Bone mesenchymal stem cells transplantation combined with mild hypothermia improves the prognosis of cerebral ischemia in rats

Bone marrow mesenchymal stem cells (BMSCs) are used as a great promising choice for the treatment of cerebral ischemia. Herein, we discuss the neuroprotective effects of the combination of BMSCs transplantation and mild hypothermia (MH) in an ischemia-reperfusion rat model. First, BMSCs were isolated using density gradient centrifugation and the adherent screening method, followed by culture, identification and labeling with DAPI. Second, adult male SD rats were divided into 5 groups: sham group (surgery without blockage of middle cerebral artery), model group (middle cerebral artery occlusion (MCAO) was established 2h prior to reperfusion), BMSCs group (injection of BMSCs via the lateral ventricle 24h after MCAO), MH group (mild hypothermia for 3h immediately after MCAO) and combination therapy group (combination of BMSCs and MH). Finally, the modified neurological severity score (mNSS) test was performed to assess behavioral function at different time points (before MCAO, before transplantation, at day 1, day 5 and day 10 after transplantation). After that, the brain was subjected to TTC staining, and the homing and angiogenesis were evaluated by immumofluorescence and immunohistochemistry. Immunofluorescence staining and Western Blot analysis were performed to calculate the percentage of the infarct area and explore glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF). Our results showed that the combination therapy significantly decreased mNSS scores (P<0.01) and reduced the percentage of the infarct area (P<0.01) than a single treatment. Moreover, the expression of GFAP and VEGF increased significantly in the combination therapy group (at day 5, day 10 after transplantation; at all time points after transplantation, respectively) compared to the single treatment groups. Taken together, it was suggested that the combination of BMSCs transplantation and MH can significantly reduce the percentage of the infarct area and improve functional recovery by promoting homing and angiogenesis, which may be a beneficial treatment for cerebral ischemia.

骨髓间充质干细胞（Bone marrow mesenchymal stem cells, BMSCs）是治疗脑缺血极具前景的候选方案。本研究探讨了骨髓间充质干细胞移植联合亚低温（mild hypothermia, MH）在缺血再灌注大鼠模型中的神经保护作用。首先，采用密度梯度离心法结合贴壁筛选法分离骨髓间充质干细胞，随后进行培养、鉴定并以DAPI进行标记。其次，将成年雄性SD大鼠分为5组：假手术组（仅行手术但不阻塞大脑中动脉）、模型组（缺血再灌注前2小时建立大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）模型）、BMSCs移植组（大脑中动脉闭塞后24小时经侧脑室注射骨髓间充质干细胞）、亚低温组（大脑中动脉闭塞后立即行3小时亚低温处理）以及联合治疗组（骨髓间充质干细胞移植联合亚低温治疗）。最后，在不同时间点（大脑中动脉闭塞前、移植前、移植后第1天、第5天及第10天）采用改良神经功能缺损评分（modified neurological severity score, mNSS）评估大鼠行为功能。随后取脑组织进行TTC染色，并通过免疫荧光与免疫组化技术评估细胞归巢与血管生成情况。采用免疫荧光染色与蛋白质印迹（Western Blot）分析计算脑梗死体积百分比，并检测胶质纤维酸性蛋白（glial fibrillary acidic protein, GFAP）与血管内皮生长因子（vascular endothelial growth factor, VEGF）的表达水平。本研究结果显示，相较于单一治疗组，联合治疗组的改良神经功能缺损评分显著降低（P<0.01），脑梗死体积百分比亦显著减少（P<0.01）。此外，相较于单一治疗组，联合治疗组的胶质纤维酸性蛋白与血管内皮生长因子表达水平分别在移植后第5天、第10天与移植后所有时间点显著升高。综上，骨髓间充质干细胞移植联合亚低温可通过促进细胞归巢与血管生成，显著降低脑梗死体积百分比并改善功能恢复，有望成为治疗脑缺血的有效方案。