Investigating the Mechanism and Identity of a Spreading Depolarization Activator
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Authors
Hellas, Julia
Date
Type
thesis
Language
eng
Keyword
Stroke , Spreading depolarization , Ischemia
Alternative Title
Abstract
Stroke leads to irreversible brain injury and is the second global leading cause of death. The brain is metabolically demanding, requiring steady oxygen- and glucose-rich blood for normal function. During stroke, where cerebral blood flow stops, neurons cannot produce adenosine triphosphate (ATP), causing rapid failure of the ATP-dependent sodium/potassium transporter (Na+/K+ pump), and promoting spreading depolarization (SD) which is a propagating wave of electrical inactivation traversing the higher brain that can recur to worsen neuronal injury. SD injury can cause neurological deficits, yet there are no pharmacological treatments. Fundamentally, we do not understand the molecular events driving either the spread or the depolarization.
At picomolar concentrations, marine poison palytoxin (PLTX) converts the Na+/K+ pump into an open Na+/K+ channel, thereby inducing SD in brain slices, red blood cell swelling, and hemolysis. We hypothesize that an endogenous, PLTX-like SD activator (SDa) is released by stressed grey matter that initiates and drives SD. While there are hints in the literature suggesting such a substance promotes SD during metabolic stress, it has not been rigorously examined.
We first captured an SDa sample by exposing brain slices from one rodent to oxygen/glucose deprived (OGD) artificial cerebrospinal fluid (aCSF) at 34ºC. This ‘Post-SD aCSF’ evoked SD in naïve slices with 78-82% frequency (n=16 slices). In contrast, ‘Pre-SD aCSF’, bathing slices prior to OGD, evoked SD in 0% of naïve slices (n=18 slices). Freezing did not reduce SDa activity but heating to 100 ºC for 5 min lowered SD frequency to 27% (n=24 slices). We then ruled out that pH changes or released K+ were responsible. Finally, we built upon previous work from the Andrew lab where trace amounts of PLTX seemed to ‘prime’ the Na+/K+ pump for opening, facilitating hemolysis. We found that this could be replicated in slices.
The isolated Post-SD aCSF should serve as a reasonably purified SDa solution, given the absence of cellular disruption/extraction procedures. A stronger understanding of the activity and identity of an endogenous SDa and improved understanding of the mechanisms driving SD will help elucidate novel targets for inhibiting recurrent SD in clinical populations suffering brain ischemia.
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ProQuest PhD and Master's Theses International Dissemination Agreement
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Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.