Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): CONTRAST CONSORTIUM (CVON, Dutch Heart Foundation) Background Incomplete microvascular reperfusion (IMR) could be a detrimental factor impairing regained functionality in stroke patients despite early recanalization. However, the presence of this phenomenon remains to be ascertained. Gyrencephalic large-animal models of acute ischemic stroke (AIS) such as swine are gaining attention as a next step in translation of preclinical to clinical stroke research, with brain anatomy and hemostasis more similar to humans. However, the occurrence and relevance of IMR in swine models of AIS is not well described yet, since most models do not include recanalization. Purpose We aimed to study the presence of IMR after brain ischemia in a translational swine model of AIS. Methods Anesthetized female Yorkshire-Landrace pigs (n=23) underwent craniotomy to occlude the right-sided middle cerebral arteries with aneurysm clips. Clips were released after 1 (n=5), 2 (n=5), and 4 (n=5) hours of occlusion to allow recanalization for 4 hours (recanalized AIS, n=15) or left in place for 8 hours (non-recanalized AIS, n=5). Three animals underwent the craniotomy without vessel clipping (Sham, n=3). 3D angiography was used to confirm occlusion and recanalization. Brains were collected after sacrifice, stained with TTC and then processed for histologic analysis. Infarct size was determined by TTC staining and expressed as percentage of the infarcted hemisphere. Histologic sections of the upper quadrant of the left and right hemispheres were immuno-stained with CD61 to visualize platelet aggregates in the microvasculature. Intravascular platelet aggregates (Figure 2A) were quantified using automated software analysis (Orbit) (CD61+ aggregates/mm2). Results All animals survived until the end of the procedure. Figure 1 shows differences in infarct size depending on the duration of occlusion (p=0.006). Figure 2 depicts an overall higher presence of platelet (CD61+) aggregates in the right hemisphere (infarct) compared to the left hemisphere (intact) (Figure 2B). There was a significant difference in platelet aggregates between left and right hemisphere but groupwise comparison showed only a trend (p=0.075) for more aggregates in the right hemisphere, compared to left, after 4 hours occlusion. Linear regression model could not identify whether infarct size and duration of occlusion were independent predictors of the number of platelet aggregates in the right hemisphere (p=0.191). Conclusions IMR occurs after brain ischemia in a swine model of AIS as a result of microvascular platelet aggregates. Further analysis and experiments are needed to elucidate which predictors can explain intravascular platelet aggregation.Infarct SizePlatelet aggregates

中文翻译：

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猪再通急性缺血性卒中模型的不完全微血管再灌注


资金致谢 资金来源类型： 公共补助金 – 仅国家预算。主要资金来源：对比联盟（CVON，荷兰心脏基金会） 背景 尽管早期再通，但不完全微血管再灌注 (IMR) 可能是损害中风患者功能恢复的有害因素。然而，这种现象是否存在仍有待确定。急性缺血性中风（AIS）的环脑大型动物模型（例如猪）作为临床前研究向临床中风研究转化的下一步正在受到关注，其大脑解剖结构和止血功能与人类更加相似。然而，AIS 猪模型中 IMR 的发生和相关性尚未得到很好的描述，因为大多数模型不包括再通。目的 我们旨在研究 AIS 转化猪模型中脑缺血后 IMR 的存在。方法对麻醉的雌性约克夏长白猪（n=23）进行开颅手术，用动脉瘤夹封堵右侧大脑中动脉。闭塞 1 (n=5)、2 (n=5) 和 4 (n=5) 小时后松开夹子，以允许再通 4 小时（再通 AIS，n=15）或留在原处 8 小时（未再通的 AIS，n=5)。三只动物接受了开颅手术，但没有夹闭血管（假手术，n=3）。 3D 血管造影用于确认闭塞和再通。处死后收集大脑，用 TTC 染色，然后进行组织学分析。通过TTC染色测定梗塞​​大小并表示为梗塞半球的百分比。左半球和右半球上象限的组织学切片用 CD61 进行免疫染色，以可视化微脉管系统中的血小板聚集体。 使用自动软件分析 (Orbit) 对血管内血小板聚集体（图 2A）进行定量（CD61+ 聚集体/mm2）。结果所有动物均存活至手术结束。图 1 显示了梗死面积的差异取决于闭塞持续时间 (p=0.006)。图 2 显示了与左半球（完整）相比，右半球（梗死）中血小板 (CD61+) 聚集体的总体存在量较高（图 2B）。左半球和右半球之间的血小板聚集物存在显着差异，但分组比较仅显示出在闭塞 4 小时后，与左半球相比，右半球有更多聚集物的趋势 (p=0.075)。线性回归模型无法确定梗死面积和闭塞持续时间是否是右半球血小板聚集数量的独立预测因子（p=0.191）。结论 在 AIS 猪模型中，微血管血小板聚集导致脑缺血后发生 IMR。需要进一步的分析和实验来阐明哪些预测因素可以解释血管内血小板聚集。梗塞大小血小板聚集