| Wada et al. found that repeated hyperbaric oxygen (HBO) preconditioning induced an ischemic tolerance in gerbil hippocampus in 1996, and this result proved that hyperbaric oxygen preconditoning was one of methods to prevent the ischemia-reperfusion injury. Subsequently a lot of research was consistent with the results reported by Wada. HBO preconditioning is capable of inducing ischemic tolerance not only in the brain but also in other organs, such as the liver, heart, and spinal cord. Ischemia-reperfusion injury was prevented by HBO preconditioning in HBO pretreated organs. The mechanism of HBO preconditioning was unclear, whereas HBO preconditioning could effectively prevent ischemia-reperfusion damage. Our lab investigated the ischemic tolerance induced by HBO preconditioning on spinal cord in past years to clarify the mechanism of HBO preconditioning and make it be applied in clinic as soon as quickly. We found that: (1) repeated hyperbaric oxygen preconditioning induced tolerance against spinal cord ischemia in rabbits; (2) the primary ingredient acted in the ischemic tolerance induced by HBO preconditioning was hyper-oxygen but not hyperbaria; (3) the dimethylthiourea (DMTU), a potent oxygen free radical scavenger, abolished the ischemic tolerance in spinal cord induced by hyperbaic oxygen preconditioning; (4) reactive oxygen species (ROS) generated in HBO process triggers the cascade in cellular events leading to increased antioxidant enzyme activities, which scavenge the ROS and protect spinal cord from ischemia-reperfusion injury; (5) the mechanism of ischemic tolerance induced by HBO preconditioning may partly be related to the attenuation of mitochondria ATPase activity decrease after ischemia, thus protected the function of mitochondria. According to the results of our previous in vivo studies, we found that the inducing of ischemic tolerance was partly be letated to the generation of ROS in HBO pretreatment process. Reactive oxygen species (ROS) generated in HBO process triggers the cascade in cellular events leading to increased antioxidant enzyme activities and protected the function of mitochondria, which protect spinal cord from ischemia-reperfusion injury. We know that a lot of physiological reactions occur in HBO pretreatment process. However, it is unknown whether the tolerance is the direct effect of HBO preconditioning on neurons or is the effect secondary to other changes in whole body. In other words, we want to know that if HBO preconditioning could directly act on neurons and protect the cell by trigger a series of cellular events. To answer these questions and further clarify the mechanism of the HBO preconditioning-induced ischemic tolerance, we established an in vitro HBO treatment system for primary cultured spinal cord neurons.Hydrogen peroxide (H2O2)-mediated oxidative injury in the cultured spinal cord neurons was undertaken in this study to simulate the damage induced by ischemia-reperfusion. The aim of current study is to determine: (1) if the tolerance of HBO preconditioning against oxidative injury could be induced in primary cultured spinal cord neurons; (2) if so, what might be responsible for the tolerance induction.Based on these questions, primary cultured spinal cord neurons were used for model. First, we established methods for the culture of primary embryonic mouse spinal cord neurons and for identify of the cultured neurons. Pathology, general hematoxylin-eosin (H&E) stain and immunohistochemistry stain were used to observe the morphologic character of cultured neurons. Methyl thiazoleterazolium (MTT) assay and comet assay (single cell gel electrophoresis) were undertaken to investigate the effect of HBO preconditioning on oxidative injury in primary cultured spinal cord neurons.To observe the level of antioxidant enzymes in cultured spinal cord neurons pretreated with HBO, western blot and reverse transcriptase polymerase chain reaction (RT-PCR) were used. To further clarify the mechanism of HBO preconditioning-induced ischemic tolerance, we used the MTT assay and comet assay to investigate the effect of tin-mesoporphyrin IX (SnMP), a specific inhibitor to HO-1 activity, on HBO preconditioning-induced tolerance. The main results of present study as follows:I) A stable method for the culture of primary embryonic mouse spinal cord neurons: The cultured embryonic mouse spinal cord neurons in 10% fetal bovine serum (FBS) DMEM media survived over 10 days. The morphological features of cultured neurons were in good condition during their growth. During 4～6 days, the cell viability was stable and the morphological features were best compared with that of neurons at different culture-time. The cultured spinal cord neurons were identified by immunocytochemical staining withβ-tubulin antibodies. The purity of cultured spinal cord neurons was over 90%.II) Hydrogen peroxide (H2O2) induced oxidative injury in the cultured spinal cord neurons: Treated with H2O2 at doses of 25μmol/L, 50μmol/L, 100μmol/L and 200μmol/L respectively for 1 hour, the neuron viability compared with that of control were 81±9.85%, 53±9.03%, 34±6.61% and 21±5.13%, respectively. The tail moment values which shown the DAN damage in cultured neurons of different doses groups were 0.235±0.051, 0.455±0.089, 0.538±0.079 and 0.762±0.112, and that in control group was 0.056±0.011. Compared with control group, the tail moment in groups treated with H2O2 at different doses increased significantly (P<0.05, n = 3, respectively). H2O2 treatment induced serious injury in the cultured spinal cord neurons. Especially treated with 50μmol/L H2O2, the cell viability decreased about half of that in control group. Therefore, in the following experiment a dose of 50μmol/L for H2O2 was used at various time points after HBO pretreatment.III) The protective effect of HBO preconditioning against oxidative injury in cultured spinal cord neurons:1) The effect of HBO preconditioning on neuron viability: At various time points after HBO pretreatment (1h, 4h, 8h, 12h, 24h), the cellular viabilities of primary cultured neurons shown as optical densities in H2O2 group were significantly decreased compared with that of control (P<0.05). At 1 hour after HBO preconditioning, there was no significant difference between H2O2 group and HBO+ H2O2 group (P>0.05). At 4 hour after HBO preconditioning, the optical density in H2O2 group was 0.121±0.011 and the optical density in HBO+ H2O2 group was 0.251±0.073, which was increased significantly (P<0.01 versus H2O2 group). The optical density of HBO+ H2O2 group increased obviously began 4 hours after HBO pretreatment, reached peak at the time of 12 hours (0.432±0.056), and lasted at least 24 hours as long as we observed.2) The effect of HBO preconditioning on DNA damage of neurons: H2O2 treatment induced DNA damage in cultured neurons. At various time points after HBO pretreatment (1h, 4h, 8h, 12h, 24h), the DNA damage of primary cultured neurons shown as the tail moment values in H2O2 group were significantly increased compared with that of control (P<0.01). At 1 hour after HBO preconditioning, there was no significant difference between H2O2 group and HBO+ H2O2 group (P>0.05). At 4 hour after HBO preconditioning, the tail moment in H2O2 group was 0.515±0.085 and in HBO+ H2O2 group was 0.381±0.096, which was decreased significantly (P<0.01 versus H2O2 group). The tail moment of HBO+ H2O2 group decreased obviously began 4 hours after HBO pretreatment, and lasted at least 24 hours as long as we observed.IV) HBO preconditioning induced the expression of HO-1 in cultured neurons:1) HBO preconditioning induced expression of HO-1 protein in cultured neurons: Equal loading was verified by immunoblotting ofβ-actin. The level of HO-1 mRNA in each group is denoted as a ratio to that ofβ-actin. The expression of HO-1 protein was little before HBO treatment, which was only 0.15 fold compared withβ-actin. At 1 hour after HBO treatment, there was no significant difference between control and HBO group (P>0.05). The level of HO-1 protein was increased significantly (0.66 fold, P<0.01 versus control) at 4 hours after HBO treatment, reached peak at the time of 8 hours (1.44 fold), and lasted at least 24h in our observed duration. However, there was no clear difference in the expression of SOD between control and HBO group. The level of SOD protein was about the same as that ofβ-actin.2) HBO preconditioning induced increased level of HO-1 mRNA in cultured neurons: The level of HO-1 mRNA in each group is denoted as a ratio to that ofβ-actin. The level of HO-1 mRNA was low before HBO treatment, which was 0.55 compared with that ofβ-actin. The level of HO-1 mRNA was increased significantly (1.09, P<0.01 versus control) at 1 hour after HBO treatment, reached peak at the time of 8 hours (1.88). Subsequently, the level of HO-1 mRNA decreased gradually. The level ofβ-actin mRNA had no difference between groups at various time points.V) SnMP abolished the protective effect induced by HBO preconditioning in cultured neurons: In control group, the optical density and the tail moment value of spinal cord neurons treated with 10μmol/L SnMP had no significant change compared with that of the neurons untreated with SnMP (P>0.05). In H2O2 group, results were the same as that of control group. In HBO+ H2O2 group, the optical density and the tail moment value of spinal cord neurons treated with SnMP had obvious change. The optical density of neurons without SnMP treatment was 0.351±0.054, and the optical density of neurons with SnMP treatment was 0.203±0.041. There was significant difference between two groups (P<0.01). The tail moment of neurons without SnMP treatment was 0.208±0.066, and the optical density of neurons with SnMP treatment significantly increased (0.507±0.098, P<0.01 versus the neurons without SnMP treatment).In conclusion, our work has demonstrated that H2O2 induced oxidative injury in primary cultured spinal cord neurons. Preconditioning with HBO induced tolerance against the oxidative injury via increased expression of antioxidant enzyme in cultured neurons. Both the level of HO-1 protein and mRNA increased after HBO preconditioning, and the increasing of HO-1 level was parallel with the protective effect induced by HBO preconditioning in cultured neurons. SnMP, a specific HO-1 inhibitor, abolished cellular protection against the H2O2-mediaed injury. These results suggest that HO-1 play a role for the HBO-induced tolerance against oxidative injury at cellular level. Further studies may focus on the approach by which HBO preconditioning induces the expression of HO-1. |
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