缺氧致新生小鼠脑发育异常的形态学研究

Morphological study on abnormal brain development inducedby hypoxia in neonatal mice

目的:建立新生小鼠缺氧模型,观察缺氧后大脑中认知相关脑区主要神经细胞类型在发育期的变化特征。方法:新生2 d的仔鼠在10%氧环境连续饲养5 d,之后放回正常氧环境,并在发育阶段不同时间点取材。利用免疫荧光组织化学比较小鼠缺氧后胼胝体(CC)和运动皮质(M1)中少突胶质细胞密度、成熟少突胶质细胞的比率、髓鞘蛋白水平的变化,以及前扣带回皮质(ACC)、海马(Hippo)、感觉皮质(S1)中兴奋性神经元、抑制性神经元的表达变化。进一步比较ACC中不同类型抑制性中间神经元、小胶质细胞、星形胶质细胞的密度变化特征。同时,还检测了缺氧对突触蛋白表达的影响。结果:免疫荧光定量检测结果显示:与对照组相比,缺氧小鼠CC、M1中髓鞘蛋白表达水平,成熟少突胶质细胞的比率均降低;ACC中兴奋性神经元的数量无显著差异,而ACC、Hippo以及S1的γ-氨基丁酸(GABA)标记的抑制性神经元的数量显著降低;ACC中小清蛋白阳性(PV~+)神经元、生长抑素阳性(SST~+)神经元和血管活性肠多肽阳性(VIP~+)神经元的数量均显著减少;缺氧小鼠ACC中囊泡膜谷氨酸转运体1(VGluT1)标记的兴奋性突触点的数量和桥尾蛋白(gephyrin)标记的抑制性突触点的数量均显著减少。虽然星形胶质细胞和小胶质细胞的数量无显著差异,但缺氧损伤以后小胶质细胞被激活。结论:慢性缺氧将导致发育期小鼠出现以少突胶质细胞和中间神经元发育受阻,以及突触形成障碍为主要特征的改变。这些结果为探索脑智发育异常相关疾病的神经机制提供了重要实验依据。

Objective:Neonatal mice hypoxia model was established to observe the responses of the main neural cell types in cognition-related brain areas.Methods:Pups at postnatal day 2(P2)were subjected to 10%oxygen for succeeding 5 days,and harvested at different development stage for histologic study.Immunofluorescence histochemistry was used to compare the changes of oligodendrocyte density,mature oligodendrocyte ratio and myelin protein level in corpus callosum(CC)and motor cortex(M1)after hypoxia,as well as the expression changes of excitatory and inhibitory neurons in anterior cingulate cortex(ACC),hippocampus(Hippo)and sensory cortex(S1).Furthermore,the density changes of different types of inhibitory intermediate neurons,microglia and astrocytes in ACC were compared.At the same time,the effect of hypoxia on the expression of synaptic proteins was also detected.Results:Quantitative immunofluorescence results showed lower myelin protein levels and mature oligodendrocyte ratio in CC and M1 of hypoxic mice compared with control mice.There was no significant difference in the number of excitatory neurons in ACC,but the number of gamma-aminobutyric acid(GABA)neurons in ACC,Hippo,and S1 were significantly reduced,especially parvalbumin neuron,ssomatostatin neurons,and vasoactive intestinal polypeptide neurons in ACC.The number of excitatory synapses labeled by vesicular glutamate transporter 1(VGluT1)and inhibitory synapses labeled by gephyrin were significantly decreased in ACC of hypoxic mice.Although there was no significant difference in astrocyte and microglia numbers,microglia were activated after hypoxic injury.Conclusion:Chronic hypoxia will lead to changes in the development of oligodendrocytes and interneurons,impair synapse formation.These results provide an important experimental basis for exploring the neural mechanism of diseases related to abnormal brain intelligence development.