The Effects And Mechanism Of Hyperbaric Oxygen On Cerebral Cortex Injury In MCAO Rats Based On ERK1/2Pathway

Objective: The blood and oxygen supply is very low in marginal zone of brainischemia infarct area, and will induce neuron death. Hyperbaric oxygen (HBO) wasused to raise the oxygen level in this condition, but its outcomes were debated. Thepresent study aimed to investigate the effect of HBO on brain damage in a rat modelof transient focal cerebral ischemia/reperfusion (MCAO/R) and the mechanism action.Autophagy flux was detected, and apoptosis was discussed for mechanismdemonstration, MAPK pathway, oxidative stress and Mcl-1expression were detectedfor autophagy and apoptosis evaluation.Method: Male Wistar rats, which had suffered1.5h of transient middle cerebralartery occlusion (tMCAO) with Longa’s neuron score of3, were given pure oxygen at3.0atmospheres absolute (ATA), for60minutes after the third hour of reperfusion.After24hours of reperfusion, rats were killed and brains were removed and studied.Infarct ratio and brain damage were revealed by2,3,5-triphenyltetrazolium chloride(TTC) and hematoxylin and eosin(H&E) staining. Tissue in infarct area marginal zoneof cortex was taken for examination. RT-PCR and Western blotting were used toanalysis the mRNA and protein expression, including autophagy protein (LC3,Beclin1, and p62), apoptosis regulating protein (Bax, Mcl-1), anti-oxidative enzyme(HO1, NQO1, GLSM) and downstream of MAPK pathway Extracellularsignal-regulated kinase1/2(ERK1/2).Result: The infarct ratio in HBO group increased remarkably when compared withthe MCAO group (p<0.01) after TTC stain. We could observe further tissuerarefaction and neuron soma pyknosis in HBO group through H&E stain in paraffinsection of brain. Results above demonstrate that HBO enhance the MCAO braindamage.In HBO group, LC3transformation and p62content were in higher levelcompared with MCAO group (p<0.01).The content of autophagy initiate proteinBeclin1in two groups both decreased at the condition of same expression level oftranscription, and have no signification between the groups. This information suggests that Beclin1was used and autophagy actived in HBO and MCAO group, moreover,autophagy pathway in HBO may be interrupted.In our experiment, we found that the downstream of MAPK pathwayextracellular signal-regulated kinase1/2(ERK1/2) expression and its activation wereup-regulated apparently in HBO group. All three anti-oxidative enzymes (HO1,NQO1, GLSM) raised strongly in HBO group versus MCAO group. This informationsuggests that ERK1/2activation may cause of further oxidative stress in HBO group.Administration of the ERK1/2inhibitor U0126not only decreases the infarct ratio intwo groups but also reduces the autophagy protein LC3and p62contents in HBOgroup. Those results indicate that ERK1/2actived by oxidative stress in HBO groupinterrupt the autophagy pathway, and then enhance the MCAO damage.Additionally, Mcl-1transcription in HBO group were increased versus MCAOgroup, moreover, MCAO group increased versus sham (p<0.01). But the proteincontents of Mcl-1decreased both in MCAO and HBO group compared with sham(p<0.01), and no difference between the two. These indicate that the degradation ofMcl-1is more severe in HBO group. While Bax/Mcl-1in HBO group is higher thanthat of MCAO group, means HBO could promote apoptosis.Conclusion: HBO enlarges the area of brain damage. Because of autophagy pathwaywas interrupted by activation of ERK1/2via oxidative stress, and other hand,apoptosis may be promoted by degradation of Mcl-1during HBO treatment. Thisstudy indicated that combination with ERK1/2inhibitor could decrease the risk ofHBO treatment for brain ischemia.