Multiple Effects Of Hyperbaric Oxygen, 2ME2 And Cystamine On Related Factors And They Take Hippocampus Protection From Apoptosis After Global Ischemia-reperfusion

[OBJECTIVE]①To study the change regularity about the himppocampus neurons apoptosis, and to explore the role and function of the Hypoxia-inducible factor-1α(HIF-1α) after global ischemic-reperfusion.②To explore if severe hypoxia induces the over-expression of HIF-1αand p53, and then the over-expression activate caspase-9 and caspase-3 (apoptosis pathway) to evoking neurons apoptosis.③We want to test if HBO to protect neurons by enhancing the oxygen giving, further more we can prove the HBO take a protection function on neurons from apoptosis due to HBO depress the over-expression of the downstream genes of HIF-1α.[MATERIALS AND METHOD]Materials Male SD rats (n=78) were randomly divided into 13 groups:1 sham group,6 groups of global ischemia-hypotension (GI), and 6 groups of HBO treatment after global ischemia-hypotension (GI+HBO). Transient 2 vessel occlusion (2VO) global ischemia was produced by using the method originally described by Smith et al. (1984) with some modifications (Sugawara et al.,1999) as previously described by us (Zhou et al.,2003). HBO (3 ATA for 2 h) was applied at 1 h after global ischemia-hypotension. Rats were sacrificed at 6,12,24,48, and 96 h and 7 days. Rats were sacrificed at 6,12,24,48, and 96 h and 7 days.Nissl staining and cell counting:The CA1 area in the hippocampus from each animal was captured and Imaging-Pro-Plus (OLYMPUS) was used to perform quantitative analysis of cell numbers. The number of Nissl staining cells in each region was counted, and the densities of the cells in the CA1 regions were averaged to obtain a mean density value for each animal.Immunohistochemistry staining will provide information of the distribution of positive cells to HIF-1αand other apoptotic genes in the CA1 regions of brain.Double fluorescence labeling provides unique information regarding the cell types that are positive to HIF-1αand other apoptotic genes and if the same neuron is positive to both HIF-1α and other apoptotic genes.TUNEL staining provides information of DNA fragmentation, a unique phenomenon of apoptosis.Western blots will provide information of quantified changes of HIF-la and other apoptotic genes in neuronal tissues. Samples from four animals from each group were used for Western blot study.[Statistical analysis]All the data in this study were expressed as mean F standard deviation (SD). Differences in data between the control and each group of global ischemia, with or without HBO treatment, were compared by using one-way ANOVA and then, if a significant variance was found, the Tukey-Kramer multiple comparison procedure. A value of P< 0.05 was considered statistically significant.[RESULT]Nissl staining and cell counting:Neuronal tissue loss in the hippocampus and to a lesser degree in the ischemic cortex was observed at 24 h, progressed at 48 h, and was maximum at 7 days after global ischemia. HBO treatment reduced brain tissue loss in all time periods as shown in picture.Immunohistochemistry staining:At 12 h after global ischemic-hypotension insult, strong TUNEL-positive staining appeared in hippocampus and to a much lesser degree in the cortex. The higher magnification demonstrated TUNEL staining in the nuclei. Progressive TUNEL staining occurred from brain samples at 24-48 h after global ischemia-hypotension. In samples from (GI+HBO) rats few TUNEL cells were noticed (especially at 12-24 h) either in the hippocampus or in cortex. Fluorescence staining showed HIF-la positive cells (red color in the nuclei) in the CA1 region of the hippocampus from samples collected at 24 h after global ischemia-hypotension. Double fluorescences staining of the same slide with TUNEL howed positive staining in the nuclei (green color). Merging two composites into Fig.2M3 demonstrated a colocalization of TUNEL with HIF-lain the nuclei of neurons in the hippocampus (yellow color) indicating HIF-1a expressed in apoptotic cells. As shown in Fig. 2M, both TUNEL and HIF-la stained only nuclei but not cytosol in the neurons.The immunohistochemistry expression of p53, caspase-9, and caspase-3:In the normal cortex and hippocampus, negative immunohistochemical staining was obtained for p53, caspase-9, and caspase-3. At 24 h after global ischemic-hypotension insult, strong staining of p53, caspase-9, and caspase-3 was observed in the hippocampus and cortex. Higher magnification in inserts demonstrated p53, caspase-9, and caspase-3 positive neurons in the hippocampus and cortex.Double labeling of neurons in hippocampus CA1 regions at 24 h after global ischemia-hypotension revealed positive costaining (yellow) in the nuclei of neurons to p53 (green) and HIF-1α(red). In addition, costaining of caspase-9 (green) and caspase-3 (green) in the cytosol and the costaining of HIF-1α(red) in the nuclei were observedExpression of caspase-8 and bcl-2 in Western blotting:Contrary to the observations mentioned above, we have obtained unexpected results on caspase-8 and bcl-2. The protein expression of caspase-8 increased significantly after global ischemia-hypotension at 6 h, peaked at 12 h, decayed and plateaued at 24 h to 7 days in the ischemic cortex when compared to the normal controls (P< 0.05, GI vs. control, ANOVA). HBO treatment did not alter the initial peak but further potentiated the level of caspase-8 from 12 h to 7 days (P< 0.05, GI+HBO vs. GI). More interestingly, the protein expression of bcl-2 was increased significantly at 6 h, continued to increase and peaked at 7 days after global ischemia-hypotension (P< 0.05, GI vs. control, ANOVA). HBO reduced markedly the expression of bcl-2 at all time points (P< 0.05, GI+HBO vs. GI) and reduced the level of bcl-2 to almost control level (P> 0.05 vs. control) except at 24 h (P< 0.05 vs. control).[CONCLUSION]Global ischemia-hypotension (10 min ischemia,30-35 mm Hg) produced a marked increase of HIF-1αexpressions in the hippocampus and cortex at 6 h and peaked at 48-96 h. The expressions of p53, caspase-9, and caspase-3 were all increased in a similar time course. These molecular changes were accompanied by massive cell loss in the hippocampal regions and to a lesser degree in the cortex, with features of apoptosis. HBO treatment reduced expressions of HIF-1α, p53, caspase-9, and caspase-3 and decreased cell death. The protein levels of proapoptotic caspase-8 and antiapoptotic bcl-2 was increased after global ischemia-hypotension and HBO potentiated the expression of caspase-8 and decreased expression of bcl-2. These results indicate that HBO has multiple actions on apoptotic genes even though the overall effect of HBO was decreased HIF-1αexpression and reduced apoptosis after global ischemia-hypotension. Survivin is an anti-apoptotic gene that could decrease the apoptosis by depressing the expression of caspase-3. Hypoxia-inducible factor-la (HIF-la) is a transcription factor specifically activated by hypoxia.2-methoxyestradiol (2ME2) is an estradiol derivative and a known HIF-la inhibitor.2ME2 decreased apoptosis by inhibiting HIF-la. The aim of the present study was to investigate if survivin is involved in the anti-apoptotic effect of 2ME2. Male adult SD rats were used to make the global ischemia (GI) model. Ten minutes after GI, 2ME2 was injected intraperitoneally (16mg/kg weight). Rats were sacrificed at 6,12,24,48, and 96 h and 7 days. GI produced a marked increase of HIF-1a expressions in the hippocampus at 6 h and peaked at 48-96 h. The expression of survivin and caspase-3 were increased lightly in a similar time course. These molecular changes were accompanied by massive cell loss and apoptosis in the hippocampal regions.2ME2 treatment reduced expression of HIF-la, increased survivin expression and decreased the expression of caspase-3. These results indicate that survivin was involved in the anti-apoptotic effect of 2ME2 treated following GI.2ME2 may decrease HIF-la expression, up-regulate the survivin expression, inhibited the expression of caspase-3 and finally reduce apoptosis after GI. [Objective]①To study the change regularity about the himppocampus neurons apoptosis, and to explore the interreaction among tTG, NPM, P53 and caspase-3 after global ischemic-reperfusion.②Using the cystamine as the inhibitor of tTG to study the affection of cystamine on tTG and its downstream factors NPM and P53 after global ischemia-reperfusion.③To explore neuroprotection mechanisms of cystamine through studying the interreaction between tTG, NPM, P53 and caspase-3 used CYS after global ischemia-reperfusion.[Materials and Methods]The experimental GI model was established by 4-vessel occlusion method and confirmed successfully by an electroencephalogram (EEG) record.104 adult male Sprague-Dawley rats were randomly divided into sham group (n=8), global cerebral ischemia-reperfusion group(GI, n=48) and global cerebral ischemia-reperfusion+CYS treatment group (GI+CYS, n=48), and the two latter GI group and GI+CYS group respectivly including six subsets each with 8 rats.CYS(150mg/kg/day)were administered to rats in GI+CYS group by abdominal cavity injection one time a day. The rats in sham group (n=8) were treated with lml saline by abdominal cavity injection and sacrificed 3 days later.The cerebral hippocampus tissues were obtained to make frozen section at 3 days after sham operation in sham group and at 6 hours,12 hours, 1day,3 days,5 days and 7 days after ischemia-reperfusion in GI and GI+CYS groups. The expressions of tTG, NPM, p53, caspase-3 proteins were detected by Immunohistochemistry(SP), Western blot and TUNEL technology was used to detect neuronal apoptosis. The positive neurons were analysesed by light microscopy and Image-Proplus 5.0 image analysis system. Western blot analysis was quantified by densitometry.All data were presented as mean±standard deviation (SD). Data analysis was performed using SPSS13.0 statistical software. AP value less than 0.05 was considered statistically significant. [Results]TUNEL staining:Compared with the sham group, the number of apoptotic cells in subsets 1d, 3d,5d and 7d of GI group and subsets 3d,5d and 7d of GI+CYS group were significantly increased (p<0.05). The number of apoptotic cells in subsets 1d,3d,5d,7d of GI+CYS group were significantly less than of GI group. (p<0.05)Immunohistochemistry:The expression of tTG,NPM, p53:Compared with the sham group,the expression of tTG and p53 were significantly increased, NPM was significantly decreased in hippocampus at 12h, 1d and 3d time points subsets of GI group(P<0.01).Compared with the corresponding subsets of GI group, the expression of tTG and p53 of GI+CYS groups were significantly decreased, the expression of NPM of GI+CYS groups in subsets 1d,3d,5d and 7d was significantly enhanced(P<0.01) after global cerebral ischemia-reperfusion. Expression of Caspase-3:Compared with the sham group, the caspase-3 immunopositive cells were significantly increased in the injured hippocampus in 6h, 12h, 1d,3d,5d and 7d GI subset groups(P<0.01). Compared with the corresponding subsets of GI group, the expression level of caspase-3 in 1d,3d,5d and 7d subsets of GI+CYS were significantly decreased. (P<0.01)Western blot analysis:the expression of tTG were in GI group were thinnest at 6h and 12h time points, and then the expression became thick at Id time point, the expression raised significantly at 3d,5d,7d time points after global ischemia-reperfusion. Compare with sham group, the different of the raised expression of tTG at 3d,5d,7d time points is significant(P<0.05).In GI+CYS groups, the protein expression level of tTG began raised at Id, expressed thinnest at3d,5d,7d time points, the protein expression were all thick comparing with corresponding time points in hippocampus after cerebral ischemia-reperfusion, the different is significant (p<0.05).To compare with sham group, the protein expression level of NPM were thinnest at 6h time point, the protein expression to recover thickness at 12h time point, and get to the control level at 1d time point, finally, the expression kept a higher level at 3d,5d and 7d after global ischemia-reperfusion. After treatment, the NPM protein expression of GI+CYS groups began to enhance at 6h time point, to get the peak at 1 d time point, the expression kept a higher level at 3d,5d and 7d. After global ischemia-reperfusion, the NPM protein expression level of GI+CYS groups were higher than that of NPM in GI groups, the different are significant (p<0.05). After global ischemia-reperfusion, the p53 protein expression level began to enhance significantly at 6h,to get the peak at 12h and 1d,and began to decrease from 3d,finly get to normal level at 7d; the p53 protein expression level of GI+CYS groups were lower than that of p53 at 6h,12h and 1d in GI groups, the different are significant (p<0.05) After global ischemia-reperfusion,for GI group groups, the protein expression level of caspase-3 were thicker at 6h,12h, 1d time points, and it bagan to decrease at 3d,but that kept a higher level at 3d,5d and 7d time points. After global ischemia-reperfusion and CYS treatment, the caspase-3 expression level of GI+CYS groups were all lower than that of GI groups, the different are significant for corresponding time points (p<0.05)[Conclusion]Global cerebral ischemia-reperfusion can induce the up-regulation of the expression of tTG, p53 and caspase-3, and does down-regulation of the expression of NPM, which lead to injury neurons to apoptosis by tTG, NPM, p53 and caspase-3 cell signal transmission pathway and producing effector molecule. CYS can depress the expression of tTG, the expression inhibition of tTG induce the over-expression of NPM, and the later reducing the mitochondrial level of p53 and depress the expression of caspase-3 (may be indirectly), which might be one of the mechanisms that CYS protects hippocampus neurons from apoptosis after global brain ischemia-reperfusion.