Evoked and spontaneous pain assessment during tooth pulp injury

Heather Lynn Rossi,Claire H Mitchell,Brian Schmidt,Saumitra Pitake,Ishmail Abdus-Saboor,Jennifer Gibbs,Lily Pachanin See,William Foster

doi:10.1038/s41598-020-59742-5

Abstract

Injury of the tooth pulp is excruciatingly painful and yet the receptors and neural circuit mechanisms that transmit this form of pain remain poorly defined in both the clinic and preclinical rodent models. Easily quantifiable behavioral assessment in the mouse orofacial area remains a major bottleneck in uncovering molecular mechanisms that govern inflammatory pain in the tooth. In this study we sought to address this problem using the Mouse Grimace Scale and a novel approach to the application of mechanical Von Frey hair stimuli. We use a dental pulp injury model that exposes the pulp to the outside environment, a procedure we have previously shown produces inflammation. Using RNAscope technology, we demonstrate an upregulation of genes that contribute to the pain state in the trigeminal ganglia of injured mice. We found that mice with dental pulp injury have greater Mouse Grimace Scores than sham within 24 hours of injury, suggestive of spontaneous pain. We developed a scoring system of mouse refusal to determine thresholds for mechanical stimulation of the face with Von Frey filaments. This method revealed that mice with a unilateral dental injury develop bilateral mechanical allodynia that is delayed relative to the onset of spontaneous pain. This work demonstrates that tooth pain can be quantified in freely behaving mice using approaches common for other types of pain assessment. Harnessing these assays in the orofacial area during gene manipulation should assist in uncovering mechanisms for tooth pulp inflammatory pain and other forms of trigeminal pain.

Highlights

Injury of the tooth pulp is excruciatingly painful and yet the receptors and neural circuit mechanisms that transmit this form of pain remain poorly defined in both the clinic and preclinical rodent models
We chose to assess the Toll-like Receptor 4 (Tlr4), transient receptor potential channels vanilloid 1 and ankyrin 1 (Trpv[1] and Trpa1), and the mas-related G protein coupled receptor D (Mrgprd), because all are found in neurons that innervate the dental pulp[7,17,18,19,20,21] and could be involved in the development of either spontaneous or mechanical pain in the context of infection and injury
We found that the PAMP/DAMP family member Tlr[4] was upregulated in dental pulp injury (DPI)-treated versus sham-treated mice (Fig. 1C,E,I), as were the associated nociceptive channels TrpV1 (Fig. 1C,D,H), and TrpA1 (Fig. 1C,F,J)

Summary

Introduction

Injury of the tooth pulp is excruciatingly painful and yet the receptors and neural circuit mechanisms that transmit this form of pain remain poorly defined in both the clinic and preclinical rodent models. Recent elegant work in freely behaving mice used both VFH stimulation of the whisker pad and optogenetic www.nature.com/scientificreports activation of trigeminal nociceptors to uncover a craniofacial neural circuit for pain[12], demonstrating feasibility of the VFH approach for the study of oral pain Another approach thought to measure spontaneous pain in rodents is a paradigm called the Mouse Grimace Scale (MGS), that interrogates facial expressions including the positioning of the mouse nose, cheek, ear, eye, and whiskers[13,14]. Because the mice can decide if they want to expose their faces to the stimuli, we were able to record the threshold in which mice are no longer inquisitive enough to tolerate facial stimuli, and this tolerance threshold was able to segregate injured versus sham mice These two assays present a different time course following injury, indicating putative spontaneous pain early and throughout the 6-day observation period, while mechanical allodynia and stimulation intolerance is delayed. The behavioral assays we have defined here to assess pain-like behavior in the mouse should make it easier for researchers to adopt these approaches to aid in uncovering mechanisms for tooth pain

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Conclusion