Under normal cellular conditions there is a constant balancing act between the creation of reactive oxygen species (ROS) and their elimination by the antioxidant system including enzymes such as superoxide dismutase, glutathione peroxidase, and catalase. Abnormally low levels of ROS can impede cellular signaling and some normal intracellular reactions, while overly high levels of ROS create oxidative stress which can lead to the improper oxidation of lipids, proteins and DNA resulting eventually resulting in apoptosis, necrosis, and other cellular damage.
It has been demonstrated that acute hypoxia induces increased production of ROS in the brain by altering the activity of the cytochrome chain responsible for mitochondrial oxidative phosphorylation. This results in a decrease in ATP synthesis and an increase in ROS while decreasing the activity of the normal cellular antioxidant system. The resulting oxidative stress initiates apoptosis, which contributes significantly to the neuronal cell death observed in hypoxia. Recovery from hypoxia, and the survival of affected neurons is dependent on reoxygenation but little study has been done concerning the effects of reoxygenation on oxidative stress levels and the ROS/antioxidant system balance.
A 2017 study by Coimbra-Costa et al. examined the effects of reoxygenation on a variety of oxidative stress parameters and various components of the antioxidant system. Rats were exposed to hypoxia by breathing a gas mixture of 7% oxygen and 93% nitrogen for 6 hours, followed by reoxygenation in normal room air for either 24 or 48 hours. Animals were sacrificed and brain tissue was collected for a series of tests relating to oxidative stress levels and the functions of the antioxidant system. Oxidation levels in the brain samples were assessed through the TBARS test for lipid peroxidation, Barsotti’s method for the measurement of advanced oxidation protein products and the measurement of NO via the Griess reaction.
Measurement of the antioxidant activity in the brain samples was determined through the ratio of reduced (GSH) to oxidized (GSSH) glutathione and the activity of superoxide dismutase (SOD) (Arbor Assays Superoxide Dismutase Colorimetric Activity Kit). Expression levels and localization of various oxidative stress and antioxidant system proteins were established via western blot and immunohistochemical staining.
Ultimately, the Coimbra-Costa study determined oxidative stress could be considered the main damaging event induced by hypoxia. However, unlike what has been observed in ischemia, reoxygenation after hypoxia does not induce further oxidative damage. Rather increased blood flow not only promotes the resupply of oxygen, it also removes harmful waste products such as lactate and hydrogen that might otherwise cause brain damage. The oxidative stress induced by the acute hypoxia stage of the experiment was reversed after 24 hours of reoxygenation at normal O2 levels, but the apoptosis triggered in response to the oxidative stress remained. The study found apoptosis after hypoxia was more prevalent in the hippocampus than the cortex and may be responsible for the type of impaired brain functions, such as spatial reference memory issues, often reported after hypoxic brain injury.
The brain is particularly vulnerable to the effects of excess ROS because it already has low catalase and glutathione peroxidase activity relative to other tissue types, but high metabolic activity and oxygen consumption. The brain is also rich in lipids with unsaturated fatty acids that can react with ROS and generate peroxyl radicals that oxidize the lipid membranes. The potential for damage due to elevated ROS levels experienced during hypoxia is therefore high. Fortunately, upon reoxygenation, the remediation of the majority the oxidative stress impact, except for the ROS mediated apoptosis, is relatively quick. Therefore, it’s likely if ways can be found to reduce the level of ROS induced apoptosis in the throes of a hypoxic event, long term prognosis may be significantly improved.
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