scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of Pirt-EGFPf mice in neuropathic pain condition

Abstract

Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons, which differ in soma size and neurochemical properties, may play different roles in nerve regeneration and pain. However, details about transcriptomic changes in different neuronal subtypes under maladaptive neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain that mimics the etiology of clinical conditions. Therefore, we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons 7 days after sciatic CCI. By using Pirt-EGFPf mice that selectively express enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed a loss of marker genes in injured neurons of 12 standard neuronal clusters, and the emergence of four prominent CCI-induced clusters at this peak-maintenance phase of neuropathic pain. Importantly, a portion of injured neurons from a subset of the 12 standard clusters (NP1, PEP5, NF1, and NF2) were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated subtype-specific transcriptomic perturbations in these clusters, but not others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in primary sensory neurons after nerve injury. Collectively, these findings may contribute to the identification of new target genes and development of DRG neuron subtype-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.