Improvement of rat islet viability during transplantation: validation of pharmacological approach to induce VEGF overexpression:

Delayed and insufficient revascularization during islet transplantation deprives islets of oxygen and nutrients, resulting in graft failure. Vascular endothelial growth factor (VEGF) could play a critical role in islet revascularization. We aimed to develop pharmacological strategies for VEGF overexpression in pancreatic islets using the iron chelator deferoxamine (DFO), thus avoiding obstacles or safety risks associated with gene therapy. Rat pancreatic islets were infected in vivo using an adenovirus (ADE) encoding human VEGF gene (4.10(8) pfu/pancreas) or were incubated in the presence of DFO (10 mumol/L). In vitro viability, functionality, and the secretion of VEGF were evaluated in islets 1 and 3 days after treatment. Infected islets or islets incubated with DFO were transplanted into the liver of syngenic diabetic rats and the graft efficiency was estimated in vivo by measuring body weight, glycemia, C-peptide secretion, and animal survival over a period of 2 months. DFO induced transient VEGF overexpression over 3 days, whereas infection with ADE resulted in prolonged VEGF overexpression lasting 14 days; however, this was toxic and decreased islet viability and functionality. The in vivo study showed a decrease in rat deaths after the transplantation of islets treated with DFO or ADE compared with the sham and control group. ADE treatment improved body weight and C-peptide levels. Gene therapy and DFO improved metabolic control in diabetic rats after transplantation, but this effect was limited in the presence of DFO. The pharmacological approach is an interesting strategy for improving graft efficiency during transplantation, but this approach needs to be improved with drugs that are more specific

Instant blood-mediated inflammatory reaction during islet transplantation: the role of Toll-like receptors signaling pathways:

The instant blood-mediated inflammatory reaction (IBMIR) leads to massive destruction of transplanted islets. Islet isolation and time of culture may elicit the release of potent activators of Toll-like receptors (TLRs) signaling pathways during IBMIR. This work sought to evaluate the role of TLR signaling pathways to mediate inflammatory reactions. Isolated rat pancreatic islets were cultured for 12, 24, or 48 hours. Their viability was assessed by fluorescein diacetate/propidium iodide and their functionality, by glucose stimulation tests. Endotoxin levels were quantified using the Limulus Amebocyte Lysate assays. After RNA extraction and reverse transcription, we performed polymerase chain reaction (PCR) arrays. Samples obtained immediately after isolation were defined as controls. Eighty-four genes belonging to the TLR signaling pathways, were compared with control samples. After culture, islets were viable and functional with low endotoxin levels (< 0.1 endotoxin units/mL) showed TLR activation not due to exogenous contamination. Analysis of PCR arrays highlighted significant up-regulation of TLR-2. After 24 hours of culture, TLR-2 was up-regulated to 6.8 +/- 0.6-fold (P < .001) compared with controls but decreased to 4.3 +/- 1.4-fold after 48 hours. In the same way, expression of myeloid differentiation primary response gene 88 (Myd88) was significantly up-regulated (3.2 +/- 0.4-fold [P < .001]) compared with controls. After 12 hours of culture, interleukin-10 gene expression was significantly up-regulated at 11.6 +/- 3.7- fold (P < .05), reaching 17.5 +/- 8.3 after 24 hours. Finally, the cyclo-oxygenase-2 gene expression was up-regulated to 509 +/- 67.1-fold (P < .05) after 12 hours of culture. These data confirmed the implication of TLR signaling pathways in early inflammatory events

Perfluorocarbon emulsions prevent hypoxia of pancreatic beta-cells

As oxygen carriers, perfluorocarbon emulsions might be useful to decrease hypoxia of pancreatic islets before transplantation. However, their hydrophobicity prevents their homogenisation in culture medium. To increase the surface of contact between islets and Perfluorooctyl bromide (PFOB), and consequently oxygen delivery, we tested effect of a PFOB emulsion in culture medium on beta-cell lines and rat pancreatic islets. RINm5F beta-cell line or pancreatic rat islets were incubated for 3 days in the presence of PFOB emulsion in media (3.5% w/v). Preoxygenation of the medium was performed before culture. Cell viability was assessed by apoptotic markers (Bax and Bcl-2) and by staining (fluoresceine diacetate and propidium iodide). beta-Cell functionality was determined by insulin release during a glucose stimulation test and. Hypoxia markers, HIF-1alpha and VEGF, were studied at days 1 and 3 using RT-PCR, Western blotting, and ELISA. PFOB emulsions preserved viability and functionality of RINm5F cells with a decrease of HIF-1alpha and VEGF expression. Islets viability was preserved during 3 days of culture. Secretion of VEGF was higher in untreated control (0.09 +/- 0.041 mug VEGF/mg total protein) than in PFOB emulsion incubated islets (0.02 +/- 0.19 mug VEGF/mg total protein, n = 4, p < 0.05) at day 1. At day 3, VEGF secretion was increased as compared to day 1 in control (0.23 +/- 0.04 mug VEGF/mg total protein) but it was imbalance by the presence of PFOB emulsion (0.09 +/- 0.03 mug VEGF/mg total protein, n = 5, p < 0.05). While insulin secretion was maintained in response to a glucose stimulation test until day 3 when islets were incubated in the presence of PFOB emulsion preoxygenated (0.81 +/- 0.16 at day 1 vs. 0.75 +/- 0.24 at day 3), the ability to secrete insulin in the presence of high glucose concentration was lost in islets controls (0.51 +/- 0.18 at day 1 vs. 0.21 +/- 0.13 at day 3). Atmospheric oxygen delivery by PFOB emulsion might be sufficient to decrease islets hypoxia. However, to improve islets functionality, overoxygenation is needed. Finally, maintenance of islet viability and functionality for several days after isolation could improve the outcome of islets transplantation

Impact of Diffusion-Weighted Imaging Lesion Volume on the Success of Endovascular Reperfusion Therapy

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