Peripheral Purinergic Modulation in Pediatric Orofacial Inflammatory Pain Affects Brainstem Nitroxidergic System: A Translational Research

Abstract

Physiology of orofacial pain pathways embraces primary afferent neurons, pathologic changes in the trigeminal ganglion, brainstem nociceptive neurons, and higher brain function regulating orofacial nociception. The goal of this study was to investigate the nitroxidergic system alteration at brainstem level (spinal trigeminal nucleus), and the role of peripheral P2 purinergic receptors in an experimental mouse model of pediatric inflammatory orofacial pain, to increase knowledge and supply information concerning orofacial pain in children and adolescents, like pediatric dentists and pathologists, as well as oro-maxillo-facial surgeons, may be asked to participate in the treatment of these patients. The experimental animals were treated subcutaneously in the perioral region with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a P2 receptor antagonist, 30 minutes before formalin injection. The pain-related behavior and the nitroxidergic system alterations in the spinal trigeminal nucleus using immunohistochemistry and western blotting analysis have been evaluated. The local administration of PPADS decreased the face-rubbing activity and the expression of both neuronal and inducible nitric oxide (NO) synthase isoforms in the spinal trigeminal nucleus. These results underline a relationship between orofacial inflammatory pain and nitroxidergic system in the spinal trigeminal nucleus and suggest a role of peripheral P2 receptors in trigeminal pain transmission influencing NO production at central level. In this way, orofacial pain physiology should be elucidated and applied to clinical practice in the future.

Figure 1

Time course of face-rubbing activity observed after subcutaneous injection of saline (CTR), formalin (FORM), PPADS (25 mg/kg), and formalin (PPADS+FORM) into the right upper lip. The mean number of seconds that each mouse spent rubbing was plotted for each 3 minutes block over the 60 minutes postinjection observation period. The experiments were performed in triplicate. Data represent mean ± S.D.; ^∗p < 0.05 versus CTR animals; °p < 0.05 versus FORM animals.

Figure 2

Double-label confocal images of spinal trigeminal nucleus stained neurons for NeuN (green) and nNOS (red). The nuclei were stained in blue (DAPI). Bar: 5 μm.

Figure 3

nNOS-positive neurons in the ipsilateral spinal trigeminal nucleus: (a) subnucleus caudalis (Sp5C) of saline-treated animals (CTR), (b) Sp5C of formalin-treated animals (FORM), (c) Sp5C of PPADS and formalin-treated animals (PPADS+FORM), (a′) subnucleus oralis (Sp5O) of saline-treated animals (CTR), (b′) Sp5C of formalin-treated animals (FORM), and (c′) Sp5C of PPADS and formalin-treated animals (PPADS+FORM). Arrows indicate nNOS-positive neurons. Bar 50 μm. Time course of nNOS immunopositive neurons in the ipsilateral spinal trigeminal nucleus in saline-treated animals (CTR), formalin-treated animals (FORM) and PPADS (25 mg/kg), and formalin-treated animals (PPADS+FORM) after (d) 3, (e) 6, (f) 12, and (g) 24 h from formalin injection. The experiments were performed in triplicate. Data represent mean ± S.D.^∗p < 0.05 vs. CTR; °p < 0.05: FORM vs. PPADS+FORM.

Figure 4

iNOS-positive neurons in the ipsilateral spinal trigeminal nucleus: (a) subnucleus caudalis (Sp5C) of saline-treated animals (CTR), (b) Sp5C of formalin-treated animals (FORM), (c) Sp5C of PPADS and formalin-treated animals (PPADS+FORM), (a′) subnucleus oralis (Sp5O) of saline-treated animals (CTR), (b′) Sp5C of formalin-treated animals (FORM), and (c′) Sp5C of PPADS and formalin-treated animals (PPADS+FORM). Arrows indicate iNOS-positive neurons. Bar 50 μm. Statistical evaluation of immunopositive neurons in the ipsilateral trigeminal nucleus in saline-treated animals (CTR), formalin-treated animals (FORM) and PPADS (25 mg/kg), and formalin-treated animals (PPADS+FORM) after 3 h from formalin injection. The experiments were performed in triplicate. Data represent mean ± S.D.^∗p < 0.05 vs. CTR; °p < 0.05: FORM vs. PPADS+FORM.

Figure 5

Statistical evaluation of nNOS (a) and iNOS (b) expression in the brainstem in saline-treated animals (CTR), PPADS-treated animals (PPADS), formalin-treated animals (FORM), and PPADS- and formalin-treated animals (PPADS+FORM) after 3 h from formalin injection. The experiments were performed in triplicate. Values are mean ± S.D. and represent the IOD (integrated optical density); ^∗p < 0.05 vs. CTR animals; °p < 0.05 vs. FORM animals.