Immune activity plays an important role in the development of dive related injury and disease in human divers. Activation of inflammatory processes, particularly through the alternate pathway of the complement system, is integral in the development of decompression sickness in the presence of gas emboli. Without this activity it is possible for non-symptomatic bubbles to exist post decompression. In recent decades, decompression sickness-like injuries have been reported in several species of marine mammals, raising new questions about their susceptibility to such conditions. This work is aimed to address the hypothesis that one mechanism through which marine mammals normally avoid dive-related disease and injury is through a less reactive immune system. To do this: 1) enzyme immune-assays for detection of beluga (C5a and Factor Bb) and harbor seal (C5a) complement components were validated, 2) changes in these proteins following exposures to nitrogen bubbles were measured and 3) changes in these proteins following exposures to pressure followed by nitrogen bubbles were measured. Beluga serum samples (n = 3) were exposed to 0.06 ml min-1, 0.2 ml min-1 or 0.5 ml min-1 nitrogen for either 30 seconds, 3 minutes or 30 minutes. Harbor seal samples (n = 3) were exposed to either 0.2 ml min-1 or 0.5 ml min-1 nitrogen for either 30 seconds or 3 minutes. For combined effects, beluga serum was exposed to 1000 psi (∼ 680 m) for 5 minutes with 2 minutes for compression and decompression prior to exposure to either 0.06 ml min-1 or 0.02 ml min-1 nitrogen. Paired T-tests or Repeated Measures ANOVA were used to compare control measures of complement proteins with values after experimental exposures (α = 0.05). Belugas showed no significant changes in C5a following exposures to nitrogen, except for the 30 second exposure to 0.5 ml min-1 where a significant increase was detected (p = 0.001). In contrast, harbor seals showed significant increases in C5a following both the 30 second and 3 minute exposures to both 0.2 ml min (p = 0.002) and 0.5 ml min-1 (p = 0.001). No significant changes in Factor Bb were detected, however the 3 minute and 30 minute exposures to 0.5 ml min-1 resulted in a large increase in this protein. Exposure to pressure appeared to cause an increase in C5a in belugas, though this did not appear to alter the response of beluga complement to nitrogen. Individual animals however displayed quite different responses to pressure experiments, with one individual showing much larger changes than the other two. While this data is preliminary, and increasing sample numbers may change the patterns reported here, this study is, to our knowledge, the first to investigate marine mammal complement in regards to dive-related conditions. Results suggest that belugas may be less reactive to nitrogen bubbles than both harbor seals and humans, as similar exposures have been reported to activate complement and increase C5a in humans. The differences observed between the response of belugas and harbor seals may suggest species specific differences in susceptibility to dive injuries. Such information is important for understanding the emergence of dive-related injuries in marine mammals.
The authors would like to thank the Research and Veterinary Services department at the Mystic Aquarium, as well as the beluga husbandry staff and staff of the animal rescue clinic for helping to provide samples. Thank you to Dr. Cara Field for use of the pressure chamber for pressure exposures. The authors also thank MyBioSource for helping develop assays for measuring marine mammal complement proteins. This work is funded by the Office of Naval Research, Award # N00014-15-1-2203.
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