动脉内脑局部低温联合3K3A-APC保护缺血/再灌注损伤神经血管单元微血管屏障的协同机制研究

Ischemia and reperfusion injury (I/R) remains to be an unresolved problem accompanied with the acute recanalization during cerebral infarction. I/R may cause significant damages including invalid reperfusion, cerebral edema, and hemorrhagic transformation. Although therapeutic hypothermia (TH) has been considered as the most promising neuroprotectant, it was scarcely used clinically because it was not facilitated to conduct to fulfill the requirement of treatment time window. In addition, use of 3K3A-Activated Protein C (3K3A-APC), an engineered form of Activated protein C shows multiple neuroprotective effects including stabilization of the brain endothelial barrier. Intriguingly, our preliminary data show that the combined treatment with TH and 3K3A-APC significantly improves outcome as compared to individual treatment in the rat model of middle cerebral artery thread occlusion and recanalization (MCAO/R). Nevertheless, the underlying mechanisms remain to be determined. Our current hypothesis is that the combined treatment (intra-arterial local hypothermia + 3K3A-APC) enhances the protection of microvascular barrier in the neurovascular unit (NVU); TH promotes the activation of the non-canonical cleavage of PAR1 pathway initiated by 3K3A-APC, and then inhibits the NF-κB pathway and its downstream targets, MMP-9 and inflammatory cytokines (e.g. IL-1ß, TNF-α and IL-6). To test the hypothesis, we will use the rat model of MCAO/R and in vitro cell culture with the human brain endothelial cell line (HBMECs) that were pretreatment by oxygen glucose deprivation/reoxygenation. We will determine the synergistic effects on the outcome of combining TH and 3K3A-APC treatment, and further investigate the underlying mechanisms by employing MRI, histochemistry, Western blot and genetic manipulation with the CRISPR/Cas9 technology. This study will provide important experimental evidence and critical insights into the application of combined treatment with local TH and 3K3A-APC in the treatment of acute cerebral infarction.

缺血/再灌注损伤是急性脑梗死血管再通治疗面临的难题。低温是很有希望的神经保护措施，因实施困难，临床转化受限；3K3A-APC是活化蛋白C的基因工程变异体，有稳定血管内皮屏障等多重神经保护作用。我们前期研究发现二者联合治疗大鼠缺血/再灌注模型（MCAO/R）优于单一治疗，但机制需进一步研究。为此我们提出假说：动脉内脑局部低温联合3K3A-APC对缺血/再灌注损伤的神经血管单元微血管屏障有协同保护作用；低温可能促进3K3A-APC 介导的PAR-1非经典剪切信号通路，抑制NF-κB上调MMP-9和炎症因子表达。为验证这一假说，我们选用大鼠MCAO/R、氧糖剥脱/复氧的人脑血管内皮细胞系模型，采用磁共振影像、组织病理、Western blot、基因敲除等手段，探讨两者的协同疗效及其分子机制。本研究将为脑局部低温联合3K3A-APC治疗急性脑梗死提供重要的理论和实验依据。

脑缺血再灌注损伤（ischemic reperfusion injury, IRI）是一个复杂的病理生理级联反应过程，单一的神经保护药物很难全面阻断这种网络式的级联损伤反应。低温则可抑制IRI网络中的大多数病理生理反应，被认为是最有希望的神经保护措施。但全身性低温因副作用明显，在急性脑梗死治疗中一直难以实现转化。随着血管内取栓治疗的广泛开展，动脉内脑局部低温（intra-arterial hypothermia, IAH）已成为可能，但其具体实施条件仍不清楚。本研究采用正交设计方案（L9[3^4]），在MCAO/R大鼠中探索出IAH的最佳干预条件：4℃生理盐水，2/3颈内动脉血流速度（2/3 VICA），持续灌注20分钟。IAH开始后10 min内脑局部温度（Tb）快速降至35℃以下的亚低温水平，并可维持60-80min，但核心体温（Tcore）下降小于1.0 ℃；颈静脉回流血温度（Tjvb）与Tb存在显著相关性（Rho=0.999;P <0.01），可用于指导IAH的具体实施。IAH明显减少脑梗死体积，减轻脑水肿，改善神经功能缺损以及减轻神经血管单元微血管屏障的破坏从而发挥神经保护效应，其机制可能与上调冷诱导RNA结合蛋白（CIRP）表达，抑制NF-KB，降低MMP-9和MMP-2表达有关。结合实验条件并根据炎症与氧化应激是IRI的两大主要损伤机制，我们研究动脉内联合灌注炎症抑制药（米诺环素）和自由基清除剂（依达拉奉）对MCAO/R大鼠进行干预是否优于单药治疗。研究结果显示与单药比较，两种药物联合可明显减少脑梗死体积，减轻脑水肿，改善神经功能缺损；并可加强对炎症及氧化应激反应的抑制作用，从而保护神经血管单元微血管屏障的完整性；这种保护效应主要与抑制小胶质细胞活化，调节NF-κB通路有关。总之，本课题探索出最佳IAH条件为进一步研究低温保护IRI及其机制提供了新途径；而动脉内米诺环素联合依达拉奉治疗的研究也为干预级联网络式的IRI提供了新思路。本项目的研究成果为IAH联合多种神经保护剂的临床转化提供了重要实验依据和发展思路，具有潜在的临床转化运用前景。针对IRI不同时相的分子网络机制，采用IAH联合多种神经保护药的鸡尾酒式疗法很可能是将来神经保护的希望所在。