小鼠脑出血后不同时间点小胶质细胞极化状态的实验研究

目的 观察小鼠脑出血后不同时间点小胶质细胞M1及M2型的转化，为促炎型M1型小胶质细胞向抗 炎修复型M2型小胶质细胞的转化，减轻脑出血后神经功能损伤提供理论依据。 方法 选取健康雄性ICR小鼠48只，随机分为假手术组、脑出血组，每组按术后时间点不同随机 分为1 d、3 d、7 d三个时间点，每个时间点8只。通过立体定位仪用微量注射器向尾状核注射Ⅳ型胶 原酶0.5 U制备脑出血模型，假手术组注射等量生理盐水。各组于术后对应时间点参照改良Garcia 评分量表进行神经功能缺损评分后灌注取脑，采用蛋白免疫印迹检测M1型小胶质细胞标志物肿瘤 坏死因子α（tumor necrosis factor-α，TNF-α）、白细胞介素6（interleukin-6，IL-6），M2型小胶质细胞 标志物脑源性神经营养因子（brai n-derived neurotrophic factor，BDNF）、胰岛素样生长因子1（insulinlike growth factor 1，IGF-1）的含量；采用免疫荧光染色标记小胶质细胞M1型（Iba1+CD80）、M2型 （Iba1+CD206），评价出血后血肿周围组织小胶质细胞活化状态。 结果 脑出血组1 d、3 d、7 d各时间点Garci a评分均较假手术组低，TNF-α、IL-6、BDNF及I GF-1的蛋白 表达量均较假手术组增多（均P＜0.01）。脑出血后1 d时M1型高于M2型小胶质细胞数量（38.33±1.53 vs 23.00±3.00，P =0.01）；3 d时M1型同样高于M2型（66.33±3.06 vs 57.33±2.52，P =0.02）；7 d时M1 型低于M2型（33.67±1.15 vs 52.33±0.58，P＜0.01）。 结论 脑出血急性期（1～3 d）以M1型小胶质细胞为主，脑出血亚急性期（7 d）以M2型小胶质细胞 为主。     

小胶质细胞在出血性脑卒中发病机制中的研究进展

出血性脑卒中(intracerebral haemorrhage,ICH)是脑卒中致死率最高的类型,目前临床对其仍缺乏有效的治疗方法。小胶质细胞是ICH后第一个产生免疫应答的中枢神经系统细胞。ICH急性期脑损伤后,小胶质细胞可被诱导为经典的M1型(促炎作用)或补充替代的M2型(抗炎作用),其中,M1型抑制中枢神经系统的修复,M2型通过分泌抗炎因子和神经营养因子来促进组织的再生和修复。同时,小胶质细胞与星形胶质细胞、神经元、少突胶质细胞以及T淋巴细胞生理病理上具有紧密联系,其M1型和M2型极化与其他神经细胞产生不同的交互作用,这些均在ICH的病理过程中具有至关重要的作用。基于此,本文对ICH后小胶质细胞和其他神经细胞的相互作用的关键分子机制进行综述。     

小鼠脑出血后不同时间点小胶质细胞极化状态的实验研究

目的 观察小鼠脑出血后不同时间点小胶质细胞M1及M2型的转化，为促炎型M1型小胶质细胞向抗
炎修复型M2型小胶质细胞的转化，减轻脑出血后神经功能损伤提供理论依据。
方法 选取健康雄性ICR小鼠48只，随机分为假手术组、脑出血组，每组按术后时间点不同随机
分为1 d、3 d、7 d三个时间点，每个时间点8只。通过立体定位仪用微量注射器向尾状核注射Ⅳ型胶
原酶0.5 U制备脑出血模型，假手术组注射等量生理盐水。各组于术后对应时间点参照改良Garcia
评分量表进行神经功能缺损评分后灌注取脑，采用蛋白免疫印迹检测M1型小胶质细胞标志物肿瘤
坏死因子α（tumor necrosis factor-α，TNF-α）、白细胞介素6（interleukin-6，IL-6），M2型小胶质细胞
标志物脑源性神经营养因子（brai n-derived neurotrophic factor，BDNF）、胰岛素样生长因子1（insulinlike
growth factor 1，IGF-1）的含量；采用免疫荧光染色标记小胶质细胞M1型（Iba1+CD80）、M2型
（Iba1+CD206），评价出血后血肿周围组织小胶质细胞活化状态。
结果 脑出血组1 d、3 d、7 d各时间点Garci a评分均较假手术组低，TNF-α、IL-6、BDNF及I GF-1的蛋白
表达量均较假手术组增多（均P＜0.01）。脑出血后1 d时M1型高于M2型小胶质细胞数量（38.33±1.53
vs 23.00±3.00，P =0.01）；3 d时M1型同样高于M2型（66.33±3.06 vs 57.33±2.52，P =0.02）；7 d时M1
型低于M2型（33.67±1.15 vs 52.33±0.58，P＜0.01）。
结论 脑出血急性期（1～3 d）以M1型小胶质细胞为主，脑出血亚急性期（7 d）以M2型小胶质细胞
为主。     

小胶质细胞在创伤性脑损伤后癫痫中的作用研究进展

创伤性脑损伤(TBI)是一种严重威胁人们生命健康的疾病, 而TBI后癫痫(PTE)是其严重的后遗症之一。由于PTE发生的病理生理机制尚未阐明, 目前尚无有效的预防和治疗方法。TBI触发了强烈且持久的炎症级联反应, 提示神经炎症可能与PTE的发病机制有关。而小胶质细胞, 作为大脑中最常见的免疫细胞, 在神经炎症中发挥着重要作用。小胶质细胞在TBI后被激活, 可表达促炎或抗炎等表型。不同的极化状态与各种促炎或抗炎型介质的释放有关, 且不同程度上影响着大脑的修复和PTE的发生与发展。鉴于不同表型的小胶质细胞在神经炎症中发挥的作用不同, 调节小胶质细胞的极化方向可能对TBI和PTE患者的治疗具有重要意义。本文综述了TBI后不同时间点小胶质细胞的表型和功能, 深入探究小胶质细胞极化的信号通路和导致癫痫发生的潜在机制, 以及总结了防治TBI和PTE的药物研究进展, 以期为PTE动物实验的临床转化奠定基础。     

小胶质细胞激活与脑出血后继发性脑损伤机制的研究进展

最近研究发现,小胶质细胞激活参与了脑出血后的继发性损伤。激活的小胶质细胞大量分泌促炎性因子、基质金属蛋白酶和血红素加氧酶-1等,这些物质加重了脑出血后的炎症反应和氧化应激,导致明显的脑水肿和神经功能障碍。应用药物抑制小胶质细胞激活,减少上述物质的表达,明显减轻了脑出血大鼠的脑水肿和提高了行为学评分。本文对小胶质细胞激活参与脑出血继发性损伤的机制作一综述,以期为脑出血的治疗提供新的思路。     

小胶质细胞在中枢神经系统创伤后的双重作用及调控机制

小胶质细胞是中枢神经系统的固有免疫细胞,在脑或脊髓创伤后的神经炎症反应中起关键作用。神经系统损伤后小胶质细胞可提供神经保护因子,清除细胞碎片并调控神经修补过程。而另一方面,小胶质细胞会产生高水平的促炎及细胞毒性介质从而阻碍CNS修复,促使神经元失能及细胞死亡。小胶质细胞的双重特性可能与其损伤后的表型及功能反应有关。本综述探讨近年来有关脑和脊髓损伤后小胶质细胞活化表型的研究,以及小胶质细胞在神经元、血管、少突胶质细胞生长及再生中的可能发挥的作用。并简述已知的调控表型转换的分子机制,着重探讨可以影响小胶质细胞活化状态的治疗途径。了解小胶质细胞表型调控机制有助于我们增加神经系统损伤恢复的知识,并提供新的治疗策略。     

microRNAs调控小胶质细胞极化在神经炎症中的作用

microRNAs(miRNAs)是一类长度约为19-25个核苷酸稳定的内源性小分子非编码RNA,小胶质细胞是广泛分布于中枢神经系统的常驻免疫细胞,miRNAs与小胶质细胞的极化有着密切的关系。本文综述近年来促炎型/抗炎型miRNAs调控小胶质细胞M1型/M2型极化及其在相关炎症介导的神经系统疾病中的研究进展,充分阐明小胶质细胞极化过程中miRNAs调控机制,为寻找治疗与小胶质极化相关炎症介导的神经系统疾病的新靶点提供了有力的理论依据。     

小胶质细胞与恶性胶质瘤相互关系的研究进展

近年来,大量研究显示小胶质细胞与胶质瘤的发生及发展有着密切的关系。恶性胶质瘤细胞不但能通过分泌细胞因子,作用于小胶质细胞表面相应的受体促进其向胶质瘤中浸润,还能够诱导其向M2型极化,参与胶质瘤免疫抑制微环境的形成。同样,小胶质细胞也能够促进胶质瘤细胞的增殖、迁移、药物耐受等恶性生物学行为。本文就小胶质细胞与恶性胶质瘤之间相互关系的研究做一综述,以期为恶性胶质瘤的研究及治疗提供新的思路。     

芬戈莫德调控小胶质细胞表型转化对少突胶质前体细胞的影响

目的 研究芬戈莫德(FTY720)对原代培养的小胶质细胞表型的影响及干预后的小胶质细胞对少突胶质前体细胞分化成熟的影响。方法 体外培养原代小胶质细胞及少突胶质前体细胞(OPCs),采用RT-PCR法检测FTY720干预后小胶质细胞M1及M2型指标的变化; ELISA法检测小胶质细胞上清TNF-α、TGF-β、IL-1β、IL-13分泌量的变化; 将干预后的小胶质细胞上清液加入纯化后的少突胶质前体细胞中采用Olig2与MBP共染免疫荧光染色方法观察FTY720对少突胶质前体细胞分化成熟的影响。结果 向M2型转化; FTY720干预后的小胶质细胞促进少突胶质前体细胞的分化成熟。结论 FTY720通过促进小胶质细胞向M2型转化,从而促进少突胶质前体细胞的分化。     

小胶质细胞/巨噬细胞在胶质母细胞瘤中作用的研究进展

胶质母细胞瘤（GBM）是成人最常见的颅内恶性肿瘤，具有极强的侵袭性。此外，由于化疗药物难以通过血脑屏障和胶质母细胞瘤的耐药性，及对放疗敏感性较差等特点，故预后极差。其中肿瘤微环境的改变起到了至关重要的作用，在微环境中胶质瘤相关的小胶质细胞/巨噬细胞（GAMs）的作用正逐渐被重视。GAMs不仅有中枢系统的常驻小胶质细胞，还有来自外周的巨噬细胞。GAMs还有截然不同的两种极化类型，即抑制肿瘤生长的M1表型和促进肿瘤生长的M2表型。并且GAMs不单单和肿瘤细胞具有联系，还与微环境中其他非癌性脑细胞也有互动。该文将从GAMs的来源、极化、与肿瘤微环境中各种细胞间的相互影响阐述其在GBM中的作用，并且从靶向治疗的角度探讨其最新的研究进展。     

Endocannabinoids drive the acquisition of an alternative phenotype in microglia

The ability of microglia to acquire diverse states of activation, or phenotypes, reflects different features that are determinant for their contribution to homeostasis in the adult CNS, and their activity in neuroinflammation, repair or immunomodulation. Despite the widely reported immunomodulatory effects of cannabinoids in both the peripheral immune system and the CNS, less is known about how the endocannabinoid signaling system (eCBSS) influence the microglial phenotype. The general aim of the present study was to investigate the role of endocannabinoids in microglia polarization by using microglia cell cultures. We show that alternative microglia (M2a) and acquired deactivated microglia (M2c) exhibit changes in the eCB machinery that favor the selective synthesis of 2-AG and AEA, respectively. Once released, these eCBs might be able to act through CB1 and/or CB2 receptors in order to influence the acquisition of an M2 phenotype. We present three lines of evidence that the eCBSS is critical for the acquisition of the M2 phenotype: (i) M2 polarization occurs on exposure to the two main endocannabinoids 2-AG and AEA in microglia cultures; (ii) cannabinoid receptor antagonists block M2 polarization; and (iii) M2 polarization is dampened in microglia from CB2 receptor knockout mice. Taken together, these results indicate the interest of eCBSS for the regulation of microglial activation in normal and pathological conditions.     

NADPH oxidase isoform expression is temporally regulated and may contribute to microglial/macrophage polarization after spinal cord injury

Spinal cord injury (SCI) results in both acute and chronic inflammation, as a result of activation of microglia, invasion of macrophages and activation of the NADPH oxidase (NOX) enzyme. The NOX enzyme is a primary source of reactive oxygen species (ROS) and is expressed by microglia and macrophages after SCI. These cells can assume either a pro- (M1) or anti-inflammatory (M2) polarization phenotype and contribute to tissue response to SCI. However, the contribution of NOX expression and ROS production to this polarization and vice versa is currently undefined. We therefore investigated the impact of SCI on NOX expression and microglial/macrophage polarization over time in a mouse model of contusion injury. Adult C57Bl/6 mice were exposed to a moderate T9 contusion SCI and tissue was assessed at acute, sub-acute and chronic time points for NOX isoform expression and co-expression with M1 and M2 microglia/macrophage polarization markers. Two NOX isoforms were increased after injury and were associated with both M1 and M2 markers, with an M1 preference for NOX2 acutely and NOX4 chronically. M2 cells were primarily found at acute time points only; the peak of NOX2 expression was associated with the decline in M2 polarization. In vitro, NOX2 inhibition shifted microglial polarization toward the M2 phenotype. These results now show that microglial/macrophage expression of NOX isoforms is independent of polarization state, but that NOX activity can influence subsequent polarization. These data can contribute to the therapeutic targeting of NOX as a therapy for SCI.     

Microglia and macrophages differentially modulate cell death after brain injury caused by oxygen‐glucose deprivation in organotypic brain slices

Macrophage can adopt several phenotypes, process call polarization, which is crucial for shaping inflammatory responses to injury. It is not known if microglia, a resident brain macrophage population, polarizes in a similar way, and whether specific microglial phenotypes modulate cell death in response to brain injury. In this study, we show that both BV2‐microglia and mouse bone marrow derived macrophages (BMDMs) were able to adopt different phenotypes after LPS (M1) or IL‐4 (M2) treatment in vitro, but regulated cell death differently when added to mouse organotypic hippocampal brain slices. BMDMs induced cell death when added to control slices and exacerbated damage when combined with oxygen–glucose deprivation (OGD), independently of their phenotype. In contrast, vehicle‐ and M2‐BV2‐microglia were protective against OGD‐induced death. Direct treatment of brain slices with IL‐4 (without cell addition) was protective against OGD and induced an M2 phenotype in the slice. In vivo, intracerebral injection of LPS or IL‐4 in mice induced microglial phenotypes similar to the phenotypes observed in brain slices and in cultured cells. After injury induced by middle cerebral artery occlusion, microglial cells did not adopt classical M1/M2 phenotypes, suggesting that another subtype of regulatory phenotype was induced. This study highlights functional differences between macrophages and microglia, in response to brain injury with fundamentally different outcomes, even if both populations were able to adopt M1 or M2 phenotypes. These data suggest that macrophages infiltrating the brain from the periphery after an injury may be cytotoxic, independently of their phenotype, while microglia may be protective.     

Complex Changes in the Innate and Adaptive Immunity Accompany Progressive Degeneration of the Nigrostriatal Pathway Induced by Intrastriatal Injection of 6-Hydroxydopamine in the Rat

We investigated changes in innate and adaptive immunity paralleling the progressive nigrostriatal damage occurring in a neurotoxic model of Parkinson’s disease (PD) based on unilateral infusion of 6-hydroxydopamine (6-OHDA) into the rat striatum. A time-course analysis was conducted to assess changes in morphology (activation) and cell density of microglia and astrocytes, microglia polarization (M1 vs. M2 phenotype), lymphocyte infiltration in the lesioned substantia nigra pars compacta (SNc), and modifications of CD8+ and subsets of CD4+ T cell in peripheral blood accompanying nigrostriatal degeneration. Confirming previous results, we observed slightly different profiles of activation for astrocytes and microglia paralleling nigral neuronal loss. For astrocytes, morphological changes and cell density increases were mostly evident at the latest time points (14 and 28 days post-surgery), while moderate microglia activation was present since the earliest time point. For the first time, in this model, we described the time-dependent profile of microglia polarization. Activated microglia clearly expressed the M2 phenotype in the earlier phase of the experiment, before cell death became manifest, gradually shifting to the M1 phenotype as SNc cell death started. In parallel, a reduction in the percentage of circulating CD4+ T regulatory (Treg) cells, starting as early as day 3 post-6-OHDA injection, was detected in 6-OHDA-injected rats. Our data show that nigrostriatal degeneration is associated with complex changes in central and peripheral immunity. Microglia activation and polarization, Treg cells, and the factors involved in their cross-talk should be further investigated as targets for the development of therapeutic strategies for disease modification in PD.     

Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke

Acute AMPK activation exacerbates ischemic brain damage experimentally. Paradoxically, the clinical use of an AMPK activator metformin reduces the incidence of stroke. We investigated whether post-stroke chronic metformin treatment promotes functional recovery and tissue repair via an M2-polarization mechanism following experimental stroke. Mice were randomly divided to receive metformin or vehicle daily beginning at 24 h after middle cerebral artery occlusion (MCAO). Neurological deficits were monitored for 30 days following MCAO. To characterize the polarization of the microglia and infiltrating macrophages, the expression of the M1 and M2 signature genes was analyzed with qPCR, ELISA and immunohistochemistry. Post-MCAO angiogenesis and neurogenesis were examined immunohistochemically. An in vitro angiogenesis model was employed to examine whether metformin promoted angiogenesis in a M2 polarization-dependent manner. Post-stroke chronic metformin treatment had no impact on acute infarction but enhanced cerebral AMPK activation, promoted functional recovery and skewed the microglia/macrophages toward an M2 phenotype following MCAO. Metformin also significantly increased angiogenesis and neurogenesis in the ischemic brain. Consistently, metformin-induced M2 polarization of BV2 microglial cells depended on AMPK activation in vitro. Furthermore, treatment of brain endothelial cells with conditioned media collected from metformin-polarized BV2 cells promoted angiogenesis in vitro. In conclusion, post-stroke chronic metformin treatment improved functional recovery following MCAO via AMPK-dependent M2 polarization. Modulation of microglia/macrophage polarization represents a novel therapeutic strategy for stroke.     

Neurokinin Receptor 1 (NK1R) Antagonist Aprepitant Enhances Hematoma Clearance by Regulating Microglial Polarization via PKC/p38MAPK/NFκB Pathway After Experimental Intracerebral Hemorrhage in Mice

Hematoma clearance is an important therapeutic target to improve outcome following intracerebral hemorrhage (ICH). Recent studies showed that Neurokinin receptor-1 (NK1R) inhibition exerts protective effects in various neurological disease models, but its role in ICH has not been explored. The objective of this study was to investigate the role of NK1R and its relation to hematoma clearance after ICH using an autologous blood injection mouse model. A total of 332 adult male CD1 mice were used. We found that the expression levels of NK1R and its endogenous ligand, substance P (SP), were significantly upregulated after ICH. Intraperitoneal administration of the NK1R selective antagonist, Aprepitant, significantly improved neurobehavior, reduced hematoma volume and hemoglobin levels after ICH, and promoted microglia polarization towards M2 phenotype. Aprepitant decreased phosphorylated PKC, p38MAPK, and NFκB p65, and downregulated M1 markers while upregulating M2 markers after ICH. Intracerebroventricular administration of the NK1R agonist, GR73632 or PKC agonist, phorbol 12-myristate 13-acetate (PMA) reversed the effects of Aprepitant. To demonstrate the upstream mediator of NK1R activation, we performed thrombin injection and found that it increased SP. Inhibiting thrombin suppressed SP and decreased M1 markers while increasing M2 microglia polarization. Thus, NK1R inhibition promoted hematoma clearance after ICH by increasing M2 microglial polarization via downregulating PKC/p38MAPK/NFκB signaling pathway, and thrombin may be a key upstream mediator of NK1R activation. Therapeutic interventions inhibiting NK1R signaling may be a new target for the treatment of ICH.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01077-8.     

Inhibition of Mer exacerbates early brain injury by regulating microglia/macrophage phenotype after subarachnoid hemorrhage in mice

BackgroundPolarization of microglia/macrophages toward the pro-inflammatory phenotype is a crucial contributor to neuroinflammation after subarachnoid hemorrhage (SAH). Mer belongs to the TAM receptor tyrosine kinases family, which is known to play a significant role in the resolution of inflammation. However, the effect and mechanism of Mer after SAH remain unclear. In this study, we explored the effect of Mer on modulating the microglia/macrophage phenotype and neuroinflammation and possible potential mechanism after SAH. Method: Endovascular perforation model of SAH was performed. There are 3 parts in this study. Firstly, the time course of Mer expression was determined within 72 hours after SAH. Secondly, the effect of Mer downregulation on brain water content, neurological function, and microglial polarization was evaluated at 24 h after SAH. Thirdly, the neuroprotective effects of pharmacological Mer agonist were assessed. Result: The expression of Mer increased after SAH, and was prominently localized in microglia/macrophages. Treatment with Mer siRNA increased pro-inflammatory phenotype and decreased anti-inflammatory phenotype of microglia/macrophage, thus resulted in exacerbation of neurological deficits and brain edema after SAH. Mechanistically, the downregulation of Mer inhibited the downstream anti-inflammatory signals, SOCS1/SOCS3, by decreasing phosphorylated STATs. Conclusion: Mer is involved in the microglia/macrophage polarization and inflammation resolution after SAH, and that mechanism, at least in part, may contribute to the involvement of the STATs/SOCSs pathway.