Immature rat brain slices exposed to oxygen-glucose deprivation as an in vitro model of neonatal hypoxic-ischemic encephalopathy
Immature rat brain slices exposed to oxygen-glucose deprivation as an in vitro model of neonatal hypoxic-ischemic encephalopathy
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DOI：
10.1016/j.jneumeth.2005.01.005
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发表时间：
2005-06-30
影响因子：
3
通讯作者：
Lizasoain, I
中科院分区：
文献类型：
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作者：
Fernández-López, D;Martínez-Orgado, J;Lizasoain, I
为了分析暴露于氧-糖剥夺（OGD）的未成熟大鼠脑片是否可能再现导致新生儿缺氧缺血性脑病（NHIE）中神经元死亡的主要病理生理事件，从7日龄Wistar大鼠获得500 μ m厚的脑片，并在含氧生理溶液中孵育。OGD组25只，除氧10-30 min，然后将切片置于正常培养液中。在对照组中，培养基组成保持不变（CG，n = 30）。每30分钟获得培养基样品，持续3小时。取脑片进行尼氏染色，光镜下观察海马CA 1区和皮质神经元的损伤情况。此外，通过分光光度法测定释放至培养基的LDH，定量神经元死亡。此外，培养基谷氨酸（Glu）水平通过HPLC测定，TNF α水平通过ELISA测定，而诱导型一氧化氮合酶表达通过对切片匀浆进行Western印迹测定。确定了最佳的OGD时间为20 min。OGD后，海马和皮质神经元数量明显减少。LDH释放在30分钟时达到最大，此时其是CG的5倍。此外，在OGD期结束时，培养基Glu浓度是CG水平的200倍。Glu与LDH释放量呈线性关系。最后，在OGD后3小时，观察到NOS的显著诱导以及TNF α释放的增加。总之，OGD似乎是一种可行的和可重复的体外模型，导致神经元损伤，这是在NHIE中发现的生理病理学相似。(c)2005 Elsevier B. V.保留所有权利。
To analyze whether exposure to oxygen-glucose deprivation (OGD) of immature rat brain slices might reproduce the main pathophysiologic events leading to neuronal death in neonatal hypoxic-ischemic encephalopathy (NHIE), 500 mu m-thick brain slices were obtained from 7-day-old Wistar rats, and incubated in oxygenated physiological solution. In OGD group, oxygen and glucose were removed from the medium for 10-30 min (n = 25); then, slices were re-incubated in normal medium. In control group the medium composition remained unchanged (CG, n = 30). Medium samples were obtained every 30 min for 3 h. To analyze neuronal damage, slices were stained with Nissl and CA1 area of hippocampus and cortex were observed under microscopy. In addition, neuronal death was quantified as LDH released to the medium determined by spectrophotometry. Additionally, medium glutamate (Glu) levels were determined by HPLC and those of TNF alpha by ELISA, whereas inducible nitric oxide synthase expression was determined by Western blot performed on slices homogenate. Optimal OGD time was established in 20min. After OGD, a significant decrease in the number of neurones in hippocampus and cortex was observed. LDH release was maximal at 30min, when it was five-fold greater than in CG. Furthermore, medium Glu concentrations were 200 times greater than CG levels at the end of OGD period. A linear relationship between Glu and LDH release was demonstrated. Finally, 3 h after OGD a significant induction of NOS as well as an increase in TNF alpha release were observed. In conclusion, OGD appears as a feasible and reproducible in vitro model, leading to a neuronal damage, which is physiopathologically similar to that found in NHIE. (c) 2005 Elsevier B.V. All rights reserved.