Cerebral hypoxia-ischemia stimulates cytokine gene expression in perinatal rats.
OBJECTIVE
We tested the hypothesis that cerebral hypoxia-ischemia selectively stimulates interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) gene expression in brain regions susceptible to irreversible injury in perinatal rats.


METHODS
To elicit focal hypoxic-ischemic brain injury, 7-day-old perinatal (P7) rats were subjected to right carotid artery ligation followed by 3 hours of 8% O2 exposure and were killed 0 to 48 hours after hypoxia. Regional tissue IL-1 beta and TNF-alpha mRNA content were measured by reverse transcription followed by polymerase chain reaction amplification (RT-PCR) in samples prepared from cortex and hippocampus of the lesioned and contralateral hemispheres. cDNAs were amplified with primers specific for IL-1 beta, TNF-alpha, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which served as an internal control. The RT-PCR products were subjected to Southern blot analysis and hybridized with 32P-labeled gene-specific probes. Radioactivity was measured in excised bands, and results were normalized on the basis of levels of GAPDH expression.


RESULTS
In unlesioned P7 brain, IL-1 beta mRNA was barely detectable. In lesioned forebrain, there was a marked, transient stimulation of IL-1 beta mRNA expression, peaking at 4 hours after hypoxia. Hybridization signal was increased 16- to 30-fold over values from contralateral hemisphere samples in three independent assays (P < .05 comparing values in left and right cortex and in left and right hippocampus with the Kruskal-Wallis ranking test); by 24 hours after hypoxia, levels returned to normal. Similar transient increases in TNF-alpha mRNA expression were detected. In a closely related model of perinatal brain injury elicited by focal intracerebral N-methyl-D-aspartate injection, there was a corresponding acute stimulation of IL-1 beta and TNF-alpha mRNA expression at 4 hours after injection.


CONCLUSIONS
These results suggest that IL-1 beta and TNF-alpha may play important roles in the response of the developing brain to acute hypoxic-ischemic injury.
Szaflarski
J
J
Department of Pediatrics, University of Michigan, Ann Arbor 48109-0646, USA.
Burtrum
D
D
Silverstein
F S
FS
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D015139:Blotting, Southern; D001921:Brain / Q000187:drug effects / Q000276:immunology*; D002540:Cerebral Cortex / Q000276:immunology; D003342:Corpus Striatum / Q000276:immunology; D017931:DNA Primers; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis; D006624:Hippocampus / Q000187:drug effects / Q000276:immunology; D002534:Hypoxia, Brain / Q000276:immunology*; D007375:Interleukin-1 / Q000096:biosynthesis*; D002546:Ischemic Attack, Transient / Q000276:immunology*; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000494:pharmacology; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D017931:DNA Primers; D007375:Interleukin-1; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016202:N-Methylaspartate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases