METHYLENE BLUE PROTECTS ASTROCYTES FROM HYPOXIA-REOXYGENATION INJURY BY IMPROVING CELLULAR BIOENERGETICS

In ischemic stroke, which is caused by a clot that blocks a brain blood vessel, a portion of the brain is deprived of oxygen and nutrients that are carried in the blood. When this clot is removed either by time or drugs, the affected region is flooded with an overabundance of nutrients and oxygen to a degree that is larger than the cells in the region can process and this unbalances the system and causes further damage. In our current study, we are testing a chemical compound called Methylene Blue to see if it can improve the cells’ ability to handle this excess of nutrients and oxygen available to them at the time of clot dissolution and thereby improve their survival after ischemic stroke. Purpose (a): Ischemic Stroke inflicts a double blow to the affected brain region by characteristically presenting a period of acute ischemia during which the cells are completely deprived of valuable nutrients (Glucose & oxygen) resulting in cell death, however as the blood flow is restored (Spontaneously/surgically) the surviving cells are exposed to an overwhelming levels of glucose and oxygen resulting in reperfusion injury which further aggravates the cellular injury inflicted by ischemia. Methylene Blue (MB) is a heterocyclic aromatic compound shown to function as an alternative electron carrier and improve glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen in the brain. In our current study we aim to delineate if MB is protective in astrocyte against hypoxia-reoxygenation injury and determine its underlying mechanism. Methods (b): Primary astrocytes cultures isolated from day old C57BL6 were used in the current study. Protective role of Methylene blue (MB) in primary astrocyte cultures was evaluated in an in vitro model of cellular hypoxia (0.1% O2, 6h) and re-oxygenation (24h). The effect of MB on glucose uptake was determined by using the 2-NBDG assay. Oxygen sensitive dye Tris (2,2′-bipyridyl) dichloro Ruthenium(II) hexahydrate and Fluorescence Life Time Imaging (FLTI) was used to determine the effect of MB on intracellular oxygen concentration. ATP assay was used to determine the effect of MB on cellular energy status. Results (c): Results from cell viability assay showed that MB treatment significantly protected astrocytes from hypoxia-reoxygenation induced cell death. MB treatment significantly increased cellular glucose uptake in primary astrocyte cultures. FLTI showed that MB significantly increased intracellular oxygen concentration in primary astrocytes. Astrocytes treated with MB also had significantly higher ATP concentration compared to non-treated cells. Conclusions (d): Methylene Blue protects astrocytes against hypoxia-reoxygenation injury by improving astrocyte bioenergetics.