Gerrit H. J. Wolters

The capsular overgrowth on microencapsulated pancreatic islet grafts in streptozotocin and autoimmune diabetic rats

Abstracts This study investigates whether capsular overgrowth on alginate‐polylysine microencapsulated islets is influenced by (1) the presence of islet tissue, (2) MHC incompatibility between donor and recipient, or (3) the presence of autoimmune diabetes. Encapsulated Albino Oxford (AO, n= 6, isografts) and Lewis (n= 6, allografts) rat islets, and encapsulated human islets (n= 5, xenografts) were implanted intraperitoneally into streptozotocin‐dia‐betic AO rats. Also, encapsulated AO islets were implanted into autoimmune diabetic Bio Breeding/Organon (BB/O) rats (n= 5, allografts). Five isografts, five allografts, and three xenografts in AO recipients and five allografts in BB/O recipients resulted in normoglycemia. Two weeks after implantation, islets containing capsules were retrieved by peritoneal lavage, after which all animals that had become normoglycemic after transplantation returned to a state of hyperglycemia. Recovery rates of the capsules of these successful grafts, expressed as percentages of the initially implanted graft volume, varied from 72%± 7% to 80%± 9%. The associated pericapsular infiltrates (PCI) were similar in all groups and varied from 3.2%± 1.4% to 8.3%+ 2.6% Similar recovery rates and PCI were also found with empty capsules. However, the recovery rates of recipients with graft failures were lower and showed more PCI. Immunohistological staining of PCI showed no differences in the types of cells in the PCI on capsules with or without islets. We conclude that this early PCI is a capsule‐induced foreign body reaction that is not influenced by MHC incompatibility or by the presence of autoimmune diabetes, and it should be avoided by improving the biocompatibility of the capsules.

Different Roles of Class I and Class II Clostridium Histolyticum Collagenase in Rat Pancreatic Islet Isolation

Crude Clostridium histolyticum collagenase was purified by gel filtration and fractionated by anion exchange chromatography into class I with high collagen digestion activity (CDA) and low FALGPA (2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine) hydrolysis activity (FHA), class II with low CDA and high FHA, and a fraction called class WI with intermediate activities. The roles of these collagenase classes in rat pancreatic islet isolation were investigated. Dissociations were carried out with 360 mg of pancreatic tissue in 10 ml of buffer containing 10% (wt/vol) albumin to suppress endogenous proteolytic activity, 100 U of C. histolyticum neutral protease, and one or two purified collagenase(s). For purified nonfraction-ated (PNF) collagenase, 2.6 mg of enzyme containing 2.4 U CDA and 38.0 U FHA was used, and for the separate classes, comparable amounts of activity were added. PNF collagenase dissociated the tissue completely in 32 min and yielded 5.0 ± 0.4 μl islet tissue/g pancreas. Class I collagenase alone dissociated pancreatic tissue extremely slowly and incompletely; only a few islets were released (0.7 ± 0.2 μl/g pancreas). Class II collagenase alone dissociated the tissue adequately in 50 min, and a high islet yield of 5.7 ± 0.6 μl/g was obtained. With class I/II, a similar dissociation time (47 min) and islet yield (5.5 ± 0.3 μl/g) were obtained. Combining class I and class II collagenase resulted in a more rapid dissociation (32 min) and a higher islet yield (7.1 ± 0.8 μl/g) than that obtained with PNF collagenase (P < 0.05). Similar results were obtained when purified class I collagenase was replaced by recombinant class I collagenase (32 min, islet yield 6.1 ± 0.3 μl/g). We conclude that class U collagenase plays a major role in pancreas dissociation and islet isolation and that class I collagenase acts synergistically with class II collagenase.

The Efficacy of Intraperitoneal Pancreatic Islet Isografts in the Reversal of Diabetes in Rats

The peritoneal cavity is of renewed interest for pancreatic islet transplantation, since it is the preferable site for transplantation of immunoisolated islets. In this study we investigated the minimum islet graft volume needed to restore normoglycemia after free intraperitoneal isogenic transplantation in streptozotocin diabetic rats. Furthermore, graft function was tested by measuring glucose and insulin response to an intravenous glucose load and spontaneously ingested carbohydrate-rich meal. Three graft volumes were used: 8.0–10.0 (group A); 4.0–5.0 (group B); and 2.0–2.3 μl (group C); 1 μl contained about 300 islets. All 10 rats in group A and 7 out of 9 rats in group B became normoglycemic for at least 6 months posttransplant, with blood glucose levels not significantly different from normal control animals. Only 3 out of 9 animals in group C became

Rat Islet Isograft Function. Effect of Graft Volume and Transplantation Site

Islet isograft function was analyzed after transplantation of 4 well-defined endocrine volumes (12.5%, 25%, 50%, and 100% of the endocrine volume in the normal adult rat pancreas) to 3 different sites (kidney, liver, and spleen). Graft function was tested in unanesthetized, unstressed rats by the responses to glucose infusion and to a meal. All animals with grafts > or = 25% of the endocrine volume of the rat pancreas returned to normoglycemia after transplantation. The minimal graft volume for restoring normoglycemia is probably between 12.5% and 25%, since also a small number of grafts of 12.5% were successful. At 1 month, basal glucose and insulin levels were similar to those of controls in rats with grafts to the spleen, but higher in rats with grafts to the kidney or liver. Irrespective of the transplantation site, recipients had higher glucose and lower insulin levels than controls in response to glucose infusion. In response to a meal, however, only the first-phase insulin response was reduced, but the total insulin output during the entire test was similar to that of controls. Graft performance was found to be graft-size dependent. Results of tests performed at 2 months showed a tendency of increasing responsiveness compared with the results of tests at 1 month.

Histochemical analysis of the role of class I and class II Clostridium histolyticum collagenase in the degradation of rat pancreatic extracellular matrix for islet isolation

To understand why class II Clostridium histolyticum collagenase is much more effective than class I in the isolation of rat pancreatic islets, we analyzed the role of these collagenases in pancreatic tissue dissociation. Crude collagenase was purified and then fractionated into class I and II with different enzyme activities and protein compositions. Pancreatic tissue was incubated with either class I, class II, or class I + II, with or without added protease, under conditions that eliminated endogenous proteolytic activity. The degradation of pancreatic extracellular matrix was monitored by selective histochemical staining of tissue samples. Class I and II showed similar capacities to degrade glycoproteins and degraded about one-third of the glycoproteins during 120 min of incubation. The degradation of collagens by class I and II was relatively more effective, 80 to 95% of the collagens being removed in 120 min, and also class dependent. Both in the presence and absence of protease, class II was more effective at degrading collagens than class I, but this difference in efficacy was less apparent than with islet isolation. Class I + II degraded collagens faster and more complete than did the individual classes, indicating a synergistic effect of class I and II. Evaluation of collagen degradation at various pancreatic locations did not show a selective degradation of collagens by any of the collagenase classes. The present data offer a partial explanation for the major role of class II in islet isolation.

Peri-insular presence of collagenase during islet isolation procedures

Reportedly, higher islet yields are obtained by ductal collagenase administration and subsequent digestion of the pancreas than by the chopped tissue collagenase digestion technique. However, the exact mechanism of islet isolation is not known. This study aims to understand the underlying mechanism of a favorable effect of ductal collagenase administration. To this end, we investigated if the higher yields can be explained by a different distribution of the collagenase enzymes in the pancreatic tissue after ductal application as compared to during chopped tissue digestion. India ink was used to mimic and visualize the distribution of collagenase in histological sections of pancreases of several species. Ink particles were seen around and even within the islets both after ductal application and during chopped tissue collagenase digestion. Thus, collagenase enzymes are not restricted to the exocrine tissue compartment with either technique. In view of this observation, we compared the efficacy of both techniques in islet isolation procedures in paired experiments in rats. Both techniques gave similar islet yields to those reportedly obtained with the ductal collagenase method. However, with either technique, the islet yield was only approximately 50% of the endocrine volume of the pancreas, indicating that a substantial loss of islet tissue had occurred. We conclude that, irrespective of the route of collagenase administration, collagenase enzymes are present in the peri-insular space during islet isolation procedures. This is pertinent in view of the finding that both methods have similar islet yields in rats and that collagenase digestion, as such, is associated with loss of islet tissue.

Calcium, zinc and other elements in islet and exocrine tissue of the rat pancreas as measured by histochemical methods and electron-probe micro-analysis, Effects of fasting and tolbutamide.

SummaryFasting for 24 or 72 h causes a strong decrease of pancreatic islet calcium content as detected by glyoxal-bis-(2-hydroxyanil), (GBHA). There is strong evidence that GBHA only detects ionized calcium and not total calcium (Wolters et al., 1979). Fasting does not influence the zinc content as detected by dithizone (DZN), and aldehyde-fuchsin (AF) staining intensity is only slightly decreased. After degranulation of islets by tolbutamide (which reduced the insulin content of the pancreas to 10% of the control value) the staining intensities of GBHA, DZN and AF were strongly depressed.Calcium (as well as other elements) were also measured by electron-probe micro-analysis (EPMA). It appeared that 24 or 72 h of fasting did neither affect the total content of Ca nor of Na, P, S, and K of the islets significantly. In exocrine tissue the Ca content increased gradually as a result of fasting. Thus, after 72 h of fasting the Ca content was 25% higher than in fed controls. On the other hand after 72 h of fasting the K content appeared to be decreased. EPMA revealed that after degranulation of islets the Ca content decreased markedly (35%). S appeared to be decreased by only 14%, whereas the content of the other elements was not changed.The results show that GBHA-detectable Ca is only a part of EPMA-detectable Ca. The GBHA-Ca “pool” which contains ionized Ca, is subjected to changes when the animals are fasted, the total Ca content as measured by EPMA does not change. Thus, at least two distinguishable pools of Ca exist within the islets (GBHA-detectable and not-GBHA-detectable). It is suggested that as a result of fasting Ca passes from one pool to another.

Evaluation of the glyoxal-bis-(2-hydroxyanil)-method for staining of calcium in model gelatin films and pancreatic islets

SummaryThe nature of tissue calcium, detectable with glyoxal-bis-(2-hydroxyanil), (GBHA), was investigated using gelatin films as model. The results indicate that in the films the procedure detects only the calcium fraction which was ionized in the original gelatin solution. The GBHA staining intensity (absorbance) appeared to be linear with the amount of ionized calcium in the range from 0 to 2 μg/cm2. The method allows detection of amounts of ionized calcium as low as 0.15 μg/cm2 or 0.0015 pg/μ2.For the measurement of calcium in pancreatic tissue of fed rats, the tissue was subjected to freeze-substitution at −80°C in acetone containing 1% oxalic acid. Adjacent sections were stained with either GBHA or aldehyde-fuchsin (AF). Exocrine tissue hardly stained with GBHA whereas islet tissue stained intensely. For GBHA as well as for AF a variation in staining intensity (visual evaluation) between islets was observed. Islet GBHA- and AF-staining intensities did not correlate. The AF-staining intensity but not the GBHA-staining intensity decreased with increasing islet diameter. Also in pancreatic islet tissue the GBHA method appears to be very sensitive and reproducible and small differences in islet GBHA-staining intensity can be detected. The results indicate that between islets differences in ionized calcium content exist. These differences do not correlate with the degree of B-cell granulation.

Effects of calcium manipulation and glucose stimulation on a histochemically detectable mobile calcium fraction in isolated rat pancreatic islets

SummaryPancreatic B-cell calcium as histochemically detectable with glyoxal bis (2-hydroxyanil)=GBHA was studied in isolated islets of fed rats. GBHA has previously been shown by us to detect an ionized or readily ionizable Ca-fraction (GBHA-Ca). In the presence of Ca++ (2.5 mM), high glucose (15 mM) induced a rapid decrease (30%) of islet GBHA-Ca followed by a rise between 30 and 60 min to levels above the initial value. At low glucose (0 or 2.5 mM) GBHA-Ca showed a slight and gradual decline under these conditions. Omission of Ca++ at low glucose rapidly decreased (30%) islet GBHA-Ca. This decrease was markedly inhibited by high glucose, although glucose did not induce insulin secretion under these conditions. Preincubation in the absence of Ca++ (15 min) depleted islet GBHA-Ca, but partial restoration occurred during subsequent incubation with Ca++ at low glucose. By contrast, high glucose completely restored GBHA-Ca within 5 min, followed by a decline and a subsequent rise. Reintroduction of Ca++ also rapidly restored the glucose-induced insulin secretion. These results indicate that islet GBHA-Ca represents a mobile Ca-fraction which is dependent on extracellular Ca++ and which responds very rapidly to glucose stimulation. It is suggested that changes of GBHA-Ca in the B-cells may reflect changes in the Ca pool involved in the insulin secretory mechanism.

Effects of thioacetamide on pancreatic islet B-cell function

Thioacetamide (0.01–1.3 mM) fails to exert any significant immediate effect upon insulin release from rat isolated islets. However, when administered (4 µmol/g body wt) intraperitoneally 24 h before sacrifice, it reduced food intake and body weight and affected the secretory response of isolated islets to several secretagogues, despite unaltered insulin content of such islets. This coincided with a decrease in d-[U-14C]glucose oxidation, total islet calcium content and the ionized calcium content of secretory granules in islet B-cells, and changes in both 133Ba and 45Ca net uptake. Likewise, in islets prepared from thioacetamide-injected rats and prelabeled with 45Ca before perifusion, the cationic and insulin secretory responses to d-glucose or gliclazide, but not to the association of Ba2+ and theophylline in the absence of extracellular Ca2+, often differed from that otherwise found in islets prepared from control rats. These findings are interpreted as indicative of an impaired capacity of Ca2+ sequestration by intracellular organelles in the islet B-cells of thioacetamide-treated rats.