MicroRNA-195 Prevents Hippocampal Microglial Polarization Towards The M1 Phenotype Induced By Chronic Brain Hypoperfusion Through Regulating CX3CL1/CX3CR1 Signalling

Abstract Background Microglial polarization was found respond dynamically to acute brain hypoxia induced by stroke and traumatic brain injury (TBI). However, studies on the process of microglial polarization during chronic cerebral ischaemia (CCI) are limited. Our goal is to investigate the influence of CCI on microglial polarization following chronic brain hypoperfusion (CBH) and exploit potential molecular mechanisms.Methods CBH model was performed by bilateral carotid artery ligation (2VO) in rats. Using stereotaxic injection technique, lenti-pre-miR-195 and anti-miR-195 oligonucleotide fragments (lenti-pre-AMO-195) were injeted into CA1 region of the hippocampus to construct animal models with high or low expression of miR-195. Immunofluorescence staining and flow cytometry were conducted to examine the status of microglial polarization. In vitro, Transwell co-culture system was taken to investigate the role of miR-195 on neuronal-microglial communication through CX3CL1-CX3CR1 signalling. Quantitative real-time PCR was used to detect the level of miR-195 and inflammatory factors. The protein levels of CX3CL1 and CX3CR1 were evaluated by both western blot and immunofluorescence staining.Results CBH induced by 2VO initiated microglial activation in the rat hippocampus from 1 week to 8 weeks, as evaluated by increased Iba-1 immunofluorescence, that the balance between microglial polarization towards the M1 and M2 phenotypes was shifted towards the M1 phenotype and that the expression of CX3CL1 and CX3CR1 was increased at 8 w following CBH. An in vitro study in a Transwell co-culture system demonstrated that transfection of either primary cultured neonatal rat neurons (NRNs) or microglial BV2 cells with AMO-195 induced M1 polarization of BV2 cells and increased CX3CL1 and CX3CR1 expression and that these effects were reversed by miR-195 mimics. Furthermore, overexpression of miR-195 induced by lenti-pre-miR-195 prevented the changes triggered by knockdown of endogenous miR-195 induced by lentiviral vector-mediated expression of lenti-pre-AMO-195 and 2VO surgery.Conclusions Our findings conclude that downregulation of miR-195 in the hippocampus is involved in CBH-induced microglial polarization towards M1 phenotype by governing communication between neurons and microglia through the regulation of CX3CL1 and CX3CR1 signalling. This indicates that miR-195 may provide a new strategy for clinical prevention and treatment of CBH.