CAJ | 학술논문

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、12h、1d、2d、3d、4d、5d、6d和7d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5d时点后开始恢复，6和7d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。目的：研究高壓氧（ HBO）對新生大鼠缺氧缺血性腦損傷（ HIBD）1週內腦皮質細胞線粒體功能的影響，探討HBO對HIBD可能的保護作用及其機製。方法新生SD大鼠360隻分為正常對照組、HIBD組和HIBD+HBO組，每組120隻。HIBD組和HIBD+HBO組結扎左側頸總動脈後暴露于8％ O2+92％ N2低氧環境中2 h製備HIBD模型。HIBD+HBO組在缺氧缺血後立即予HBO榦預（壓力為2 ATA，每次持續60 min，每日1次，連續7 d），HIBD組不予HBO榦預，正常對照組不予結扎左側頸總動脈和HBO榦預。以HIBD模型建立後設為缺氧缺血後0 h時點，3組于0 h、2 h、4 h、6 h、12h、1d、2d、3d、4d、5d、6d和7d時點斷頭處死（各組各時點n=10），取損傷側腦皮質製備單細胞懸液，予細胞線粒體膜電勢（ΔΨm）標記物囉丹明123（Rho123）孵育，用流式細胞儀檢測Rho123的平均熒光彊度（MFL），併以該MFL值作為ΔΨm值。結果①正常對照組腦皮質細胞ΔΨm值為（4.66±0.80）MFL，HIBD組各時點腦皮質細胞ΔΨm值均低于正常對照組相應時點，且最低為0 h時點[（2.85±0.56）MFL]，各時點差異均有統計學意義（ P＜0.05）；②HIBD組及HIBD+HBO組腦皮質細胞ΔΨm均呈現降低-恢複-再降低的變化規律，兩組ΔΨm初次降低時間均為缺氧缺血後0 h時點，初次恢複時間均為缺氧缺血後2~12 h，再次降低的時間均為缺氧缺血後1~4 d，HIBD+HBO組ΔΨm的再次降低程度更明顯且最低為缺氧缺血後3 d時點[（2.62±1.03）MFL]；③HIBD組腦皮質細胞ΔΨm在再次降低後未再迴複，而HIBD+HBO組ΔΨm在再次降低後，于缺氧缺血後5d時點後開始恢複，6和7d時點ΔΨm值逐漸趨近但低于正常對照組水平，差異無統計學意義（ P＜0.05）。結論 HBO在HIBD後0 h至3 d內不能改善缺氧缺血損傷側腦皮質細胞的線粒體功能，HIBO後過早開始HBO治療可能導緻受損腦皮質細胞的進一步損傷，但HBO可能在HIBD後5~7 d內可通過改善腦皮質線粒體功能促進HIBD受損細胞功能恢複。
목적：연구고압양（ HBO）대신생대서결양결혈성뇌손상（ HIBD）1주내뇌피질세포선립체공능적영향，탐토HBO대HIBD가능적보호작용급기궤제。방법신생SD대서360지분위정상대조조、HIBD조화HIBD+HBO조，매조120지。HIBD조화HIBD+HBO조결찰좌측경총동맥후폭로우8％ O2+92％ N2저양배경중2 h제비HIBD모형。HIBD+HBO조재결양결혈후립즉여HBO간예（압력위2 ATA，매차지속60 min，매일1차，련속7 d），HIBD조불여HBO간예，정상대조조불여결찰좌측경총동맥화HBO간예。이HIBD모형건립후설위결양결혈후0 h시점，3조우0 h、2 h、4 h、6 h、12h、1d、2d、3d、4d、5d、6d화7d시점단두처사（각조각시점n=10），취손상측뇌피질제비단세포현액，여세포선립체막전세（ΔΨm）표기물라단명123（Rho123）부육，용류식세포의검측Rho123적평균형광강도（MFL），병이해MFL치작위ΔΨm치。결과①정상대조조뇌피질세포ΔΨm치위（4.66±0.80）MFL，HIBD조각시점뇌피질세포ΔΨm치균저우정상대조조상응시점，차최저위0 h시점[（2.85±0.56）MFL]，각시점차이균유통계학의의（ P＜0.05）；②HIBD조급HIBD+HBO조뇌피질세포ΔΨm균정현강저-회복-재강저적변화규률，량조ΔΨm초차강저시간균위결양결혈후0 h시점，초차회복시간균위결양결혈후2~12 h，재차강저적시간균위결양결혈후1~4 d，HIBD+HBO조ΔΨm적재차강저정도경명현차최저위결양결혈후3 d시점[（2.62±1.03）MFL]；③HIBD조뇌피질세포ΔΨm재재차강저후미재회복，이HIBD+HBO조ΔΨm재재차강저후，우결양결혈후5d시점후개시회복，6화7d시점ΔΨm치축점추근단저우정상대조조수평，차이무통계학의의（ P＜0.05）。결론 HBO재HIBD후0 h지3 d내불능개선결양결혈손상측뇌피질세포적선립체공능，HIBO후과조개시HBO치료가능도치수손뇌피질세포적진일보손상，단HBO가능재HIBD후5~7 d내가통과개선뇌피질선립체공능촉진HIBD수손세포공능회복。
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. 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.