Spreading depolarizations (SDs) that occur in stroke and traumatic brain injury patients exacerbate the neuronal damage in metabolically compromised tissue. SDs are waves of sustained neuronal and glial depolarization that actively propagate a breakdown of ion gradients through the injured brain. The ion fluxes during SD are followed by a rapid accumulation of water inside neurons and astrocytes causing cytotoxic edema. Pyramidal neurons lack functional aquaporins. This implies that passive osmotically obligated water entry after the ionic movements during SD is unlikely. Using pharmacological inhibition, we have identified several chloride-coupled cotransporters that mediate ion and water fluxes and can participate in neuronal swelling. Recently mice with conditional deletion of these transporter proteins were generated offering unique opportunity to identify the exact molecular conduits of water influx into neurons during SD, and this will be studied in Aim 1. Cytotoxic edema distorts intracellular organelles. Mitochondria play a variety of functional roles in neurons, from metabolic support and neuroprotection to the release of cytokines that trigger apoptosis. In dendrites, the mitochondrial structure is closely linked to their function, and fragmentation (fission) of the normally elongated mitochondria indicates loss of their function under pathological conditions. To date, fragmentation of mitochondria was studied either in dissociated cultured neurons or brain slices, but not in the intact living brain. Using real-time in vivo 2-photon microscopy, we quantified mitochondrial fragmentation during brain injury. We demonstrated that alterations in neuronal mitochondria morphology occurs within minutes of injury onset and can be reversible in traumatic and ischemic injuries. Impact of SD on mitochondrial structure could be one of the major factors affecting mitochondrial fragmentation during noxious conditions, and it will be examined in aims 2 and 3.
The specific aims are: 1) To identify neuronal chloride cotransporters that are involved in the onset of SD-induced neuronal swelling. 2) To test the hypothesis that SD is the triggering mechanism of rapid neuronal mitochondrial fragmentation. 3) To test the hypotheses that SD facilitates mitochondrial fission and delays fusion in metabolically compromised cortical tissue. The proposal integrates a variety of classic and state of the art technologies; viral expression, mouse genetics, in vivo 2- photon microscopy coupled with electrophysiology and laser speckle imaging, as well as ultrastructural studies with serial section transmission electron microscopy. A novel automatic analysis algorithm, based on supervised statistical computer learning will be used to quantify the degree of dendritic mitochondrial fragmentation. Model of transient global ischemia (BCCAO) will be used to evoke SD. In a model of focal stroke (photothrombotic occlusion) and the healthy cortex, SD will be evoked by KCl microinjection. The results will bring new insight to the development of cytotoxic edema during the stroke and address how and under what conditions the mitochondrial organelles are affected by SD.
Neurologic emergencies such as ischemic and hemorrhagic stroke, head trauma, cardiac arrest, and asphyxiation-hypoxia are the top causes of mortality globally. Two of the major pathogenic events that cause acute brain damage during these clinical conditions are the spreading depolarizing waves and the associated brain swelling that course across the cortex injuring brain cells. Detailed understanding of how this injury evolves will help to design treatments for brain recovery in these neurologic conditions.
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| Vardjan, Nina; Horvat, Anemari; Anderson, Jamie E et al. (2016) Adrenergic activation attenuates astrocyte swelling induced by hypotonicity and neurotrauma. Glia 64:1034-49 |
| Kim, Ki Jung; Iddings, Jennifer A; Stern, Javier E et al. (2015) Astrocyte contributions to flow/pressure-evoked parenchymal arteriole vasoconstriction. J Neurosci 35:8245-57 |
| Steffensen, Annette B; Sword, Jeremy; Croom, Deborah et al. (2015) Chloride Cotransporters as a Molecular Mechanism underlying Spreading Depolarization-Induced Dendritic Beading. J Neurosci 35:12172-87 |
