P-306

Anesthetic preconditioning protects beta-cells from oxygen free radical-induced death

K. Fukazawa¹, E. Zahr², C. Ricordi³, A. Pileggi², E. Pretto, Jr.^1
1 University of Miami / Department of Anesthesiology, Division of Transplant and Vascular Anesthesia, Jackson Memorial Hospital – and University of Miami Transplant Institute, Miami, USA; ² University of Miami / Diabetes Research Institute, Departments of Surgery, Microbiology & Immunology, Miami, USA; ³ University of Miami / Diabetes Research Institute, Departments of Surgery, Medicine, Microbiology & Immunology, and Biomedical Engineering; Jackson Memorial Hospital – and University of Miami Transplant Institute , Miami, USA

Objective: Islet cell transplantation is a promising therapeutic modality for the treatment of insulin-dependent diabetes that is limited by reduced islet viability due to their susceptibility to oxidative stress during isolation. Volatile anesthetics are cytoprotective against ischemia/reperfusion injury (anesthetic pre-conditioning or APC). In this study we investigated the effectiveness of Isoflurane (Iso)to improve islet cell survival after induction of oxidative stress with hydrogen peroxide (H₂O₂).

Methods: Murine insulinoma beta-TC3 cells were incubated for 48h then subdivided into4 groups, with 3 series of cell cultures per group repeated in triplicate.Group 1 were sham cultured beta-TC3 cells; group 2 cells were subdivided into those pre-treated with 0.5 mM Iso for 15 min or 60 min without H₂O₂,followed by washout of Iso; group 3 cells were exposed only to 0.3mM H₂O₂for 3h; group 4 cells were pre-treated with Iso for 15min or 60min as in 2,followed by Iso washout then exposure to 0.3mM H₂O₂ for3h. Cells were then harvested and assayed for viability via FACS analysis (Live/Dead far red, and TMRE, mitochondrial fluorescent dye to detect apoptosis). Data was analyzed using ANOVA with post hoc Tukey for multiple comparisons.

Results: H₂O₂ decreased beta-TC3cell survival to 44±13% compared to controls (p<0.01). Pretreatment with Iso significantly improved beta-TC3 cell survival compared to cells exposed to H₂O₂ alone (98±2% vs. 44±13% after 15min, p<0.01; and 105±0% vs. 44±13%,p<0.01 after 60min).

Conclusions: Our findings suggest that in this experimental model APC of islet cells with Iso protects them against apoptosis by H₂O₂ induced oxidative stress. Since islet isolation also induces oxidative stress, Iso pre-treatment may better preserve viability of islet cells thus reducing the amount of pancreatic tissue required and increasing the success of islet cell transplantation.

P-306

Anesthetic preconditioning protects beta-cells from oxygen free radical-induced death

K. Fukazawa¹, E. Zahr², C. Ricordi³, A. Pileggi², E. Pretto, Jr.^1
1 University of Miami / Department of Anesthesiology, Division of Transplant and Vascular Anesthesia, Jackson Memorial Hospital – and University of Miami Transplant Institute, Miami, USA; ² University of Miami / Diabetes Research Institute, Departments of Surgery, Microbiology & Immunology, Miami, USA; ³ University of Miami / Diabetes Research Institute, Departments of Surgery, Medicine, Microbiology & Immunology, and Biomedical Engineering; Jackson Memorial Hospital – and University of Miami Transplant Institute , Miami, USA

Objective: Islet cell transplantation is a promising therapeutic modality for the treatment of insulin-dependent diabetes that is limited by reduced islet viability due to their susceptibility to oxidative stress during isolation. Volatile anesthetics are cytoprotective against ischemia/reperfusion injury (anesthetic pre-conditioning or APC). In this study we investigated the effectiveness of Isoflurane (Iso)to improve islet cell survival after induction of oxidative stress with hydrogen peroxide (H₂O₂).

Methods: Murine insulinoma beta-TC3 cells were incubated for 48h then subdivided into4 groups, with 3 series of cell cultures per group repeated in triplicate.Group 1 were sham cultured beta-TC3 cells; group 2 cells were subdivided into those pre-treated with 0.5 mM Iso for 15 min or 60 min without H₂O₂,followed by washout of Iso; group 3 cells were exposed only to 0.3mM H₂O₂for 3h; group 4 cells were pre-treated with Iso for 15min or 60min as in 2,followed by Iso washout then exposure to 0.3mM H₂O₂ for3h. Cells were then harvested and assayed for viability via FACS analysis (Live/Dead far red, and TMRE, mitochondrial fluorescent dye to detect apoptosis). Data was analyzed using ANOVA with post hoc Tukey for multiple comparisons.

Results: H₂O₂ decreased beta-TC3cell survival to 44±13% compared to controls (p<0.01). Pretreatment with Iso significantly improved beta-TC3 cell survival compared to cells exposed to H₂O₂ alone (98±2% vs. 44±13% after 15min, p<0.01; and 105±0% vs. 44±13%,p<0.01 after 60min).

Conclusions: Our findings suggest that in this experimental model APC of islet cells with Iso protects them against apoptosis by H₂O₂ induced oxidative stress. Since islet isolation also induces oxidative stress, Iso pre-treatment may better preserve viability of islet cells thus reducing the amount of pancreatic tissue required and increasing the success of islet cell transplantation.