CNS-oxygen toxicity and blood glucose levels in MnSOD enzyme knockdown mice.

Many studies have been conducted in the search for the mechanism underlying CNS-oxygen toxicity (OT), which may be fatal when diving with a closed-circuit apparatus. We investigated the influence of hyperbaric oxygen (HBO) on blood glucose level (BGL) in Mn-superoxide dismutase (SOD2) knockdown mice regarding CNS-OT in particular under stress conditions such as hypoglycemia or hyperglycemia. Two groups of mice were used: SOD2 knockdown (Heterozygous, HET) mice and their WT family littermates. Animals were exposed to HBO from 2 up to 5 atmosphere absolute (ATA). Blood samples were drawn before and after each exposure for measurement of BGL. The mice were sacrificed following the final exposure, which was at 5 ATA. We used RT-PCR and Western blot to measure levels of glucose transporter 1 (GLUT1) and hypoxia inducible factor (HIF)1a in the cortex and hippocampus. In the hypoglycemic condition, the HET mice were more sensitive to oxidative stress than the WT. In addition, following exposure to sub-toxic HBO, which does not induce CNS-OT, BGL were higher in the HET mice compared with the WT. The expression of mRNA of GLUT1 and HIF-1a decreased in the hippocampus in the HET mice, while the protein level decreased in the HET and WT following HBO exposure. The results suggest that the higher BGL following HBO exposure especially at SOD2 HET mice is in part due to reduction in GLUT1 as a consequence of lower HIF-1a expression. This may add part to the puzzle of the understanding the mechanism leading to CNS-OT.

The transition from day-to-night activity is a risk factor for the development of CNS oxygen toxicity in the diurnal fat sand rat (Psammomys obesus).

Performance and safety are impaired in employees engaged in shift work. Combat divers who use closed-circuit oxygen diving apparatus undergo part of their training during the night hours. The greatest risk involved in diving with such apparatus is the development of central nervous system oxygen toxicity (CNS-OT). We investigated whether the switch from day-to-night activity may be a risk factor for the development of CNS-OT using a diurnal animal model, the fat sand rat (Psammomys obesus). Animals were kept on a 12:12 light-dark schedule (6 a.m. to 6 p.m. at 500 lx). The study included two groups: (1) Control group: animals were kept awake and active during the day, between 09:00 and 15:00. (2) Experimental group: animals were kept awake and active during the night, between 21:00 and 03:00, when they were exposed to dim light in order to simulate the conditions prevalent during combat diver training. This continued for a period of 3 weeks, 5 days a week. On completion of this phase, 6-sulphatoxymelatonin (6-SMT) levels in urine were determined over a period of 24 h. Animals were then exposed to hyperbaric oxygen (HBO). To investigate the effect of acute melatonin administration, melatonin (50 mg/kg) or its vehicle was administered to the animals in both groups 20 min prior to HBO exposure. After the exposure, the activity of superoxide dismutase, catalase and glutathione peroxidase was measured, as were the levels of neuronal nitric oxide synthase (nNOS) and overall nitrotyrosylation in the cortex and hippocampus. Latency to CNS-OT was significantly reduced after the transition from day-to-night activity. This was associated with alterations in the level of melatonin metabolites secreted in the urine. Acute melatonin administration had no effect on latency to CNS-OT in either of the groups. Nevertheless, the activity of superoxide dismutase and catalase, as well as nitrotyrosine and nNOS levels, were altered in the hippocampus following melatonin administration. On the basis of these results, we suggest that a switch from diurnal to nocturnal activity may represent an additional risk factor for the development of CNS-OT. Utilizing a diurnal animal model may contribute to our understanding of the heightened risk of developing CNS-OT when diving with closed-circuit oxygen apparatus at night.

Alteration of blood glucose levels in the rat following exposure to hyperbaric oxygen.

Findings regarding blood glucose level (BGL) on exposure to hyperbaric oxygen (HBO) are contradictory. We investigated the influence of HBO on BGL, and of BGL on latency to central nervous system oxygen toxicity (CNS-OT). The study was conducted on five groups of rats: Group 1, exposure to oxygen at 2.5 atmospheres absolute (ATA), 90 min/day for 7 days; Group 2, exposure to oxygen once a week from 2 to 6 ATA in increments of 1 ATA/wk, for a period of time calculated as 60% of the latency to CNS-OT (no convulsions); Group 3, exposure to 6 ATA breathing a gas mixture with a pO2 of 0.21; Group 4, received 10 U/kg insulin to induce hypoglycemia before exposure to HBO; Group 5, received 33% glucose to induce hyperglycemia before exposure to HBO. Blood samples were drawn before and after exposures for measurement of BGL. No change was observed in BGL after exposure to oxygen at 2.5 ATA, 90 min/day for 7 days. BGL was significantly elevated after exposure to oxygen at 6 ATA until the appearance of convulsions, and following exposure to 4, 5, and 6 ATA without convulsions (P < 0.01). No change was observed in BGL after exposure to 6 ATA breathing a gas mixture with a pO2 of 0.21. Hypoglycemia shortened latency to CNS oxygen toxicity, whereas hyperglycemia had no effect. Our results demonstrate an influence of HBO exposure on elevation of BGL, starting at 4 ATA. This implies that BGL may serve as a marker for the generation of CNS-OT.

Hyperbaric oxygen preconditioning protects rats against CNS oxygen toxicity.

We examined the hypothesis that repeated exposure to non-convulsive hyperbaric oxygen (HBO) as preconditioning provides protection against central nervous system oxygen toxicity (CNS-OT). Four groups of rats were used in the study. Rats in the control and the negative control (Ctl-) groups were kept in normobaric air. Two groups of rats were preconditioned to non-convulsive HBO at 202 kPa for 1h once every other day for a total of three sessions. Twenty-four hours after preconditioning, one of the preconditioned groups and the control rats were exposed to convulsive HBO at 608 kPa, and latency to CNS-OT was measured. Ctl- rats and the second preconditioned group (PrC-) were not subjected to convulsive HBO exposure. Tissues harvested from the hippocampus and frontal cortex were evaluated for enzymatic activity and nitrotyrosine levels. In the group exposed to convulsive oxygen at 608 kPa, latency to CNS-OT increased from 12.8 to 22.4 min following preconditioning. A significant decrease in the activity of glutathione reductase and glucose-6-phosphate dehydrogenase, and a significant increase in glutathione peroxidase activity, was observed in the hippocampus of preconditioned rats. Nitrotyrosine levels were significantly lower in the preconditioned animals, the highest level being observed in the control rats. In the cortex of the preconditioned rats, a significant increase was observed in glutathione S-transferase and glutathione peroxidase activity. Repeated exposure to non-convulsive HBO provides protection against CNS-OT. The protective mechanism involves alterations in the enzymatic activity of the antioxidant system and lower levels of peroxynitrite, mainly in the hippocampus.

Do hyperbaric oxygen-induced seizures cause brain damage?

It is commonly accepted that hyperbaric oxygen-induced seizures, the most severe manifestation of central nervous system oxygen toxicity, are harmless. However, this hypothesis has not been investigated in depth. We used apoptotic markers to determine whether cells in the cortex and hippocampus were damaged by hyperbaric oxygen-induced seizures in mice. Experimental animals were exposed to a pressure of 6 atmospheres absolute breathing oxygen, and were randomly assigned to two groups sacrificed 1h after the appearance of seizures or 7 days later. Control groups were not exposed to hyperbaric oxygen. Caspase 9, caspase 3, and cytochrome c were used as apoptotic markers. These were measured in the cortex and the hippocampus, and compared between the groups. Levels of caspase 3, cytochrome c, and caspase 9 in the hippocampus were significantly higher in the hyperbaric oxygenexposed groups compared with the control groups 1 week after seizures (p<0.01). The levels of two fragments of caspase 9 in the cortex were higher in the control group compared with the hyperbaric oxygen-exposed group 1h after seizures (p<0.01). Hyperbaric oxygen-induced seizures activate apoptosis in the mouse hippocampus. The reason for the changes in the cortex is not understood. Further investigation is necessary to elucidate the mechanism underlying these findings and their significance.

Is glucose-6-phosphate dehydrogenase deficiency a risk factor for hyperbaric oxygen exposure?

Divers and patients lacking glucose-6-phosphate dehydrogenase (G6PD) may face a serious threat of central nervous system oxygen toxicity (CNS-OT) during exposure to hyperbaric oxygen (HBO), due to the important part played by G6PD in cellular redox balance. Our objective was to investigate G6PD deficiency as a risk factor for CNS-OT. We exposed G6PD-deficient (G6PDdef) and wild type (WT) mice to HBO at 405 kPa. Latency to CNS-OT was measured by observing the animal and monitoring the time to appearance of convulsions. Changes in glutathione peroxidase (GPx) and catalase activity were measured in red blood cells, and levels of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) and 3-nitrotyrosine (NT) were measured in extracts of whole brain tissue by Western blot analysis. Unexpectedly, latency to CNS-OT was more than twice as long in G6PDdef mice compared with WT (36.9 ± 15.4 and 15.6 ± 13.2 min, respectively, P < 0.005). No significant differences were found in GPx and catalase activity or in protein levels of eNOS. However, nNOS and NT levels were lower in G6PDdef mice compared with WT (50.6%, P < 0.01 and 52.8%, P < 0.05, respectively). Our results suggest that the enhanced resistance of G6PDdef mice to HBO is due in part to a reduction in nNOS and NT levels in the brain. We conclude that G6PD deficiency at the level of the animals in the present study may not be a risk factor for developing CSN-OT, but this remains to be verified for human subjects.

Energetic efficiency in trained and sedentary rats after exposure to normobaric and hyperbaric oxygen.

INTRODUCTION: Contradictory results have been obtained regarding the beneficial effect of hyperbaric oxygen (HBO) on exercise performance. The purpose of this study was to investigate the effect of different combinations of pressure and time in hyperoxia on the energetic efficiency of trained and sedentary rats. METHODS: At the end of the training period, rats were exposed to one of three protocols: 1) 100% normobaric oxygen for 24 h; 2) HBO at 2 ATA for 4 h; 3) HBO at 2.5 ATA for 6 h. After the hyperoxic exposures, V(O2max) was evaluated and compared with preexposure values. RESULTS: The slope of the linear section of the oxygen consumption-velocity curve in the trained rats was significantly steeper after exposure to either 100% normobaric oxygen for 24 h or HBO at 2 ATA for 4 h than before the exposure. The opposite was found for the sedentary rats. After exposure to HBO at 2.5 ATA for 6 h, the slope of the oxygen consumption-velocity curve in the trained rats did not differ from the pre-exposure slope. However, the highest velocity these rats reached was lower than their maximum velocity before this exposure. In the sedentary rats, the slope of the oxygen consumption-velocity curve was found to be steeper after the 2.5 ATA exposure compared with the preexposure slope. CONCLUSIONS: Our results suggest that exposure to 100% normobaric oxygen for 24 h and HBO at 2 ATA for 4 h induces a reduction in the energetic efficiency of trained rats, but improves energetic efficiency in sedentary rats.

Hyperoxia may reduce energetic efficiency in the trained rat.

BACKGROUND: Several studies have been conducted in recent years in the attempt to improve running performance by the use of hyperbaric oxygen, but there is disagreement as to whether this has any beneficial effect. The purpose of this study was to measure the effect of 24 h breathing 100% O2 in normobaric conditions on energetic efficiency in the trained rat. METHODS: Experiments were carried out on trained rats whose oxygen consumption was evaluated during the training period and on its completion. At the end of the training period, the rats were divided into two groups: 1) rats exposed to air (21% O2) in normobaric conditions; and 2) rats exposed to 100% O2 in normobaric conditions. In addition, two groups of sedentary rats were used: 3) sedentary rats exposed to air (21% O2) in normobaric conditions; and 4) sedentary rats exposed to 100% O2 in normobaric conditions. Energetic efficiency was estimated by measuring O2 consumption at submaximal exercise (45 m.min-1, 10 degrees incline). RESULTS: Training alone reduced O2 consumption by 18% during submaximal exercise. Exposure to 100% oxygen for 24 h in normobaric conditions reversed the effect of complete training by elevating the O2 consumption by 17%, which was close to the oxygen consumption of the rats during the incomplete training period. CONCLUSIONS: Our results suggest that prolonged exposure to hyperoxia induces a reduction in the energetic efficiency of the trained rat. The relevance of these findings to sports and diving is discussed.