| Rapid changes in morphology and in inducible heat shock protein 70 (iHSP 70), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) were investigated after cerebral ischemia/reperfusion injury in newborn pigs. We also examined whether ischemia-induced changes involved indomethacin-sensitive mechanisms. In anesthetized piglets, increased intracranial pressure was used to produce 5 or 10 min of global cerebral ischemia. Pial arteriolar blood flow was monitored visually through a cortical window. Cessation of cerebral blood flow was verified with a microsphere technique. At 2-12 hrs after ischemia, various brain regions were sampled for morphology, immunohistochemistry, and immunoblot analysis. Morphology of untreated and time control brains were virtually identical. Six hrs after ischemia, cresyl violet and/or hematoxylin and eosin staining revealed cellular damage in cortical layers II/III and VI, dentate granular, subgranular, and hippocampal CA3 layers and cerebellar Purkinje cells. No cellular injury was detected with indomethacin pretreatment (5 mg/kg, i.v.). Immunoblots revealed differential expression of iHSP 70 in untreated brains. Cerebellum contained the highest levels followed by medulla, thalamus, hippocampus, and parietal cortex. iHSP 70 immunoreactivity (IR) was localized to cerebellar granular, dentate granular and subgranular layers, and cortical layers II/III. Increased iHSP 70 IR was detected in parietal cortex and hippocampus 6 hrs after ischemia. Using immunoblot data, calculation of percent change from control revealed a significant increase in iHSP 70 in parietal cortex (111 {dollar}pm{dollar} 39%, n = 6), hippocampus (195 {dollar}pm{dollar} 69%, n = 8), and cerebellum (136 {dollar}pm{dollar} 45%, n = 7) (p {dollar}<{dollar} 0.05, mean {dollar}pm{dollar} SE). Indomethacin pretreatment significantly (p {dollar}<{dollar} 0.05) attenuated the ischemia-induced increase in iHSP 70 as shown by the percent change from control values in parietal cortex and hippocampus (7 {dollar}pm{dollar} 30% and 89 {dollar}pm{dollar} 30%, respectively; n = 5). Indomethacin pretreatment had no effect on ischemia-induced increases in iHSP 70 in cerebellum. Immunohistochemistry localized eNOS only to the vascular wall and nNOS primarily to neurons. Whereas eNOS levels tended to be higher 4 hrs after ischemia, immunoblot analysis revealed a significant increase compared to time controls only at 6 hrs. Calculation of percent change from control revealed ischemia-induced eNOS increases in parietal cortex (117 {dollar}pm{dollar} 43%, n = 5) and hippocampus (40 {dollar}pm{dollar} 16%, n = 4) (p {dollar}<{dollar} 0.05, mean {dollar}pm{dollar} SE). The increase in eNOS levels was also apparent on tissue sections as an increase in immunoreactivity intensity. Indomethacin pretreatment abolished the ischemia-induced increase in eNOS in all three regions. In contrast, changes in nNOS levels were not detected after either ischemia or ischemia with indomethacin pretreatment. Thus, the HSP 70 and eNOS responses are rapid, specific, and involve indomethacin-sensitive mechanisms which are region-dependent. |
