Materials and Methods: Animals:

1. LOWER DECOMPRESSION SICKNESS RISK IN PIGS SUPPLIED WITH H2-METABOLIZING • MICROBES DURING DIVES IN H2. • A. Fahlman, S.R. Kayar, W. Lin, and W.B. Whitman. Naval Med. Res. Center, Bethesda, MD 20889 and Dept. of Microbiology, Univ. of Georgia, Athens, GA 30602. 354.1 Materials and Methods: Animals: -Yorkshire pigs (Sus scrofa), castrated males, n=34, body mass range 17-22 kg Groups: 1) Untreated controls 2) Surgical controls*: injected with 60 mL saline into caecum and large intestine 3) Treated animals*: injected with varying volumes (12-83 mL) and activities (200- 2200 mol CH4/min) of Methanobrevibacter smithii into the caecum and large intestine *Surgery performed under anesthesia, abdomen opened to allow access to caecum and large intestine. Animals studied immediately after recovery. Dive simulation: -Chamber pressurized to 24 bar (21.6-22.9 bar PH2, 0.3-0.5 bar PO2) for 3 hr. -Decompression rate 0.9 bar/min to 11 bar; animals observed for 1 hr for DCS - Euthanized in chamber on confirmation of DCS or at end of hour. Measurements: -Chamber gases analyzed by gas chromatography for H2, O2, He, N2, and CH4 -CH4 output rate (mol CH4/min) from the chamber was used as an indicator of the CH4 production rate from pigs. -Severe symptoms of DCS included: walking difficulties, fore and/or hind limb paralysis, falling, convulsions. Abstract: Pigs supplied with H2-metabolizing microbes (n=14; 18.90.9 kg) had a 40% lower incidence of decompression sickness (DCS) compared to control animals (n=10; 19.61.6 kg), following exposure to elevated pressures of H2. Animals received caecal injections of either Methanobrevibacter smithii (12-83 mL, activity 200-2200 mmol CH4/min) or 60 mL of saline, 1-2 h prior to experiments. To simulate a H2 dive, animals were placed in a dry hyperbaric chamber, compressed to 24 bar (21.6-22.9 bar H2, 0.3-0.5 bar O2) for 3 h, then decompressed to 11 bar at 0.9 bar/min, and observed for 1 h for severe symptoms of DCS. Chamber concentrations of O2, N2, He, H2, and CH4 were measured by gas chromatography throughout the dive. The CH4 release rate in treated animals (9.7-23.4 mmol/min) was used to indicate the microbial activity of reducing the tissue burden of H2. Treated animals had a significantly lower DCS incidence (P<0.05) than control animals (6/14 vs. 7/10), and a significantly higher mean CH4 release rate (P<0.001) of 14.43.6 mmol/minvs. 6.72.5 mmol/min. Increasing the washout rate of the inert gas by metabolizing H2 decreased the DCS risk in a pig model during H2 dives.(Supported by NMRDC work unit #61153N MR04101.00D-1103; animal use guidelines of NIH Pub. # 92-3415, 1992). Treated Total activity injected was positively correlated (P<0.01) with average CH4 output rate from chamber during the last hour at 24 bar. Control Introduction: The amount of gas breathed by a diver that becomes dissolved in the diver’s tissues is a function of the pressure of the gas and the duration of elevated pressure. Decompression sickness (DCS) is believed to be caused by a rapid pressure decrease, resulting in the possible formation of gas bubbles. Currently the only method of avoiding DCS is to carefully control the decompression rate according to existing tables. However, even when the ascent rates are meticulously followed, divers can get DCS. Hydrogen is suitable as a diving gas for extremely deep dives due to its low density, making it easy to breathe. Additionally, a novel method for safer decompressions from hydrogen dives involves the use of H2-metabolizing microbes (Kayar et al., Am. J. Physiol. 275: R677-682, 1998). These microbes convert some of the dissolved H2 into methane (4 H2 + CO2 => CH4 + 2 H2O). The conversion could potentially reduce the time for safe decompression or decrease the incidence of DCS by reducing the body burden of excess inert gas released upon decompression. Hypothesis: Increased H2 wash-out, by microbial removal of the inert gas, decreases DCS incidence in pigs. Results: 1) Varying quantities of CH4 were detected in the chamber during the last hour at 24 bar from animals without M. smithii (6.6 ± 2.9 moles CH4/min, mean ± stdev), indicating an existing native gut flora of methanogens. 2) Chamber CH4 output from treated animals (10.0 ± 5.1 moles CH4/min) was significantly higher during the last hour at 24 bar than from non- treated animals (P<0.01), showing that injection of methanogens increased the CH4 output. 3) There was a positive correlation ( P<0.01) between increasing methanogenic activity injected into the pig and CH4 output from the chamber, supporting a dose response effect. 4) The DCS incidence in the untreated control group (9/10) was not significantly different from the surgical control group (7/10, P > 0.29, Fisher’s Exact test). 5) Animals injected with methanogens had a 43 % (6/14) incidence of DCS which was significantly lower than the 80% (16/20) DCS rate of the control animals (P<0.05, Fisher’s Exact test). ‡ Treated in He Conclusion: -Injection of methanogenic microbes reduces the DCS incidence in pigs during simulated H2 dives Decompression sickness incidence for untreated control (UC), surgical control (SC), and treated (T) pigs at 24 bar. ‡Incidence significantly different from untreated animals (P<0.05, 1-tailed 2-test) Error bars represent 95% binomial confidence limits. Average chamber CH4 release rate during dives to 24 bar with varying PH2 for a representative treated animal, an untreated control animal and for one treated animal in He.

2. Points of Contact: NMRI-Principal Investigator Dr. Susan Kayar, E-mail: kayars@nmripo.nmri.nnmc.navy.mil Phone:(301)295-5903 Research Assistant Andreas Fahlman, E-mail: fahlmana@nmripo.nmri.nnmc.navy.mil Phone:(301)295-5867