Korean J Pain.  2022 Jul;35(3):261-270. 10.3344/kjp.2022.35.3.261.

The ability of orexin-A to modify pain-induced cyclooxygenase-2 and brain-derived neurotrophic factor expression is associated with its ability to inhibit capsaicin-induced pulpal nociception in rats

• Shahsavari F
• Abbasnejad M
• Esmaeili-Mahani S
• Raoof M

• KMID: 2530981
• DOI: http://doi.org/10.3344/kjp.2022.35.3.261

Abstract

Background
The rostral ventromedial medulla (RVM) is a critical region for the management of nociception. The RVM is also involved in learning and memory processes due to its relationship with the hippocampus. The purpose of the present study was to investigate the molecular mechanisms behind orexin-A signaling in the RVM and hippocampus’s effects on capsaicin-induced pulpal nociception and cog-nitive impairments in rats.
Methods
Capsaicin (100 g) was applied intradentally to male Wistar rats to induce inflammatory pulpal nociception. Orexin-A and an orexin-1 receptor antagonist (SB-334867) were then microinjected into the RVM. Immunoblotting and immunofluorescence staining were used to check the levels of cyclooxygenase-2 (COX-2) and brain-derived neurotrophic factor (BDNF) in the RVM and hippocampus.
Results
Interdental capsaicin treatment resulted in nociceptive responses as well as a reduction in spatial learning and memory. Additionally, it resulted in decreased BDNF and increased COX-2 expression levels. Orexin-A administration (50 pmol/1 μL/rat) could reverse such molecular changes. SB-334867 microinjection (80 nM/1 μL/rat) suppressed orexin’s effects.
Conclusions
Orexin-A signaling in the RVM and hippocampus modulates capsaicininduced pulpal nociception in male rats by increasing BDNF expression and decreasing COX-2 expression.

Keyword

Brain-Derived Neurotrophic Factor; Capsaicin; Cognitive Dysfunction; Cyclooxygenase 2; Facial Pain; Nociceptin; Orexins; Pulpitis; Rostral Ventromedial Medulla

Figure

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  Fig. 1 Experimental procedure. RVM: rostral ventromedial medulla, MWM: Morris water maze.

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  Fig. 2 Coronal section through the rostral ventromedial medulla (RVM) adapted from the atlas of Paxinos and Watson. 4V: ventricle 4, PY: pyramidal trac.

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  Fig. 3 Effect of intra-rostral ventromedial medulla administration of orexin-A on the total time of nociceptive behaviors in capsaicin-treated rats. The data is shown as mean ± standard error of the mean. Cont: control, Caps: capsaicin, ORX: orexin-A, SB: SB-334867. ***P < 0.001 vs. control or Caps-vehicle group, +++P < 0.001 vs. Caps or artificial cerebrospinal fluid-treated group (aCSF), and ###P < 0.001 vs. (ORX 50 + Caps)-treated group.

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  Fig. 4 Effect of intra-rostral ventromedial medulla orexin-A administration on escape latency (A) and travelled distance (B) in capsaicin-treated rats in the Morris water maze task. The data is shown as mean ± standard error of the mean. Cont: control, Caps: capsaicin, ORX: orexin-A, SB: SB-334867. **P < 0.01 and ***P < 0.001 vs. control group, +P < 0.05 and +++P < 0.001 vs. Caps-treated group, and ###P < 0.001 vs. (ORX 50 + Caps)-treated group.

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  Fig. 5 Effect of intra-rostral ventromedial medulla orexin-1 receptors agonist and antagonist on cyclooxygenase-2 (COX-2) expression in the hippocampus of capsaicin-treated rats. The data is shown as mean ± standard error of the mean. β-actin uses as an internal control. Cont: control, Caps: capsaicin, ORX: orexin-A, SB: SB-334867. *P < 0.05 and ***P < 0.001 vs. control group, +++P < 0.001 vs. Caps-treated group, and #P < 0.05 vs. (ORX 50 + Caps)-treated group.

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  Fig. 6 Effect of intra-rostral ventromedial medulla orexin-1 receptors agonist and antagonist on brain-derived neurotrophic factor (BDNF) expression changes in the hippocampus of capsaicin-treated rats. Data is shown as mean ± standard error of the mean. β-actin is used as an internal control. Cont: control, Caps: capsaicin, ORX: orexin-A, SB: SB-334867. *P < 0.05 vs. control group, +++P < 0.001 vs. Caps-treated group, ###P < 0.001 vs. (ORX 50 + Caps)-treated group.

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  Fig. 7 Immunofluorescence detection of cyclooxygenase-2 (COX-2) in the rostral ventromedial medulla (RVM) of rats. The data is shown as mean ± standard error of the mean. (A) COX-2 staining in RVM cells (green in color), propidium iodide staining to indicate the nucleus of cells (blue in color) in control, capsaicin, capsaicin plus orexin-A (50 pmol/1 μL/rat) and SB-334867 (80 nmol/1 μL/rat) treated groups. (B) Comparison of the number of COX-2 positively stained cells in the RVM sections of the experimental groups. Cont: control, Caps: capsaicin, ORX: orexin-A, SB: SB-334867, DAPI: 4′,6-diamidino-2-phenylindole. ***P < 0.01 vs. control group, +++P < 0.001 vs. Caps-treated group, ##P < 0.01 vs. (ORX 50 + Caps)-treated group.

• 

  Fig. 8 Immunofluorescence detection of brain-derived neurotrophic factor (BDNF) in the rostral ventromedial medulla (RVM) of rats. The data is shown as mean ± standard error of the mean. (A) BDNF staining in RVM cells (green in color), propidium iodide was stained to indicate the cell’s nucleus (blue in color) in control, capsaicin, capsaicin plus orexin-A (50 pmol/1 μL/rat) and SB-334867 (80 nmol/1 μL/rat) treated groups. (B) Comparison of the number of BDNF-positive stained cells in the RVM sections of the experimental groups. Cont: control, Caps: capsaicin, ORX: orexin-A, SB: SB-334867, DAPI: 4′,6-diamidino-2-phenylindole. **P < 0.01 vs. the control group and +P < 0.05 vs. the Caps-treated group.

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