创伤性脑损伤大鼠脑组织中α-酮戊二酸脱氢酶的活性变化及意义

Changes and significance ofα-ketoglutarate dehydrogenase complex activity in brain tissues of rats with traumatic brain injury

目的探讨创伤性脑损伤(TBI)大鼠脑组织中α-酮戊二酸脱氢酶(α-KGDHC)活性变化及意义。方法将80只Sprague Dawley大鼠随机分为假手术组、TBI组、TBI-溶剂对照组、TBI-3-甲基-2-氧基戊酸(KMV)组,每组20只。TBI组、TBI-溶剂对照组和TBI-KMV组大鼠建立中度液压TBI模型,假手术组大鼠给予相同的手术步骤,但不实施液压冲击。TBI前24 h,TBI-KMV组大鼠侧脑室注射10μL KMV,TBI-溶剂对照组大鼠侧脑室注射10μL生理盐水,注射速度为lμL·min^-1;假手术组和TBI组大鼠侧脑室不做注射。分别于TBI后24、72 h及7 d时,每组随机选取5只大鼠,采用Shapira和Wahl脑损伤神经功能评分法进行神经功能评分,Morris水迷宫实验检测大鼠学习记忆功能;Morris水迷宫实验后快速断头处死大鼠,完整取出脑组织,迅速分离出损伤侧大脑皮层、海马及丘脑,采用比色法检测脑组织中α-KGDHC活性。TBI后72 h,每组随机选取5只大鼠,应用40 g·L^-1多聚甲醛溶液灌注固定,断头取脑组织,采用Fluoro-Jade染色液对坏死神经元进行染色并计数。结果TBI后24、72 h及7 d,TBI组和TBI-溶剂对照组大鼠神经功能评分显著低于假手术组(P<0.05),TBI-KMV组大鼠神经功能评分显著低于TBI-溶剂对照组和TBI组(P<0.05),TBI-溶剂对照组与TBI组大鼠神经功能评分比较差异无统计学意义(P>0.05)。TBI后24、72 h及7 d,TBI组和TBI-溶剂对照组大鼠登台时间及登台累计距离显著长于假手术组(P<0.05),TBI-KMV组大鼠登台时间及登台累计距离显著长于TBI-溶剂对照组和TBI组(P<0.05),TBI-溶剂对照组与TBI组登台时间及登台累计距离比较差异无统计学意义(P>0.05)。TBI后24、72 h及7 d,TBI组和TBI-溶剂对照组大鼠损伤侧大脑皮层、海马及丘脑组织中α-KGDHC活性显著低于假手术组(P<0.05),TBI-KMV组大鼠大脑皮层、海马及丘脑组织中α-KGDHC活性显著低于TBI组和TBI-溶剂对照组(P<0.05),TBI-溶剂对照组与TBI组大鼠大脑皮层、海马及丘脑组织中α-KGDHC活性比较差异无统计学意义(P>0.05)。TBI后72 h,TBI组和TBI-溶剂对照组大鼠损伤侧大脑皮层、海马和丘脑坏死神经元数显著多于假手术组(P<0.05),TBI-KMV组大鼠损伤侧大脑皮层、海马和丘脑坏死神经元数显著多于TBI组和TBI-溶剂对照组(P<0.01),TBI组与TBI-溶剂对照组大鼠损伤侧大脑皮层、海马和丘脑坏死神经元数比较差异无统计学意义(P>0.05)。结论TBI大鼠脑组织中α-KGDHC活性降低,抑制α-KGDHC活性可加重脑损伤,α-KGDHC可能参与了TBI和继发性脑损伤的发生与发展。

Objective To investigate the changes and significance ofα-ketoglutarate dehydrogenase complex(α-KGDHC)activity in brain tissues of rats with traumatic brain injuries(TBI).Methods Eighty Sprague Dawley rats were randomly divided into sham operation group,TBI group,TBI-solvent control group and TBI-α-keto-β-methyl-n-valeric acid(KMV)group,with twenty rats in each group.The TBI models of rats in the TBI group,TBI-solvent control group and TBI-KMV group were established by moderate hydraulic impact.The rats in the sham operation group were given the same procedure,but no hydraulic shock was applied.At 24 hours before TBI,10μL KMV was injected into the lateral ventricles of rats in the TBI-KMV group,and 10μL normal saline was injected into the lateral ventricles of rats in the TBI-solvent control group,the injection speed was 1μL·min^-1.The rats in the sham operation group and TBI group did not receive lateral ventricle injection.At 24,72 hours and 7 days after TBI,5 rats in each group were randomly selected,and the neurological function was scored by Shapira and Wahl brain injury neural function scoring method,and the learning and memory function of rats were examined by Morris water maze test.After Morris water maze test,the rats were decapitated and killed quickly,and the brain tissues were taken out completely,and the damaged cerebral cortex,hippocampus and thalamus were separated rapidly.The activity ofα-KGDHC in brain tissues was detected by colorimetry.At 72 hours after TBI,5 rats in each group were randomly selected,then the rats were perfused and fixed with 40 g·L^-1 paraformaldehyde solution,and the brain tissues were taken out after decapitation.The necrotic neurons were stained and counted with Fluoro-Jade staining solution.Results At 24,72 hours and 7 days after TBI,the neurological function score of rats in the TBI group and TBI-solvent control group was significantly lower than that in the sham operation group(P<0.05),the neurological function score of rats in the TBI-KMV group was significantly lower than that in the TBI-solvent control group and TBI group(P<0.05),and there was no significant difference in the neurological function score of rats between the TBI-solvent control group and TBI group(P>0.05).At 24,72 hours and 7 days after TBI,the time and the cumulative distance of arriving platform of rats in the TBI group and TBIsolvent control group were significantly longer than those in the sham operation group(P<0.05),the time and the cumulative distance of arriving platform of rats in the TBI-KMV group were significantly longer than those in the TBI-solvent control group and TBI group(P<0.05),and there was no significant difference in the time and the cumulative distance of arriving platform of rats between the TBI-solvent control group and TBI group(P>0.05).At 24,72 hours and 7 days after TBI,the activity ofα-KGDHC in impaired cerebral cortex,hippocampus and thalamus tissues of rats in the TBI group and TBI-solvent control group was significantly lower than that in the sham operation group(P<0.05),the activity ofα-KGDHC in impaired cerebral cortex,hippocampus and thalamus tissues of rats in the TBI-KMV group was significantly lower than that in the TBI group and TBI-solvent control group(P<0.05),and there was no significant difference in the activity ofα-KGDHC in impaired cerebral cortex,hippocampus and thalamus tissues of rats between the TBI-solvent control group and TBI group(P>0.05).At72 hours after TBI,the number of necrotic neurons in cerebral cortex,hippocampus and thalamus of rats in the TBI group and TBI-solvent control group was significantly higher than that in the sham operation group(P<0.05),the number of necrotic neurons in cerebral cortex,hippocampus and thalamus of rats in the TBI-KMV group was significantly more than that in the TBI group and TBI-solvent control group(P<0.05),and there was no significant difference in the number of necrotic neurons in cerebral cortex,hippocampus and thalamus of rats between the TBI-solvent control group and TBI group(P>0.05).Conclusion activity ofα-KGDHC in brain tissue of TBI rats decreased,inhibiting the activity ofα-KGDHC can aggravate brain injury.α-KGDHC may be involved in the occurrence and development of TBI and secondary brain injury.