Diabetes is one of the significant risk factors for ischemic stroke. Hyperglycemia exacerbates the pathogenesis of stroke, leading to more extensive cerebral damage and, as a result, to more severe consequences. However, the mechanism whereby the hyperglycemic status in diabetes affects biochemical processes during the development of ischemic injury is still not fully understood. In the present work, we record for the first time the real-time dynamics of HO in the matrix of neuronal mitochondria in vitro in culture and in vivo in the brain tissues of rats during development of ischemic stroke under conditions of hyperglycemia and normal glucose levels. To accomplish this, we used a highly sensitive HyPer7 biosensor and a fiber-optic interface technology. We demonstrated that a high glycemic status does not affect the generation of HO in the tissues of the ischemic core, while significantly exacerbating the consequences of pathogenesis. For the first time using Raman microspectroscopy approach, we have shown how a sharp increase in the blood glucose level increases the relative amount of reduced cytochromes in the mitochondrial electron transport chain in neurons under normal conditions in awake mice.