Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice

The evolution of brain injury was examined in mice subjected to focal cereb ral ischemia as induced by 30 min of intraluminar thread occlusion of the m iddle cerebral artery. followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by IH-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, h eat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunoc ytochemistry, and correlated with the degree of DNA fragmentation, as asses sed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intr aluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a sho rt-lasting postischemic hyperperfusion to approximately 120%. In non-ischem ic control animals, fractional protein synthesis values of 0.81 +/- 0.26 an d 0.94 +/- 0.23 were obtained. Thread occlusion resulted in a suppression o f protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04 +/- 0.08 in caudate-putamen and 0.14 +/- 0.1 9 in somatosensory cortex. P < 0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71 +/- 0.40 and 0.63 +/- 0.26. respect ively). but remained suppressed in the caudate-putamen (0.14 +/- 0.22 and 0 .28 +/-0.28). Regional ATP levels did not show any major disturbances at th e reperfusion times examined. Thread occlusion resulted in a transient incr ease of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 It after ischemia, which suggests that spreading d epressions were elicited in the tissue. At the same time. c-jun and hsp72 m RNAs were elevated only in ischemic, brain areas showing inhibition of prot ein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the cauda te-putamen, where protein synthesis rates were still reduced. and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did n ot increase in the cortex. where protein synthesis recovered. After 24 h an d 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in th e cortex. In summary, the suppression of protein synthesis is reversible in the ische mia-resistant cortex following 30 min of thread occlusion in mice, but pers ists in the vulnerable caudate-putamen, In the caudate-putamen, apoptotic p rograms are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor i nfluencing tissue survival after transient focal ischemia. (C) 2001 IBRO. P ublished by Elsevier Science Ltd. All rights reserved.