Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • We hypothesize that HBO increases GABA

    2022-06-22

    We hypothesize that HBO2 increases GABA activity at the GABAA receptor via a nitric oxide dependent mechanism. Blotting for the phosphorylated β3 subunit of the GABAA receptor should theoretically be increased when there is more GABA activity at the receptor (McDonald et al., 1998). We expected that more phosphorylation at the GABAA receptor would occur after a pain stimulus due to increased activation of inhibitory post-synaptic processes at the lumbar level of the spinal cord. An increase in phosphorylation at the β3 subunit has been linked to both increased and decreased receptor function of the GABAA receptor (Brandon et al., 2000, McDonald et al., 1998). This effect may be partially caused by the protein kinase that phosphorylated the subunit. Specifically, a major site of regulation on the β3 subunit of the GABAA receptor is the serine 408/409 phosphorylation site. PKA can differentially regulate phosphorylation to either increase activation or decrease activation based on whether both serines 408 and 409 are phosphorylated (potentiation of GABAA activity) or whether just 409 is phosphorylated (inhibition of GABAA receptor activity) (McDonald et al., 1998). Our antibody was specific for phosphorylation of both serine 408 and 409 so it is reasonable to infer that a decrease in expression can be linked to inhibition of activity and an increase (or restoration) can be linked to potentiation of GABAA receptor activity. Our results support the conclusion that glacial acetic difluprednate and HBO2 separately inhibit GABAA receptor activity. However, when combined, HBO2 reversed the decreased activity. These results support our behavioral finding that blockade of the GABAA receptor reduces antinociceptive activity of HBO2 in the acetic acid test. Glacial acetic acid caused a decrease in nNOS expression that was partially prevented by HBO2. This supports earlier findings from our lab that blocking NO could antagonize the antinociceptive effect of HBO2 because NO release being at levels seen prior to the pain stimulus would be required for antinociception under HBO2 (Ohgami et al., 2009, Quock et al., 2011). We expected HBO2 to further increase this level and that this would coincide with an increase in nNOS levels. Similarly, we expected nNOS expression to be increased after HBO2. While nNOS expression was higher under HBO2 when the animals were exposed to glacial acetic acid, levels of expression were decreased, although not significantly, compared to control. In neuropathic and inflammatory pain models, NO is hypothesized to play a role in central sensitization (Wu et al., 2001). However, NO has also been found to have an antinociceptive role in pain (Schmidtko et al., 2009). Our results could indicate that, while HBO2 does prevent or decrease expression of the phosphorylated β3 subunit of the GABAA receptor in the presence of a noxious stimulus, this effect does not coincide with an increase in nNOS above normal levels. Therefore, we cannot conclude that the effects of antinociceptive effects of HBO2 involve a pathway involving both nNOS and GABA. However, it should be important to note that we only blotted for one subunit of the GABAA receptor and our results might not hold for phosphorylation at any of the other receptor subunits. In addition, we may have a low density of β3 subunits in the lumbar spinal cord. This is not likely because immunohistochemical studies have found that β3 subunits are widespread in the dorsal horn of the spinal cord, although not in the motor neurons (Bohlhalter et al., 1996). We also did not examine whether either of the other two NOS isoforms—endothelial NOS (eNOS) or inducible NOS (iNOS)—might increase under these same conditions. If so, this might compensate for the lower amounts of available NO from nNOS. Indeed, prior studies with knockout mice indicate that this is likely as inhibition of any one of the three isoforms can result in a compensatory increase in the other two isoforms (Boettger et al., 2007). It is also possible that we did not wait sufficiently long for major changes in nNOS expression to occur. Since nNOS is post-transcriptionally regulated, it may take longer than the time elapsed in this study for significant changes to occur so we may not be collecting samples at the time of maximum change. Indeed, changes in nNOS levels and NO activity were generally seen at later time points in previous studies. The maximum time varies, but other studies have found it to be at least 30 min after injections of acetic acid (Larson et al., 2000, Shi et al., 2005, Wu et al., 2001). Animals in this study were sacrificed immediately after decompression and 30 min did not elapse from the time of the initial injection to the time of sacrifice.