Protective effects of insulin during ischemia-reperfusion injury in hamster cheek pouch microcirculation

Objective: The effects of insulin ( 0.18 nM - 0.18 muM) on reduced capillary perfusion, microvascular permeability increase and leukocyte adhesion induced by ischemia-reperfusion injury were investigated in the hamster cheek pouch microcirculation. To gain insight into the insulin's mechanism of action, the effects of its higher concentration ( 0.18 muM) were investigated after inhibition of tyrosine kinase ( TK), nitric oxide synthase ( NOS), protein kinase C ( PKC), phosphatidylinositol 3- kinase and K-(ATP)(+) channels, alone or in combination. Two concentrations for each inhibitor were used. Methods: Microcirculation was visualized by fluorescence microscopy. Perfused capillary length, microvascular permeability, leukocyte adhesion to venular walls, vessel diameter and capillary red blood cell velocity were assessed by computer- assisted methods. Measurements were made at baseline ( B), after 30 min of ischemia ( I), and after 30 min of reperfusion ( R). Results: In control animals, perfused capillary length decreased by 63 +/- 5% of baseline at R. Microvascular permeability increased at I and R, while leukocyte adhesion was most pronounced in V1 postcapillary venules at R. Insulin dose- dependently preserved capillary perfusion at R ( - 28 +/- 6 and - 15 +/- 6% of baseline), but was unable to prevent the increase in permeability at I ( 0.25 +/- 0.05 and 0.29 +/- 0.06 Normalized Grey Levels, NGL) and R ( 0.49 +/- 0.10 and 0.53 +/- 0.09 NGL), according to the concentrations. Adhesion of leukocytes was observed mostly in V3 venules at R ( 9 +/- 2 and 10 +/- 2/100 mum venular length, with the lower and higher concentration, respectively). Nitric oxide synthase inhibition by N-G- nitro- L- arginine-methyl ester prior to insulin did not affect capillary perfusion at R ( - 18 +/- 3% of baseline with higher concentration), but prevented permeability increase ( 0.20 +/- 0.04 NGL, according to higher concentration) and reduced leukocyte adhesion in V3 venules at R ( 1.5 +/- 1.0/100 mum of venular length, with higher concentration). Blockade of K-(ATP)(+) channels by glibenclamide prior to insulin decreased perfused capillary length at R ( - 58 +/- 6% of baseline with higher concentration), attenuated leakage at R ( 0.30 +/- 0.04 NGL, according to higher concentration) and caused leukocyte adhesion mainly in V1 venules at R ( 9.0 +/- 1.5/100 mum of venular length, with higher concentration). Inhibition of either TK, PKC or phosphatidylinositol 3- kinase did not affect microvascular responses to insulin. Simultaneous inhibition of TK and NOS did not increase protection. Conclusions: Insulin prevents ischemia-reperfusion injury by promoting capillary perfusion through an apparent activation of K-(ATP)(+) channels and increase in nitric oxide release. Copyright (C) 2005 S. Karger AG, Basel.