Mishra, Anusha2011-08-182011-08-182011-07https://hdl.handle.net/11299/113254University of Minnesota Ph.D. dissertation. July 2011. Major: Neuroscience. Advisor: Eric A. Newman. 1 computer file (PDF); x, 75 pages.Neurovascular coupling is a process by which neuronal activity leads to localized increases in blood flow in the central nervous system. When neurovascular coupling results in hyperperfusion of the neural tissue, the response is termed functional hyperemia and serves to satisfy the increased energy demand of active neurons. In brain slices, high [O2] alters neurovascular coupling, decreasing activity-dependent vasodilations and increasing vasoconstrictions. However, in vivo, hyperoxia has no effect on neurovascular coupling. In order to resolve these conflicting reports of O2 modulation, I examined neurovascular coupling in both ex vivo and in vivo rat retina preparations. In the ex vivo retina, 100% O2 reduced the amplitude of light-evoked arteriole vasodilations by 3.9-fold and increased the amplitude of vasoconstrictions by 2.6-fold when compared to responses in atmospheric [O2] (21%), consistent with slice data. Oxygen exerted its effect by decreasing vasodilatory prostaglandin signaling and increasing vasoconstrictory 20-hydroxyeicosatetraenoic acid signaling. However, in vivo, hyperoxia (breathing 100% O2) had no effect on light-evoked arteriole vasodilations or on blood flow. We found that the differing effects of O2 arise because retinal pO2 increases to a much greater extent in the ex vivo preparation (to 548 mmHg) than in vivo (to 53 mmHg; Yu et al. Am J Physiol 267:H2498-H2507). When retinal pO2 was raised to 53 mmHg in the ex vivo retina, no change in neurovascular coupling was observed. These results demonstrate that although O2 can modulate neurovascular signaling pathways when pO2 is raised high enough, such levels are not attained in vivo, even when an animal breaths 100% O2. Functional hyperemia can also be modulated by pathological conditions. It is diminished in the retinas of diabetic patients, possibly contributing to the development of diabetic retinopathy. I investigated the mechanism responsible for this loss in a streptozotocin-induced rat model of type 1 diabetes. Here I show that light-evoked arteriole dilation was reduced by 58% in these diabetic rats at 7 month survival time. The diabetic retinas showed neither a decrease in the thickness of the retinal layers nor an increase in neuronal loss, although signs of early glial reactivity were observed. Functional hyperemia is believed to be mediated, at least in part, by glial cells and we found that glial-evoked vasodilation was reduced by 60% in diabetic animals. An upregulation of inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry, and inhibition of iNOS restored both light- and glial-evoked dilations to control levels. These findings suggest that high NO levels resulting from iNOS upregulation alters glial control of vessel diameter and may underlie the loss of functional hyperemia observed in diabetic retinopathy. I further tested whether inhibiting iNOS reverses the loss of flicker-induced vasodilation in diabetic rat retinas in vivo. Flicker-evoked arteriolar dilations were diminished by 61% in diabetic animals, compared to non-diabetic controls. Treating diabetic animals with aminoguanidine (an iNOS inhibitor), either acutely via IV injection or long-term in drinking water, restored flicker-induced arteriole dilations in diabetic rats to control levels. The amplitude of the electroretinogram b-wave was similar in control and diabetic animals, suggesting that the deficit in functional hyperemia was not due to a reduction in neuronal activity. These findings demonstrate that inhibiting iNOS with AG is effective in preventing the loss of, and restoring, normal flicker-induced vasodilation in the diabetic rat retina. Treatment with iNOS inhibitors early in the course of diabetes has the potential to slow the progression of retinopathy by maintaining normal neurovascular coupling.en-USDiabetic retinopathyFunctional hyperemiaGlial cellsNeurovascular couplingOxygenRetinaNeuroscienceThesis or Dissertation