Capsaicin-evoked iCGRP release from human dental pulp: A model system for the study of peripheral neuropeptide secretion in normal healthy tissue

Abstract

The mechanisms underlying trigeminal pain conditions are incompletely understood. In vitro animal studies have elucidated various targets for pharmacological intervention; however, a lack of clinical models that allow evaluation of viable innervated human tissue has impeded successful translation of many preclinical findings into clinical therapeutics. Therefore, we developed and characterized an in vitro method that evaluates the responsiveness of isolated human nociceptors by measuring basal and stimulated release of neuropeptides from collected dental pulp biopsies. Informed consent was obtained from patients presenting for extraction of normal wisdom teeth. Patients were anesthetized using nerve block injection, teeth were extracted and bisected, and pulp was removed and superfused in vitro. Basal and capsaicin-evoked peripheral release of immunoreactive calcitonin gene-related peptide (iCGRP) was analyzed by enzyme immunoassay. The presence of nociceptive markers within neurons of the dental pulp was characterized using confocal microscopy. Capsaicin increased the release of iCGRP from dental pulp biopsies in a concentration-dependent manner. Stimulated release was dependent on extracellular calcium, reversed by a TRPV1 receptor antagonist, and desensitized acutely (tachyphylaxis) and pharmacologically by pretreatment with capsaicin. Superfusion with phorbol 12-myristate 13-acetate (PMA) increased basal and stimulated release, whereas PGE2 augmented only basal release. Compared with vehicle treatment, pretreatment with PGE2 induced competence for DAMGO to inhibit capsaicin-stimulated iCGRP release, similar to observations in animal models where inflammatory mediators induce competence for opioid inhibition. These results indicate the release of iCGRP from human dental pulp provides a novel tool to determine the effects of pharmacological compounds on human nociceptor sensitivity.

Variability in Capsaicin-stimulated Calcitonin Gene-related Peptide Release from Human Dental Pulp

“…In agreement with a previous study we found no significant effect for any of these individual factors (21). This finding is important because it suggests that excluding subjects meeting these specific criteria would be unlikely to reduce the observed variability in the primary outcome, i.e.…”

“…Previous studies using human dental pulp from extracted teeth have completely dissociated the dental pulp from the surrounding hard tissues (21,22). In this study, the use of ex vivo tooth slices maintains the gross anatomical relationships present in the pulpo-dentin complex, specifically the orientation of the nerve endings to dentinal tubules and odontoblasts.…”

“…The activation of TRPV1 in neurons can stimulate the release of neurotransmitters, including calcitonin gene-related neuropeptide (CGRP), a pro-inflammatory vasodilator, which contributes to peripheral and central sensitization in the trigeminal sensory system (12–16). The use of capsaicin-stimulated CGRP release as a primary endpoint is a well-established model for evaluating peripheral afferent activation, and the ability to use human tissue, as described in this study, provides a powerful translational model to test the effects of novel pharmacological compounds on human pulpal nociceptor activity (17–20). In this study we characterized a model in which capsaicin-stimulated CGRP release was measured from dental pulp within human tooth slices.…”

Serotonergic neuromodulation of peripheral nociceptors

“…Nociceptors innervating the human dental pulp express TRPV1. These neurons also contain CGRP [48]. Furthermore, 5-HT receptor protein is present in human pulp as reported by western blot [49].…”

The neuroanatomy of sexual dimorphism in opioid analgesia

“…For example, using a recently-developed in vitro superfusion method to measure proinflammatory peptide release from human dental pulp from extracted teeth [30], Loyd et al [67] reported sex differences in inflammation-induced proinflammatory peptide release that was dependent on stage of menstrual cycle. Specifically, inflammatory mediator-evoked proinflammatory peptide release was highest in amenstrual females and females in the last week of menses [67].…”