P-选择素缺陷型小鼠短暂性脑缺血后血脑屏障通透性降低：治疗中风的未来治疗靶点。

Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke.

机构信息

Department of Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada.

出版信息

BMC Neurosci. 2010 Feb 2;11:12. doi: 10.1186/1471-2202-11-12.

DOI:10.1186/1471-2202-11-12

PMID:20122276

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2829030/

Abstract

BACKGROUND

The link between early blood- brain barrier (BBB) breakdown and endothelial cell activation in acute stroke remain poorly defined. We hypothesized that P-selectin, a mediator of the early phase of leukocyte recruitment in acute ischemia is also a major contributor to early BBB dysfunction following stroke. This was investigated by examining the relationship between BBB alterations following transient ischemic stroke and expression of cellular adhesion molecule P-selectin using a combination of magnetic resonance molecular imaging (MRMI), intravital microscopy and immunohistochemistry. MRMI was performed using the contrast, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated to Sialyl Lewis X (Slex) where the latter is known to bind to activated endothelium via E- or P selectins. Middle cerebral artery occlusion was induced in male C57/BL 6 wild-type (WT) mice and P-selectin-knockout (KO) mice. At 24 hours following middle cerebral artery occlusion, T1 maps were acquired prior to and following contrast injection. In addition to measuring P- and E-selectin expression in brain homogenates, alterations in BBB function were determined immunohistochemically by assessing the extravasation of immunoglobulin G (IgG) or staining for polymorphonuclear (PMN) leukocytes. In vivo assessment of BBB dysfunction was also investigated optically using intravital microscopy of the pial circulation following the injection of Fluorescein Isothiocyanate (FITC)-dextran (MW 2000 kDa).

RESULTS

MRI confirmed similar infarct sizes and T1 values at 24 hours following stroke for both WT and KO animals. However, the blood to brain transfer constant for Gd DTPA (Kgd) demonstrated greater tissue extravasation of Gd DTPA in WT animals than KO mice (P < 0.03). In the P selectin KO mice, Delta T1 stroke -Delta T1 contralateral control cortex, decreased significantly in the Gd-DTPA(sLeX) group compared to Gd-DTPA, indicative of sLeX mediated accumulation of the targeted contrast agent. Regarding BBB function, in the P-selectin KO mice compared to WT control mice, there was an attenuation in the extravasation of IgG (P < 0.001), a trend for decreased FITC extravasation and less infiltration of PMN leukocytes (P < 0.001) thereby supporting the observed increase in Kgd permeability in stroke brain of WT compared to KO mice.

CONCLUSION

P-selectin expression contributes to enhanced BBB dysfunction at 24 hours after transient focal cerebral ischemia.

摘要

背景

急性中风中早期血脑屏障（BBB）破裂与内皮细胞激活之间的联系仍未得到明确界定。我们假设 P-选择素是急性缺血时白细胞募集早期阶段的一种介质，也是中风后早期 BBB 功能障碍的主要原因。通过使用磁共振分子成像（MRMI）、活体显微镜检查和免疫组织化学检查，结合检查短暂性缺血性中风后 BBB 改变与细胞黏附分子 P-选择素的表达之间的关系，对这一假设进行了研究。MRMI 使用与唾液酸化路易斯 X（Slex）结合的对比剂钆二乙三胺五乙酸（Gd-DTPA）进行，后者已知通过 E-或 P 选择素与激活的内皮结合。在雄性 C57/BL6 野生型（WT）小鼠和 P-选择素敲除（KO）小鼠中诱导大脑中动脉闭塞。在大脑中动脉闭塞后 24 小时，在注射对比剂之前和之后采集 T1 图谱。除了测量脑匀浆中的 P-和 E-选择素表达外，还通过评估免疫球蛋白 G（IgG）的外渗或多形核（PMN）白细胞的染色来免疫组织化学确定 BBB 功能的改变。还通过注射荧光素异硫氰酸酯（FITC）-葡聚糖（MW 2000 kDa）后对软脑膜循环进行活体显微镜检查，从光学角度研究了 BBB 功能的体内评估。

结果

MRI 证实 WT 和 KO 动物在中风后 24 小时的梗死大小和 T1 值相似。然而，Gd DTPA（Kgd）的血脑转移常数显示 WT 动物比 KO 小鼠的 Gd DTPA 有更多的组织外渗（P <0.03）。在 P 选择素 KO 小鼠中，与 Gd-DTPA 相比，Gd-DTPA(sLeX)组的 T1 卒中-ΔT1 对侧对照皮质明显降低，表明靶向对比剂的 sLeX 介导积累。关于 BBB 功能，与 WT 对照小鼠相比，P-选择素 KO 小鼠的 IgG 外渗减少（P <0.001），FITC 外渗减少趋势，PMN 白细胞浸润减少（P <0.001），从而支持与 KO 小鼠相比，WT 中风脑的 Kgd 通透性增加。