Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992), pp. 1409-1417

Role of Ischemia in Acute Pancreatitis Hemorrhagic Shock Converts Edematous Pancreatitis to Hemorrhagic Pancreatitis in Rats TAKAHISA KYOGOKU, MD, TADAO MANABE, MD, and TAKAYOSHI TOBE, MD

Ischemia has been considered to play a role in the development o f acute pancreatitis. The aim o f this study was to investigate the effect o f ischemia, caused by hemorrhagic shock, on cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by the intravenous infusion o f a supramaximally stimulating dose o f cerulein (10 I~g/kg/hr) for 6 hr. Hemorrhagic shock was induced by the removal o f blood until the mean arterial blood pressure reached35 mm Hg. This level was maintained for 30 min, after which time all the blood was reinfused. Hemorrhagic shock alone induced no morphological change in the pancreas. However, after the induction o f hemorrhagic shock in animals treated with cerulein, hemorrhage and parenchymal necrosis were frequently observed in the pancreas. Seven o f 20 rats (35%) receiving cerulein plus hemorrhagic shock had died by 48 hr after the start o f cerulein infusion, whereas none o f the rats in the cerulein or shock group died during this experiment. Cathepsin B activity in the pancreas o f the cerulein plus shock group was significantly higher than in the other groups at 48 hr. These results suggest that ischemia may be a contributing factor in the pathogenesis o f acute pancreatitis. KEY WORDS: experimental pancreatitis; cerulein; hemorrhagic shock; cathepsin B; rat.

The pathogenesis of acute pancreatitis remains an unsolved problem, and many different theories have been reported for many years. Recently, circulatory disturbance or ischemia has been reported to be one of the pathogenetic factors in acute pancreatitis. Clinical studies have documented the occurrence of acute pancreatitis after cardiac surgery, which may be due to pancreatic ischemia following secondary obstruction of blood vessels and hypoperfusion (1). In addition, autopsies of patients dying after shock Manuscript received August 13, 1991~ revised manuscript received December 31, 1991; accepted January 13, 1992. From the First Department of Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan. This study was supported by a grant, Scientific Research B-03454319, from the Ministry of Education, Science and Culture, and a grant from the Ministry of Health and Welfare of Japan. Address for reprint requests: Dr. T. Kyogoku, First Department of Surgery, Faculty of Medicine, Kyoto University, 54Shogoin Kawara-cho, Sakyo-ku, Kyoto, 606, Japan.

have demonstrated a high incidence of acute pancreatitis or pancreatic necrosis that could be correlated with the severity and duration of shock (2-6). In experimental studies, pancreatic edema caused by duct ligation and hyperstimulation has been considered to lead to parenchymal necrosis in the presence of temporary arterial obstruction (7, 8). Pfeffer et al (9) and Redha et al (10) produced hemorrhagic-necrotizing pancreatitis by the intraarterial injection of microspheres in dogs and rats. Broe et al (11) showed that 2 hr of total ischemia can produce significant injury in the isolated ex vivo perfused canine pancreas. Printz et al (12) demonstrated that hemorrhagic shock worsened the microscopic evidence of pancreatitis induced by supramaximal secretagogue stimulation in rats. Klar et al (13) demonstrated that a pharmacological vasoconstrictor, phenylephrine, increased severity of cerulein-induced pancreatitis. More-

Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

0163-2116/92/0900-1409506.50/09 1992PlenumPublishingCorporation

1409

KYOGOKU E T A L over, m a n y authors h a v e shown histological vascular alterations in acute hemorrhagic pancreatitis (14, 15) and impairment of pancreatic microcirculation and reduction of pancreatic perfusion in acute pancreatitis (16). Acute e d e m a t o u s pancreatitis was induced in rats b y the infusion of a supramaximally stimulating dose o f cerulein (17). H o w e v e r , this pancreatitis is reversible and not fatal. In clinical cases, treatment of severe fatal hemorrhagic-necrotizing pancreatitis is m o r e important. H o w e v e r , the factors that convert mild e d e m a t o u s pancreatitis to severe hemorrhagic-necrotizing pancreatitis h a v e not yet been clarified. In the present study, our aim was to determine w h e t h e r hemorrhagic shock had morphologic and e n z y m a t i c influences on cerulein-induced acute pancreatitis in rats and to clarify the role of ischemia and microcirculatory disturbance in the d e v e l o p m e n t of severe hemorrhagic pancreatitis. Particular interest was focused on morphologic changes o f the pancreatic microvasculature.

MATERIALS AND METHODS Experimental Protocol. Male Sprague-Dawley rats (Japan SLC Inc., Shizuoka, Japan) weighing 170-240 g were given free access to standard rat chow and water before the experiments, After an overnight fast, they were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg body weight), and PE-50 cannulas (Clay Adams, Parsippany, New Jersey) were placed in both femoral arteries and veins and tunneled subcutaneously to exit at the base of the tail. The catheter placed in the right femoral vein allowed continuous administration of physiologic saline containing heparin (5 units/ml) at a rate of 0.40 ml/hr. Arterial blood pressure was monitored via the catheter placed in the right femoral artery, and the left femoral artery was used for the removal and reinfusion of blood. The animals were housed in individual cages without restraints. Acute pancreatitis was induced by the intravenous infusion of supramaximally stimulating doses of 10 I~g/ kg/hr of cerulein (Ceosunin, Kyowa Hakko, Ltd., Tokyo, Japan) dissolved in physiologic saline over a period of 6 hr. Seventy-five minutes after the start of cerulein infusion, blood was removed from the femoral artery into a syringe containing 300 units of heparin until the mean arterial blood pressure (MABP) reached 35 mm Hg (in about 10 min). This level was carefully maintained after 30 min by the removal or reinfusion of small amounts of blood, After 30 min of hypotension, all the blood was reinfused (in about 5 min). After cerulein infusion for 6 hr, the animals were given standard chow ad libitum and infused with heparin-treated saline at a rate of 0.20 ml/hr. The animals were divided into the following four groups: (A) control--infused with physiologic saline without the induction of hemorrhagic shock; (B) shockm infused with physiologic saline with the induction of

1410

hemorrhagic shock; (C) cerulein--infused with a supramaximal dose of cerulein without the induction of hemorrhagic shock; (D) cerulein plus shock--infused with a supramaximal dose of cerulein with the induction of hemorrhagic shock. At 6 and 24-hr after the start of the infusion of saline or cerulein, blood was drawn through the catheter in the femoral artery and rapidly centrifuged at 2000g for 15 rain. Serum was collected and stored at - 4 0 ~ C for the measurement of serum amylase and lipase activities. Survival rates were recorded for each group (N = 20) 24 and 48 hr after the start of the infusion. Other rats in each group were killed by decapitation 6, 24, and 48 hr after the start of the infusion. The pancreas was removed rapidly, trimmed of fat and lymph nodes, and weighed. The pancreas was examined histologically, and the deoxyribonucleic acid (DNA), amylase, and cathepsin B contents were measured. Assays. A portion of pancreatic tissue was homogenized in ice-cold 50 mM phosphate buffer (pH 7.4) for 1 min in a Polytron (Kinematica, Luzern, Switzerland). The homogenates were centrifuged at 10,000g for 30 min at 4~ C, and the supernatant was used for DNA, amylase, and cathepsin B assay. DNA was measured fluorometrically with Bisbenzimide H 33258 Fluorochrome (Calbiochem-Behring Corp., La Jolla, California) by the method of LaBarca and Paigen (18) with calf thymus DNA (type I, Sigma Chemical Co., St. Louis, Missouri) as the standard. Amylase activity was determined by a chromogenic method with the Phadebas amylase test (Pharmacia Diagnostics AB, Uppsala, Sweden) (19). Lipase activity was determined by the BALB-DTNB method with Lipase Kit S (Dainippon Pharmaceutical Co., Osaka, Japan) (20). Cathepsin B activity was measured with the substrate CB Z - a r g i n y l - a r g i n i n e - 2 - n a p h t h y l a m i d e ( B a c h e m Feinchemikalien AG, Bubendorf, Switzerland), as described by McDonald and Ellis (21). The degree of pancreatic edema was evaluated by a comparison of the weight of one part of the pancreas obtained immediately after killing the animal (wet weight) with that of the same sample after incubation at 70~ C for 48 hr (dry weight). Histologie Examination. A portion of the pancreatic tissue was fixed in 10% formalin solution, embedded in paraffin and cut into sections. The sections were stained with hematoxylin and eosin and examined under a light microscope by a blinded observer. Interstitial edema was scored as follows: 0 = absent, 1 = interlobular septa were expanded, 2 = intralobular septa were expanded, and 3 = individual acini were separated. Inflammatory infiltration was scored as follows: 0 = absent, 1 = less than 20 neutrophils per intermediate power field (IPF, at • magnification), 2 = 20-50 neutrophils per IPF, 3 = more than 50 neutrophils per IPF. Parenchymal hemorrhage was scored as follows: 0 = absent, 1 = 1-2 foci per slide, 2 = 3-5 foc i per slide, 3 = more than 5 foci per slide. Parenchymal necrosis was graded according to the approximate percentage of the involved area: 0 = absent, 1 = less than 5%, 2 = 5-20%, 3 = more than 20%. The grading of vacuolization was based on the percentage of acinar cells with cytoplasmic vacuoles in the examined field: 0 = absent, 1 = less than 20%, 2 = 20-50%, 3 = more than 50%. Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

ROLE OF ISCHEMIA IN ACUTE PANCREATITIS TABLE 1. SERUM AMYLASE AND LIPASE ACTIVITIES IN CERULEIN-INDUCED ACUTE PANCREATITIS WITH OR WITHOUT HEMORRHAGIC SHOCK*

mmH

120 | =

100

it ii

0- 80 ~

i i

h !i

i i

...... "...... ........

~

..... *

""~af

_.--it ...... " .....

60 H

~

4O

~

20

II

Shock

Caerutein Infusion I

i

0

2

I

i

4

6

,fJ

I

24

Time (Hours)

Fig 1. Time course of mean arterial blood pressure in ceruleininduced acute pancreatitis with or without hemorrhagic shock. Rats were infused for 6 hr with saline with or without cerulein. Seventy-five minutes after the start of cerulein infusion, hemorrhagic shock was induced at a mean arterial blood pressure of 35 mm Hg for 30 min. O 9 controls (N = 9); O---.----Q,shock (N = 14);A---A, cerulein (N = 15); &----&, cerulein plus shock (N = 21). *P < 0.01 vs cerulein group. **P < 0.01 vs control, shock, and cerulein group, t P < 0.05 vs shock and cerulein group.

Statistical Analysis. The results were expressed as the mean -+ SE. In the statistical analysis of blood pressure and biochemical data, we used one-way analysis of variance. Survival data for the different groups were compared by the • method with Yates' correction. Histological data were evaluated by the Mann-Whitney U test. Significance for all statistical analysis was P < 0.05. RESULTS The maximum volume of blood withdrawn during h y p o t e n s i o n w a s 38.1 -+ 0.9 m l / k g b o d y w e i g h t in t h e s h o c k g r o u p a n d 35.8 -+ 0.5 m l / k g b o d y w e i g h t in t h e c e r u l e i n p l u s s h o c k g r o u p . M A B P a t t h e s t a r t o f t h e e x p e r i m e n t w a s 103 -- 2 m m H g in t h e c o n t r o l a n d 106 -+ 2 m m H g in t h e c e r u l e i n g r o u p , a n d it r e m a i n e d u n c h a n g e d d u r i n g t h e o b s e r v a t i o n p e r i o d . M A B P in t h e s h o c k g r o u p h a d d e c r e a s e d t o 93 -- 1 m m H g 4 h r a f t e r t h e r e i n f u s i o n o f b l o o d , b u t b y 24 h r it h a d r e t u r n e d to t h e n o r m a l c o n t r o l l e v e l (106 -+ 2 m m Hg). I n t h e cerulein plus shock group, MABP was significantly l o w e r 6 a n d 24 h r a f t e r t h e s t a r t o f c e r u l e i n i n f u s i o n (79 -- 4 a n d 95 +-- 4 m m H g ) t h a n in t h e s h o c k g r o u p ( F i g u r e 1). N o n e o f t h e r a t s in t h e c e r u l e i n o r s h o c k g r o u p d i e d d u r i n g this e x p e r i m e n t . H o w e v e r , five o f 20 (25%) a n d s e v e n o f 20 (35%) r a t s in t h e c e r u l e i n p l u s s h o c k g r o u p d i e d 24 a n d 48 hr, r e s p e c t i v e l y , a f t e r the start of cerulein infusion. S e r u m a m y l a s e a n d l i p a s e a c t i v i t i e s in t h e c e r ulein and cerulein plus shock groups had increased markedly by 6 hr after the start of cerulein infusion, Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

Time after start of cerulein infusion 6 hr Control (N = 7) Shock (N = 16) Cerulein (N = 12) Cerulein + shock (N = 15) 24 hr Control (N = 8) Shock (N = 13) Cerulein (N = 13) Cerulein + shock (N = 16)

Amylase activity (IU/liter)

Lipase activity (IU/liter)

2,750 --- 280 2,600 --- 200 47,960 • 3,330 a,b

16 • 1.2 18 --- 1.2 3,950 • 278 a,b

68,510 • 11,200 a,b

5,480 - 740 a,b

3,430 • 170 3,430 • 510 3,110 • 160

17 • 1.2 15 • 1.0 29 • 2.6

8,350 • 2,090 a,b,c

50 - 8.2 a,b,c

*Rats were infused for 6 hr with saline with or without cerulein; 75 min after the start of cerulein infusion, hemorrhagic shock was induced at a mean arterial blood pressure of 35 mm Hg for 30 min. Each value represents mean • SE. Significant difference (P < 0.05) vs a, control; b, shock; and c, cerulein group.

b u t t h e r e w a s n o significant d i f f e r e n c e b e t w e e n t h e m . T h e s e v a l u e s h a d r e t u r n e d to c o n t r o l l e v e l s in t h e c e r u l e i n g r o u p b y 24 hr, b u t in t h e c e r u l e i n p l u s s h o c k g r o u p t h e y r e m a i n e d e l e v a t e d e v e n a t 24 hr. T h e r e w a s n o c h a n g e in s e r u m a m y l a s e a n d l i p a s e a c t i v i t i e s in t h e s h o c k g r o u p ( T a b l e 1). T h e d r y to w e t w e i g h t r a t i o o f t h e p a n c r e a s w a s s i g n i f i c a n t l y d e c r e a s e d in t h e c e r u l e i n g r o u p 6 h r after the start of cerulein infusion. Pancreatic e d e m a w a s a l s o o b s e r v e d at 6 h r in t h e c e r u l e i n p l u s s h o c k g r o u p . H o w e v e r , t h e r a t i o w a s n o t so l o w a s in t h e c e r u l e i n g r o u p ( F i g u r e 2).

r

O (~)

-

-

1

r--~ **

r - - i **

30.

g:

6' h

Fig 2.

2~- h

l[ 48 h

D r y - w e t weight ratio of the pancreas. Rats were infused

for 6 hr with saline with or without cerulein. Seventy-five minutes after the start of cerulein infusion, hemorrhagic shock was induced at a mean arterial blood pressure of 35 mm Hg for 30 min. Open bar, control; stippled bar, shock; hatched bar, cerulein; shaded bar, cerulein plus shock. There were five or six rats in each group. Significant difference between groups: *P < 0.05; **P < 0.01.

1411

KYOGOKU ET AL (xl03 IU/mg) 40

r

r-

-

1

r-~,

**

,

I

i

I

n

*~

r - - i r - - 1

50"

40" o

2o-

"~

30"

m E

6h

24h

48h

Fig 3. Amylase content in the pancreas. Rats were infused for 6 hr with saline with or without cerulein. Seventy-five minutes after the start of cerulein infusion, hemorrhagic shock was induced at a mean arterial blood pressure of 35 mm Hg for 30 min. Open bar, control; stippled bar, shock; hatched bar, cerulein; shaded bar, cerulein plus shock. There were five or six rats in each group. Significant difference between groups: *P < 0.05; **P < 0.01.

In the shock group, the amylase content in the pancreas was increased at 24 hr and decreased at 48 hr. In the cerulein and cerulein plus shock groups, it was lower than in the control group, but there was no significant difference between them (Figure 3). Cathepsin B activity in the pancreas was significantly higher 6 hr after the start of cerulein infusion in the shock (8.68 --- 1.82 units/mg DNA) and cerulein plus shock groups (10.03 -+ 1.97 units/mg DNA) than in the control group (2.82 - 0.20 units/mg DNA). At 24 and 48 hr after the start of cerulein infusion, it was significantly higher in both the cerulein (48.14 --- 3.43 and 16.34 -+ 2.64 units/mg DNA) and the cerulein plus shock groups (35.02 --6.24 and 34.61 - 7.01 units/mg DNA) than in the control group (4.08 - 0.78 and 1.84 +-- 0.75 units/mg DNA). In the cerulein group, it reached a maximum at 24 hr and was lower at 48 hr. In the cerulein plus shock group, however, it remained higher (Figure 4). Histologic examination 6 hr after the start of cerulein infusion revealed interstitial edema and inflammatory cell infiltration in the interstitium of the pancreas with cytoplasmic vacuoles of various sizes in the acinar cells in rats treated with cerulein alone. Parenchymal necrosis was rarely observed except for some cell degeneration at the periphery of the lobules. After hemorrhagic shock had been induced in animals infused with a supramaximally stimulating dose of cerulein, hemorrhage and parenchymal necrosis of the pancreas were frequently observed. On the other hand, the degree of interstitial edema and vacuolization was decreased. Hem-

1412

0

, 6h

24h

, 48h

Fig 4. Ca~epsin B activity in the pancreas. Rats were infused for 6 hr with saline with or without cerulein. Seventy-five minutes after the start of cerulein infusion, hemorrhagic shock was induced at a mean arterial blood pressure of 35 mm Hg for 30 rain. Open bar, control; stippled bar, shock; hatched bar, cerulein; shaded bar, cerulekn plus shock. There were six rats in each group. Significant difference between groups: *P < 0.05; **P < 0.01.

orrhage was observed in 11 of the 15 rats treated with cerulein plus shock, but its degree varied from focal to massive. Hemorrhage was not observed in any other organs except the pancreas and gastric mucosa in the cerulein plus shock group. Parenchymal necrosis in the cerulein plus shock group was focal lobular or sublobular necrosis. Hemorrhage and parenchymal necrosis were still present at 24 and 48 hr in the cerulein plus shock group (Figure 5). Venous congestion, thrombosis, and accumulation of granulocytes in veins were seen in the cerulein plus shock group at 6 hr (Figure 6a), and degeneration of arterial walls was frequently observed in the cerulein plus shock group at 24 and 48 hr (Fig. 6b). There were no morphological abnormalities in the pancreas in the shock group, except for a few small vacuoles in some acinar cells and dilatation of acinar lumens at 48 hr (Table 2). DISCUSSION Acute pancreatitis is divided into three groups by pathological definition: edematous pancreatitis, hemorrhagic pancreatitis, and necrotizing pancreatitis. It is said that hemorrhagic-necrotizing pancreatitis is the most severe and has a high mortality rate. It is likely that there are many different mechanisms by which acute pancreatitis progresses from mild edematous pancreatitis to severe necrotizing pancreatitis. Recently, there has been an increasing interest in circulatory changes or ischemia as one of the factors that aggravate acute pancreatitis (22). Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

ROLE OF ISCHEMIA IN ACUTE PANCREATITIS

Fig 5. Microscopic view of the pancreas in the cerulein plus shock group 48 hr after the start of the cerulein infusion. Interlobular hemorrhage and focal lobular necrosis are evident (H&E, original magnification x70).

Rats infused with a supramaximally stimulating dose of cerulein develop hyperamylasemia, pancreatic edema, and inflammatory cell infiltrations in the pancreas (17). However, this edematous pancreatitis is reversible and not fatal. The present study shows that mild edematous pancreatitis develops into severe hemorrhagic-necrotizing pancreatitis after the induction of hemorrhagic shock in animals treated with a supramaximally stimulating dose of cerulein. Hemorrhagic shock was induced by removing blood until the MABP reached 35 mm Hg. This level was maintained for 30 min; then all the blood was returned. None of the animals with induced hemorrhagic shock alone died during this experiment. We chose this degree of hypotension, 35 mm Hg, because several animals died during the shock period if the MABP was maintained at 30 mm Hg, and hemorrhage of the pancreas was rare if the MABP was maintained at 40 mm Hg. When we Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

maintained the hypotension for more than 30 min, we could not achieve more severe changes in the pancreas (data not shown). There were no morphological changes in the pancreas of rats with temporary hypotension for 30 min. Spormann et al (8) showed that temporary occlusion of the main pancreatic arteries for up to 1 hr caused slight histological changes in the pancreas. Jones and Trump (23) demonstrated that the pancreas seems to survive total ischemia longer than many other organs. In the present study, hypotension superimposed upon cerulein-induced edematous pancreatitis led to the development of hemorrhagic-necrotizing pancreatitis with a high mortality rate when hemorrhagic shock was induced 75 min after the start of cerulein infusion. However, hemorrhage and necrosis of the pancreas were not observed if rats were infused with a supramaximal dose of cerulein immediately after the induction of hemorrhagic shock (data not shown). These findings suggest that ischemia can-

1413

KYOGOKU ET AL

Fig 6. Microscopic view of the pancreas in the cerulein plus shock group 6 hr (a) and 48 hr (b) after the start of the caerulein infusion. (a) Venous congestion and accumulation of granulocytes are visible in veins (H&E, original magnification • (b) Degeneration of arterial walls is visible (H&E, original magnification x240).

not be an initiating factor but may be an aggravating factor in the development of hemorrhagic pancreatitis. It is interesting to speculate on the mechanism by which hemorrhagic shock converts edematous pancreatitis into hemorrhagic pancreatitis. In our experimental model, parenchymal hemorrhage in the pancreas was frequently observed. This fact indi-

1414

cates that the blood vessel changes are more striking in this model than in cerulein-induced pancreatitis because parenchymal hemorrhage is thought to result from the disruption of damaged vessels in the pancreas. Therefore, microcirculatory disturbance in the pancreas is considered to be the main cause of the development of hemorrhagic pancreatitis. Our histologic study showed venous dilataDigestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

ROLE OF ISCHEMIA IN ACUTE PANCREATITIS TABLE 2. HISTOLOGIC CHANGES IN CERULEIN-[NDUCED ACUTE PANCREATITIS WITH OR WITHOUT HEMORRHAGIC SHOCK*

Time after start of cerulein infusion 6 hr Control (N = 3) Shock (N = 6) Cerulein ( N = 14) Cerulein + shock (N = 15) 24 hr Control (N = 5) Shock (N = 5) Cerulein (N = 5) Cerulein + shock (N = 5) 48 hr Control (N = 3) Shock (N = 10) Cerulein (N = 10) Cerulein + shock (N = 9)

Interstitial edema

Inflammatory infiltration

Hemorrhage

Vacuolization 0 0 1-3 (2.6 - 0.2) 0-2 (1.1 --- 0.2)a

0 0 2-3 (2.4 --- 0.1) 1-2 (1.7 --- 0.1)a

0 0 1-2 (1.2 --- 0.1) 0-3 (1.4 -+ 0.2)

0-3 (1.5 -+ 0.3)a

0 0 0-1 (0.3 --- 0.1) 0-3 (1.6 --- 0.3)a

0 0-1 (0.2 -+ 0.2) 0-1 (0.8 --- 0.2) 0-2 (1.0 +- 0.3)

0 0

0 0

0 0

0 0

0-2 (1.0 +- 0.3) 1-2 (1.4 --_ 0.2)

0 0-2 (1.4 -+ 0.4)a

0-1 (0.2 --- 0.2) 0-2 (1.2 -+ 0.3)a

1 (1.0 --- 0.0) 0-2 (1.0 - 0.3)

0 0

0 0

0 0

0-3 (1.6 -+ 0.3) 1-3 (2.0 -+ 0.3)

0-1 (0.3 +- 0.1) 0-1 (0.6 +- 0.2)

0-2 (0.6 -+ 0.2) 0-2 (1.2 ~- 0.3)

0 0-1 (0.5 -+ 0.2) 0-1 (0.8 +- 0.1) 1-2 (1.1 -- 0.1)

0 0-1 (0.0 --- 0.1) 0-1 (0.9 - 0.1) 0-1 (0.8 -- 0.1)

0 0 0

Parenchymal necrosis

*Rats were infused for 6 hr with saline with or without cerulein; 75 rain after the start o f cerulein infusion, hemorrhagic shock was induced at a mean arterial blood pressure o f 35 mm Hg for 30 min. Details o f the grading o f morphologic changes are described in Materials and Methods. Each value in parentheses represents mean --- SE. a, significant difference (P < 0.05) vs cerulein group at each time.

tion and congestion, microthrombi, and accumulation of granulocytes in the lumen and even within the walls of veins at 6 hr, and fibrinoid degeneration of the arterial walls at 24 and 48 hr. This study also showed that venous changes such as dilatation and congestion were more remarkable than arterial changes in the early stage of pancreatitis. In the later stage, however, arterial wall degeneration seemed to push forward the circulatory disturbance of the pancreas. Robert et al (24) and Gurll (25) reported that blood flow appears to be more severely impaired in the pancreas in hemorrhagic shock than in the other organs. Rao et al (14) showed that vasculitis and fibrinoid necrosis of small blood vessels preceded the inflammatory cell infiltrate in hemorrhagic pancreatitis induced in rats by the closed duodenal loop technique. Anderson (26) reported that venous stasis could convert edematous pancreatitis to fatal pancreatic necrosis. In the present study hemorrhagic shock additively increased the circulatory disturbance in the pancreas. This finding suggests that the pancreas is more susceptible to microcirculatory disturbance induced by hemorrhagic shock if activation of digestive enzymes and/or inflammatory cell infiltration in the interstitium of the pancreas has been Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

induced by the infusion of a supramaximal stimulating dose of cerulein. The mechanism of microcirculatory disturbance induced by hemorrhagic shock remains obscure. Recent studies indicate that oxygen-derived free radicals contribute to the etiology of postischemic tissue injury in many organs (27, 28). The hypoxanthine-xanthine oxidase system in the capillary endothelial cells (29) and peripheral polymorphonuclear leukocytes seem to be the major sources of oxygen-derived free radicals. During the ischemic period, ATP is catabolized to hypoxanthine and NAD-reducing xanthine dehydrogenase is converted to the oxygen radical-producing xanthine oxidase. During reperfusion, molecular oxygen reacts with hypoxanthine and xanthine oxidase to produce superoxide anion and hydrogen peroxide. On the other hand, polymorphonuclear leukocytes are activated by several chemotactic factors, accumulate in the microcirculatory system, and release several chemical mediators that cause endothelial cell injury, increased vascular permeability, stasis and sludging of blood cells, formation of microthrombi, and finally degeneration of vascular walls (28). In our experiments, we frequently observed the adherence of polymorphonuclear leukocytes to

1415

KYOGOKU E T A L

microvascular endothelium and the degeneration of vascular walls. However, further experiments are necessary to determine the nature of the chemical mediators that play a role in the microcirculatory disturbance of the pancreas in our experimental model. In this study the total activity of cathepsin B at 48 hr was statistically higher in the cerulein plus shock group than in the cerulein group. The lysosomal enzyme, cathepsin B, is known to be capable of activating trypsinogen (30), and then trypsin can activate other digestive enzymes. Spath et al (31) demonstrated that fragility of lysosomes was enhanced and disruption of pancreatic lysosomal membranes occurred during hemorrhagic shock. It has been reported that digestive zymogens and lysosomal hydrolases become colocalized within cytoplasmic organelles and that this colocalization might be an important event in the development of pancreatitis (32). Although Printz et al (12) reported that hemorrhagic shock did not alter the subcellular redistribution of cathepsin B in the early stage of cerulein-induced pancreatitis, increased cathepsin B activity may play some role in the development of severe hemorrhagic pancreatitis. Our present data show that systemic blood pressure in the cerulein plus shock group decreased significantly. Many investigators have demonstrated that cardiac output, systemic blood pressure, and pancreatic blood flow are decreased in pancreatitis (33, 34). In this manner, mild pancreatitis could develop into severe pancreatitis if systemic circulatory failure and microcirculatory disturbance of the pancreas are not counteracted. Breaking this vicious circle would seem to be an effective way of preventing the conversion from mild to severe pancreatitis. REFERENCES 1. Feiner H: Pancreatitis after cardiac surgery. Am J Surg 131:684-688, 1976 2. Jones RT, Garcia JH, Mergner WJ, Pendergrass RE, Valingorsky JM, Trump BF: Effects of shock on the pancreatic acinar cell. Arch Pathol 99:634-644, 1975 3. Warshaw AL, O'Hara PJ: Susceptibility of the pancreas to ischemic injury in shock. Ann Surg 188:197-201, 1978 4. Gmaz-Nikulin E, Nikulin A, Plamenac P, Hegewald G, Gaon D: Pancreatic lesions in shock and their significance. J Pathol 135:223-236, 1981 5. Becker V, Stollhoff K: Shock and terminal pancreatitis. Pathol Res Pract 179:512-516, 1985 6. Takahashi T, Yaginuma N: Ischemic injury of the human pancreas. Its basic patterns correlated with the pancreatic

1416

microvasculature. Pathol Res Pract 179:645-651, 1985 7. Popper HL, Necheles H, Russell KC: Transition of pancreatic edema into pancreatic necrosis. Surg Gynecol Obstet 87:79-83, 1948 8. Spormann H, Sokolowski A, Letko G: Effect of temporary ischemia upon development and histological patterns of acute pancreatitis in the rat. Pathol Res Pract 184:507-513, 1989 9. Pfeffer RB, Lazzarini-Robertson A, Safadi D, Mixter G, Secoy CF, Hinton JW: Gradations of pancreatitis, edematous, through hemorrhagic, experimentally produced by controlled injection of microspheres into blood vessels in dogs. Surgery 51:764-769, 1962 10. Redha F, Uhlschmid G, Ammann RW, Freiburghaus AU: Injection of mierospheres into pancreatic arteries causes acute hemorrhagic pancreatitis in the rat: A new animal model. Pancreas 5:188-193, 1990 11. Broe PJ, Zuidema GD, Cameron JL: The role of ischemia in acute pancreatitis: Studies with an isolated perfused canine pancreas. Surgery 91:377-382, 1982 12. Printz H, Saluja A, Leli U, Sengupta A, Steer M: Effects of hemorrhagic shock, aspirin, and ethanol on secretagogueinduced experimental pancreatitis. Int J Pancreatol 6:207217, 1990 13. Klar E, Rattner DW, Compton C, Stanford G, Chernow B, Warshaw AL: Adverse effect of therapeutic vasoconstrictors in experimental acute pancreatitis. Ann Surg 214:168174, 1991 14. Rao SS, Watt IA, Donaldson LA, Crocket A, Joffe SN: A serial histologic study of the development and progression of acute pancreatitis in the rat, Am J Pathol 103:39-46, 1981 15. Brackett KA, Crocket A, Joffe SN: Ultrastructure of early development of acute pancreatitis in the rat. Dig Dis Sci 28:74-84, 1983 16. Klar E, Endrich B, Messmer K: Microcirculation of the pancreas. A quantitative study of physiology and changes in pancreatitis. Int J Microcirc Clin Exp 9:85-101, 1990 17. Lampel M, Kern HF: Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Arch A 373:97-117, 1977 18. LaBarca C, Paigen K: A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344-352, 1980 19. Ceska M, Birath K, Brown B: A new and rapid method for the clinical determination of e~-amylase activities in human serum and urine. Clin Chim Acta 26:437-444, 1969 20. Kurooka S, Okamoto S, Hashimoto M: A novel and simple colorimetrie assay for human serum lipase. J Biochem 81:361-369, 1977 21. McDonald JK, Ellis S: On the substrate specificity of cathepsin BI and B2 including a new fluorogenic substrate for cathepsin B1. Life Sci 17:1269-1276, 1975 22. Kiar E, Messmer K, Warshaw AL, Heffarth C: Pancreatic ischaemia in experimental acute pancreatitis: Mechanism, significance and therapy. Br J Surg 77:1205-1210, 1990 23. Jones RT, Trump BF: Cellular and subcellular effects of ischemia on the pancreatic acinar cell. Virchows Arch B 19:325-336, 1975 24. Robert JH, Toledano AE, Toth LS, Premus G, Dreiling DA: Hypovolemic shock, pancreatic blood flow, and pancreatitis. Int J Pancreatol 3:283-292, 1988 25. Gurll NJ: Organ blood flow and metabolism in shock: Overview. Prog Clin Biol Res 299:3-8, 1989 Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

R O L E O F I S C H E M I A IN A C U T E P A N C R E A T I T I S 26. Anderson MC: Venous stasis in the transition of edematous pancreatitis to necrosis. JAMA 183:534-537, 1963 27. McCord JM: Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312:159-163, 1985 28. Granger DN: Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol 255:HI269H1275, 1988 29. Jarasch ED, Bruder G, Heid HW: Significance of xanthine oxidase in capillary endothelial cells. Acta Physiol Scand 548(suppl):39-46, 1986 30. Figarella C, Miszczuk-Jamska B, Barrett AJ: Possible lysosomal activation of pancreatic zymogens. Biol Chem HoppeSeyler 369(suppl):293-298, 1988

Digestive Diseases and Sciences, Vol. 37, No. 9 (September 1992)

31. Spath JA, GorczynskiRJ, Lefer AM: Pancreatic perfusionin the pathophysiology of hemorrhagic shock. Am J Physiol 226:443-451, 1974 32. Saluja A, Hashimoto S, Saluja M, Powers RE, Meldolesi J, Steer ML: Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis. Am J Physiol 253:G508-G516, 1987 33. Papp M, Makara GB, Hajtman B, Csaki L: A quantitative study of pancreatic blood flow in experimental pancreatitis. Gastroenterology 51:524-527, 1966 34. Goodhead B: Acute pancreatitis and pancreatic blood flow. Surg Gynecol Obstet 129:331-340, 1969

1417