The role of Tlr7 in cutaneous and neuropathic pain states
Creators: La Caprara, Olivia R
Publisher: name: University of Alberta Library
Year: 2023

<p>The immune system interacts reciprocally with the nervous system in order to accomplish facets of innate immunity via various mechanisms including toll-like receptors (TLRs). TLR activity leads to the release of cytokines which can effect acute and chronic pain states by promoting afferent neuron hypersensitivity. TLR7 is one such receptor that has been implicated in pathways related to inflammation and pain in mice, making it a relevant subject for understanding how these processes are modulated. This project assesses the function of TLR7 with regard to murine nociception in an acute cutaneous inflammatory, chronic cutaneous inflammatory, and neuropathic autoimmune model.<br>
To assess the acute cutaneous inflammatory response, mice were placed in a clear plexiglass container after being injected with 1% formalin into the left hindpaw and observed for nocifensive responses over a period of 60 minutes. To model a chronic cutaneous inflammatory pain state, 0.1 ml of Complete Freund’s Adjuvant (CFA) was injected into the left hindpaw and the von Frey assay was performed over 28 days, until recovery of normal mechanical sensitivity was observed. A mouse model of Multiple Sclerosis (MS), Experimental Autoimmune Encephalomyelitis (EAE), was generated in mice using a subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG) to induce a neuropathic pain state in an autoimmune context. Mechanical hypersensitivity was assessed using the von Frey assay over a period of 14 days, at which point accurate responses were no longer reliable due to advanced disease progression, and mice were observed for clinical motor function for 21 days to score disease severity. <br>
The formalin assay showed an increase in overall pain behaviour in Tlr7-/- mice compared to wildtypes, with a significant difference in the female group. In both sexes, wildtype mice recovered from mechanical hypersensitivity induced by CFA while Tlr7-/- mice remained hypersensitive. EAE disease onset occurred at a similar time point in both Tlr7-/- and wildtype mice, although the Tlr7-/- group demonstrated consistently higher clinical scores over the course of 21 days post-induction. Von Frey testing revealed that while both Tlr7-/- and wildtype mice developed tactile hypersensitivity by 14 days post-induction, this hypersensitivity was accelerated in Tlr7-/- mice. Tlr7-/- mice of both sexes exhibited significant reduction of their paw withdrawal thresholds several days before the wildtype controls. <br>
Following these experiments, the spinal cord (SC) and dorsal root ganglion (DRG) were assessed for Tlr7 RNA and ionized calcium binding receptor molecule 1 (Iba1) protein expression in EAE, while SC cFos and hind paw cluster of differentiation (CD) 45, Iba1, and CD4 protein expression levels were measured in the formalin and CFA responses. For the CFA model specifically, the SC was explored further in terms of peptidergic calcitonin gene-related peptide (CGRP) and non-peptidergic isolectin-B4 (IB4) protein expression levels for nociceptor density and projection length within the dorsal horn. In all models, certain significant changes were found in the SC and hind paw which indicate greater inflammation in Tlr7-/- tissue. With regard to localizing Tlr7, RNAScope combined with immunohistochemistry (IHC) showed that it is colocalized with neurons in the DRG, and thus far we are not able to detect meaningful differences in expression level of Tlr7 in EAE. <br>
In order to test the effect of exogenous agonism on TLR7 and the resolution of mechanical hypersensitivity after cutaneous inflammatory insult, we injected imiquimod into the mouse hind paw following an initial CFA injection. While both vehicle and treatment groups ultimately showed a trend toward recovery, the treatment group demonstrated significantly less mechanical hypersensitivity altogether throughout the course of the experiment in addition to displaying a quicker recovery to baseline levels, suggesting that TLR7 agonism facilitates pain resolution.<br>
Overall, we show that TLR7 is a critical regulator of the resolution of mechanical hypersensitivity and the progression of disease course in the neuropathic pain model of EAE and in cutaneous inflammatory models. We propose a resolutionary functional role of TLR7 and the potential to consider it a novel therapeutic target for clinical studies.</p>