Isoflurane administration during i t procedures can complica

Isoflurane administration during i.t. procedures can complicate the interpretation of our results since isoflurane has been shown to have both neuroprotective and neurotoxic effects (Jiang et al., 2017). Isoflurane pretreatment has been implicated as having protective effects in ischemia in animal models (Jiang et al., 2017). In animal models of neural injury in neonatal rats, isoflurane was shown to decrease the hypoxia-related increase in the release of amino L-Phenylephrine neurotransmitters while elevating levels of GABA (Zhao et al., 2016). However, in the above experiment, animals were exposed to varying concentrations of isoflurane for a prolonged period — 6 h (Zhao et al., 2016). In our experiments, animals were exposed to isoflurane at a concentration of 2.5% isoflurane for no more than 5 min at a time. In addition, all our animals receiving i.t. injections received isoflurane so any effect of isoflurane on GABA concentrations would be consistent across all animals receiving i.t. injections. It should be noted, however, there are other possible explanations for these results. A previous study using a chronic constriction injury (CCI) model of neuropathic pain found that HBO2 was effective at decreasing CCI-induced allodynia (Fu et al., 2017). Furthermore, while CCI increased apoptosis of GABAergic neurons in the dorsal horn, HBO2 administration inhibited this effect which protects the balance between excitation and inhibition in the spinal cord (Fu et al., 2017). The results of this paper implicate a neuroprotective effect of HBO2 on GABAergic neurons in the dorsal horn of the spinal cord in relief of neuropathic pain (Fu et al., 2017). While our experiments did not investigate neuroprotective effects of HBO2, they do implicate GABA as having a role in HBO2-induced antinociception which is in agreement with the prior experiment. HBO2-induced antinociception could be caused by interactions between the opioidergic and GABAergic pathways that are induced by changes caused by HBO2. Still other possibilities could include supraspinal serotonergic and/or noradrenergic pathways that recruit local GABAergic inhibitory neurons in the spinal cord (Millan, 2002). These data indicate that the pathways involved in HBO2-induced antinociception are complex and interactive. More research is needed to distinguish exactly how various pathways interact in HBO2 induced antinociception. The western blotting results indicate an interaction between the noxious stimulant for abdominal constrictions, glacial acetic acid, and HBO2 in expression of the phosphorylated β3 subunit of the GABAA receptor in the lumbar but not thoracic spinal cord. Glacial acetic acid and HBO2 independently decreased expression of the phosphorylated β3 subunit of the GABAA receptor; however, in the presence of both, expression of the GABAA receptor subunit was restored to control levels. Isoflurane was not given to animals involved in western blotting eliminating the possibility that elevated GABA levels related to isoflurane exposure could explain these results (Zhao et al., 2016). This could indicate that, in the presence of an acute pain stimulus, expression is reduced, but this decrease is reversed by HBO2. In contrast to the GABAA receptor, HBO2 did not fully restore nNOS expression in the spinal cord to control levels. In the presence of noxious stimulation, nNOS expression in the spinal cord is significantly decreased. HBO2 did slightly increase expression of nNOS in the lumbar spinal cord in the presence of pain, but this effect was not statistically significant. HBO2 alone itself decreased nNOS expression compared to control, and this is not significantly affected by the presence or absence of noxious stimulation, although the reduction in nNOS in the lumbar spinal cord was not as great as that in the presence of pain. These results indicate that, in the lumbar spinal cord, HBO2 increased the expression of the phosphorylated β3 subunit of the GABAA receptor. We focused on this receptor because phosphorylation at this site could indicate increased activity of the receptor (McDonald et al., 1998). Thus, restoration of phosphorylation of this site by HBO2 and painful stimuli could indicate that GABA activity at the GABAA receptor in lumbar spinal cord is restored when HBO2 is producing antinociception. However, this effect is not dependent upon nNOS pathways because, while nNOS expression is also decreased when pain is present, it is not restored by HBO2.