Colantuoni A, Lapi D, Paterni M, Marchiafava P L
Department of Neuroscience, Federico II University Medical School, Naples, Italy.
J Vasc Res. 2005 Jan-Feb;42(1):55-66. doi: 10.1159/000083092. Epub 2005 Jan 3.
The effects of insulin (0.18 nM-0.18 microM) 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 microM) 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.
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).
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 microm 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 microm 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 microm 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.
Insulin prevents ischemia-reperfusion injury by promoting capillary perfusion through an apparent activation of K+(ATP) channels and increase in nitric oxide release.
在仓鼠颊囊微循环中研究胰岛素（0.18 nM - 0.18 μM）对缺血 - 再灌注损伤诱导的毛细血管灌注减少、微血管通透性增加和白细胞黏附的影响。为深入了解胰岛素的作用机制，在单独或联合抑制酪氨酸激酶（TK）、一氧化氮合酶（NOS）、蛋白激酶C（PKC）、磷脂酰肌醇3 - 激酶和K⁺（ATP）通道后，研究了其较高浓度（0.18 μM）的作用。每种抑制剂使用两种浓度。
通过荧光显微镜观察微循环。采用计算机辅助方法评估灌注的毛细血管长度、微血管通透性、白细胞对小静脉壁的黏附、血管直径和毛细血管红细胞速度。在基线（B）、缺血30分钟（I）后和再灌注30分钟（R）后进行测量。
在对照动物中，再灌注时灌注的毛细血管长度降至基线的63±5%。微血管通透性在缺血时和再灌注时增加，而再灌注时白细胞黏附在V1后微静脉中最为明显。胰岛素在再灌注时剂量依赖性地维持毛细血管灌注（分别为基线的 - 28±6%和 - 15±6%），但根据浓度不同，无法预防缺血时（归一化灰度水平分别为0.25±0.05和0.29±0.06）和再灌注时（归一化灰度水平分别为0.49±0.10和0.53±0.09）通透性的增加。再灌注时白细胞黏附主要见于V3小静脉（较低和较高浓度时分别为9±2和10±2/100微米小静脉长度）。在胰岛素给药前用N⁃硝基⁃L⁃精氨酸甲酯抑制一氧化氮合酶，对再灌注时的毛细血管灌注无影响（较高浓度时为基线的 - 18±3%），但可预防通透性增加（较高浓度时归一化灰度水平为0.20±0.04），并减少再灌注时V3小静脉中的白细胞黏附（较高浓度时为1.5±1.0/100微米小静脉长度）。在胰岛素给药前用格列本脲阻断K⁺（ATP）通道，可使再灌注时灌注的毛细血管长度减少（较高浓度时为基线的 - 58±
