Research on the effects of hyperbaric oxygen on biphasic analgesic through nNOS/NO/γ-GABA pathways in mice

Abstract

Objective To study the analgesic effect of repeated hyperbaric oxygen (HBO) exposures and explore the mechanism involving neural nitric oxide synthase (nNOS), nitric oxide (NO) and γ-aminobutyric acid (GABA). Methods The animal pain model was established and the animals were randomly divided into the HBO group, the hyperbaric air (HBA) group, the normobaric air (NBA) group and the normobaric oxygen (NBO) group, and were exposed repeatedly to either HBO or air. The chamber was ventilated with 100% O2 for 5 min, then, the chamber was pressurized to 0.35 MPa at a rate of 0.10 MPa/min. At the pressure of 0.35 MPa, the chamber was again ventilated with oxygen/air for 60 minutes, and then, was decompressed at a rate of 0.10 MPa/min. The animals were exposed in the chamber one session a day for a succession of 4 days. Analgesic effect was evaluated by abdominal contraction test, and nitrate reductase assay was used to determine the expression levels of NO and NOS in the brain tissue and the spinal cord. NOS inhibitors were given by i. c.v injection to measure the effect of NOS on the analgesic effect of HBO. The nNOS+ neurons and glatamic acid decarboxylase(GAD) positive (GAD+ ) neurons in the periaqueductal gray (PAG) were labeled by fluorescopy. Results Repeated HBO treatment induced a biphasic analgesic effect, including: (1) early analgesia which was displayed an hour after HBO exposure and lasted for about 8 hours; (2) late analgesia which was displayed one day after HBO exposure, reached peak one week later and lasted for about 3 weeks. Three hours after the termination of last HBO exposure, medication of the non-specific NOS inhibitor N′-Nitro-L-arginine-methyl ester hydrochloride (L-NAME) and nNOS inhibitor S-methyl-L-thiocitrulline (SMTC) could obviously inhibit early analgesic effect. L-NAME and SMTC could significantly inhibit late analgesia. One hour after HBO exposure, the levels of NO and nNOS in the brain tissue and spinal cord were considerably elevated. The late analgesic effect of HBO significantly decreased, when CGP35348 was injected in the lateral ventricle 7 days after HBO treatment. Immunofluorescence indicated that there was a co-localization between nNOS+ neurons and GAD+ neurons in the PAG. Conclusions Repeated 4 HBO exposures induced a double-phased analgesia. Initial analgesic effect displayed one hour after HBO treatment, involving activation of nNOS, while late analgesic effect emerged one day after HBO exposure, with the interaction between nNOS and GABAB receptors. Key words: Hyperbaric oxygen; Analgesia; nNOS/NO/γ-GABA pathways