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Experimental Studies |
1 From the Department of Diagnostic Imaging, Yale University School of Medicine, PO Box 208042, New Haven, CT 06520-8042 (M.G.T.); and the Transplantation and Autoimmunity Branch, National Institute of Diabetes and Digestive and Kidney Diseases (B.H., D.B., S.S., N.P., D.M.H.); Radiology Department, Warren Grant Magnuson Clinical Center (Z.N., R.C.); and Surgery Service, Veterinary Resources Program, Office of Research Services (J.B.), Department of Health and Human Services, National Institutes of Health, Bethesda, Md. From the 2002 RSNA scientific assembly. Received October 31, 2002; revision requested January 13, 2003; final revision received May 12; accepted May 20. Address correspondence to M.G.T. (e-mail: michael.tal@yale.edu).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS: First, temporary embolization of the hepatic and gastric arteries was performed in 14 adult nonhuman primates (six cynomolgus, five rhesus, and three pigtail macaques). After embolization was confirmed with angiography, streptozotocin was injected at a dose of 50–70 mg/kg into the celiac artery and branches supplying the pancreas. The macaques then were given intravenous injections of arginine and glucose, and blood levels of insulin and C peptide were measured with an enzyme-linked immunosorbent assay to determine whether diabetes had been induced.
RESULTS: All but one of the macaques developed persistent long-term C peptide–negative diabetes after the streptozotocin injection. One macaque did not develop diabetes after the initial injection and was given a second dose of streoptozotocin, which did induce diabetes. None of the macaques showed any symptoms of hepatic or renal injury, and only one died (of gastric dilatation 5 days after the procedure).
CONCLUSION: Streptozotocin injection after temporary embolization of the hepatic and gastric arteries is a safe and reproducible method for inducing C peptide–negative diabetes in adult nonhuman primates in preparation for preclinical investigation of type 1 diabetes treatments.
© RSNA, 2004
Index terms: Animals • Diabetes mellitus • Experimental study • Pancreas, interventional procedures, 770.1264 • Pancreas, transplantation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The further development of islet transplantation therapy would benefit from a reliable and safe way of inducing insulinopenic diabetes in the adult nonhuman primate model (4,5,8–10). To our knowledge, the spontaneous development of type 1 diabetes mellitus has not been reported in nonhuman primates (11,12). Methods previously used to inducediabetes in nonhuman primates include total pancreatectomy and systemic administration of various doses of streptozotocin (4,5,11,13–24). However, total pancreatectomy is associated with high surgical morbidity and mortality, and the exocrine pancreatic deficiency that is induced with this method results in reduced absorption and inconsistent drug levels. The administration of a low dose of streptozotocin (20–50 mg/kg) is unreliable because it inconsistently induces C peptide–negative diabetes; approximately 20%–50% of primates that have been given this treatment have not become diabetic. The use of higher streptozotocin doses (140–184 mg/kg) has been effective in inducing C peptide–negative diabetes but is associated with major systemic side effects and therefore has generally been limited to juvenile primates (23,24).
Because islets constitute only approximately 2% of the pancreatic cell mass and yet consume 20% of the arterial blood supply (25,26), we hypothesized that diabetes could be induced by creating a high local arterial streptozotocin concentration with the selective injection of the drug into the pancreatic arterial system. Furthermore, because the agent has a relatively short half-life, we hypothesized that intraarterial injection of streptozotocin into the various arteries supplying the head, body, and tail of the pancreas—like local arterial injections of chemotherapeutic agents in the treatment of liver tumors (27)—would prevent the systemic accumulation of toxic concentrations of the drug. We developed and tested the technique in this study for the induction of long-term C peptide–negative diabetes in adult nonhuman primates.
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MATERIALS AND METHODS |
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Animals
Six cynomolgus macaques aged 4–6 years, three pigtail macaques aged 8–10 years, and five rhesus macaques aged 2 years were obtained from the National Institutes of Health primate facility (Poolesville, Md). Serologic testing was performed to ensure that the animals were negative for herpes B, simian retrovirus, simian T-cell leukemia/lymphoma virus, and simian immunodeficiency virus. The animals had a continuous water supply and were fed a regular primate diet supplemented with fresh fruits twice daily.
Diabetes Induction
Vascular access.—Each animal’s abdomen was cleaned, shaved, and prepared in a sterile manner. Two methods of vascular access were used. Initially, ultrasonographic (US) examination of the groin was performed to identify the common femoral vein and artery. If the common femoral artery was deemed large enough (>2 mm in diameter), percutaneous access was attempted with US guidance. Percutaneous access was performed in five primates by using a 5-cm 21-gauge needle. In the other nine primates, surgical exposure of the common femoral artery was performed. All procedures were performed by one of three authors (M.G.T., B.H., Z.N.). After exposure of the common femoral artery, two 2–0 silk sutures were placed under the artery for hemostatic control. The femoral artery was then accessed with a 5-cm 21-gauge needle. When arterial blood flow was identified in the needle, an 0.018-inch floppy guide wire (Cook, Bloomington, Ind) was advanced through the needle and into the aorta. In some cases, passage of the floppy guide wire into the aorta was not feasible and fluoroscopically guided manipulation of an 0.018-inch Glide wire (Cook) was necessary. After the guide wire was properly placed in the aorta, the needle was removed and a 4-F coaxial catheter (Cook) was advanced into the aorta. The inner dilator was removed and contrast material was injected under fluoroscopic guidance to confirm the position of the catheter. A 0.035-inch floppy guide wire (Betson; Cook) was then advanced through the dilator and the dilator was exchanged for a 4-F pigtail catheter (Angiodynamics, Queensbury, NY).
Visceral angiography.—Injection of contrast material into the aorta was performed through the pigtail catheter. The visceral vessels were identified (Fig 1). Then the pigtail catheter was exchanged for either a 4-F angled Glide catheter or a 4-F Rim catheter (Angiodynamics), depending on the anatomic configuration of the celiac trunk. The Rim catheter was trimmed at the midpoint of the secondary curve in the catheter to enable stable positioning in animals with acute downward angulation of the celiac axis. The catheter was selectively placed in the celiac artery, and iohexol 240 mg/mL (Omnipaque; Nycomed, Princeton, NJ) was injected to enable identification of the celiac artery and its splenic, hepatic, and gastric branches (Fig 2).
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After injection of contrast material into the celiac artery, a 3-F microcatheter (Renegade; Boston Scientific, Natick, Mass) was advanced over an 0.018-inch guide wire (Transcend; Boston Scientific) into the hepatic artery and then further advanced beyond the gastroduodenal artery into the proper hepatic artery (Fig 3). After injection of contrast material to confirm correct microcatheter placement, several 1–2-mm clots were injected via a 1-mL syringe. Cessation of flow to the liver was confirmed with another injection of contrast material (Fig 4). The catheter was then advanced to the left gastric artery, and embolization of that artery was performed with the same technique until complete occlusion was achieved.
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Vascular closure.—In the primates in which percutanous access was achieved, homeostasis was achieved by manual compression for 15 minutes. In the primates in which vascular access was gained by surgical cutdown, a 6–0 nylon suture was placed in a circle (purse-string configuration) around the access site in the outer wall of the artery. After the catheter was removed, the suture was tightened to provide vascular closure and to enable uninterrupted blood flow to the lower extremity. The fascial layers were then sutured with 4–0 and 2–0 absorbable sutures. Buprenorphine hydrochloride (Reckit Colman Pharmaceuticals, Richmond, Va) was administered postoperatively by intramuscular injection at a dose of 0.05 mg/kg every 12 hours for postoperative analgesia.
Follow-up after Streptozotocin Injection
Primates were followed up closely for the first 24 hours after injection in order to avoid severe hypoglycemia as a result of beta cell death. Ondansetron (Zofran; GlaxoSmithKline, Philadelphia, Pa) was administered intravenously at a dose of 0.15 mg/kg at 0 and 4 hours after streptozotocin injection. Fluid (100 mL of normal saline solution) also was given intravenously at 0, 4, and 8 hours after the procedure. Repeat injections of 50% dextrose were given as needed to control hypoglycemia. Primates were followed up with weekly blood tests of liver and kidney function to determine any toxic effects on the liver and kidneys.
Assessment of Diabetes Induction
The success of diabetes induction was assessed by means of daily blood glucose measurements. Obliteration of endogenous insulin production was confirmed by periodic measurements of arginine-stimulated C peptide levels and by the results of intravenous glucose tolerance testing. Fasting and postprandial blood glucose levels were monitored daily at 8:00 AM, noon, and 6:00 PM by testing a drop of blood from the primate’s tail with an electronic measurement system (Glucometer Elite; Bayer, Elkhart, Ind).
The C peptide secretory response to intravenous arginine provides an accurate reflection of beta cell mass (29). After an overnight fast, blood samples of 1.0 mL each were collected at 5 and 0 minutes before the intravenous infusion of 2 g arginine (as a 10% solution) into the cephalic vein. Subsequent samples were collected from the contralateral femoral artery at 2, 3, 4, 5, 7, 9, 10, and 15 minutes after the arginine infusion. Restraint for blood sampling and arginine stimulation procedures was achieved with an intramuscular injection of 10 mg ketamine per kilogram of body weight (Fort Dodge Laboratories, Fort Dodge, Iowa). The intravenous glucose tolerance test is an accepted method for assessing insulin resistance, glucose disposition, and islet function (30), and it is employed in the follow-up of patients after islet transplantation (2). After an overnight fast, the primates were anesthetized as described for the arginine stimulation test. Two intravenous lines were placed in the extremities, and 0.3 g of glucose (as 50% dextrose solution) per kilogram of body weight was injected over 30 seconds. Blood samples were taken at 1 minute prior to glucose injection, immediately after injection, and at 1, 3, 5, 10, 15, 20, 30, 60, and 180 minutes after injection (30,31), and insulin and C peptide levels were measured with an enzyme-linked immunosorbent assay (ALPCO Mercodia, Windham, NH).
Diabetic primates were given injections of insulin glargine (Lantus; Aventis, Bridgewater, NJ) at a dose of 2–3 units per kilogram of body weight in the morning and regularly throughout the day as needed.
Of the 14 primates, one died of gastric dilatation 7 days after diabetes induction, seven were allocated to a separate study and underwent islet transplantation via the portal vein, and six were kept in a diabetic state and given insulin for diabetes control.
Postmortem Examination, Collection of Tissue Specimens, and Tissue Processing
Complete necropsies (performed by N.P.) were performed on five macaques (four cynomolgus and one pigtail) 4–12 hours after death. The five selected for necropsy included the one that died of gastric dilatation and four that were euthanized after rejection of subsequent islet transplants. All major organ systems were sampled, including all four liver lobes and the kidneys. The liver lobes were sliced at 0.5–1.0-cm intervals to facilitate complete fixative penetration. Tissues were immersion fixed in 10% buffered formalin with ionized zinc (Z-Fix; Anatech, Springfield, Va) for 5 days and then routinely processed with paraffin infiltration and embedded in paraffin.
Histopathologic and Immunochemical Analyses
Formalin-fixed paraffin-embedded tissue blocks were cut with a rotary microtome into slices 4–6 µm thick, which were placed on positively charged glass slides (Superfrost/Plus; A. Daigger, Wheeling, Ill), dried, and stained with hematoxylin-eosin for histopathologic examination within 3 days. After histopathologic examination, the specimen slides were cleared of hematoxylin-eosin stain, rehydrated with decreasing reagent alcohols and water, flushed with a buffered wash (APK; Ventana Medical Systems, Tucson, Ariz), and immunostained the same day. Immunohistochemical analysis was performed by using an automated system (NEXES; Ventana Medical Systems). All endogenous biotin was blocked (Endogenous Biotin Blocking Kit; Ventana Medical Systems). Primary polyclonal antibodies were incubated 32 minutes (rabbit negative control, Ventana Medical Systems; insulin and somatostatin, Cell Marque, Ventana Medical Systems; and glucagon, 1/3,900 polyclonal rabbit antihuman, DAKO, Carpinteria, Calif). Immunostaining was completed by using a kit that included an endogenous peroxidase inhibitor (Basic DAB Detection Kit; Ventana Medical Systems). Slides were counterstained with hematoxylin; washed; blued with 1% acid alcohol and 1% ammonium hydroxide, in that order; dehydrated with increasing reagent alcohols; cleared with xylene; and sealed with Cytoseal 60 (Microm; Portsmouth, NH). Photomicrographs were generated by using imaging software (Flashpoint; Cary, NC) and video capture.
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RESULTS |
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The primates were followed up weekly for toxic effects on the liver and kidneys, and no abnormalities were detected. Results of repeated arginine stimulation tests and intravenous glucose tolerance tests confirmed the persistence of C peptide–negative diabetes throughout the follow-up period (
10 months after induction). The primates that did not undergo islet transplantation were given regular doses of insulin (2–3 U/kg) to avoid ketosis from persistently brittle diabetes.
Survival
One of the macaques in our study died 7 days after diabetes induction. The cause of death as determined at autopsy was gastric dilatation. This primate’s procedure was unique in one respect. After vascular access was achieved, heparin (50 U/kg) was given intravenously before blood was set aside for autologous clot formation. For that reason, a mixture of gelatin sponge and blood was used to embolize the proper hepatic and gastric arteries. The ischemia induced in the stomach by the resultant clot may have contributed to gastric dilatation and the animal’s subsequent death. This primate, however, had normal liver and renal function after diabetes induction, and blood glucose was well controlled with insulin.
Seven diabetic primates received insulin therapy for approximately 6 months before being allocated to different islet transplantation studies. These primates were given pancreatic islets via the portal vein. Four of the seven subsequently rejected the islets and were euthanized and designated for necropsy. In the other three primates, diabetes was reversed by the islet transplantation. The six other primates that did not undergo islet transplantation had ongoing diabetes that was controlled with insulin.
Histopathologic Findings
Full immunochemical and histopathologic analyses were performed in tissues from five primates. Pancreatic immunostaining revealed intact alpha and delta cells but near complete obliteration of beta cells (Fig 6). We detected no histologic evidence of injury to the liver or kidneys.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Investigators in prior studies also used streptozotocin to induce diabetes in adult primates, with various results (12). In these studies, streptozotocin was administered intravenously via a peripheral vein at a dose of 30–60 mg per kilogram of body weight. A resultant permanent diabetic state (although not always C peptide negative) was reported in 50%–80% of primates. Even the use of high doses (144–184 mg/kg) of peripherally administered streptozotocin did not always result in C peptide–negative diabetes (24). Furthermore, the peripheral intravenous administration of a high dose of streptozotocin is usually reserved for juvenile macaques because older primates may not tolerate the procedure (24).
Because streptozotocin not only destroys pancreatic beta cells but also has toxic effects on the kidneys and liver, in our study a high dose of streptozotocin was injected selectively into the arteries (celiac, gastroduodenal, and splenic) that supply the pancreas, while the pancreatic blood supply was isolated to decrease the exposure of the liver to streptozotocin. Embolization of the proper hepatic artery allowed the maintenance of blood flow and streptozotocin infusion into the gastroduodenal artery and the proximal branches of the celiac artery. We postulated that high streptozotocin concentrations might damage the gastric mucosa, particularly the endocrine cells in the gastric wall. Therefore, the gastric artery was embolized in order to prevent streptozotocin from flowing in high concentration to the stomach.
Splenic arterial angiograms clearly depicted the continuous flow of blood from the smallest and most distal branches of the splenic artery to the pancreatic tail. If we had embolized the splenic artery, part of the pancreatic tail would have been isolated from streptozotocin. In view of this fact, and given that the spleen has no known endocrine role that can be adversely affected by high streptozotocin concentration, we decided not to embolize the splenic artery. In so doing, we realized that some streptozotocin would be infused into the spleen and thus wasted; nevertheless, this method would ensure that the distal pancreatic tail would be exposed to streptozotocin.
Practical application: The reported method for inducing C peptide–negative diabetes in adult nonhuman primates by using interventional radiology techniques is both safe and reproducible. Investigators at large animal centers with access to interventional radiology equipment may find the model developed in this study useful in preclinical testing of diabetes treatments.
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FOOTNOTES |
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