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Refractory nerve agent induced seizures: the role of GABA(A) receptors and oxidative stress

Date

2023-04-27

Journal Title

Journal ISSN

Volume Title

Publisher

ORCID

Type

Thesis

Degree Level

Masters

Abstract

Introduction: Organophosphate nerve agents act as potent acetylcholinesterase inhibitors which can cause refractory seizure activity in exposed casualties. The mechanism of these refractory seizures is poorly understood, thus creating a treatment challenge when selecting the appropriate anticonvulsant and neuroprotection, as survivors tend to show neurological deficits. Oxidative stress may play a role in neuropathology of nerve agent exposures, specifically via free radical production by NADPH oxidases (NOX2 and NOX4) and saturation of endogenous antioxidant mechanisms. Furthermore, previous research indicates GABA(A) receptor trafficking has a role in refractory seizure activity and that GABA neurotransmission may be modulated by oxidative stress. N-acetylcysteine (NAC) is a well characterized, effective, and widely available antioxidant compound. The first aim of this study assessed the role of oxidative stress in nerve agent, specifically soman, induced brain injury and its effect on GABA(A) receptor distribution. The second aim of this study was to investigate the effect of antioxidant NAC on seizure activity, oxidative markers and GABA(A) receptor levels. Methods: Rats were pre-treated with HI-6 and atropine to ensure survival 30 minutes prior to an 85 µg/kg soman injection or saline control, end points for brain dissection and collection were at 3, 24, and 48 hours. The second objective of this study used the same methods with the addition of 100 mg/kg and 300 mg/kg NAC pre-treatment 60 minutes prior to exposure. Western blot was used to analyze hippocampus and cortex samples prepared as homogenates and synaptoneurosomes to assess GABA(A)α1 subunit levels, NOX changes, and brain injury biomarkers. Confocal imaging was used to qualitatively assess gliosis and brain damage in the hippocampus CA1 and dentate gyrus regions. Biochemical assays were used to quantify oxidative stress and antioxidant function. Results: Rats exhibited either convulsive or nonconvulsive seizures induced by soman. GABA(A)α1 subunit densities decreased at 3 hours in synaptoneurosomes but not in homogenates, indicating decreased GABA(A) receptor availability on post-synaptic membranes. At 24 and 48 hours, GABA(A)α1 returned to control levels in the hippocampus and cortex. Oxidative stress parameters do not change significantly in any of the six biochemical assays used and Western blot brain injury markers were not significantly elevated. Gliosis appeared as early as 3 hours in severe seizure brains and persisted until at least 48 hours. NAC preserves GABA(A)α1 expression at the post-synaptic density at 100 mg/kg doses but does not reduce seizure severity. Conclusions: Oxidative stress is insignificant to neurological damage observed at the end points analyzed. This study provides the first in vivo evidence that GABA(A) receptor internalization occurs following soman exposure independently of oxidative stress and supports the hypothesis for the role of GABA(A) receptor downregulation as an important mechanism for refractory nerve agent induced seizures. Although oxidative stress was not observed in exposed animals, the administration of NAC prevented the downregulation of synaptic GABA(A) receptors which could provide an improvement of seizure arrest using diazepam. Further studies determining the adjunct efficacy of NAC and the mechanism of action will have to be conducted.

Description

Keywords

nerve agent, GABA, refractory, organophosphate, oxidative stress

Citation

Degree

Master of Science (M.Sc.)

Department

Anatomy, Physiology, and Pharmacology

Program

Anatomy, Physiology, and Pharmacology

Part Of

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DOI

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