二氮嗪对缺氧缺血性脑损伤新生大鼠不同脑区神经保护作用研究

Study of neuroprotective effect of diazoxide on different brain regions of neonatal rats with hypoxic-ische- mic encephalopathy

目的 探讨ATP敏感性钾通道（KATP通道）开放剂二氮嗪对宫内窒息所致新生大鼠缺氧缺血性脑损伤不同脑区的神经保护作用.方法 采用夹闭妊娠21 d的SD大鼠子宫动静脉方法制作宫内窒息模型,仅分离而不夹闭子宫动静脉剖宫产新生鼠为对照组,夹闭孕鼠子宫动静脉剖宫产新生鼠采用随机数字法随机分为窒息组、二氮嗪组、二氮嗪＋格列苯脲组及溶剂组,每组16只.窒息组和对照组不予以药物干预,其余各组分别在宫内窒息后并行剖宫产后0、12、24h腹腔内注射相应干预药物.新生鼠生后72 h处死,以2,3,5-氯化三苯基四氮唑染色方法检测脑梗死面积,以HE染色方法观察皮质、海马、小脑、丘脑和脑干的组织病理学变化并计算脑损伤梯度评分,以反转录-聚合酶链反应方法检测不同脑区KATP通道亚单位mRNA表达量.结果 窒息组脑梗死面积（51.7±10.4）％与对照组（0.3±0.1）％、二氮嗪组（17.0±2.7）％与窒息组（51.7±10.4）％、二氮嗪＋格列苯脲组（31.0±8.5）％与二氮嗪组（17.0±20.7）％比较差异均有统计学意义（P＜0.05）,窒息组脑梗死面积大,经二氮嗪干预后脑梗死面积减小；窒息组内五部分脑区神经细胞损伤梯度评分差异有统计学意义（P＜0.05）,其中海马（4.60±0.52）和皮质（4.50±0.53）损伤最严重,其次是小脑（4.20±0.63）；表达量海马和皮质,Kir6.2mRNA在二氮嗪组（2.56 ±0.88、2.48±0.41）与对照组（3.27±0.38、3.06±0.81）比较差异无统计学意义（P＞0.05）.结论 新生SD大鼠宫内窒息后海马和皮质损伤最严重,其次是小脑,最后是丘脑和脑干.二氮嗪对五个脑区均有神经保护作用,且对损伤最严重脑区海马及皮质的神经保护作用最强.

Objective To investigate the protective effects of diazoxide,a KATP channel opener,on different brain regions of neonatal rats with hypoxic-ischemic brain damage （HIBD） induced by intrauterine asphyxia.Methods The intrauterine asphyxia model was performed by clamping the uterine veins and arteries of the pregnant rats at the 21st day of gestation and the pups were bom by cesarean section.They were randomly divided into four groups：hypoxia-ischemia （HI） group,diazoxide （Dia） group,diazoxide ＋ glibenclamide （Dia ＋ Gli） group and solvent （NaOH） group.The neonatal rats in control group were born after the uterine vessels of the pregnant rats were isolated but not clamped.No drugs were used in HI and control group; otherwise,the intervention medicine was injected intraperitoneally at 0 h,12 h and 24 h after birth in other three groups.The neonatal rats were sacrificed 72 h after birth.The cerebral infarction areas were measured by TTC staining,the histopathologic scores were calculated after HE staining in cortex,hippocampus,cerebellum,thalamus and brainstem and the mRNA of the subunits of the KATP channel were detected by RT-PCR in different brain regions.Results The differences of the cerebral infarction areas between the HI group and control group,Dia group and HI group and Dia ＋ Gli group and Dia group were significant （P 〈0.05）.The infarction areas in HI group were high and those were decreased after the treatment of Diazoxide.The histopathologic scores were significantly different among the five brain regions in HI group （P 〈 0.05）：hippocampus （4.60 ± 0.52） and cortex （4.50 ± 0.53） were the most serious injured regions,the next was cerebellum （4.20 ± 0.63）.In hippocampus and cortex,the light density of Kir6.2 mRNA in Dia group（2.56 ± 0.88 and 2.48 ± 0.41） had no significant difference compared with that in control group（3.27 ± 0.38 and 3.06 ± 0.81）.Conclusions Hippocampus and cortex are the most seriously injured regions after HIBD induced by intrauterine asphyxia in neonatal rats.Diazoxide,a KATP channel opener,has neuroprotective effect on the five brain regions,especially on the hippocampus and cortex.