DOI: 10.34945/f5730s
title: hM4Di-mediated chemogenetic attenuation of acute nociceptive signaling enhances locomotor function and prevents the development of thermal hypersensitivity following moderate T10 spinal cord contusion injury in female Sprague-Dawley rats

description: STUDY PURPOSE: Identifying novel therapeutic approaches to promote recovery of neurological functions following spinal cord injury (SCI) remains a great unmet need. Nociceptive signaling in the acute phase of SCI has been shown to inhibit recovery of locomotor function and promote the development of chronic neuropathic pain. We therefore hypothesized that inhibition of nociceptive signaling in the acute phase of SCI might improve long-term functional outcomes in the chronic phase of injury. To test this hypothesis, we took advantage of a selective strategy utilizing AAV6 to deliver inhibitory (hM4Di) DREADDs to nociceptors of the L4-L6 dorsal root ganglia, in order to evaluate the effects of transient nociceptor silencing on long-term sensory and motor functional outcomes in a rat thoracic contusion SCI model. DATA COLLECTED: Abbreviated Methods: (More detailed information can be found in the Methodology document.) Female Sprague-Dawley rats (200-250g) were used for this study. AAV6-hM4Di or AAV6-YFP (8x10^12 genome copies/mL) was injected into bilateral sciatic nerves 4 weeks prior to SCI/sham. Either thoracic (T10) contusion SCI (150 kydnes, 1 s dwell, Infinite Horizon Impactor device) or sham surgery was performed at Day 0. Immediately following SCI, either clozapine-N-oxide (CNO; hM4Di agonist) or vehicle (0.05% DMSO) was injected intraperitoneally. Beginning on the day of surgery, either CNO or vehicle + 5 mM saccharine was administered in drinking water for 14 days. Following CNO or vehicle treatment from 0-14 days post-SCI, we conducted behavioral assessments until 70 days post-SCI, then performed histological assessments of lesion severity and axon plasticity. Behavioral assessments include open field locomotor (BBB) testing, CatWalk gait analysis, Hargreaves (thermal sensitivity) testing of hindpaws, von Frey (mechanical reactivity) testing of hindpaws, and acetone (cold allodynia) testing of hindpaws. All behavioral testing except for BBB was performed weekly beginning at 2 weeks post-SCI and continuing until 10 weeks post-SCI. BBB data were collected at 1, 3, 7 days post-SCI then weekly until 10 weeks post-SCI. At the study endpoint, animals were transcardially perfused and spinal cord and DRG tissue was collected for histological analysis. The following histological measures were assessed: Lesion volume based on GFAP immunoreactivity, grey matter sparing based on NeuN labeling, white matter sparing based on beta-tubulin labeling, intensity of beta-tubulin immunoreactivity, density of CGRP+ fibers in the T10 and L4 spinal cord, density of IB4-binding fibers in the T10 and L4 spinal cord, density of synaptophysin+ punctae on ChAT+ motor neurons in the L4 spinal cord. Detailed histological analysis methods are described in the Methodology file. Results: -Immunohistochemistry results show highly selective expression of hM4Di within small diameter nociceptors including CGRP+ and IB4-binding neurons. -A two-way repeated measures ANOVA identified a significant effect of time x treatment (P&lt;0.0001) in the thermal hindlimb paw withdrawal scores between SCI-YFP and SCI-hM4Di animals. At the first time point tested following SCI (14 DPI), animals in the SCI-YFP group showed significantly reduced withdrawal latencies to a thermal stimulus relative to naïve controls, and this continued until 35 DPI (Tukey's multiple comparisons test, P&lt;0.05 at each time point). -No significant differences in von Frey mechanical reactivity scores, or acetone cold allodynia scores, were observed between SCI-YFP and SCI-hM4Di treatment groups. -A repeated measures ANOVA identified a significant effect of treatment on open field locomotor (BBB) scores (P=0.0489), indicating that acute nociceptor silencing improved long-term locomotor recovery. -Based on a pLDA analysis of CatWalk data, we found that compared to the naïve and sham groups, both SCI groups exhibited significantly lower pLDA scores at all timepoints post-SCI (P &lt; 0.0002 in all cases); however, the pLDA scores of SCI groups were not significantly different to each other. -Lesion volume, calculated by drawing regions of interest around GFAP+ reactive glial cell layers immediately surrounding the lesion cavity, was not significantly different between SCI-YFP and SCI-hM4Di subjects. -We found that density of both CGRP+ and IB4-binding axons did not differ significantly between SCI groups. DATA USAGE NOTES: