断乳前丰富环境暴露促进海马神经发生及其机制研究

Preweaning exposure to enriched environment induces hippocampal neurogenesis:experiment withrats

目的探讨断乳前丰富环境暴露对海马神经发生的影响及其可能机制。方法 36只10日龄的 SD 大鼠随机分为对照组和丰富环境组,每组18只。10～24日龄时两组大鼠隔日予以BrdU 50 mg/kg 腹腔注射,丰富环境组大鼠每日予以丰富环境暴露20 min。24日龄时丰富环境暴露结束后两组各取6只大鼠取海马提核蛋白行钙调蛋白和磷酸化 CREB Western 印迹检测。63日龄时处死其余大鼠取脑行冰冻切片,取前囟后3.6 mm 部位切片行尼氏染色,计数右侧海马齿状回细胞数目。BrdU 免疫组化后计算右侧海马 DG 区 BrdU 阳性细胞数目。免疫荧光双标后计算 BrdU 阳性细胞分化为神经元(BrdU/NeuN 共表达)和星性胶质细胞(BrdU/GFAP 共表达)的比率。结果 Western印迹检测结果显示与对照组比较,丰富环境组大鼠海马核蛋白的钙调蛋白(0.605±O.035 vs 0.245±0.035,t=-10.182,P=0.01)和磷酸化 CREB(0.485±0.007 vs 0.220±0.014,t=-23.702,P=0.002)水平较高。尼氏染色后计数前囟后3.6 mm 部位右侧海马齿状回细胞数目,结果显示丰富环境组细胞数目明显多于对照组(1580±72 vs 1375±62,t=-7.461,P<0.01)。丰富环境组大鼠右侧海马区 BrdU 阳性细胞数明显多于对照组(5363±487 vs 2984±318,t=-14.177,p<0.01),且神经元分化率(85.0%±2.8%vs 80.2%±2.8%,t=-4.166,P<0.01)和星形胶质细胞分化率也高于对照组(4.0%±0.5%vs 2.6%±0.6%,t=-6.493,P<0.01)。结论断乳前丰富环境暴露可以促进海马神经发生,其机制有可能与丰富环境暴露促进海马钙调蛋白和 CREB 活化有关。

Objective To explore the effects of preweaning exposure to enriched environment on hippocampal neurogenesis and the underlying mechanisms. Methods Thirty-six 10-day-old SD rats were randomly divided into the 2 equal groups ： control group and enriched environment group （ EE group. From the age of 10 days to 24 days the rats received intraperitoneal injection of bromodeoxyuridine （ BrdU ） 50 mg/kg every other day to label the newly proliferated cells in vivo, and the rats in EE group were daily exposed to enriched environment for 20 minutes. Six rats of each group were sacrificed whren they were 24 days of age. Nuclear protein of the hippocampus was extracted to undergo Western blotting to detect the levels of calmodulin and phosphorylated CREB （cAMP response element binding）. Other rats were sacrificed at the age of 63 days. Coronal cryostat sections of brain were cut. Sections at the level 3.6 mm posterior to the bregma were obtained and stained with methyl aniline blue and the number of cells in the hippocampal dentate gurus （DG） of the right hemisphere were counted using × 400 microscope. BrdU immunohistochemistry and double immunofluorescence labeling with BrdU/NeuN or BrdU/GFAP were done, and the numbers of BrdU-labeled cells and ratios of neurons and astrocytes differentiated from BrdU-labeled cells were calculated. Results The levels of calmodulin and phosphorylated CREB in the hippocampal nuclear extract of the EE group were 0. 065 ± 0. 035 and 0. 485 ± 0. 007 respectively, both significantly higher than those of the control group （0. 245 ± 0. 035 and 0. 220 ± 0. 014 respectively, P = 0.01 and P = 0. 002）. The number of cells in the DG area of right hippocampus 3.6 mm posterior to bregma of the EE group was 1580 ± 72, significantly higher than that of the control rats （ 1375 ± 62,t = - 7. 461,P 〈 0.01 ）. The number of BrdU labeled cells of the EE group was 5363 ± 487, significantly higher than that of the control group （2984 ± 318, t = - 14. 177, P 〈 0.01 ）. The ratio of neurons of the EE group was 85.0% ± 2.8%, significantly higher than that of the control group （80.2% ± 2.8%, t = -4. 166, P 〈0.01 ）. The differentiation rate of astrocytes of the EE group was 4.0% ± 0.5%, significantly higher than that of the control group （2.6% ± 0.6%, t = -6.493, P〈0.01）. Conclusion Preweaning exposure to enriched environment can induce neurogenesis. The underlying mechanism may be that enriched environment induces the activation of calmodulin and CREB in hippocampus.