Effect of the silk protein sericin on cryopreserved rat islets

Background/purpose

Cryopreservation is necessary for the long-term storage of islet cells and to increase the practicality of clinical islet transplantation. Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal health problems, such as bovine spongiform encephalopathy and viral infections. The aim of this study was to develop a new serum-free freezing medium by examining the effectiveness of the silk protein sericin, which is produced by Bombyx mori.

Methods

Islets prepared from Lewis rats by collagenase digestion and Histopaque gradient centrifugation, followed by culture in medium containing 0.1% sericin for 3?days, were cryopreserved using 0.1, 0.5, 1, 2, and 5% sericin or FBS. DMSO (1, 4, 7, 10, and 15%) was added to the medium as a cryoprotectant. After thawing, on days 1, 4, 7, and 14, viable islets were counted in order to evaluate their survival. Insulin secretion was measured in vitro by a static incubation test on day 4. The in vivo function of cultured islets was tested by syngeneic transplantation. Islets were evaluated histologically and immunohistochemically after transplantation.

Results

There were no significant differences between freezing medium containing 1% sericin and that containing 10% FBS with regard to the survival rate of islets and stimulated insulin secretion. Following transplantation, islets rapidly reversed hyperglycemia and maintained normal glycemic control. In addition, the use of 7% DMSO as a cryoprotectant with sericin showed the same results as higher DMSO concentrations with FBS.

Conclusion

The present results showed that serum-free medium containing sericin is useful for both cryopreservation and cell culture.     

The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells

Aims/hypothesis

The Edmonton Protocol for islet transplantation has provided hope for type 1 diabetic patients. However, this protocol requires lifelong immunosuppression, specifically sirolimus, a cellular antiproliferate. The effect of sirolimus on human pancreatic ductal cells (HDCs) is not known. This may be important since HDCs are believed to be islet precursors. Since neonatal porcine islets (NPIs), which contain many ductal precursor cells, could be a potential clinical source of islets, we also tested the effects of sirolimus on this tissue.

Methods

HDCs (n=4), NPIs (n=9) and human islets (n=5) were cultured with and without sirolimus (20 ng/ml) for 6 days.

Results

HDCs and NPIs cultured with sirolimus showed a 50 and 28% decrease, respectively, in cell number relative to control (p<0.05). Control cultures expanded 1.65- and 2.44-fold relative to time 0. Decreases in cell number of sirolimus-treated HDCs were not due to apoptosis as measured by TUNEL staining. No functional effects on human islets or NPIs were observed following static incubation with high glucose. Treatment of syngeneically transplanted and naïve BALC/c mice with sirolimus resulted in altered OGTT profiles with prolonged elevation of hyperglycaemia and weight gain. There was no difference in graft and organ insulin content between treatment groups.

Conclusions/interpretation

Our results indicate that sirolimus decreases ductal cell numbers in culture and alters glucose-stimulated insulin secretion in vivo. The administration of sirolimus to islet transplant recipients is likely to impair graft function as a result of decreasing ductal neogenesis and induction of insulin resistance.     

The glucagon-like peptide-1 receptor agonist exenatide restores impaired pro-islet amyloid polypeptide processing in cultured human islets: implications in type 2 diabetes and islet transplantation

Aims/hypothesis

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), is associated with beta cell death in type 2 diabetes as well as in cultured and transplanted human islets. Impaired prohIAPP processing due to beta cell dysfunction is implicated in hIAPP aggregation. We examined whether the glucagon-like peptide-1 receptor (GLP-1R) agonist exenatide can restore impaired prohIAPP processing and reduce hIAPP aggregation in cultured human islets and preserve beta cell function/mass during culture conditions used in clinical islet transplantation.

Methods

Isolated human islets (n?=?10 donors) were cultured with or without exenatide in normal or elevated glucose for 2 or 7 days. Beta cell apoptosis, proliferation, mass, function, cJUN N-terminal kinase (JNK) and protein kinase B (PKB) activation and amyloid formation were assessed. ProhIAPP, its intermediates and mature hIAPP were detected.

Results

Exenatide-treated islets had markedly lower JNK and caspase-3 activation and beta cell apoptosis, resulting in higher beta/alpha cell ratio and beta cell area than non-treated cultured islets. Exenatide improved beta cell function, manifested as higher insulin response to glucose and insulin content, compared with non-treated cultured islets. Phospho-PKB immunoreactivity was detectable in exenatide-treated but not untreated cultured islets. Islet culture caused impaired prohIAPP processing with decreased mature hIAPP and increased NH₂-terminally unprocessed prohIAPP levels resulting in higher release of immature hIAPP. Exenatide restored prohIAPP processing and reduced hIAPP aggregation in cultured islets.

Conclusions/interpretation

Exenatide treatment enhances survival and function of cultured human islets and restores impaired prohIAPP processing in normal and elevated glucose conditions thereby reducing hIAPP aggregation. GLP-1R agonists may preserve beta cells in conditions associated with islet amyloid formation.     

Clinical islet transplantation in Japan

Introduction

The results of clinical islet transplantation in Japan are, here in, reported and discussed its efficacy and problems.

Methods

Since the first islet transplantation was performed in 2004, 65 islet isolations and 34 islet transplantations to 18 type 1 diabetic patients have been performed in Japan.

Results

Following islet transplantation, patients experienced decreased insulin requirements and lower hemoglobin A1C levels, and positive serum C-peptide levels. All patients achieved stabilized blood glucose levels and the disappearance of hypoglycemic unawareness. Although three patients achieved insulin independency for a limited period, persistent islet graft function was difficult to maintain. Overall islet graft survival was 86.5% at 6 months, 78.7% at 1 year, and 62.9% at 2 years after the first islet transplantation. In our institution, we carried out 23 islet isolations and six islet transplantations to four patients. Although insulin independency was not achieved, all patients showed a disappearance of hypoglycemic unawareness.

Conclusions

Using data from the Japanese Trial of Islet Transplantation, the effectiveness of islet transplantation was shown even when using the pancreata from non-heart-beating donors. Although there are a number of problems to be solved and further improvement is needed, we can state that the introduction of clinical islet transplantation offers hope for type 1 diabetic patients.     

Mesenchymal stromal cells improve transplanted islet survival and islet function in a syngeneic mouse model

Aims/hypothesis

Islet transplantation is used therapeutically in a minority of patients with type 1 diabetes. Successful outcomes are hampered by early islet beta cell loss. The adjuvant co-transplantation of mesenchymal stromal cells (MSCs) has the promise to improve islet transplant outcome.

Methods

We used a syngeneic marginal islet mass transplantation model in a mouse model of diabetes. Mice received islets or islets plus 250,000 MSCs. Kidney subcapsule, intra-hepatic and intra-ocular islet transplantation sites were used. Apoptosis, vascularisation, beta cell proliferation, MSC differentiation and laminin levels were determined by immunohistochemical analysis and image quantification post-transplant.

Results

Glucose homeostasis after the transplantation of syngeneic islets was improved by the co-transplantation of MSCs together with islets under the kidney capsule (p?=?0.01) and by intravenous infusion of MSCs after intra-hepatic islet transplantation (p?=?0.05). MSC co-transplantation resulted in reduced islet apoptosis, with reduced numbers of islet cells positive for cleaved caspase 3 being observed 14 days post-transplant. In kidney subcapsule, but not in intra-ocular islet transplant models, we observed increased re-vascularisation rates, but not increased blood vessel density in and around islets co-transplanted with MSCs compared with islets that were transplanted alone. Co-transplantation of MSCs did not increase beta cell proliferation, extracellular matrix protein laminin production or alpha cell numbers, and there was negligible MSC transdifferentiation into beta cells.

Conclusions/interpretation

Co-transplantation of MSCs may lead to improved islet function and survival in the early post-transplantation period in humans receiving islet transplantation.     

Correlation between angiogenesis and islet graft function in diabetic mice: magnetic resonance imaging assessment

Background/purpose

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to evaluate neovascularization after intravenous injection of gadolinium, where contrast leaks out of new vessels and remains within the tissues. We examined the relationship between DCE-MRI and metabolic parameters such as blood glucose, serum insulin and glucose tolerance test (GTT) after intraportal islet transplantation.

Methods

Streptozotocin-induced diabetic BALB/c mice (n = 15) received syngeneic intraportal islet transplantation (500 islet equivalent). Blood glucose, serum insulin and GTT were evaluated till postoperative day (POD) 14. Liver DCE-MRI was performed at POD 3, 7 and 14. Correlations between DCE-MRI and metabolic parameters were examined using regression analysis.

Results

Eight mice achieved normoglycemia after intraportal transplantation. At POD 3 a significant but moderate correlation between DCE-MRI and blood glucose was found. No DCE-MRI or metabolic parameters correlated at POD 7. However, at POD 14 strong or moderate correlations between DCE-MRIs were found: negative correlations with blood glucose (R ² = 0.86) and GTT (R ² = 0.48) but a positive correlation with serum insulin (R ² = 0.32).

Conclusion

We report that DCE-MRI can reflect the metabolic and functional condition of the transplanted islets.     

Green-tea polyphenol (-)-epigallocatechin-3-gallate provides resistance to apoptosis in isolated islets

Background/Purpose

Apoptosis resulting from disruption of the normal cell-matrix relationship (anoikis) during islet isolation, and the reactive oxygen and nitrogen species generated following hypoxia/reoxygenation (H/R) can lead to a loss of islet tissue in culture and the reduced survival of transplanted pancreatic islets. The aim of this study was to investigate the effect of (-)-epigallocatechin-3-gallate (EGCG), a well-known antiapoptotic agent, on inhibiting anoikis and H/R injury in an in vitro islet culture system.

Methods

Islets were isolated from F344 rats and cultured under normal or H/R condition with/without EGCG.

Results

EGCG inhibited apoptosis and lactate-dehydrogenase leakage from anoikis and H/R in a dose-dependent manner. Further, EGCG prevent increases in 8-hydroxy-2′-deoxyguanosine content and inhibited the decline of insulin secretory function induced by H/R.

Conclusions

These results suggest that the addition of EGCG to an islet culture system may improve the survival rate of isolated islets and reduce the loss of functional islet mass that compromises the stable reversal of diabetes after islet transplantation.

     

Better vascular engraftment and function in pancreatic islets transplanted without prior culture

Aims/hypothesis Recent studies suggest that donor endothelial cells may contribute to islet graft revascularisation. Since islet endothelial cells disappear during culture, we hypothesised that transplantation of islets without prior culture is beneficial for their engraftment.Methods Cultured (4–7 days) or freshly isolated islets (<4 h after donor pancreas extirpation) were syngeneically transplanted into Wistar–Furth rats and C57Bl/6 mice beneath the renal capsule. Islet graft revascularisation was evaluated by measuring vascular density, blood flow and tissue oxygen tension. Islet graft function was investigated by a minimal islet mass model in inbred mice (C57Bl/6).Results Four days after implantation, the partial pressure of oxygen (pO₂) in the transplanted cultured islets was less than 10 mmHg (1.33 kPa), but tended to be higher in grafts composed of freshly isolated islets. The pO₂ in the grafts of freshly isolated islets had more than doubled 4 weeks later, whereas the pO₂ in the grafts of cultured islets remained at values similar to those recorded 4 days after transplantation. Transplanted freshly isolated islets also had a higher vascular density than transplanted cultured islets (40 vs 25% of that in endogenous islets) when investigated 1 month post-implantation. When applying a minimal islet mass model in inbred mice, 200 freshly isolated islets cured alloxan-diabetic mice in all cases, whereas only 33% of the group receiving similar numbers of cultured islets were cured.Conclusions/interpretation Transplantation of pancreatic islets without prior culture is beneficial for their vascular engraftment and function.     

Pancreatic duct cells as a source of VEGF in mice

Aims/hypothesis

Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking.

Methods

We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo.

Results

Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA.

Conclusions/interpretation

Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.     

Withaferin A inhibits pro-inflammatory cytokine-induced damage to islets in culture and following transplantation

Aims/hypothesis

Beta cell death triggered by pro-inflammatory cytokines plays a central role in the pathogenesis of type 1 diabetes and loss of transplanted islets. The nuclear factor κB (NF-κB) signalling pathway is a key regulator of beta cell stress response, survival and apoptosis. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, has been demonstrated to be a potent, safe, anti-inflammatory molecule that can inhibit NF-κB signalling. Therefore, we evaluated the ability of WA to protect mouse and human islets from the damaging effects of pro-inflammatory cytokines in vitro and following intraportal transplantation.

Methods

Mouse and human islets were treated with a cytokine cocktail, and NF-κB activation was measured by immunoblots, p65 nuclear translocation and chromatin immunoprecipitation of p65-bound DNA. Intraportal transplantation of a marginal mass of syngeneic mouse islets was performed to evaluate the in vivo protective effect of WA.

Results

Treatment with WA substantially improved islet engraftment of syngeneic islets (83% for infusion with 200 islets + WA; 0% for 200 islets + vehicle) in a mouse model of diabetes, compared with marginal graft controls with superior islet function in WA-treated mice confirmed by glucose tolerance test. Treatment of human and mouse islets with WA prevented cytokine-induced cell death, inhibited inflammatory cytokine secretion and protected islet potency.

Conclusions

WA was shown to be a strong inhibitor of the inflammatory response in islets, protecting against cytokine-induced cell damage while improving survival of transplanted islets. These results suggest that WA could be incorporated as an adjunctive treatment to improve islet transplant outcome.     

Effects of immunosuppression on alpha and beta cell renewal in transplanted mouse islets

Aims/hypothesis

Immunosuppressive drugs used in human islet transplantation interfere with the balance between beta cell renewal and death, and thus may contribute to progressive graft dysfunction. We analysed the influence of immunosuppressants on the proliferation of transplanted alpha and beta cells after syngeneic islet transplantation in streptozotocin-induced diabetic mice.

Methods

C57BL/6 diabetic mice were transplanted with syngeneic islets in the liver and simultaneously abdominally implanted with a mini-osmotic pump delivering BrdU alone or together with an immunosuppressant (tacrolimus, sirolimus, everolimus or mycophenolate mofetil [MMF]). Glycaemic control was assessed for 4 weeks. The area and proliferation of transplanted alpha and beta cells were subsequently quantified.

Results

After 4 weeks, glycaemia was significantly higher in treated mice than in controls. Insulinaemia was significantly lower in mice treated with everolimus, tacrolimus and sirolimus. MMF was the only immunosuppressant that did not significantly reduce beta cell area or proliferation, albeit its levels were in a lower range than those used in clinical settings.

Conclusions/interpretation

After transplantation in diabetic mice, syngeneic beta cells have a strong capacity for self-renewal. In contrast to other immunosuppressants, MMF neither impaired beta cell proliferation nor adversely affected the fractional beta cell area. Although human beta cells are less prone to proliferate compared with rodent beta cells, the use of MMF may improve the long-term outcome of islet transplantation.     

Institutional indications for islet transplantation after total pancreatectomy

Background/Purpose

This study was designed to establish institutional indications for pancreatic islet transplantation by examining patients with total pancreatectomy as candidates for islet allotransplantation.

Methods

In 12 patients who underwent total pancreatectomy, we compared pre-and postoperative plasma glucose level, body mass index, HbA1c, and daily insulin use; we examined candidacy for islet allotransplantation based on the guidelines of Japan’s islet transplantation registry.

Results

Eight of the 12 patients with total pancreatectomy were operated for intraductal papillary mucinous neoplasm. At our institution, the 5-year survival of patients with intraductal papillary mucinous neoplasm was far better (76.3%) than that of patients with pancreatic cancer. Postoperatively, plasma glucose level, HbA1c, and daily insulin use were increased in all patients with total pancreatectomy. Of the 12 patients treated with total pancreatectomy, 4 (intraductal papillary mucinous neoplasm, n = 2; islet cell tumor, n = 1; and acute pancreatitis due to arteriovenous malformation, n = 1) showed deteriorated diabetic control and therefore were considered to be candidates for islet allotransplantation according to the guidelines.

Conclusions

Islet allotransplantation could be indicated for patients with favorable postoperative survival who have had a total pancreatectomy for either benign or neoplastic disease.     

The anterior chamber of the eye as a clinical transplantation site for the treatment of diabetes: a study in a baboon model of diabetes

Aims/hypothesis

The aim of this study was to provide evidence that the anterior chamber of the eye serves as a novel clinical islet implantation site.

Methods

In a preclinical model, allogeneic pancreatic islets were transplanted into the anterior chamber of the eye of a baboon model for diabetes, and metabolic and ophthalmological outcomes were assessed.

Results

Islets readily engrafted on the iris and there was a decrease in exogenous insulin requirements due to insulin secretion from the intraocular grafts. No major adverse effects on eye structure and function could be observed during the transplantation period.

Conclusions/interpretation

Our study demonstrates the long-term survival and function of allogeneic islets after transplantation into the anterior chamber of the eye. The safety and simplicity of this procedure provides support for further studies aimed at translating this technology into the clinic.     

Human multipotent adult progenitor cells enhance islet function and revascularisation when co-transplanted as a composite pellet in a mouse model of diabetes

Aims/hypothesis

Hypoxia in the initial days after islet transplantation leads to considerable loss of islet mass and contributes to disappointing outcomes in the clinical setting. The aim of the present study was to investigate whether co-transplantation of human non-endothelial bone marrow-derived multipotent adult progenitor cells (MAPCs), which are non-immunogenic and can secrete angiogenic growth factors during the initial days after implantation, could improve islet engraftment and survival.

Methods

Islets (150) were co-transplanted, with or without human MAPCs (2.5?×?10⁵) as separate or composite pellets, under the kidney capsule of syngeneic alloxan-induced diabetic C57BL/6 mice. Blood glucose levels were frequently monitored and IPGTTs were carried out. Grafts and serum were harvested at 2 and 5 weeks after transplantation to assess outcome.

Results

Human MAPCs produced high amounts of angiogenic growth factors, including vascular endothelial growth factor, in vitro and in vivo, as demonstrated by the induction of neo-angiogenesis in the chorioallantoic membrane assay. Islet–human MAPC co-transplantation as a composite pellet significantly improved the outcome of islet transplantation as measured by the initial glycaemic control, diabetes reversal rate, glucose tolerance and serum C-peptide concentration compared with the outcome following transplantation of islets alone. Histologically, a higher blood vessel area and density in addition to a higher vessel/islet ratio were detected in recipients of islet–human MAPC composites.

Conclusions/interpretation

The present data suggest that co-transplantation of mouse pancreatic islets with human MAPCs, which secrete high amounts of angiogenic growth factors, enhance islet graft revascularisation and subsequently improve islet graft function.

     

Transplantation sites for human and murine islets

Aims/hypothesis

Beta cell replacement is a potential cure for type 1 diabetes. In humans, islet transplants are currently infused into the liver via the portal vein, although this site has disadvantages. Here, we investigated alternative transplantation sites for human and murine islets in recipient mice, comparing the portal vein with quadriceps muscle and kidney, liver and spleen capsules.

Methods

Murine islets were isolated from C57BL6/J mice and transplanted into syngeneic recipients. Human islets were isolated and transplanted into either severe combined immunodeficiency (SCID) or recombination-activating gene 1 (RAG-1) immunodeficient recipient mice. All recipient mice were 8–12 weeks of age and had been rendered diabetic (defined as blood glucose concentrations ≥20 mmol/l on two consecutive days before transplantation) by alloxan tetrahydrate treatment. Islets were transplanted into five different sites (portal vein, quadriceps muscle, kidney, liver and spleen capsules). Blood glucose concentrations were monitored twice weekly until mice were killed. Dose–response studies were also performed to determine the minimum number of islets required to cure diabetes (‘cure’ is defined for this study as random fed blood glucose of <15 mmol/l).

Results

For transplantation of murine islets into the different sites, the kidney yielded 100% success, followed by muscle (70%), portal vein (60%), spleen capsule (29%) and liver capsule (0%). For human islets, transplantation into the kidney cured diabetes in 75–80% of recipient mice. Transplantation into muscle and portal vein had intermediate success (both 29% at 2000 islet equivalents), while transplantation into liver and spleen capsule failed (0%). With increased islet mass, success rates for muscle grafts improved to 52–56%.

Conclusions/interpretation

For both human and murine islets, equivalent or superior glucose lowering results were obtained for transplantation into skeletal muscle, compared with the portal vein. Unfortunately, kidney grafts are not feasible in human recipients. Skeletal muscle offers easier access and greater potential for protocol biopsies. This study suggests that human trials of muscle as a transplant site may be warranted.

     

Transplantation sites for porcine islets

Aims/hypothesis

Xenotransplantation has great potential to provide beta cell replacement and thereby provide a cure for large numbers of people with type 1 diabetes. Crucial to the success of xenotransplantation is establishment of the most viable sites for transplantation.

Methods

We compared porcine islet tissue transplanted into kidney, liver and spleen in pig recipients as assessed by blood glucose levels and IVGTT.

Results

Kidney was the superior site for porcine islet tissue transplantation, followed by liver then spleen. This was demonstrated by IVGTTs showing significant difference between the peak glucose levels: 22.8 ± 2.9 mmol/l for kidney compared with 26.8 ± 1.3 mmol/l for spleen and 24.7 ± 1.7 mmol/l for liver.

Conclusions/interpretation

Kidney grafts are not as feasible in humans and liver results were relatively poorer than spleen. For islet transplantation to be viable and successful in the longer term, there remains a need for future investigation of alternative sites.

     

Influence of Liver Histopathology on Transaminitis Following Total Pancreatectomy and Autologous Islet Transplantation

Background

In type 1 diabetics undergoing allogenic islet transplants, transaminitis and portal vein thrombosis (PVT) after transhepatic portal infusion of islets may be related to infusion pressure and the purity of islets. Complications of intraoperative portal infusion of islets in patients with chronic pancreatitis undergoing a total pancreatectomy (TP) and autologous islet transplant (AIT) and the relationship to liver histopathology have not been examined.

Aim

The purpose of this study was to examine complications of intraportal infusion of autologous islets after TP.

Methods

Data on 26 TP-AIT patients were analyzed.

Results

Infusion of islets [mean 304,473 ± 314,557 islet equivalents, median volume 300 mL (50–600)] resulted in mean postinfusion PV pressure of 9.15 ± 10.09 cmH₂O which correlated with infused islets equivalents (r ² = 33.6, P = 0.002) and volume (r ² = 30.4, P = 0.005). Of 23 patients undergoing liver biopsy, 8 (35 %) were normal, 10 (43 %) had steatosis, and 5 (22 %) periportal fibrosis. Peak alanine aminotransferase (ALT; median 1 day after infusion) differed among the three histologic groups (P = 0.025). The difference in ALT was statistically significant between steatosis (showed the greatest increase) and the other two groups, but not between the normal and fibrosis groups. No correlation was found between the portal pressure increase at infusion and other variables. Two patients that developed PVT on day 1 had the highest infusion pressures; a third occurred on day 5.

Conclusion

Preexisting liver pathology is a contributing factor in the rise in liver enzymes but does not correlate with development of PV thrombosis.     

Implanted islets in the anterior chamber of the eye are prone to autoimmune attack in a mouse model of diabetes

Aims/hypothesis

Type 1 diabetes is an autoimmune disease resulting from the destruction of insulin-producing beta cells. Along with advances in generating replacement beta cells for treating diabetes, there is also increasing demand for non-invasive tools to evaluate the recurrence of autoimmune attack on transplanted tissue. Here, we examined the anterior chamber of the eye as a potential islet transplant site, and also evaluated whether in vivo imaging of the islets transplanted in the eye could enable real-time visualisation of autoimmune processes underway in the pancreas.

Methods

Syngeneic islet equivalents were transplanted into the eye or kidney capsule of streptozotocin-induced diabetic C57BL/6 mice to compare islet dose (25–125 islet equivalents) and function across transplant sites. Autoimmune attack of syngeneic islets was evaluated in the pancreas and eye tissues of NOD and NOD-severe combined immunodeficient (SCID) mice given diabetogenic splenocytes.

Results

Islet transplantation in the eye decreased fasting plasma glucose levels and increased weight gain and survival in an islet-dose-dependent manner. Even 50 islets in the eye reduced blood glucose levels, whereas ≥200 islets were required in the kidney for a similar effect. Autoimmune destruction of pancreatic islets in the eye mirrored that in the pancreas and could be visualised in real time by non-invasive imaging.

Conclusions/interpretation

We found that far fewer islets were required to restore normoglycaemia when transplanted into the anterior chamber of the eye vs the kidney capsule. However, our results suggest that islets are not protected against autoimmune attack in the eye, making this a suitable site for visualising autoimmune processes against transplanted tissue.     

Quantification of islet size and architecture

Human islets exhibit distinct islet architecture particularly in large islets that comprise of a relatively abundant fraction of α-cells intermingled with β-cells, whereas mouse islets show largely similar architecture of a β-cell core with α-cells in the periphery. In humans, islet architecture is islet-size dependent. Changes in endocrine cell mass preferentially occurred in large islets as demonstrated in our recent study on pathological changes of the pancreas in patients with type 2 diabetes.¹ Kilimnik G, Zhao B, Jo J, Periwal V, Witkowski P, Misawa R, et al. Altered islet composition and disproportionate loss of large islets in patients with type 2 diabetes. PLoS One 2011; 6:e27445; http://dx.doi.org/10.1371/journal.pone.0027445; PMID: 22102895 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] The size dependency of human islets in morphological changes prompted us to develop a method to capture the representative islet distribution in the whole pancreas section combined with a semi-automated analysis to quantify changes in islet architecture. The computer-assisted quantification allows detailed examination of endocrine cell composition in individual islets and minimizes sampling bias. The standard immunohistochemistry based method is widely applicable to various specimens, which is particularly useful for large animal studies but is also applied to a large-scale analysis of the whole organ section from mice. In this article, we describe the method of image capture, parameters measured, data analysis and interpretation of the data.