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ABSTRACTBackground Standard methods assessing pain in rodents are often observer dependent, potentially resulting in biased outcomes. Advanced dynamic weight bearing (ADWB) offers an observer-independent approach that can provide objective, reliable data in preclinical pain research.Aims The aim of this study was to characterize the use of ADWB in assessing murine responses to allyl isothiocyanate (AITC)-induced hyperacute hypersensitivity and identify best practices for use of the device.Methods Male C57BL/6J mice received intraplantar injections of saline or 0.1% AITC solution and were assessed using the ADWB system; simultaneous observer-dependent durations of paw licking and biting were measured. ADWB data were analyzed using the proprietary software from Bioseb and correlated to observer-dependent results, with parameters assessed to optimize data collected.Results ADWB detected pain-directed changes in weight and surface area distribution in AITC-treated mice, with paw weight and surface area placement correlating to paw licking and biting. Optimization of adjustable threshold parameters allowed for reduced coefficients of variability and increased duration of validated data.Conclusions The ADWB assay provides an efficient and unbiased measure of chemical-induced hyperacute hypersensitivity in mice. ADWB detection parameters influence amount of validated data and variability, a consideration for data analysis in future studies.

Spinal cord injury (SCI) is a devastating injury to the central nervous system in which 60 to 80% of patients experience chronic pain. Unfortunately, this pain is notoriously difficult to treat, with few effective options currently available. Patients are also commonly faced with various compounding injuries and medical challenges, often requiring frequent hospitalization and antibiotic treatment. Change in the gut microbiome from the “normal” state to one of imbalance, referred to as gut dysbiosis, has been found in both patients and rodent models following SCI. Similarities exist in the bacterial changes observed after SCI and other diseases with chronic pain as an outcome. These changes cause a shift in the regulation of inflammation, causing immune cell activation and secretion of inflammatory mediators that likely contribute to the generation/maintenance of SCI pain. Therefore, correcting gut dysbiosis may be used as a tool towards providing patients with effective pain management and improved quality of life.

Circulating immune cells, which are recruited to the site of injury/disease, secrete various inflammatory mediators that are critical to nociception and pain. The role of tissue-resident immune cells, however, remains poorly characterized. One of the first cells to be activated in peripheral tissues following injury are γδT cells, which serve important roles in infection, disease, and wound healing. Using a mouse line lacking these cells, we sought to identify their contribution to inflammatory pain. Three distinct models of peripheral inflammatory pain were used: intraplantar injection of formalin (spontaneous inflammatory pain), incisional wound (acute inflammatory pain), and intraplantar injection of complete Freund's adjuvant (chronic inflammatory pain). Our results show that absence of γδT cells does not alter baseline sensitivity, nor does it result in changes to mechanical or thermal hypersensitivity after tissue injury. Myeloid cell recruitment did show differential changes between models of acute and chronic inflammatory pain. These results were consistent in both male and female mice, suggesting that there are no sex differences in these outcomes. This comprehensive characterization suggests that γδT cells do not contribute to basal sensitivity or the development and maintenance of inflammatory pain.

Skin-resident γδ T cells play an important role in maintaining the immune barrier at the epithelial surface. Their roles in wound healing, regulation of immune response to injury, and reepithelialization have been characterized extensively in the mouse, though their function in human skin remains largely unknown. Human skin-resident γδ T cells sparsely populate the skin and are often small and rounded in appearance. Those in the mouse ear and back, which line the dermal barrier, are highly arborized cells with many processes extending from the cell body. To date, these cells have been studied primarily in the mouse ear and back; however, it is important to further identify and characterize γδ T cells in other body sites to better understand their function and study their contribution to injury and disease. We developed a novel method to visualize these cells in the skin (whole-mount and cryosections) that when combined with flow cytometry allowed us to assess differences in skin-resident γδ T cell numbers, morphology, and activation state in the ear, back, and footpad (chosen for their importance in immunological and pain research). In comparing cell length, number of dendritic processes, and expression of the activation marker CD69, we found that γδ T cell morphology and activation states vary significantly among the three tissue environments. Specifically, γδ T cells in the footpad are smaller, have fewer processes, and show the highest levels of activation compared to back- and ear-resident cells. Our observations suggest that our understanding of skin-resident γδ T cell functionality, drawn from the experiments performed in the ear and back tissue, may not be applicable to all skin environments. The footpad-resident cells also more closely resemble γδ T cells in human skin, suggesting that cells in this tissue environment may serve as a better translational model when studying γδ T cell function/activity.