GASTROENTEROLOGY 1992;102:550-562

Soybean Trypsin Inhibitor and Cerulein Accelerate Recovery of Cerulein-Induced Pancreatitis in Rats GRAZYNA

JURKOWSKA,

GILLES

GRONDIN,

SERGE

MASS&

and JEAN MORISSET Centre de Recherche SW les Mecanismes de SBcrBtion, DBpartement de Biologie, Facult6 des Sciences, and DQpartement de Pathologie, Facult6 de Mbdecine, Universitb de Sherbrooke, Sherbrooke, QuBbec, Canada

The role of exogenous and endogenous cholecystokinin has been studied in the process of pancreatic regeneration after acute pancreatitis. A mild form of pancreatitis was induced in rats by suhcutaneous cerulein at 12 pg - kg-‘, three times a day for 2 days. After 3 days of rest, the cerulein-treated rats were divided into four groups: rats with acute pancreatitis fed 20% casein, who received no treatment; rats fed 50% casein; rats fed 20% casein supplemented with 1% soybean trypsin inhibitor (SBTI); an d rats fed 20% casein who received 1 pgskg-’ of subcutaneous cerulein, three times a day. Controls were fed 20% casein plus saline suhcutaneously. Rats were killed after 5,10,or 20 days of treatment. Pancreatitis resulted in significant decreases in pancreatic weight and contents of protein, amylase, chymotrypsin, RNA and DNA. During the regenerative process, 1 pg -kg-l of cerulein increased all parameters to control values within 5 days and induced pancreatic growth thereafter. SBTI restored the pancreas to normal after 10 days with cellular hypertrophy; the 50% casein diet gave a response similar to SBTI without hypertrophy. It can he concluded that cerulein and SBTI can accelerate pancreatic regeneration after an attack of acute pancreatitis.

A

lthough acinar cells of the adult exocrine pancreas have a relatively low resting mitotic activthey can divide ity,‘,’ under certain circumstances rapidly. These include such situations as postnatal developmenP5 and responses to the administration of certain hormones and peptides,2*3s”‘0 partial resection,“~” toxic injury,13*‘* duct ligation injury,’ and acute inflammation.‘5~‘6 It is generally assumed that an attack of acute pancreatitis with injury and loss of acinar tissue is followed by full recovery of the structure and function of the exocrine pancreas.17 This concept implies a regenerative capacity of the pancreas. Spontaneous

tissue degeneration in acute pancreatitis and a great reparative ability of the pancreas has been described after hormone-induced acute pancreatitis.‘5~‘“~‘8-20 However, focal pancreatic atrophy with slight fibrosis was observed late after hormone-induced” and bile-induced acute pancreatiti? in experimental animals. Similar phenomena have also been described in humans. ” After a single episode of acute pancreatitis, investigatorP observed four cases of pancreatic atrophy on serial computed tomography examinations, and three of the patients ultimately developed pancreatic insufficiency. High doses of cerulein (a skin amphibian decapeptide structurally and functionally related to cholecystokinin [CCK]) given intravenously (IV) or subcutaneously (SC) have been reported to cause acute interstitial pancreatitis.15*23-25 After 2 days of SC injections of cerulein (12 pg. kg-‘), there was evident depletion of pancreatic protein, digestive enzymes, and total DNA; this effect persisted even after 3 and 13 days of recovery.23 The regenerative response of the damaged pancreas is probably triggered by complex mechanisms involving gastrointestinal hormones and peptides; among these, CCK seems to be the most important. Indeed, exogenous CCK and its analogues (cholecystokinin octapeptide [CCK-81 and cerulein) stimulate pancreatic growth in suckling, growing, and adult rats.7,8*‘0,26-28 Similarly, pancreatic growth was also observed in response to endogenous CCK released after feeding trypsin inhibitors2g-32 and pancreaticobiliary diversion.33 CCK also accelerates pancreatic regeneration after Dl-ethionine degeneration of the pancreas in rats being fed a protein-free diet.6 Almost nothing is known about the rate and degree of pancreatic regeneration occurring spontaneously after acute pancreatitis. In view of the fact that 0 1992by the American Gastroenterological Association 0016-5085/92/$3.00

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February 1992

CCK stimulates pancreatic growth and can accelerate pancreatic regeneration,6 the present study was undertaken to determine whether exogenous and endogenous CCK can affect pancreatic tissue recovery at different time points after an onset of acute pancreatitis. Materials and Methods Male Sprague-Dawley rats (n = 114) weighing 218of the experiment were used in this study. Pancreatitis was induced by SC injections of cerulein, 12 pg. kg body wt-’ every 8 hours for 2 days. The CCK analogue was dissolved in gelatin to prolong its absorption.34 Control rats received saline in gelatin SC. During the induction of pancreatitis and the next 3 days without treatment, all rats were fed a 20% casein diet. Beginning from day 6, cerulein-treated rats were divided into four groups for different treatments (diet + injections SC): rats with acute pancreatitis (API rats) were given a 20% casein diet and were injected with saline, AP II rats were on a 50% casein diet + saline, AP III rats were on a 20% casein diet supplemented with 1% soybean trypsin inhibitor (SBTI) + saline, AP IV rats were on a 20% casein diet + cerulein, 1 pg. kg body wt-’ every 8 hours SC. Control rats were fed a 20% casein diet and given saline. Each SC subcutaneous injection consisted of 0.2 mL containing 75% gelatin + 25% saline in control or 75% gelatin f 25% saline or a stock solution of cerulein in the pancreatitis groups (AP I-IV). The last injection was given at 16 hours on the day preceding decapitation. Food consumption and body weight were monitored after the first 2 and 5 days and later on every other day for the rest of the experiment. Rats were fed ad libitum at all times. After an overnight fast with free access to water, rats were weighed and killed after 2,5,10,15, or 25 days of the experiment (this means after AP induction, AP induction + 3 days of recovery, and after AP induction + 3 days of recovery and 5, 10, or 20 days of treatment, respectively). The pancreas was excised and weighed after being trimmed free of fat, mesentery, and lymph nodes. The pieces of tissue were homogenized using a motor-driven ground-glass homogenizer in 0.6N perchloric acid for nucleic acid determinations, or in ice-cold 0.1 mol/L TrisHCl buffer (pH 8.0) for protein, amylase, and chymotrypsin assays. DNA and RNA were extracted according to the method of Mainz and a1.35DNA was determined according to the method of Volkin and Cohn3” using calf thymus DNA as the standard. RNA was hydrolyzed overnight in 0.3N KOH and measured by determining the absorption at 260 nm of the final O.lN perchloric acid extract according to the method of Munro and Fleck.37 Serum and tissue amylase activity were measured according to the method of Bernfeld,38 chymotrypsin according to Hummel’s method,39 and protein according to the method of Lowry et aL40 The diets containing 20% and 50% casein were obtained from ICN Nutritional Biochemicals (Cleveland, OH). SBTI was obtained from Worthington Biochemicals (Freeland, NJ, Code SIC); 1 mg inhibited 0.8 mg bovine trypsin. The cerulein was a gift of Professor de Castiglione, Farmitalia Corporation (Milan, Italy). For microscopy, 220 g at the beginning

three mals mol/L ature

fragments of the pancreatic tissue from three aniper group were fixed in 2% glutaraldehyde in 0.1 cacodylate buffer (pH 7.4) for 1 hour at room temperand processed as previously described.4’ Statistical

Analysis

Results were analyzed using unpaired two-tailed Student’s test. A value of P < 0.05 was considered significant.

Results Amylasemia Evidence of acute pancreatitis caused by this treatment comes from high values of serum amylase concentration observed only 2 hours after the first cerulein injection, 70.4 ? 4.2 U vs. 13.6 + 1.9 U in the controls. After 1 day of treatment, serum amylase levels were still elevated at 24.2 f 4.0 U. However, after 2 and 5 days, serum amylase levels were back to control values at 15.2 f 2.1 U and 14.6 * 1.9 U, respectively. Body Weights The body weights of rats with acute pancreatitis decreased significantly by 6% and 11% compared with their paired controls after 2 days (235 f 3.7 g) and 5 days (258.7 & 7.2 g) of the experiment. Later, body weights increased with time (10, 15, and 25 days) in all groups with no significant differences among the treatment groups and their respective controls at each of the later time points of the experiment. Control rats ate an average of 22.2 * 1.3 g of food during the first 10 days of the experiment; during the last 15 days, their daily food consumption increased to 26.8 -rt 2.1 g. The rats with acute pancreatitis significantly reduced their food consumption by 40% during cerulein-induced pancreatitis (first 2 days) but were up to control consumption after 5 days. Pancreatic Weight and Total Protein, Enzyme, and Nucleic Acid Contents After Induction of Acute Pancreatitis and During

the Recuperative

Period

Pancreatic weight. After 2 days of injection of a supramaximal dose of cerulein, pancreatic wet weight was significantly reduced (by 33%), and this decrease was amplified to 58% after the next 3 days of rest (Figure Protein,

1).

amylase, and chymotrypsin contents. Acute pancreatitis after 2 days resulted in significant decreases of SO%, lOO%, and 90% in total pancreatic protein, amylase, and chymotrypsin contents, respectively. Total protein and amylase levels remained low for the next 3 days of recovery (Figure

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Pancreatic Weight and Total Enzyme and Nucleic Acid Contents During the Treatment Periods After Acute Pancreatitis

IO00 j

800

r

z * 600

ii 200 t

0

Control 5

2 Ap5 dw

Groups

Pancreatic weight. In the acute pancreatitis groups (AP I), signs of regeneration were evident as the pancreatic tissue weight of these rats recovered towards their respective control values, as indicated by significant decreases of 36%, 29%, and 25% after 5, 10, and 20 days of treatment (Figure 3A-C). How-

100

r: Groups

F

I;

80 60 ??

w

40 20 Broups

0

600-1

If gj

t

T

2 500 Y E 400

w

E 300 k 200 E 100 0

100

Groups

8o 60

40 20 Groups

0 Groups

Figure 1. Effect of acute pancreatitis and 3 days of rest on pancreatic weight and total contents of protein, amylase, and chymotrypsin. Values are means + SE of six rats per group. Pancreatitis induction is described in Table 1 note. *Significantly different from respective control value. “Significantly different from the acute pancreatitis group after 2 days.

1) compared with their respective controls, whereas total chymotrypsin recovered to control values. Nucleic acid contents and RNA/DNA ratio. Cerulein-induced acute pancreatitis after 2 days resulted in significant decreases in total pancreatic content of DNA (39%) and RNA (85%) and the RNA/ DNA ratio (76%). After 3 days of recovery, total RNA content and its concentration (RNA/DNA) slightly improved, whereas tota DNA content further decreased to 53% of its control value, an indication of further loss of pancreatic tissue (Figure 2).

??

2 AP 5 days

Groups

Figure 2. Effect of acute pancreatitis and 3 days of rest on total pancreatic contents of RNA and DNA and RNA/DNA ratio. Values are means + SE of six rats per group and are expressed as percentage differences with their respective controls. Pancreatitis induction is described in Table 1 note. *Significantly different from respective control. Control values: 14.2+ 1.7mg and 15.0f 2.2mg for total RNA at days 2 and 5,4.2 +_0.3mg and 4.5+ 0.9mg for total DNA, and 3.4 f 0.3and 3.3 f 0.6 for RNA/DNA ratio.

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above the control value. Cerulein was the most efficient treatment of all because it induced full tissue recovery to the control value after only 5 days of treatment (Figure 3A). Furthermore, the CCK analogue induced significant increases of 30% and 59%, respectively, in pancreatic weight above control values after 10 and 20 days of treatment (Figure 3B and C). The potent trophic effect of cerulein can be better appreciated when increases in pancreatic weight are compared with those in the API group. Indeed, significant increases of 82% and 112% above AP I values were observed after 10 and 20 days of treatment (Figure 3B and C). Furthermore, cerulein induced pancreatic growth more than all the other groups after 20 days. Total Protein, Amylase, Contents

”

Control

API

API1

APIll

APIV

Qroups

**

??

2000

??

1

****

.T

C

_

Control

API

API1

APIll

APlV

sroups

Figure 3. Effects of different treatments on pancreatic weight. Values are means + SE of six rats per group after 5 (A),10 (B), and 20 (C) days of treatment. The different groups and treatments are described in Table 1 note. Significantly different from control (*), AP II I**), AP III (a**), and AP IV (ww).

ever, full recovery was not observed at the end of treatment (Figure 3C). Feeding the animals a 50% casein diet resulted in a complete recovery of the pancreatic tissue after 10 days (Figure 3B) but did not induce tissue growth above the control value if prolonged to 20 days (Figure 3C). The addition of SBTI to the 20% casein diet (AP III) resulted in a significant increase of 28% in tissue weight compared with AP I rats after 5 days (Figure 3A); a recovery to control values was observed after 10 days (Figure 3B), and growth continued after 20 days (Figure 3C) as shown by tissue weight significantly increasing by 26.5%

and Chymotrypsin

Total protein levels. Changes in total protein content after the different treatments and length of time (Figure 4A-C) are a mere image of those changes observed in pancreatic weight. Indeed, in the AP I group, total protein contents were still lower by 28% after 20 days of treatment compared with controls (Figure 4C). The 50% casein diet (AP II) restored total protein contents to control values after 10 days of feeding (Figure 4B) but did not increase levels to above control values after 20 days of treatment (Figure 4C). On the contrary, SBTI addition to the 20% casein diet completely restored total protein levels after only 5 days of feeding (Figure 4A), causing a 58% increase above control values after 20 days (Figure 4C). In response to cerulein (AP IV), acute pancreatitis tissues significantly increased their total protein content by 30% above control values after only 5 days of treatment (Figure 4A). Further increases of 59.8% and 96.6% were observed after 10 and 20 days of treatment (Figure 4B and C). Total amylase levels. As shown in Figure 5, the recovery of total amylase content in the AP I groups was progressive with time after the 3-day resting period, as indicated by the reduction of 50% and 33% after 5 and 10 days of treatment compared with control values (Figure 5A and B). After 20 days, no significant differences could be observed with the control (Figure 5C). The 50% casein diet (AP II) did not restore total amylase contents after the three time periods studied; on the contrary, the situation became worse with time compared with control values. A significant decrease of 50% was observed after 5 days (Figure 5A) compared with a 57% decrease after 20 days (Figure 5C). The SBTI supplement (AP III) completely restored total amylase contents to control values after 10 days of feeding (Figure 5B) but did not cause any significant increase above the con-

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(Figure 6A). Among the three other treatments (AP II, III, and IV), they all significantly increased total chymotrypsin contents above control values within 5 days and their order of efficacy was AP IV > AP III > AP II (Figure 6A). This same efficacy among these three groups remained until the end of the experiment [Figure 6B and C) with cerulein remaining the most potent throughout. Total Nucleic Acid Contents

Control

API

API1

APIll

APIV

woups

mained 300

ff

1

Total RNA levels. Total RNA contents rebelow control values by 31% in the AP I 300

A

1

-

c(

2

200

B

8 G

A

0 r; 200

9 Y

J 100

100 $ t-l Control

API

API1

APIll

APIV

0

Groups

Control

AP

I

AP II

AP III

AP IV

eraups

??

I

-

C

“0 c

: 300 Y { 200 f

Control

API

API1

APIII

APIV

doups

B

400

100 n ” Control

AP II

AP I

AP Ill

AP lV

6rws

Figure 4. Effects of different treatments on total protein contents. Values are means + SE of six rats per group after 5 (A), 10 and 20 (C) days of treatment. The different groups and treat(B), ments are described in Table 1 note. Significantly different from control(*), AP II(w), AP III(w*), and AP IV (*::*).

C trol level 10 days later (Figure 5C). On the contrary, cerulein totally eliminated the reduction of amylase induced by acute pancreatitis within 5 days of treatment (Figure 5A) and was responsible for significant increases of 64% and 86% above control values after 10 and 20 days of treatment (Figure 5B and C). Total chymotrypsin levels. The fastest recovery to control values without treatment was observed with this proteolytic enzyme (AP I). Indeed, total chymotrypsin contents had already returned to control values within 5 days after the resting period

COfllWJl

AP

I

AP II

AP Ill

AP IV

Groups

Figure 5. Effects of different treatments on total amylase contents. Values are means f SE of six rats per group after 5 (A), 10 (B), and 20 (C) days of treatment. The different groups and treatments are described in Table 1 note. Significantly different from control (*), AP II (*:), AP III (**a), and AP IV (we*).

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??

3000

sY 5

2000

s & E

1000

A

5 0

Control

API

API1

APIll

APlV

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555

tained values constantly lower than the control after 5 days of treatment for the AP I (-26%), AP II (-34%), and AP III groups (-23%). During this same period, cerulein (AP IV) successfully restored total DNA contents to control value (Figure 8A). Over the next 5 days, total DNA contents were comparable to control values in all groups but AP IV, showed levels of DNA by 28% above the control level (Figure 8B). In the last 10 days of treatment, cerulein alone further manifested its trophic effect by causing an increase of 59%

Oroups

120

-I

A

100 f

B

ii w

80 6o 40 20

il

Control

API

API1

APIll

APIV

Control

API

APIt

APIll

Control

API

APtI

APIll

API’.’

uroups

Groups

6000 s Y : r k

5000

E

2000

U

1000

C

4000 3000

APIV

OrouPs

n ”

Control

API

API1

APIll

APIV

Groups

Figure 6. Effects of different treatments on total chymotrypsin contents. Values are means f SE of six rats per group after 5 (A), 10 (B), and 20 (C) days of treatment. The different groups and treatments are described in Table 1 note. Significantly different from control (*), AP II (**), AP III (***), and AP IV (MW).

group after 5 days of treatment (Figure 7A); however, total recovery was observed after 10 days of treatment (Figure 7B). The 50% casein regimen (AP II), SBTI supplement (AP III), and cerulein treatment (AP IV) all succeeded in restoring total RNA contents to control values within 5 days (Figure 7A). Only the SBTI supplement and cerulein caused significant increases of 33% and 59% after 10 days and 68% and 115% after 20 days of treatment compared with the respective control values (Figure 7B and C). Total DNA levels. Total DNA contents sus-

300 ??

h

200

a tX3 ‘; w

100

0

C

Control

API

API1

APIll

APIV

sroups

Figure 7. Effects of different treatments on RNA contents. Values are means + SE of six rats per groups after 5 (A), 10 (B), and 20 (C) days of treatment and are expressed as percentage of the control groups. The different groups and treatments are described in Table 1 note. Significantly different from control (:), AP II (w), AP III (**:), and AP IV (we:). Total RNA control values are 16.6 t 2.5 mg, 18.3 + 1.8 mg, and 21.2 + 2.5 mg, respectively, after 5 (A), 10 (B), and 20 (C) days of treatment.

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in the total DNA content above the control values (Figure 8C). Total DNA contents in AP IV rats were significantly higher than those in AP I groups at each time point of the treatment.

Table 1. Effects of Different Treatments Pancreatic Tissue Hyperplasia Hypertrophy

Treatment groups

Hyperplasia

Index

Using total DNA contents as a measure number, it can be seen that cerulein-induced

of cell acute

Hyperplasia DNA

Day of treatment

5 10 20

Hypertrophy RNA/DNA

Protein/DNA

Chymotrypsin/ DNA

Amylase/DNA

”

Control

Control

API

API

API1

API1

APIll

APIII

API’/

APIV

Broups

Groups

API

AP II

AP III

AP IV

0.74 0.8 0.9

0.7 0.96 1.09

0.76 0.94 1.05

o.9ga 1.28’.’ 1.560fb

0.94 0.97 1.03

1.2 1.4O.b 1.6°.b 1.2 1.2’~~ 1.45aJJ

1.2 1.25’ 1.3’ 1.3b 1.6” 1.2O

7.30-b 5.6nfb 5.2’~~ 0.5 1.1 1.2’

10 20

0.8 0.76

1.2 1.1 1.05 1.07 0.95 0.7

5

1.1 1.1 0.8 0.5 0.9 0.6

5.2a,b 2.9”.b 1.4a 0.5 0.6 0.4

5 10 20

0

on Indices of and Cellular

5

10 20 5 10 20

0.9

13.3a.b 6.1’1~ 7.2a*b 0.9O 1.3 l.la

NOTE. Values are ratios of treatment groups to control groups after 5, 10, and 20 days of treatment of six animals per group. Acute pancreatitis was induced by SC injection of cerulein, 12 pg. kg-’ three times a day for 2 days, followed by a z-day resting period. On the 6th day, control rats were fed 20% casein and were given SC injections of saline three times a day for 5 (day lo), 10 (day 15), and 20 (day 25) days. Pancreatitic rats were (a) fed 20% casein plus SC saline (AP I); (b) fed 50% casein plus SC saline (AP II); (c) fed 20% casein supplemented with 1% SBTI plus SC saline (AP III); or (d) fed 20% casein plus SC cerulein, 1 pgakg-’ three times a day (AP IV). These animals were killed after 5 (day lo), 10 (day 13, and 20 (day 25) days of treatment. Hyperplasia represents total DNA content of the pancreas. Cellular hypertrophy was established by dividing total contents of RNA, protein, chymotrypsin, and amylase by the total content of DNA. “Values significantly greater than those of the AP I group at P < 0.05.

bValues significantly greater than 1, therefore significantly different from controls at P < 0.05.

pancreatitis resulted in significant pancreatic cell loss, which remained for 5 days after the 3-day resting period (Table 1; AP I). This loss of pancreatic cells was not improved by feeding a 50% casein diet (AP II) or a supplement of 1% SBTI with the 20% casein diet

-

Control

API

API1

APIll

APIV

Broups

Figure 8. Effects of different treatments on DNA contents. Values are means f SE of six rats per groups after 5 (A), 10 (B), and 20 (C) days of treatment and are expressed as percentage of the control groups. The different groups and treatments are described in Table 1 note. Significantly different from control (*), AP II (w), AP III (we), and AP IV (*es*). Total DNA control values are 4.6 f 0.3 mg, 5.1 f 0.5 mg, and 5.5 f 0.6 mg, respectively, after 5 (A), 10 (B), and 20 (C) days of treatment.

(AP III). However, within this short period cerulein restored the number of pancreatic control values (AP IV). Upon prolongation

of time, cells to

of treatments to 10 and 20 days, all groups recovered to control values. Cerulein, after recovery to the control value by 5 days, induced pancreatic growth as shown by the sustained increases in total DNA levels of 28% and 56% above control values after 10 and 20 days of treatment. Compared with AP I, cerulein caused significant hyperplasia at each time point of treatment.

February1992

Hypertrophy A critical examination of the data in Table 1 reveals that SBTI added to the 20% casein diet (AP III) was an efficient treatment in causing pancreatic cellular hypertrophy. Indeed, of the four criteria presented, SBTI significantly increased three above control values after 10 and 20 days of treatment. Cerulein also showed a hypertrophic effect (Table 1). It is also important to point out that SBTI and cerulein (AP III and IV) resulted in significant increases in RNA, protein, and chymotrypsin concentrations above levels for untreated pancreatitis (AP I) after 10 and 20 days of treatment. However, amylase concentrations in treated groups never exceeded control values at any time during the treatment. Histology of the Pancreas During Acute Pancreatitis Induction and Regeneration After 2 days, cerulein-induced pancreatitis was well established as evidenced by an extensive acinar cell loss and the presence of mononuclear cell infiltration although the lobular architecture remains recognizable (Figure 9A and B). At higher magnification, many cells can be seen to be in the process of being transformed or already transformed in tubular complexes called ductlike structures. Such structures are composed of a variable number of acinar cells with or without zymogen granules and some tubular complexes empty of zymogen granules (Figure SC and D). The connective tissue is infiltrated with polymorphonuclear leucocytes, lymphocytes, and macrophages (Figure 9B-D). However, the endocrine tissue, blood vessels, and intralobular and interlobular pancreatic ducts are normal (Figure 9A and B). These histopathologic modifications are typical of all cerulein-treated animals as evaluated from light-microscopy slides stained with H&E (data not shown). After a 3-day resting period, a large improvement was observed in the overall pancreatic structure, as seen in Figure 9E. Indeed, few tubular complexes are present, and many small pancreatic acini can be identified, whereas the connective tissue remains infiltrated with inflammatory cells. The control pancreas shows normal acinar cells grouped in acini and forming lobules interconnected by a thin layer of connective tissue without any inflammatory cells (Figure 9F). Five days into the treatment period, the pancreata in the AP I group showed major improvements compared with 5 days earlier. Indeed, as shown in Figure lOA, compared with Figure 9E, the acini are larger, with acinar cells having more zymogen granules and the connective tissue spaces free of inflammatory cells. The periacinar and perilobular spaces still ap-

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pear much wider than normal (Figure 1OA vs. Figure 9F). The morphology of the pancreas from the 50% casein diet treatment (AP II) is comparable with that of AP I (data not shown). The major effect of SBTI feeding (Figure 10B) was to increase the zymogen granule content of the acinar cells, although the larger periacinar and perilobular spaces are still present. Exogenous cerulein was the most potent regenerative agent, because after only 5 days all the alterations reminiscent of pancreatitis had disappeared (Figure 1OC). Furthermore, the CCK analogue produced larger acini with acinar cells richer in zymogen granules than controls (Figure 1OC vs. Figure 9F). After 20 days of treatment, the pancreata in the pancreatitis group (AP I) and those fed the 50% protein diet (AP II) (Figure 100) were comparable with controls (Figure 9F), because the wider periacinar and perilobular spaces previously observed had reverted to normal sizes. SBTI feeding (AP III; Figure 1OE) rendered the pancreatic tissue comparable with that after 5 days of cerulein treatment (Figure 1OC) with larger acini richer in zymogen granules than the controls (Figure 9F). The cerulein treatment (AP IV) resulted in larger pancreatic acini loaded with zymogen granules (Figure 1OF) and smaller periacinar spaces than controls (Figure 9F). Discussion This study presents, for the first time, data on pancreatic tissue recovery at different time periods up to 23 days after acute pancreatitis induction by a supramaximal dose of cerulein. The regenerative process of the pancreatic tissue was accelerated by endogenous release of CCK or exogenous cerulein, a CCK analogue. Endogenous CCK release was achieved by feeding the animals a high-protein diet (50%) or SBTI (1% supplement to the diet) as previously documented by Green et a1.,32*42 Liddle et a1.,43 and Rosewicz et al.@ The temporary reduction in food intake observed during the first 2 days of the experiment may explain the reduced body weight during that period and the following 3 days. The induction of acute pancreatitis was performed according to the method of Wood et a1.23with six SC injections of a supramaximal dose of cerulein, 12 pg - kg-* every 8 hours for 2 days. This treatment caused gross edema of the pancreas within 30 minutes after a single injection of cerulein. The other characteristic symptoms of acute pancreatitis were (a) elevated serum amylase levels, (b) depletion of pancreatic digestive enzymes and total DNA contents, and (c) infiltration with inflammatory cells.23J4 Our present data corroborate similar elevated serum amylase levels and tissue biochemical and morpho-

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Figure 9. Pancreatic histology of normal pancreas of pancreatic tissue after 2 days of cerulein-induced pancreatitis and after a I-day resting period. Cerulein was given SC at a dose of 12 pg - kg-l three times a day for 2 days. (A-D) Pancreatic tissue 2 days after cerulein injection. (A) Arrowheads represent a lobular structure; asterisks, blood vessels; large arrow, some endocrine tissue; small arrow, an interlobular duct (original magnification X175).(B) Asterisks represent periactnar and perilobular spaces; orrowIieo&, some imlammatory cells (origblal magnification X449). (C) Arzuws represent tubular complexes with normal acinar cells; a&risk, a periacinar space (or&bud magnification X890). (0) Arrows represent ductlike structure of tubular complexes; mzuwhefIds, imlammatory cellq osterisks, a perilobular space (original magnihcation X8W). (E) Pancreatic tissue after a 3dayrestingperiod.Afznwrepresents some tubular complexes; periacinar the asterisks, and perilobular spaces (original magnification X500).(F) Normal pancreatic tissue. Arrows represent periacinar space (original magnification X800).

logical changes as indicated in Figures 1, 2, and 9. These figures also indicate that injury to the pancreatic tissue is sustained during a s-day resting period after the end of the cerulein injections, as shown by further decreases in pancreatic weights and total DNA contents. In our model, however, we did not observe obvious morphological signs of local pancreatic atrophy as observed by Niederau et al.‘O Four and 14 days after cerulein-induced pancreatitis in mice, nor the extensive pancreatic atrophy reported by Svensson et al.‘l 6 weeks after bile-induced pan-

creatitis in the dog and also in some cases of human pancreatitis.” Regeneration of the pancreatic tissue after an episode of acute pancreatitis occurs, but this process seems different according to the model used to induce the pathology. Recovery following repeated necrohemorrhagic pancreatitis20*21 remains questionable. However, in the different models of ceruleininduced edematic pancreatitis in which the CCK analogue was either given IV during 12 hours’5,1s or SC and structural over 3 hours,” the biochemical

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Figure 10. Pancreatic histology 10 and 25 days after the induction of cerulein-induced pancreatitis following different treatments. (A) API group treated for 5 days with saline. Arrows represent the periacinar space; asterisk, the perilohular space (original magnification X880). (B) AP III group fed SBTI for 5 days. Arrows represent the periacinar space; asterisk, the perilohular space (original magnification X800). (Cl AP IV group treated 5 days with SC cerulein, 1 pg - kg-’ three times a day. Arrows represent the periacinar space (0riginaI magnification X800). (D) AP II group with rats fed a 50% casein diet for 20 days. Arrows represent the periacinar space (original magnification X800). (El AP III group fed SBTI for 20 days. Arrows represent the periacinar space (original magnification X8tM).(F) AP IV group treated 20 days with SC cerulein, 1 pg. kg-’ three times a day. Arrows represent the periacinar space (original magnification X800).

changes were comparable with those described in our study. In these other models, total recovery to normal architecture without any sign of fibrosis or atrophy of the gland was achieved within 9-12 daysI or 14-21 days? after induction of acute pancreatitis. Such differences may result from the dose, route, frequency, or time of cerulein administration and the experimental animal chosen. In our model, regeneration of the pancreatic tissue in the API group proceeded slowly and was not quite

complete 23 days after pancreatitis induction. Although wider periacinar and perilobular spaces previously observed reverted to normal sizes (Figure lOD), pancreatic weights and total protein contents had not reached control values (Figures 3 and 4). However, the nucleic acid levels (Figures 7 and 8) were totally restored within 13 days after the induction of pancreatitis. Such spontaneous regeneration of the pancreatic tissue after cerulein-induced acute pancreatitis has been previously observed by Adler

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et all5 and Elsasser et al.‘” and was associated with increased mitotic activity and rates of DNA synthesis. In the first 2 days of recovery, centroacinar cells showed the most activity, whereas acinar cells only showed signs of increased DNA synthesis and mitotic activity 3-6 days after induction of pancreatitis.16 In the model presented in this study, DNA synthesis was increased by 247% at the end of pancreatitis induction and by 306% 3 days later; by day 40, thymidine incorporation into DNA was still greater than control values by 71% (unpublished data). These data indicate that the regenerative process is already underway when the pancreatic tissue is still challenged by a supramaximal dose of cerulein. The observation that RNA and DNA contents reached control values before total protein levels and tissue weights strongly suggests that the first priority of the gland is to restore the total number of cells. Furthermore, increased plasma CCK levels have been reported in chronic pancreatitis in humans45; if such increases in plasma CCK levels were to be observed in our model, the hormone might also be responsible for the higher rates of DNA synthesis and mitotic indices. Chymotrypsin, however, seems to be an exception to this rule of tissue priority, because its content was already restored to the control value after the 3-day resting period (Figure 1). This rapid induction of chymotrypsin is probably the result of a specific induction of its gene expression by endogenous CCK release, as previously shown by Rosewicz et al. in normal rats.44 As pointed out earlier, endogenous cholecystokinin release was achieved by feeding animals a 50% casein diet or a 20% casein diet supplemented with 1% SBTI (groups AP II and III). With both regimens, a complete recovery of the pancreatic tissue was obtained after 10 days of feeding because pancreatic weight and total protein, RNA, DNA, and chymotrypsin contents were comparable with or above control values (Figures 3B-8B). At this time, the only difference between these two treatments is the cellular hypertrophy as indicated in Table 1. Indeed, feeding SBTI for 10 days resulted in significant increases above control values in RNA, protein, and chymotrypsin concentrations expressed on a DNA basis, whereas the 50% casein diet only increased chymotrypsin concentration. By extending feeding to 20 days, SBTI developed as a more potent growth factor than the 50% casein diet, because it amplified pancreatic cellular hypertrophy (Table 1) and induced larger acini richer in zymogen granules (Figure 10E vs. Figure 10D). This difference may result from a more sustained elevation of plasma CCK levels in response to SBTI than casein as previously shown by Liddle et a1.43 The beneficial effect of a relatively high protein

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content (50%) in the diet in our study is intermediary with that observed in normal rats4’ Indeed, feeding rats a 75% casein diet for 7 days following IO days of a 5% casein diet also resulted in specific increases in pancreatic weight, and protein, DNA, and chymotrypsin contents compared with the 5% casein group.42 The lack of effect of the 50% casein diet on amylase recovery in our study results from the high protein contents in the diet coupled with its low carbohydrate content.46 Contrary to what was observed by Schick et a1.,47 the cerulein treatment in this study was associated with significant increases in total amylase contents after 10 and 20 days of treatment. However, these results are hardly comparable because Schick et al. studied amylase synthesis in the course of a 24-hour cerulein-infusion treatment without any measure of total pancreatic amylase content. Furthermore, over that 24-hour period, a total of 6 pg - kg-’ of cerulein was infused IV, twice the amount given SC in this study at 8-hour intervals. The cerulein-induced increases in total amylase content observed in this study are not new findings, because it has been previously reported by our group and others.27-31*34 Even though a suggested endogenous release of CCK caused by a high-protein diet and SBTI was able to restore the damaged pancreatic tissue levels to normal and caused cellular hypertrophy in the case of SBTI, neither of these two treatments succeeded in initiating pancreatic hyperplasia above the control level. On the contrary, within 5 days of treatment, cerulein (AP IV) given at a physiological dose34,46 completely restored pancreatic acinar cell structure (Figure 1OC vs. Figure 10A) and all the pancreatic parameters studied to control values (Figures 3-8A) with pancreatic hyperplasia compared with the AP I group (Table 1). Furthermore, upon continuation of treatment to 10 or 20 days (Figure 8B and C), cerulein induced hyperplasia of the pancreatic tissue compared with control values with minimal cellular hypertrophy, as indicated by a specific effect only on chymotrypsin concentration (Table 1). This specific effect of cerulein on increased cell numbers is similar to that observed in rapidly growing pancreata before weaning” and differs from its action on normal grown pancreata in which both hyperplasia and hypertrophy were observed. 27,34To our knowledge, this is the first description of a beneficial effect of a cerulein treatment on pancreatic recovery after acute inflammation. Majumdar et a1.6 have reported complete recovery of total DNA content after ethionineinduced pancreatic degeneration with infusion of CCK-8 for 7 days, but pancreatic weight and protein contents remained under control values. The beneficial effects of cerulein and CCK-8” on pancreatic regeneration do not agree with those given in other

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reports in which these two CCK analogues were reported to worsen the mortality and morbidity in bile and diet-induced pancreatitis.4g-51 The most important difference in our protocol resides in the fact that the cerulein treatment was begun 3 days after the induction of a mild form of acute pancreatitis, whereas in some other instances4’ the cerulein treatment was started during the acute phase of inflammation and used extremely high doses of cerulein, 50 pg. kg-’ (total dose over 9 hours), which by itself will induce acute pancreatitis rather than stimulate pancreatic regeneration. Finally, the recent observation that L-364,718, a potent CCK-receptor antagonist, given at a dose of 1.0 mg. kg-’ twice a day, was able to inhibit spontaneous pancreatic regeneration after cerulein-induced pancreatitis suggests that endogenous CCK is involved in the regeneration process (unpublished results). In conclusion, this study has established for the first time that (a) spontaneous regeneration of the pancreatic tissue after cerulein-induced pancreatitis has not been completely achieved 23 days after its induction, (b) feeding a high-protein (50%) diet for IO days accelerated recovery, because it completely restored the gland to its normal weight with its cellular components, (c) the SBTI supplement to the diet was responsible for full recovery of the pancreatic tissue within 10 days of treatment and was also associated with pancreatic cellular hypertrophy, and (d) cerulein was the most efficient factor for total recovery from acute pancreatitis, because the gland became normal within 5 days of treatment. Furthermore, this CCK analogue was responsible for further tissue growth if its administration was prolonged to IO and 20 days. Finally, these data suggest that endogenous CCK release and an exogenous CCK analogue may represent efficient treatments to accelerate pancreatic regeneration following mild acute pancreatitis. References

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42. Green GM, Levan VH, Liddle RA. Plasma cholecystokinin and pancreatic growth during adaptation to dietary protein. Am J Physiol 1986;251:G70-G74. 43. Liddle RA, Green GM, Conrad CK, Williams JA. Proteins but not amino acids, carbohydrates, or fats stimulate cholecystokinin secretion in the rat. Am J Physiol 1986;251:G243-G248. 44. Rosewicz S, Dunbor-Lewis L, Wang XY, Liddle RA, Logsdon CD. Pancreatic digestive enzyme gene expression: effects of CCK and soybean trypsin inhibitor. Am J Physiol 1989; 256:G733-G738. 45. Schafmayer A, Backer HD, Werner M, Folsch UR, Creutzfeldt W. Plasma cholecystokinin levels in patients with chronic pancreatitis. Digestion 1985;32:136-139. 46. Reboud JP, Ben Abdeljlil A, Desnuelle P. Variations de la teneur en enzyme du pancreas de rat en fonction de la composition des regimes. Biochim Biophys Acta 1962;58:326-337. 47. Schick J, Kern H, Scheele G. Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis. J Cell Biol 1984;993:1569-1574. 48. Sarfati PD, Green GM, Brazeau P, Morisset J. Presence of somatostatin-like immunoreactivity in rat pancreatic juice: a physiological phenomenon. Can J Physiol Pharmacol 1986; 64:539-544. 49. Evander A, Ihse I, Lundquist I. Influence of hormonal stimulation on acute experimental pancreatitis in the rat. Eur Surg Res 1981;13:257-268. 59. Evander A, Ihse I, Lundquist I. Influence of gastrointestinal hormones on the course of acute experimental pancreatitis. Hepatogastroenterology 1982:29:161-166. 51. Niderau C, Liddle RA, Ferrell LD, Grendell JH. Beneficial effects of cholecystokinin receptor blockade and inhibition of proteolytic enzyme activity in experimental acute hemorrhagic pancreatitis in mice. Evidence for cholecystokinin as a major factor in the development of acute pancreatitis. J Clin Invest 1986;78:1056-1063.

Received May 1,199O. Accepted May 28, 1991. Address requests for reprints to: Jean Morisset, Ph.D., Centre de Recherche sur les Mecanismes de Secretion, Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Quebec, Canada JlK 2Rl. Supported by grant MA-7320 from the Medical Research Council of Canada and grant 733 from le Ministere de 1’Education du Quebec. The authors acknowledge the technical assistance of M. Vanier and the secretarial assistance of A. Rousseau.