A novel method for tracking nitrogen kinetics in vivo under hyperbaric conditions using radioactive nitrogen-13 gas and positron emission tomography.

Decompression sickness (DCS) is caused by gaseous nitrogen dissolved in tissues forming bubbles during decompression. To date, no method exists to identify nitrogen within tissues, but with advances in positron-emission tomography (PET) technology, it may be possible to track gaseous radionuclides into tissues. We aimed to develop a method to track nitrogen movement in vivo and under hyperbaric pressure that could then be used to further our understanding of DCS using nitrogen-13 (13N2). A single anesthetized female Sprague-Dawley rat was exposed to 625 kPa, composed of air, isoflurane, and 13N2 for 10 min. The PET scanner recorded 13N2 during the hyperbaric exposure with energy windows of 250-750 keV. The PET showed an increase in 13N2 concentration in the lung, heart, and abdominal regions, which all reached a plateau after ∼4 min. This showed that it is possible to gain noninvasive in vivo measurements of nitrogen kinetics through the body while at hyperbaric pressures. Tissue samples showed radioactivity above background levels in the blood, brain, liver, femur, and thigh muscle when assessed using a γ counter. The method can be used to evaluate an array of challenges to our understanding of decompression physiology by quantifying nitrogen load through γ counts of 13N2, and signal intensity of the PET. Further development of the method will improve the specificity of the measured outcomes, and enable it to be used with larger mammals, including humans.NEW & NOTEWORTHY This article describes a method for the in vivo quantification and tracking of nitrogen through the mammalian body whilst exposed to hyperbaric pressure. The method has the potential to further our understanding of decompression sickness, and quantitatively evaluate the effectiveness of both the treatment and prevention of decompression sickness.

Effects of oxygen-prebreathing on tissue nitrogenation in normobaric and hyperbaric conditions.

BACKGROUND: Breathing pure oxygen causes nitrogen washout from tissues, a method commonly deployed to prevent decompression sickness from hypobaric exposure. Theoretically prebreathing oxygen increases the capacity for nitrogen uptake and potentially limits supersaturation during dives of short duration. We aimed to use 13N2, a radioactive nitrogen isotope, to quantify tissue nitrogen following normobaric and hyperbaric exposures. METHODS: Twenty Sprague Dawley rats were divided in 4 conditions; normobaric prebreathe, normobaric control, hyperbaric prebreathe, hyperbaric control. Prebreathed rats breathed oxygen for 1 h prior to the experiment whilst controls breathed air. Normobaric rats breathed air containing 13N2 at 100 kPa for 30 min, whereas hyperbaric rats breathed 13N2 at 700 kPa before being decompressed and sedated using air-isoflurane (without 13N2 for a few minutes). After euthanization, blood, brain, liver, femur and thigh muscle were analyzed by gamma counting. RESULTS: At normobaria prebreathing oxygen resulted in higher absolute nitrogen counts in blood (p = .034), as well as higher normalized counts in both the liver and muscle (p = .034). However, following hyperbaric exposure no differences were observed between conditions for any organ (p>.344). Both bone and muscle showed higher normalized counts after hyperbaria compared to normobaria. CONCLUSIONS: Oxygen prebreathing caused nitrogen elimination in normobaria that led to a larger "sink" and uptake of 13N2. The lack of difference between conditions in hyperbaria could be due to the duration and depth of the dive mitigating the effect of prebreathing. In the hyperbaric conditions the lower counts were likely due to off-gassing of nitrogen during the sedation procedure, suggest a few minutes was enough to off-gas in rodents. The higher normalized counts under hyperbaria in bone and muscle likely relate to these tissues being slower to on and off-gas nitrogen. Future experiments could include shorter dives and euthanization while breathing 13N2 to prevent off-gassing.

Downregulation of osteoprotegerin in colorectal cancer cells promotes liver metastasis via activating tumor-associated macrophage.

Osteoprotegerin (OPG) is a secreted cytokine that functions as a decoy receptor for receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL). Anti-RANKL treatment for bone metastasis has been widely accepted for solid tumors. However, the mechanism of OPG-RANKL-RANK signaling in systemic colorectal cancer (CRC) metastasis remains unclear. In this study, we investigated the relevance and function of OPG expression in CRC liver metastasis. First, we performed in silico analysis using The Cancer Genome Atlas public database and found that lower OPG expression in CRC was associated with poor overall survival. Immunohistochemistry analyses using resected specimen from patients with CRC in our institute confirmed the result. Patient-matched primary CRC and liver metastases showed a significant downregulation of OPG expression in metastatic lesions. In CRC cell lines, OPG expression did not suppress cell proliferation and migration. However, OPG expression inhibited macrophage migration by suppressing the RANKL-RANK pathway. Moreover, in vivo mouse liver metastasis models showed that OPG expression in CRC cells suppressed liver metastases. In addition, treatment with an anti-RANKL neutralizing antibody also suppressed liver metastases. These results showed that downregulation of OPG expression in CRC cells promotes liver metastasis by activating tumor-associated macrophage, which can become a candidate for targeted therapy with anti-RANKL neutralizing antibody for CRC liver metastasis.