Biomarkers related to gas embolism: Gas score, pathology, and gene expression in a gas bubble disease model.

Fish exposed to water supersaturated with dissolved gas experience gas embolism similar to decompression sickness (DCS), known as gas bubble disease (GBD) in fish. GBD has been postulated as an alternative to traditional mammals' models on DCS. Gas embolism can cause mechanical and biochemical damage, generating pathophysiological responses. Increased expression of biomarkers of cell damage such as the heat shock protein (HSP) family, endothelin 1 (ET-1) or intercellular adhesion molecule 1 (ICAM-1) has been observed, being a possible target for further studies of gas embolism. The GBD model consisted of exposing fish to supersaturation in water with approximately 170% total dissolved gas (TDG) for 18 hours, producing severe gas embolism. This diagnosis was confirmed by a complete histopathological exam and the gas score method. HSP70 showed a statistically significant upregulation compared to the control in all the studied organs (p <0.02). Gills and heart showed upregulation of HSP90 with statistical significance (p = 0.015 and p = 0.02, respectively). In addition, HSP70 gene expression in gills was positively correlated with gas score (p = 0.033). These results suggest that gas embolism modify the expression of different biomarkers, with HSP70 being shown as a strong marker of this process. Furthermore, gas score is a useful tool to study the abundance of gas bubbles, although individual variability always remains present. These results support the validity of the GBD model in fish to study gas embolism in diseases such as DCS.

Evidence of a hormonal reshuffle in the cecal metabolome fingerprint of a strain of rats resistant to decompression sickness.

On one side, decompression sickness (DCS) with neurological disorders lead to a reshuffle of the fecal metabolome from rat caecum. On the other side, there is high inter-individual variability in terms of occurrence of DCS. One could wonder whether the fecal metabolome could be linked to the DCS-susceptibility. We decided to study male and female rats selected for their resistance to decompression sickness, and we hypothesize a strong impregnation concerning the fecal metabolome. The aim is to verify whether the rats resistant to the accident have a fecal metabolomic signature different from the stem generations sensitive to DCS. 39 DCS-resistant animals (21 females and 18 males), aged 14 weeks, were compared to 18 age-matched standard Wistar rats (10 females and 8 males), i.e., the same as those we used for the founding stock. Conventional and ChemRICH approaches helped the metabolomic interpretation of the 226 chemical compounds analyzed in the cecal content. Statistical analysis shows a panel of 81 compounds whose expression had changed following the selection of rats based on their resistance to DCS. 63 compounds are sex related. 39 are in common. This study shows the spectral fingerprint of the fecal metabolome from the caecum of a strain of rats resistant to decompression sickness. This study also confirms a difference linked to sex in the metabolome of non-selected rats, which disappear with selective breeding. Results suggest hormonal and energetic reshuffle, including steroids sugars or antibiotic compounds, whether in the host or in the microbial community.

Hypobaric hypoxia induced renal damage is mediated by altering redox pathway.

Systemic hypobaric hypoxia is reported to cause renal damage; nevertheless the exact pathophysiological mechanisms are not completely understood. Therefore, the present study aims to explore renal pathophysiology by using proteomics approach under hypobaric hypoxia. Six to eight week old male Sprague Dawley rats were exposed to hypobaric hypoxia equivalent to altitude of 7628 metres (pO2-282mmhg) at 28°C and 55% humidity in decompression chamber for different time intervals; 1, 3, and7 days. Various physiological, proteomic and bioinformatic studies were carried out to examine the effect of chronic hypobaric hypoxia on kidney. Our data demonstrated mild to moderate degenerative tubular changes, altered renal function, injury biomarkers and systolic blood pressure with increase in duration of hypobaric hypoxia exposure. Renal proteomic analysis showed 38 differential expressed spots, out of which 25 spots were down regulated and 13 were up regulated in 7 dayhypobarichypoxic exposure group of rats as compared to normoxia control. Identified proteins were involved in specific molecular changes pertinent to endogenous redox pathways, cellular integrity and energy metabolism. The study provides an empirical evidence of renal homeostasis under hypobaric hypoxia by investigating both physiological and proteomics changes. The identification of explicit key proteins provides a valuable clue about redox signalling mediated renal damage under hypobaric hypoxia.

Evidence of Heritable Determinants of Decompression Sickness in Rats.

INTRODUCTION: Decompression sickness (DCS) is a complex and poorly understood systemic disease caused by inadequate desaturation after a decrease of ambient pressure. Strong variability between individuals is observed for DCS occurrence. This raises questions concerning factors that may be involved in the interindividual variability of DCS occurrence. This study aimed to experimentally assess the existence of heritable factors involved in DCS occurrence by selectively breeding individuals resistant to DCS from a population stock of Wistar rats. METHODS: Fifty-two male and 52 female Wistar rats were submitted to a simulated air dive known to reliably induce about 63% DCS: compression was performed at 100 kPa·min up to 1000 kPa absolute pressure before a 45-min long stay. Decompression was performed at 100 kPa·min with three decompression stops: 5 min at 200 kPa, 5 min at 160 kPa, and 10 min at 130 kPa. Animals were observed for 1 h to detect DCS symptoms. Individuals without DCS were selected and bred to create a new generation, subsequently subjected to the same hyperbaric protocol. This procedure was repeated up to the third generation of rats. RESULTS: As reported previously, this diving profile induced 67% of DCS, and 33% asymptomatic animals in the founding population. DCS/asymptomatic ratio was not initially different between sexes, although males were heavier than females. In three generations, the outcome of the dive significantly changed from 33% to 67% asymptomatic rats, for both sexes. Interestingly, survival in females increased sooner than in males. CONCLUSIONS: This study offers evidence suggesting the inheritance of DCS resistance. Future research will focus on genetic and physiological comparisons between the initial strain and the new resistant population.

Evidence of cell damages caused by circulating bubbles: high level of free mitochondrial DNA in plasma of rats.

Bubble formation can occur in the vascular system after diving, leading to decompression sickness (DCS). DCS signs and symptoms range from minor to death. Too often, patients are admitted to a hyperbaric center with atypical symptoms, as bubbles cannot be detected anymore. In the absence of a relevant biomarker for humans, the therapeutic management remains difficult. As circulating DNA was found in the blood of healthy humans and animals, our study was made to correlate the extracellular mitochondrial DNA (mDNA) concentration with the occurrence of clinical DCS symptoms resulting from initial bubble-induced damages. Therefore, 109 rats were subjected to decompression from a simulated 90-m sea water dive, after which, 78 rats survived (71.6%). Among the survivors, 15.6% exhibited typical DCS symptoms (DCS group), whereas the remaining 56% showed no detectable symptoms (noDCS group). Here, we report that the symptomatic rats displayed both a circulating mDNA level (DNADCS → 2.99 ± 2.62) and a bubble grade (median Spencer score = 3) higher than rats from the noDCS group (DNAnoDCS → 1.49 ± 1.27; Spencer score = 1). These higher levels could be correlated with the platelet and leukocyte consumption induced by the pathogenic decompression. Rats with no detectable bubble had lower circulating mDNA than those with higher bubble scores. We determined that in rats, a level of circulating mDNA >1.91 was highly predictive of DCS with a positive-predictive value of 87.3% and an odds ratio of 4.57. Thus circulating mDNA could become a relevant biomarker to diagnose DCS and should be investigated further to confirm its potential application in humans.

Neuroprotective role of the TREK-1 channel in decompression sickness.

Nitrogen supersaturation and bubble formation can occur in the vascular system after diving, leading to death and nervous disorders from decompression sickness (DCS). Bubbles alter the vascular endothelium, activate platelets, and lead to focal ischemia with neurological damage mediated by the mechanosensitive TREK-1 neuronal potassium ion channel that sets pre- and postsynaptic resting membrane potentials. We report a neuroprotective effect associated with TREK-1. C57Bl6 mice were subjected to decompression from a simulated 90 msw dive. Of 143 mice that were wild type (WT) for TREK-1, 51.7% showed no DCS, 27.3% failed a grip test, and 21.0% died. Of 88 TREK-1 knockouts (KO), 26.1% showed no DCS, 42.0% failed a grip test, and 31.8% died. Mice that did not express TREK-1 had lower DCS resistance and were more likely to develop neurological symptoms. We conclude that the TREK-1 potassium channel was neuroprotective for DCS.

Acclimation to decompression: stress and cytokine gene expression in rat lungs.

Previous studies demonstrated that animals exposed to repeated compression-decompression stress acclimated (i.e., developed reduced susceptibility) to rapid decompression. This study endeavored to characterize inflammatory and stress-related gene expression and signal transduction associated with acclimation to rapid decompression. Rats were divided into four groups: 1) control-sham: pressure naïve rats; 2) acclimation-sham: nine acclimation dives [70 feet seawater (fsw), 30 min]; 3) control-dive: test dive only (175 fsw, 60 min); and 4) acclimation-dive: nine acclimation dives and a test dive. After the test dive, rats were observed for decompression sickness (DCS). Expression of 13 inflammatory and stress-related genes and Akt (or PKB, a serine/threonine protein kinase) and MAPK phosphorylation of lung tissue were examined. The expression of immediate early gene/transcription factor early growth response gene 1 (Egr-1) was observed in both control and acclimation animals with DCS but not in animals without DCS. Increased Egr-1 in control-dive animals with DCS was significantly greater than in acclimation-dive animals with DCS. TNF-α, IL-1ß, IL-6, and IL-10 were significantly elevated in control-DCS animals. Acclimation-DCS animals had increased TNF-α, but there was no change in IL-1ß, IL-6, and IL-10. High levels of Akt phosphorylation were observed in lungs of acclimation-sham, acclimation-dive, and control-dive animals; phosphorylated ERK1/2 was only observed in animals with DCS. This study suggests that activation of ERK1/2 and upregulation of Egr-1 and its target cytokine genes by rapid decompression may play a role in the initiation and progression of DCS. It may be that the downregulated expression of these genes in animals with DCS is associated with previous exposure to repeated compression-decompression cycles. This study represents an initial step toward understanding the molecular mechanisms associated with acclimation to decompression.

Oxygen pretreatment as protection against decompression sickness in rats: pressure and time necessary for hypothesized denucleation and renucleation.

Pretreatment with HBO at 300-500 kPa for 20 min reduced the incidence of decompression sickness (DCS) in a rat model. We investigated whether this procedure would be effective with lower oxygen pressures and shorter exposure, and tried to determine how long the pretreatment would remain effective. Rats were pretreated with oxygen at 101 or 203 kPa for 20 min and 304 kPa for 5 or 10 min. After pretreatment, the animals were exposed to air at 1,013 kPa for 33 min followed by fast decompression. Pretreatment at 101 or 203 kPa for 20 min and 304 kPa for 10 min significantly reduced the number of rats with DCS to 45%, compared with 65% in the control group. However, after pretreatment at 304 kPa for 5 min, 65% of rats suffered DCS. When pretreatment at 304 kPa for 20 min was followed by 2 h in normobaric air before compression and decompression, the outcome was worse, with 70-90% of the animals suffering DCS. This is probably due to the activation of "dormant" micronuclei. The risk of DCS remained lower (43%) when pretreatment with 100% O(2) at normobaric pressure for 20 min was followed by a 2 h interval in normobaric air (but not 6 or 24 h) before the hyperbaric exposure. The loss of effectiveness after a 6 or 24 h interval in normobaric air is related to micronuclei rejuvenation. Although pretreatment with hyperbaric O(2) may have an advantage over normobaric hyperoxia, decompression should not intervene between pretreatment and the dive.

Decompression from saturation using oxygen: its effect on DCS and RNA in large swine.

INTRODUCTION: The use of hyperbaric oxygen (HBO) to expedite decompression from saturation has not been proven and may increase risk of toxicity to the pulmonary system. To evaluate any benefit of HBO during decompression, we used a 70-kg swine model of saturation and examined lung tissue by microarray analysis for evidence of RNA regulation. METHODS: Unrestrained, non-sedated swine were compressed to 132 fsw (5 ATA) for 22 h to achieve saturation. Animals then underwent decompression on air (AirD) or HBO (HBOD) starting at 45 fsw (2.36 ATA). Animals were evaluated for Type I and Type II decompression sickness (DCS) for 24 h. Control (SHAM) animals were placed in the chamber for the same duration, but were not compressed. Animals were sacrificed 24 h after exposure and total RNA was isolated from lung samples for microarray hybridizations on the Affymetrix platform. RESULTS: There was no evidence of Type I DCS or severe cardiopulmonary DCS in any of the animals; abnormal gaits were noted only in the HBOD group (4/9).Three genes (nidogen 2, calcitonin-like receptor, and pentaxin-related gene) were significantly up-regulated in both the AirD and HBOD groups compared to controls. Three other genes (TN3, platelet basic protein, and cytochrome P450) were significantly down-regulated in both groups. CONCLUSIONS: HBO during decompression from saturation did not reduce the incidence of DCS. Gene regulation was apparent and similar in both the AirD and HBOD groups, particularly in genes related to immune function and cell signaling.