Department of Plastic Surgery, Cleveland Clinic, Cleveland, OH 44195, USA.
Microvasc Res. 2012 Mar;83(2):213-22. doi: 10.1016/j.mvr.2011.11.005. Epub 2011 Dec 6.
Tissue ischemia and reperfusion (I/R) affects blood flow restoration and oxygen delivery to the damaged tissues contributing to tissue morbidity and microcirculatory compromise. Pulsed acoustic cellular expression (PACE) technology is known to support tissue neovascularization. The aim of this study was to test PACE conditioning mechanism of action on microcirculatory hemodynamics in ischemia-reperfusion injury model.
34 rat cremaster muscle flaps were monitored under intravital microscopy system in 4 experimental groups: 1) non-ischemic controls (n=10), 2) 5h ischemia without conditioning (n=8), 3) pre-ischemic (5h) PACE conditioning (n=8), 4) post-ischemic (5h) PACE conditioning (n=8). Standard microcirculatory hemodynamics of RBC velocity, vessel diameters and functional capillary perfusion were recorded for 2h after I/R. Immunohistochemistry assessed expression of proangiogenic factors: VEGF and vWF, whereas real-time PCR assessed proangiogenic (VEGF, eNOS) and proinflammatory factors (iNOS; chemokines: CCL2, CXCL5 and chemokine receptor CCR2).
Pre-ischemic PACE conditioning (group 3) resulted in increased RBC velocity of second (A-2) and third order arterioles (A-3) and venule (V-1) by 40%, 15% and 24% respectively comparing to ischemic group without conditioning (p<0.05). Post-ischemic PACE conditioning (group 4) revealed: 1) increase in RBC velocity in second (A-2) and third order arterioles (A-3) by 65% and 31% respectively comparing to ischemia without conditioning (group 2), 2) 33% increase in first order arterioles diameter (A-1) (p<0.05) compared to ischemic controls, 3) 21% increase in number of functional capillaries compared to ischemia without conditioning (group 2) (P<0.05). Immunostaining assays showed that PACE postconditioning up-regulated proangiogenic factors vWF and VEGF protein expression. This correlated with increased gene expression of VEGF (up to 180%). In contrast, gene expression of proinflammatory factors (iNOS, CCL2, CXCL5) decreased compared to ischemic controls. Pre-ischemic PACE conditioning decreased gene expression of proinflammatory chemokines (CCL2 and CXCL5), compared to ischemic controls without conditioning.
As expected 5h ischemia resulted in deterioration of microcirculatory hemodynamics confirmed by decreased vessels diameters and RBC velocities. This was alleviated by pre- and post-ischemic PACE conditioning which improved functional capillary density and stimulated angiogenesis as confirmed by up-regulated VEGF expression. Furthermore, post-ischemic PACE conditioning correlated with decreased expression of early proinflammatory factors (iNOS, CCL2, CXCL5). Both types of PACE conditioning ameliorated deleterious effect of ischemia-reperfusion injury on microcirculatory hemodynamics of muscle flaps.
组织缺血再灌注（I/R）会影响受损组织的血流恢复和氧气输送，导致组织发病率和微循环受损。脉冲声细胞表达（PACE）技术已知可支持组织新生血管形成。本研究旨在测试 PACE 调节作用对缺血再灌注损伤模型中微循环血液动力学的影响。
在 4 个实验组中，通过活体显微镜系统监测 34 只大鼠提睾肌皮瓣：1）非缺血对照（n=10），2）无预处理的 5 小时缺血（n=8），3）缺血前（5 小时）PACE 预处理（n=8），4）缺血后（5 小时）PACE 预处理（n=8）。在 I/R 后 2 小时记录 RBC 速度、血管直径和功能性毛细血管灌注的标准微循环血液动力学。免疫组织化学评估促血管生成因子：VEGF 和 vWF 的表达，而实时 PCR 评估促血管生成（VEGF、eNOS）和促炎因子（iNOS；趋化因子：CCL2、CXCL5 和趋化因子受体 CCR2）。
缺血前 PACE 预处理（第 3 组）使第二级（A-2）和第三级动脉（A-3）以及小静脉（V-1）的 RBC 速度分别增加 40%、15%和 24%，与未预处理的缺血组相比（p<0.05）。缺血后 PACE 预处理（第 4 组）显示：1）与未预处理的缺血组（第 2 组）相比，第二级（A-2）和第三级动脉（A-3）的 RBC 速度分别增加 65%和 31%，2）第一级动脉（A-1）直径增加 33%（p<0.05）与缺血对照组相比，3）与未预处理的缺血组（第 2 组）相比，功能性毛细血管数量增加 21%（p<0.05）。免疫染色检测显示 PACE 后处理上调了促血管生成因子 vWF 和 VEGF 蛋白表达。这与 VEGF 基因表达增加（高达 180%）相关。相比之下，与缺血对照组相比，促炎因子（iNOS、CCL2、CXCL5）的基因表达降低。与未预处理的缺血对照组相比，缺血前 PACE 预处理降低了促炎趋化因子（CCL2 和 CXCL5）的基因表达。
正如预期的那样，5 小时的缺血导致血管直径和 RBC 速度降低，从而导致微循环血液动力学恶化。缺血前和缺血后 PACE 预处理可改善功能毛细血管密度并刺激血管生成，这可通过上调 VEGF 表达得到证实。此外，缺血后 PACE 预处理与早期促炎因子（iNOS、CCL2、CXCL5）的表达降低相关。两种类型的 PACE 预处理均减轻了缺血再灌注损伤对肌皮瓣微循环血液动力学的有害影响。
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