Steward O, Kelley M S, Torre E R
Department of Neuroscience, University of Virginia School of Medicine, Charlottesville 22908.
Exp Neurol. 1993 Dec;124(2):167-83. doi: 10.1006/exnr.1993.1187.
The present study evaluates the temporal relationships between increases in glial fibrillary acidic protein (GFAP) mRNA, GFA protein levels, and GFAP immunostaining in the hippocampus of adult rats following unilateral lesions of the entorhinal cortex (EC). GFAP mRNA levels were assessed at 12 h, 1, 2, 4, 6, 8, 10, 12, 14, and 30 days postlesion by dot blot assays using 35S-labeled cRNA probes against the mRNA. Animals were also prepared for in situ hybridization during the peak of GFAP mRNA expression (2 days postlesion) to explore the nature of individual differences in the spatial extent of the increases. GFA protein levels were assessed by Western blot and dot immunoblot techniques in a separate group of animals prepared at 1, 2, 4, 6, 8, and 10 days postlesion and by immunostaining at 1, 2, 4, 6, and 8 days postlesion. The dot blot analyses of GFAP mRNA levels confirmed previous studies, in that we observed dramatic increases in the levels of GFAP mRNA in the hippocampus ipsilateral to the EC lesions. The increases were biphasic, with a large peak in mRNA levels at 1-2 days postlesion (about 10-fold greater than control) and a second peak at 6-8 days. In most animals, the increases were predominantly ipsilateral to the lesion. However, in some animals, there were also large increases on the contralateral side. In situ hybridization experiments revealed two different spatial patterns of increased gene expression, one in which the increases in GFAP mRNA occurred bilaterally and one in which increases were restricted primarily to the hippocampus ipsilateral to the lesion. Immunochemical measures revealed that GFA protein levels increased gradually in the hippocampus ipsilateral to the lesion, reaching a peak at about 2-fold higher than control at 4 days postlesion, and then remained near this level until at least 10 days postlesion. In the contralateral hippocampus, GFA protein levels were increased to about the same extent as on the ipsilateral side at 1, 2, and 4 days postlesion, but then began to decline, returning to near control levels by 8 days. Increases in immunostaining occurred with about the same time course as the increases in GFA protein levels as measured immunochemically. These results define the temporal relationship between increases in GFAP mRNA and increases in GFA protein, providing new insights into the regulation of gene expression in reactive astrocytes.
本研究评估了成年大鼠内嗅皮质（EC）单侧损伤后海马中胶质纤维酸性蛋白（GFAP）mRNA增加、GFA蛋白水平增加与GFAP免疫染色之间的时间关系。通过使用针对mRNA的35S标记cRNA探针的斑点印迹分析，在损伤后12小时、1、2、4、6、8、10、12、14和30天评估GFAP mRNA水平。在GFAP mRNA表达高峰（损伤后2天）期间，还对动物进行了原位杂交，以探讨增加的空间范围个体差异的性质。在另一组损伤后1、2、4、6、8和10天制备的动物中，通过蛋白质印迹和斑点免疫印迹技术评估GFA蛋白水平，并在损伤后1、2、4、6和8天通过免疫染色进行评估。GFAP mRNA水平的斑点印迹分析证实了先前的研究，即我们观察到EC损伤同侧海马中GFAP mRNA水平显著增加。增加是双相的，损伤后1-2天mRNA水平有一个大峰值（比对照高约10倍），6-8天有第二个峰值。在大多数动物中，增加主要在损伤同侧。然而，在一些动物中，对侧也有大幅增加。原位杂交实验揭示了基因表达增加的两种不同空间模式，一种是GFAP mRNA增加双侧发生，另一种是增加主要局限于损伤同侧的海马。免疫化学测量显示，损伤同侧海马中GFA蛋白水平逐渐增加，在损伤后4天达到比对照高约2倍的峰值，然后至少在损伤后10天保持在该水平附近。在对侧海马中，损伤后1、2和4天GFA蛋白水平增加到与同侧大致相同的程度，但随后开始下降，到8天时恢复到接近对照水平。免疫染色的增加与免疫化学测量的GFA蛋白水平增加的时间进程大致相同。这些结果定义了GFAP mRNA增加与GFA蛋白增加之间的时间关系，为反应性星形胶质细胞中基因表达的调节提供了新的见解。
