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. 2019 Apr 20;20(8):1951.
doi: 10.3390/ijms20081951.

Dose-Dependent Effect of Hyperbaric Oxygen Treatment on Burn-Induced Neuropathic Pain in Rats

Zong-Sheng Wu  1 , Sheng-Hua Wu  2   3   4 , Su-Shin Lee  5   6   7   8 , Cen-Hung Lin  9 , Chih-Hau Chang  10 , Jing-Jou Lo  11 , Chee-Yin Chai  12 , Ching-Shuang Wu  13 , Shu-Hung Huang  14   15   16   17
Affiliations

Dose-Dependent Effect of Hyperbaric Oxygen Treatment on Burn-Induced Neuropathic Pain in Rats

Zong-Sheng Wu et al. Int J Mol Sci. .

Abstract

Hyperbaric oxygen treatment (HBOT) has been used to reduce neuropathic pain. Melatonin and opioid receptors are involved in neuropathic pain, but it is not known if HBOT works through these pathways to achieve its antinociceptive effect. We divided anesthetized rats into two treatment and three sham groups. The two treatment groups received third-degree burns on their right hind paws, one treated in a hyperbaric chamber for a week and the other for two weeks. We evaluated the mechanical paw-withdrawal threshold (MWT) and expression of melatonin receptor 1 (MT1), melatonin receptor 2 (MT2), μ (MOR) and κ (KOR) opioid receptor, brain-derived neurotrophic factor (BDNF), Substance P, and calcitonin gene-related peptide (CGRP) in cuneate nucleus, dorsal horn, and hind paw skin by immunohistochemical, immunofluorescence assays and real-time quantitative polymerase chain reaction (RT-PCR). The group receiving one-week HBOT had increased expressions of MT1, MT2, MOR and KOR and decreased expressions of BDNF, Substance P, and CGRP. Their mechanically measured pain levels returned to normal within a week and lasted three weeks. This anti-allodynia effect lasted twice as long in those treated for two weeks. Our findings suggest that increasing the duration of HBOT can reduce burn-induced mechanical allodynia for an extended period of time in rats. The upregulation of melatonin and opioid receptors observed after one week of HBOT suggests they may be partly involved in attenuation of the mechanical allodynia. Downregulation of BDNF, substance P and CGRP may have also contributed to the overall beneficial effect of HBOT.

Keywords: cuneate nucleus; hyperbaric oxygen; melatonin; neuropathic pain; opioid receptor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a,b) Behavior test results of each group (n = 6 rats per group). (a) 1 week of hyperbaric oxygen treatment (HBOT), (b) 2 weeks of HBOT. The mechanical withdrawal threshold (MWT) increased significantly in the burned rats receiving HBOT compared to the burn with sham HBOT group. (*** p < 0.001; black bar represents treatment period of hyperbaric oxygen, w = weeks).
Figure 2
Figure 2
(a,b) Immunohistochemical (IHC) results of melatonin receptor 1 (MT1), melatonin receptor 2 (MT2), μ (MOR) and κ (KOR) opioid receptor in cuneate nucleus of medulla oblongata. MT1, MT2, MOR, and KOR expression significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The expression of MT1, MT2, MOR and KOR were not upregulated after two-weeks HBOT. (*** p < 0.001, original magnification: 200×, w = weeks, SB = sham burn, SH = sham HBOT, MO = medulla oblongata, CN = cuneate nucleus).
Figure 2
Figure 2
(a,b) Immunohistochemical (IHC) results of melatonin receptor 1 (MT1), melatonin receptor 2 (MT2), μ (MOR) and κ (KOR) opioid receptor in cuneate nucleus of medulla oblongata. MT1, MT2, MOR, and KOR expression significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The expression of MT1, MT2, MOR and KOR were not upregulated after two-weeks HBOT. (*** p < 0.001, original magnification: 200×, w = weeks, SB = sham burn, SH = sham HBOT, MO = medulla oblongata, CN = cuneate nucleus).
Figure 3
Figure 3
(a,b) Immunohistochemical (IHC) results of MT1, MT2, MOR, and KOR in the right dorsal horn of the spinal cord. MT1, MT2, MOR, and KOR expression significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The expression of MT1, MT2, MOR and KOR were not upregulated after two-weeks HBOT. (c) RT-PCR results of MT1 and MT2 relative quantification. The mRNA expression significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The mRNA expression increased in Burn 4w with HBOT 2w compared with Burn 4w with sham HBOT 2w. (*** p < 0.001, original magnification: 200×, w = weeks, SB = sham burn, SH = sham HBOT).
Figure 3
Figure 3
(a,b) Immunohistochemical (IHC) results of MT1, MT2, MOR, and KOR in the right dorsal horn of the spinal cord. MT1, MT2, MOR, and KOR expression significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The expression of MT1, MT2, MOR and KOR were not upregulated after two-weeks HBOT. (c) RT-PCR results of MT1 and MT2 relative quantification. The mRNA expression significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The mRNA expression increased in Burn 4w with HBOT 2w compared with Burn 4w with sham HBOT 2w. (*** p < 0.001, original magnification: 200×, w = weeks, SB = sham burn, SH = sham HBOT).
Figure 3
Figure 3
(a,b) Immunohistochemical (IHC) results of MT1, MT2, MOR, and KOR in the right dorsal horn of the spinal cord. MT1, MT2, MOR, and KOR expression significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The expression of MT1, MT2, MOR and KOR were not upregulated after two-weeks HBOT. (c) RT-PCR results of MT1 and MT2 relative quantification. The mRNA expression significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The mRNA expression increased in Burn 4w with HBOT 2w compared with Burn 4w with sham HBOT 2w. (*** p < 0.001, original magnification: 200×, w = weeks, SB = sham burn, SH = sham HBOT).
Figure 4
Figure 4
Immunofluorescence (IF) results of MT1 expression in NeuN positive cells and MT2 expression in NeuN positive cells in the right dorsal horn of the spinal cord. MT1 and MT2 expression in NeuN positive cells significantly increased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. The expressions of MT1 and MT2 were not upregulated after two-weeks HBOT. (*** p < 0.001, arrows indicate stained cells, original magnification: 200×, w = weeks, SB = sham burn, SH = sham HBOT).
Figure 5
Figure 5
Immunohistochemical (IHC) results of glial fibrillary acidic protein (GFAP) in right dorsal horn of spinal cord. GFAP expression was significantly decreased in Burn 4w with HBOT 1w compared with Burn 4w with sham HBOT 1w. (*** p < 0.001, original magnification: 200×, for upper part, original magnification: 400× for lower part, w = weeks, SB = sham burn, SH = sham HBOT).
Figure 6
Figure 6
Immunohistochemical (IHC) results of brain-derived neurotrophic factor (BDNF) in right dorsal horn of spinal cord. BDNF expression was significantly decreased in Burn 4w with HBOT compared with Burn 4w with sham HBOT. (*** p < 0.001, original magnification: 200×, for upper part, original magnification: 400× for lower part, w = weeks, SB = sham burn, SH = sham HBOT).
Figure 7
Figure 7
Immunohistochemical (IHC) results of Substance-P and calcitonin gene-related peptide (CGRP) in right hind paw skin. Expressions of Substance P and CGRP were significantly decreased in Burn 4w with HBOT compared with Burn 4w with sham HBOT. (*** p < 0.001, arrows indicate stained cells, original magnification: 200×, w = weeks, SB = sham-burn, SH = sham HBOT).
Figure 8
Figure 8
One-week HBOT upregulated MT1, MT2, MOR, and KOR expression in the cuneate nucleus of medulla oblongata and in the right dorsal horn of the spinal cord. HBOT downregulated BDNF in the right dorsal horn and Substance P and CGRP in right hind paw skin. HBOT alleviated burn-induced neuropathic pain via this pain transmission pathway (MO = medulla oblongata, CN = cuneate nucleus).
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