神经细胞铁死亡在蛛网膜下腔出血大鼠脑组织中的表达及其作用

Expression and role of neuronal ferroptosis in brain tissue of rats with subarachnoid hemorrhage

目的 探究神经细胞铁死亡在蛛网膜下腔出血(subarachnoid hemorrhage, SAH)大鼠早期脑损伤(early brain injury, EBI)期间的表达情况及其神经功能损害的关系。方法 48只健康雄性SD大鼠被随机分为4组:假手术组(Sham组)、蛛网膜下腔出血组(按造模后取材时间不同分为3个亚组:SAH 6 h组、SAH 24 h组、SAH 72 h组),每组12只。用血管内穿刺法进行SAH造模,Sham组大鼠仅行血管穿刺,但不刺破血管。参照Garcia评分表检测各组大鼠的神经功能评分,干湿重法脑组织含水量;HE染色观察神经细胞形态并统计坏死数量,普鲁士蓝染色观察脑组织中铁沉积情况;吸光光度法检测各组大鼠脑组织铁含量、脂质过氧化物丙二醛(malondialdehyde, MDA)含量及谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)活性,标准曲线法检测谷胱甘肽(glutathione, GSH)含量。结果 与Sham组比较,SAH 6 h、24 h、72 h组大鼠的神经功能评分显著降低(P<0.05)。与Sham组比较,SAH 24 h、72 h组大鼠脑组织含水量明显升高(P<0.05),但SAH 6 h组差异无统计学意义(P>0.05)。与Sham组比较,SAH 24 h、72 h组大鼠脑组织海马CA1区神经元细胞坏死数明显增多(P<0.05),SAH 6 h组神经元细胞坏死数量差异无统计学意义(P>0.05)。普鲁士蓝染色结果显示,在Sham组脑皮质组织中,没有发现明显的铁沉积,SAH 6 h组脑皮质组织中有少量铁沉积;与Sham组比较,SAH 24 h、72 h组大鼠脑组织中铁沉积明显增多(P<0.05)。与Sham组比较,SAH 24 h、72 h组大鼠脑组织中铁含量、MDA含量明显增加(P<0.05),GSH含量和GPX4活性显著降低(P<0.05)。结论 神经细胞铁死亡在SAH大鼠EBI期间出现,且呈时间依赖性,72 h最为严重;其可能与神经功能损害有关。

Objective To investigate the expression of neuronal ferroptosis during EBI in SAH rats and its correlation to neurological impairment.Methods Forty-eight healthy male SD rats were randomly divided into four groups:Sham group,Subarachnoid Hemorrhage(SAH)group with three subgroups based on different time points after modeling(SAH 6 h,SAH 24 h,SAH 72 h),with 12 rats in each group.SAH was induced using an endovascular perforation method,while the Sham group underwent only vascular puncture without vessel perforation.Neurological function scores were evaluated using the Garcia scoring system,and brain tissue water content was measured using the wet-dry method.HE staining was used to observe neuronal morphology and count the number of necrotic cells.Prussian blue staining was performed to observe iron deposition in brain tissues.The absorbance spectrophotometry was used to measure brain tissue iron content,malondialdehyde(MDA)content,and glutathione peroxidase 4(GPX4)activity,while the standard curve method was used to measure glutathione(GSH)content.Results Compared to the Sham group,the neurological function scores of rats in the SAH 6 h,24 h,and 72 h groups were significantly decreased(P<0.05).Brain tissue water content in the SAH 24 h and 72 h groups was significantly increased compared to the Sham group(P<0.05),but no significant difference was found in the SAH 6 h group(P>0.05).The number of necrotic neurons in the hippocampal CA1 region of brain tissue was significantly higher in the SAH 24 h and 72 h groups compared to the Sham group(P<0.05),while there was no significant difference in the SAH 6 h group(P>0.05).Prussian blue staining showed no significant iron deposition in the cortical tissue of the Sham group,but there was a slight iron deposition in the cortical tissue of the SAH 6 h group.Compared to the Sham group,iron deposition in brain tissue was significantly increased in the SAH 24 h and 72 h groups(P<0.05).Furthermore,compared to the Sham group,brain tissue iron content and MDA levels were significantly increased in the SAH 24 h and 72 h groups(P<0.05),while GSH content and GPX4 activity were significantly decreased(P<0.05).Conclusion Neurocytic ferroptosis occurs during the early brain injury period in rats with SAH and shows time dependency,with the most severe effects observed at 72 hours.This process may be related to neurological dysfunction.