Fig. 7: The hyperglycemic shift to necroptosis is driven by mitochondrial ROS during neonatal hypoxia-ischemia brain injury.

Normal or hyperglycemic neonatal (p10) C57BL/6 mice were subjected to regional cerebral hypoxia-ischemia and sacrificed at 24 h of reperfusion. A Nonreducing SDS–PAGE and immunoblot of contralateral (cn) and ipsilateral (ip) tissue. There is a robust increase in the high MW, oxidized species of RIP1 in ipsilateral tissue from hyperglycemic mice. B Isolated brain tissue was stained with triphenly-tetrazolium (TTC) to measure infarct volume. Infarct volume is increased in hyperglycemic mice but is prevented by mitochondrial complex I deactivator, mitoSNO. N = 7 per mouse group. Graphed values represent mean ± standard deviation. One-way ANOVA with Fisher’s post hoc. C Representative images corresponding to quantitative results shown in B. D Reducing SDS–PAGE of contralateral and ipsilateral tissue. RIP1 increases while apoptotic markers, caspases-3, -6, -7, and PARP1 cleavage decrease in hyperglycemic mice. These trends are reversed by mitoSNO. E Immunoprecipitation of RIP1 from brain tissue. RIP1 is phosphorylated (p-RIP1) and RIP3 and MLKL co-precipitate with RIP1 in hyperglycemic mice. This did not occur in hyperglycemic mice treated with mitoSNO.