Online

Pappas, Anthony C.

Neuroscience Graduate Program

2016

PhD

Deficits within the brain microcirculation contribute to poor patient outcome following aneurysmal subarachnoid hemorrhage (SAH). However, the underlying pathophysiology is not well understood. Intra-cerebral (parenchymal) arterioles are encased by specialized glial processes, called astrocyte endfeet. Ca²⁺ signals in the endfeet, driven by the ongoing pattern of neuronal activity, regulate parenchymal arteriolar diameter and thereby influence local cerebral blood flow. In the healthy brain, this phenomenon, called neurovascular coupling (NVC), matches focal increases in neuronal activity with local arteriolar dilation. This ensures adequate delivery of oxygen and other nutrients to areas of the brain with increased metabolic demand. Recently, we demonstrated inversion of NVC from vasodilation to vasoconstriction in brain slices obtained from SAH model animals. This pathological change, which would restrict blood flow to active brain regions, was accompanied by an increase in the amplitude of spontaneous Ca²⁺ events in astrocyte endfeet. It is possible that the emergence of higher amplitude endfoot Ca²⁺ events shifts the polarity of NVC after SAH by elevating levels of vasoactive agents (e.g. K⁺ ions) within the perivascular space. In the first aim of this dissertation we tested whether altered endfoot Ca²⁺ signaling underlies the inversion of NVC after SAH. Brain injury is often associated with increased levels of extracellular purine nucleotides (e.g. ATP). A recent study found that ATP levels in the cerebrospinal fluid of aneurysmal SAH patients were roughly 400-fold higher than that of non-SAH controls. Astrocytes express a variety of purinergic (P2) receptors that, when activated, could trigger a spike in intra-cellular Ca²⁺. It is possible that enhanced signaling via astrocyte P2 receptors underlies the change in endfoot Ca²⁺ signaling after SAH. In the second aim of this dissertation we determined the role of purinergic signaling in the generation of high-amplitude spontaneous endfoot Ca²⁺ events after SAH. Parenchymal arteriolar diameter and endfoot Ca²⁺ dynamics were recorded simultaneously in fluo-4-loaded rat brain slices using combined infrared-differential interference contrast and multi-photon fluorescence microscopy. We report that SAH led to a time-dependent emergence of spontaneous endfoot high-amplitude Ca²⁺ signals (eHACSs) that were only present in brain slices exhibiting inversion of NVC. Depletion of intracellular Ca²⁺ stores abolished spontaneous endfoot Ca²⁺ signals, including eHACSs, and restored arteriolar dilation in SAH brain slices to two downstream elements in the NVC signaling cascade, (1) increased endfoot Ca²⁺ and (2) elevated extracellular K+. We next tested the role of purinergic signaling in the generation of SAH-induced eHACSs by recording endfoot activity before and after treatment with the broad-spectrum purinergic receptor antagonist, suramin. Remarkably, suramin selectively abolished eHACSs and restored vasodilatory NVC in SAH brain slices. Desensitization of Ca²⁺-permeable ionotropic purinergic (P2X) receptors had no effect on eHACSs after SAH. However, eHACSs were selectively blocked using a cocktail of inhibitors targeting Gq-coupled purinergic (P2Y) receptors. Collectively, our results support a model in which SAH leads to an emergence of P2Y receptor-mediated eHACSs that cause inversion of NVC. Further, we identify the FDA-approved drug, suramin, as a potential therapy to be used in the treatment of aneurysmal SAH.