A Study On The Mechanism Of Hyperbaric Oxygen Treatment After Acute Cerebral Ischemia

Objective: Improving tissue oxygenation is considered an important strategy for treating ischemic stroke. HBO therapy has been proven successful when applied early after cererbral ischemia in our previous study. We thus in the current study explored the pathologic changes concerning cell death in both the ischemic core and penumbra after HBO therapy to further investigate the mechanism. The change of the glucose utilization and mitochondrial function under HBO conditions was further investigated to search for the possible pathway of neuroprotection.Methods: We applied an intraluminal MCA occlusion model in rats under controlled conditions. We designed to evaluate the effect of HBO therapy (3 ATA, 60 minutes) on focal ischemia when applied at different times (3 and 12 hours) after ischemia. We then examined the effect of HBO therapy on apoptosis and necrosis both in the cortical penumbra area and ischemic core after ischemia, indicated by HE, TUNEL staining; activated caspase-3, caspase-9, Bcl-2, Bax immunoreactivity; the Annexin V/PI double staining and transmissional electron microscope. Glucose utilization in the ischemic area with ¹⁸FDG-micro-PET and the mitochondrial respiratory function, activities of the mitochondrial respiratory chain complex and contents of ATP in mitochondria isolated from the cortical penumbra area and ischemic core were also obserbed. Furthermore, we measured the contents of free radicals such as H₂O₂ and O₂(?), malondialdehyde (MDA) and antioxidant enzyme activities such as SOD and GSH-PX.Results: We clearly demonstrated that early HBO therapy significantly improved the neurological deficits and reduced infarct volume of brain both 24 h and 7 d after ischemia. We also demonstrated that HBO therapy decreased the expression of activated caspase-3, 9 and DNA fragmentation visualized with TUNEL staining and increased the expression of Bcl-2 along the ischemic penumbra, while it increased the activated caspase-3, 9 positive cells and enhanced the ratio of apoptotic to necroticcells in the ischemic core. Furthermore, glucose utilization in the ischemic area underwent a severe decrease during 1h-3 h after MCAO, while the early HBO treatment significantly attenuated the decrease in cerebral metabolic rate of glucose in the ischemic core of cortex compared to controls. HBO treatment also significantly enhanced the activities of mitochondrial respiratory chain complex I and IV in the ischemic core and enhanced the complex IV activity in the ischemic penumbra, finally increased the mitochondrial respiratory control rate and the mitochondrial ATP level. Meanwhile, we found that HBO treatment increased both the content of O₂(?) and the activity of SOD in the ischemic core and penumbra, while the content of MDA was only decreased in the ischemic penumbra.Conclusion: (1) HBO treatment had neuroprotective effects on focal cerebral ischemia when initiated early after ischemia. It is better to treat the stroke patients as earlier as possible in the clinical practice. (2) HBO therapy inhibited neuronal apoptosis in the penumbra but increased the ratio of apoptotic to necrotic cells in the ischemic core. Such change might explain the neuroprotective effect of HBO. (3) The early exposure of HBO can partially reverse the downward trend for the glucose utilization in the ischemic core and facilitate the production of mitochondrial ATP both in the ischemic core and penumbra, which might contribute to the reported change of cell death pattern in the ischemic area after early HBO therapy.