Neuronal and astroglial injury and recovery from stroke-induced depolarizations

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DESCRIPTION (provided by applicant): In ischemic stroke, cessation of blood flow results in depletion of local brain energy reserves. In areas of severe metabolic compromise decreased ATP leads to failure of the sodium-potassium pump, resulting in anoxic depolarization (AD) propagating from the ischemic core. Prolonged AD causes acute injury to neurons manifested by cellular swelling along with dendritic beading and spine loss, becoming terminal injury in the absence of reperfusion. Shorter duration (<5 min) recurring peri-infarct depolarizations (PIDs) initiate at the edge of the core and cause damage in the ischemic penumbra for hours to days after stroke. AD and PIDs are variations of a common spreading depolarizing process that depends upon the degree of local metabolic compromise (AD in the core where blood flow is <10%, PID in the penumbra where blood flow is 20-40%). Though important for maintaining ionic balance, preventing neurotoxicity, and providing trophic support for neurons, the dynamic contributions of astrocytes to the early stages of ischemic injury resulting from these spreading depolarizations are not well understood. The proposed research will clearly define the extent of neuronal and astroglial injury after AD and PIDs and recovery during reperfusion. The experimental approach will involve real-time in vivo 2-photon laser scanning microscopy at the level of single neurons and astrocytes simultaneously with electrophysiological recordings of AD and PIDs in two acute mouse models of stroke. The specific aims are: 1) To test the hypothesis that the anoxic depolarization component of ischemic injury affects astrocytes concurrently with neurons. 2) To test the hypothesis that PIDs acutely damage astrocytes and neurons in the penumbra, resulting in expansion of the ischemic core. In Aim 1, transient common carotid artery occlusion will be used to assess neuronal and astroglial injury and recovery concurrently with changes in the cortical direct current (DC) potential (which signifies AD) and blood flow. I hypothesize that AD acutely injures astrocytes in addition to neurons during the early stages of ischemia. During reperfusion, healthy astrocytes are needed to restore the ionic gradients that allow neurons to repolarize. Photothrombotic microcirculatory occlusion will be used in Aim 2 to create an ischemic lesion surrounding a penumbra-like "area at risk". Astrocytes and neurons in this area will be imaged as PIDs propagate within the penumbra, creating further mismatch between blood flow and metabolic demand. I hypothesize that neurons and astrocytes damaged by recurring PIDs are able to recover as long as adequate blood flow is available. Over time, energy needs for repolarization are no longer met by the diminishing energy supply, resulting in terminal injury and penumbral recruitment into the infarct core detected at the cellular level of resolution. The proposed work should implicate astrocytes as a novel target for clinical stroke therapy. The local anesthetic dibucaine will be used in an attempt to inhibit damage caused by PIDs in a subset of experiments, potentially increasing the therapeutic window after stroke. PUBLIC HEALTH RELEVANCE: As a result of this work, I anticipate to provide novel information about the dynamic relationship between astrocytes and neurons during the early events of stroke. Restoration of neuronal networks facilitated by astroglial recovery may provide a novel avenue for stroke therapy in the clinic.