Hyperglycemia Accentuates Persistent “Functional Uncoupling” of Cerebral Microvascular Nitric Oxide and Superoxide Following Focal Ischemia/Reperfusion in Rats

Hyperglycemia worsens outcome in stroke patients and in a variety of stroke models, most prominently following prolonged ischemia and reperfusion. Vascular dysfunction has been associated with this worsened outcome, manifested by reduced reperfusion cerebral blood flow (CBF), as well as increased hemorrhagic transformation, edema, and mortality. The phenomenon of “uncoupling” of endothelial nitric oxide synthase (eNOS) has been associated with hyperglycemia in the periphery and results in a dysfunctional eNOS-generating superoxide radical (\( {\text{O}}_2^{ - } \)) in lieu of nitric oxide (NO). A net result of NOS uncoupling is reduction in blood flow and generation of a pro-thrombotic, inflammatory vascular phenotype that could explain many of the features of hyperglycemic stroke. Because the sources of increased \( {\text{O}}_2^{ - } \) and mechanisms of reduced NO are difficult to identify under in vivo pathological conditions, we have termed the presence of perivascular excess of \( {\text{O}}_2^{ - } \) expression relative to NO as “functional uncoupling.” We hypothesized that hyperglycemia would induce a state of functional uncoupling in the cerebral microvasculature that would be exacerbated by transient focal ischemia. We examined the relative expression of systemically injected radical sensitive dyes in cerebral microvessel profiles in the basal and 24 h post-stroke brain in a rat model of middle cerebral artery occlusion and recanalization (MCAO/R) in controls and after hyperglycemia. We focused on the peri-infarct region because of its importance in penumbra extension and edema. Differential expression of fluorescent dyes sensitive to \( {\text{O}}_2^{ - } \) and NO in microvessel profiles were assessed in the peri-infarct region. Hyperglycemia was induced by streptozotocin 48 h prior to MCAO/R. We found that hyperglycemia resulted in an increase in \( {\text{O}}_2^{ - } \) relative to NO, a pattern consistent with functional uncoupling. This ratio was accentuated 24 h after MCA/O in both groups. Hyperglycemic rats showed a synergistic increase in the \( {\text{O}}_2^{ - } \)/NO ratio as well as reduced acute reperfusion CBF, increased infarct size, and enhanced expression of nitrotyrosine. While effects of hyperglycemia on oxidative radicals is well known, we showed for the first time that hyperglycemia synergistically worsened functional uncoupling in the peri-infarct microvasculature and that it persisted for the 24-h duration of the experiment. Persistent generation of microvascular \( {\text{O}}_2^{ - } \) and reduction in NO expression suggest potential late therapeutic targets to restore microvessel function and improve vascular outcomes in hyperglycemic stroke.