Transcriptional Profiling of Non-injured Nociceptors After Spinal Cord Injury Reveals Diverse Molecular Changes

Abstract

Traumatic spinal cord injury (SCI) has devastating implications for patients, including a high predisposition for developing chronic pain distal to the site of injury. Chronic pain develops weeks to months after injury, consequently, patients are treated after irreparable changes have occurred. Nociceptors are central to chronic pain; however, the diversity of this cellular population presents challenges to understanding mechanisms and attributing pain modalities to specific cell types. To begin to address how peripheral sensory neurons below the injury level may contribute to the below-level pain reported by SCI patients, we examined SCI-induced changes in gene expression in lumbar dorsal root ganglia (DRG) below the site of injury. SCI was performed at the T10 vertebral level, with injury produced by a vessel clip with a closing pressure of 15 g for 1 min. Alterations in gene expression produce long-term sensory changes, therefore, we were interested in studying SCI-induced transcripts before the onset of chronic pain, which may trigger changes in downstream signaling pathways and ultimately facilitate the transmission of pain. To examine changes in the nociceptor subpopulation in DRG distal to the site of injury, we retrograde labeled sensory neurons projecting to the hairy hindpaw skin with fluorescent dye and collected the corresponding lumbar (L2-L6) DRG 4 days post-injury. Following dissociation, labeled neurons were purified by fluorescenceactivated cell sorting (FACS). RNA was extracted from sorted sensory neurons of naïve, sham, or SCI mice and sequenced. Transcript abundances validated that the desired population of nociceptors were isolated. Cross-comparisons to data sets from similar studies confirmed, we were able to isolate our cells of interest and identify a unique pattern of gene expression within a subpopulation of neurons projecting to the hairy hindpaw skin. Differential gene expression analysis showed high expression levels and significant transcript changes 4 days post-injury in SCI cell populations relevant to the onset of chronic pain. Regulatory interrelationships predicted by pathway analysis implicated changes within the synaptogenesis signaling pathway as well as networks related to inflammatory signaling mechanisms, suggesting a role for synaptic plasticity and a correlation with pro-inflammatory signaling in the transition from acute to chronic pain.

Profiling sensory neuron microenvironment after peripheral and central axon injury reveals key pathways for neural repair

“…2b). The number of DE genes we detected after SCI was similar to those observed in FACS-sorted nociceptors after compression SCI 37 . However, a small proportion of neurons (5%) which include both nociceptors (TrkA positive) and LTMRs (TrkB positive), expressed Atf3 after SCI ( Fig.…”

Ascending dorsal column sensory neurons respond to spinal cord injury and downregulate genes related to lipid metabolism

“…Nonetheless, our analysis revealed many DE genes (> 500) in ascending DRG sensory neurons. This is significantly more than the DE genes observed (< 100) in FACS-sorted nociceptors after compression SCI 14 , which are not directly injured by SCI 37 . This would suggest that injury to a projection neuron which receives primary input from DRG neurons leads to a much smaller change in gene expression than direct injury to the ascending DRG neuron itself.…”

“…Whether and how DRG sensory neurons alter their transcriptome after SCI remains unclear because most analyses have used whole DRG after SCI 3 , 4 , 13 , which includes uninjured nociceptors 14 and non-neuronal cells 15 . In this study, we took advantage of a mouse line that labels DRG sensory neurons ascending the spinal cord to unravel their transcriptional response to SCI.…”

Ascending dorsal column sensory neurons respond to spinal cord injury and downregulate genes related to lipid metabolism

“…Nonetheless, our analysis revealed many DE genes (>500) in ascending sensory neurons. This is significantly more than the DE genes observed (<100) in FACS-sorted nociceptors after compression SCI 14 , which are not directly injured by SCI 34 . This would suggest that injury to a projection neuron which receives primary input from DRG neurons leads to a much smaller change in gene expression than direct injury to the ascending neuron itself.…”

“…Whether and how sensory neurons alter their transcriptome after SCI remains unclear because most analyses have used whole DRG after SCI 3,4,13 , which includes uninjured nociceptors 14 and non-neuronal cells 15 . In this study, we took advantage of a mouse line that labels sensory neurons ascending the spinal cord to unravel their transcriptional response to SCI.…”