Fingolimod promotes angiogenesis and attenuates ischemic brain damage via modulating microglial polarization.

Microglial activation plays a crucial role in the pathology of ischemic stroke. Recently, we demonstrated that fingolimod (FTY720) exerted neuroprotective effects via immunomodulation in ischemic white matter damage induced by chronic cerebral hypoperfusion, which was accompanied by robust microglial activation. In this study, we assessed the pro-angiogenic potential of FTY720 in a murine model of acute cortical ischemic stroke. The photothrombotic (PT) method was used to induce cortical ischemic stroke in mice. We evaluated cortical damage, behavioral deficits, microglial polarization, and angiogenesis to identify the neuroprotective effects and possible molecular mechanisms of FTY720 in acute ischemic stroke. In vivo, a reduction in neuronal loss and improved motor function were observed in FTY720-treated mice after PT stroke. Immunofluorescence staining revealed that robust microglial activation and the associated neuroinflammatory response in the peri-infarct area were ameliorated by FTY720 via its ability to polarize microglia toward the M2 phenotype. Furthermore, both in vivo and in vitro, angiogenesis was enhanced in the microglial M2 phenotype state. Behaviorally, a significant improvement in the FTY720-treated group compared to the control group was evident from days 7 to 14. Our research indicated that FTY720 treatment promoted angiogenesis via microglial M2 polarization and exerted neuroprotection in PT ischemic stroke.

Shang K ，He J ，Zou J ，Qin C ，Lin L ，Zhou LQ ，Yang LL ，Wu LJ ，Wang W ，Zhan KB ，Tian DS ... -  《-》

Fingolimod Protects Against Ischemic White Matter Damage by Modulating Microglia Toward M2 Polarization via STAT3 Pathway.

White matter (WM) ischemic injury, a major neuropathological feature of cerebral small vessel diseases, is an important cause of vascular cognitive impairment in later life. The pathogenesis of demyelination after WM ischemic damage are often accompanied by microglial activation. Fingolimod (FTY720) was approved for the treatment of multiple sclerosis for its immunosuppression property. In this study, we evaluated the neuroprotective potential of FTY720 in a WM ischemia model. Chronic WM ischemic injury model was induced by bilateral carotid artery stenosis. Cognitive function, WM integrity, microglial activation, and potential pathway involved in microglial polarization were assessed after bilateral carotid artery stenosis. Disruption of WM integrity was characterized by demyelination in the corpus callosum and disorganization of Ranvier nodes using Luxol fast blue staining, immunofluorescence staining, and electron microscopy. In addition, radial maze test demonstrated that working memory performance was decreased at 1-month post-bilateral carotid artery stenosis-induced injury. Interestingly, FTY720 could reduce cognitive decline and ameliorate the disruption of WM integrity. Mechanistically, cerebral hypoperfusion induced microglial activation, production of associated proinflammatory cytokines, and priming of microglial polarization toward the M1 phenotype, whereas FTY720 attenuated microglia-mediated neuroinflammation after WM ischemia and promoted oligodendrocytogenesis by shifting microglia toward M2 polarization. FTY720's effect on microglial M2 polarization was largely suppressed by selective signal transducer and activator of transcription 3 (STAT3) blockade in vitro, revealing that FTY720-enabled shift of microglia from M1 to M2 polarization state was possibly mediated by STAT3 signaling. Our study suggested that FTY720 might be a potential therapeutic drug targeting brain inflammation by skewing microglia toward M2 polarization after chronic cerebral hypoperfusion.

Qin C ，Fan WH ，Liu Q ，Shang K ，Murugan M ，Wu LJ ，Wang W ，Tian DS ... -  《-》

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 24h after middle cerebral artery occlusion (MCAO). Neurological deficits were monitored for 30days 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.

Jin Q ，Cheng J ，Liu Y ，Wu J ，Wang X ，Wei S ，Zhou X ，Qin Z ，Jia J ，Zhen X ... -  《-》

Deficiency in the voltage-gated proton channel Hv1 increases M2 polarization of microglia and attenuates brain damage from photothrombotic ischemic stroke.

Microglia become activated during cerebral ischemia and exert pro-inflammatory or anti-inflammatory role dependent of microglial polarization. NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) production in microglia plays an important role in neuronal damage after ischemic stroke. Recently, NOX and ROS are consistently reported to participate in the microglial activation and polarization; NOX2 inhibition or suppression of ROS production are shown to shift the microglial polarization from M1 toward M2 state after stroke. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent ROS generation in the brain. However, the effect of Hv1 proton channel on microglial M1/M2 polarization state after cerebral ischemia remains unknown. In this study, we investigated the role of microglial Hv1 proton channel in modulating microglial M1/M2 polarization during the pathogenesis of ischemic cerebral injury using a mouse model of photothrombosis. Following photothrombotic ischemic stroke, wild-type mice presented obvious brain infarct, neuronal damage, and impaired motor coordination. However, mice lacking Hv1 (Hv1(-/-)) were partially protected from brain damage and motor deficits compared to wild-type mice. These rescued phenotypes in Hv1(-/-) mice in ischemic stroke is accompanied by reduced ROS production, shifted the microglial polarization from M1 to M2 state. Hv1 deficiency was also found to shift the M1/M2 polarization in primary cultured microglia. Our study suggests that the microglial Hv1 proton channel is a unique target for modulation of microglial M1/M2 polarization in the pathogenesis of ischemic stroke. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent generation of reactive oxygen species (ROS) in the brain. ROS participate in microglial activation and polarization. However, the effect of Hv1 on microglial M1/M2 polarization state after cerebral ischemia remains unknown. Hv1 deficiency was found to shift the microglial polarization from M1 to M2 state in ischemic stroke accompanied by reduced ROS production. Our study suggests that the microglial Hv1 proton channel is a unique target for modulation of microglial M1/M2 polarization in the pathogenesis of ischemic stroke.

Tian DS ，Li CY ，Qin C ，Murugan M ，Wu LJ ，Liu JL ... -  《-》

Berberine Facilitates Angiogenesis Against Ischemic Stroke Through Modulating Microglial Polarization via AMPK Signaling.

Zhu J ，Cao D ，Guo C ，Liu M ，Tao Y ，Zhou J ，Wang F ，Zhao Y ，Wei J ，Zhang Y ，Fang W ，Li Y ... -  《-》