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. 2020 Jun 3:13:1323-1333.
doi: 10.2147/JPR.S239831. eCollection 2020.

P2Y2 Receptors Mediate Masseter Muscle Mechanical Hypersensitivity in Rats

Petra Tariba Knežević  1   2 , Robert Vukman  1   2 , Mia Uhač  3 , Davor Illeš  4 , Daniela Kovačević Pavičić  1   2 , Sunčana Simonić-Kocijan  1   2
Affiliations

P2Y2 Receptors Mediate Masseter Muscle Mechanical Hypersensitivity in Rats

Petra Tariba Knežević et al. J Pain Res. .

Abstract

Purpose: P2Y2 receptors (P2Y2Rs) are among the various receptors that play an important role in nociception. The goal of this research was to investigate possible P2Y2R expression changes in the trigeminal ganglion (TRG) in bilateral masseter muscle (MM) hypersensitivity following unilateral MM inflammation. The impact of unilateral intramasseteric administration of P2Y2R antagonist on bilateral MM hypersensitivity was also explored.

Materials and methods: Bilateral MM hypersensitivity was provoked by unilateral intramasseteric injection of complete Freund's adjuvant (CFA). The head withdrawal threshold (HWT) was assessed bilaterally 4 days later. Bilateral TRG and MM isolation were followed, and quantitative real-time polymerase chain reaction (qRT-PCR) and histopathological analysis were carried out on these tissues, respectively. The involvement of P2Y2Rs in nocifensive behavior was evaluated by administering two doses of P2Y2R antagonist AR-C118925 (0.2 or 1 mg/100 μL) in inflamed MM 4 days post-CFA administration. Bilateral HWT was assessed at different time points following antagonist injection.

Results: qRT-PCR analysis demonstrated P2Y2R up-regulation in TRG ipsilateral to the site of CFA administration. Compared to the controls, both doses of AR-C118925 injected ipsilateral to the TRG increased the bilateral HWT at 30, 60, 90, and 120 minutes after antagonist administration.

Conclusion: The findings suggest that P2Y2Rs may affect MM inflammatory hypersensitivity owing to its up-regulation in the TRG in MM inflammatory pain states.

Keywords: facial pain; masticatory muscles; temporomandibular disorders; trigeminal ganglion.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Experimental design timelines. (A) Phase one of the experiment – evaluation of CFA injection on nocifensive behavior, masseter muscle (MM) tissue histopathology and TRG tissue qRT-PCR. (B) Phase two – evaluation of AR-C118925/DMSO injection on nocifensive behavior in sham rats. (C) Phase two – evaluation of AR-C118925/DMSO administration on nocifensive behavior in unilateral MM inflammation-induced rats. Abbreviations: CFA, complete Freund’s adjuvant; HWT, head withdrawal threshold; MM, masseter muscle; TRG, trigeminal ganglion; qRT-PCR, quantitative real-time polymerase chain reaction; DMSO, dimethyl sulfoxide.f
Figure 2
Figure 2
Histopathological analysis of the masseter muscle (MM) tissue (hematoxylin and eosin staining, 20x). (A) Complete Freund’s adjuvant (CFA)-injected MM presents signs of inflammation. (B) MM contralateral to CFA injection shows no signs of inflammation. (C) MM of control rats shows no signs of inflammation. Abbreviations: CFA, complete Freund’s adjuvant; MM, masseter muscle.
Figure 3
Figure 3
Relative levels of P2Y2 mRNA expression in the trigeminal ganglion (TRG). (A) Relative levels of P2Y2 mRNA expression in the TRG ipsilateral to complete Freund’s adjuvant (CFA) injection. (B) Relative levels of P2Y2 mRNA expression relative levels in the TRG contralateral to CFA administration. Note: *p˂0.05. Abbreviations: TRG, trigeminal ganglion; CFA, complete Freund’s adjuvant.
Figure 4
Figure 4
The effect of unilateral intramasseteric administration of AR-C118925 or dimethyl sulfoxide (DMSO) on baseline head withdrawal threshold (HWT). (A) The effect on baseline HWT of AR-C118925/DMSO ipsilateral injection. (B) The effect on baseline HWT of AR-C118925/DMSO contralateral injection. Abbreviations: DMSO, dimethyl sulfoxide; HWT, head withdrawal threshold.
Figure 5
Figure 5
The effect of unilateral administration of AR-C118925 or dimethyl sulfoxide (DMSO) on head withdrawal threshold (HWT) in rats injected with complete Freund’s adjuvant (CFA). (A) The effect on HWT of ipsilateral CFA and AR-C118925/DMSO injection. (B) The effect on HWT of contralateral CFA and AR-C118925/DMSO injection. Notes: *p˂0.05, ***p≤0.001 for Dlow group, ^p˂0.05, ^^p≤0.01, ^^^p≤0.001 for Dhigh group. Abbreviations: DMSO, dimethyl sulfoxide; HWT, head withdrawal threshold; CFA, complete Freund’s adjuvant; Dlow, 0.2 mg/100 μL dose of AR-C118925; Dhigh, 1 mg/100 μL dose of AR-C118925.
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