未成熟新生鼠缺氧缺血损伤后大脑皮质和海马脑源性神经营养因子的变化

Expression changes of brain-derived neurotrophic factors in cortex and hippocampus after hypoxia-ischemia injury in immature rats

目的观察未成熟新生鼠缺氧缺血（hypoxia—ischemia，HI）损伤后大脑皮质和海马脑源性神经营养因子（brain．derivedneurotrophicfactor，BDNF）的变化，为临床早产儿HI脑损伤提供动物实验依据。方法3日龄清洁级SD大鼠，分为试验组和对照组。试验组幼鼠分离左侧颈总动脉、结扎离断，缺氧处理；对照组幼鼠分离左侧颈总动脉，不结扎离断也不进行缺氧处理。术后3、7和14d分别取材进行免疫组织化学染色和Westernblot实验。海马层面脑切片进行焦油紫（cresylfastvidet，CV）染色，观察大脑皮质和海马损伤情况，观察模型制作是否成功。进行BDNF免疫组织化学染色，统计大脑皮质和海马单位面积阳性细胞数量。利用Westernblot法检测大脑皮质和海马BDNF蛋白相对表达量。结果CV染色结果：Ⅲ损伤后，损伤侧大脑皮质和海马均有不同程度受损，面积减小，证实HI模型制作成功。免疫组织化学染色结果示大脑皮质和海马BDNF阳性细胞变化规律：与损伤对侧和对照组比较，损伤侧阳性细胞数术后3d减少（海马：t=-3．751、-2．920，P均〈0．05；皮质：t=-3．225、-2．298，P均〈0．05），术后7d（海马：t=4．136、2．256，P均〈0．05；皮质：t=3．924、2．838，P均〈0．05）、14d（海马：t=3．051、2．719，P均〈0．05；皮质：t=3．256、2．624，P均〈0．05）增多。Westernblot结果示大脑皮质和海马部位BDNF蛋白表达量变化规律：（1）海马：与损伤对侧比较，术后3d试验组损伤侧BDNF蛋白表达量减少（t=-3．388，P〈0．05）；与损伤对侧、对照组比较，术后14d损伤侧蛋白表达量增加（t=4．874、4．646，P均〈0．05）。（2）皮质：与损伤对侧、对照组比较，术后3d损伤侧蛋白表达量减少（t=-7．386、-3．256，P均〈0．05）；与对照组比较，术后7d损伤侧蛋白表达量增加（t=4．439，P〈0．05）；与损伤对侧、对照组比较，术后14d损伤侧蛋白表达量增加（t=24．161、3．942，P均〈0．05）。结论HI损伤早期大脑皮质和海马BDNF阳性细胞数和蛋白表达量均减少，而恢复期BDNF阳性细胞数量和蛋白表达量升高，BDNF可能在未成熟新生鼠HI脑损伤的恢复中起一定作用。

Objective To study the effect of hypoxia-ischemia （HI） on the brain-derived neurotrophic factor （BDNF） expression in the brain cortex and the hippoeampus of immature rats, and to provide new therapeutic strategies for HI brain injury. Methods Three-day-old rats were divided into 2 groups. One group of rat pups were subjected to the left carotid artery ligation followed by 60 mL/L oxygen for 2.5 hours（ HI-treated rats）. The other group of rat pups were only subjected to the left carotid artery separation without ligation and 60 mL/L oxygen （sham-treated rats）. The brain tissues were prepared at 3,7 and 14 d after treatment. Cresyl fast videt（CV） staining was used to evaluate the damage of the cortex and the hippocampus and check whether the models were successfully made. Immunostaining was used to determine the changes in BDNF positive cells in the brain cortex and the hippocampus after HI. Western blot a- nalysis was used to evaluate the expression of BDNF protein in the brain cortex and the hippocampus after HI. Results Models were successfully made. CV staining showed that there was brain damages and area loss in the cortex and the hippoeampus after HI. BDNF immunostaining showed that the number of BDNF-positive cells was significantly de- creased in the cortex （ t = - 3. 225, - 2. 298, all P 〈 0.05 ） and the hippocampus （ t = - 3. 751, - 2. 920, all P 〈 0.05 ） in the damaged side of the brain compared to the eontralateral side in the rats treated with HI and the sham-trea- ted rats at 3 d after surgery, while increased at 7 d （ t = 3. 924,2. 838, all P 〈 0.05 for cortex ; t = 4.136,2. 256, all P 〈 0.05 for hippocampus ） and 14 d （ t = 3. 256,2. 624, all P 〈 0.05 for cortex; t = 3.051,2.719, all P 〈 0.05 for hippo- campus） after surgery. Western blot analysis showed protein expressions of BDNF： （ 1 ） Hippocampus：the protein ex- pressions of BDNF were significantly decreased in damaged side of the brain compared to the contralateral side of rats treated with HI at 3 d（ t = -3. 388 ,P 〈 0.05 ） after surgery ,while increased compared to the contralateral side of rats treated with HI and the sham-treated rats at 14 d（ t =4. 874,4. 646, all P 〈 O. 05） after surgery. （2）Cortex：the protein expression of BDNF was significantly decreased in damaged side of the brain compared to the contralateral side of rats treated with HI and the sham-treated rats at 3 d （ t = - 7. 386, - 3. 256, all P 〈 0.05 ） after surgery, compared to the sham-treated rats at 7 d（t =4. 439 ,P 〈0.05） and the contralateraI side of rats treated with HI and the sham-treated rats 14 d（ t = 24. 161,3. 942 ,all P 〈 0.05 ） after surgery. Conclusions The number of BDNF-positive cells and pro- tein expressions are decreased in the cortex and the hippocampus at the early stage of HI injury, and increased at the late stage. BDNF may play a role in the healing stage of HI brain injury.