小鼠急性缺血性脑中风后小胶质细胞增生来源的研究
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摘要
小胶质细胞是中枢神经系统(Central nervous system, CNS)重要的免疫效应细胞,它们通过监测微环境的变化,参与维持中枢神经系统的稳态平衡。缺血性脑中风能引起小胶质细胞的迅速激活,活化小胶质细胞在形态、抗原表达、功能行为等方面发生改变,尤其是他们在损伤区发生细胞增殖和聚集现象,小胶质细胞的这些反应被称为“小胶质细胞增生”。目前对这些增殖的小胶质细胞的来源仍有争议,有研究者认为小胶质细胞增生主要是通过小胶质细胞的自我更新得以维持。而另外一些报道称骨髓来源的一些祖细胞参与了小胶质细胞增生的过程,但这些研究都是基于辐射骨髓移植动物模型,而辐射和骨髓移植可能会带来血脑屏障的破坏等人为的影响,因此这些现象在生理条件下能否发生仍不清楚。
本论文利用中风这一通常病理条件下既有血脑屏障破坏的的疾病模型,研究了小胶质细胞在缺血后增生的主要来源。为了避免辐射等人为因素对动物正常生理活动的影响,本实验建立了两种血液嵌合模型,并利用双光子成像技术研究了缺血性脑中风后亚急性期内增殖小胶质细胞的起源和动态变化,以及诱导其激活和增殖的影响因素。本论文的研究结果表明,正常健康动物体内血液细胞无法通过完整的血脑屏障。然而脑中风血脑屏障受损后,少量血液来源的Cx3crlGFP/+细胞能迁移进入大脑实质。虽然这些迁入的细胞与脑实质内小胶质细胞一样有荧光标记,但是它们表现出与本地小胶质细胞不同的表型特征和动力学反应。缺血损伤后进入脑实质的血源细胞数量很少,前5天发生迁移的细胞数量增加,随后逐渐减少。由于这些迁移细胞无法进行自我增殖,并且随着损伤时间的推移逐渐发生凋亡,因此血源迁移细胞无法长期存活。与此相反,免疫组织化学染色和双光子活体成像结果显示,缺血激活的本地小胶质细胞持续向损伤区聚集并能进行分裂增殖,而且这些细胞在损伤后一周内保持持续增加的趋势。因此,本地小胶质细胞的自我增殖是小胶质细胞增生的主要来源。另外,研究还发现血源Cx3erlGFP/+细胞的迁移和本地小胶质细胞的激活和增殖与损伤区血脑屏障的开放范围有关。本论文的研究结果揭示了血源Cx3erlGFP/+迁移细胞和本地小胶质细胞代表着两类不同的细胞种群,这两类细胞可能具有不同的功能和治疗潜力。
Microglia are the main immunocompetent cells in the central nervous system (CNS) which continuously monitor the microenvironment to maintain the homeostasis. Ischemic stroke induces rapid activation of microglia. Activated microglia alter their morphology, cell surface antigen expression and functional behaviour, and their numbers increase markedly at the site of ischemia. These responses of microglia are collectively termed "microgliosis". The main source of microgliosis remains controversial. Some studies suggest that microgliosis are maintained through self-expansion, but others suggest that bone marrow-derived progenitors may contribute to microgliosis. However, these reports are based on experiments in which animals suffered lethal irradiation and their bone marrow was artificially replaced with exogenous cells. It is unclear whether a similar phenomenon could be observed under physiological conditions.
Here we investigated the origin and kinetics of reactive microglia using a photothrombosis stroke model, which is characterized by pathological disruption of blood brain barrier (BBB) permeability without intervention of irradiation or transplantation. To avoid the potential influence of irradiation or transplantation, we established a model of blood chimera using parabiotic animals and examined the source and dynamics of activated microglia in subacute phase after ischemic stroke by using in vivo two-photon microscopy. We found there was no infiltration of blood-derived cells with an intact BBB in healthy mice. However, a small population of blood-derived Cx3crlGFP/+cells were detected in the lesion sites of cerebral parenchyma when the BBB integrity was damaged after ischemia. Although these infiltrating cells also expressed Green fluorescence protein (GFP), they displayed different phenotypes and kinetics from reactive microglia. The number of infiltrating cells increased in the first5days after stroke and then subsequently decreased. We found these Cx3crlGFP/+infiltrating cells did not proliferate and were gradually lost through apoptosis. In contrast, immunohistology and in vivo imaging revealed that activated microglia renewed themselves and were recruited to ischemic area continuously, and their number increased in the first week after stroke. These results indicated that microglial proliferation rather than infiltration of Cx3crlGFP/+circulating cells is the main source of microgliosis after ischemic stroke. In addition, we found that the permeability of BBB was associated with the migration of Cx3crlGFP/+infiltrating cells and the activation and proliferation of resident microglia. Together, our data suggest that the Cx3crlGFP/+infiltrating cells and reactive microglia may represent two distinct populations of cells with different function and therapeutic potential for the treatment of stroke.
本论文利用中风这一通常病理条件下既有血脑屏障破坏的的疾病模型,研究了小胶质细胞在缺血后增生的主要来源。为了避免辐射等人为因素对动物正常生理活动的影响,本实验建立了两种血液嵌合模型,并利用双光子成像技术研究了缺血性脑中风后亚急性期内增殖小胶质细胞的起源和动态变化,以及诱导其激活和增殖的影响因素。本论文的研究结果表明,正常健康动物体内血液细胞无法通过完整的血脑屏障。然而脑中风血脑屏障受损后,少量血液来源的Cx3crlGFP/+细胞能迁移进入大脑实质。虽然这些迁入的细胞与脑实质内小胶质细胞一样有荧光标记,但是它们表现出与本地小胶质细胞不同的表型特征和动力学反应。缺血损伤后进入脑实质的血源细胞数量很少,前5天发生迁移的细胞数量增加,随后逐渐减少。由于这些迁移细胞无法进行自我增殖,并且随着损伤时间的推移逐渐发生凋亡,因此血源迁移细胞无法长期存活。与此相反,免疫组织化学染色和双光子活体成像结果显示,缺血激活的本地小胶质细胞持续向损伤区聚集并能进行分裂增殖,而且这些细胞在损伤后一周内保持持续增加的趋势。因此,本地小胶质细胞的自我增殖是小胶质细胞增生的主要来源。另外,研究还发现血源Cx3erlGFP/+细胞的迁移和本地小胶质细胞的激活和增殖与损伤区血脑屏障的开放范围有关。本论文的研究结果揭示了血源Cx3erlGFP/+迁移细胞和本地小胶质细胞代表着两类不同的细胞种群,这两类细胞可能具有不同的功能和治疗潜力。
Microglia are the main immunocompetent cells in the central nervous system (CNS) which continuously monitor the microenvironment to maintain the homeostasis. Ischemic stroke induces rapid activation of microglia. Activated microglia alter their morphology, cell surface antigen expression and functional behaviour, and their numbers increase markedly at the site of ischemia. These responses of microglia are collectively termed "microgliosis". The main source of microgliosis remains controversial. Some studies suggest that microgliosis are maintained through self-expansion, but others suggest that bone marrow-derived progenitors may contribute to microgliosis. However, these reports are based on experiments in which animals suffered lethal irradiation and their bone marrow was artificially replaced with exogenous cells. It is unclear whether a similar phenomenon could be observed under physiological conditions.
Here we investigated the origin and kinetics of reactive microglia using a photothrombosis stroke model, which is characterized by pathological disruption of blood brain barrier (BBB) permeability without intervention of irradiation or transplantation. To avoid the potential influence of irradiation or transplantation, we established a model of blood chimera using parabiotic animals and examined the source and dynamics of activated microglia in subacute phase after ischemic stroke by using in vivo two-photon microscopy. We found there was no infiltration of blood-derived cells with an intact BBB in healthy mice. However, a small population of blood-derived Cx3crlGFP/+cells were detected in the lesion sites of cerebral parenchyma when the BBB integrity was damaged after ischemia. Although these infiltrating cells also expressed Green fluorescence protein (GFP), they displayed different phenotypes and kinetics from reactive microglia. The number of infiltrating cells increased in the first5days after stroke and then subsequently decreased. We found these Cx3crlGFP/+infiltrating cells did not proliferate and were gradually lost through apoptosis. In contrast, immunohistology and in vivo imaging revealed that activated microglia renewed themselves and were recruited to ischemic area continuously, and their number increased in the first week after stroke. These results indicated that microglial proliferation rather than infiltration of Cx3crlGFP/+circulating cells is the main source of microgliosis after ischemic stroke. In addition, we found that the permeability of BBB was associated with the migration of Cx3crlGFP/+infiltrating cells and the activation and proliferation of resident microglia. Together, our data suggest that the Cx3crlGFP/+infiltrating cells and reactive microglia may represent two distinct populations of cells with different function and therapeutic potential for the treatment of stroke.
引文
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