高压氧对缺氧缺血1周内新生大鼠脑皮质细胞线粒体膜电势的影响

Effect of hyperbaric oxygenation on mitochondrial membrane potential of cortex neuronal cells of neonatal rats in the first week after hypoxic ischemic brain damage

目的研究高压氧(HBO)对新生大鼠缺氧缺血性脑损伤(HIBD)1周内脑皮质细胞线粒体功能的影响,探讨HBO对HIBD可能的保护作用及其机制。方法新生SD大鼠360只分为正常对照组、HIBD组和HIBD+HBO组,每组120只。HIBD组和HIBD+HBO组结扎左侧颈总动脉后暴露于8%O2+92%N2低氧环境中2 h制备HIBD模型。HIBD+HBO组在缺氧缺血后立即予HBO干预(压力为2 ATA,每次持续60 min,每日1次,连续7 d),HIBD组不予HBO干预,正常对照组不予结扎左侧颈总动脉和HBO干预。以HIBD模型建立后设为缺氧缺血后0 h时点,3组于0 h、2 h、4 h、6 h、12 h、1 d、2 d、3 d、4 d、5 d、6 d和7 d时点断头处死(各组各时点n=10),取损伤侧脑皮质制备单细胞悬液,予细胞线粒体膜电势(ΔΨm)标记物罗丹明123(Rho123)孵育,用流式细胞仪检测Rho123的平均荧光强度(MFL),并以该MFL值作为ΔΨm值。结果①正常对照组脑皮质细胞ΔΨm值为(4.66±0.80)MFL,HIBD组各时点脑皮质细胞ΔΨm值均低于正常对照组相应时点,且最低为0 h时点[(2.85±0.56)MFL],各时点差异均有统计学意义(P<0.05);②HIBD组及HIBD+HBO组脑皮质细胞ΔΨm均呈现降低-恢复-再降低的变化规律,两组ΔΨm初次降低时间均为缺氧缺血后0 h时点,初次恢复时间均为缺氧缺血后2~12 h,再次降低的时间均为缺氧缺血后1~4 d,HIBD+HBO组ΔΨm的再次降低程度更明显且最低为缺氧缺血后3 d时点[(2.62±1.03)MFL];③HIBD组脑皮质细胞ΔΨm在再次降低后未再回复,而HIBD+HBO组ΔΨm在再次降低后,于缺氧缺血后5 d时点后开始恢复,6和7 d时点ΔΨm值逐渐趋近但低于正常对照组水平,差异无统计学意义(P<0.05)。结论 HBO在HIBD后0 h至3 d内不能改善缺氧缺血损伤侧脑皮质细胞的线粒体功能,HIBO后过早开始HBO治疗可能导致受损脑皮质细胞的进一步损伤,但HBO可能在HIBD后5~7 d内可通过改善脑皮质线粒体功能促进HIBD受损细胞功能恢复。

Objective The initial insult of hypoxic-ischemic( HI)brain damage( HIBD)is the deprivation of oxygen( O2 ) to the brain cells,followed by a cascade of brain cell damage including mitochondrial dysfunction. Theoretically,hyperbaric oxygenation( HBO)could affect the recovery of mitochondrial function in HIBD by greatly increasing the O2 delivery diffusion gradient. The objective of this study was to prove the hypothesis that HBO may reduce HI-induced brain injury via affecting brain cell mitochondrial function,and to understand the changing patterns of mitochondrial function following HBO treatment in the first week after HI. Methods In the present study,HIBD rat model and flow cytometer were used to explore the change of ΔΨm,the indicator of mitochondrial function of cortex neuronal cells of neonatal rats after HIBD. Neonatal Sprague Dawley( SD)rat pups were randomly divided into normal control,HIBD,and HIBD+HBO groups. The end of HI was considered to be 0 h time point. The HBO treatment was given at 0h time point,and then once a day for consecutive 7 days( in 24 h intervals). Animals were euthanized at 0,2,4,6,12 h time points(in order to study theΔΨm changes at the very early stage after a single dose of HBO treatment),and at 2,3,4,5,6,and 7 d time points( in order to study the ΔΨm changes after a series of HBO treatment). Results The change of ΔΨm of the ipsilateral cortex in both HIBD and HIBD+HBO groups showed fluctuating change pattern.&nbsp;Within 2 h to 12 h after HI insult,ΔΨm of HIBD group recovered to some extent,but ΔΨm of HIBD+HBO group recovered to almost normal level. A secondary drop of ΔΨm was observed in both groups at 1 -4 d after HI insult. The secondary drop of HIBD+HBO group was more severe than that of HIBD group. There was a secondary recovery of ΔΨm observed in HIBD+HBO group in 5-7 d after HI insult,but not in HIBD group. TheΔΨm of HIBD+HBO group recovered again to almost normal level at 6 d time point. TheΔΨm of HIBD group in 2-7 d after HI stayed at low level,showing slowly decreasing tendency. Conclusion HBO in the early stage after HI might not be a good therapy to improve the mitochondrial function in the cerebral cortex. The secondary recovery observed in HIBD + HBO group indicated that HBO treatment may protect HI-induced brain damage by improving neural cell mitochondrial function in the cerebral cortex during sub-acute stage after HI.