Upregulation of ERK phosphorylation in rat dorsal root ganglion neurons contributes to oxaliplatin-induced chronic neuropathic pain

Toyoaki Maruta; Takayuki Nemoto; Koutaro Hidaka; Tomohiro Koshida; Tetsuro Shirasaka; Toshihiko Yanagita; Ryu Takeya; Isao Tsuneyoshi

doi:10.1371/journal.pone.0225586

. 2019 Nov 25;14(11):e0225586. doi: 10.1371/journal.pone.0225586

Upregulation of ERK phosphorylation in rat dorsal root ganglion neurons contributes to oxaliplatin-induced chronic neuropathic pain

Toyoaki Maruta ^1,^*,^#, Takayuki Nemoto ^2,^#, Koutaro Hidaka ¹, Tomohiro Koshida ¹, Tetsuro Shirasaka ¹, Toshihiko Yanagita ³, Ryu Takeya ⁴, Isao Tsuneyoshi ¹

Editor: Ferenc Gallyas Jr⁵

PMCID: PMC6876879 PMID: 31765435

Abstract

Oxaliplatin is the first-line chemotherapy for metastatic colorectal cancer. Unlike other platinum anticancer agents, oxaliplatin does not result in significant renal impairment and ototoxicity. Oxaliplatin, however, has been associated with acute and chronic peripheral neuropathies. Despite the awareness of these side-effects, the underlying mechanisms are yet to be clearly established. Therefore, in this study, we aimed to understand the factors involved in the generation of chronic neuropathy elicited by oxaliplatin treatment. We established a rat model of oxaliplatin-induced neuropathic pain (4 mg kg^-1 intraperitoneally). The paw withdrawal thresholds were assessed at different time-points after the treatment, and a significant decrease was observed 3 and 4 weeks after oxaliplatin treatment as compared to the vehicle treatment (4.4 ± 1.0 vs. 16.0 ± 4.1 g; P < 0.05 and 4.4 ± 0.7 vs. 14.8 ± 3.1 g; P < 0.05, respectively). We further evaluated the role of different mitogen-activated protein kinases (MAPKs) pathways in the pathophysiology of neuropathic pain. Although the levels of total extracellular signal-regulated kinase (ERK) 1/2 in the dorsal root ganglia (DRG) were not different between oxaliplatin and vehicle treatment groups, phosphorylated ERK (p-ERK) 1/2 was up-regulated up to 4.5-fold in the oxaliplatin group. Administration of ERK inhibitor PD98059 (6 μg day^-1 intrathecally) inhibited oxaliplatin-induced ERK phosphorylation and neuropathic pain. Therefore, upregulation of p-ERK by oxaliplatin in rat DRG and inhibition of mechanical allodynia by an ERK inhibitor in the present study may provide a better understanding of intracellular molecular alterations associated with oxaliplatin-induced neuropathic pain and help in the development of potential therapeutics.

Introduction

Oxaliplatin, a platinum-based drug, is used as the first-line chemotherapy for metastatic colorectal cancer. Unlike other platinum anticancer agents, oxaliplatin does not result in significant renal impairment and ototoxicity. However, oxaliplatin is associated with acute and chronic peripheral neuropathies [1, 2]. Oxaliplatin-induced acute neuropathy is characterized by acral paresthesia that is enhanced by exposure to cold. Furthermore, cumulative oxaliplatin dose can cause chronic neuropathy, which includes pain, paresthesia, hypoesthesia, dysesthesia, and changes in proprioception. Therefore, oxaliplatin-induced neuropathic pain is a major clinical side-effect that can influence the treatment as well as the quality of life.

Pain results from the activation of a subset of sensory neurons termed nociceptors. Under physiological conditions, activation of unmyelinated (C-fiber) and myelinated (Aδ-fiber) nociceptive afferent fibers indicates potential tissue damage, which is reflected in the high thresholds of nociceptors for mechanical, thermal, and chemical stimuli; these neurotransmissions are attributed to ion channels, neurotransmitters, and intracellular signaling [3, 4]. These conditions change dramatically in neuropathic pain states, including chemotherapy-induced peripheral neuropathy (CIPN). Understanding the changes that occur in neuropathic pain is vital to identify new therapeutic targets and develop novel analgesics [4]. Recently, it has been reported that oxaliplatin-induced acute paresthesia is induced by voltage-dependent sodium channel (Na_V1.6) dysfunction [5–7] and upregulation of transient receptor potential (TRP) channels, TRPM8 and TRPA1 [8–11], which are temperature-sensitive channels. However, the pathophysiology of oxaliplatin-induced neuropathic pain as a chronic neuropathy has not yet been clearly established.

Mitogen-activated protein kinases (MAPKs) signaling cascade is known to be involved in the regulation of cellular functions such as cell differentiation, proliferation, and apoptosis [12, 13]. MAPKs, such as extracellular signal-regulated kinase (ERK), p38 kinase, and c-jun N-terminal kinase (JNK), have been linked with the development of pain [12, 13]. Furthermore, it has recently been reported that the modulation of MAPKs activation is associated with oxaliplatin-induced apoptosis in cultured dorsal root ganglion (DRG) neurons [14, 15].

Therefore, the aim of the current study was to understand the factors involved in the generation of chronic neuropathy elicited by oxaliplatin treatment. We investigated whether MAPKs were modulated by oxaliplatin in the rat DRG and found that oxaliplatin treatment up-regulates ERK phosphorylation in rat DRG and induced chronic neuropathic pain. We also demonstrated that administration of an ERK inhibitor inhibits oxaliplatin-induced neuropathic pain. Thus, our study suggests a novel mechanism by which oxaliplatin treatment can influence MAPKs signaling and contribute to chronic neuropathy.

Materials and methods

Animals

Six-week-old male Sprague Dawley rats (Kudo, Japan) weighing approximately 200–250 g were used in the study. All rats were individually housed in a temperature- and humidity-controlled environment with a 12-hour light-dark cycle and were permitted free access to food and water. The study was conducted in strict accordance with the guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). The experiments were approved by the Experimental Animal Care and Use Committee of University of Miyazaki (Permit Number: 2015–528). All efforts were made to minimize the number of animals used and their suffering.

Pharmacological treatments

In the first series of experiments, we investigated the intracellular molecular alterations in DRG. Oxaliplatin (4 mg kg^-1 of body weight; Sigma-Aldrich, St. Louis, MO, USA) or vehicle (5% glucose) was injected intraperitoneally (i.p.) twice a week for 4 weeks [16]. Oxaliplatin was prepared in 5% glucose to a final concentration of 2 mg ml^-1. von Frey test was conducted before and 1 week after each oxaliplatin or vehicle treatment. On day 28, at the end of the last behavioral test, L4-L6 DRGs were dissected from each group and intracellular molecules including ERK were measured using western blot analysis.

In the second series of experiments, we investigated the effects of ERK inhibitor on oxaliplatin-induced neuropathy. Rats were anesthetized with an intraperitoneal injection of a combination anesthetic (0.375 mg kg^-1 of medetomidine, 2.0 mg kg^-1 of midazolam, and 2.5 mg kg^-1 of butorphanol). A PE-10 polyethylene catheter (Becton-Dickinson, Sparks, MD, USA) was inserted into the subarachnoid space through the atlanto-occipital membrane and pushed to the region of lumbar enlargement [17]. An osmotic pressure pump (ALZET model 2004, DURECT, Cupertino, CA, USA; total volume of the pump: 200 μl, drug infusion: 0.25 μl hour^-1 for 4 weeks) was connected to the catheter and placed subcutaneously on the back [18]. Immediately after surgery, the operating surgeon regularly observed animals until they were ambulatory. Furthermore, the animals' appearance, movement, and appetite were observed daily for one week after surgery. ERK inhibitor PD98059 (6 μg day^-1; Sigma-Aldrich, St. Louis, MO, USA) or vehicle [20% dimethyl sulfoxide (DMSO)] was injected intrathecally (i.t.) for 4 weeks with the osmotic pressure pump. PD98059 was dissolved in 20% DMSO to a final concentration of 1 μg μl^-1. One week after the pump placement, oxaliplatin (4 mg kg^-1) or vehicle (5% glucose) was injected i.p. twice a week for 3 weeks. The sham-operated (Sham) mice underwent a similar surgical procedure except for pump placement and drug treatments. von Frey test was conducted before pump placement, and before and 1 week after each oxaliplatin or vehicle treatment. On day 28 of pump placement, at the end of the last behavioral test, L4-L6 DRGs were dissected from each group and intracellular levels of various molecules were measured using western blot analysis.

von Frey test

Mechanical sensitivity was examined by testing the paw withdrawal threshold using the von Frey (VF) filaments (Stoelting, Wood Dale, IL, USA). Briefly, each rat was placed in a 20 cm × 20 cm suspended chamber on a metallic mesh floor. After an acclimation period of 30 minutes, a series of calibrated VF filaments were applied perpendicularly to the plantar surface of the right and left hind paws with sufficient force to bend the filament for 5 seconds. Brisk withdrawal or paw flinching was considered as a positive response. In the absence of a response, the filament of next greater force was applied. After a positive response, the filament of next lower force was applied. The tactile stimulus producing a 50% likelihood of withdrawal response was calculated using the up-down method [19]. von Frey test was conducted before an osmotic pressure pump placement, and before and 1 week after each intraperitoneal oxaliplatin or vehicle treatment for 4 weeks.

Western blot analysis

On day 28 of intraperitoneal treatment or pump placement, all rats were euthanized with sevoflurane exposure and the L4-L6 DRGs from each group were quickly dissected for further analysis. Briefly, the collected DRGs were mechanically homogenized in ice-cold lysis buffer composed of 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, and 20 mM Tris-HCl, pH 7.5, with added Protease and Phosphatase Inhibitor Cocktails (Roche Diagnostics, Mannheim, Germany) and centrifuged at 12000 rpm and 4°C for 10 minutes. The supernatant was collected and stored at -80°C until use. The total protein content was determined in each sample using the Bradford method-based protein assay kit, with bovine serum albumin (BSA) as standard (Aproscience, Naruto, Japan). The supernatants were solubilized in 2× SDS electrophoresis sample buffer and heated at 98°C for 5 minutes. Equal amount of proteins (7.0–7.5 μg per lane) were separated by SDS-12% polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Merck Millipore, Burlington, MA, USA). The membrane was then incubated with a blocking solution [5% BSA in Tween-Tris-buffered saline (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20)] and further incubated overnight at 4°C in Can Get Signal Solution-1 (TOYOBO, Osaka, Japan) with rabbit anti-ERK polyclonal antibody (1:2000, K-23, Santa Cruz, Dallas, TX, USA), mouse anti-p-ERK monoclonal antibody (1:2000, E-4, Santa Cruz), rabbit anti-p38 monoclonal antibody (1:2000, D13E1, Cell Signaling Technology, MA, USA), rabbit anti-p-p38 monoclonal antibody (Thr180/Tyr182) (1:2000, D3F9, Cell Signaling Technology), rabbit anti-JNK polyclonal antibody (1:2000, D-2, Santa Cruz), mouse anti-p-JNK monoclonal antibody (1:2000, Santa Cruz), rabbit anti-BDNF polyclonal antibody (1:2000, ab226843, Abcam, Cambridge, UK) or mouse anti-β-actin monoclonal antibody (1:2000, A1978, Sigma-Aldrich, St. Louis, MO, USA). After repeated washing, the immunoreactive bands were developed using Can Get Signal Solution-2 with horseradish peroxidase-conjugated anti-rabbit antibody (1:5000, GE Healthcare Japan Corporation, Tokyo, Japan) or anti-mouse antibody (1:5000, Santa Cruz), then visualized using an enhanced chemiluminescence detection system reagent (Amersham ECL-prime, GE Healthcare Japan Corporation), and captured in a LAS-3000 Luminoimage analyzer (Fuji Film, Tokyo, Japan). A commercially available molecular weight marker (Amersham ECL rainbow marker–full range, GE Healthcare Japan Corporation), consisting of proteins of molecular weight 12 to 225 kDa, was used as a reference for each molecular weight. The densities of protein blots were quantified using ImageJ [20] and the protein levels were normalized to β-actin levels.

Statistical analysis

For behavioral experiments, the hindpaw data within each group were analyzed using one-way repeated measures analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. Comparisons between two means of the hindpaw data and western blot data were performed by Welch’s test and Student’s t-test, respectively. The results were presented as mean ± SEM (for von Frey test) or ± SD (for western blot analysis). P < 0.05 was considered as significant. The statistics software used was JMP 11 (SAS Institute, Inc., Cary, NC, USA) for Macintosh.

Results

Mechanical allodynia in a rat model of oxaliplatin-induced neuropathic pain

The oxaliplatin treatment (4 mg kg^-1, twice a week for 4 weeks) induced increased pain behavior in the rat model [16]. Fig 1 shows that the paw withdrawal thresholds measured with VF filaments to the non-noxious mechanical stimulus at 3 and 4 weeks after oxaliplatin treatment were significantly lower than the vehicle treatment (4.4 ± 1.0 g vs. 16.0 ± 4.1 g; P = 0.046 and 4.4 ± 0.7 g vs. 14.8 ± 3.1 g; P = 0.027, respectively).

Oxaliplatin treatment up-regulates ERK phosphorylation in rat DRG neurons

The modulation of MAPKs activation, including ERK, p38 kinase, and JNK pathways, have not only been linked with the development of pain [12, 13], but also with the oxaliplatin-induced apoptosis in DRG [14, 15]. Western blot analyses of different MAPKs in the DRG of oxaliplatin-treated rats as compared to vehicle-treated rats are illustrated in Figs 2–4. Although no difference was observed in the total ERK1/2 levels between oxaliplatin and vehicle treatment groups, p-ERK1/2 was found to be up-regulated up to 4.5-fold (447.6 ± 273.6%, P = 0.0029) in DRG of oxaliplatin-induced neuropathic pain rat model (Fig 2). On the other hand, no change was observed in the phosphorylation and protein levels of p38 and JNK between oxaliplatin and vehicle treatment groups (Figs 3 and 4).

Fig 2 — (A) The ratio of p-ERK to ERK expression was significantly increased in DRG of oxaliplatin treated rats. (B) No difference was observed in the protein levels of ERK between oxaliplatin and vehicle treatment groups. Comparisons between two groups of the blots were performed by Student’s t-test. All data are calculated as mean ± SD of 5 animals. ** P < 0.001, compared to the vehicle.

Fig 4 — There was no difference in JNK phosphorylation and protein levels between oxaliplatin and vehicle treatment groups. Comparisons between two groups of the blots were performed by Student’s t-test. All data are calculated as mean ± SD of 5 animals.

Fig 3 — There was no difference in p38 phosphorylation and protein levels between oxaliplatin and vehicle treatment groups. Comparisons between two groups of the blots were performed by Student’s t-test. All data are calculated as mean ± SD of 5 animals.

Oxaliplatin treatment increases brain-derived neurotrophic factor (BDNF) levels in rat DRG

BDNF is not only a nerve growth factor, but also a neurotransmitter of nociceptive fibers in the dorsal horn of the spinal cord. In the spinal nerve ligation (SNL) model of neuropathic pain, BDNF expression was found to be up-regulated in the rat spinal dorsal horn [21]. BDNF expression was also up-regulated in DRG in lumbar 5 ventral root transection model of neuropathic pain [21].

In our study, BDNF levels were increased in DRG of oxaliplatin-induced neuropathic pain rat model (115.8 ± 23.4%, P = 0.047) (Fig 5).

Effects of ERK inhibitor on oxaliplatin-induced neuropathic pain

Fig 6 shows that ERK inhibitor PD98059 (6 μg day^-1) injected intrathecally inhibited oxaliplatin-induced mechanical allodynia. The paw withdrawal thresholds in the oxaliplatin and PD98059 treatment group were mostly maintained from baseline and were significantly higher than the oxaliplatin and vehicle treatment group at 3 weeks after oxaliplatin treatment (OX + PD98059: 15.2 ± 2.9 g and Sham: 16.2 ± 2.6 g vs. OX + Veh: 4.8 ± 0.8 g; P = 0.021 and P = 0.01, respectively). Concomitantly, PD98059 also inhibited oxaliplatin-induced upregulation of ERK phosphorylation in DRG (OX + PD98059: 147.6 ± 47.6%,vs. OX + Veh: 402.9 ± 251.2%, P = 0.0094) (Fig 7).

Fig 7 — (A) The ratio of p-ERK to ERK expression was significantly increased in DRG of oxaliplatin treated rats, which was inhibited by PD98059. (B) No difference was observed in the protein level of ERK among sham, oxaliplatin + vehicle (20% DMSO), and oxaliplatin + PD98059 treatment groups. Comparisons between two groups of the blots were performed by Student’s t-test. All data are calculated as mean ± SD of 5 animals. ** P < 0.01, compared with OX + Veh.

Discussion

Platinum-based drugs are the first-line chemotherapy for different cancers. Platinum derivatives such as oxaliplatin and cisplatin act as cytotoxins on tumor cells by forming platinum-DNA adducts, thus leading the tumor cells to programmed cell death. These platinum derivatives induce Chemotherapy-Induced Peripheral Neuropathy (CIPN) as one of the clinical side-effects [1, 2]. In an in-vitro study, treatment of cultured DRG neurons from E15 rat embryos with toxic doses of oxaliplatin or cisplatin induced a dose-dependent neuronal apoptosis by phosphorylating and inactivating the anti-apoptotic protein Bcl-2 and increasing the levels of the pro-apoptotic protein Bax [14].

Furthermore, studies have shown that these platinum derivatives modulate different MAPKs [13]. MAPKs are vital for intracellular signal transduction and play critical roles in regulation of neural plasticity and inflammatory responses [12, 13]. This family of kinases consists of three key members: ERK, p38, and JNK. Accumulating evidence shows that the activation of MAPKs can induce the synthesis of pronociceptive mediators via distinct molecular and cellular mechanisms, resulting in the enhancement and prolongation of pain [12, 13]. The platinum derivatives phosphorylate and activate p38 while they reduce the levels of active and total JNK. Both oxaliplatin and cisplatin have shown to activate ERKs during early stages (4–8 hours after treatment), although they behave differently at later stages [14]. Moreover, by using specific inhibitors of the different MAPKs, it has been demonstrated that the platinum-induced neuronal apoptosis is mediated by early p38 and ERK1/2 activation [14]. In in-vivo studies, oxaliplatin has shown to increase p38 phosphorylation at 0.5 and 4 hours after the treatment [22], or protein kinase C (PKC) phosphorylation, ERK1/2 phosphorylation, and c-fos expression on day 14 after the treatment [23], in the spinal cord of oxaliplatin-induced neuropathy mouse model. These results and our findings suggest a role for MAPKs including ERK in the generation and development of oxaliplatin-induced peripheral neuropathy.

Electrophysiological studies in patients undergoing oxaliplatin-treatment demonstrated nerve hyperexcitability in both peripheral motor [24] and sensory axons [25]. Furthermore, electrophysiological in-vitro studies in isolated peripheral nerve segments indicated that the hyperexcitability is characterized by an increase in the duration of the compound A-fiber action potential and the emergence of after-activity persisting over several tens of milliseconds [26–29]. A modulating effect on both voltage-dependent sodium channels and delayed rectifier potassium channels has been demonstrated during oxaliplatin administration to the myelinated axons in frog nerves [30] and to the neuronal cells in cell culture [31]. Thus, abnormal Na⁺ channel and/or K⁺ channel function has been highlighted as a possible mechanism of oxaliplatin-induced neuropathy. However, focusing on Na⁺ channel subtypes, Na_V1.7, Na_V1.8, and Na_V1.9, expressed in the DRG, conditional knockout mice established using the Na_V1.7^Advill line, which eliminates Na_V1.7 expression in all the DRG neurons, and the Na_V1.7^Wnt1 line, which lacks Na_V1.7 expression in the DRG and sympathetic ganglion neurons, both Na_V1.7^Advill and Na_V1.7^Wnt1 mice developed mechanical and cold allodynia normally following oxaliplatin treatment [32]. In addition, global deletion of Na_V1.3, Na_V1.8, or Na_V1.9 also did not attenuate either mechanical or cold allodynia in oxaliplatin-induced neuropathy. Furthermore, in our study, the expression levels of Na_V1.7, Na_V1.8, and Na_V1.9 were not altered in the DRG of oxaliplatin treated rats compared to non-treated rats (S1 Fig and S2 Fig). These findings suggest that the expression of the voltage-dependent Na⁺ channel subtypes Na_V1.3, Na_V1.7, Na_V1.8, and Na_V1.9 is not required for the development of oxaliplatin-induced neuropathy.

So, how does abnormal Na⁺ current lead to the development of nerve hyperexcitability? Firstly, Na⁺ channel subtypes other than Na_V1.3, Na_V1.7, Na_V1.8, and Na_V1.9 could contribute to oxaliplatin-induced nerve hyperexcitability. Indeed, a recent study revealed that the expression of Na_V1.6 was dramatically increased in the DRG in oxaliplatin-induced CIPN model rats [33]. Furthermore, the agomir of miR-30b, a microRNA implicated in neuropathic pain, cancer, and neurodegenerative diseases, can downregulate Na_V1.6 and alleviate oxaliplatin-induced mechanical allodynia and cold hypersensitivity [33]. Secondly, the cytokines and chemokines associated with altering intracellular signaling could modify Na⁺ current, which is conducive to nerve hyperexcitability. Recently accumulated evidence has shown that oxaliplatin treatment increases pro- and anti-inflammatory cytokines and chemokines [34–40]. Oxaliplatin injection enhanced the mRNA levels of cytokines including tissue necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and chemokines including monocyte chemoattractant protein-1 (MCP-1, also referred to as C-C chemokine ligand (CCL) 2) and monocyte inflammatory protein-1 (MIP-1α, also referred to as CCL3) in the spinal dorsal horn [34]. Melatonin attenuates pain hypersensitivity by inhibition of TNF-α in oxaliplatin-induced neuropathy [33]. CCL2 and its receptor CCR2 have also been shown to be increased in the DRG after oxaliplatin administration, in parallel with the development of mechanical hypersensitivity [35, 36]. Wang et al. have reported that oxaliplatin treatment up-regulates NF-κB and induces neuronal hyperexcitability in DRG [37]. This neuronal hyperexcitability was inhibited by NF-κB inhibitors. Oxaliplatin-induced pain has also been shown to be accompanied with the upregulation of PI3K-mTOR and mTOR-mediated signals as well as IL-1β, IL-6, and TNF-α in DRG. As PI3K or mTOR signal was inhibited, mechanical and cold hypersensitivity were attenuated in oxaliplatin treated rats, and the levels of proinflammatory cytokines also decreased [38]. The upregulation of pro-inflammatory cytokines and membrane pro-inflammatory cytokine receptors in the midbrain periaqueductal gray, which has an inhibitory or excitatory control on pain transmission via the rostral ventromedial medulla, projecting to the spinal dorsal horn, of oxaliplatin treated rats is likely to impair the descending inhibitory pathways in regulation of pain transmission and thereby, contribute to the development of neuropathic pain after the administration of chemotherapeutic oxaliplatin [39]. These reports suggest that the mechanism of development of oxaliplatin-induced neuropathy resembles inflammatory pain. Furthermore, Huang et al. and Liu et al. have reported an increase in the levels of TNF-α, NF-κB, and phosphorylation of ERK in the spinal cord and DRG of an oxaliplatin-induced peripheral neuropathy rat model and a lumber disk herniation rat model [36, 40]. Previous studies have demonstrated that TNF-α enhances TTX-R Na⁺ currents via TNFR1 and the p38 pathway in the cultured DRG neurons within 1 minute of the onset of TNF-α application (peak effect within 3–5 minutes) [41], as well as via the p38 pathway in the uninjured DRG neurons after L5-ventral root transection (VRT) in vivo [42]. The phosphorylated ERK1 lowers the activation threshold, making it easier to open Na_V1.7 channel in response to weak stimuli [43]. We have also reported that veratridine-induced ²²Na⁺ influx was inhibited by the inhibitors of ERK and p38, indicating that the basal constitutive activities of ERK and p38 may prime Na_V1.7 to open [44]. These findings suggest that oxaliplatin-induced neuroinflammation and inflammatory mediators would evolve abnormal Na⁺ channel currents via MAPK including ERK phosphorylation, and thus, lead to the development of neuropathy.

Conclusions

Oxaliplatin administration induces chronic mechanical allodynia in rats. The phosphorylation of ERK is upregulated in the DRG of oxaliplatin-induced neuropathic pain rat model, whereas other MAPKs, p38, and JNK are not altered. ERK inhibitor impedes mechanical allodynia by inhibiting oxaliplatin-induced upregulation of ERK phosphorylation. Thus, the findings from the present study may provide a better understanding of the intracellular molecular alterations in the development of oxaliplatin-induced neuropathic pain and help in designing effective therapeutics.

Supporting information

S1 Fig. The protein levels of Na_V1.7, Na_V1.8, and Na_V1.9 in oxaliplatin-treated rat DRG.

Typical western blots of Na_V1.7, Na_V1.8, and Na_V1.9 are shown. These images suggested that there was no difference in Na_V1.7, Na_V1.8, and Na_V1.9 protein levels between oxaliplatin and vehicle treatment groups.

(TIFF)

Click here for additional data file.^{(1MB, tiff)}

S2 Fig. The mRNA levels of Na_V1.7, Na_V1.8, and Na_V1.9 in oxaliplatin-treated rat DRG.

Typical polymerase chain reaction (PCR) gel images of Na_V1.7, Na_V1.8, and Na_V1.9 are shown. These images suggested that there was no difference in Na_V1.7, Na_V1.8, and Na_V1.9 mRNA expression levels between oxaliplatin and vehicle treatment groups.

(TIFF)

Click here for additional data file.^{(1MB, tiff)}

S3 Fig. Expanded views of western blots and PCR used in Figs 2–5, 7, S1 and S2.

(TIFF)

Click here for additional data file.^{(1.4MB, tiff)}

Acknowledgments

This study is attributed to the Department of Anesthesiology, Faculty of Medicine, University of Miyazaki. The authors would like to thank Noriko Hidaka, Mio Kurogi, and Toshiko Watanabe for their technical and secretarial assistance in this study. The authors would like to thank Editage (www.editage.jp) for English language editing.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was supported by a Grant for Clinical Research from University of Miyazaki Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0225586.r001

Decision Letter 0

Ferenc Gallyas, Jr

22 Aug 2019

PONE-D-19-17881

Upregulation of ERK phosphorylation in rat dorsal root ganglion neurons contributes to oxaliplatin-induced chronic neuropathic pain

PLOS ONE

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Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: In the current manuscript, the authors investigate the factors underlying the neuropathy development following oxaliplatin treatment in rats. This phenotype is seen after 3-4 weeks of treatment. The authors propose that upregulation of ERK phosphorylation in rat DRG neurons is responsible for generating this pain phenotype. They use ERK inhibition in combination with the evaluation of mechanical pain and western blot analysis to show that this mechanism may be responsible for the oxaliplatin-induced neuropathic pain. The data are conciseness and supportive, the study is well written and the conclusions are broadly reasonable. To improve the paper the authors should consider the following points:

1. The authors need to increase the sample size to ascertain that this time course of the incidence of mechanical allodynia in oxaliplatin group respect to vehicle group is not related to the small sample size -i.e. n=5 per group. The control group show a high variability These results will provide crucial information to the interpretation of the current data set (Fig1).

2. The authors should include more information that clarifies and justifies their statistical analyses. Specify the statistical methods performed in the figure legends and include the significance values in manuscript text. Please also provide all vehicle group values (mean +/- SD) which are used to relative the oxaliplatin group values, and F and P-values for each individual factor for all statistic performed.

3. For the ERK inhibition experiments on oxaliplatin-induced neuropathic pain, important further controls include sham rats that receive vehicle and ERK inhibitor in behavioral test and western blot. While this represents significant additional experiments, it would help interpret the existing main findings.

4. There have been previous reports of acute oxaliplatin administration already contributes to mechanical hyperalgesia (peripheral neuropathic pain) in DRG involving different mechanisms (Huang et al., 2018; Illias et al., 2018) - perhaps this should be integrated in their discussion.

Reviewer #2: This paper studied the factors contributing to the induction of chemotherapy-induced peripheral neuropathy (CIPN), using the platinum based chemotherapeutic agent, oxaliplatin. The authors used a rat model of oxaliplatin-induced neuropathic pain to evaluate the role of different mitogen-activated protein kinase (MAPK) pathways in the dorsal root ganglia of the lumbar spinal cord segment. The authors were specifically interested in studying the expression of ERK in the DRG, and the effect oxaliplatin treatment had on ERK phosphorylation in relation to the onset of mechanical allodynia. The results from these experiments report that oxaliplatin induces phosphorylation of EDK, and that delivering an ERK inhibitor inhibited oxaliplatin-induced ERK phosphorylation and blocked the onset of mechanical allodynia.

This paper provides great insight on the pathology underlying the development of cancer induced neuropathic pain. A lot of research in the pain field has focused on other models of chronic pain, such as diabetic neuropathy and peripheral nerve injury, but mechanisms underlying cancer therapeutic induced neuropathic pain are still not understood. I believe that these findings would contribute to furthering our knowledge on the pathology driving chronic pain caused by anti-cancer agents. There are questions and comments that should be addressed, before moving forward.

Abstract/Introduction:

• Lines 29-30: The statement "we aimed to understand the factors involved in the development and maintenance of chronic neuropathy elicited by oxaliplatin treatment" is misleading. The pathology underlying, both, the development and maintenance of chronic pain is a complex, multi-phase process involving changes in peripheral and central nervous system. The design of this study focused on the mechanism driving the onset of mechanical allodynia in relation to the effect oxaliplatin treatment had on the expression of p-ERK within the DRG, rather than the process of transitioning from acute to chronic pain. Clarifying the aim will strengthen the conclusions made by the authors, and help readers better interpret the data presented in each experiment. This statement is also made again in lines 68-70.

• Lines 49-60: Background information is introduced only on sodium ion channels and TRP channels, indicating their role in temperature hypersensitivity. A brief overview of current literature on mechanoreceptors in pain models should be introduced to reinforce that the author's are specifically studying mechanical allodynia because it's pathophysiology is poorly understood in this model of chronic pain.

Methods:

• Lines 87-112: Why was there a difference in duration of treatment between experiments 1 and 2? Do you have data comparing p-ERK/ERK expression in animals receiving treatment for 3 weeks versus animals receiving treatment for 4 weeks?

• Lines 107-108: The sham procedure is unclear. Did sham animals receive any i.p. or i.t. treatment, or were they naïve?

Results:

• The figure legends indicate values are expressed as mean +/- SD, but the values aren't reported in the results sections, figure legends, or on the graphs.

• The number of animals assigned to each treatment group should be reported on all the graphs.

• Lines 228-241: The mechanical allodynia measurements from the sham group need to be included in Figure 6 and incorporated into the statistical analysis. The sham behavioral data should be presented in this figure in order compare all three groups in Figure 7.

Discussion:

• Lines 285-289: The authors expand on the findings from previous studies concerning the involvement of voltage dependent sodium channel subtypes in the pathophysiology of oxaliplatin-induced neuropathy. The author's findings from the experiments on Na1.7, Na1.8, and Na1.9 expression in the DRG are consistent with the literature, but recent studies have reported that Na1.6 expression in the DRG contributes to the onset of oxaplatin induced neuropathic pain. The reports characterize Na1.6 in the DRG for its role in pain behavior as well as having abnormal spontaneous neuronal activity following nerve injury. Evidence from these studies show that directly targeting Na1.6 in the DRG alleviates oxaplatin induced mechanical allodynia and cold hypersensitivity. These studies should be reviewed and interpreted in the context of this study in the final paragraph of the discussion.

Overall, I thought this was a very well executed study that will contribute to the CIPN research community. After these points are addressed, I believe the manuscript will be ready to move forward in the review process as it satisfies the PLOS ONE criteria for publication.

**********

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PLoS One. 2019 Nov 25;14(11):e0225586. doi: 10.1371/journal.pone.0225586.r002

Author response to Decision Letter 0

2 Oct 2019

Dear Reviewer #1 (Manuscript ID: PONE-D-19-17881; Title: Upregulation of ERK phosphorylation…)

Thank you for your valuable comments on our paper. In response to your comments, we have extensively revised our paper, as shown below in a point-by-point manner.

Points 1 & 2. Reviewer #1 pointed out that the sample size of the behavioral experiments, especially in the vehicle group, were small and that more information about the data and statistical analysis should be provided.

Considering this, we reviewed our statistical analysis. First, we must apologize for a miscalculation of SEM from SD. SEM = SD / √n (n = number of sample size), so SD / √5 was correct, but we miscalculated by using SD / √3. We have now revised all the SEM data.

Second, in the comparisons between two groups of the hind paw data from the behavioral experiments, the F test shows P < 0.05, thus in this revised version of the manuscript we have used Welch’s test, not Student’s t-test. In addition, we have added the statistical methods in the figure legends, and we have provided the P-value in manuscript text when there are significant differences.

Regarding sample size, we do not think that we need more samples. We agree that if the sample size of vehicle group were increased, the SD and SEM would likely be narrower (because variability would become low). However, we think that as there were already statistical significances, we should minimize the number of animals used in each experiment.

Reviewer #1 stated that we should provide all vehicle group values (mean ± SD) which are used to relative the oxaliplatin group values. In our present study, western blot data were calculated as percentages of the control blot density, which was expressed as 100% (control meant vehicle or sham group in each experiment) on each membrane. The blot data were presented as means of these percentages ± SD. We consider this to be a common way to present expression analysis from western blots. For example, in figure 2 (A), the p-ERK level of oxaliplatin group was 447.6 ± 273.6% as a percentage of the vehicle, whereas the densities of protein blots quantified by ImageJ in the vehicle vs. oxaliplatin were 0.31 ± 0.19 vs. 0.96 ± 0.37, respectively. However, we consider that the calculation of densities is inaccurate in our present study, because we did not use an internal standard (e.g. the same DRG sample) on each membrane. Thus, we could not normalize the densities of the blots on each membrane.

Point 3. Reviewer #1 pointed out that for the ERK inhibition experiments, sham rats receiving vehicle (5% glucose) i.p. and ERK inhibitor i.t. (Veh + PD98059) should be used, because it would help interpret our findings.

Of course, we also considered whether a Veh + PD98059 group was needed or not when designing the experiments. We researched previous studies in which similar ERK inhibition experiments were performed. Some studies (marked below with*) did not use a Veh + PD98059 group. When there was a Veh + inhibitor treatment group, the ERK inhibitor did not affect the behavioral test (marked below with**). Furthermore, in our present study, Figure 6 & 7 show that the ERK inhibitor clearly inhibits ERK phosphorylation in DRG and oxaliplatin-induced mechanical allodynia. Therefore, we decided not to use a Veh + PD98059 group.

*(1) Cao Y, Li K, Fu KY, Xie QF, Chiang CY, Sessle BJ. Central sensitization and MAPKs are involved in occlusal interference-induced facial pain in rats. J Pain. 2013;14:793-807. (2) Wang XW, Li TT, Zhao J, Mao-Ying QL, Zhang H, Hu S, Li Q, Mi WL, Wu GC, Zhang YQ, Wang YQ. Extracellular signal-regulated kinase activation in spinal astrocytes and microglia contributes to cancer-induced bone pain in rats. Neuroscience. 2012;217:172-181. (3) Yoon SY, Kwon SG, Kim YH, Yeo JH, Ko HG, Roh DH, Kaang BK, Beitz AJ, Lee JH, Oh SB. A critical role of spinal Shank2 proteins in NMDA-induced pain hypersensitivity. Mol Pain. 2017;13:1744806916688902.

**(1) Sanna MD, Mello T, Ghelardini C, Galeotti N. Inhibition of spinal ERK1/2-c-JUN signaling pathway counteracts the development of low doses morphine-induced hyperalgesia. Eur J Pharmacol. 2015;764:271-277. (2) Xing F, Kong C, Bai L, Qian J, Yuan J, Li Z, Zhang W, Xu JT. CXCL12/CXCR4 signaling mediated ERK1/2 activation in spinal cord contributes to the pathogenesis of postsurgical pain in rats. Mol Pain. 2017;13:1744806917718753.

Point 4. Reviewer #1 pointed out that the reports of Huang et al. 2018 and Illias et al. 2018 should be integrated into the discussion. Therefore, we added a discussion of these reports (lines 331-333 and lines 347-349). We also added these reports to reference list (35 and 36).

We felt that Reviewer #1 emphasized acute oxaliplatin administration. There are various oxaliplatin administration methods that produce oxaliplatin-induced mechanical allodynia. In some methods, single or short administration of oxaliplatin produces mechanical allodynia in the early phase, such as thermal hyperalgesia. We chose a method similar to the clinical administration and mechanical allodynia onset for humans. In our present report, we did not refer to the variation of mechanical allodynia due to the differences in the oxaliplatin administration methods.

Dear Reviewer #2 (Manuscript ID: PONE-D-19-17881; Title: Upregulation of ERK phosphorylation…)

Thank you for your valuable comments on our paper. In response to your comments, we have extensively revised our paper, as shown below point-by-point manner.

Abstract/Introduction:

Lines 29-30 & 68-70: We agree your suggestion and revise the sentence from “development and maintenance” to “generation” (line 30 and line 78).

Lines 49-60: Reviewer #2 pointed out that mechanoreceptors in pain model should be introduced to reinforce that we studied oxaliplatin-induced mechanical allodynia, not thermal hyperalgesia. However, neuropathic pain is caused not only by mechanoreceptors, so we added some sentences about the potential mechanisms of neuropathic pain (lines 57-64) in the introduction and added the following reports to the references (3. Baron R, Binder A, Wasner G. Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol. 2010;9:807-819. and 4. St John Smith E. Advances in understanding nociception and neuropathic pain. J Neurol. 2018;265:231-238.).

Lines 87-112: Reviewer #2 pointed out the difference in treatment duration between experiment 1 and 2. We used an ALZET osmotic pressure pump for continuous injection of PD98059. We could find an ideal pump for our experiments (size, infusion rate, and infusion duration), however, this pump can only continue to infuse the drug for 4 weeks. Therefore, we had to choose the data in animals receiving oxaliplatin treatment for 3 weeks, in which the paw withdrawal thresholds were significantly lower than vehicle treatment.

Lines 107-108: Reviewer#2 pointed out that the sham procedure is unclear. However, we already described “The sham-operated (Sham) mice underwent a similar surgical procedure except for pump placement and drug treatments” in Method section (line 119-120). Therefore, the sham-operated mice (Sham) did not receive any i.p. or i.t. treatment.

Results:

We changed the sentences from “Values are expressed” to “All data are calculated” in each figure legend. We also added the values in manuscripts text when there are significant differences.

The number of animals assigned to each treatment group are already described in the figure legends, but not reported on the graphs. Therefore, we added the number on each graph.

Lines 228-241: We did not show the sham group in Figure 6, because the hind paw data of the sham group was not altered compared with the OX + PD98059 group and we were afraid that Figure 6 would become too busy and difficult to interpret. However, we agree with the reviewer's comment and have added the sham group in Figure 6.

Discussion:

Lines 285-289: We found a recent report that the expression of NaV1.6 in DRG was increased in oxaliplatin-induced CIPN model rats. We review this study in the discussion (lines 317-324) and have added this report to the references (33. Li L, Shao J, Wang J, Liu Y, Zhang Y, Zhang M, Zhang J, Ren X, Su S, Li Y, Cao J, Zang W. MiR-30b-5p attenuates oxaliplatin-induced peripheral neuropathic pain through the voltage-gated sodium channel NaV1.6 in rats. Neuropharmacology. 2019;153:111-120.).

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PLoS One. doi: 10.1371/journal.pone.0225586.r003

Decision Letter 1

Ferenc Gallyas, Jr

29 Oct 2019

PONE-D-19-17881R1

Upregulation of ERK phosphorylation in rat dorsal root ganglion neurons contributes to oxaliplatin-induced chronic neuropathic pain

PLOS ONE

Dear Dr. MARUTA,

Please address the statistical analysis issues raised by reviewer#1.

We would appreciate receiving your revised manuscript by Dec 13 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

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An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Ferenc Gallyas, Jr., Ph.D., D.Sc.

Academic Editor

PLOS ONE

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Reviewers' comments:

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Reviewer #1: (No Response)

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6. Review Comments to the Author

Reviewer #1: Comments to authors:

In the revised manuscript, the authors have adequately revised the manuscript and addressed important questions regarding the experimental results and analysis. But if possible, I would still like to address some point that were raised in the review:

1) In the behavioral experiments, in the comparisons between time points of the hind paw data is recommendable to perform a repeated measures ANOVA.

2) In western blot experiments, I agree with the authors that calculating data as percentages of control blot density (expressed as 100%) is a common way of representation. What I pointed out in the previous review is to express somehow the variance within the control group, since although it is considered 100%, it is an average of measures with SD. The protein density data quantified in ImageJ for experimental and control groups would support the levels of significance expressed in the figures (above suggested supplementary table). Also, because I understand that the statistical analysis has been carried out with these data. According to the authors, I consider that methodologically the most adequate quantification has not been performed due to the absence of internal standard. However, if it is possible to normalize the density of protein blots of each membrane with respect to the background of each of them. Have you been considered in the analysis?

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Reviewer #2: Yes: Zach LaMacchia

PLoS One. 2019 Nov 25;14(11):e0225586. doi: 10.1371/journal.pone.0225586.r004

Author response to Decision Letter 1

4 Nov 2019

Dear Reviewer #1 (Manuscript ID: PONE-D-19-17881; Title: Upregulation of ERK phosphorylation…)

Thank you for your valuable comments on our paper. In response to your comments, we have extensively revised our paper, as shown below in a point-by-point manner.

Points 1. Reviewer #1 pointed out that in the comparisons between time points of the hind paw data is recommendable to perform a repeated measures ANOVA.

So, we used one-way repeated ANOVA. OX group in Figure 1 and OX + Vehicle group in Figure 6 were significantly changed according to time (p = 0.0003 and p = 0.0039, respectively). We revised the sentences in statistical methods and Figure legends (lines 175, 195, and 260).

Point 2. Reviewer #1 pointed out that to express somehow the variance within the control group, since although it is considered 100%, it is an average of measures with SD; and if it is possible to normalize the density of protein blots of each membrane with respect to the background of each of them.

Of course, the density of protein blots in control group must have the variance. We tried to use the background density as an internal standard. For example, in figure 2 (A), the p-ERK level of oxaliplatin group was 447.6 ± 273.6% as a percentage of the vehicle, whereas the densities of protein blots quantified by ImageJ in the vehicle vs. oxaliplatin were 0.31 ± 0.19 vs. 0.96 ± 0.37 (p = 0.0006), respectively; and when using the background density as an internal standard, the densities of protein blots in the vehicle vs. oxaliplatin were 0.46 ± 0.32 vs. 1.39 ± 0.65 (p = 0.003), respectively. This way to use the background density as an internal standard seems good and we are grateful for Reviewer #1’s valuable advice. However, we have some concerns that this way might depend on how to set background and we do not know whether there is consensus in the use of the background density as an internal standard. We consider that using same sample as an internal standard on each membrane is better, if the variance of control is needed to be expressed. So, in this study, we would like to keep to express the calculating data as percentages of control blot density (expressed as 100%).

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PLoS One. doi: 10.1371/journal.pone.0225586.r005

Decision Letter 2

Ferenc Gallyas, Jr

8 Nov 2019

Upregulation of ERK phosphorylation in rat dorsal root ganglion neurons contributes to oxaliplatin-induced chronic neuropathic pain

PONE-D-19-17881R2

Dear Dr. MARUTA,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0225586.r006

Acceptance letter

Ferenc Gallyas, Jr

13 Nov 2019

PONE-D-19-17881R2

Upregulation of ERK phosphorylation in rat dorsal root ganglion neurons contributes to oxaliplatin-induced chronic neuropathic pain

Dear Dr. MARUTA:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. The protein levels of Na_V1.7, Na_V1.8, and Na_V1.9 in oxaliplatin-treated rat DRG.

(TIFF)

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S2 Fig. The mRNA levels of Na_V1.7, Na_V1.8, and Na_V1.9 in oxaliplatin-treated rat DRG.

(TIFF)

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S3 Fig. Expanded views of western blots and PCR used in Figs 2–5, 7, S1 and S2.

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Upregulation of ERK phosphorylation in rat dorsal root ganglion neurons contributes to oxaliplatin-induced chronic neuropathic pain

Toyoaki Maruta

Takayuki Nemoto

Koutaro Hidaka

Tomohiro Koshida

Tetsuro Shirasaka

Toshihiko Yanagita

Ryu Takeya

Isao Tsuneyoshi

Roles

Abstract

Introduction

Materials and methods

Animals

Pharmacological treatments

von Frey test

Western blot analysis

Statistical analysis

Results

Mechanical allodynia in a rat model of oxaliplatin-induced neuropathic pain

Fig 1. Paw withdrawal test (von Frey test) for mechanical allodynia induced by oxaliplatin.

Oxaliplatin treatment up-regulates ERK phosphorylation in rat DRG neurons

Fig 2. Upregulation of ERK phosphorylation by oxaliplatin in rat DRG.

Fig 4. JNK phosphorylation and protein levels in rat DRG.

Fig 3. p38 phosphorylation and protein levels in rat DRG.

Oxaliplatin treatment increases brain-derived neurotrophic factor (BDNF) levels in rat DRG

Fig 5. Upregulation of BDNF by oxaliplatin in rat DRG.

Effects of ERK inhibitor on oxaliplatin-induced neuropathic pain

Fig 6. Inhibition of oxaliplatin-induced mechanical allodynia by ERK inhibitor PD98059.

Fig 7. Effect of ERK inhibitor PD98059 on oxaliplatin-induced upregulation of ERK phosphorylation in rat DRG.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Ferenc Gallyas, Jr

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Author response to Decision Letter 0

Decision Letter 1

Ferenc Gallyas, Jr

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Author response to Decision Letter 1

Decision Letter 2

Ferenc Gallyas, Jr

Roles

Acceptance letter

Ferenc Gallyas, Jr

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Associated Data

Supplementary Materials

Data Availability Statement

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