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. 2021 Jun 3;20(1):793-803.
doi: 10.1007/s40200-021-00820-4. eCollection 2021 Jun.

Minocycline alleviates nociceptive response through modulating the expression of NR2B subunit of NMDA receptor in spinal cord of rat model of painful diabetic neuropathy

Che Aishah Nazariah Ismail  1   2 , Anis Kausar Ghazali  3 , Rapeah Suppian  4 , Che Badariah Abd Aziz  1 , Idris Long  4
Affiliations

Minocycline alleviates nociceptive response through modulating the expression of NR2B subunit of NMDA receptor in spinal cord of rat model of painful diabetic neuropathy

Che Aishah Nazariah Ismail et al. J Diabetes Metab Disord. .

Abstract

Background: It has been reported that neuropathic pain can be overcome by targeting the NR2B subunit of N-methyl-D-aspartate receptors (NR2B). This study aimed to investigate the effects of minocycline on phosphorylated and total expression of NR2B in the spinal cord of rats with diabetic neuropathic pain.

Methods: A total of 32 Sprague-Dawley male rats were randomly assigned into four groups (n = 8); control healthy, control diabetic (PDN), and PDN rats that received 80 µg or 160 µg intrathecal minocycline respectively. The rats were induced to develop diabetes and allowed to develop into the early phase of PDN for two weeks. Hot-plate and formalin tests were conducted. Intrathecal treatment of minocycline or normal saline was conducted for 7 days. The rats were sacrificed to obtain the lumbar enlargement region of the spinal cord (L4-L5) for immunohistochemistry and western blot analyses to determine the expression of phosphorylated (pNR2B) and total NR2B (NR2B).

Results: PDN rats showed enhanced flinching (phase 1: p < 0.001, early phase 2: p < 0.001, and late phase 2: p < 0.05) and licking responses (phase 1: p < 0.001 and early phase 2: p < 0.05). PDN rats were also associated with higher spinal expressions of pNR2B and NR2B (p < 0.001) but no significant effect on thermal hyperalgesia. Minocycline inhibited formalin-induced flinching and licking responses (phase 1: p < 0.001, early phase 2: p < 0.001, and late phase 2: p < 0.05) in PDN rats with lowered spinal expressions of pNR2B (p < 0.01) and NR2B (p < 0.001) in a dose-dependent manner.

Conclusion: Minocycline alleviates nociceptive responses in PDN rats, possibly via suppression of NR2B activation. Therefore, minocycline could be one of the potential therapeutic antinociceptive drugs for the management of neuropathic pain.

Keywords: Formalin test; Minocycline; NR2B subunit of N-methyl-D-aspartate; Painful diabetic neuropathy; Thermal hyperalgesia.

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Conflict of interest statement

Conflicts of InterestNone.

Figures

Fig. 1
Fig. 1
Thermal hyperalgesia as indicated by hot-plate test in the groups. The data is expressed as mean ± standard error of mean (S. E. M.) (n = 12). No significant difference was detected between the groups
Fig. 2
Fig. 2
Formalin-induced nociceptive flinching responses in the groups. The values are expressed as mean ± standard error of mean (S. E. M.) (n = 12). *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control group, #p < 0.05, ##p < 0.01 and ###p < 0.001 compared with PDN group, §§§p < 0.001 compared with (M 80) group
Fig. 3
Fig. 3
The total number of phosphorylated NR2B subunit of NMDAR (pNR2B) (A) and mean relative pNR2B protein level (B) in the ipsilateral region of spinal cord in the groups. The values are expressed in mean ± standard error of mean (S. E. M.). Minocycline significantly lowered the expressions of pNR2B in dose-dependent manner in the spinal cord of PDN rats. Arrows indicate the expression of pNR2B (400X magnification). *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control group, ##p < 0.01 and ###p < 0.001 compared to PDN group and §§p < 0.01 compared to (M 80) group
Fig. 4
Fig. 4
The total number of phosphorylated NR2B subunit of NMDAR (pNR2B) (A) and mean relative pNR2B protein level (B) in the contralateral region of spinal cord in the groups. The values are expressed in mean ± standard error of mean (S. E. M.). The reduced pNR2B expressions was detected in minocycline-treated groups in dose-dependent manner in the spinal cord of PDN rats. Arrows indicate the expression of pNR2B (400X magnification). *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control group, ##p < 0.01 and ###p < 0.001 compared to PDN group
Fig. 5
Fig. 5
The total number of NR2B subunit of NMDAR (NR2B) (A) and mean relative NR2B protein level (B) in the ipsilateral region of spinal cord in the groups. The values are expressed in mean ± standard error of mean (S. E. M.). Minocycline significantly reduced the expressions of NR2B in dose-dependent manner in the spinal cord of PDN rats. Arrows indicate the expression of NR2B (400X magnification). *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control group, ##p < 0.01 and ###p < 0.001 compared to PDN group and §§p < 0.01 compared to M 80 group
Fig. 6
Fig. 6
The total number of NR2B subunit of NMDAR (NR2B) (A) and mean relative NR2B protein level (B) in the contralateral region of spinal cord in the groups. The values are expressed in mean ± standard error of mean (S. E. M.). Minocycline significantly lowered the expressions of NR2B in dose-dependent manner in the spinal cord of PDN rats. Arrows indicate the expression of NR2B (400X magnification). *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control group, #p < 0.05 and ###p < 0.001 compared to PDN group
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