The Protective Effect Of Inonsine On Oligodendrocytes Injured By Chemical Hypoxia

Inosine is a kind of small molecules widely existing in organism, good protection of inosine to variety of cells has been demonstrated. In the past few years, studies about the protective effects of inosine on the central nervous system suggested that inosine can not only regulate immune cell function, promote the growth of neurons axon widely after injury, but also protect neural cells after injury. In vitro, inosine can protect the ROC-1 cells, astrocytes and neuronal activity against chemical hypoxia injury. In vivo, the treatment of inosine to cerebral ischemia injury in rats can reduce the neural cells apoptosis and death, inhibit platelet accumulation, the degradation of inositol phosphate and calcium ions formation, and reduce cerebral ischemia damage degree, minimize the cerebellum infarction volume and inhibit post-glutamatergic synaptic effect, so as to promote neurological recovery in rats. However, ROC-1 cells are a hybrid cell lines generated by fusion of rat oligodendrocytes and rat C6 glioma cells, which has both the characteristics of oligodendrocyte and astrocyte, so it is uncertain whether inosine has protective effect on oligodendroytes injured by hypoxia. In addition, ROC-1 is a kind of immortal tumor cell lines and can not really reflect the more sensitivity of oligodendrocytes to pathological damage. Finally, although inosine treatment of ischemic rat models can reduce the neural cell apoptosis, stimulate nerve function recovery, but it is still not clear about the effects of inosine on oligodendrocytes jeopardised by hypoxia in vivo. Therefore, it is greatly important to investigate whether inosine has protective effect on oligodendrocytes injured by hypoxia in vitro and in vivo.This research comprises three parts. The first part is to confirm whether inosine has protective effects for differentiated oligodendrocytes in chemical hypoxic environments; The second part is to research the effects of inosine on oligodendrocyte precursor cells with proliferation capacity in chemical hypoxic environments; The third part is to investigate the influence of inosine on locomotor function recovery and the number of survival oligodendrocytes surrounding necrotic area of the rat seven days after spinal cord contusion injury.In the first part, we investigated the effects of inosine on primarily cultured rat oligodendrocytes injured by rotenone mediated chemical hypoxia, using MTT assay, PI-Hoechst staining, TUNEL, ATP assay, real-time PCR and immune fluorescence staining. Results showed that rotenone exposure for 24 h caused different injury degrees to immature and mature oligodendrocytes. Therefore, to reach roughly similar damage degrees, we used different concentration of rotenone to injure immature oligodendrocyte and mature oligodendrocyte, as 5μM rotenone for immature oligodendrocytes and 20μM rotenone for mature oligodendrocytes. For both stages of oligodendrocytes, inosine was treated 30 min prior to rotenone exposure. The results showed that 10 mM inosine can significantly reduce the immature oligodendrocytes death and apoptosis, and improve a damaged immature oligodendrocytes activity in dose-dependence; while 10 mM inosine reduced mature oligodendrocytes death, but did not reduce mature oligodendrocytes apoptosis. Meanwhile pretreatment of 10 mM inosine raised the viability of damaged mature oligodendrocytes. To clear of peroxynitrite and supply ATP are the possible mechanisms of the protective effects of inosine on immature and mature oligodendrocytes. The differences between immature and mature OLs in susceptibility to hypoxia and inosine protection were possibly correlated with the difference of adenosine receptors on different stage of oligodendrocytes.In the second part, we assessed the effects of inosine on oligodendrocyte precursor cells (OPCs) injured by chemical hypoxia through morphological observation, combining with PI dyeing, immune fluorescence staining and MTT method.Results showed that 20μM rotenone significantly induced OPCs death, viability, and decrease of total cell number and NG2 antigen expression, but did not induce OPCs apoptosis. Ten mM inosine pretreatment to damged OPCs did not improve OPCs survival and OPCs viability damaged by rotenone, but it can increase NG2 expression in damaged cells. In addition, it is worth noting that 10 mM inosine significantly reduce the viability of OPCs in the normal culture condition (the control group).In the third part, we observed the influence of intraperitoneal injection of inosine to rats with spinal cord contusion injury on locomotor function recovery and the number of oligodendrocytes adjacent to necrotic area of the spinal cord through the behavior evaluation and immunohistochemical staining.The results showed that intraperitoneal injection of inosine (75mg/kg, once every eight hours) to rat with spinal cord contusion did not improve locomotor function and the number of oligodendrocytes adjacent to necrotic area of rat. In shor, in vitro we really observed that the survival and activity of both immature oligodendrocytes and mature oligodendrocytes mediated by chemical hypoxia were obviously improved by inosine and the survival and activity of oligodendrocyte precursor cells was not improved. To clear of peroxynitrite and supply ATP are the possible mechanisms of the protective effects of inosine on immature and matue oligodendrocytes. And inosine hasn't protective effect on OPCs injured by rotenone. However, it was not observed that treatment of inosine to rat with spinal cord contusion improved locomotor function and the number of oligodendrocytes adjacent to necrotic area of rat. The possible reasons of which were administration dosage, administration way, administration time and the time length of observation may be the curative effect of inosine treatment to rat with spinal cord injury.