TY - JOUR
T1 - Laser capture microdissection and analysis of amplified antisense RNA from distinct cell populations of the young and aged rat brain
T2 - Effect of traumatic brain injury on hippocampal gene expression
AU - Shimamura, Megumi
AU - Garcia, Jeanna M.
AU - Prough, Donald S.
AU - Hellmich, Helen
N1 - Funding Information:
Supported in part by the Department of Anesthesiology and grants from the University of Texas Medical Branch Sealy Center on Aging and John Sealy Memorial Endowment Fund for Biomedical Research #2558-01 (to H.L.H).
PY - 2004/3/17
Y1 - 2004/3/17
N2 - 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.
AB - 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.
KW - Aging
KW - Disorders of the nervous system
KW - Hippocampal gene expression
KW - Laser capture microdissection
KW - RNA amplification
KW - Trauma
KW - Traumatic brain injury
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U2 - 10.1016/j.molbrainres.2003.11.015
DO - 10.1016/j.molbrainres.2003.11.015
M3 - Article
C2 - 14992815
AN - SCOPUS:1342285555
SN - 0169-328X
VL - 122
SP - 47
EP - 61
JO - Molecular Brain Research
JF - Molecular Brain Research
IS - 1
ER -