高压氧吸氧持续时间与脑出血大鼠模型神经细胞损伤因子水平的关系

Relationship between duration of hyperbaric oxygen inhalation and nerve cell injury factors in rat models of intracerebral hemorrhage

目的研究高压氧(HBO)吸氧持续时间与脑出血(ICH)大鼠模型神经细胞损伤因子水平的关系。方法健康清洁级成年雄性SD大鼠150只,采用随机数字表法分为正常对照组、ICH组和4个HBO组(A组、B组、C组和D组),每组25只。正常对照组大鼠不作处理,ICH组和HBO组大鼠以胶原酶诱导法建立ICH模型。4个HBO组于ICH造模后6 h进行HBO干预,吸氧持续时间分别为40、60、80、100 min。每组分别于第1、3、5、7、14天各处死5只大鼠取材。观察各组大鼠不同时间点的脑组织含水量及病理学变化,采用免疫组化法和实时荧光定量PCR法检测各组大鼠不同时间点血肿周边脑组织中B淋巴细胞瘤-2基因(Bcl-2)和Bcl-2相关X蛋白(Bax)表达及其mRNA水平。结果第1天,ICH组、4个HBO组大鼠的脑组织含水量均高于正常对照组(P<0.05)。除正常对照组外,其他各组大鼠的脑组织含水量均呈先增后减的趋势,ICH组大鼠在第3天达到最大值,4个HBO组大鼠于第5天达到最大值。正常对照组大鼠脑组织血管结构清晰,细胞核及核膜清晰可见,未见明显组织肿胀、细胞坏死以及胶质细胞增生;ICH组大鼠血肿周围脑组织水肿,局部脑组织可见胶质细胞明显增生;4个HBO组大鼠血肿周围脑组织水肿逐渐减轻,胶质细胞增生,细胞坏死减轻。与正常对照组相比,相同时间点其他各组大鼠血肿周边脑组织的Bcl-2表达及其mRNA水平显著增加(均P<0.05);与ICH组相比,相同时间点4个HBO组大鼠血肿周边脑组织的Bcl-2表达及其mRNA水平显著增加(P<0.05);第3、5、7、14天,A组、B组大鼠血肿周边脑组织的Bcl-2表达及其mRNA水平显著低于C组、D组(P<0.05)。与正常对照组相比,相同时间点其他各组大鼠血肿周边脑组织的Bax表达及其mRNA水平显著增加(均P<0.05);与ICH组相比,相同时间点4个HBO组大鼠血肿周边脑组织的Bax表达及其mRNA水平显著减少(P<0.05);相同时间点B组、C组大鼠血肿周边脑组织的Bax表达及其mRNA水平显著低于A组、D组(P<0.05)。结论HBO吸氧持续时间与ICH大鼠模型神经细胞的Bcl-2、Bax表达及其mRNA水平密切相关,而HBO最佳吸氧持续时间为80 min,可通过上调Bcl-2表达及其mRNA水平、下调Bax表达及其mRNA水平,减少ICH大鼠血肿周围脑组织的细胞凋亡。

Objective To study the relationship between the duration of hyperbaric oxygen(HBO)inhalation and nerve cell injury factors in rat models of intracerebral hemorrhage(ICH).Methods A total of 150 healthy and clean adult male SD rats were divided into normal control group,ICH group,and four HBO subgroups(group A,B,C,and D),with 25 rats in each group.Collagenase-induced ICH model was established in the ICH group and the HBO subgroups.The four HBO subgroups were intervened by HBO at 6 h after ICH modeling,and the durations of oxygen inhalation were 40,60,80,and 100 min,respectively.Five rats in each group were sacrificed on day 1,3,5,7,and 14 after treatment.Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the expressions and mRNA levels of B-lymphocytoma-2 gene(Bcl-2)and Bcl-2-associated X protein(Bax)in peripheral brain tissues of hematoma in each group at different time points.Results On the 1st day,the water contents of brain tissues in the ICH group and the four HBO subgroups were higher than that in the normal control group(P<0.05).Except for the normal control group,the water contents in the brain tissues of other groups increased first and then decreased.The water contents in the brain tissues of the ICH group reached the maximum value on the 3rd day,while those of the four HBO subgroups reached their maximum values on the 5th day.In the normal control group,the cerebral vascular structure of rats was clear,the nucleus and nuclear membrane were clearly visible,and no obvious tissue swelling,cell necrosis,and glial cell proliferation were observed.In the ICH group,brain edema occurred around the hematoma,and obvious glial cell proliferation was observed in local brain tissues.In the four HBO subgroups,brain tissue edema around hematoma was gradually reduced,glial cell proliferation and cell necrosis were reduced.Compared with the normal control group at each time point,the expressions of Bcl-2 and its mRNA levels in the brain tissues around the hematoma in other groups increased significantly(all P<0.05).Compared with the ICH group at each time point,the expressions of Bcl-2 and its mRNA levels in brain tissues around hematoma in the four HBO subgroups increased significantly(P<0.05).On the 3rd,5th,7th,and 14th day,the expressions of Bcl-2 and its mRNA levels in the brain tissues around the hematoma in HBO subgroup A and B were significantly lower than those in subgroup C and D(P<0.05).Compared with the normal control group at each time point,the expressions of Bax and its mRNA levels in brain tissues around hematoma in other groups increased significantly(all P<0.05).Compared with the ICH group at each time point,the expressions of Bax and its mRNA levels in brain tissues around hematoma in the four HBO subgroups decreased significantly(P<0.05).At the same time points,the expressions of Bax and its mRNA levels in brain tissues around hematoma in HBO subgroup B and C were significantly lower than those in subgroup A and D(P<0.05).Conclusion The duration of HBO inhalation was closely related to the expressions and mRNA levels of Bcl-2 and Bax in the ICH rat model’s neurons,and the optimal duration of HBO inhalation is 80 min.The reduction of apoptosis of brain tissue cells around hematoma in ICH rats can be achieved by up-regulating the Bcl-2 expression and its mRNA level and down-regulating the Bax expression and its mRNA level.