| Retinal ganglion cell (RGC) death is the end result of practically all diseases of the optic nerve, including glaucomatous optic neuropathy. Optic nerve and retina are the most easily accessible parts of the central nervous system (CNS), and lying outside the skull. The optic nerve is a white matter tract of the diencephalon composed principally of the axons of RGC. Therefore, optic nerve injury has been used to model brain axonal injury. Optic nerve crush (ONC) is a well established model of delayed RGC death. The neurodegenerative process after ONC may be similar to the process of secondary degeneration described in the context of glaucomatous optic neuropathy. After ONC, a fraction of RGCs undergo early necrosis, whereas others are subjected to delayed apoptotic death and will always be relatively numerous. Even after a complete transection many RGCs are still able to survive for long periods after the insult.Hyperbaric oxygen (HBO) therapy has been traditionally applied in the treatment of carbon monoxide poisoning, stroke, air embolism and decompression sickness. Recently, HBO preconditioning (HBO-PC) has been shown to have neuroprotective effects against ischemic injury in gerbil hippocampus, rabbit spinal cord, transient brain infarct in mouse and rat or in primary cultured spinal cord neurons. However the mechanism is not fully understood and more evidence is needed for HBO treatment to be accepted clinically. The majority of studies examined HBO preconditioning effects on a delayed brain injury. HBO preconditioning should have a powerful anti-apoptotic effect as apoptosis is a dominant form of hippocampal cell death after global cerebral ischemia.In the present study, on the assumption that the processes of secondary degeneration of neurons after ONC are likely to involve pathways and mediators similar to those in brain trauma, we investigate whether the administration of HBO-PC offers neuroprotection to retinal ganglion cells by reducing ONC-induced caspase-dependent apoptosis. We hope to provide experimental evidence for the clinical application of HBO preconditioning.Part I The protective effect of HBO preconditioning on optic nerve crush injury in ratsObjective:Establishing a reliable, convenient and reproducible animal model with optic nerve crush (ONC), we investigate whether the HBO protocol is of protective effect on ONC injury.Methods:HBO preconditioning (HBO-PC) was conducted four times by given 100% oxygen at 2.5 atmosphere absolute (ATA) for 1 h every 12 h interval for 2 days prior to ONC. Rats were euthanized at 1 or 2 weeks after ONC. RGC density was counted by Hematoxylin and Eosin (HE) staining in the retina and retrograde labeling with FluoroGold application to the superior colliculus. Visual function was assessed by flash visual evoked potentials (FVEP).Results:The number of HE-stained cells in the ganglion cell layer of sham group was 89.5±9.4 cells/HPF at 1 week and 92.7±9.1 cells/HPF at 2 weeks. More HE-stained cells in the ganglion cell layer were observed in HBO-PC group than in no HBO-PC group (69.3±7.4 vs 56.6±6.5 cells/HPF at 1 week, P＜0.01; 62.3±6.1 vs 46.1±6.6 at 2 weeks, P<0.01). Therefore, HBO-PC had a statistically significant rescue effect in retinas when compared with ONC group. Moreover, we applied retrograde labeling with fluorogold to count the number of surviving RGCs at 2 weeks after ONC. The average RGC density in retinas of sham-operated group was 1650±216/mm2. At 2 weeks after ONC, the RGC densities of HBO-PC+ONC group and ONC group decreased to 995±158/mm2 and 478±101/mm2, respectively (p<0.01). The FVEP measurements illustrate that the HBO-PC+ONC group had significantly improved visual function as compared to the corresponding ONC group at both 1 and 2 weeks. The latency of the P1 wave at 1 week was 90±4ms,94±4ms, and 108±6ms for the control, HBO-PC+ONC (p>0.05 vs control), and ONC groups (p<0.01 vs control). At 2 weeks after operation, the latency of the P1 wave was 92±7ms,117±12ms,169±15ms for the control, HBO-PC+ONC (p<0.05 vs control, p＜0.001 vs ON-crushed), and ONC groups (p<0.001 vs control). The amplitude of the P1 wave was 17±2μv,18±3μv, and 12±2μv for the control, HBO-PC+ONC (p>0.05 vs control), and ONC groups (p<0.01 vs control) at 1 week,21±3μv,14±2μv, and 7±1μv for the control, HBO-PC+ONC (p<0.05 vs control, p<0.001 vs ONC), and ONC groups (p<0.001 vs control) at 2 weeks.Conclusion:The RGC density in the retinas of ONC, HBO-PC-treated rats was significantly higher than that of the corresponding ONC rats. FVEP measurements indicated a significantly better preserved latency and amplitude of the P1 wave of in the HBO-PC group. These observations demonstrate that HBO-PC appears to be neuroprotective against ONC injury.PartⅡHyperbaric oxygen preconditioning promotes survival of retinal ganglion cells by inhibition of mitochondrial apoptosis in a rat model of optic nerve crushObjective:To investigate the influence of HBO preconditioning on mitochondrial apoptosis. Methods:The animal models were made and the HBO was performed according to the protocol introduced in Part I. The terminal deoxynucleotidyl transferase-mediated Dutp nick end labeling (TUNEL) assay was performed on paraffin-embedded sections according to the manufacture instructions. The activity of caspase-3 and-9 were measured with caspase-3 and caspase-9/CPP32 Fluorometric Assay Kit. The protein expression levels of cytochourome c, Bcl-2, BclxL and Bax were detected by Western Blot.Results:At 2 weeks after operation, TUNEL assay illustrated that TUNEL-positive cells/HPF of 1.8±1.6 cells in sham operated group,7.2±1.3 cells in HBO-PC+ONC group and 19.1±4.2 cells in ONC group (p<0.01 vs HBO-PC+ONC group) in the RGC layer. Caspase-9 activity in HBO-PC+ONC group were higher than that of ONC group at indicated time points (4.15±1.33-fold vs 11.25±3.26-fold at 1 week; 2.25±0.79 vs 6.29±1.95 at 2 weeks, P＜0.01). Similarly, the measurements of caspase-3 activity showed a 2.62±0.45-fold and 3.22±0.67-fold increase in HBO-PC+ONC group and ONC group respectively at 1 week (p<0.05); a 1.72±0.34-fold and 2.82±0.51-fold increase in HBO-PC+ONC group and ONC group respectively at 2 weeks (p<0.01). Western blot analysis of retina samples at 24 h after ONC showed that HBO-PC reduced the protein expression of cytochourome c (P<0.05), HBO-PC enhanced the anti-apoptotic protein levels of Bcl-2 and BclxL.The protein level of Bax remains constant in all groups after ONC.Conclusion:The inhibition of mitochondrial apoptosis may be one of the mechanisms of neuroprotection induced by HBO preconditioning in a rat model of optic nerve crush. PartⅢMechanism of neuroprotection induced by hyperbaric oxygen preconditioning involves upregulation of hypoxia-inducible factor-1 and erythropoietin in a rat model of optic nerve crushObjective:To investigate whether the protective effect induced by HBO preconditioning involves upregulation of hypoxia-inducible factor-1 and erythropoietin in retina.Methods:HBO was performed according to the protocol introduced in Part I. RT-PCR and Western blot were used to analyze the HIF-1 and EPO mRNA level, the HIF-la, EPO, ERK and HSP70 protein level using samples taken from the retinas of the experimental groups 12 h after the last HBO pretreatment without optic nerve crush injury.Results:The HIF-1 mRNA and HIF-la protein level were significantly increased 12 h after the last HBO pretreatment (13.38±1.97-fold and 1.86±0.24-fold vs control, P＜0.01) in rat retinas, while the EPO mRNA and protein level were also significantly increased (5.55±1.03-fold and 3.01±0.33-fold vs control, P＜0.01). Moreover, EPO activates downstream effectors, such as ERK and HSP70, which protein level was also significantly increased (1.64±0.11-fold and 1.81±0.12-fold vs control, P＜0.01).Conclusion:HBO preconditioning can induce the protective effect on ONC injury, which may probably involve up-regulation of HIF-1 and its target gene EPO.
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