| Perioperative brain ischemia/reperfusion (I/R) injury is not rare. And the result is often devastating. The proof of benefits for brain I/R injury treatment is very limited and lack of solid evidence support compared to I/R injuries of other organs, as heart and lung. At the same time, the mortality rate of perioperative stroke is as high as 33%-60% varies with surgery types. Thus, the effort of finding effective prevention or treatment strategies against brain I/R injury and successfully translating into clinical practice is the major issue we face in perioperative brain protection research.Ever since the report of Wada et al. in 1996 that showed hyperbaric oxygen (HBO) preconditioning could induce ischemic tolerance in gerbils’forebrain, abundant researches have been done on HBO preconditioning. Studies showed that HBO preconditioning could also induce ischemia tolerance and reduce I/R injury in the heart, liver and spinal cord. However, the mechanism of HBO perconditioning is far from clear elucidation. Our previous study showed that repeated HBO perconditioning could mimic ischemia perconditioning and induce robust protection against brain I/R injury, of which innate antioxidant pathway activation is involved. such as raised activity of antioxidant enzymes like CAT and SOD, and enhancecd heme oxygenase-1 (HO-1) transduction and expression. And the use of HO-1 specific inhibitor tin-mesoporphyrin IX (SnMP) could reverse the protective effect of HBO perconditioning. Thus far, we have only tested protective effect of HBO perconditioning against brain I’R injury in animal experiments, whether HBO perconditioning could exhibit direct protection on cortical neurons against I/R injury is not clear. To answer the question above and further elucidate the mechanism of HBO perconditioning induced brain ischemic tolerance, we adopt primary cortical neuron cultures, using the model of oxygen glucose deprivation (OGD) simulating I/R injury to study the effect of HBO perconditioning, and further explore the mechanism of that effect.Part I Repeated HBO perconditioning protected cultured cortical neuron from oxygen-glucose deprivation injuryAim To evaluate the effect of HBO perconditioning aginst oxygen and glucose deprivation (OGD) injury in cultured neonatal rat’s cortical neurons. Method The OGD model was established in cultured neonatal rat’s cortical neuron. After assessment of purity of the neurons, they were divided into Sham group (neurons without HBO perconditioning or OGD),OGD group (neurons were exposed to OGD only) and HBO group (neurons received 0.35Mpa 100% O2,2h/d for Shamsecutive 5d prior to OGD exposure). After 24 h period of reoxygenation, the cells were examined for MTT and LDH assay to detect cell death and injury. Flow cytometry was carried out to assess neuron apoptosis. SOD and CAT Shamcentration were also determined to study th effect of HBO perconditioning on neurons. Result (1) : The OGD model was successfully established in cultured neonatal rats’cortical neurons.(2) OD value of the MTT assay were significantly decreased in OGD group compared with that in HBO and sham groups (p<0.05), however, there are no statistical differences between HBO group and sham group. (3) HBO perconditioning alleviated apoptosis in cortical neurons (2.69%) compared to OGD group (4.71%) or Sham group (2.69%) at 24h after reoxygenation (P<0.05) according to flow cytometry detection. (4) Compared to OGD group, HBO perconditioning decreased the release of LDH at 24h after reoxygenation (P<0.05). (5) No significant changes of SOD activity was found among groups in the time points of day1-day5 before OGD. At 24h after reoxygenation, SOD activity of group OGD was lower than that of the other two groups (P<0.05). In HBO group, CAT activity increased as HBO perconditioning repeated. CAT activity dropped after OGD in OGD group(P<0.05), but did not change in HBO group or Sham group. Shamclusion Oxygen and glucose deprivation induced neuronal injury, and repeated HBO perconditioning protected neurons against OGD injury.Part II PPARγpathway is involved in the mechanism of protective effect of repeated HBO perconditioning against oxygen-glucose deprivation injury in cultured cortical neuronsExperiment I Changes of PPARγin the protective effect of repeated HBO perconditioning on cortical neurons against oxygen-glucose deprivation injuryAim Investigate the changes of PPAR signals in repeated HBO perconditioning protected cortical neuron of neonatal rat from oxygen and glucose deprivational injury. Method The OGD model was established in cultured neonatal rats’cortical neurons. After assessment of purity of the neurons, the plates of neuron were divided into sham group (neurons avoided from the treatment of both OGD and HBO perconditioning), OGD group (neurons were exposed to OGD only) and HBO groups (0.35MPa,2h,5d). 2h, 24 h and 48h after reoxygenation, the level of PPARγprotein and mRNA were examined by Western Blot and RT-PCR. Result PPARγmRNA and protein expression in HBO group began to increase at 2h after reoxygenation and peaked at 24h, decreased at 48h compared to the previous time points in the same group, but was still significantly more than that of OGD group and sham group (P<0.05). Shamclusion Repeated HBO perconditioning protected cortical neurons from oxygen-glucose deprivation injury by up-regulating PPARγ.Experiment II Effect of PPARγagonist and antagonist on the neuronal protection induced by repeated HBO perconditioning in cultured cortical neurons against oxygen-glucose deprivation injuryAim To investigate the role of PPARγin neuroprotection induced by repeated HBO perconditioning against OGD injury in cultured neonatal rats’ cortical neurons and the effects of PPARγagonist and antagonist on the protective effect of HBO perconditioning. Method The OGD model was established in cultured neonatal rats’cortical neuron. After assessment of purity of the neurons, the plates of neurons were divided into sham group (neurons avoided from the treatment of both OGD and HBO perconditioning), OGD group (neurons were exposed to OGD only), HBO group, (0.35MPa,2h,5d), PPARγagonist group (ACT) and PPARγantagonist group (IHB). The agonist rosiglitazone or antagonist GW966 were added in the medium by Shamcentration of 10μM respectively at 24h after the last session of HBO perconditioning. Thirty minutes later, OGD was applied, medium was changed back to normal after reoxygenation. At 24 h after reoxygenation, MTT, LDH and flow cytometry assay were deteced, and the level of PPARγprotein and mRNA were examined by Western Blot or RT-PCR. Result (1) OD value were significantly decreased in OGD group and IHB group compared with that in HBO group (p<0.05), however, there are no statistical differences between HBO group and ACT group. (2) LDH release were significantly increased in OGD and IHB groups compared with that in HBO group, Sham group and ACT group (p<0.05). There are no statistical differences between HBO group and ACT group. (3) Apoptotic neurons were observed but only a few in groups HBO (3.01%), Sham (2.82%) and ACT (2.69%). However the apoptotic neurons were significantly increased in OGD (4.71%) group and IHB (3.47%) group at 24h after OGD. No differences was found between group HBO and group ACT in apoptotic neuron numbers. (4) Expression of PPARγmRNA and protein were increased in ACT group 2 h and 24 h after reoxygenation compared with that in other groups, however expression of PPARγmRNA and protein were decreased in IHB group at 24 h after reoxygenation compared to any other goups(P<0.05).Shamclusion PPARγagonist protected cortical neurons from OGD injury by up-regulating mRNA and protein expression of PPARγ. PPARγantagonist partialy reversed the protective effect.Conclusion1. Repeat HBO perconditioning protect neuron from injury induced by oxygen glucose deprivation.2. HBO perconditioning induced ischemic tolerance partialy by increaseing the expression of PPARγand improved the ability of oxygen radical clearance.3. PPARγagonist improved neuroprotection of HBO perconditioning by increasing PPARγmRNA and protein expression, and PPARγantgonist partialy reversed this protective effect. |
PDF Full Text Request