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Single cell RNA sequencing analysis of mouse dorsal root ganglions (DRGs) with and without spared nerve injury.

ABSTRACT: We report here a systematic approach to characterize the transcriptional responses of different cells types in the dorsal root ganglion (DRG) to peripheral nerve injury using single cell RNA sequencing (scRNAseq). We compare scRNAseq datasets of lumbar DRGs form naïve mice with corresponding datasets from mice subjected to spared nerve injury (SNI) 7 or 14 days prior to analysis. SNI surgery was performed in the left and right hindleg and development of mechanical allodynia was monitored by von Frey testing relative to control mice and the baseline level. Mice were perfused with PBS prior to extraction of L3 and L4 DRGs and DRGs were enzymatically and mechanically dissociated to single cell suspension before scRNAseq. Analysis of transcriptional changes in this nerve injury-paradigm reveals a differential response at 7 days versus 14 days post injury, suggesting dynamic gene modulation over time.

ORGANISM(S): Mus musculus

PROVIDER: GSE174430 | GEO | 2021/09/01

REPOSITORIES: GEO

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Project description:Peripheral nerve injury alters the expression of hundreds of proteins in dorsal root ganglia (DRG). Targeting some of these proteins has led to successful treatments for acute pain, but not for sustained postoperative neuropathic pain. The latter may require targeting multiple proteins. Since a single microRNA (miR) can affect the expression of multiple proteins, here, we describe an approach to identify chronic neuropathic pain-relevant miRs. We used two variants of the spared nerve injury (SNI): Sural-SNI and Tibial-SNI and found distinct pain phenotypes between the two. Both models induced strong mechanical allodynia, but only Sural-SNI rats maintained strong mechanical and cold allodynia, as previously reported. In contrast, we found that Tibial-SNI rats recovered from mechanical allodynia and never developed cold allodynia. Since both models involve nerve injury, we increased the probability of identifying differentially regulated miRs that correlated with the quality and magnitude of neuropathic pain and decreased the probability of detecting miRs that are solely involved in neuronal regeneration. We found seven such miRs in L3-L5 DRG. The expression of these miRs increased in Tibial-SNI. These miRs displayed a lower level of expression in Sural-SNI, with four having levels lower than those in sham animals. Bioinformatics analysis of how these miRs could affect the expression of some ion channels supports the view that, following a peripheral nerve injury, the increase of the 7 miRs may contribute to the recovery from neuropathic pain while the decrease of four of them may contribute to the development of chronic neuropathic pain. The approach used resulted in the identification of a small number of potentially neuropathic pain relevant miRs. Additional studies are required to investigate whether manipulating the expression of the identified miRs in primary sensory neurons can prevent or ameliorate chronic neuropathic pain following peripheral nerve injuries. To identify the miRs that were differentially dysregulated between Tibial-SNI and Sural-SNI, we first performed 12 microarrays in a limited number of samples (in four individual DRGs per group: Sham, Tibial-SNI and Sural-SNI; two L3-DRG and two L4-DRG). Then, miRs identified as having differential expression were corroborated with real time qRT-PCR in RNA isolated from individual DRGs (L3, L4 and L5) derived from 4 rats per group (not presented here, but in the manuscript).

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Single cell RNA sequencing analysis of mouse dorsal root ganglions (DRGs) with and without spared nerve injury.

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