| Purpose It is well known that, matured neurons are not able to regenerate from severe injury. So emphases are recently focused on enhancement of the regeneration ability of neurons, improvement of the regrowth of their axons and dendritic trees or reinforcement of their tolerance to certain degree of injury. B vitamins are usually applied to certain optic nerve disease induced by glaucoma, diabetic retinopathy or other eye disorders. Recent researches suggests that, such kind of drugs maintain the normal physical function in nerve system by demonstrate their biochemical activity of coenzymes and/or some special functions. But few studies focus on the effects of B vitamins on retinal ganglion cells (RGCs), also very limit proof focus on neuroprotective effects of these drugs to low glucose injured retinal neurons. Furthermore, some present discoveries indicated that the induction and hyper-expression of heat shock protein 70 (HSP70) can enhance the tolerance of certain injury, which suggest us to seek for some new ways of treating the damage of retinal neurons. The purpose of this study is to prove the efficiency of B vitamins as the routine optic nerve protectors, so as to guide their clinical applying. Here we also observed the induction and expression of HSP70, and its protection on low glucose or excitotoxicity injured retinal neurons, so that to elucidate the new methods of neuroprotection?enhancing the tolerance of retinal neurons to certain injury.Methods Neonatal rat retinal neurons in vitro culture system were established at the beginning of the study. Then we observed the effects of B vitamins, including vitamin B1 (thiamin), B6 (pyridoxine), B12 and its derivation mythylcobalamin, on the survival of retinal neurons as well as the regeneration of their axons. Neuroprotection of the above vitamins on low glucose injured retinal neurons was also studied at the same time. Finally, the expression of HSP70 in Muller cells and retinal neurons was detected. Its protection on glucose deprived or glutamate induced retinal neuron injury was proved.The reactivity of retinal neurons on a various kind of changes can be relative exactly evaluated since non-neurons were inhibited in this study. Cell viability were detected by monotetrazolium (MTT) chromatometry, which indirectly reflects the survival ability of cells. Lactate dehydrogenase (LDH) released by retinal neurons was analyzed after low glucose insults to evaluate the degree of cellinjury. Immunocytochemical methods and corresponding antibodies were applied to identify RGCs and Muller cells. The results were analyzed statistically.Neonatal rat retinal neurons, RGCs and Muller cells in vitro culture systems were set up initially to carry on lateral researches. Researches on the neurotrophic and neuroprotective effects of B vitamins were divided into three stages. The neurotropic effects of B vitamins on retinal neurons were first proved. The most effective concentration of each drug was confirmed by detection of cells viability. Vitamins in these concentrations were added into culture media at the beginning of culturing. The cultures were terminated at various periods. Cells were collected to undergo HE staining and immunocytochemistry reaction against Thy-1 to identify RGCs. Axons length of retinal neurons and RGCs were measured by microruler under microscope respectively to evaluate the neurotropic effects of B vitamins in improving the regeneration and growth of axons for retinal neurons and RGCs. Finally, we observed the reaction of retinal neurons when exposed to the insult of glucose deprivation. The neuroprotection of B vitamins were also evaluated at the same time.Furthermore, neonatal rat retinal Muller cells culture system was established together with retinal neuron cultures and treated by hyperthermia to induce the expression of HSP70. The efficiency of HSP70 in enhancing the tolerance of retinal neurons to low glucose and glutamate induced cell injury.Results Cultured retinal neurons showed the tendency to grow in clusters. In...
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