Materials and Methods: Animals:

1. DECOMPRESSION SICKNESS RISK CORRELATED WITH ACTIVITY OF H2-METABOLIZING MICROBES • INJECTED IN PIGS PRIOR TO DIVES IN H2. • A Fahlman, SR Kayar, WJ Becker, WC Lin, and WB Whitman. Naval Medical Research Center, Bethesda, MD 20889-5607 and Dept. of Microbiology, Univ. of Georgia, Athens, GA 30602. • . Abstract: BACKGROUND: Biochemical decompression, a novel approach for decreasing decompression sickness (DCS) risk by increasing the tissue washout rate of the diluent gas, was tested in an animal model during simulated H2 dives. Logistic regression was used to show that the DCS risk was negatively correlated with the biochemical activity used to decrease the tissue gas burden. METHODS: To increase the washout rate of H2, we used a H2-metabolizing microbe (Methanobrevibacter smithii) that converts H2 to CH4. Pigs (Sus scrofa, 19.41.3 kg) were either untreated controls (n=10), saline-injected controls (n=10), or injected with M. smithii into the large intestine (n=14; varying total activity 200-2200 mmol CH4/min). The pigs were placed in a dry hyperbaric chamber and compressed to 24 bar (20.6-22.9 bar H2, 0.3-0.5 bar O2). Chamber gases (O2, N2, He, H2, and CH4) were monitored using gas chromatography throughout the dive. After 3 h, the pigs were decompressed to 11 bar at 0.9 bar/min, and observed for severe symptoms of DCS for 1 h. RESULTS: Pigs with M. smithii had a 46% lower incidence of DCS compared to controls (6/14 vs. 16/20). Using logistic regression techniques, we showed that the DCS risk rate decreased with increasing total activity of microbes injected (P = 0.015, Log-Likelihood ratio = 5.965, 1 df). CONCLUSION: Reducing the tissue concentration of the inert gas significantly reduced the risk of DCS in a pig model. (NMRDC #61153N MR04101.00D-1103). 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. Statistical analysis: -Variables: body mass, PH2, %H2, pre- and post decompression CH4 output rate, activity injected, and ambient temperature, were tested for the univariate case for significance against outcome. - Only variables with a P<0.25 (Wald’s test) were considered and tested for the multivariate case. - Backward and forward steps were used to test for the multivariate case. Total activity injected was positively correlated (P<0.01) with average CH4 output rate from chamber during the last hour at 24 bar. Introduction: 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 H2 dissolved in the diver’s tissues into methane (4 H2 + CO2 => CH4 + 2 H2O). The conversion decreases the incidence of DCS by reducing the body burden of excess H2 released upon decompression (Fahlman et al., FASEB J. 13:A408, 1999). In this study, we further hypothesized that increasing H2 wash-out rate by increasing the methanogenic activity supplied to the animal will decrease the DCS incidence in a dose-dependent manner. Results: 1) Animals injected with methanogens had a 43 % incidence of DCS which was significantly lower than the 80% DCS incidence of the untreated and surgical control animals. 2) There was a positive correlation between increasing methanogenic activity injected into the pig versus CH4 output from the chamber. 3) Body mass, pre- and post-decompression CH4 output rate, and activity injected were the only univariate variables with a P<0.25 (Wald’s test) and further considered for the multivariate case. 4) Activity injected was the only significant variable, and there was a negative correlation between activity injected versus DCS incidence (P<0.02, likelihood ratio test). ‡ CONCLUSION: -Injection of methanogenic microbes reduces the DCS incidence in pigs during simulated H2 dives. - The DCS incidence decreased with increasing methanogenic activity injected, supporting a dose -response effect. Decompression sickness incidence for untreated control (UC), surgical control (SC), and treated (T) pigs at 24 bar. ‡Incidence in T (6/14) was significantly lower than in UC and SC animals (P<0.05, 1-tailed 2-test). The DCS incidence in the UC group (9/10) was not significantly different from that of the SC group (7/10, P > 0.29, Fisher’s Exact test). Error bars represent 95% binomial confidence limits. Points of Contact: NMRI-Principal Investigator Dr. Susan Kayar, E-mail: kayars@nmripo.nmri.nnmc.navy.mil Research Assistant Andreas Fahlman, E-mail: fahlmana@nmripo.nmri.nnmc.navy.mil