S1PR调节剂治疗脑出血的时间窗及机制研究

Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke, causing high mortality, morbidity, and disability. Secondary injury and the development of perihematomal edema (PHE) exacerbate tissue damage and neurologic deterioration over an extended window of hours to days after the onset of ICH. Currently, there are no effective treatments to prevent or reverse PHE and secondary damage. The inflammatory responses following ICH contribute to the genesis of PHE and aggravate its mass effect. Thus targeting inflammation could be a viable approach to attenuate PHE and second injury during ICH. Fingolimod, a sphingosine 1-phosphate receptor (S1PR) modulator, has been approved for relapsing-remitting multiple sclerosis in 2010. Because fingolimod inhibits the egress of several lymphocyte subpopulations bearing S1PRs and limit their recirculation, we reasoned that fingolimod may reduce PHE and improve the outcome of patients with ICH. For this purpose, our group recently completed a proof-of-concept study examining the safety and efficacy of fingolimod in patients with ICH. We found that three dosages of oral fingolimod reduced circulating lymphocytes and PHE, attenuate neurologic deficits, and promote recovery. To advance S1PR modulation as a potential therapy of ICH, several questions must be addressed. First, edema grows rapidly during the first day, but continue to progress at a slow pace for 2 weeks. It remains unclear when immune modulation should be initiated and how long such interventions should last to achieve better clinical outcome in ICH patients. Second, the mechanims by S1PR modulator ameliorates ICH- induced brain injury, whether additional beneficial effect on the BBB may come from the nonimmunological mechanism of action of S1P1 modulator; Third, because S1PR1 are broadly expressed in endothelial cells and astrocytes, two of the principal cellular components of the BBB, it is important to identify S1PR1 is sufficient and/or necessary for S1PR modulator to provide beneficial effects in ICH. We will adopt two experimental models of ICH induced by injection of autologous blood or collagenase, and utilize young and aged mice to address this hypothesis. Our specific aims are: (1) To determine the optimal timing for S1PR modulation in experimental ICH. (2) To determine how S1PR modulations attenuate brain injury after ICH. (3) To assess novel S1PR1 modulators with superior efficacy and safety profile for clinical translation. The outcome of our study will improve the understanding of immunology in ICH, bridge the gap between preclinical and clinical studies and advance immune modulation as a potential viable approach for ICH treatment.

脑出血后脑部炎性反应促进脑水肿的形成，由于脑水肿的占位效应引起脑疝导致死亡或加重患者的残疾程度；脑水肿的大约2周达到高峰，因此给临床干预提供了可能性。但是目前无控制脑水肿生成的药物。在前期临床试验中，我们发现给发病72小时内的脑出血患者3天非选择S1PR的免疫调节剂fingolimod，患者的血脑屏障渗出受到抑制，脑水肿减轻，临床症状改善。为了更好促进S1PR的调节剂的脑出血临床转化，3个问题需要明确：最佳治疗时间窗；是否有中枢作用；是否S1PR1依赖。为了回答这些问题，我们会利用药物在不同时间删除免疫细胞来挑选S1PR调节剂治疗脑出血的最佳治疗时间；另外用免疫细胞缺陷小鼠来验证S1PR治疗的作用是否与中枢作用相关；最后用血管内皮细胞和星形胶质细胞的S1PR1 的条件敲除小鼠来判断这种作用是否S1PR1依赖。通过该实验将深化对脑出血的免疫炎性机制的认识，促进免疫干预脑出血的临床转化.

脑卒中分为出血性脑卒中和缺血性脑卒中，对于出血性脑卒中，脑出血后脑部炎性反应促进脑水肿的形成，由于脑水肿的占位效应引起脑疝导致死亡或加重患者的残疾程度。但目前尚无控制脑水肿生成的药物。在前期临床试验中，我们发现给发病72小时内的脑出血患者连续使用3天的S1PR的免疫调节剂fingolimod，患者的血脑屏障渗出受到抑制，脑水肿减轻，临床症状改善。为促进S1PR的免疫调节剂的脑出血临床转化，通过扫描SWI和T2序列观测脑出血小鼠模型的脑水肿体积的动态演变过程发现脑内水肿体积在3天时达到高峰，3-7天水肿体积消退明显，14天后基本消失，确立最佳观测时间点为造模后第3天。进一步研究发现fingolimod可通过降低外周免疫细胞抑制脑出血小鼠模型的急性期炎症细胞，改善出血后的脑水肿。在第1天和第3天通过核磁观测脑出血小鼠脑部水肿体积，发现免疫缺陷小鼠水肿体积有小于野生小鼠的趋势，这证明S1PR的调节剂可能通过中枢作用抑制脑水肿。.对于缺血性脑卒中，脑梗死后脑部炎性反应参与血管损伤和凝血作用，促进炎性血栓形成，由于炎性血栓的梗阻效应引起病损区小血管再灌注障碍导致梗死体积扩大，加重患者的残疾程度。但目前无控制炎性血栓形成的药物。对此，我们首先建立了超小型超顺磁性氧化铁( USPIO)增强磁敏感加权成像 (USPIO-SWI)体系，发现当 USPIO剂量为16.8mg /kg时，USPIO-SWI序列可清楚成像大鼠脑内小动脉，且此剂量下评估eMCAO大鼠脑动脉造影再灌注水平是可行的。 使用大鼠脑栓塞模型，从24小时内的梗死体积扩大和神经功能评分2个角度明确了免疫调节剂降低外周免疫细胞具有增强tPA溶栓的治疗作用。利用流式细胞术和组织免疫荧光染色技术进一步明确微血栓形成与病损区的小血管前向和后向灌注的变化相关。本研究回答了脑卒中后的免疫炎性形成的关键问题，也为脑卒中治疗提供了新的干预途径。