Graduation Year

2021

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Integrative Biology)

Major Professor

Jay B. Dean, Ph.D.

Committee Member

Dominic D'Agostino, Ph.D.

Committee Member

Paula Bickford, Ph.D.

Committee Member

Byeong Cha, Ph.D.

Keywords

caudal solitary complex, Sprague-Dawley, seizures, Superoxide

Abstract

Hyperbaric oxygen (HBO2) is a frequently encountered condition in undersea medicine and hyperbaric oxygen therapy (HBOT). The risk of CNS oxygen toxicity (CNS-OT) seizures limits its use in hyperbaric medicine and limits bottom time for diving operations. In this study, we sought to understand the role of reactive oxygen species (ROS) in two mitigation strategies for CNS-OT; ketone metabolic therapy (KMT), which is known to delay onset of CNS-OT seizures, and mitochondria targeted antioxidant therapy (MitoTAT), which has never been tested under HBO2 conditions. We specifically focused on superoxide anions, one of the early reduction products of molecular oxygen, and the caudal solitary complex (cSC), an oxygen-sensitive subcortical nuclei that is an integral part of cardio-respiration control. First, we tested the effects of KMT on superoxide production in the caudal solitary complex rat medullary tissue slices loaded with the fluorogenic dye dihydroethidium (DHE) to study changes in superoxide production at various levels of hyperoxia (0.4 to 0.95/1.95/4.95 ATA O2) before and during treatment with ketone salts (2-5 mM). We tested the hypothesis that KMT provides neuroprotection against CNS-OT in part through decreased production of superoxide during exposure to HBO2. We found that superoxide production was inhibited with the addition of ketone salts at a concentration of 5 mM during exposure to all levels of hyperoxia; however, it was not decreased significantly at 2-2.5 mM. We also discovered that not all cells in the cSC are equally O2-sensitive in terms of their rate of superoxide production. In the second part of this study, we tested the hypothesis that MitoTAT using Mitoquinol (MitoQ) increases the latency time to seizure (LSz) in freely behaving rats; MitoQ inhibits mitochondrial superoxide production. Initially, up-and-down acute toxicity testing indicated a maximal safe dose of 22mg/kg (i.p.) at 1 ATA air. Liquid chromatography and dual mass spectrometry (LC/MS/MS) indicated that MitoQ (i.p.) in rat blood serum peaked after 60min and remained elevated for 3hrs. Remarkably, when pretreated with MitoQ (22mg/kg i.p.) and exposed to 5 ATA O2, seizures either took longer to occur or did not occur by 60min; however, most rats died within 24hr of co-exposure to MitoQ + HBO2. A second up-and-down acute toxicity test was conducted using 11 to 25mg/kg plus 5 ATA O2. Rats dosed with >14mg/kg of MitoQ and exposed to 5 ATA O2 did not survive post-dive, whereas rats treated with <14mg/kg MitoQ survived but also seized as quickly as controls. Our findings indicate that KMT works in part to delay seizure genesis during exposure to HBO2 by significantly reducing superoxide production. Moreover, MitoTAT using MitoQ delays seizure genesis; however, is toxic when taken at >14mg/kg plus HBO2. Possible explanations for this acute toxicity are presented.

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