Pancreatic problems: An experimental model

Pancreatic Problems: An Experimental Model By Patricia M. Davidson, C. Meeham, D.G. Young, and A.K. Foulis Glasgow, Scotland 9 AIIoxan, a cytotoxic agent specific to beta cells of the pancreas, has previously been reported as a t r e a t m e n t for nesidioblastosis. However, the limited knowledge of the effects of AIIoxan on the pancreas, due to absence of an adequate animal model, was the stimulus to the present study. Thirty-one rabbits w e r e subjected to laparotomy and temporary vascular isolation of the tail of the pancreas. AIIoxan (200 m g / k g 5% solution) was injected into a peripheral vein and was followed, after 4 minutes, by dextrose to counteract its effect. Autopsy results showed specific beta cell death in the pancreas apart from the isolated tail. 9 1986 by Grune & Stratton. Inc. INDEX W O R D S : AIIoxan; nesidioblastosis.

'ESIDIOBLASTOSIS is a group of morphologic

features in the pancreas representing an increase N in the amount of endocrine tissue 1 and possible maldis-

tribution and malregulation of the endocrine cells. 2 This results in the clinically recognizable syndrome of inappropriate insulin production and subsequent hypoglycemia. Alloxan (mesoxalyl urea), a cytotoxic agent specific to insulin-producing cells, has been reported as a therapeutic option in the treatment of this condition .3 The development of an animal model, in which there is only partial loss of insulin production and in which the surviving animals are not insulin-dependent, could be used to study the effects of Alloxan on the pancreas with regard to its use in the management of nesidioblastosis. This could be achieved by temporarily isolating the tail of the pancreas from the systemic circulation during intravenous administration of Alloxan, thus allowing survival of insulin-producing cells in this protected area. MATERIALS AND METHODS A total of 31 New Zealand white male rabbits were included in the study. These animals were fasted for 12 hours preoperatively and one hour prior to laparotomy 0.5 mL of Hypnorm, a neurolept analgesic, was given intramuscularly. The animals were anaesthetized with a combination of Halothane (1% to 2%) and Nitrous Oxide. Antibiotic prophylaxis (cephotaxime) was administered preoperatively and a nasogastric tube was passed prior to laparotomy. An upper left transverse abdominal incision was used. The spleen and tail of the pancreas were identified and delivered through the incision following division of the lienorenal ligament. The vascular supply to the tail of the pancreas via the splenic artery and the short gastric arteries was occluded by positioning two vascular clamps at right angles (Fig 1), producing temporary isolation of the tail of the pancreas from the systemic circulation. Following vascular occlusion 200 mg/kg of a 5% freshly prepared solution of Alloxan (mesoxalyl urea) was injected into a peripheral

Journal of Pediatric Surgery, Vol 21, No 3 (March), 1986: pp 255-256

vein and, after 4 minutes, 0.5 g/kg of dextrose (25%) was injected. The vascular clamps were released 2 minutes later, after a total of 6 minutes occlusion time. The abdomen was then closed. During the 24-hour postoperative period the animals were nursed in a warm environment and allowed free access to water. Blood glucose levels were checked intermittently. A total of 10 g/kg/24 hours of dextrose was administered to correct hypoglycemic episodes either as a 50% solution orally or as a 15% solution (diluted with 0.9% saline plus 40 mmol/L KCL) subcutaneously if the nasogastric tube was blocked. Those animals that did not die during the study were killed at 8 weeks. An autopsy was performed on every animal. The pancreases were fixed in formalin and embedded in paraffin. Sections were stained by standard immunoperoxidase techniques using antisera to insulin, glucagon, somatostatin, and pancreatic polypeptide. Sections of liver, kidney, and lung were sampled and stained by haematoxylin and eosin.

RESULTS

The first 17 rabbits in the study were operated on with a nonstandard technique during evolution of the protocol; 3 survived. The 14 deaths included 3 surgical deaths (bleeding, wound dehiscence), 3 deaths from aspiration during anesthesia, and 8 deaths from either metabolic or toxic effects of Alloxan. The histologic findings in these early deaths showed beta cell necrosis in both head and tail of the pancreas. Liver damage (periportal necrosis, perivenular fat) and kidney damage (acute tubular necrosis) were present in some animals. One rabbit, which did not receive IV dextrose prior to release of the clamps, developed diabetes mellitus. In 2 of the 3 animals killed electively, beta cells were absent in the head of the pancreas but there was normal islet tissue in the tail. The third rabbit (given 100 mg/kg Alloxan) showed no histologic abnormalities. Exocrine tissue and the endocrine cells secreting glucagon, somatostatin, and pancreatic polypeptide were unaffected in both head and tail. Of the 14 rabbits that were operated on in the standard fashion described, 7 survived and 7 died. The 7 deaths included 2 surgical deaths and 5 deaths as a consequence of the metabolic or toxic effects of Allox-

From the Western Infirmary, the Royal Infirmary, and the Royal Hospital for Sick Children, Glasgow, Scotland. Presented at the 32nd Annual Congress of the British Association of the Paediatric Surgeons, Vienna, Austria, July 17-19, 1985. Address reprint requests to D.G. Young, FRCS, DTM&H, Reader in Paediatric Surgery, Royal Hospital for Sick Children, Yorkhill, Glasgow, G3 8S J, Scotland. 9 1986 by Grune & Stratton, Inc. 0022 3468/86/2103-0021503.00/0

255

256

DAVIDSON ET AL ~

Stomach

Table 2. Blood Values of Glucose and Insulin Before and A f t e r

Oral Glucose Load in a Postoperative Animal

, / t

Pancreas ~

~

k~'N~

~,~ ~

J ~t' ' ' f \~ / ~ , ~:~ T

Short gastric

--

:~ I --I ~'~ ) ~ ' / . . . . . . ,

~

"~.'~" Clamp (~'j/"

ligament

Splenic artery and vein

an. The histologic findings in the early deaths (<48 hours; Table 1) showed beta cell necrosis in the head of the pancreas and, in one case, also in the tail. Mitotic activity could be seen in the surviving beta cells in the tail of the pancreas of the 2 rabbits that died within 5 days of the experiment. In 4 animals there was evidence of liver damage (periportal necrosis, perivenular fat) and kidney damage (acute tubular necrosis). The 7 surviving rabbits remained healthy and put on weight. They were not insulin-dependent and showed a normal insulin response to a glucose load (1.7 g/kg; Table 2). These animals were subsequently killed and all showed virtual total loss of beta cells in the head of Table 1. Fate of Islet Cells in the Head and Tail of Pancreas in Surviving and Dead Rabbits Head of Pancreas

Alive 7 Dead 7

Tail of Pancreas

NormalIslets AbnormalIslets NormalIslets Abnormal Islets 0 0

7 7

Preprandial

Postprandial

5.3 retool// 4.4 m/~/L

9.8 mmol/L

26.6 m#/L

the pancreas with normal islet tissue in the tail (Table 1). The exocrine tissue and other endocrine cells were unaffected and no other histologic abnormalities were seen.

DISCUSSION

Fig 1. The surgical procedure: showing vascular clamps in position across the tail of the pancreas, splenic vessels, and short gastric vessels to occlude the blood supply to the tail of pancreas.

Number

Glucose Insulin

7 6

0 1

Alloxan is cytotoxic to the insulin-secreting beta cells of the pancreas.4 It is thought to act by irreversibly binding to the glucose receptor on the cell membrane. 5 This study confirms that beta cells are destroyed by Alloxan and that glucagon; somatostatin; and pancreatic polypeptide-producing cells are unaffected. Alloxan has a short half-life 6 and the binding to the receptor can be partially counteracted by the use of intravenous dextrose, hence its administration prior to release of the vascular clamps. However, Alloxan in the dose administered (200 m g / k g ) produced toxic side-effects on the liver and kidneys as well as beta cell death. A lower dose may not destroy all the beta cells but, in treatment of nesidioblastosis in humans, it would be appropriate to administer a small starting dose and repeat as necessary until the desired result was achieved, thus minimizing toxicity. Regeneration of the pancreas following surgery in the neonatal period has been reported. 7 In this study, following the death of a substantial part of the beta cell population, there was evidence of beta cell regeneration as indicated by mitotic activity seen in the pancreases of the 2 animals that died after 5 days. This observation requires further studies.

REFERENCES 5. Zawalian WS, Beidler LM: Glucose and alloxan interactions 1. Heitz PU, KloppelG, Hacki WH, et al: Nesidioblastosis:The in the pancreatic islets. Am J Physiol 224:963-965, 1973 pathologic basis of persistent hyperinsulinemic hypoglycemia in infants. Diabetes 26:632-642, 1977 6. Dulin WE, Sorer MG: Chemically and hormonally induced 2. Gould VE, Memoli VA, Dardi E, et ah Nesidiodysplasia and diabetes, in Volk BW, Wellmann KF (eds): The Diabetic Pancreas. nesidioblastosis of infancy. Scan J Gastroenterol 16:129-142, 1981 New York, Plenum Press, 1977, pp 425-465 3. Davidson PM, Young DG, Logan RW, et al: Alloxan therapy 7. Aynsley-GreenA, Polak JM, BloomSR, et al: Nesidioblastosis for nesidioblastosis. J Pediatr Surg 19:87-89, 1984 of the pancreas: Definition of the syndromeand the management of 4. Rerup CC: Drugs producing diabetes through damage of the the severe neonatal hyperinsulinaemic hypoglycemia. Arch Dis insulin secreting cells in Pharmacological Reviews Vol 22. BaltiChild 56:496-508, 1981 more, Williams and Wilkins, 1970, pp 485-518