Brief ischemia induced tolerance to subsequent ischemia in the hippocampal neurons. Male Mongolian gerbils were subjected to 2 min of ischemia in an awake condition. This ischemic insult only rarely produced neuronal damage in the gerbil brain. One day (n = 9), 2 days (n = 9), or 4 days (n = 10) following the first brief ischemia, the animals (double-ischemia group) were subjected to the second ischemia for 5 min. The single-ischemia group received a sham procedure instead of the first ischemia and was identically subjected to the second ischemia 1 day (n = 9), 2 days (n = 10), and 4 days (n = 13) following the sham procedure. One week following the second ischemia, all gerbils were perfusion fixed and the neuronal density in the hippocampal CA1 sector was measured. In double-ischemia groups, the neuronal density per 1-mm length of the pyramidal cell layer was 103.4 +/- 93.1 (SD) in the 1-day subgroup, 125.6 +/- 64.2 in the 2-day subgroup, and 176.2 +/- 93.7 in the 4-day subgroup, while the density in normal gerbils was 254.7 +/- 18.6. The average neuronal density in the single-ischemia group was much lower than that in the double-ischemia group (whole control group: 10.9 +/- 27.4). Immunostaining using monoclonal antibody raised against 70-kDa heat-shock protein revealed an increase in 70-kDa heat-shock protein in the CA1 area following 2 min of ischemia. Very brief ischemia induces heat-shock proteins and, presumably, thereby renders neurons more tolerant to subsequent metabolic stress.