肠道菌群失调对血脑屏障的调控作用及其在恒河猴脑缺血再灌注损伤中的影响研究

Disruption of Blood-Brain barrier (BBB) plays an important role in pathophysiological process of ischemic stroke. However, the mechanism of its regulation remains elusive. Using a new method developed in our lab for serial sampling of cisterna magna CSF, we are able to determine the temporal change of BBB permeability after ischemia/reperfusion by calculating the albumin ratio (AR) between CSF and blood. Combining AR with dynamic contrast enhanced MRI (DCE-MRI), we can monitor the temporal and spatial change of BBB permeability during ischemia stroke and reperfusion. Moreover, the longitudinal study will enable us to perform correlation analysis between BBB permeability and any potential regulatory factors. Our previous data showed monkey gut microbiota disturbance induced by antibiotics could cause a reversible increase of BBB permeability. Considering the strong correlation between the severity of BBB permeability and the stroke outcome, we hypothesize that gut microbiota dysbiosis induced by antibiotics could have a negative effect on stroke outcome via opening of BBB. To test this hypothesis, we are going to treat rhesus monkeys with broad-spectrum antibiotics within the first 24 hours after ischemia and reperfusion, determine the temporal-spatial change of BBB permeability, and do correlation analysis between gut microbiota diversity, BBB permeability, and stroke outcome. If the correlations that we anticipate do exist, we will try to re-induce the gut microbiota by fecal transplantation or oral administration of short chain fatty acids (SCFA). The current study will help to elucidate the possible regulatory effect of gut microbiota on BBB permeability and the outcome of ischemic stroke. Moreover, it could provide support for evaluating the risk-benefit ratio of preventative antibiotic therapy (PTA) for stroke patients.

血脑屏障在缺血再灌注损伤中扮演了重要角色，但是其调控机制却不够明晰。利用能在清醒状态下对恒河猴枕大池脑脊液动态采集的自创方法，测定脑脊液/血清白蛋白比值；辅助以动态增强扫描核磁共振测定容积转运常数，我们可以对恒河猴血脑屏障通透性的时间变化和空间分布做精准的检测。我们的前期数据显示抗生素可诱导恒河猴肠道菌群失调，并导致血脑屏障通透性的可逆性增加。而血脑屏障通透性和脑卒中预后具有较强相关关系，提示肠道菌群的紊乱极有可能通过破坏血脑屏障而对脑卒中预后造成负面影响。在本研究中，我们拟对缺血再灌注损伤恒河猴在再灌注24小时内进行抗生素干预，降低肠道菌群多样性，测定其对BBB通透性的调节以及预后的最终影响。如果该影响确实存在，我们将使用动物自体粪便移植以及短链脂肪酸灌胃等方法尝试逆转。本研究有助于阐明肠道微生态对BBB调节的因果关系，并为脑卒中相关感染的预防性抗生素治疗提供风险效益评估依据。

血脑屏障损伤是缺血性脑卒中发病的重要病理生理过程，然而其具体调控机制不详。近年来研究发现肠道微生态失调可能与BBB调控有关。该课题主要目标是通过健康恒河猴肠道菌群失调和血脑屏障通透性的关系，确认菌群失调对缺血性脑卒中预后的影响，以及SCFA灌胃的干预作用。在课题的执行过程中，我们发现抗生素诱导的健康恒河猴和小鼠肠道菌群失调可暂时增大BBB的通透性。给予恒河猴和大鼠MCAO模型口服抗生素阿莫西林克拉维酸钾，均可发现脑损伤较MCAO模型更为严重。在肠道菌群失调的情况下，口服给予SCFA，尤其是乙酸和丙酸，可以在一定程度上缓解BBB的通透性升高以及MCAO结局。基于此，我们的假说基本得到验证。在技术上，我们完善了BBB通透性的时间和空间动态检测、脑脊液的动态采集等创新方法，完成了申报书中计划的绝大部分关键实验。但由于项目执行期间恒河猴的价格大幅增长，超过申报前的十倍有余，使得我们仅完成两例恒河猴MCAO模型实验，改为通过啮齿类动物实验完成研究内容。本项目所用到的模型和技术的新方法，在产学研创新合作方面做出了很好的成绩，进行了大量临床前新药评价服务，在市场中极具竞争力。在基础研究方面，作为BBB的中枢外调节因素，肠道菌群失调可成为缺血性脑卒中发病的新机制，靶向肠道菌群的SCFA有望成为改善血脑屏障通透性以及缺血性脑卒中预后的干预手段，为缺血性脑卒中的诊断和治疗提供理论性实验基础。