中風期靜脈放血對於中風損傷之探討

Abstract

中風位居台灣十大死因第三名，同時也是造成殘障的主要原因。目前缺血性中風的主要治療策略為打通栓塞的腦內動脈，避免腦細胞因缺血而壞死。現時最有效的藥物為組織胞漿素原活化劑 (tPA)，而此藥物是唯一個獲得美國食品藥物局(FDA)核准的血栓溶解劑。但是並非每位患者皆能對tPA的血栓溶解作用產生反應，無法疏通血管的結果進一步使得病人在臨床的預後表現較差。血液稀釋為中風治療中的一種非標準治療方式，在臨床上通常是針對血溶比過高的病人進行。在動物實驗中，透過給予血漿代用劑進行血液稀釋被證實能夠減少中風後的組織受損程度。靜脈放血會導致低血容量性的血液稀釋效果，但它是否對缺血性中風俱有保護作用仍不清楚。本篇的研究目的即是在探討中風期的靜脈放血對於缺血性中風的影響。利用永久性栓塞的中風模式(permanent middle cerebral artery occlusion, pMCAO)，我們發現自發性高血壓大鼠(Spontaneously hypertensive rat, SHR) 在中風後三小時進行靜脈放血 0.8 mL，能顯著降低中風引起之梗死(infarction)及腦水腫情形，並改善中風後的神經功能缺損及運動功能障礙。在中風的正常血壓Wistar老鼠中進行靜脈放血，也同樣觀察到組織受損減少及神經功能缺損的改善。進一步探討靜脈放血保護性作用的可能機制，我們發現與中風後發炎反應相關的發炎介質: iNOS 及 COX-2之表現，在靜脈放血治療之大鼠中有減少的現象，表示中風後發炎反應有被抑制的情況。利用蛋白質陣列系統(Proteome array)和酵素免疫分析法(ELISA)分析中風後自發性高血壓大鼠之血清中發炎介質，我們推測巨噬細胞移動抑制因子(Macrophage migration inhibitory factor, MIF)在靜脈放血的保護作用中可能扮演了調控性的角色。因此，我們將MIF (10μg/kg) 以靜脈注射方式打入中風後的自發性高血壓大鼠，發現其加劇了與中風損傷相關的血腦障蔽破損。另外，皮下給予MIF拮抗劑：ISO-1 (3 mg/kg) 則能抑制中風引發之梗死(infarction)。這兩者都顯示MIF在缺血性中風俱有傷害性的作用。綜合以上的結論，在中風發作後3小時進行靜脈放血0.8 mL，能夠減少梗死及改善神經功能缺損。此神經保護作用可能是透過改變血清中發炎因子之平衡，而抑制中風後的發炎反應。我們的研究結果顯示了在無法疏通血管的情況下，靜脈放血對於缺血性中風後損傷能提供保護的作用，然而針對其保護機制及臨床之應用，有待更多後續的研究。

Stroke is a leading cause of long-term disability worldwide, and thrombolytic agent, tissue plasminogen activator (tPA), remains the only FDA-approved therapy for ischemic stroke. Unfortunately, some patients respond poorly to tPA and result in a worsen clinical outcome. Hemodilution using plasma expander has been reported to reduce brain infarction in experimental stroke model. Phlebotomy causes hypovolemic-hemodilting effect, but whether it offers protection after ischemic stroke remains unclear. Using permanent occlusion model, we aimed to evaluate the therapeutic potential of phlebotomy for ischemic stroke. Male spontaneously hypertensive rats (SHR) were subjected to permanent MCA occlusion (pMCAO) to produce cerebral ischemia, and the treatment group was subjected to phlebotomy by drawing 0.8 ml of whole blood after the MCA occlusion. Our results show that phlebotomy at 3 h after MCA occlusion significantly reduced stroke-induce infarction and brain edema compared with control group. However, phlebotomy at 6 h failed to demonstrate neuroprotection. Functional evaluation by modified neurological severity score and cylinder test also shows improvement in behavioral deficit in phlebotomy group. The reduction in infarction and the improvement in neurological score were also seen in Wistar rats under same treatment condition using three-vessels occlusion model. Assessment by immunoblot revealed that inflammatory responses were decreased in phlebotomy-3h group as the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the inflammatory mediators implicated in post-stroke inflammation, was reduced. We therefore aimed to investigate the possible mechanisms for the protective effect of phlebotomy. Analysis of the cytokine profile in serum after the occlusion using proteome profiler array and ELISA led us to presume Macrophage migration inhibitory factor (MIF) as a possible mediator of the protection by phlebotomy. The administration of MIF (10μg/kg, i.v.) aggravated the disruption of blood-brain barrier related to stroke injury; whereas its antagonist, ISO-1 (3 mg/kg, s.c.), inhibited stroke-induced infarction, indicating that MIF plays an important role in ischemic stroke. In conclusion, phlebotomy of whole blood offers protection when conducted 3 h after stroke onset by reducing infarction and improving neurological function and may exert its protective effect through the suppression of inflammatory cascade by attenuating the balance of inflammatory mediators in serum. These findings suggest that phlebotomy may offer protection after ischemic stroke, especially in tPA non-responders.