Langenbecks Arch. Chir, 341,297-301 (1976)
Langenbecks Archiv for Chirurgie © by Springer-Verlag 1976
Prevention of Stress Ulcer by Somatostatin in Rats P. Mattes, H. H. Lauterbach, and S. Raptis Department of General Surgery, Center for Operative Medicine and Department of Endocrinology and Metabolism, Center for Internal Medicine and Pediatrics, University of Ulm, Federal Republic of Germany
Summary. In animal experiments it was examined, to what extent prophylactic application of Somatostatin is able to prevent the stress-induced ulcer of the rat. 32 male Wistar rats were exposed to a combined immobilisation-hypoxia-stress. Somatostatin was given intraperitoneally as bolus and intravenously as a continuous infusion. The results indicated that Somatostatin lowers significantly the incidence rate of stress-induced gastric ulcers in the rat.
Key words: Somatostatin - Stress ulcer. Somatostatin is k n o w n to reduce not only the release of gastrin but basal and stimulated gastric secretion as well [1,2, 8, 9]. First clinical application of Somatostatin in hemorrhaging stress ulcers of risk patients caused a cessation of hemorrhage and fast healing of the ulcer [6]. It is the aim of the present study to examine in animal experiments to what extent the prophylactic application of Somatostatin is able to prevent the stress-induced ulcer of the rat.
Materials and Methods 32 male Wistar rats weighing 200 g each were kept fasting for 40 h, but had free access to water, The animals were randomized into 4 groups: Group l (n = 10): 250 p.g Somatostatin was given as bolus injection in 1 ml 0.9% NaCI. Group 2 (n = 10) (control-group): 1 ml NaCI was given intraperitoneally. Group 3 (n = 6): 250/,tg Somatostatin in 1 ml 0,9% NaC1 was given as a bolus injection and followed by an i.v. infusion of 500 lag Somatostatin over 6 h. Group 4 (n = 6): 1 ml 0.9% NaC1 was given as a bolus injection i.v. followed by 5.4 ml 0.9% NaCI as a 6 h i.v. infusion.
Adress for offprint requests: Priv.-Doz. Dr. med. P. Mattes and Dr. reed. H. H. Lauterbach, Department of General Surgery, University of Ulm, Steinh6velstr. 9, D-7900 Ulm/Donau, Federal Republic of Germany
298
P. Mattes et al.
Stress model was a combined immobilisation-hypoxia-stress [3, 13]. Preliminary tests in groups of 10 animals each produced in 100% mucous membrane lesions after 6-8 h immobilisationhypoxia-stress. The rate of ulcers increased with duration of stress. After immobilisation by wrapping up the animals in plaster of Paris bandages the rats were incubated for 8 h (group I and 2), respectively 6 h (group 3 and 4) in an infant incubator at 14% oxygen content. The atmosphere was produced by introducing nitrogen and was measured contiously with an oxymeter. At the end of the experiment the animals were killed by decapitation. The stomachs were extracted, sliced open at the greater curvature, evaluated for their blood content, rinsed with Jores solution i and fixed. Evaluation of the stress-induced mucous membrane lesions was done macroscopically according to quantity and size. These two factors were combined into an ulcer index modified after Brodie [3]. The erosions were classified in size order with a valuation Figures 1-3 according to their longest diameter. size order l: 0 - I mm diameter size order 2 : 1 - 2 mm diameter size order 3 : 2 - 3 mm diameter. For each stomach the valuation figure was multiplied by the quantity of erosions occuring in this size order. These figures were added for each animal and defined as ulcer index (J). The mean value of this index existing for each animal was compared with the control group. The values are mean values + SEM. Calculation of significance was done according to the disconnected t-test. The erosions were evaluated by an examiner to whom the previous treatment of the animals was unknown.
Results T h e c o m b i n e d i m m o b i l i s a t i o n - h y p o x i a - s t r e s s o v e r 6 - 8 h i n d u c e d i n all a n i m a l s of t h e c o n t r o l g r o u p 2 a n d 4 e r o s i o n s i n t h e g l a n d u l a r a r e a of t h e s t o m a c h . A f t e r t h e e n d of t h e e x p e r i m e n t all c o n t r o l a n i m a l s e x h i b i t e d p a r t l y f r e s h , c o a g u l a t e d b l o o d in t h e s t o m a c h . P r o p h y l a c t i c a p p l i c a t i o n o f 250 lag S o m a t o s t a t i n i.p. d e c r e a s e d size as well as q u a n t i t y of t h e e r o s i o n s d u r i n g a n 8 h stress as c o m p a r e d t o t h e c o n t r o l g r o u p (Fig. 1 a a n d b). T h e u l c e r i n d e x r e p r e s e n t i n g b o t h size a n d q u a n t i t y of e r o s i o n s , a m o u n t e d t o 7.5 + 4 . 3 7 in t h e t r e a t e d a n i m a l s , as c o m p a r e d t o 2 6 . 4 + 1 6 . 6 8 i n t h e c o n t r o l a n i m a l s ( T a b l e 1). T h e d i f f e r e n c e s a r e h i g h l y s i g n i f i c a n t ( P < 0 . 0 0 5 ) .
Table 1. Mean values, standard deviation of ulcer index and number of ulcers per size order and significance computation of the 8-h experiment (n = number of animals; J = ulcer index per animal) Test group
n
Number of ulcers per size order
J
1 I Somatostatin 250 lag i.p. II Control group
10
7.50 _+ 4.37
2
3
6.90 + 4.20
0.30 +_ 0.67
0
19.50 _+ 9.22
2.00 + 2.26
0.70 _+ 0.74
P < 0.005 10
26.40 _+ 16.68
1 Preserving solution for anatomical preparations: Sal carolinum factitium 50 g, formaldehyd soiutus 50 cm a, concentrated, watery chloralhydrate solution 50 cm a, water 1000 cm 3
Prevention of Stress Ulcer by Somatostatin in Rats
299
Fig.la and b. Stomach resection preparation after a 6-h immobilisation-hypoxia-stress, a Without Somatostatin: multiple hemorrhagic mucous membrane lesions up to 2 mm diameter in the glandular area of the stomach, b With Somatostatin: some scattered mucous membrane lesions up to 1 mm in diameter
Table 2. Mean values, standard deviation of ulcer index and number of ulcers per size order and sig-
nificance computation of the 6-h experiment (n = number of animals; J = ulcer index per animal) Test group
n
Number of ulcers per size order
J
1
III Somatostatin a 250 pg + 500 ~tg IV Control group
6
0.83 + 0.98
2
3
0.50 + 0.83
0.16 + 0.40
0
6.66 + 4.84
2.16 + 1.94
0.66 + 1.03
P < 0.0025 6
13.00 _+ 5.69
250 ~tg i.v. as bolus and 500 ~tg i.v. in 5.4 ml 0.9 ~ NaCI as a 6-h infusion
I n t r a v e n o u s application o f 250 lag S o m a t o s t a t i n as bolus and 500 lag S o m a tostatin as a 6 h c o n t i n u o u s infusion i n d u c e d a r e d u c t i o n of size a n d q u a n t i t y of s t o m a c h e r o s i o n s also as c o m p a r e d to t h e u n t r e a t e d group. In g r o u p 3 and 4 with stress d u r a t i o n of 6 h the ulcer i n d e x a m o u n t e d to 0.83 + 0.98 in the animals with S o m a t o s t a t i n , to 13.0 + 5.69 in the c o n t r o l animals ( T a b l e 2). T h e d i f f e r e n c e is statistically highly significant as well ( P < 0.0025). E r o s i o n s of size o r d e r 3 w e r e n o t f o u n d in any o n e of the animals t r e a t e d w i t h S o m a t o s t a t i n ( T a b l e 2). N o b l o o d c o u l d b e t r a c e d in the s t o m a c h s of the t r e a t e d animals in contrast to t h e c o n t r o l animals. A l l animals in g r o u p 1 - 4 regardless of
300
P. Mattes et al.
the foregoing treatment, exhibited a bilious imbibition of the mucous membrane of the stomach.
Discussion In an previous paper we have been able to show the therapeutic effect of Somatostatin in stress ulcer hemorrhage [6]. The present study demonstrates that prophylactic Somatostatin application in immobilisation-hypoxia-stress of the rat is capable of lowering conspicuously the incidence rate of stress-induced ulcers. The protective effect of Somatostatin manifests itself predominantly in a reduction of size and quantity of stress ulcers. While the control animals always exhibited signs of upper gastro-intestinal hemorrhage, erosions of the Somatostatin-treated animals produced no hemorrhage. Pathogenesis of the stress ulcer has not been completely clarified as yet. Provoking factors, apart from a bilious reflux [12], may possibly be a decreased regeneration capacity of the mucous membrane in consequence of a reduced mucosa blood flow [10,11]. A n increased acid production could not be demonstrated in the immobilisationstress of the rat [7], though acid reduction and neutralisation are defense mechanisms against stress ulcer [3]. The mode of action of Somatostatin in the prophylaxis of the stress-induced ulcer cannot be elucidated by our experiments. It may be assumed, though, that the protective effect is, at least partially, based on an acid reduction. Bilious reflux does not seem to be influenced by Somatostatin. In all animals a bilious reflux was evidenced. Lankisch et al. [5] were able to observe a cessation of the gall-bladder contraction and thus reduction of bilious flow after application of Somatostatin, we however did not detect any decrease of bile production in patients with a T-drainage [4]. Further studies have to clarify to what extent Somatostatin, apart from a reduction of aggressive factors, induces an activation of protective factors in the genesis of stress ulcers.
Literatur 1. Arnold, R, Creutzfeldt, W.: Hemmung der pentagastrin-induzierten Siiuresekretion des Magens beim Menschen durch Sornatostatin. Dtsch. rned. Wschr. 100, 1014-1016 (1975) 2. Bloom, S. R., Mortimer, Ch., Thorner, M, O., Berser, G.M., Hall, R., Gomez-Pan, A., Roy, V. M., Russell, R. C. G., Coy, D. H., Kastin, A. J.: Inhibition of gastrin and gastric-acid secretion by growth hormon release inhibiting hormone. Lancet 1974 II, 1106-1109 3. Brodie, D. A., Hanson, H. M.: A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38, 353-360 (1960) 4. Dollinger, H. C., Mattes, P., Raptis, S.: unver6ffentlicht 5. Lankisch, P. G., Arnold, R., Creutzfeldt, W.: Wirkung yon Sornatostatin auf die Betazol-stirnulierte Magensekretion, die Carhachol-stirnulierte Pankreassekrefion und die Gallenblasenkontraktion des Menschen. Dtsch. ined. Wschr. 100, 1797-1800 (1975) 6. Mattes, P,, Raptis, S., Hell, Th., Rasche, H., Scheck, R.: Extended Somatostatin treatment of patient with bleeding ulcer. Horrn. Metab. Res. 7, 508-511 (1975) 7. Menguy, R.: Effects of restraint stress on gastric secretion in the rat. Arner. J. dig. Dis. 5, 911-916 (1960)
Prevention of Stress Ulcer by Somatostatin in Rats
301
8. Raptis, S., Dollinger, H. C., von Berger, L., Schlegel, W., Schrfder, K.E., Pfeiffer, E. F.: Effects of Somatostatin on gastric secretion and release in man. Digestion 13, 15-26 (1975) 9. Raptis, S., Thum, Ch., yon Berger, L., Schr6der, K. E., Meissner, C., Schlegel, W.: Inhibition of gastrin secretion by somatostatin. Acta endocr. (Kbh.), Suppl. 193, 74 (1975) 10. Schellerer, W., Schwille, P. O., Hermanek, P., Samberger, N. M., Reitzenstein, M., Scholz, D., Wagner, W.: Neue Vorstellungen zur Pathogenese des Stress-Ulcus. Langenbecks Arch. Chir., Suppl. Chir. Forum 91, 91-95 (1974) 11. Sehellerer, W.: The role of mucosal flow in the pathogenesis of stress ulcers. Acta hepato-gastroent. 21, 138-141 (1974) 12. Schumpelick, V.: Die Funktion der Galle beim Stress-Ulcus der Ratte. Dtsch. reed. Wschr. 100, 1398-1399 (1975) 13. Zumtobel, V.: Auswirkungen des akuten generalisierten Sauerstoffmangels auf die Magenschleimhaut. (-.Habilitationsschrift) M/inchen 1974
Received April 15, 1976
Langenbecks Archiv for Chirurgie © by Springer-Verlag 1976
Prevention of Stress Ulcer by Somatostatin in Rats P. Mattes, H. H. Lauterbach, and S. Raptis Department of General Surgery, Center for Operative Medicine and Department of Endocrinology and Metabolism, Center for Internal Medicine and Pediatrics, University of Ulm, Federal Republic of Germany
Summary. In animal experiments it was examined, to what extent prophylactic application of Somatostatin is able to prevent the stress-induced ulcer of the rat. 32 male Wistar rats were exposed to a combined immobilisation-hypoxia-stress. Somatostatin was given intraperitoneally as bolus and intravenously as a continuous infusion. The results indicated that Somatostatin lowers significantly the incidence rate of stress-induced gastric ulcers in the rat.
Key words: Somatostatin - Stress ulcer. Somatostatin is k n o w n to reduce not only the release of gastrin but basal and stimulated gastric secretion as well [1,2, 8, 9]. First clinical application of Somatostatin in hemorrhaging stress ulcers of risk patients caused a cessation of hemorrhage and fast healing of the ulcer [6]. It is the aim of the present study to examine in animal experiments to what extent the prophylactic application of Somatostatin is able to prevent the stress-induced ulcer of the rat.
Materials and Methods 32 male Wistar rats weighing 200 g each were kept fasting for 40 h, but had free access to water, The animals were randomized into 4 groups: Group l (n = 10): 250 p.g Somatostatin was given as bolus injection in 1 ml 0.9% NaCI. Group 2 (n = 10) (control-group): 1 ml NaCI was given intraperitoneally. Group 3 (n = 6): 250/,tg Somatostatin in 1 ml 0,9% NaC1 was given as a bolus injection and followed by an i.v. infusion of 500 lag Somatostatin over 6 h. Group 4 (n = 6): 1 ml 0.9% NaC1 was given as a bolus injection i.v. followed by 5.4 ml 0.9% NaCI as a 6 h i.v. infusion.
Adress for offprint requests: Priv.-Doz. Dr. med. P. Mattes and Dr. reed. H. H. Lauterbach, Department of General Surgery, University of Ulm, Steinh6velstr. 9, D-7900 Ulm/Donau, Federal Republic of Germany
298
P. Mattes et al.
Stress model was a combined immobilisation-hypoxia-stress [3, 13]. Preliminary tests in groups of 10 animals each produced in 100% mucous membrane lesions after 6-8 h immobilisationhypoxia-stress. The rate of ulcers increased with duration of stress. After immobilisation by wrapping up the animals in plaster of Paris bandages the rats were incubated for 8 h (group I and 2), respectively 6 h (group 3 and 4) in an infant incubator at 14% oxygen content. The atmosphere was produced by introducing nitrogen and was measured contiously with an oxymeter. At the end of the experiment the animals were killed by decapitation. The stomachs were extracted, sliced open at the greater curvature, evaluated for their blood content, rinsed with Jores solution i and fixed. Evaluation of the stress-induced mucous membrane lesions was done macroscopically according to quantity and size. These two factors were combined into an ulcer index modified after Brodie [3]. The erosions were classified in size order with a valuation Figures 1-3 according to their longest diameter. size order l: 0 - I mm diameter size order 2 : 1 - 2 mm diameter size order 3 : 2 - 3 mm diameter. For each stomach the valuation figure was multiplied by the quantity of erosions occuring in this size order. These figures were added for each animal and defined as ulcer index (J). The mean value of this index existing for each animal was compared with the control group. The values are mean values + SEM. Calculation of significance was done according to the disconnected t-test. The erosions were evaluated by an examiner to whom the previous treatment of the animals was unknown.
Results T h e c o m b i n e d i m m o b i l i s a t i o n - h y p o x i a - s t r e s s o v e r 6 - 8 h i n d u c e d i n all a n i m a l s of t h e c o n t r o l g r o u p 2 a n d 4 e r o s i o n s i n t h e g l a n d u l a r a r e a of t h e s t o m a c h . A f t e r t h e e n d of t h e e x p e r i m e n t all c o n t r o l a n i m a l s e x h i b i t e d p a r t l y f r e s h , c o a g u l a t e d b l o o d in t h e s t o m a c h . P r o p h y l a c t i c a p p l i c a t i o n o f 250 lag S o m a t o s t a t i n i.p. d e c r e a s e d size as well as q u a n t i t y of t h e e r o s i o n s d u r i n g a n 8 h stress as c o m p a r e d t o t h e c o n t r o l g r o u p (Fig. 1 a a n d b). T h e u l c e r i n d e x r e p r e s e n t i n g b o t h size a n d q u a n t i t y of e r o s i o n s , a m o u n t e d t o 7.5 + 4 . 3 7 in t h e t r e a t e d a n i m a l s , as c o m p a r e d t o 2 6 . 4 + 1 6 . 6 8 i n t h e c o n t r o l a n i m a l s ( T a b l e 1). T h e d i f f e r e n c e s a r e h i g h l y s i g n i f i c a n t ( P < 0 . 0 0 5 ) .
Table 1. Mean values, standard deviation of ulcer index and number of ulcers per size order and significance computation of the 8-h experiment (n = number of animals; J = ulcer index per animal) Test group
n
Number of ulcers per size order
J
1 I Somatostatin 250 lag i.p. II Control group
10
7.50 _+ 4.37
2
3
6.90 + 4.20
0.30 +_ 0.67
0
19.50 _+ 9.22
2.00 + 2.26
0.70 _+ 0.74
P < 0.005 10
26.40 _+ 16.68
1 Preserving solution for anatomical preparations: Sal carolinum factitium 50 g, formaldehyd soiutus 50 cm a, concentrated, watery chloralhydrate solution 50 cm a, water 1000 cm 3
Prevention of Stress Ulcer by Somatostatin in Rats
299
Fig.la and b. Stomach resection preparation after a 6-h immobilisation-hypoxia-stress, a Without Somatostatin: multiple hemorrhagic mucous membrane lesions up to 2 mm diameter in the glandular area of the stomach, b With Somatostatin: some scattered mucous membrane lesions up to 1 mm in diameter
Table 2. Mean values, standard deviation of ulcer index and number of ulcers per size order and sig-
nificance computation of the 6-h experiment (n = number of animals; J = ulcer index per animal) Test group
n
Number of ulcers per size order
J
1
III Somatostatin a 250 pg + 500 ~tg IV Control group
6
0.83 + 0.98
2
3
0.50 + 0.83
0.16 + 0.40
0
6.66 + 4.84
2.16 + 1.94
0.66 + 1.03
P < 0.0025 6
13.00 _+ 5.69
250 ~tg i.v. as bolus and 500 ~tg i.v. in 5.4 ml 0.9 ~ NaCI as a 6-h infusion
I n t r a v e n o u s application o f 250 lag S o m a t o s t a t i n as bolus and 500 lag S o m a tostatin as a 6 h c o n t i n u o u s infusion i n d u c e d a r e d u c t i o n of size a n d q u a n t i t y of s t o m a c h e r o s i o n s also as c o m p a r e d to t h e u n t r e a t e d group. In g r o u p 3 and 4 with stress d u r a t i o n of 6 h the ulcer i n d e x a m o u n t e d to 0.83 + 0.98 in the animals with S o m a t o s t a t i n , to 13.0 + 5.69 in the c o n t r o l animals ( T a b l e 2). T h e d i f f e r e n c e is statistically highly significant as well ( P < 0.0025). E r o s i o n s of size o r d e r 3 w e r e n o t f o u n d in any o n e of the animals t r e a t e d w i t h S o m a t o s t a t i n ( T a b l e 2). N o b l o o d c o u l d b e t r a c e d in the s t o m a c h s of the t r e a t e d animals in contrast to t h e c o n t r o l animals. A l l animals in g r o u p 1 - 4 regardless of
300
P. Mattes et al.
the foregoing treatment, exhibited a bilious imbibition of the mucous membrane of the stomach.
Discussion In an previous paper we have been able to show the therapeutic effect of Somatostatin in stress ulcer hemorrhage [6]. The present study demonstrates that prophylactic Somatostatin application in immobilisation-hypoxia-stress of the rat is capable of lowering conspicuously the incidence rate of stress-induced ulcers. The protective effect of Somatostatin manifests itself predominantly in a reduction of size and quantity of stress ulcers. While the control animals always exhibited signs of upper gastro-intestinal hemorrhage, erosions of the Somatostatin-treated animals produced no hemorrhage. Pathogenesis of the stress ulcer has not been completely clarified as yet. Provoking factors, apart from a bilious reflux [12], may possibly be a decreased regeneration capacity of the mucous membrane in consequence of a reduced mucosa blood flow [10,11]. A n increased acid production could not be demonstrated in the immobilisationstress of the rat [7], though acid reduction and neutralisation are defense mechanisms against stress ulcer [3]. The mode of action of Somatostatin in the prophylaxis of the stress-induced ulcer cannot be elucidated by our experiments. It may be assumed, though, that the protective effect is, at least partially, based on an acid reduction. Bilious reflux does not seem to be influenced by Somatostatin. In all animals a bilious reflux was evidenced. Lankisch et al. [5] were able to observe a cessation of the gall-bladder contraction and thus reduction of bilious flow after application of Somatostatin, we however did not detect any decrease of bile production in patients with a T-drainage [4]. Further studies have to clarify to what extent Somatostatin, apart from a reduction of aggressive factors, induces an activation of protective factors in the genesis of stress ulcers.
Literatur 1. Arnold, R, Creutzfeldt, W.: Hemmung der pentagastrin-induzierten Siiuresekretion des Magens beim Menschen durch Sornatostatin. Dtsch. rned. Wschr. 100, 1014-1016 (1975) 2. Bloom, S. R., Mortimer, Ch., Thorner, M, O., Berser, G.M., Hall, R., Gomez-Pan, A., Roy, V. M., Russell, R. C. G., Coy, D. H., Kastin, A. J.: Inhibition of gastrin and gastric-acid secretion by growth hormon release inhibiting hormone. Lancet 1974 II, 1106-1109 3. Brodie, D. A., Hanson, H. M.: A study of the factors involved in the production of gastric ulcers by the restraint technique. Gastroenterology 38, 353-360 (1960) 4. Dollinger, H. C., Mattes, P., Raptis, S.: unver6ffentlicht 5. Lankisch, P. G., Arnold, R., Creutzfeldt, W.: Wirkung yon Sornatostatin auf die Betazol-stirnulierte Magensekretion, die Carhachol-stirnulierte Pankreassekrefion und die Gallenblasenkontraktion des Menschen. Dtsch. ined. Wschr. 100, 1797-1800 (1975) 6. Mattes, P,, Raptis, S., Hell, Th., Rasche, H., Scheck, R.: Extended Somatostatin treatment of patient with bleeding ulcer. Horrn. Metab. Res. 7, 508-511 (1975) 7. Menguy, R.: Effects of restraint stress on gastric secretion in the rat. Arner. J. dig. Dis. 5, 911-916 (1960)
Prevention of Stress Ulcer by Somatostatin in Rats
301
8. Raptis, S., Dollinger, H. C., von Berger, L., Schlegel, W., Schrfder, K.E., Pfeiffer, E. F.: Effects of Somatostatin on gastric secretion and release in man. Digestion 13, 15-26 (1975) 9. Raptis, S., Thum, Ch., yon Berger, L., Schr6der, K. E., Meissner, C., Schlegel, W.: Inhibition of gastrin secretion by somatostatin. Acta endocr. (Kbh.), Suppl. 193, 74 (1975) 10. Schellerer, W., Schwille, P. O., Hermanek, P., Samberger, N. M., Reitzenstein, M., Scholz, D., Wagner, W.: Neue Vorstellungen zur Pathogenese des Stress-Ulcus. Langenbecks Arch. Chir., Suppl. Chir. Forum 91, 91-95 (1974) 11. Sehellerer, W.: The role of mucosal flow in the pathogenesis of stress ulcers. Acta hepato-gastroent. 21, 138-141 (1974) 12. Schumpelick, V.: Die Funktion der Galle beim Stress-Ulcus der Ratte. Dtsch. reed. Wschr. 100, 1398-1399 (1975) 13. Zumtobel, V.: Auswirkungen des akuten generalisierten Sauerstoffmangels auf die Magenschleimhaut. (-.Habilitationsschrift) M/inchen 1974
Received April 15, 1976
