Lapi D, Marchiafava P L, Colantuoni A
Department of Physiology and Biochemistry, University of Pisa, Pisa, Italy.
J Vasc Res. 2008;45(2):89-102. doi: 10.1159/000109818. Epub 2007 Oct 12.
The aim of the study was to assess the rat pial microvessel alterations due to transient bilateral common carotid artery occlusion (BCCAO) and to investigate the mechanism of 10% hypertonic glycerol neuroprotection. Our suggestion was that 10% glycerol solution infusion could dilate pial arterioles through nitric oxide release and/or stimulation of ATP-sensitive potassium (K(ATP)) channels. Therefore, we studied the effects of hypertonic glycerol after inhibition of nitric oxide synthase, with N(G)-nitro-L-arginine-methyl ester or N(G)-nitro-L-arginine, or K(ATP) channels with glibenclamide.
Pial microcirculation of male Wistar rats was visualized by a fluorescent microscopy technique through an open cranial window, using fluorescein isothiocyanate bound to dextran (molecular weight 70 kDa). BCCAO was induced for 30 min and reperfusion lasted 60 min. The arterioles were classified according to the Strahler ordering scheme. Permeability increase was quantified by normalized grey levels (NGL). Leucocytes were stained with rhodamine 6G. Perfused capillary length and capillary red blood cell (RBC) velocity were measured by computer-assisted methods.
The arterioles were assigned 5 orders of branchings, from order 1 (diameter 16.0 +/- 2.5 microm) to order 5 (62.0 +/- 5.0 microm). BCCAO caused inhomogenous changes in diameter of arterioles and leakage of fluorescent dextran, that was further enhanced by reperfusion (0.45 +/- 0.05 NGL, p < 0.01). Adhesion of leukocytes to venules was marked and capillary perfusion was reduced by 39.2 +/- 6.0% of baseline as well as capillary RBC velocity. 10% glycerol solution caused an increase in diameter of all arterioles within 25 +/- 2 min of administration (by 20 +/- 5% in order 4, 25 +/- 4% in order 3 and 18 +/- 3% in order 2; p < 0.01). Leakage (0.19 +/- 0.03 NGL, p < 0.01), leukocyte adhesion (2.0 +/- 1.0/100 microm of venular length, p < 0.01) and capillary occlusion (reduction by 13.0 +/- 5.5% of baseline) were prevented compared with controls. Capillary RBC velocity increased compared with controls. N(G)-nitro-L-arginine-methyl ester or N(G)-nitro-L-arginine infused prior to glycerol caused vasoconstriction and reduced the protective effects of hypertonic glycerol on permeability increase. The number of adherent leukocytes and perfused capillary length decreased, while capillary RBC velocity was higher than baseline. Glibenclamide prior to 10% glycerol solution blunted glycerol-induced vasodilatation, but did not affect protection by hypertonic glycerol on blood-brain barrier disruption, leukocyte adhesion and capillary perfusion, preserving high capillary RBC velocity. Papaverine (20 mg/kg body weight) induced an increase in arteriolar diameter, enhancing interstitial edema; adhesion of leukocytes was marked as well as capillary occlusion, while capillary RBC velocity increased.
10% glycerol solution was able to prevent microvascular alterations due to BCCAO protecting cerebral tissue. The effects appear to be due to hyperosmolality causing stimulation of K(ATP) channels, increase in vessel wall shear stress and release of nitric oxide.
本研究旨在评估短暂双侧颈总动脉闭塞（BCCAO）引起的大鼠软脑膜微血管改变，并探讨10%高渗甘油神经保护的机制。我们推测10%甘油溶液输注可通过释放一氧化氮和/或刺激三磷酸腺苷敏感性钾（K(ATP)）通道来扩张软脑膜小动脉。因此，我们研究了用N(G)-硝基-L-精氨酸甲酯或N(G)-硝基-L-精氨酸抑制一氧化氮合酶后，以及用格列本脲抑制K(ATP)通道后高渗甘油的作用。
通过荧光显微镜技术，经开放的颅骨窗口，使用与葡聚糖（分子量70 kDa）结合的异硫氰酸荧光素，观察雄性Wistar大鼠的软脑膜微循环。诱导BCCAO 30分钟，再灌注持续60分钟。根据斯特拉勒分级方案对小动脉进行分类。通过归一化灰度值（NGL）定量通透性增加。用罗丹明6G对白细胞进行染色。通过计算机辅助方法测量灌注的毛细血管长度和毛细血管红细胞（RBC）速度。
小动脉分为5个分支等级，从1级（直径16.0±2.5微米）到5级（62.0±5.0微米）。BCCAO导致小动脉直径不均匀变化以及荧光葡聚糖渗漏，再灌注使其进一步加重（0.45±0.05 NGL，p<0.01）。白细胞与小静脉的黏附明显，毛细血管灌注减少至基线的39.2±6.0%，毛细血管RBC速度也降低。10%甘油溶液在给药后25±2分钟内使所有小动脉直径增加（4级增加20±5%，3级增加25±4%，2级增加18±3%；p<0.01）。与对照组相比，渗漏（0.19±0.03 NGL，p<0.01）、白细胞黏附（2.0±1.0/100微米小静脉长度，p<0.01）和毛细血管闭塞（减少至基线的13.0±5.5%）均得到预防。与对照组相比，毛细血管RBC速度增加。在甘油给药前输注N(G)-硝基-L-精氨酸甲酯或N(G)-硝基-L-精氨酸导致血管收缩，并降低了高渗甘油对通透性增加的保护作用。黏附白细胞数量和灌注的毛细血管长度减少，而毛细血管RBC速度高于基线。在10%甘油溶液给药前使用格列本脲减弱了甘油诱导的血管舒张，但不影响高渗甘油对血脑屏障破坏、白细胞黏附和毛细血管灌注的保护作用，保持了较高的毛细血管RBC速度。罂粟碱（20毫克/千克体重）诱导小动脉直径增加，加重间质水肿；白细胞黏附明显，毛细血管闭塞，而毛细血管RBC速度增加。
10%甘油溶液能够预防BCCAO引起的微血管改变，保护脑组织。其作用似乎是由于高渗性刺激K(ATP)通道、增加血管壁剪切应力和释放一氧化氮。
