Normobaric hyperoxia therapy exerts its neuroprotective effect through an increase in tissue pO2 and a decrease in free radical generation, caspases and MMP expression in the ischemic penumbra

Stroke is a leading cause of death and disability. However, current treatments are ineffective. Oxygen therapy aimed at increasing tissue pO2 has been used as a potential treatment modality. Unfortunately, most experiments with oxygen therapy were conducted without monitoring the actual interstitial pO2 level that is essential for penumbral tissue to survive, and also a critical parameter for evaluation of the efficiency of oxygen therapy. Using the unique capability of in vivo electron paramagnetic resonance (EPR) oximetry to measure localized interstitial pO2, we have conducted EPR-guided oxygen therapy by monitoring penumbral pO2 in a rat model of 90-min transient ischemia while normobaric hyperoxia is administered. Our results reveal that penumbral pO2 level can be modulated by changing the percentage of oxygen content in the breathing gas, and that 95% O2 given to rats is capable of raising penumbral interstitial pO2 close to the physiological (pre-ischemic) value during ischemia. However, 95% O2 also caused an increase in penumbra pO2 to a level that was twice as high as the pre-ischemic level when given during reperfusion. Oxygen therapy with 95% O2, begun immediately after ischemia and continued for 90-min significantly reduced infarction volume by 40% and improved neurological function. Oxygen therapy given upon reperfusion also reduced infarction volume by 15%, but it was not statistically significant. In order to investigate the possible molecular mechanism for the observed neuroprotective effect of hyperoxia treatment, we have studied the effects of oxygen therapy in the ischemic penumbra. We focused specifically on the generation of free radicals and the activation of matrix metalloproteinase (MMP) and caspase, two well characterized mechanisms leading to cerebral injury that are regulated through free radical generation. Our results demonstrate that 95% O2 treatment administered during the 90-min ischemia decreased the formation of 8-hydroxy-2'-deoxyguanine, a biomarker of oxidative DNA damage. Furthermore, 90-min ischemia dramatically increased the MMP-2 and 9 expression at 24 hrs post reperfusion, while hyperoxia treatment decreased penumbral MMP-9 expression by 50%. Interestingly, there was no difference in MMP-2 expression between the oxygen treated and non-treated groups. Similarly, 90-min cerebral ischemia induced approximately a 100% increase in 22 kD cleaved caspase-8 and a 25% increase in 43 kD cleaved caspase-8 in the penumbra, as compared to the contralateral side during normoxia. Hyperoxia treatment significantly decreased caspase activation in the penumbra. These results establish for the first time that normobaric hyperoxia oxygen therapy exerts its neuroprotective effect during ischemia through the increase of penumbral tissue pO2, which leads to a reduction in free radical generation, and consequently, caspases and MMPs expression.