Transcription Repressor Hes1 Contributes to Neuropathic Pain Development by Modifying CDK9/RNAPII-Dependent Spinal mGluR5 Transcription

Diverse transcriptional controls in the dorsal horn have been observed in pain hypersensitivity. However, the understanding of the exact causes and mechanisms of neuropathic pain development is still fragmentary. Here, the results demonstrated nerve injury decreased the expression of spinal hairy and enhancer of split 1 (Hes1), a transcriptional repressor, and enhanced metabotropic glutamate receptor subtype 5 (mGluR5) transcription/expression, which was accompanied with behavioral allodynia. Moreover, nerve injury decreased Hes1 levels and reciprocally increased cyclin dependent kinase-9 (CDK9) levels and recruited CDK9 to phosphorylate RNA polymerase II (RNAPII) in the promoter fragments of mGluR5, thereby enhancing mGluR5 transcription/expression in the dorsal horn. These effects were also induced by intrathecally administering naïve rats with Hes1 small interfering RNA (siRNA). Conversely, Hes1 overexpression using intrathecal lentiviral vectors in nerve injury rats produced reversal of pain behavior and reversed protein expressions, phosphorylation, and coupling to the promoter segments in the dorsal horn. Collectively, the results in this study indicated nerve injury diminishes spinal Hes1-dependent suppression of CDK9-dependent RNAPII phosphorylation on the mGluR5 promoter that possibly enhances mGluR5 transcription/expression for neuropathic pain development.


Introduction
In contrast to most mammalian basic helix-loop-helix (bHLH) transcription factors site-specifically activating gene expression [1], hairy and enhancer of split (Hes) is a unique bHLH [2] that represses genes transcription [3,4] to regulate neural morphology and functions [5]. Among the Hes1-7 families, Hes1 is recognized as a regulator for the transcription of synaptic proteins and thereby crucially impacts synaptic transmission and plasticity development [6]. Down-regulated Hes1 expression increases synapses integration in rats' dentate gyrus accompanied with progression of memory-associated plasticity [7], and conversely, reagent-induced Hes1 expression in the rat hippocampus results in learning and memory deficits [8]. Nevertheless, though mounting evidence has shown that spinal pain-associated plasticity relies on molecular processes mediating learning and memory in brain regions [9,10], the potential involvement of Hes1-suppressed genes transcription in the spinal plasticity underlying pain hypersensitivity has yet to be established.
Cyclin dependent kinase-9 (CDK9), an elongation factor [11], phosphorylates RNA polymerase II (RNAPII) to initiate transcription elongation of genes [12]. CDK/RNAPII-dependent elongation persuades gene transcription, mediating the development of plasticity, as evidenced by the depletion of specific CDK isoforms, which induces heterochromatin enrichment that results in learning defects in Drosophila [13], and the stalling of RNAPII at specific gene promoters of tegmentum neurons incites plasticity underlying morphine rewards in rats [14]. Notably, in a subset of genes, Hes1 suppresses transcription elongation by hindering the recruitment of CDK9 and the following RNAPII phosphorylation [15]. One of our publications recently linked CDK9-dependent RNAPII phosphorylation to the transcription of pain plasticity-associated genes [16]; Hes1 may contribute to pain by impacting the CDK9/RNAPII-initiated transcription of genes essential for spinal plasticity.
In addition to widely mediating physiological neurotransmission [17], spinal glutamatergic synapses also crucially underlie the pathophysiological machinery of pain hypersensitivity [18]. By focusing on the dorsal horn, the first relaying point for pain neurotransmission, our laboratory has linked metabotropic glutamate receptor subtype 5 (mGluR5), a member of the glutamatergic receptor family, to plasticity underlying neuropathic pain development not only because experimental neuropathic injury upregulated mGluR5 expression in the dorsal horn [19], but also because enhanced mGluR5 transcription in rodent dorsal horn neurons provoked allodynia, a pain sensation caused by innocuous stimulation that is a clinical sign of neuropathic pain [20]. Since Hes1 represses transcription of glutamatergic receptors and thereby reduces synaptic efficacy in cortical neurons [6], it is hypothesized that neuropathic injury-induced pain hypersensitivity involves impedance of spinal Hes1-suppressed mGluR5 transcription by prompting interactions between CDK9/RNAPII and mGluR5 promoters that enhance mGluR5 transcription. Results in this study provided a molecular, histological, behavioral, and genetic basis supporting the above hypothesis, and additionally revealed that reciprocal Hes1-CDK9 expression regulates RNAPII phosphorylation on promoter loci that impact spinal mGluR5 transcription, mediating neuropathic pain.

SNL Decreased Spinal Hes1 and Provoked Behavioral Allodynia
To characterize the role of spinal Hes1 in neuropathic pain, the expression profile of Hes1 in the dorsal horn following spinal nerve ligation (SNL), a procedure mimicking neuropathic injury [21,22], was initially examined. In dorsal horn samples ipsilateral to the injury, Western blot analyses revealed SNL gradually reduced the Hes1 level, which began to fall at day 3, plateaued at day 7, and was maintained at a relatively constant level until days 14 and 21 after operation (Figure 1a; 0.46 ± 0.02, 0.24 ± 0.03, 0.30 ± 0.02, and 0.31 ± 0.02). Moreover, withdrawal threshold data collected by testing animals' responses to von Frey hair demonstrated SNL induced tactile allodynia as evidenced by decrements in the mechanical threshold of rats' ipsilateral hind paw, with a time course matching the spinal Hes1 diminishing (Figure 1b; 2.79 ± 0.58, 0.93 ± 0.13, 1.25 ± 0.12, and 1.24 ± 0.24 g). To further strengthen the role of Hes1 in the spinal machinery mediating the development of neuropathic pain, spinal slice dissected at day 7 after operation (a time-point at which SNL animals displayed maximal behavioral allodynia) were labeled with Hes1-specific antibody. Images in (Figure 1c,d) illustrate SNL decreased Hes1-positive neurons in the ipsilateral, but not contralateral, dorsal horn compared with the sham operation. These results indicate neuropathic injury provokes behavioral allodynia accompanied with decreased Hes1 in the ipsilateral dorsal horn.

Knockdown of Spinal Hes1 Expression Provokes Tactile Allodynia
Based on above observations, the involvement of spinal Hes1 in allodynia was further tested by establishing a rat model in which spinal Hes1 expression was focally knocked down by daily intrathecal administration of antisense small interfering RNA (siRNA) targeted to the Hes1 mRNA. The siRNA protocol efficiently trimmed down spinal Hes1 expression without provoking any observable motor deficits in naïve animals as evidenced by Hes1 siRNA (1, 3, and 5 µg; 10 µL) dose-dependently reducing Hes1 levels in dorsal horn samples (Figure 2a; 0.56 ± 0.04, 0.30 ± 0.03, and 0.13 ± 0.02), but did not exhibit effects on the performance time measured by the rota-rod test ( Figure 2b). Intriguingly, knockdown of spinal Hes1 expression provoked allodynia in naïve animals as Hes1 siRNA (5 and 10 µL) decreased the mechanical threshold at days 2, 3, and 4 after treatment (Figure 2c; 5.19 ± 0.75, 2.35 ± 0.43, and 2.22 ± 0.42 g). These data provide a genetic basis supporting the notion that down-regulated Hes1 expression is an essential factor for the spinal machinery underlying the development of tactile allodynia.

Spinal Hes1 Overexpression Ameliorates SNL-Provoked Allodynia
After identifying that the role of reduced Hes1 expression in spinal mechanisms mediates the development of neuropathic allodynia, whether an enhancement of spinal Hes1 levels is sufficient to ameliorate behavioral allodynia caused by neuropathic injury was tested next. For this purpose, we intrathecally injected animals with lentiviral vectors encoding the rat Hes1 protein (Hes1 vector) or encoding GFP alone (control vector) on day 7 after SNL, and performed behavior and protein analyses on day 14 after vector injection ( Figure 3a). Western blot analyses confirmed the efficacy and specificity of our protocol as gene transfer enhanced Hes1 expression in the dorsal horn of naïve rats (Figure 3b; 0.91 ± 0.06). Moreover, administration of Hes1 vector to SNL rats ameliorated the established allodynia and reversed the associated Hes1 down-regulation in the dorsal horn (Figure 3c, d; 7.75 ± 0.57 and 0.52 ± 0.04 g), implying focal Hes1 overexpression in the spinal cord is sufficient to ameliorate neuropathic injury-induced behavioral allodynia.

Spinal Hes1 Overexpression Ameliorates SNL-Provoked Allodynia
After identifying that the role of reduced Hes1 expression in spinal mechanisms mediates the development of neuropathic allodynia, whether an enhancement of spinal Hes1 levels is sufficient to ameliorate behavioral allodynia caused by neuropathic injury was tested next. For this purpose, we intrathecally injected animals with lentiviral vectors encoding the rat Hes1 protein (Hes1 vector) or encoding GFP alone (control vector) on day 7 after SNL, and performed behavior and protein analyses on day 14 after vector injection ( Figure 3a). Western blot analyses confirmed the efficacy and specificity of our protocol as gene transfer enhanced Hes1 expression in the dorsal horn of naïve rats (Figure 3b; 0.91 ± 0.06). Moreover, administration of Hes1 vector to SNL rats ameliorated the established allodynia and reversed the associated Hes1 down-regulation in the dorsal horn (Figure 3c, d; 7.75 ± 0.57 and 0.52 ± 0.04 g), implying focal Hes1 overexpression in the spinal cord is sufficient to ameliorate neuropathic injury-induced behavioral allodynia.

Discussion
The data in the current study demonstrated the involvement of signal-induced recruitment of CDK9 and subsequent RNAPII phosphorylation, a well-recognized mechanism mediating gene elongation to initiate the transcription cycle, as a novel pathogenic pathway for the development of neuropathic pain by modifying glutamatergic mGluR5 transcription and expression in the dorsal horn. In addition, by demonstrating that SNL downregulated spinal Hes1 expression that diminished its suppression of the recruitment of CDK9 and RNAPII as well as the subsequent phosphorylation of RNAPII on the mGluR5 promoter segments, these data for the first time demonstrated that Hes1, a transcription repressor, contributes to neuropathic pain development by impacting the spinal CDK9/pRNAPII-dependent mGluR5 transcription. Results in the current study defined Hes1 as a transcription repressor, whose impacts on synaptic plasticity are essential for the neural processing of pain by hampering signal-induced recruitment of CDK9/RNAPII-dependent gene transcription. These results provide a novel insight into the spinal mechanism leading to pain hypersensitivity, and thereby, raise a possible strategy to develop a medical treatment for neuropathic pain relief, which remains a challenge in clinical scenarios, by targeting the pre-transcription initiation steps of gene cycles.
Hes is a family of transcription factors that regulate neuronal function and morphology [5]. Studies have linked Hes1, a member of the Hes1-7 family, to neural plasticity underlying learning and memory [6] and to the pathogenesis of Alzheimer's disease [24]. Analogous to studies that reveal that pain-associated plasticity that occurs in spinal neurons shares similar characteristics with the plasticity underlying learning and/or memory in brain regions [10], results in the current study provide evidence supporting the role of Hes1 in the spinal plasticity underlying pain hypersensitivity following neuropathic injury. Data in this study are consistent with a study that investigated Hes1 mRNA expression following diabetic neuropathy [25]. Nevertheless, in addition to Hes1, the potential contribution of other Hes families to pain-associated plasticity in the spinal neuraxis needs further studies to be elucidated because a genome-wide mapping analysis of brain tissue dissected from post-mortem Huntington's disease patients revealed that the hyper-methylation of Hes4 promoter sequences was strikingly correlated with the measure of striatal degeneration and age-of-onset [26].
At the start of the transcription cycle, the positive transcriptional elongation factor (p-TEFb), which is comprised of cyclin T1 and CDK9, is recruited to and hence phosphorylates RNAPII on the promoter sequence of specific genes to facilitate gene elongation [27]. Data in the current study provide support for the role of the CDK9/pRNAPII cascade as a novel pathogenic pathway for the progression of spinal plasticity underlying neuropathic pain development by up-regulating transcription of mGluR5 in the dorsal horn. In addition to linking CDK9/pRNAPII-dependent mGluR5 transcription to the spinal plasticity mediating allodynia caused by neuropathic insults, the results in this study demonstrated that SNL-induced spinal CDK9/pRNAPII expression and coupling to the mGluR5 promoter, as well as mGluR5 transcription in the dorsal horn, were all reversed by focal overexpression of spinal Hes1. Together with our observation that neuropathic injury down-regulated spinal Hes1 expression, these data suggest that by hampering CDK9/RNAPII-regulated mGluR5 transcription, Hes1 acts as a suppressor to regulate the spinal plasticity underlying neuropathic pain development. This proposal is further supported by the fact that focal knockdown of Hes1 expression in naïve animals provoked CDK9 expression, RNAPII phosphorylation, coupling of CDK9 and pRNAPII to the mGluR5 promoter, and mGluR5 transcription in the dorsal horn, which resembles the sequelae caused by experimental neuropathic injury. Results in the current study are consistent with studies on immune cells that demonstrated that Hes1 selectively inhibits signal-induced recruitment of CDK9 that prevents RNAPII phosphorylation to impede transcription elongation of genes encoding chemokine, crucial for neutrophils' chemotaxis [15]. Moreover, as such a mechanism widely works in various systems/organs, and pain-associated spinal plasticity shares molecular mechanisms with forms of plasticity occurring in brain regions, the potential involvement of Hes1-diminished CDK9/RNAPII-dependent transcription elongation in plasticity underlying brain functions, such as learning, memory, anxiety, and stress, is a really interesting issue that needs to be studied.
It is noteworthy, in this study, that focal knockdown of Hes1 expression resulted in an enhanced abundance of CDK9, and vice versa, i.e., a genetically reduced CDK9 level is accompanied with increased Hes1 expression in the dorsal horn of naïve rats, results that revealed a reciprocal change between the amount of spinal Hes1 and CDK9. Such a reciprocal interaction was also evidenced by observations that in the dorsal horn, SNL decreased Hes1 associated with enhancement of CDK9 expression; and lentivirus-induced Hes1 overexpression conversely reversed the SNL-reduced Hes1 expression and led to decreased CDK9 levels. Results in this study are somewhat different from that obtained from immune cells, in which knockout of Hes1 expression in macrophages did not globally alter cellular CDK9 levels [15]. Although the detailed mechanisms involved remain unclear, several potential causes might underlie this discrepancy. First, the samples explored were mainly in the dorsal horn, whereas their study investigated bone marrow-derived macrophages in culture; whether Hes1 exhibits diverse interactions with CDK9 in different cell types is unclear. Second, while focally knocked down/overexpressed spinal Hes1 expression was restricted to specific lumbar segments in the current study, their experiments generated knockout mice in which Hes1 expression was systemically deficient. The possibility that in the present in vivo preparations, Hes1 synthesized from organs/systems other than the dorsal horn could alter spinal Hes1-CDK9 interactions cannot be excluded, for such a systemic effect does not occur in cultures of homogenous cells that have no unnecessary impacts coming from other organs/systems. Moreover, in contrast to their study, which assayed Hes1-repressed chemokine transcription by treating macrophages with lipopolysaccharides, the current study investigated the impact of neuropathic insults on the Hes1-suppressed receptor transcription. Whether Hes1-CDK9 interactions vary when encountering different challenges, or alternatively, a reciprocal Hes1-CDK9 relationship occurs specifically in the nervous system when facing neural damage needs further studies to be elucidated because peripheral nerve injury in rats was demonstrated to downregulate ZAS3, a zinc finger protein, accompanied with reciprocal transcription of NF-kappa B-dependent genes in the dorsal root ganglion [28]. In addition, emerging evidence has demonstrated that spinal mGluR5 is a key mediator of neuroplasticity underlying pain hypersensitivity [29,30], and previous laboratory has reported that experimental neuropathic injury provoked behavioral allodynia along with enhanced dorsal horn mGluR5 expression, and conversely, administering neuropathic animals with an mGluR5-specific antagonist ameliorated behavioral allodynia [19,31]. The obtained results demonstrated that Hes1 contributes to pain-associated spinal plasticity through enhancement of mGluR5 transcription in the dorsal horn. However, a study show that restoring endocannabinoid signaling allows mGluR5 activation to increase infralimbic output and hence inhibit pain behaviors [32]. Nevertheless, the clear-cut mechanism in the spinal cord underlying this discrepancy needs further investigation.

Neuropathic Pain Rat Model
A total of 429 male Sprague-Dawley rats weighing 200 to 250 g (age: 7-8 weeks) were used in the study; and rats were subjected to spinal nerve ligation (SNL) and sham operation, respectively. Animal procedures in this study were reviewed and approved by the Institutional Review Board of Taipei Medical University, Taipei, Taiwan (LAC-2017-0385) and Biosafety Committee, Mackay Medical College, New Taipei, Taiwan (B1060014). All animal experimental procedures were performed under the guidelines of the International Association for the Study of Pain [33]. In each group, 7 rats were used for the behavioral tests; 6 rats were used for Western blotting; 5 rats were used for the quantitative reverse-transcription PCR (RT-PCR), chromatin immunoprecipitation-qPCR (ChIP), and immunohistochemistry. The investigators in this study were blinded to the treatment groups. Five rats that showed neurological deficits after implantation of an intrathecal catheter were excluded from the statistical analysis.
Spinal nerve ligation (SNL) was used as the neuropathic pain model in rats [22,34,35]. Animals were anesthetized with isoflurane (induction, 5%; maintenance, 2% in room air), and the left L5-L6 spinal nerves were isolated and ligated tightly with 6-0 silk suture. Age-matched sham-treated control rats were used as the control group, the surgical procedures were identical to the nerve-ligated animals, except that the nerves were not ligated.

Intrathecal Catheter Implantation
To implant an intrathecal catheter, rats were anesthetized with isoflurane (induction, 5%; maintenance, 2% in room air) and inserted PE-10 tubing the dorsal aspect of lumbar enlargement of the rats' spinal cord, as described previously [34]. Then, the outer part of the catheter was plugged and immobilized into the skin on closure of the incision made. After 3 days of recovery, rats displaying signs of neurological dysfunction were discarded and excluded from further experiments.

Behavioral Studies
To quantify tactile allodynia, rats were placed in individual plastic boxes on a mesh floor and habituated for 1 h. A series of calibrated von Frey filaments (Stoelting, Wood Dale, IL) were then applied to the plantar surface of the hind paw of animals with sufficient force to bend the filaments for 5 s to measure the paw-withdrawal threshold (up-down method) [21]. Motor coordination was evaluated in rats by the rota-rod apparatus (Panlab Harvard Apparatus, Barcelona, Spain). Rats were trained to three training trials at 3 to 4 h intervals on 2 separate days. The rod was set to accelerate from 4 to 30 rpm over a 180 s period. Three measurements were obtained at intervals of 5 min and were averaged for each test.

Western Blot Analysis
Rats' dorsal horn (L4-5) samples were immediately removed, dissected, and homogenized in 25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS with a complete protease inhibitor mixture (Roche). Samples were then put on ice for 1 h with shaking. The lysates were centrifuged at 14,000 rpm for 20 min at 4 • C. The supernatant was carefully collected, and the protein concentration was measured using a BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Identical amounts of samples were loaded and separated by SDS-PAGE and electrophoretically transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk or BSA in TBS containing 0.1% Tween-20 for 1 h and then incubated with one of the following primary antibodies at 4 • C for 1 h. The primary antibodies were as follows: Anti-Hes1 (rabbit, 1:1000, Genetex, Irvine, CA, USA), anti-mGluR5 (rabbit, 1:1000, Millipore, Billerica, Massachusetts), anti-CDK9 (rabbit, 1:1000, Cell Signaling, Beverly, MA, USA), anti-pRNAPII (rabbit, 1:1000, Abcam, Cambridge, USA), and anti-GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then washed and incubated with peroxidase-conjugated goat anti-rabbit IgG (1:8000, Jackson ImmunoResearch, West Grove, PA, USA) or goat anti-mouse IgG (1:8000, Jackson ImmunoResearch) for 1 h at room temperature. The protein bands were visualized with an enhanced chemiluminescence detection kit (ECL Plus, Millipore) and then subjected to densitometric analysis using Science Lab 2003 software (Fuji, Tokyo, Japan). The protein band intensity was quantified by Images J.

Immunofluorescence Analysis
To test the specificity of the antibody used in the immunofluorescence, the molecular weight of the antibody was first checked using western blot analysis. In addition, the specificity of the antibody was further confirmed by the western blot analysis of samples treated with siRNA targeting Hes1 and CDK9 [16] and pRNAPII-selective inhibitor [16]. Rats were sacrificed and perfused intracardially with PBS followed by 4% paraformaldehyde in PBS (pH 7.4). The spinal cord samples were removed, post-fixed in the same fixative at 4 • C for 4 h, and cryoprotected in 30% sucrose in PBS overnight at 4 • C. The tissues were cut to a 30-µm thickness using a cryostat and were mounted on glass slides. The sections were washed and incubated with 5% BSA in PBS to block nonspecific binding. The sections were then incubated with rabbit anti-Hes1 (1:400, Genetex, Irvine, CA, USA) overnight (4 • C). Subsequently, the sections were rinsed in PBS and incubated with goat/donkey secondary antibodies conjugated to Alexa Fluor 488 (1:1500, Invitrogen) and Alexa Fluor 594 (1:1500, Invitrogen) were used (1 h, 37 • C). A Mix-n-Stain antibody labeling kit (Biotium, Hayward, CA, USA) were used to examine the interaction between Hes1 (1:400, Genetex, Irvine, CA, USA), CDK9 (rabbit, 1:1000, Cell Signaling, Beverly, MA, USA), and pRNAPII (rabbit, 1:400, Abcam, Cambridge, USA). Finally, these sections were rinsed in PBS, mounted on slides, dried, and cover-slipped. The sections were detected by a camera-coupled device (X-plorer; Diagnostic Instruments, Inc., USA) through a fluorescent microscope (LEICA DM2500, Germany).

Chromatin Immunoprecipitation-qPCR
Chromatin immunoprecipitation (ChIP) assays were performed with a commercial ChIP Kit (Millipore, Billerica, Massachusetts). Fresh dorsal horn samples were rapidly removed from anesthetized animals and cut into small pieces (1-2 mm 3 ) using razor blades. The samples were then incubated in fresh 1% paraformaldehyde in PBS buffer, and gently agitated (10 min) at room temperature to crosslink proteins to DNA. After being washed, the samples were incubated in lysis buffer on ice. Subsequently, the tissues were sheared by sonication to generate chromatin fragments with an average length of 200 to 1000 base pairs. In total, 1% of the sonicated chromatin was saved as an input control for qPCR. The chromatin was pulled down for 2 h at room temperature using the following antibodies: Rabbit anti-Hes1 (rabbit, 1:1000, Genetex, Irvine, CA, USA), anti-CDK9 (rabbit, 1:1000, Cell Signaling, Beverly, MA, USA), pRNAPII (rabbit, 1:1000, Abcam, Cambridge, USA), or an equivalent amount of control IgG. Using protein G magnetic beads at 4 • C for overnight, the protein-DNA immunocomplexes were precipitated. After the beads were washed, they were re-suspended in ChIP elution buffer, incubated with proteinase K at 62 • C for 2 h, and then incubated at 95 • C for 10 min to reverse the crosslinks between the protein and DNA. ChIP signals were quantified by quantitative PCR analysis with a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, USA). The specific primer pairs for the mGluR5 promoter region are described below.
Real-time PCR was performed on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). TaqMan Universal PCR Master Mix (2X) and TaqMan gene expression assay probes were GAPDH (Rn99999916_s1, Applied Biosystems, Carlsbad, CA, USA) and mGluR5 (Rn.PT.58.36061021, IDT, Coralville, IA, USA). Reactions (total volume, 20 uL) were comprised by incubation at 95 • C for 20 s, followed by 40 cycles of 1 s at 95 • C and 20 s at 60 • C, followed by a DNA melting curve for the determination of amplicon specificity. The expression level of the target mRNA was normalized to expression of GAPDH mRNA and analyzed by the standard 2 − CT method [36].
The Hes1-specific siRNA and the missense siRNA were administered intrathecally for 4 consecutive days in animal rats (daily for 4 days in naïve rats or daily from days 3 to 6 after SNL). For the treatment siRNA, rats were anesthetized with isoflurane (induction, 5%; maintenance, 2% in room air).

Statistical Analysis
All data are expressed as the mean ± SEM. Data in this study were analyzed using SigmaPlot 10.0 (Systat Software) or Prism 6.0 (GraphPad). Paired two-tailed Student's t-test was used to compare the means of groups. One-way ANOVA was used followed by Tukey's post-hoc test to compare more than two groups. Two-way ANOVA followed by Tukey's post-hoc test was used to assess changes in values for serial measurements over time. In all analyses, significance was set at p < 0.05.

Conclusions
By modifying glutamatergic mGluR5 transcription and expression in the dorsal horn, signal-induced recruitment of CDK9 and subsequent RNAPII phosphorylation contribute to a novel pathogenic pathway for the development of neuropathic pain. SNL downregulated spinal Hes1 expression and its suppression of the CDK9 and RNAPII recruitment and RNAPII phosphorylation on the mGluR5 promoter segments to mediate the development of neuropathic pain.

Conflicts of Interest:
The authors declare no competing to this study.

Abbreviations
Hes 1 hairy and enhancer of split 1 CDK9 Cyclin dependent kinase-9 RNAPII RNA polymerase II phosphorylation mGluR5 metabotropic glutamate receptor subtype 5 SNL Spinal nerve ligation ChIP-qPCR Chromatin immunoprecipitation-quantitative polymerase chain reaction siRNA small-interfering RNA