Hyperoxic reperfusion after global cerebral ischemia promotes inflammation and long-term hippocampal neuronal death

Abstract

In this study we tested the hypothesis that long-term neuropathological outcome is worsened by hyperoxic compared to normoxic reperfusion in a rat global cerebral ischemia model. Adult male rats were anesthetized and subjected to bilateral carotid arterial occlusion plus bleeding hypotension for 10 min. The rats were randomized to one of four protocols: ischemia/normoxia (21% oxygen for 1 h), ischemia/hyperoxia (100% oxygen for 1 h), sham/normoxia, and sham/hyperoxia. Hippocampal CA1 neuronal survival and activation of microglia and astrocytes were measured in the hippocampi of the animals at 7 and 30 days post-ischemia. Morris water maze testing of memory was performed on days 23-30. Compared to normoxic reperfusion, hyperoxic ventilation resulted in a significant decrease in normal-appearing neurons at 7 and 30 days, and increased activation of microglia and astrocytes at 7, but not at 30, days of reperfusion. Behavioral deficits were also observed following hyperoxic, but not normoxic, reperfusion. We conclude that early post-ischemic hyperoxic reperfusion is followed by greater hippocampal neuronal death and cellular inflammatory reactions compared to normoxic reperfusion. The results of these long-term outcome studies, taken together with previously published results from short-term experiments performed with large animals, support the hypothesis that neurological outcome can be improved by avoiding hyperoxic resuscitation after global cerebral ischemia such as that which accompanies cardiac arrest.

FIG. 1.

Promotion of hippocampal neuronal death by hyperoxic reperfusion after global cerebral ischemia. Rats were perfusion-fixed at 7 days (B) and 30 days (C) reperfusion after 10-min global cerebral ischemia was induced by bilateral carotid artery occlusion and bleeding hypotension. Normal-appearing neurons in the CA1 subregion were quantified using a stereological approach on cresyl violet–stained sections, as exemplified by the images shown in A, which were generated from the brains from animals perfusion-fixed at 30 days reperfusion. Examples of neurons exhibiting normal and abnormal morphologies are shown by the white and black arrows, respectively (scale bar = 25 μm; values represent the means ± standard error for n = 6–8 animals per group [7 days], and n = 10 per group [30 days]; *p < 0.05, **p < 0.01, ***p < 0.001).

FIG. 2.

Microglial activation in the hippocampal CA1 subregion following global cerebral ischemia. Rats were perfusion-fixed at 7 days (B) and 30 days (C) reperfusion after 10 min of global cerebral ischemia. Sections were then stained using Hoechst (blue) and primary antibody to Iba-1 (red) and visualized using fluorescent immunodetection (A), or using nickel-3,3′-diaminobenzidine (Ni-DAB), for stereologic quantification (B and C). Ionized calcium binding adaptor protein-1 (Iba-1)-immunoreactive cells were classified as resting, hypertrophic, or activated (scale bars for images obtained at 20 × magnification = 25 μm; values represent the means ± standard error for n = 6–8 animals per group [7 days], and n = 9–10 animals per group [30 days]; *p < 0.05, **p < 0.01, ***p < 0.001). Color image is available online at www.liebertonline.com/neu.

FIG. 3.

Astrocyte activation in the hippocampal CA1 subregion following global cerebral ischemia. Rats were perfusion-fixed at 7 days (B) and 30 days (C) reperfusion after 10 min of global cerebral ischemia. Sections were then stained using Hoechst (blue) and primary antibody to glial fibrillary acidic protein (green) and visualized using fluorescent immunodetection (A), or nickel-3,3′-diaminobenzidine (Ni-DAB), for stereologic quantification (B and C). GFAP-immunoreactive cell activation was morphologically classified as normal, moderate, or intense (scale bars for images obtained at 20 × magnification = 25 μm; values represent the means ± standard error for n = 6–8 animals per group [7 days], and 9–10 animals per group [30 days]; *p < 0.05, **p < 0.01, ***p < 0.001). Color image is available online at www.liebertonline.com/neu.

FIG. 4.

Effects of global cerebral ischemia and normoxic and hyperoxic reperfusion on Morris water maze measures of learning and memory. Latency to reach the hidden platform and the percentage of the swim path in the target quadrant during training are shown in panels A and C, respectively. Results from the visible platform tests are provided in panels B and D, respectively. The insert in panel C shows the significant main interaction effect between sham versus ischemic experimental groups. Experimental groups were sham/hyperoxic (SH; n = 5), sham/normoxic (SN; n = 5), ischemic/hyperoxic (IH; n = 10), and ischemic/normoxic (IN; n = 10). Each point represents the mean ± standard error.

FIG. 5.

Hyperoxic reperfusion reduces the percentage of time that post-ischemic rats spend searching for the platform in the correct quadrant during the probe test. Values represent the mean ± standard error for sham/hyperoxic (n = 5), sham/normoxic (n = 5), ischemic/hyperoxic (n = 10), and ischemic/normoxic (n = 10; *p < 0.05).