Laser microdissection reveals regional and cellular differences in GFAP mRNA upregulation following brain injury, axonal denervation, and amyloid plaque deposition

Abstract

Astrocytes are one of the major cell types responding to central nervous system injury. Upregulation of the astrocytic intermediate filament molecule glial fibrillary acidic protein (GFAP) is a key event associated with this reaction. To study the response of astrocytes to different types of brain lesions, GFAP mRNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in mouse brain following injury, axonal denervation (entorhinal cortex lesion), and amyloid plaque deposition (APP23 transgenic mice). Analysis of tissue areas surrounding a lesion revealed a 21-fold increase of GFAP mRNA in tissue surrounding an injury site, a 6-fold increase in denervated tissue areas, and a 5-fold increase in plaque containing tissue. To this GFAP mRNA increase, astrocytic proliferation and migration as well as an increase of cellular GFAP mRNA expression within astrocytes could have contributed. To determine the degree of GFAP mRNA upregulation in individual astrocytes, an immunofluorescence protocol was developed to harvest astrocytes selectively by laser microdissection and preserve intact RNA. qRT-PCR analysis of GFAP mRNA in microdissected astrocytes revealed an 82-fold increase in astrocytes surrounding an injury site, a 30-fold increase in astrocytes located in a denervation zone, and an 18-fold increase in astrocytes surrounding an amyloid plaque. These data demonstrate that GFAP mRNA is strongly upregulated within individual reactive astrocytes in response to a lesion. Because astrocytic GFAP mRNA upregulation differs among the three lesioning paradigms, we conclude that the lesion type is an important determinant of postlesional astrocytic reactivity.