Dose-dependent neuronal injury after traumatic brain injury.
The Fluoro-Jade (FJ) stain reliably identifies degenerating neurons after multiple mechanisms of brain injury. We modified the FJ staining protocol to quickly stain frozen hippocampal rat brain sections and to permit systematic counts of stained, injured neurons at 4 and 24 h after mild, moderate or severe fluid percussion traumatic brain injury (TBI). In adjacent sections, laser capture microdissection was used to collect uninjured (FJ negative) CA3 hippocampal neurons to assess the effect of injury severity on mRNA levels of selected genes. Rats were anesthetized, intubated, mechanically ventilated and randomized to sham, mild (1.2 atm), moderate (2.0 atm) or severe (2.3 atm) TBI. Four or 24 h post-TBI, ten frozen sections (10 microm thick, every 15th section) were collected from the hippocampus of each rat, stained with FJ and counterstained with cresyl violet. Fluoro-Jade-positive neurons were counted in hippocampal subfields CA1, CA3 and the dentate gyrus/dentate hilus. At both 4 and 24 h post-TBI, numbers of FJ-positive neurons in all hippocampal regions increased dose-dependently in mildly and moderately injured rats but were not significantly more numerous after severe injury. Although analysis of variance demonstrated no overall difference in expression of mRNA levels for heat shock protein 70, bcl-2, caspase 3, caspase 9 and interleukin-1beta in uninjured CA3 neurons at all injury levels, post hoc analysis suggested that TBI induces increases in neuroprotective gene expression that offset concomitant increases in deleterious gene expression.
Hellmich
Helen Lee
HL
Department of Anesthesiology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0830, USA. hhellmic@utmb.edu
Capra
Bridget
B
Eidson
Kristine
K
Garcia
Jeanna
J
Kennedy
Deborah
D
Uchida
Tatsuo
T
Parsley
Margaret
M
Cowart
Jeremy
J
DeWitt
Douglas S
DS
Prough
Donald S
DS
D000704:Analysis of Variance; D000818:Animals; D001930:Brain Injuries / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism / Q000473:pathology*; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D008297:Male; D042282:Microdissection / Q000379:methods; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D009930:Organic Chemicals; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000097:blood / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D015599:Trauma Severity Indices
D005452:Fluoresceins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D007375:Interleukin-1; D009930:Organic Chemicals; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C435731:fluoro jade; C506435:Casp9 protein, rat; D053453:Caspase 9; D020169:Caspases