In situ hybridization (ISH) measurements of c-fos and hsp70 expression were made in brain slice studies of hypoxia, with or without fructose-1,6-bisphosphate (FBP) pretreatment. Each experiment used eighty 350 μm thick cerebrocortical slices, obtained from twenty 7-day old rats. Thirty minute periods of hypoxia were followed by 8 h of hyperoxic perfusion. Slices were removed at eight predetermined times, and processed for ISH and immunohistochemistry. In three of six hypoxia experiments, slices were pretreated for 60 min with 2 mM FBP, a condition known to maintain ATP level in brain slices during hypoxia. In three other hypoxia experiments slices received no pretreatment. In two control experiments slices were perfused for 11.5 h without hypoxia. In control experiments, hsp70 mRNA was barely detectable in slices at all times, although moderate c-fos mRNA expression occurred at 1 h after decapitation. Hypoxia produced a modest but statistically significant increase in c-fos mRNA and hsp70 mRNA induction 4 h following reoxygenation. At all times after hypoxia, FBP pretreatment reduced expression of c-fos and hsp70 mRNA. The absence of hsp70 mRNA in control slices suggests that intracellular protein denaturation was minimal in this preparation. In slices made hypoxic, the decrease in c-fos and hsp70 mRNA caused by FBP pretreatment suggests ameliorated progression towards injury. Immunohistochemistry showed no HSP70 protein at any time following hypoxia, with or without FBP pretreatment, presumably due to delayed HSP70 protein synthesis, or to a block in translation, as observed in vivo in other studies.
- brain slice
- FBP (fructose-1,6-bisphosphate)
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