Early regional response of apoptotic activity in newborn piglet brain following hypoxia and ischemia

Abstract

Responses of selected neuroregulatory proteins that promote (Caspase 3 and Bax) or inhibit (Bcl-2, high Bcl-2/Bax ratio) apoptotic cell death were measured in the brain of piglets subjected to precisely controlled hypoxic and ischemic insults: 1 h hypoxia (decreasing FiO₂ from 21 to 6%) or ischemia (ligation of carotid arteries and hemorrhage), followed by 0, 2 and 4 h recovery with 21% FiO₂. Protein expression was measured in cortex, hippocampus and striatum by Western blot. There were no significant differences in expression of Caspase-3 between sham operated, hypoxic and ischemic groups. There were significant regional differences in expression of Bcl-2 and Bax in response to hypoxia and ischemia. The changes in Bcl-2/Bax ratio were similar for hypoxia and ischemia except for striatum at zero time recovery, with ischemia giving lower ratios than hypoxia. The Bcl-2/Bax ratio was also lower for the striatum than for the other regions of the brain, suggesting this region is the more susceptible to apoptotic injury.

Fig. 1

Comparison of cortical oxygenation during ischemia and hypoxia and different times of post-ischemic and post-hypoxic recovery in newborn piglets. Histograms of oxygen distribution in the microcirculation of the cortex were determined by the distribution of phosphorescence lifetimes, as described in Methods. The presented histograms are for control conditions, at the end of the 60 min of ischemia or hypoxia, and after 120 and 240 min of recovery. The histograms for controls and for the end of the hypoxic/ischemic periods are n = 19 and 22 for hypoxia and ischemia, respectively. For 120 min of recovery the n values were 12 and 15 for hypoxia and ischemia, respectively, and at 240 min recovery they were 7 and 7 for hypoxia and ischemia, respectively. The values are presented as the mean ± SE for the indicated number of experiments

Fig. 2

Bcl-2 expression in the frontal cortex of newborn piglets at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery. The results are means from 6 experiments ± SD. The data are expressed in % of the corresponding values (100%) for the control group of piglets. p values for significant difference from control were determined by Student t-test. p < 0.05 was considered statistically significant

Fig. 3

Bax expression in the frontal cortex of newborn piglets at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery. The results are means from 6 experiments ± SD. The data are expressed in % of the corresponding values (100%) for the control group of piglets. p values for significant difference from control were determined by Student t-test. p < 0.05 was considered statistically significant

Fig. 4

Bcl-2/Bax ratio in the frontal cortex measured at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery

Fig. 5

Bcl-2 expression in the hippocampus of newborn piglets at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery. The results are means from 6 experiments ± SD. The data are expressed in % of corresponding values (100%) for the control group of piglets. p values for significant difference from control were determined by Student t-test. p < 0.05 was considered statistically significant

Fig. 6

Bax expression in the hippocampus of newborn piglets at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery. The results are means from 6 experiments ± SD. The data are expressed in % of the corresponding values (100%) for the control group of piglets. p values for significant difference from control were determined by Student t-test. p < 0.05 was considered statistically significant

Fig. 7

Bcl-2/Bax ratio in the hippocampus measured at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery

Fig. 8

Bcl-2 expression in the striatum of newborn piglets at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery. The results are means from 6 experiments ± SD. The data are expressed in % of the corresponding values (100%) for the control group of piglets. p values for significant difference from control were determined by Student t-test. p < 0.05 was considered statistically significant

Fig. 9

Bax expression in the striatum of newborn piglets at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery. The results are means from 6 experiments ± SD. The data are expressed in % of the corresponding values (100%) for the control group of piglets. p values for significant difference from control were determined by Student t-test. p < 0.05 was considered statistically significant

Fig. 10

Bcl-2/Bax ratio in the striatum measured at 0, 2 and 4 h of post-ischemic and post-hypoxic recovery