Laser capture microdissection and analysis of amplified antisense RNA from distinct cell populations of the young and aged rat brain: effect of traumatic brain injury on hippocampal gene expression.
To explore the molecular mechanisms underlying the increased vulnerability of the aged brain to traumatic brain injury (TBI), we compared the expression of several age-related genes in the CA1, CA3 and dentate gyrus subfields of the young and aged rat hippocampus before and after lateral fluid percussion TBI. Using laser capture microdissection (LCM), we obtained hippocampal neurons and glia from the neuropil adjacent to the pyramidal and granule cell layers. Subsequently, we linearly amplified and analyzed the antisense mRNA using Northern blot and ribonuclease protection assays (RPA). Our procedures, which have not been previously applied to quantitative analysis of LCM mRNA from neural tissue, included a modified reverse transcription step to enhance full-length cDNA synthesis, thus enhancing the yield of larger components of in vitro-transcribed mRNA for downstream analysis. Northern analysis showed greater expression of two aging-associated genes, p21 and brain-derived neurotrophic factor (BDNF) in the aged hippocampus. The age-related differences in p21 and BDNF expression were particularly prominent after TBI. By quantitative RPA analysis, we found that the expression of p21, known to be induced in senescent cells, was significantly greater in the CA3 region of aged rats, an area that is selectively vulnerable to TBI. However, expression of genes associated with regenerative and repair functions was significantly decreased in aged hippocampus. Our RPA results indicate that substantial age-dependent differences in the transcriptional profile of distinct regions of the hippocampal formation may account, in part, for their differential susceptibility to brain injury.
Department of Anesthesiology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0830, USA.
Megumi
M
Shimamura
Jeanna M
JM
Garcia
Donald S
DS
Prough
Helen L
HL
Hellmich
D000367:Age Factors; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D001930:Brain Injuries / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004195:Disease Models, Animal; D005353:Fibronectins / Q000378:metabolism; D019204:GTP-Binding Proteins; D015870:Gene Expression / Q000502:physiology*; D006624:Hippocampus / Q000378:metabolism* / Q000473:pathology; D039661:Integrin beta3 / Q000378:metabolism; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods; D009457:Neuroglia / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D016372:RNA, Antisense / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D002352:Carrier Proteins; D018076:DNA, Complementary; D005353:Fibronectins; C470310:Gnl3 protein, rat; D039661:Integrin beta3; D009687:Nuclear Proteins; D016372:RNA, Antisense; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins