Mitigation of CNS oxygen toxicity seizures: evaluating the neuroprotective effects of L‐NAME versus Mitoquinone during exposure to 5 ATA O2 in freely behaving Sprague‐Dawley rats

Abstract

Hyperbaric oxygen (HBO2) is breathed in HBO2 therapy and in undersea medicine; however, what limits the use of HBO2 in these situations is the risk of CNS oxygen toxicity (CNS‐OT; i.e. generalized seizures, Sz). Thus, we are interested in developing strategies that extend the duration of the safe latency period for breathing HBO2 by delaying or abolishing Sz. Increased production of reactive O2 & N2 species is involved in the neuropathology of CNS‐OT. Accordingly, we tested the following two hypotheses in male Sprague‐Dawley rats: a single predive intra peritoneal treatment given 30min before exposure to 5 ATA O2 of either 1) N(g)‐Nitro‐L‐arginine methyl ester (L‐NAME), which inhibits nitric oxide synthase (NOS) and thus nitric oxide (·NO) production, or Mitoquinone (MitoQ), which inhibits mitochondrial superoxide (·O2‐), delays Sz genesis in freely behaving, adult rats. Each rat was dived twice, separated by ≥1 week, receiving either control saline, L‐NAME (30mg/kg ip), or MitoQ (see below) in randomized order. Each rat was exposed to 1 ATA air (10‐15min), 1ATA O2 (10‐15min), compressed (1 ATG/min) to 5 ATA O2. The latency time to first Sz (LSz1) was measured from time zero (3 ATA O2) until onset of Sz at 5 ATA O2 or 60min (MitoQ) or 75min (L‐NAME), whichever came first. For L‐NAME dives, LSz1 in control dives = 10.8 ± 2.5min (avg ± SD; n=14) whereas L‐NAME delayed Sz significantly, increasing LSz1= 31.5 ± 24.3min, including 3 rats that did not seize (O) by 75min of HBO2 (Fig. 1; P= 0.0064). An additional 3 rats were surgically implanted with radio telemetric modules (4‐ET, DSI Inc.; motor cortex (ECoG) and dorsal medulla oblongata (EBulboG). Increased ECoG and EBulboG activities were delayed and blunted in rats treated with L‐NAME vs.control dives. For MitoQ dives, animal toxicity was evaluated first using up‐and‐down toxicity testing (1 ATA air). The maximum safe dose (ip) was 22g/kg MitoQ. During exposure to HBO2, MitoQ, likewise, delayed Sz or prevented Sz (n=5). Unexpectedly, however, all rats became lethargic during the dive and 3/5 rats died within 24 hr following decompression. A second up‐and‐down toxicity test was conducted using 11‐22mg/kg plus5 ATA O2. Most rats dosed with 14‐22mg/kg MitoQ and exposed to HBO2 did not Sz before 1 hr or delayed Sz; however, 11/16 died within 24 hr following decompression. By contrast, rats treated with 11mg/kg MitoQ plus HBO2 survived; however, the LSz1 was no different compared to control dives (n=2). We conclude that L‐NAME delays Sz genesis during HBO2, presumably by decreasing ·NO production and blocking ·NO‐induced cerebral hyperemia prior to Sz during HBO2. Doses of MitoQ (14‐22mg/kg) that are non‐toxic in 1 ATA air, and delay Sz during exposure to HBO2, however, become lethal when combined with HBO2. We postulate that MitoQ + HBO2 toxicity is due to unbridled ·NO production in the face of diminished production of mitochondrial ·O2‐, which normally reacts with ·NO, and thus accelerates onset of pulmonary O2 toxicity. Cocktails of L‐NAME, ketone ester, and MitoQ are currently under study as mitigation strategies for CNS‐OT.