Pancreatic islet allograft prevents basement membrane thickening in the diabetic rat retina

Diabetologia, Mar 1988

Summary Progressive basement membrane thickening is a characteristic structural abnormality in diabetic tissues in cluding the retina. We examined the effect of pancreatic islet allotransplantation on basement membrane thickening and irregularities in retinal capillaries of the streptozotocin-diabetic rat. Diabetic animals received intraportal or intracerebral pancreatic islet allografts. Animals with functioning allografts demonstrated euglycaemia and a normal body weight gain during the 400-day post-transplantation period. The characteristic thickening of capillary basement membranes was completely prevented in animals with successful transplantation. The present findings suggest that islet allotransplantation may be a rational therapeutic approach in the treatment of diabetes mellitus and the prevention of ensuing secondary complications.

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Pancreatic islet allograft prevents basement membrane thickening in the diabetic rat retina

Diabetologia Pancreatic islet aUograft prevents basement membrane thickening in the diabetic rat retina A. A. F. S i m a 0 1 S. C h a k r a b a r t i 0 1 W.J. Tze I a n d J. Yai 0 1 0 Neuropathology Research Laboratories, Department of Pathology, University of Manitoba , Winnipeg, Manitoba , and zDepartment of Endocrinology, University of British Columbia , Vancouver, British Columbia , Canada 1 Dr. A. A. F. Sima Neuropathology Research Laboratories University of Manitoba 770 Bannatyne Avenue Winnipeg , MB, R3E OW3 Canada Summary. Progressive basement membrane thickening is a characteristic structural abnormality in diabetic tissues including the retina. We examined the effect of pancreatic islet allotransplantation on basement membrane thickening and irregularities in retinal capillaries of the streptozotocin-diabetic rat. Diabetic animals received intraportal or intracerebral pancreatic islet allografts. Animals with functioning allografts demonstrated euglycaemia and a normal body weight characteristic thickening of capillary basement membranes was completely prevented in animals with successful transplantation. The present findings suggest that islet allotransplantation may be a rational therapeutic approach in the treatment of diabetes mellitus and the prevention of ensuing gain during the 400-day post-transplantation period. The P a n c r e a t i c islet cell t r a n s p l a n t a t i o n is a r a t i o n a l app r o a c h to the t r e a t m e n t o f d i a b e t e s mellitus a n d its c o m p l i c a t i o n s , since it h a s b e e n s u g g e s t e d t h a t h y p e r - g l y c a e m i a c o n d i t i o n s the d e v e l o p m e n t o f s e c o n d a r y H e n c e , i f a p h y s i o l o g i c a l g l u c o s e h o m e o s t a s i s c a n b e Islet cell transplantation; streptozotocin-diabetic 9 Springer-Verlag1988 a c h i e v e d b y m e a n s o f p a n c r e a t i c islet t r a n s p l a n t a t i o n the s e c o n d a r y c o m p l i c a t i o n s m a y b e p r e v e n t e d or halted [ 2 ]. T h i c k e n i n g o f c a p i l l a r y b a s e m e n t m e m b r a n e s is a p r o m i n e n t a b n o r m a l i t y in the target o r g a n s p r o n e to d i a b e t i c c o m p l i c a t i o n s , including the retina. A l t h o u g h i m p r o v e m e n t o f the b l o o d retinal b a r r i e r p e r m e a b i l i t y [ 3 ], n o r m a l i s a t i o n o f the p e r i c y t e - e n d o t h e l i a l cell ratio [ 4 ], a n d p r e v e n t i o n o f retinal n e o v a s c u l a r i s a t i o n [ 5, 6 ] h a v e b e e n c l a i m e d following p a n c r e a s o r islet cell t r a n s p l a n t a t i o n , n o r e p o r t is a v a i l a b l e w i t h r e g a r d to its effect o n retinal c a p i l l a r y b a s e m e n t m e m b r a n e . H o w ever, the o c c u r r e n c e o f n e o v a s c u l a r i s a t i o n in the diab e t i c rat retina has b e e n d i s p u t e d [ 7 ]. P r e v i o u s o b s e r v a tions h a v e b e e n m a d e either following isografts [ 3, 4 ] o r allograft t r a n s p l a n t a t i o n across a w e a k h i s t o c o m p a t i b ility b a r r i e r [5]. I n this c o m m u n i c a t i o n , we r e p o r t o n retinal b a s e m e n t m e m b r a n e t h i c k e n i n g the s t r e p t o z o t o c i n ( S T Z ) - d i a b e t i c rat following islet cell t r a n s p l a n t a t i o n across a m a j o r h i s t o c o m p a t i b i l i t y barrier. Material and methods Animals Twenty-nine inbred male Lewis rats of the AC1 (AgB 4/4) strain were used. Twenty-four rats were made diabetic at the age of 4 months by intravenous injection of STZ (50 mg/kg body weight). Diabetes was defined by fasting blood glucose levels greater than 22 retool/1 on three successive occasions. Pancreatic islet cell transplantation was carried out in 19 rats 12.8+ 3.4 days (mean + SD) following induction of diabetes. Transplantation procedure Male inbred Lewis rats (Le; Ag B1/I) with a body weight of 350-500 g were used as donors of pancreatic tissue. The donor animals were anaesthetised with sodium pentobarbital (50 mg/kg of body weight) (Abbott Laboratories, Montreal, Quebec, Canada). A midline laparotomy was made and the pancreas was dissected. Pancreatic tissue was digested with collagenase and islets were hand picked under a dissecting microscope. Contaminating acinal tissue and blood vessels were removed using the single layer Hypaque-Ficoll separation technique. Clean islets collected at the interface were either cultured at 26 ~ in 5% CO2 for 1 week prior to transplantation or immediately dissociated into single pancreatic endocrine cell suspensions with a combination of EDTA and trypsin treatment. The preparation of pancreatic pseudo-islets was carried out by culturing pancreatic endocrine cells at 37 ~ in 5% CO2 for 5 days to allow cellular aggregation to occur [ 8 ]. For intraportal allotransplantation a total of 1500 pancreatic whole islets or pancreatic pseudo islets suspended in 200 !*1culture medium were injected slowly into the portal vein of t I STZ-induced diabetic AC1 recipients. For intracerebral transplantation, 1500 panGroups of rats 1 Intraportal rejected 2 Intracerebral rejected 3 Intraportal accepted 4 Intracerebral accepted 5 Diabetic non-transplanted 6 Controls A.A.F. Sima et al. : Basement membrane following islet transplantation creatic whole islets or pancreatic endocrine cells (2-3 x 106 viable cells) suspended in a 50 `al culture medium were implanted stereotaxically into the fight hemisphere of the brain of eight diabetic recipients. Blood glucose, body weight, 24-h urine volume and urine sugar were monitored daily prior to and for two weeks after allotransplantation and at regular intervals thereafter. Acceptance of allografts was defined by the presence of normoglycaemia and normal weight gain in the diabetic recipients as previously described in detail [ 9 ]. The islet allografts of five intraportally and four intracerebrally transplanted animals were challenged with skin grafts from the same donor strain 7 to 10 days following transplantation. Rejection was considered to have occurred when the blood glucose level exceeded 11 mmol/l on two consecutive days following skin grafting. Clinical monitoring All animals were housed individually in air-filtered metabolic cages and given rat-chow (Wayne Lab Blox F-6, Wayne Feed Division, Chicago, Ill, USA), and water ad libitum. Diabetic animals were monitored bi-weekly with respect to blood-sugar and body-weight. All animals were killed at 18 months of age and the following groups of animals were investigated; 1) animals with intraportal transplant (rejected) (n = 5); 2) intracerebral transplant (rejected) n = 4; 3) intraportal transplant (accepted) (n =6); 4) intracerebral transplant (accepted) (n = 4); 5) diabetic non-transplanted (n = 5), and 6) non-diabetic control rats (n = 5). All animals were anaesthetised by intraperitoneal injection of sodium pentobarbital (50 mg/kg body-weight) (Abbott Laboratories, Montreal, Quebec, Canada), and killed by whole body perfusion through the left ventricle with a 0.1 mol/1 cacodylate buffered (pH 7.4) 2.5% glutaraldehyde solution. Morphological techniques Both eyes were enucleated and post-fixed in the same glutaraldehyde fixative for 4 to 6 h at 4 ~ Qualitative and quantitative light and electron microscopic studies were performed using the right eye of each animal. The examiner was not aware of the identity of the tissues. Qualitative studies After fixation, radially oriented retinal segments (1 mm 2) were taken from the superior temporal quadrant near the optic nerve head. They were washed overnight in 0.1 tool/1 cacodylate buffer (pH 7.4). The tissues were post-fixed in cacodylate buffered 1% osmium-tetroxide (pH 7.4) for 2 h, dehydrated in graded alcohol and embedded in Epon. Semithin, toluidine blue stained sections (0.5 am) were used for light microscopic orientation. Ultrathin sections were stained with aqueous uranyl acetate and lead citrate, and examined electronmicroscopically. Quantitative structural studies Morphometric studies were performed with the aid of a HewlettPackard 9874A digitizer connected with a Hewlett-Packard 9825A desktop computer (Hewlett-Packard Co, Fort Collins, Col, USA). Basement membrane thickness (BMT) was measured according to a modification [ 10 ] of the technique described originally by Robison et al. [ 11 ]. Twenty randomly selected and transversly sectioned capillaries were photographed from each animal, 10 from the deep capillary bed, located in the outer plexiform layer and inner nuclear layer, and 10 from the superficial capillary bed, located in the nerve fibre layer and ganglion cell layer. Measurements were made from electronmicrographs enlarged to a final magnification of 16,000 times. Basement membrane area (BMA) and basement membrane length (BML) were determined as follows: BMA = T - (L + P + E); where T = total capillary area, L = lumenal area, P = pericyte profile area and E = endothelial cell profile area. B M L = length of lines delimiting the BM 2 The relative thickness of the basement membrane was expressed as area per unit length BMT (nm)= [BMA (`am2)/BML (`am)] x 1000. To ascertain that the capillaries were cross-sectioned, only those with sharp circumferential delineations of the basement membrane borders were used for the measurements. The same electronmicrographs were used to examine qualitative basement membrane abnormalities. Statistical analysis All values are expressed as mean___SD. One way analysis of variance and Student's t-test were used to determine significance of difference. A p value less than 0.05 was accepted as significant. Results Clinical observations S u c c e s s f u l i s l e t a l l o t r a n s p l a n t a t i o n r e s u l t e d i n n o r m a l i s a t i o n o f t h e b l o o d s u g a r l e v e l s i n p r e v i o u s l y d i a b e t i c animals (Fig. 1), whereas animals in w h o m allograft rejection was induced showed a return to significant hyperglycaemia (Fig.l). The characteristic decrease in body weight gain in diabetic rats was ameliorated by successful islet transplantation, whereas animals with rejected allografts showed a body weight similar to that of non-transplanted diabetic rats (Fig. 2). Quafitative basement membrane changes Basement membrane irregularities were seen in all animals except for non-diabetic control rats. These changes, although not quantified, appeared to be more common in rats with rejected grafts and in diabetic non-transplanted animals. The irregularities consisted of localised nodular thickenings and the deposition of 3oo1 oTI TI '~1176 I - 2 0 0 "iQ3 w s IW0 w W~ tO~l T (3 I.U ~OIZOe~ 0 IW-a. T WO Oe~ G .2 GR.3 GR.4 GROUPS OF RATS Basement membrane thickness (BMT) A l l a n i m a l s e x a m i n e d s h o w e d t h i c k e r b a s e m e n t m e m b r a n e s in t h e s u p e r f i c i a l c a p i l l a r i e s t h a n in t h e c a p i l l a ties of the deep bed (Table 1). Diabetic non-transplanted animals showed a significant increase in BMT in both capillary beds compared with normal control rats (p < 0.001). This was also the case in transplanted animals with rejected grafts. In contrast, both intraportal and intracerebral successful allotransplantation result Discussion ed in a complete prevention of capillary basement membrane thickening in both vascular beds of the retina (Table 1, Fig. 4). The present study has demonstrated that pancreatic islet cell allotransplantation successfully prevents basement membrane thickening in the retina of previously STZ-diabetic rats. Two simple parameters reflecting good metabolic control in diabetes mellitus are b o d y weight and the degree of hyperglycaemia. Both the characteristic body weight loss and hyperglycaemia were found to be completely normalised in animals with successful allotransplantation, while, as would be expected, unsuccessful transplantation did not show the same effects. These findings are in keeping with those of other investigators who have described improvement in body weight and a reduction of hyperglycaemia after successful pancreatic islet transplantation in STZ-diabetic rats [ 12, 13 ]. The mechanisms responsible for basement membrane thickening in diabetes are as yet not completely elucidated. Enzymatic glycation of hydroxylysine residues of collagens by glucose and galactose [ 14 ], and non-enzymatic glycation of lysine residues by the same sugars at the exposed 8-NH2 groups have been postulated [ 15 ]. Other suggested possibilities include increased synthesis of collagen or other basement membrane proteins such as laminin or fibronectin, either as a primary event or as a compensatory mechanism secondary to decreased production of basement membrane proteoglycan [ 16 ]. Increased polyol pathway activity secondary to high ambient glucose levels may play a significant role, since it has been shown that galactosaemia-induced capillary basement membrane thickening can be prevented by aldose reductase inhibitors [ 11, 17 ]. It is, however, not clear as to how an activated polyol pathway may effect the basement membrane. Thickening of capillary basement membranes in diabetes may be associated with endothelial cell damage [ 18, 19 ]. Repeated endothelial cell death and regeneration with retention of old and the addition of new layers of basement membrane may be a factor in the production of basement membrane duplication and thickening [20]. We failed to demonstrate endothelial cell abnormalities in the present study, whereas we have previously described endothelial cell degeneration in the diabetic BB-rat [ 21 ]. Although no report on retinal capillary BMT following pancreatic or islet transplantation is available, the prevention of glomerular basement membrane thickening has been reported following these procedures [ 22 ]. Previous studies in the spontaneously diabetic BB-rat and in Sprague-Dawley rats have demonA.A. F. Sima et al.: Basement membrane following islet transplantation strated that aging also causes an increase in the BMT [ 21, 23 ]. Since all animals used in this study were agematched, aging as a causative factor of basement membrane thickening in diabetic rats and those with rejected grafts can be eliminated. The remote possibility that streptozotocin per se, might have been responsible for basement membrane thickening in diabetic rats, is eliminated by the fact that successfully transplanted, previously STZ-treated rats did not develop basement membrane thickening. In the current study, increased B M T was seen, both in the superficial and deep vascular beds in animals with hyperglycaemia and was prevented in both capillary beds in animals with successful transplantation. A normally thicker basement membrane in the superficial vascular bed of the retina has been noted in both normal and diabetic rodents [ 21, 23, 24 ]. A gradient in the intravascular pressure over the two capillary systems may be one explanation for these differences [25]. Localised nodular thickening, vacuolation, and deposition of fibrillar material in basement membrane may represent degenerated and entrapped pericyte debris, so-called ghost cells [ 26 ]. The finding of projections of basement membrane materials between surrounding glial cells in diabetes have been noted by other investigators [ 24 ]. It has been suggested that this redundant basement membrane is laid d o w n as a result of disturbed Mtiller cell metabolism, which in itself may be a factor responsible for increased BMT in diabetes [29]. The result of this study has demonstrated that successful pancreatic islet cell allotransplantation prevents basement membrane thickening of retinal capillaries in STZ-diabetic rats. These observations raise hope that successful, pancreatic islet allograft may provide a physiological approach to the management of diabetes and its complications. Acknowledgements.The present study was supported by grants from the Medical Research Council of Canada and the Canadian National Institute for the Blind. 1. Frank RN ( 1984 ) On the pathogenesis of diabetic retinopathy . Ophthalmology 91 : 626 - 634 2. Sutherland DEF , Goetz FC , Ramsay RC , Fryd DS , Najarian JS ( 1983 ) Clinical and experimental kidney and pancreas transplantation . In: Little HL , Jack RL , Patz A , Forsham PH (eds) Diabetic retinopathy . Thieme-Straton, New York, pp 199 - 221 3. Krupin T , Waltman SR , Scharp DW , Oestrich C , Fieldman SD , Becker B , Ballinger WF , Lacy PE ( 1979 ) Ocular fluorophotometry in the streptozotocin diabetes mellitus in the rat: effect of pancreatic islet isograft . Invest Ophthalmol Vis Sci 18 : 1185 - 1190 4. Naeser P , Anderson A ( 1983 ) Effect of pancreatic islet implantation on the morphology of retinal capillaries in alloxan diabetic mice . Acta Ophthalmol (Copenh) 61 : 38 - 44 5. 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Diabetes 30 : 326 -334 Received: 29 July 1987 and in revised form: 31 December 1987


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A. A. F. Sima, S. Chakrabarti, W. J. Tze, J. Tai. Pancreatic islet allograft prevents basement membrane thickening in the diabetic rat retina, Diabetologia, 1988, 175-181, DOI: 10.1007/BF00276852