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

Megumi Shimamura, Jeanna M. Garcia, Donald Prough, Helen Hellmich

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)47-61
Number of pages15
JournalMolecular Brain Research
Volume122
Issue number1
DOIs
StatePublished - Mar 17 2004

Fingerprint

Laser Capture Microdissection
Antisense RNA
Hippocampus
Ribonucleases
Gene Expression
Brain-Derived Neurotrophic Factor
Brain
Messenger RNA
Population
Percussion
Neuropil
Pyramidal Cells
Dentate Gyrus
Neuroglia
Northern Blotting
Brain Injuries
Genes
Reverse Transcription
Complementary DNA
Neurons

Keywords

  • Aging
  • Disorders of the nervous system
  • Hippocampal gene expression
  • Laser capture microdissection
  • RNA amplification
  • Trauma
  • Traumatic brain injury

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

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title = "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",
abstract = "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.",
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AU - Prough, Donald

AU - Hellmich, Helen

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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.

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