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. Author manuscript; available in PMC: 2024 Nov 1.
Published in final edited form as: Life Sci. 2023 Sep 21;332:122120. doi: 10.1016/j.lfs.2023.122120

Sensory neuron-specific long noncoding RNA in small non-peptidergic dorsal root ganglion neurons selectively impairs nerve injury-induced mechanical hypersensitivity

Bing Wang 1,*, Yingping Liang 1,*, Alex Bekker 1, Huijuan Hu 1,2, Yuan-Xiang Tao 1,2,3,†
PMCID: PMC10591916  NIHMSID: NIHMS1935410  PMID: 37741322

Abstract

Aims:

Nerve injury-induced mechanical hypersensitivity is one of major clinical symptoms in neuropathic pain patients. Understanding molecular mechanisms underlying this symptom is crucial for developing effective therapies. The present study was to investigate whether sensory neuron-specific long noncoding RNA (SS-lncRNA) predominantly expressed in small non-peptidergic dorsal root ganglion (DRG) neurons repaired nerve injury-induced mechanical hypersensitivity.

Materials and Methods:

SS-lncRNA downregulation in the mas-related G protein-coupled receptor member D (Mrgprd)-expressed DRG neurons was rescued and mimicked by crossbreeding MrgprdCreERT2/+ lines with Rosa26SS-lncRNA knock-in mice and SS-lncRNAfl/fl mice, respectively, followed by tamoxifen injection.

Key findings:

Rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons significantly reversed the spinal nerve ligation (SNL)-induced reduction of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in these DRG neurons and alleviated the SNL-induced mechanical hypersensitivity, without affecting the SNL-induced heat and cold nociceptive hypersensitivities, on the ipsilateral side. Conversely, mimicking SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons reduced basal KCNN1 expression in these DRG neurons and produced the enhanced response to mechanical stimulation, but not thermal and cold stimuli, on bilateral sides. Mechanistically, SS-lncRNA downregulation caused a reduction in its binding to lysine-specific demethylase 6B (KDM6B) and consequent recruitment of less KDM6B to Kcnn1 promoter and an increase of H3K27me3 enrichment in this promoter in injured DRG.

Significance:

Our findings suggest that SS-lncRNA downregulation in small non-peptidergic sensory neurons is required specifically for nerve injury-induced mechanical hypersensitivity likely through silencing KCNN1 expression caused by KDM6B-gated increase of H3K27me3 enrichment in Kcnn1 promoter in these neurons.

Keywords: SS-lncRNA, KCNN1, KDM6B, mechanical hypersensitivity, non-peptidergic small sensory neuron, neuropathic pain

Introduction

Neuropathic pain caused by primary damage or injury in the peripheral and central nervous systems is a chronic and refractory clinical condition. It affects the quality of life for 7–10% of the population in the world (Gilron et al., 2015). Over 600 billion dollars every year are spent on neuropathic pain-related healthcare and productivity loss in the United States alone (O’Connor, 2009). Current successful managements for this disorder are highly limited (Gilron et al., 2015, O’Connor, 2009). The most prescribed medications including opioids and non-opioids (e.g., gabapentin, duloxetine, and amitriptyline) have antinociceptive effects in less than 50% of neuropathic pain patients (Gilron et al., 2015, O’Connor, 2009). Moreover, the side effects of these medications are severe to significantly impact the long-term adherence (Brady et al., 2016, Finnerup et al., 2015, Volkow and McLellan, 2016, Yang et al., 2015). In the clinic, neuropathic pain patients often complain of spontaneous ongoing pain and/or enhanced responses to noxious or innocuous stimuli (such as mechanical hypersensitivity) (Campbell and Meyer, 2006). Understanding the mechanisms of how these symptoms are caused following peripheral nerve injury may open a door for developing new therapeutic managements for neuropathic pain.

The dorsal root ganglia (DRG) are a collection of cell bodies of the primary sensory neurons. These neurons are heterogeneous by a variety of molecular criteria (Dong et al., 2001, Wang et al., 2023b) and could be activated by multiple types of noxious or innoxious stimuli (Cain et al., 2001, Lawson et al., 2008). DRG neurons specifically expressing mas-related G protein-coupled receptor member D (Mrgprd) constitute > 90% of all unmyelinated small non-peptidergic DRG neurons (Dong et al., 2001, Zylka et al., 2003, Zylka et al., 2005), whereases DRG neurons specifically expressing heat-sensitive channel TRPV1 constitute most unmyelinated small peptidergic DRG neurons (Dong et al., 2001, Dussor et al., 2008, Zylka et al., 2005). A previous study revealed that ablation of the Mrgprd-expressed DRG neurons produced specific deficits in the behavioral responses to mechanical stimuli, but not to heat or cold stimuli (Cavanaugh et al., 2009). Conversely, pharmacological ablation of the central terminals of DRG TRPV1-expressed neurons led to a loss in the behavioral response to heat stimulation, but not to mechanical or cold stimuli (Cavanaugh et al., 2009). These findings suggest that distinct subpopulations of small DRG neurons selectively mediate behavioral responses to different noxious stimulus modalities. However, the detailed mechanisms underlying these phenomena particularly under neuropathic pain conditions are still elusive.

Peripheral nerve injury-induced maladaptive changes in gene expression in the DRG neurons are considered the molecular basis for the development and maintenance of neuropathic pain (Liang et al., 2015, Lutz et al., 2014, Wu et al., 2019). Long noncoding RNAs (lncRNAs; > 200 nt) are emerging as critical regulators of gene expression (Statello et al., 2021). We recently identified sensory neuron-specific long noncoding RNA (SS-lncRNA) in the mouse and human DRGs (Wang et al., 2023a). Peripheral nerve injury downregulated SS-lncRNA expression in injured DRG (Wang et al., 2023a). Rescuing this downregulation through DRG microinjection of AAV5 expressing full-length SS-lncRNA or use of the tamoxifen-pretreated inducible conditional Rosa26SS-lncRNA/AdvillinCreERT2/+ knock-in (cKI) mice reversed a decrease of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in injured DRG and alleviated nerve injury-induced nociceptive hypersensitivities, without affecting basal responses to mechanical, heat and cold stimuli (Wang et al., 2023a). Conversely, mimicking this downregulation through DRG microinjection of AAV5 expressing a specific SS-lncRNA shRNA or use of the tamoxifen-pretreated inducible conditional SS-lncRNAfl/fl/AdvillinCreERT2/+ knock-down (cKD) mice reduced the expression of DRG KCNN1 and led to the enhanced responses to mechanical, heat and cold stimuli in naïve mice (Wang et al., 2023a). These findings suggest that DRG downregulated SS-lncRNA is required for neuropathic pain by reducing DRG KCNN1. However, due to the limitation of these strategies employed, our previous study did not clearly show whether downregulated SS-lncRNA selectively contributes to mechanical hypersensitivity following peripheral nerve injury, given that it was expressed predominantly in small non-peptidergic DRG neurons (Wang et al., 2023a).

In the present study, we examined the effect of selectively rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons by crossbreeding Rosa26SS-lncRNA knock-in (SS-KI) mice with MrgprdCreERT2/+ lines followed by tamoxifen injection on the spinal nerve ligation (SNL)-induced mechanical, heat and cold hypersensitivities. We also assessed the effect of selectively knocking down SS-lncRNA in the Mrgprd-expressed DRG neurons by crossbreeding SS-lncRNAfl/fl (SSfl/fl) mice with MrgprdCreERT2/+ lines followed by tamoxifen injection on basal responses to mechanical, heat and cold stimuli in naïve mice. Finally, we exploited the potential mechanisms underlying the observations.

Materials and Methods

Animal preparation

SS-lncRNAfl/fl (SSflf/fl) mice and Rosa26SS-lncRNA knock-in (SS-KI) mice were generated by Biocytogen INC (Worcester, MA). MrgprdCreERT2/+ mice were purchased from the Jackson Laboratory (Stock No: 031286). SSfl/fl mice or SS-KI mice were backcrossed onto a C57BL/6 background for at least 3 generations to generate heterozygotes in our facility. Then these two types of female mice were mated with male MrgprdCreERT2/+ mice to generate small non-peptidergic neuron-specific inducible conditional knockdown (cKD) mice and knock-in (cKI) mice, respectively. All mice were kept under standard housing conditions including 12/12-h dark-light cycle and food and water ad libitum. Animal procedures were approved by the Institutional Animal Care & Use Committee of Rutgers New Jersey Medical School and were consistent with the ethical guidelines of the US National Institutes of Health and the International Association for the Study of Pain. The experimenters were blind to the treatment conditions.

Chronic neuropathic pain model

The preclinical mouse model of SNL-induced neuropathic pain was carried out as stated previously (Du et al., 2022, Li et al., 2020, Pan et al., 2021, Rigaud et al., 2008, Wang et al., 2023a). Briefly, after the mice were anesthetized with 2–3% isoflurane, the unilateral forth lumbar (L4) transverse process was identified and removed. The underlying L4 spinal nerve was isolated carefully, ligated with a 7-0 silk thread and then transected just distal to the ligature. Sham mice received an identical surgery but without transection and ligation of the L4 spinal nerve.

Behavioral tests

Mechanical hypersensitivity was quantified by measuring paw withdrawal response to two calibrated von Frey filaments (0.07 and 0.4 g; Stoelting Co., Wood Dale, IL) as described (Jia et al., 2022, Zhang et al., 2022, Zhang et al., 2021). Briefly, mice were placed in a Plexiglas chamber on an elevated wire mesh screen to habituate for 30 min. Mice received 10 repeated applications (10–20 seconds apart) of each von Frey filament to the middle of the plantar surface of both hind paws. A quick withdrawal of the paw was considered as a positive response. The number of withdrawal responses within 10 applications was recorded as the percentage of withdrawal frequency.

Heat hypersensitivity was determined by measuring paw withdrawal response to heat stimulation as described (Jia et al., 2022, Zhang et al., 2022, Zhang et al., 2021). Briefly, mice were placed on a glass surface in individual Plexiglas chamber. A beam of light from a Model 336 Analgesia Meter (IITC Inc. Life Science Instruments. Woodland Hills, CA) was applied to the middle of the plantar surface of each hind paw. The length of time from the turning on light A to a quick lift of the hind paw was considered as the paw withdrawal latency. A cutoff time of 20 s was used to avoid tissue damage.

Cold hypersensitivity was detected by measuring paw withdrawal response to noxious cold (0 °C) as previously described (Jia et al., 2022, Zhang et al., 2022, Zhang et al., 2021). Briefly, mice were placed in a Plexiglas chamber on the cold aluminum plate. The paw withdrawal latency was recorded as the length of time between placement and the first sign of the mouse jumping and/or flinching. This procedure was repeated three times at 10-min intervals. A cut-off time of 20 s was used.

Spontaneous ongoing pain was examined by carrying out the conditioned place preference (CPP) test as described (Jia et al., 2022, Zhang et al., 2022, Zhang et al., 2021). Briefly, the apparatus had a pair of chambers with distinct stripes (horizontal vs vertical) on the walls and different textures (rough vs smooth) on the floor connected with a door in the middle (Med Associates Inc., St. Albans, VT). After preconditioning, each mouse was monitored for 15 minutes through photobeam detectors installed along the chamber walls to automatically record basal time spent in each chamber via MED-PC IV CPP software. Conditional training was conducted for the following 3 days with the door close. The mice first received an intrathecal injection of saline (5 μl) specifically paired with one conditioning chamber in the morning. Six hours later, lidocaine (0.8 % in 5 μl of saline) was given intrathecally paired with another conditioning chamber in the afternoon. Injection order of saline and lidocaine every day was switched. On the test day (at least 20 hours later after conditional training), the mice were placed in two chambers with doors open. The time spent in each chamber was recorded for 15 min for analysis of chamber preference. Place preference was indexed by the difference between the post-test time and preconditioning time spent in the lidocaine-paired chamber.

Locomotor activity tests, including placing, grasping, and righting reflexes, were performed before tissue collection according to the protocols described previously (Jia et al., 2022, Zhang et al., 2022, Zhang et al., 2021). For the placing reflex, the hind limbs were placed slightly lower than the forelimbs and the dorsal surfaces of the hind paws were brought into contact with the edge of a table. Then, whether the hind paws were placed on the table surface reflexively was recorded. For the grasping reflex, animals were placed on a wire grid, and then whether or not the hind paws grasped the wire was recorded. For the righting reflex, animals were placed on its back on a flat surface, and it was recorded whether the mouse could immediately assume the normal upright position. Each trial was repeated five times in a 5-min interval and the score for each test was recorded by counting times of each normal reflex.

Quantitative real-time RT-PCR assay

Total RNA extraction and quantitative real-time RT-PCR were carried out as described (Jia et al., 2022, Zhang et al., 2022, Zhang et al., 2021). Briefly, the ipsilateral L4 DRGs from 4 adult mice were collected and pooled together to achieve enough RNA. Total RNA was extracted and purified by using the RNeasy mini kit (Qiagen, Valencia, CA) and reverse-transcribed by using the Omniscript RT Kit (Qiagen) with specific RT primers or oligo (dT) primers. The quantitative real-time PCR assay was conducted by using SYBR Green real-time PCR Master Mix. Template (1 μl) was amplified in a Bio-Rad CFX96 real-time PCR system by using specific primers for SS-lncRNA (forward: 5′-GTGGGATCAACATTCCCAAC-3′ and reverse: 5′-CCATGTCCGTCTTTGGATCT-3′), Kcnn1 mRNA (forward: 5′-TTGAAAAGCGTAAACGGCTCA-3′ and reverse: 5′-CAGAGCAAAAGAGCAGAGTGA-3′) or Tuba-1a mRNA (forward: 5′-GTGCATCTCCATCCATGTTG-3′ and reverse: 5′-GTGGGTTCCAGGTCTACGAA-3′). Each sample was run in triplicate in a 20μl reaction volume containing 250 nM forward and reverse primers, 10 μl of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories), and 20 ng of cDNA. The PCR amplification consisted of 30 s at 95°C, 30 s at 60°C, 30 s at 72°C, and 5 min at 72°C for 39 cycles. Ratios of ipsilateral-side mRNA levels to contralateral-side mRNA levels were calculated using the ΔCt method (2−ΔΔCt) after normalization to Tuba-1a as it has been demonstrated to be stable even after peripheral nerve injury insult in mice as shown previously (Jia et al., 2022, Zhang et al., 2022, Zhang et al., 2021).

Western blotting analysis

After the mice were deeply anesthetized, the dorsal lumber lamina was removed. The exposed lumbar enlargement segments of spinal cord were quickly dissected and collected. The tissues were then homogenized in an ice-cold lysis buffer containing 10 mM Tris, 1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 1mM DTT, 40 μM leupeptin, phosphatase and protease inhibitor cocktails (1: 100; Sigma) and 250 mM sucrose. The lysate was centrifuged at 4 °C for 15 min at 1,000 g. The supernatant was collected for measuring the concentrations of the proteins in the samples using the Bio-Rad protein assay (Bio-Rad). After the samples were heated at 99 °C for 5 min, they were loaded onto a 4–20% precast polyacrylamide gel (Bio-Rad Laboratories), and then electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories). The membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween-20 for 1 h and incubated overnight at 4 °C with the following primary antibodies including rabbit anti-KCNN1 (1:1,000, LifeSpan BioSciences, Inc), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204, 1:800, Cell Signaling), rabbit anti-ERK1/2 (1:800, Cell Signaling), mouse anti-GFAP (1:800, Cell Signaling), rabbit anti-KDM6B (1:1,000, Cell Signaling), and rabbit anti-GAPDH (1:2,000, Santa Cruz, Dallas, TX). The proteins were detected by western peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad) and exposed using the ChemiDoc XRS System with Image Lab software (Bio-Rad). The intensity of blots was quantified with densitometry using Image Lab software (Bio-Rad).

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was conducted with the EZ ChIP Kit (Upstate/EMD Millipore) as reported previously (Du et al., 2022, Pan et al., 2021, Wang et al., 2023a). In brief, DRG homogenates were crosslinked with 1% formaldehyde for 10 min at room temperature. The reaction was terminated by the addition of 0.25 M glycine. After centrifugation, the collected pellet was lysed in the SDS lysis buffer with a protease inhibitor cocktail and sonicated until the DNA was broken into the fragments with a mean length of 200–1000 nt. The lysate was pre-cleaned with 25 μl Pierce Protein A/G Agarose beads. Immunoprecipitation was carried out at 4 °C overnight using rabbit anti-KDM6B (2 μg, CST), rabbit anti-H3K27me3 (2 μg, CST), or purified rabbit IgG. Input (10–20% of the sample for immunoprecipitation) was used as a positive control. The DNA fragments were purified and detected by quantitative real-time PCR assays with Kcnn1 promoter primers (forward: 5′-TTGAACTTGCAGCAATGCCC-3′; reverse: 5′-CACCCAGGTTCCAGGATCAC-3′).

RNA immunoprecipitation (RIP) assay

The RIP assay was conducted by using a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore) as reported previously (Du et al., 2022, Pan et al., 2021, Wang et al., 2023a). Briefly, the protein A/G magnetic beads were incubated with 2 μg of rabbit anti-KDM6B (CST) or 2 μg of purified rabbit IgG (Millipore) at RT for 30 min. Except that about 10% of the sample lysate was used as Input, the remaining lysates then were incubated with the bead-antibody complexes overnight at 4 °C. The immunoprecipitated RNAs were purified by phenol/chloroform extraction and eluted in 15 μl of RNase/DNase-free water. The relative abundance of SS-lncRNA was quantified by quantitative real-time PCR assay with the SS-lncRNA primers (forward: 5′-CCATGTCCGTCTTTGGATCT-3′; reverse: 5′-GTGGGATCAACATTCCCAAC-3′).

RNA pull-down assay

The RNA pull-down assay was performed with a Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA) according to the manufacturer’s instructions (Du et al., 2022, Pan et al., 2021, Wang et al., 2023a). The SS-lncRNA sense probe (forward: 5′-TAATACGACTCACTATAGGGCATCCTAGCCTACGGCAGAG-3′; reverse: 5′-CCATGTCCGTCTTTGGATCT-3′) and SS-lncRNA antisense probe (forward: 5′-CATCCTAGCCTACGGCAGAG-3′; reverse: 5′-TAATACGACTCACTATAGGGCCATGTCCGTCTTTGGATCT-3′) were synthesized in vitro by PCR with T7 RNA polymerase by using the Biotin RNA Labeling Mix (Roche) and purified by using the Thermo GeneJET RNA Purification Kit (Thermo Fisher, USA). The sense probe was used as a negative control. The cultured DRG neurons were homogenized and cross-linked by using 37% formaldehyde for 10 min at RT. The cross-linked chromatin was sheared by sonication. Hybridization was conducted at 37°C overnight in a hybridization oven. Biotinylated RNA was captured by using Dynabeads MyOne Streptavidin T1 beads (Invitrogen, Cat No-65601). Proteins were solubilized in Laemmli sample loading buffer and Western blot analysis was carried out to verify the binding of SS-lncRNA to KDM6B.

Statistical analysis.

Mice were randomly distributed across experimental cohorts. All results are shown as the means ± S.E.M of at least three independent experiments. Data distribution was assumed to be normal, but this was not formally tested. The data were statistically analyzed using a two-tailed, paired Student’s t-test and a one-way, two-way, or three-way ANOVA. When ANOVA showed a significant difference, pairwise comparison between means was performed using the post hoc Tukey method (Prism6 software). Significance was set at P < 0.05.

Results

Effect of rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons on SNL-induced nociceptive hypersensitivity

To determine the effect of rescuing downregulated SS-lncRNA in small non-peptidergic DRG neurons on neuropathic pain, we carried out SNL or sham surgery in male cKI mice and SS-KI mice (as controls) after intraperitoneal (i.p.) injection of tamoxifen at 1 mg/daily for 7 days (Wang et al., 2023a). Consistent with our previous study (Wang et al., 2023a), the level of SS-lncRNA was reduced by 80% in the ipsilateral L4 DRG of the tamoxifen-pretreated SS-KI mice on day 28 post-SNL as compared to that in the tamoxifen-pretreated SS-KI sham mice (Fig. 1A). This reduction was significantly turned over in the Mrgprd-expressed neurons from the ipsilateral L4 DRG of the tamoxifen-pretreated cKI mice on day 28 post-SNL (Fig. 1A). As expected, the amount of SS-lncRNA was dramatically elevated in the Mrgprd-expressed neurons from the ipsilateral L4 DRG of the tamoxifen-pretreated cKI mice on day 28 post-sham surgery (Fig. 1A).

Fig. 1.

Fig. 1.

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Rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons selectively attenuated the development of SNL-induced mechanical hypersensitivity in male mice. (A) Level of SS-lncRNA in the ipsilateral L4 DRG on day 28 after SNL or sham surgery in SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen daily for 7 consecutive days before surgery. n = 12 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test. (B-H) Paw withdrawal frequency (PWF) to 0.07 g (B, F) and 0.4 g (C, G) von Frey filaments and paw withdrawal latency (PWL) to heat (D, H) and cold (E) stimuli on the ipsilateral (A-E) and contralateral (F-H) sides at the different days as indicated before and after SNL or sham surgery in SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen (Tam) daily for 7 consecutive days before surgery. n = 12 mice/group. ** P < 0.01 versus Sham SS-KI mice and ## P < 0.01 versus the SNL SS-KI mice at the corresponding time points by three-way ANOVA with repeated measures followed by post hoc Tukey test. (I and J) Spontaneous ongoing pain as assessed by the CPP paradigm 3 weeks after SNL or sham surgery in SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen daily for 7 consecutive days before surgery. Pre: preconditioning. Post: post-conditioning. n = 10 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test. (K) Levels of p-ERK1/2, ERK1/2 and GFAP in the ipsilateral L4 dorsal horn on day 28 after SNL or sham surgery in the SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen daily for 7 consecutive days before surgery. n = 3 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test.

In line with the previous observations (Han et al., 2023, Wen et al., 2022), SNL led to mechanical hypersensitivity as evidenced by increases in paw withdrawal frequencies in response to mechanical stimuli (0.07 g and 0.4 g von Frey filaments) and heat and cold nociceptive hypersensitivity as evidenced by reductions in paw withdrawal latencies in response to heat and cold stimuli, respectively, from days 3 to 28 post-SNL surgery on the ipsilateral (but not contralateral) side of the tamoxifen-pretreated SS-KI male mice (Fig. 1B–1H). However, the tamoxifen-pretreated cKI mice displayed impaired mechanical hypersensitivity, although these mice still revealed intact heat and cold nociceptive hypersensitivities, during the observation period (Fig. 1B–1E). Both SS-KI male mice and cKI male mice showed normal locomotor activity (Table 1) and no changes in basal paw withdrawal responses to mechanical, heat, and cold stimuli on the contralateral side after SNL and on both ipsilateral and contralateral sides after sham surgery (Fig. 1F–1H).

Table 1.

Mean changes in locomotor function

Treatment groups Placing Grasping Righting
SS-KI + Sham (Male) 5(0) 5(0) 5(0)
SS-KI + SNL (Male) 5(0) 5(0) 5(0)
cKI + SNL (Male) 5(0) 5(0) 5(0)
cKI + Sham (Male) 5(0) 5(0) 5(0)
SS-KI + Sham (Female) 5(0) 5(0) 5(0)
SS-KI + SNL (Female) 5(0) 5(0) 5(0)
cKI + SNL (Female) 5(0) 5(0) 5(0)
cKI + Sham (Female) 5(0) 5(0) 5(0)
SSfl/fl (Female) 5(0) 5(0) 5(0)
cKD (Male) 5(0) 5(0) 5(0)
SSfl/fl (Female) 5(0) 5(0) 5(0)
cKD (Female) 5(0) 5(0) 5(0)
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All values are Mean (SEM). n = 7–12 mice/group. 5 trials. SS: SS-lncRNA.

SS-KI: ROSA26SS-lncRNA knock-in mice. cKI: inducible conditional ROSA26SS-lncRNA/MrgprdCre-ERT2 Knock-in mice. SSfl/fl: SS-lncRNA floxed mice. cKD: inducible conditional SS-lncRNAfl/fl /MrgprdCre-ERT2 knockdown mice.

In addition to evoked pain, we also examined whether rescuing downregulated SS-lncRNA in the Mrgprd-expressed neurons affected the SNL-induced spontaneous ongoing pain. Consistent with the previous reports (Du et al., 2022, Pan et al., 2021, Wang et al., 2023a), the evoked stimulation-independent spontaneous ongoing pain demonstrated by an increase in preference for the lidocaine-paired chamber was seen in the tamoxifen-pretreated SS-KI mice on day 28 post-SNL (Fig. 1I and 1J). In contrast, the tamoxifen-pretreated SNL cKI mice, like the tamoxifen-pretreated sham SS-KI mice or cKI mice, did not exhibit any marked preference for two chambers (Fig. 1I and 1J).

DRG neuronal hyperexcitability triggers the hyperactivation of spinal cord dorsal horn neurons and astrocytes through enhancing the release of neurotransmitters/neuromodulators in primary afferents under neuropathic pain conditions (Campbell and Meyer, 2006). To further confirm our behavioral observations above, we also examined whether rescuing downregulated SS-lncRNA in the Mrgprd-expressed neurons had an impact on the SNL-induced neuronal and astrocyte hyperactivations in the dorsal horn of male mice. Consistent with previous reports (Liang et al., 2023, Mao et al., 2019, Zhang et al., 2022, Zhang et al., 2021), the amounts of p-ERK1/2 (a marker for neuronal hyperactivation (Gao and Ji, 2009, Ji et al., 1999, Ji et al., 2002, Kawasaki et al., 2004)), but not total ERK1/2, and GFAP (a marker for astrocyte hyperactivation (Narita et al., 2006, Tanga et al., 2004, Vega-Avelaira et al., 2007)) were markedly increased in the ipsilateral L4 dorsal horn on day 28 after SNL in the tamoxifen-pretreated SNL SS-KI male mice (Fig. 1K). These increases did not occur in the tamoxifen-pretreated SNL cKI male mice (Fig. 1K). Neither SS-KI mice nor cKI mice displayed the changes in basal levels of total ERK1/2, p-ERK1/2 or GFAP in the ipsilateral L4 dorsal horn on day 28 after sham surgery (Fig. 1K).

Similar results were observed in SNL female SS-KI mice and cKI mice following tamoxifen treatment (Fig. 2A–2K; Table 1).

Fig. 2.

Fig. 2.

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Rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons selectively attenuated the development of SNL-induced mechanical hypersensitivity in female mice. (A) Level of SS-lncRNA in the ipsilateral L4 DRG on day 28 after SNL or sham surgery in SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen daily for 7 consecutive days before surgery. n = 12 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test. (B-H) Paw withdrawal frequency (PWF) to 0.07 g (B, F) and 0.4 g (C, G) von Frey filaments and paw withdrawal latency (PWL) to heat (D, H) and cold (E) stimuli on the ipsilateral (A-E) and contralateral (F-H) sides at the different days as indicated before and after SNL or sham surgery in SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen (Tam) daily for 7 consecutive days before surgery. n = 12 mice/group. ** P < 0.01 versus Sham SS-KI mice and ## P < 0.01 versus the SNL SS-KI mice at the corresponding time points by three-way ANOVA with repeated measures followed by post hoc Tukey test. (I and J) Spontaneous ongoing pain as assessed by the CPP paradigm 3 weeks after SNL or sham surgery in SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen daily for 7 consecutive days before surgery. Pre: preconditioning. Post: post-conditioning. n = 10 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test. (K) Levels of p-ERK1/2, ERK1/2 and GFAP in the ipsilateral L4 dorsal horn on day 28 after SNL or sham surgery in the SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen daily for 7 consecutive days before surgery. n = 3 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test.

Collectively, our results demonstrate that rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons specifically mitigates the SNL-induced mechanical hypersensitivity, spontaneous hypersensitivity and dorsal horn central sensitization.

Effect of knocking down SS-lncRNA in the Mrgprd-expressed DRG neurons on basal responses to mechanical, heat and cold stimuli in naïve mice

Since SS-lncRNA was expressed predominantly in small non-peptidergic DRG neurons and downregulated in injured DRG following peripheral nerve injury (Wang et al., 2023a), we next assessed whether this downregulation in small non-peptidergic DRG neurons was sufficient for nerve injury-induced nociceptive hypersensitivity, particularly in mechanical hypersensitivity. To this end, we performed behavioral tests in naïve male SSflf/fl mice and cKD mice at the different days after the first tamoxifen injection. As predicted, the level of SS-lncRNA in the Mrgprd-expressed neurons of bilateral L3/4 DRGs from the tamoxifen-pretreated male cKD mice was decreased by 91% in comparison to that from the tamoxifen-pretreated male SSflf/fl mice 28 days after the first i.p. injection of tamoxifen (Fig. 3A). Vehicle injection did not alter basal expression of SS-lncRNA in bilateral L3/4 DRGs from the tamoxifen-pretreated male cKD mice (Fig. 3A). The tamoxifen-pretreated cKD mice exhibited significant increases in paw withdrawal frequencies in response to 0.07 g and 0.4 g von Frey filament stimuli from day 4 to day 28 after the first i.p. injection of tamoxifen on the left (Fig. 3B and 3C) and right (data not shown) sides. No changes in paw withdrawal latencies in response to heat and cold stimuli were seen in the tamoxifen-pretreated cKD mice during the experimental period on the left (Fig. 3D–3E) and right (data not shown) sides. As expected, the vehicle-treated cKD mice did not show any alternations in basal responses to mechanical, heat and cold stimuli on either side (Fig. 3B–3E on the left side and data shown on the right side). Locomotor functions were normal in the tamoxifen-pretreated male SSflf/fl mice or cKD mice and the vehicle-treated male cKD mice (Table 1).

Fig. 3.

Fig. 3.

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Knocking down SS-lncRNA in the Mgrprd-expressed DRG neurons selectively produced mechanical hypersensitivity in naive male mice. (A) Level of SS-lncRNA in bilateral l3/4 DRGs from SSfl/fl mice and cKD mice 28 day after the first injection of tamoxifen or vehicle (Veh). n = 9 mice, **P < 0.01, by two-way ANOVA with post hoc Tukey test. (B-E) Paw withdrawal frequency (PWF) to 0.07 g (B) and 0.4 g (C) von Frey filaments and paw withdrawal latency (PWL) to heat (D) and cold (E) stimuli on the left side at the different days as indicated in SSfl.fl mice and cKD mice with an intraperitoneal injection of tamoxifen (Tam) or vehicle daily for 7 consecutive days. n = 9 mice, **P < 0.01 versus the SSfl/fl group at the corresponding time points by three-way ANOVA with repeated measures followed by post hoc Tukey test. (F and G) Spontaneous ongoing pain as assessed by the CPP paradigm 21 days after the first tamoxifen or vehicle (Veh) injection in SSfl.fl mice and cKD mice. Pre: preconditioning. Post: post-conditioning. n = 9 mice/group. **P < 0.01, by two-way ort three-way ANOVA with post hoc Tukey test. (H and I) Levels of p-ERK1/2, ERK1/2 and GFAP in the L3/4 dorsal horn from SSfl/fl mice and cKD mice on day 28 after first intraperitoneal injection of tamoxifen or vehicle daily for 7 consecutive days. n = 3 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test.

In addition to evoked mechanical hypersensitivity, the tamoxifen-pretreated male cKD mice revealed robust preference for the lidocaine-paired chamber 28 days after the first i.p. injection of tamoxifen (Fig. 3F and 3G), showing stimulation-independent spontaneous pain. In contrast, the tamoxifen-pretreated male SSflf/fl mice and vehicle-treated male cKD mice did not reveal any preference for either saline-paired or lidocaine-paired chamber, indicating no spontaneous ongoing pain (Fig. 3F and 3G). In addition, the levels of p-ERK1/2 (not total ERK1/2) and GFAP were increased in bilateral L3/4 spinal cord from the tamoxifen-pretreated male cKD mice, but not from the vehicle-treated male cKD mice and the tamoxifen-pretreated male SSfl/fl mice, 28 days after first i.p. injection of tamoxifen or vehicle (Fig. 3H and 3I).

The results were similar in female SSfl/fl mice and cKD mice with i.p. injection of vehicle or tamoxifen (Fig. 4A–4I).

Fig. 4.

Fig. 4.

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Knocking down SS-lncRNA in the Mgrprd-expressed DRG neurons selectively produced mechanical hypersensitivity in naive female mice. (A) Level of SS-lncRNA in bilateral l3/4 DRGs from SSfl/fl mice and cKD mice 28 days after the first injection of tamoxifen or vehicle (Veh). n = 9 mice, **P < 0.01, by two-way ANOVA with post hoc Tukey test. (B-E) Paw withdrawal frequency (PWF) to 0.07 g (B) and 0.4 g (C) von Frey filaments and paw withdrawal latency (PWL) to heat (D) and cold (E) stimuli on the left side at the different days as indicated in SSfl.fl mice and cKD mice with an intraperitoneal injection of tamoxifen (Tam) or vehicle daily for 7 consecutive days. n = 9 mice, **P < 0.01 versus the SSfl/fl group at the corresponding time points by three-way ANOVA with repeated measures followed by post hoc Tukey test. (F and G) Spontaneous ongoing pain as assessed by the CPP paradigm 21 days after the first tamoxifen or vehicle (Veh) injection in SSfl.fl mice and cKD mice. Pre: preconditioning. Post: post-conditioning. n = 9 mice/group. **P < 0.01, by two-way ort three-way ANOVA with post hoc Tukey test. (H and I) Levels of p-ERK1/2, ERK1/2 and GFAP in the L3/4 dorsal horn from SSfl/fl mice and cKD mice on day 28 after first intraperitoneal injection of tamoxifen or vehicle daily for 7 consecutive days. n = 3 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test.

Overall, these findings indicate that SS-lncRNA knockdown in the Mrgprd-expressed DRG neurons induces mechanical and spontaneous hypersensitivity and dorsal horn central sensitization.

Downregulated SS-lncRNA in the Mrgprd-expressed DRG neurons participates in the SNL-induced DRG KCNN1 reduction

SS-lncRNA bound to and activated the Kcnn1 gene promoter in the cultured DRG neurons and CAD cells, respectively (Wang et al., 2023a). We next investigated whether SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons was involved in the SNL-induced KCNN1 reduction in these DRG neurons. In line with the previous reports (Wang et al., 2023a), SNL reduced the levels of Kcnn1 mRNA and KCNN1 protein in the ipsilateral L4 DRG of both male and female tamoxifen-pretreated SS-KI mice on day 28 post-SNL (Fig. 5A and 5B). These reductions were completely reversed in both male and female tamoxifen-pretreated SNL cKI mice (Fig. 5A and 5B). Unexpectedly, the amounts of Kcnn1 mRNA and KCNN1 protein were unchanged in the ipsilateral L4 DRG of both male and female tamoxifen-pretreated cKI mice on day 28 post-sham surgery (Fig. 5A and 5B). Consistently, the levels of Kcnn1 mRNA and KCNN1 protein were significantly reduced in bilateral L3/4 DRGs of both male and female cKD mice on day 28 following the first injection of tamoxifen, as compared to those in the tamoxifen-pretreated SS-KD mice or the vehicle-treated cKD mice (Fig. 5C and 5D). These findings strongly suggest that downregulated SS-lncRNA participates in nerve injury-induced KCNN1 reduction in small non-peptidergic DRG neurons.

Fig. 5.

Fig. 5.

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Rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons reversed the SNL-induced KCNN1 decrease in injured DRG and alleviated the SNL-induced dorsal horn neuronal and astrocyte hyperactivation in male and female mice. (A and B) Levels of Kcnn1 mRNA (A) and KCNN1 protein (B) in the ipsilateral L4 DRG on day 28 after SNL or sham surgery in SS-KI mice and cKI mice with an intraperitoneal injection of tamoxifen daily for 7 consecutive days before surgery. N = 12 mice/group. **P < 0.01, by two-way ANOVA with post hoc Tukey test. (C and D) Levels of Kcnn1 mRNA (C) and KCNN1 protein (D) in the left L3/4 DRGs on day 28 from SSfl/fl mice and cKD mice 28 days after first injection of tamoxifen or vehicle (Veh). N = 12 mice/group, **P < 0.01, by two-way ANOVA with post hoc Tukey test.

SS-lncRNA downregulation reduces the recruitment of KDM6B to Kcnn1 promoter in the Mrgprd-expressed DRG neurons

Finally, we examined how downregulated SS-lncRNA participated in nerve injury-induced KCNN1 reduction in small non-peptidergic DRG neurons. KDM6B is a histone demethylase that activates gene expression by removing repressive histone H3 lysine 27 trimethylation (H3K27me3) marks from chromatin (Agger et al., 2007, De Santa et al., 2007, Hong et al., 2007). Our previous RNA pull-down and mass spectrometry assays revealed the potential binding of SS-lncRNA to KDM6B in cultured DRG neurons (Wang et al., 2023a). This binding was further verified by the RNA pull-down assay with the use of a SS-lncRNA-specific antisense RNA probe in cultured DRG neurons (Fig. 6A). Moreover, a SS-lncRNA fragment was immunoprecipitated by anti-KDM6B antibody, but not by normal purified IgG, in the ipsilateral L4 DRG of sham mice (Fig. 6B). SNL markedly decreased this immunoprecipitating activity by 74% of the value of sham group in the ipsilateral L4 DRG on day 7 post-surgery (Fig. 6B). The decrease in the binding ability of SS-lincRNA to KDM6B might be attributed to the SNL-induced DRG SS-lncRNA downregulation in injured DRG, as the levels of KDM6B mRNA and protein were increased in the ipsilateral rat L4/5 DRGs from days 3 to10 post-SNL reported previously (Li et al., 2021).

Fig. 6.

Fig. 6.

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Downregulated SS-lncRNA in the Mrgprd-expressed DRG neurons participated in the SNL-induced decrease in the binding of KDM6B to Kcnn1 promoter and increase in the occupation of H3K27me3 in Kcnn1 promoter in this subtype of neurons. (A) KDM6B was pulled down by SS-lncRNA antisense (AS) probe, but not sense (SE) probe, in cultured DRG neurons. Input: extracted total protein. n = 3 biological repeats. (B) SS-lncRNA fragment immunoprecipitated by rabbit anti–KDM6B antibody in the ipsilateral L4 DRG on day 7 after SNL or sham surgery. M: ladder marker. Input: total purified RNA. IgG: purified rabbit IgG. n = 30 mice (3 repeats)/group. **P < 0.01, by two-tailed unpaired Student’s test. (C and D) Kcnn1 promoter fragment immunoprecipitated by rabbit anti-KDM6B antibody (C) or rabbit anti-H3K27me3 antibody (D) in the ipsilateral L4 DRG on day 7 after SNL or sham surgery in SS-KI or cKI mice with an intraperitoneal injection of tamoxifen daily for 7 consecutive days before surgery. Input: total purified DNA. M: ladder marker. n = 30 mice (3 repeats)/group. ** P < 0.01, by two-way ANOVA with post hoc Tukey test. (E) Kcnn1 promoter fragment immunoprecipitated by rabbit anti-KDM6B antibody or rabbit anti-H3K27me3 antibody in the ipsilateral L4 DRG on day 10 after the first injection of tamoxifen in SSfl/fl or cKD mice. Input: total purified DNA. M: ladder marker. n = 30 mice (3 repeats)/group. **P < 0.01, by two-tailed unpaired Student’s t-test.

We further performed the ChIP assay and showed that KDM6B interacted with a fragment of the Kcnn1 gene promoter, as evidenced by the amplification of this fragment from the complexes immunoprecipitated with anti-KDM6B antibody in nuclear fraction from the ipsilateral L4 DRG of the tamoxifen-pretreated SS-KI mice on day 7 post-sham surgery (Fig. 6C). This interacting activity was reduced by 42% on day 7 post-SNL in the tamoxifen-pretreated SS-KI mice (Fig. 6C). However, this reduction was not seen in the tamoxifen-pretreated SNL cKI mice (Fig. 6C), suggesting that rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons can block the SNL-induced reduction in the binding activity between KDM6B and Kcnn1 promoter in injured DRG. Conversely, the level of H3K27me3 occupied in the Kcnn1 promoter region was increased by 1.81-fold in the nuclear fraction from the ipsilateral L4 DRG of the tamoxifen-pretreated SS-KI mice on day 7 post-SNL, as compared to that on day 7 post-sham surgery (Fig. 6D). As predicted, the tamoxifen-pretreated SNL cKI mice did not display this increase (Fig. 6D). In addition, the binding activity between KDM6B and Kcnn1 promoter was reduced by 40% and the level of H3K27me3 occupied in Kcnn1 promoter was increased by 1.84-fold in the nuclear fraction of L3/4 DRGs from the tamoxifen-pretreated cKD mice as compared to the corresponding tamoxifen-pretreated SSfl/fl mice (Fig. 6E). Our data indicate that SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons may cause a reduction of Kcnn1 transcriptional activity likely through reducing the recruitment of KDM6B to Kcnn1 promoter and subsequent elevation of repressive H3K27me3 marks in Kcnn1 promoter in these neurons.

Discussion

Neuropathic pain in clinics is characterized by spontaneous ongoing pain, mechanical hypersensitivity, heat hyperalgesia, and cold hyperalgesia. The mechanisms of how peripheral nerve injury causes these symptoms remain elusive, although neuropathic pain has been intensively investigated for several decades. In this study, we reported that rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons mitigated the SNL-induced mechanical hypersensitivity, without affecting the SNL-induced heat and cold nociceptive hypersensitivities. Moreover, knocking down SS-lncRNA expression in the Mrgprd-expressed DRG neurons caused the augmented response to mechanical, but not heat and cold, stimuli in the absence of peripheral nerve injury. Given that Mrgprd-expressed DRG neurons constitute most small unmyelinated non-peptidergic DRG neurons (Dong et al., 2001, Zylka et al., 2003, Zylka et al., 2005), our findings suggest that downregulated SS-lncRNA in small non-peptidergic DRG neurons is required specifically for nerve injury-induced mechanical hypersensitivity.

Distinct subpopulations of small unmyelinated DRG neurons specifically and selectively mediate behavioral responses to different stimuli modalities. The previous work showed that ablation of Mrgprd-expressed small unmyelinated DRG neurons led to a selective reduction of basal behavioral responses to mechanical (not heat and cold) stimuli and impaired mechanical hypersensitivity (but not heat and cold hypersensitivities) after complete Freund’s adjuvant-induced inflammation (Cavanaugh et al., 2009). Thus, small unmyelinated non-peptidergic DRG neurons likely are required for full-expression of basal mechanical responses and inflammation-induced mechanical hypersensitivity, since Mrgprd is expressed selectively in the majority of these DRG neurons (Dong et al., 2001, Zylka et al., 2003, Zylka et al., 2005). Our work further demonstrated that rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons of SNL male and female mice mitigated the SNL-induced mechanical (but not heat and cold) hypersensitivity and that selectively knocking down SS-lncRNA in the Mrgprd-expressed DRG neurons of naïve male and female mice produced the augmented response to mechanical (but not heat and cold) stimuli. It is likely that SS-lncRNA expressed in small unmyelinated non-peptidergic DRG neurons mediates the role of this subtype of DRG neurons in selective response to mechanical stimuli under normal and neuropathic pain conditions.

We further demonstrated that downregulated SS-lncRNA in the Mrgprd-expressed DRG neurons was selectively required for the SNL-induced mechanical hypersensitivity likely through its participation in the SNL-induced KCNN1 reduction in this subtype of DRG neurons. DRG KCNN1 is a key player in neuropathic pain genesis. KCNN1 is expressed predominantly in small DRG neurons (Mongan et al., 2005). KCNN1 expression was reduced in the DRG avulsed from neuropathic pain patients (Boettger et al., 2002). Consistently, SNL time-dependently downregulated the expression of Kcnn1 mRNA and KCNN1 protein in injured mouse DRG neurons (Wang et al., 2023a). SNL also reduced the apamin-sensitive Kv currents in injured rat DRG neurons (Sarantopoulos et al., 2007). Behavioral observations showed that rescuing KCNN1 reduction in injured DRG alleviated the SNL-induced nociceptive hypersensitivity and that knocking down KCNN1 in naïve DRG produced enhanced responses to noxious stimuli (Wang et al., 2023a). These findings strongly suggest a critical role of DRG KCNN1 reduction in neuropathic pain. Our previous work showed the binding of SS-lncRNA to the Kcnn1 promoter in DRG neurons (Wang et al., 2023a). This binding activity was reduced due to SS-lncRNA downregulation in injured DRG after SNL (Wang et al., 2023a). The present study revealed that SNL decreased the binding activity between SS-lncRNA and KDM6B or between KDM6B and Kcnn1 promoter in injured DRG. The histone demethylase KDM6B functions as a gene transcriptional activator by removing repressive H3K27me3 in gene promoter (Agger et al., 2007, De Santa et al., 2007, Hong et al., 2007). As expected, the level of H3K27me3 occupied in the Kcnn1 promoter was reduced in the ipsilateral L4 DRG on day 7 post-SNL. More importantly, rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons revered a decrease in binding activity between KDM6B and Kcnn1 promoter and blocked the SNL-induced increase in the occupying activity of H3K27me3 in Kcnn1 promoter. Mimicking the SNL-induced SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons reduced the binding of KDM6B to Kcnn1 promoter and increased the occupying activity of H3K27me3 in the Kcnn1 promoter in this subtype of DRG neurons from naïve mice. These findings suggest that SS-lncRNA downregulation results in the reductions in its binding to both KDM6B and Kcnn1 promoter, consequent recruitment of less KDM6B to Kcnn1 promoter, an increase of repressive H3K27me3 level occupied in Kcnn1 promoter and final silence of Kcnn1 transcription in injured small unmyelinated non-peptidergic DRG neurons. These signaling cascades may be responsible for the selective role of this subtype of DRG neurons in nerve injury-induced mechanical hypersensitivity.

In conclusion, this study highlighted the significance of SS-lncRNA expressed in small unmyelinated non-peptidergic DRG neurons in selective responses to mechanical stimuli under normal and neuropathic pain conditions and provided novel insights into the molecular mechanisms underlying nerve injury-induced mechanical hypersensitivity. In addition, the present study demonstrated the role of downregulated SS-lncRNA expressed in small unmyelinated non-peptidergic DRG neurons in nerve injury-induced spontaneous ongoing pain and dorsal horn neuronal and astrocyte hyperactivation. It should be noted that SS-lncRNA was also detected in about 15% of TRPV1-positive DRG neurons, 8% of tyrosine hydroxylase-labeled DRG neurons and 2% of neurofilament-200 labeled DRG neurons (Wang et al., 2023a). Therefore, DRG downregulated SS-lncRNA also participates in responses of DRG neurons to heat and cold stimuli in naïve mice or after peripheral nerve injury evidenced in our previous study (Wang et al., 2023a). Further identification of SS-lncRNA as a potential therapeutic target of neuropathic pain may open a new avenue for the management of this disorder.

Acknowledgments

We thank Biocytogen (Worcester, MA) for generating SS-lncRNAfl/fl mice and Rosa26SS-lncRNA knock-in mice.

Funding

This work was supported by NIH grants (R01NS111553 and RFNS113881) to Y.X.T.

Footnotes

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Competing financial interests

The authors declare no competing financial interests.

Availability of data and materials

The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

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Data Availability Statement

The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

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