Exploring the Effects of Hydroxyfasudil on Cuprizone-Induced Demyelination

Background:Multiple sclerosis(MS) is one of demyelinating diseases of the central nervous system(CNS),which characterized by oligodendrocyte loss,demyelination/remyelination,progressive neurodegeneration and peripheral immune response. The cuprizone(CPZ)-induced demyelination is widely used to investigate the demyelination/remyelination. Fasudil,an inhibitor of Rho kinase(ROCK),has been proved to have neuroprotective effects in various neurodegenerative diseases. However,the problem of clinical application obviously limits the use of Fasudil,including short-course treatment,low oral bioavailability,narrower safety window and blood pressure fluctuation. Hydroxyfasudil(HF),an active metabolite of Fasudil,is more selective than Fasudil as an inhibitor of ROCK. The aim of the present study was to observe whether HF contributes to the myelin protection and/or regeneration in a mouse model of CPZinduced demyelination,and explore the mechanisms. Materials and Methods:Male C57 BL/6 mice were divided into three groups:normal group(received rodent chow);cuprizone group(fed CPZ diet and injected with normal saline intraperitoneally);HF-treated CPZ group(which received i.p. with HF(40 mg·kg-1·day-1) after 4 weeks for consecutive 2 weeks,without CPZ withdrawal). Elevated plus-maze(EPM) test and Pole test were used to measure anxiety level and locomotor coordination of mice. To examine the extent of demyelination,slides of brain were stained with Luxol Fast Blue(LFB) and Black gold Ⅱ(BGⅡ) staining. The alternation of T cell responses,neuroinflammation and the neurotrophic factor by HF were observed. Results:The intensity of LFB and BG Ⅱ staining was obviously decreased in the corpus callosum(CC) of CPZ-fed mice,as compared to normal mice without CPZ feeding(P<0.001). HF treatment significantly attenuated CPZ-induced demyelination in the CC region(P<0.01,P<0.001). In HF group(1.11±0.01),an increased expression of MBP protein was observed in brain compared with CPZ group(0.84±0.01) by western blot(P<0.001). The immunofluorescence staining of O4 antibody also indicated that O4+ mature oligodendrocytes were higher in HF group(258.21±26.37) than that in CPZ group(94.27±9.23,P<0.001). HF ameliorated motor dysfunction of CPZ mice. HF partially restored the volume of spleens in CPZ mice,the average weight of spleens in CPZ group and HF group were(57.13±2.64)g,(65.83±3.04)g,P<0.05. In addition,HF treatment effectively decreased the titer of MOG antibody in serum,supernatants of splenocytes and extracts of brain by ELISA and dot blot(P<0.001,P<0.01). The MOG antibodies penetrated into the brain can bind to oligodendrocytes and cause the damage of oligodendrocytes. HF could prevent the infiltration of T cell,B cell and macrophage into CNS(P<0.001). Compared with Normal group,the number of Iba-1+ microglia/macrophages was significantly increased in the CC region and striatum of the mice in the CPZ model group(P<0.001). HF effectively inhibited the expression of the Iba-1+ microglia/macrophages compared with CPZ group(P<0.001). In addition,HF reduced the number of Iba-1+iNOS+ and Iba-1+NF-κB+ cells in the brain of CPZ mice(P<0.001). HF administration induced an increased BDNF immunoreactivity that co-localized with GFAP+ astrocytes. Discussion:In the present study,HF treatment decreased the damage of O4+ oligodendrocytes,accompanied with the inhibition of M1 microglia-mediated neuroinflammation and the induction of astrocyte-derived BDNF in the brain. We observed two discoveries in CPZ model. One is the appearance of splenic atrophy,and the other one is the production of MOG35-55 specific autoantibodies. Some studies indicated that the destruction of neural cells or oligodendrocytes leads to release of damage-associated molecular patterns(DAMPs) to the extra cellular environment. DAMP molecules are a relatively recently uncovered family group that trigger immune response. Another study demonstrated that debris from damaged cells in CNS may present as antigens,giving rise to autoantibodies. Therefore,it is possible that the DAMPs or myelin debris can subsequently enters the blood circulation,and potentially activate peripheral immune cells by BBB and/or CNS-draining lymph nodes. Our results demonstrated that MOG antibody was detected in CPZ-fed mice serum,supernatant of cultured splenocytes and extracts of brain. The MOG antibody was elevated in the supernatant of cultured splenocytes,revealing that the production of MOG antibodies was derived from peripheral immune cells. Our results also showed that:the level of MOG antibody in the brain homogenate of CPZ-treated mice was higher than that of normal mice brain,suggesting that antibodies can enter brain tissue. Myelin is also a potent microglia stimulus and neuroinflammation trigger. We hypothesized that the protection of myelin sheath can decrease DAMPs and myelin debris,thereby reducing antibody formation by reducing the content of DAMPs and myelin debris in blood circulation. In addition,the protection of myelin sheath also decreases the inflammatory response mediated with microglia activation,thereby preventing the damage to endothelial cells and the entry of DAMPs and myelin debris into the blood circulation. Therefore,it is possible that HF reduces the peripheral humoral immune responses by protecting myelin sheath and endothelial cells. In addition to demyelination caused by MOG antibody,microglia exert inflammatory neurotoxic function in the CPZ model. CPZ-induced demyelination is characterized by microglia responses that produce inflammatory cytokines,which have been implicated in the process of demyelination. In this study,CPZ exposure induced Iba-1+ microglia expressing iNOS and NF-κB. HF enhanced oligodendrocyte survival and improved neurological deficits possibly through reducing microglia-mediated neuroinflammation,suggesting a negative role for microglia in the demyelination/remyelination process. Our results showed that HF treatment increased the numbers of GFAP+ astrocytes in the striatum,in which astrocytes expressing BDNF were obviously enhanced,suggesting that astrocyte-derived BDNF should contribute to the protection and/or regeneration of myelin sheath. In conclusion,our results demonstrated that HF treatment improved CPZ-induced demyelination,possibly by inhibiting the production of MOG specific antibodies,the infiltration of peripheral T cells/macrophages,the formation of microglia-mediated neuroinflammation as well as the increase of astrocyte-derived BDNF in the brain. These data provide several therapeutic strategies to alleviate demyelination or other related diseases.