Suenaga Jun, Hu Xiaoming, Pu Hongjian, Shi Yejie, Hassan Sulaiman Habib, Xu Mingyue, Leak Rehana K, Stetler R Anne, Gao Yanqin, Chen Jun
Center of Cerebrovascular Disease Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
Center of Cerebrovascular Disease Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; The State Key Laboratory of Medical Neurobiology, The Institutes of Brain Science and the Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China; Geriatric Research, Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, PA 15261, USA.
Exp Neurol. 2015 Oct;272:109-19. doi: 10.1016/j.expneurol.2015.03.021. Epub 2015 Mar 31.
Most of the successes in experimental models of stroke have not translated well to the clinic. One potential reason for this failure is that stroke mainly afflicts the elderly and the majority of experimental stroke studies rely on data gathered from young adult animals. Therefore, in the present study we established a reliable, reproducible model of stroke with low mortality in aged (18month) male mice and contrasted their pathophysiological changes with those in young (2month) animals. To this end, mice were subjected to permanent tandem occlusion of the left distal middle cerebral artery (dMCAO) with ipsilateral common carotid artery occlusion (CCAO). Cerebral blood flow (CBF) was evaluated repeatedly during and after stroke. Reduction of CBF was more dramatic and sustained in aged mice. Aged mice exhibited more severe long-term sensorimotor deficits, as manifested by deterioration of performance in the Rotarod and hanging wire tests up to 35d after stroke. Aged mice also exhibited significantly worse long-term cognitive deficits after stroke, as measured by the Morris water maze test. Consistent with these behavioral observations, brain infarct size and neuronal tissue loss after dMCAO were significantly larger in aged mice at 2d and 14d, respectively. The young versus aged difference in neuronal tissue loss, however, did not persist until 35d after dMCAO. In contrast to the transient difference in neuronal tissue loss, we found significant and long lasting deterioration of white matter in aged animals, as revealed by the loss of myelin basic protein (MBP) staining in the striatum at 35d after dMCAO. We further examined the expression of M1 (CD16/CD32) and M2 (CD206) markers in Iba-1(+) microglia by double immunofluorescent staining. In both young and aged mice, the expression of M2 markers peaked around 7d after stroke whereas the expression of M1 markers peaked around 14d after stroke, suggesting a progressive M2-to-M1 phenotype shift in both groups. However, aged mice exhibited significantly reduced M2 polarization compared to young adults. Remarkably, we discovered a strong positive correlation between favorable neurological outcomes after dMCAO and MBP levels or the number of M2 microglia/macrophages. In conclusion, our studies suggest that the distal MCAO stroke model consistently results in ischemic brain injury with long-term behavioral deficits, and is therefore suitable for the evaluation of long-term stroke outcomes. Furthermore, aged mice exhibit deterioration of functional outcomes after stroke and this deterioration is linked to white matter damage and reductions in M2 microglia/macrophage polarization.
中风实验模型中的大多数成功案例在临床应用中效果不佳。这种失败的一个潜在原因是中风主要影响老年人，而大多数实验性中风研究依赖于从年轻成年动物收集的数据。因此，在本研究中，我们建立了一种可靠、可重复且死亡率低的老年（18个月）雄性小鼠中风模型，并将其病理生理变化与年轻（2个月）动物的变化进行对比。为此，对小鼠进行左侧大脑中动脉远端（dMCAO）与同侧颈总动脉闭塞（CCAO）的永久性串联闭塞。在中风期间和之后反复评估脑血流量（CBF）。老年小鼠的CBF减少更为显著且持续时间更长。老年小鼠表现出更严重的长期感觉运动缺陷，如在中风后35天内转棒试验和悬线试验中的表现恶化所示。通过莫里斯水迷宫试验测量，老年小鼠中风后的长期认知缺陷也明显更严重。与这些行为观察结果一致，dMCAO后2天和14天，老年小鼠的脑梗死面积和神经元组织损失分别显著更大。然而，神经元组织损失的年轻与老年差异在dMCAO后35天之前并未持续存在。与神经元组织损失的短暂差异相反，我们发现老年动物的白质出现显著且持久的恶化，如dMCAO后35天纹状体中髓鞘碱性蛋白（MBP）染色缺失所示。我们通过双重免疫荧光染色进一步检测了Iba-1(+)小胶质细胞中M1（CD16/CD32）和M2（CD206）标志物的表达。在年轻和老年小鼠中，M2标志物的表达在中风后约7天达到峰值，而M1标志物的表达在中风后约14天达到峰值，表明两组均存在从M2到M1表型的逐渐转变。然而，与年轻成年小鼠相比，老年小鼠的M2极化显著降低。值得注意的是，我们发现dMCAO后良好的神经学结果与MBP水平或M2小胶质细胞/巨噬细胞数量之间存在强烈的正相关。总之，我们的研究表明，远端MCAO中风模型始终会导致缺血性脑损伤并伴有长期行为缺陷，因此适用于评估长期中风结果。此外，老年小鼠中风后的功能结果会恶化，这种恶化与白质损伤以及M2小胶质细胞/巨噬细胞极化减少有关。
