Study of the immunosuppressive effect of cyclosporine in experimental studies]

Specific immunosuppressive activity of cyclosporine prepared at the National Research Centre of Antibiotics by using its original producing culture was studied. It inhibited basic cellular immune responses such as DTH and transplant vs. host and prolonged the lifespan of tumor xenografts under the kidney capsule. Cyclosporine also suppressed the humoral immunity. Its inhibitory effect on lymphocyte blast transformation induced by lectins and in mixed lymphocyte cultures was shown in vitro. Cyclosporine inhibited activity of natural killer cells and T-suppressor cells induced by concanavalin A. It was demonstrated on the models of skin allotransplantation in mice and heterotopic transplantation of the heart in rats that cyclosporine prolonged the transplant lifespan. By the main indices of the cellular immunity and in the models of the transplantation immunity, cyclosporine prepared at the National Research Centre of Antibiotics was shown to be similar to that manufactured by Sandoz.     

An in vivo model for study of the angiogenic effects of basic fibroblast growth factor

We have investigated the angiogenic effects of basic fibroblast growth factor following its implantation in slow release beads under the kidney capsule. The presence of basic fibroblast growth factor in the subcapsular space induced a marked angiogenic response maximal at 1 microgram dose per kidney. Histological examination at the site of treatment failed to reveal evidence of an inflammatory response, thus supporting the observation that basic fibroblast growth factor alone can stimulate in vivo neovascularization. Beads pretreated with saline or with human growth hormone had no angiogenic effect. Because of the readily accessible location in the retroperitoneal space, the ease of drug delivery, and the marked vascular proliferation seen in response to FGF, our results suggest that the kidney capsule is an excellent model for study of the physiological role played by FGF and related peptides in promoting angiogenesis in vivo.     

The Effects of Exendin-4 Treatment on Graft Failure: An Animal Study Using a Novel Re-Vascularized Minimal Human Islet Transplant Model

Islet transplantation has become a viable clinical treatment, but is still compromised by long-term graft failure. Exendin-4, a glucagon-like peptide 1 receptor agonist, has in clinical studies been shown to improve insulin secretion in islet transplanted patients. However, little is known about the effect of exendin-4 on other metabolic parameters. We therefore aimed to determine what influence exendin-4 would have on revascularized minimal human islet grafts in a state of graft failure in terms of glucose metabolism, body weight, lipid levels and graft survival. Introducing the bilateral, subcapsular islet transplantation model, we first transplanted diabetic mice with a murine graft under the left kidney capsule sufficient to restore normoglycemia. After a convalescent period, we performed a second transplantation under the right kidney capsule with a minimal human islet graft and allowed for a second recovery. We then performed a left-sided nephrectomy, and immediately started treatment with exendin-4 with a low (20μg/kg/day) or high (200μg/kg/day) dose, or saline subcutaneously twice daily for 15 days. Blood was sampled, blood glucose and body weight monitored. The transplanted human islet grafts were collected at study end point and analyzed. We found that exendin-4 exerts its effect on failing human islet grafts in a bell-shaped dose-response curve. Both doses of exendin-4 equally and significantly reduced blood glucose. Glucagon-like peptide 1 (GLP-1), C-peptide and pro-insulin were conversely increased. In the course of the treatment, body weight and cholesterol levels were not affected. However, immunohistochemistry revealed an increase in beta cell nuclei count and reduced TUNEL staining only in the group treated with a low dose of exendin-4 compared to the high dose and control. Collectively, these results suggest that exendin-4 has a potential rescue effect on failing, revascularized human islets in terms of lowering blood glucose, maintaining beta cell numbers, and improving metabolic parameters during hyperglycemic stress.     

Improvement of pituitary homograft under the kidney capsule in mice]

The model of hyperprolactinemia induced by pituitary homografts under the kidney capsule has been used mainly in the field of reproductive physiology. The authors report an improved method for pituitary grafting in mice. The procedure was as follows: 1. The male pituitary glands with normal saline were aspirated into a polyethylene tube. 2. Two incisions were made in the kidney capsule. 3. The polyethylene tube with pituitary glands was inserted via a large incision. 4. Blowing air into the tube, the pituitary glands were left under the kidney capsule and normal saline streamed out of a small incision. Using this method, all pituitary grafted mice became pseudopregnant.     

Effect of estrone on formation of the focus of heterotopic hematopoiesis

The effect of estrone injections on the heterotopic bone marrow organ formation was studied in (C57Bl X CBA)F1 mice by bone marrow shaft transplantation under the kidney capsule. Estrone injections resulted in femur marrow fibrosis, the increase in the weight of femur and heterotopic organ ossicle, drastic reduction in heterotopic organ cellularity. Depression of hemopoiesis in ectopic organ cannot be attributed to mechanical displacement of hemopoietic cells because a newly formed bone cavity under the kidney capsule was not closed. Thus, estrone had a detrimental effect on the creation of microenvironment in the heterotopic organ.     

Thymic transplantation in miniature swine. II. Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients

Previous studies in our laboratory have demonstrated that the presence of the thymus is essential for rapid and stable tolerance induction in allotransplant models. We now report an attempt to induce tolerance to kidney allografts by transplanting donor thymic grafts simultaneously with the kidney in thymectomized recipients. Recipients were thymectomized 3 wk before receiving an organ and/or tissues from a class I-mismatched donor. Recipients received 1) a kidney allograft alone, 2) a composite allogeneic thymokidney (kidney with vascularized autologous thymic tissue under its capsule), or 3) separate kidney and thymic grafts from the same donor. All recipients received a 12-day course of cyclosporine. Thymectomized animals receiving a kidney allograft alone or receiving separate thymic and kidney grafts had unstable renal function due to severe rejection with the persistence of anti-donor cytotoxic T cell reactivity. In contrast, recipients of composite thymokidney grafts had stable renal function with no evidence of rejection histologically and donor-specific unresponsiveness. By postoperative day 14, the thymic tissue in the thymokidney contained recipient-type dendritic cells. By postoperative day 60, recipient-type class I positive thymocytes appeared in the thymic medulla, indicating thymopoiesis. T cells were both recipient and donor MHC-restricted. These data demonstrate that the presence of vascularized-donor thymic tissue induces rapid and stable tolerance to class I-disparate kidney allografts in thymectomized recipients. To our knowledge, this is the first evidence of functional vascularized thymic grafts permitting transplantation tolerance to be induced in a large animal model.     

The effect of cyclosporine A on bile secretion in dogs

Cyclosporine A is reported to cause cholestasis, but the evidence is confounded by anesthesia and surgery used in acute experiments. To better investigate the effect of cyclosporine on the liver, bile output was directly measured in three cholecystectomized dogs by cannulating the common duct through a chronic duodenal fistula. Control studies were done 1 month after surgery. Cyclosporine in oral doses of 5, 15, and 50 mg.kg-1.d-1 was then given for consecutive 1-week periods. Twice during each study period, bile output was measured for 5 h in fasted, awake animals: 3 h to establish basal conditions, followed by 2 h of taurocholate infusions at 1 and then 2 mumols.kg-1.min-1. Under basal conditions, bile flow rose with each dose of cyclosporine, increasing 63, 127, and 179% above control with cyclosporine 5, 15, and 50 mg.kg-1,d-1, respectively. Bile flow increased similarly during taurocholic acid stimulation. Cyclosporine had no effect on bile salt or bilirubin secretion. In this chronic dog model isolated from other causes of cholestasis, cyclosporine did not induce cholestasis but rather caused a dose-related choleresis without any change in bile salt secretion.     

Experimental and finite element analysis for prediction of kidney injury under blunt impact

Kidneys are third most injured organs in abdominal trauma after liver and spleen; this study therefore is an attempt to understand the behaviour of kidneys under blunt trauma. Dynamic impact tests were performed on 20 fresh porcine kidneys to study the injury propagation in the organ, and the acceleration of the impactor was measured. A kidney model was developed with structural details like capsule and cortex. The kidney cortex was modelled with solid hexahedral elements and the capsule was modelled with quadratic shell elements. The material models for the capsule and cortex were used from the experimental data reported in our previous study. The developed model was calibrated using previous and current experimental results to reproduce the injuries of the organ in terms of acceleration of the impactor, and the injuries sustained by the organ during the experiments. The developed kidney model is observed to be robust and can be integrated with the available human body finite element models to simulate accidents and to predict or simulate injuries.     

Cyclosporine in transplantation - a history of converging timelines

Discovery and pharmacological development of cyclosporine was conducted by Jean Borel and colleagues in the 1970s. Cyclosporine is the first compound to inhibit the lymphocytes specifically and reversibly, and represents the prototype of a new generation of immunosuppressive drugs: the calcineurine inhibitors. Historical chronology of successes in clinical application of cyclosporine and development of solid-organ transplantation are retraced here, underscoring the converging timelines of this drug and these interventions. In 1978-79 the first successful results of the use of cyclosporine in kidney were reported. Cyclosporine was the first single drug able to control rejection. In 1982-83 first trials demonstrated the benefit from treatment with cyclosporine in kidney recipients compared to azathioprine and steroids. In the 1980s solid-organ transplantation entered the cyclosporine era with unhoped-for results in heart transplantation. The present review focuses also on cyclosporine-based regimen of immunosuppression, adverse side effects and safety in pregnancy in subjects under treatment with cyclosporine.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

Co-Graft of Allogeneic Immune Regulatory Neural Stem Cells (NPC) and Pancreatic Islets Mediates Tolerance,while Inducing NPC-Derived Tumors in Mice

Background

Data available on the immunomodulatory properties of neural stem/precursor cells (NPC) support their possible use as modulators for immune-mediated process. The aim of this study was to define whether NPC administered in combination with pancreatic islets prevents rejection in a fully mismatched allograft model.

Methodology/Principal Finding

Diabetic Balb/c mice were co-transplanted under the kidney capsule with pancreatic islets and GFP⁺ NPC from fully mismatched C57BL/6 mice. The following 4 groups of recipients were used: mice receiving islets alone; mice receiving islets alone and treated with standard immunosuppression (IL-2Rα chain mAbs + FK506 + Rapamycin); mice receiving a mixed islet/NPC graft under the same kidney capsule (Co-NPC-Tx); mice receiving the islet graft under the left kidney capsule and the NPC graft under the right kidney capsule (NPC-Tx). Our results demonstrate that only the co-transplantation and co-localization of NPC and islets (Co-NPC-Tx) induce stable long-term graft function in the absence of immunosuppression. This condition is associated with an expansion of CD4⁺CD25⁺FoxP3⁺ T regulatory cells in the spleen. Unfortunately, stable graft function was accompanied by constant and reproducible development of NPC-derived cancer mainly sustained by insulin secretion.

Conclusion

These data demonstrate that the use of NPC in combination with islets prevents graft rejection in a fully mismatched model. However, the development of NPC-derived cancer raises serious doubts about the safety of using adult stem cells in combination with insulin-producing cells outside the original microenvironment.     

Tolerance induction in composite facial allograft transplantation in the rat model

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.     

Prolactin inhibits the development of stress-induced ulcers in the rat

Hyperprolactinaemia, as induced by pituitary homografts under the kidney capsule, was accompanied by an inhibition of development of gastric ulcers following the application of cold-plus-restraint stress in male rats. This effect was mimicked by intracisternal administration of a low dose of the hormone. Peripheral injection of the dopamine receptor antagonist, domperidone, also inhibited the development of stress-induced ulcers. However, no effect was found after peripheral injection of another dopamine receptor antagonist, haloperidol. This latter drug appeared to antagonize the cytoprotective effect of prolactin (PRL) on stress-induced ulcers. Furthermore, peripheral injection of the prostaglandin synthesis inhibitor, indomethacin, increased the incidence of gastric ulcers in hyperprolactinaemic rats subjected to cold -plus-restraint stress. These data suggest that the cytoprotective effect of PRL on development of gastric ulcers in stressed animals may involve both central (i.e. dopamine transmission) and peripheral (i.e. prostaglandin synthesis) mechanisms.     

Cyclosporine inhibits macrophage-mediated antigen presentation

The influence of cyclosporine on antigen-specific, macrophage-dependent T cell activation was analyzed in vitro. Murine T cell activation by antigens derived from Listeria monocytogenes was monitored by the production of interleukin 2. Pretreatment (2 hr, 37 degrees C) of macrophages with cyclosporine resulted in a cell population with a markedly diminished capacity to support the activation of T lymphocytes. When cyclosporine-pretreated macrophages were added to cultures of untreated T cells and antigen, the dose of cyclosporine that produced 50% inhibition (ID50) was 1.5 micrograms/ml, and if antigen was present during the drug pretreatment, the ID50 was 0.6 micrograms/ml. Pretreatment of T cells also inhibited their subsequent activation by antigen and untreated macrophages, but a higher dose of cyclosporine was required to produce similar inhibition (ID50 = 4.4 micrograms/ml). Additional experiments focused on the mechanism of inhibition of antigen presentation when macrophages were pretreated with the drug. The addition of interleukin 1 or indomethacin to the cultures did not alter the inhibitory effect of cyclosporine. Under conditions that produced greater than 90% inhibition of antigen presentation, macrophage surface Ia expression was not altered, and the uptake and catabolism of radiolabeled antigen remained normal. Thus, cyclosporine had profound effects on antigen presentation that appear to be unrelated to decreases in interleukin 1 production, increases in prostaglandin production, decreases in Ia expression, or changes in antigen uptake and catabolism.     

Small heat shock proteins expression in rat kidneys treated with cyclosporine A alone and combined with melatonin

Small heat shock proteins (sHSPs) are cytoskeletal chaperones constitutively expressed in the normal kidney but enhanced with beneficial roles during adverse stimuli. Cyclosporine A is an immunosuppressive drug with major adverse side effect such as severe nephrotoxicity. Among possible mechanisms of cyclosporine A-induced renal damage, oxidative stress and cytoskeletal damage have been suggested. Melatonin has been successfully used as antioxidant against many renal diseases. This in vivo study was performed to shed light on the protective effect of melatonin against cyclosporine A-induced renal alterations. We treated rats with cyclosporine A alone, or combined with melatonin, and with melatonin alone (as controls) for 40 days and analysed the renal abundance and distribution of two sHSPs, HSP25 and alpha B-crystallin. These data were correlated with the histopathological effects of the treatments. Cyclosporine A induced insoluble isoforms that moved to soluble fractions after melatonin coadministration as in controls. After cyclosporine A treatment, an intense signal for sHSPs was found within the glomeruli, nucleus and cytoplasm of cortical tubules, collecting ducts and vascular wall. After melatonin supply, the staining was faint, limited to the cytoplasm of cortical tubules, similar to controls. Both fibrosis and tubular alterations significantly decreased after melatonin coadministration. In conclusion, HSP25 and alpha B-crystallin are overexpressed in the rat kidney treated with cyclosporine A but are similar to controls after combined melatonin. This could be a consequence of the cytoprotective effect of melatonin in this nephrotoxic model so that a beneficial sHSPs response isbreak unnecessary.     

Cyclosporine A-induced toxicity in two renal cell culture models (LLC-PK1 and MDCK)

Renal damage caused by therapeutic treatment with cyclosporine A has been well documented. Clinical experiences have shown that cyclosporine A nephrotoxicity is determined by interstitial fibrosis with tubular atrophy. However, the exact mechanism by which this drug causes nephrotoxicity has not yet been clarified. This study used an in vitro model in an attempt to identify the cellular mechanisms underlying kidney cyclosporine A damage. We used two cell lines with the characteristics of proximal and distal tubule cells (pig kidney proximal tubular epithelial cell line [LLC-PK1] and Madin–Darby canine kidney cell line [MDCK]. The cell lines were treated with cyclosporine A for 24h. After the treatment, the cells were stained with Trypan Blue to estimate cell viability and processed by histochemical reactions to evaluate their cellular metabolism. Four enzymes (acid phosphatase, alkaline phosphatase, lactate dehydrogenase and succinate dehydrogenase) were considered. The cell viability assay showed that the LLC-PK1 cell line was more sensitive to cyclosporine A than MDCK. Remarkably, the LLC-PK1 cells disappeared with cyclosporine A treatment. As for the hydrolytic enzymes, only acid phosphatases showed an increased positivity in the treated LLC-PK1 cells. Similarly, lactate dehydrogenase showed a different activity histochemically. No statistically significant alterations were observed in the succinate dehydrogenase reaction.The cyclosporine A-treated MDCK cell lines did not show any difference in either their hydrolytic or succinate dehydrogenase enzyme positivity with respect to the control line. In contrast, there was a significant increase in lactate dehydrogenase activity. This study allowed the possible mechanism of cyclosporine A-induced damage in renal tubular cells to be evaluated. The enzymatic changes happened rapidly (during the 24h of treatment), suggesting that this alteration was one of the steps by which cyclosporine A induced toxicity. Moreover, since acid phosphatase is a marker of protein catabolism, the variation in the activity of this enzyme, in the LLC-PK1 line only, showed that cyclosporine can induce alterations leading to cellular toxicity. The modifications in lactate dehydrogenase activity, in both lines, suggested that this drug caused cell stress, inducing the production of lactic acid from glucose in the presence of oxygen. In conclusion, cyclosporine A treatment may force LLC-PK1 and MDCK cells to use anaerobic glycolysis preferentially. Further, these enzyme alterations may represent an epiphenomenon or a consequence of cyclosporine A toxicity.     

Heterotypic induction of stomach-like glands in the urogenital sinus endoderm under the influence of stomach mesenchyme of foetal rats

Summary Urogenital sinus endoderm of 16.5-day rat foetuses was combined with stomach mesenchyme and the recombinants were either treated with testosterone and grown in vitro or cultured beneath the kidney capsule of adult male rats of the same strain. It was found that testosterone stimulated mitosis in the urogenital endoderm. In recombinants grown under the kidney capsule a stratified squamous epithelium and stomach-like glands were induced under the influence of the forestomach and glandular stomach mesenchymes. However, the induced glands expressed neither rat pepsinogen nor rat ventral prostatic antigen. They did not produce mRNA for the prostatic steroid-binding protein C1. Thus, stomach mesenchyme of rat foetuses induces organ-specific morphogenesis but not functional differentiation in the heterologous endoderm, indicating that cytodifferentiation does not always accompany morphogenesis.     

Newborn pig ovarian tissue xenografted into Severe Combined Immunodeficient (SCID) mice acquires limited responsiveness to gonadotropins

In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at ∼60-90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 ± 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins.     

甘草酸二胺肠溶胶囊联合液氮冷冻治疗斑秃的疗效

为了研究观察不同剂量的甘草酸二胺肠溶胶囊联合液氮冷冻治疗斑秃的疗效分析,以及检测甘草酸二胺肠溶胶囊对患者肝肾功能的影响,进一步阐明甘草酸二胺肠溶胶囊对斑秃治疗的效果与对人体的健康影响,本研究以仅使用液氮冷冻治疗斑秃为对照组,设低剂量甘草酸二胺肠溶胶囊联合液氮为低剂量组(50 mg/次),高剂量甘草酸二胺肠溶胶囊联合液氮为高剂量组(200 mg/次),一日3次,共治疗12周,以斑秃皮损面积及严重程度指数(PASI)评分为疗效评价。采用血液生化检验(肝,肾功检查)检测3组治疗前后的丙氨酸氯基转移酶、尿素氮和肌酐水平。研究结果表明,治疗后高剂量组总有效率(92.50%)高于低剂量组(82.50%)及对照组(55.00%)(p<0.05)。高剂量组PASI评分及治疗前后丙氨酸氯基转移酶、尿素氮、肌酐水平相对于低剂量组及对照组无统计学意义(p>0.05)。本研究初步结论表明,高剂量的甘草酸二胺肠溶胶囊联合液氮治疗斑秃疗效显著高于甘草酸二胺肠溶胶囊联合液氮及仅使用液氮冷冻治疗;甘草酸二胺肠溶胶囊安全可靠,不影响患者的肝肾功能。     

Transplantation of pancreatic islets into the kidney capsule of diabetic mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse. LOADING: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped. TRANSPLANTATION: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.