Introduction: Acidic pH is an important feature of the microenvironment of cerebral ischemic injury. Acute hyperglycemia has been used as a tool to augment intraischemic acidosis, oxidative stress, edema, and cell necrosis. In addition to classical unregulated necrosis, neurons can undergo regulated necrosis. One form of regulated cell death is RIP1-regulated necroptosis. However, the effect of acidosis on the RIP1 signaling pathway has not been well studied.
Hypothesis: The acidic microenvironment associated with hyperglycemic middle cerebral artery occlusion (MCAO) amplifies oxidative stress, which then augments RIP1-regulated necroptosis.
Methods: MCAO was produced for 1 h in male Sv/Ev129 mice by the filament technique. To produce acute hyperglycemia, mice received an ip infusion of 25% dextrose before and during MCAO to increase blood glucose into the 350-400 mg/dL range and further decrease ischemic tissue pH from 6.8 to 6.4.
Results: As expected, hyperglycemic mice had a greater infarct volume (41.4±4.9% of hemisphere) than did normoglycemic mice (28.4±5.3%). With intraventricular injection of the RIP-1 kinase inhibitor necrostatin-1, infarct volume decreased significantly in hyperglycemic mice (14.9±3.1%). Hyperglycemic MCAO increased protein carbonyls and 3-nitrotyrosine to a greater extent than normoglycemic MCAO, whereas necrostatin-1 attenuated their formation in hyperglycemic mice. Using immunoprecipitation, we found that hyperglycemic MCAO augmented the association of RIP3 with RIP1, MLKL, and PGAM5 and that these associations were blocked by necrostatin-1.
Conclusions: These results indicate that RIP1-regulated necroptosis can make a major contribution to the ischemic neuronal injury that is augmented by acute hyperglycemia. The mechanism of augmentation of oxidative stress by intraischemic acidosis appears to require RIP1 kinase activity because necrostatin-1 attenuated protein carbonyl and 3-nitrotyrosine formation. Because much of the cell death appears to result from regulated necrosis rather than classical unregulated necrosis, RIP1-regulated necroptosis may serve as a therapeutic target for cerebral ischemia associated with severe acidosis.