4(A)) showed thatpancreatic hydroproxyline values were notincreased 60 or 90 days after relief of CBPDobstruction. This negative finding suggests

that pancreatic collagen values are notincreased at those times. On the other hand, anincrease in pancreatic hydroxyproline contentwas seen five days after induction of ductalobstruction and this increase persisted duringthe initial seven days after relief of CBPDobstruction. These findings show that CBPDobstruction triggers transient collagen forma-tion and that this phase of collagen formationis followed by rapid degradation of the newlyformed collagen (Fig 4(A). These findings andconclusions are consistent with those ofElsasser et ar22 23 who found that collagen wastransiently formed as a result of fibroblastinflux during secretagogue induced pancreati-tis in the rat. We have also noted the appear-ance of spindle shaped cells suggestive offibroblasts during this phase of induction andrecovery from necrotising pancreatitis in theopossum (Fig 2(B) and (C)).The second technique that we have used to

evaluate collagen metabolism during recoveryfrom necrotising pancreatitis entails the chro-matographic measurement of the ratio of col-lagenous to non-collagenous protein in thepancreas. Using this technique, we have foundcollagen values in control samples that aresimilar to those found by others who have usedthis method to study human tissues.2425 Inaccord with our results for studies evaluatingtissue hydroxyproline values, we have alsonoted that pancreatic content of collagenousprotein is increased within five days of CBPDobstruction and that those values remain in-creased during the initial seven days after reliefof CBPD obstruction (Fig 4(B)). Thereafter,pancreatic content of collagenous proteindeclines to values below those noted forcontrol sham operated animals.The third method that we have used to

evaluate collagen metabolism during recoveryfrom necrotising pancreatitis entails immuno-cytochemistry using polyclonal antibodiesdirected against types I-V collagen. Thosestudies showed no evidence of increased pan-creatic content of collagen during induction orrecovery from necrotising pancreatitis (Fig 5).

In summary, the findings reported show thatrelief of CBPD obstruction in the opossum isassociated with rapid recovery from acutenecrotising pancreatitis. We suggest that thisrecovery is associated with acinar cell replica-tion, which peaks seven to 14 days afterrelief of obstruction. Tissue concentrations ofcollagen are transiently increased during therecovery phase but fibrosis and chronic pan-creatitis do not develop. Rather, the pancreasre-acquires its previous morphological appear-ance - that is, restituto ad integrum occurs.Using this model of necrotising pancreatitis,we could detect no evidence in support of thenecrosis-fibrosis hypothesis.

There are significant differences betweenthis opossum model of necrotising pancreatitisand human pancreatitis, particularly thatcaused by alcohol misuse. Thus, the conclu-sions reached from our experimental studiesmay or may not be applicable to the clinicalsituation and the question of whether humanacute necrotising pancreatitis can evolve into

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chronic pancreatitis must remain, to someextent, unresolved. At a minimum, however,our studies show that necrosis of pancreatictissue during severe biliary acute pancreatitisdoes not inevitably result in chronic pancrea-titis or fibrosis of the gland, or both.

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8 Runzi M, Saluja A, Lerch MM, Dawra R, Nishino H, SteerML. Early ductal decompression prevents the progressionof biliary pancreatitis: an experimental study in theopossum. Gastroenterology 1993; 105: 157-64.

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