Composition Vendor Newgen KnowledgeWorks (P) Ltd. PLoS One plos plosone PLOS One 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0323628 PONE-D-24-58742 Research Article Medicine and health sciencesClinical medicineSigns and symptomsPainNeuropathic pain Research and analysis methodsAnimal studiesExperimental organism systemsModel organismsMouse models Research and analysis methodsModel organismsMouse models Research and analysis methodsAnimal studiesExperimental organism systemsAnimal modelsMouse models Biology and life sciencesNeuroscienceBrain mappingOptogenetics Research and analysis methodsBioassays and physiological analysisNeurophysiological analysisOptogenetics Biology and life sciencesAnatomyNervous systemNeuroanatomySpinal cord Medicine and health sciencesAnatomyNervous systemNeuroanatomySpinal cord Biology and life sciencesNeuroscienceNeuroanatomySpinal cord Medicine and health sciencesClinical medicineClinical immunologyHypersensitivity Biology and life sciencesImmunologyClinical immunologyHypersensitivity Medicine and health sciencesImmunologyClinical immunologyHypersensitivity Medicine and health sciencesClinical medicineSigns and symptomsPain Research and analysis methodsAnimal studiesExperimental organism systemsAnimal models Biology and life sciencesAnatomyNervous systemNervesSpinal nerves Medicine and health sciencesAnatomyNervous systemNervesSpinal nerves Reversible neuropathic pain model created by long-term optogenetic nociceptor stimulation using light-responsive pain mice Neuropathic pain model using light-responsive pain mice Kouroki Satoshi Data curation Formal analysis Funding acquisition Investigation Resources 1 https://orcid.org/0000-0002-2660-2179 Maruta Toyoaki Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Writing – original draft 1 * Hidaka Kotaro Investigation 1 Koshida Tomohiro Investigation 1 Kurogi Mio Investigation 1 Kage Yohko Methodology 2 Miura Ayako Methodology 2 Nakagawa Hikaru Methodology 2 Yanagita Toshihiko Writing – review & editing 3 https://orcid.org/0000-0002-9987-1513 Takeya Ryu Writing – review & editing 2 Tsuneyoshi Isao Writing – review & editing 1 Department of Anesthesiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Miyazaki, Japan Department of Pharmacology, Faculty of Medicine, University of Miyazaki, Miyazaki, Miyazaki, Japan Department of Clinical Pharmacology, Faculty of Medicine, School of Nursing, University of Miyazaki, Miyazaki, Miyazaki, Japan Cheron Julian Editor Johns Hopkins University, UNITED STATES OF AMERICA

The authors have declared that no competing interests exist.

 * E-mail: mmctm2@yahoo.co.jp 3052025 2025 20 5 e0323628 212025 1042025 2025 Kouroki et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Neuropathic pain has a significant social impact, with high morbidity and reduced productivity, the underlying mechanisms of neuropathic pain remain poorly understood, and effective therapeutic strategies remain elusive. The development of animal models of neuropathic pain that stimulate only the nociceptors and not the other sensory receptors or motor nerves is desirable for elucidating the complex pathogenesis of neuropathic pain. We have previously reported the generation of NaV1.7−channelrhodopsin-2 (ChR2), NaV1.8−ChR2, and NaV1.9−ChR2 mice. Optogenetics was employed in these light-responsive pain mice for generating nociceptive pain by specifically exciting the spinal dorsal root ganglion neurons, in which the respective Na+ channels are expressed through exposure to blue light. This study aimed to compare the neuropathic pain produced by the prolonged exposure of light-responsive pain mice to blue light. A reversible neuropathic pain state was established persisting for a minimum of 24 hours when each light-responsive pain mouse was irradiated with light of an intensity that consistently elicited pain. Furthermore, the mice also showed pain sensitivity to light irradiation and mechanical stimulation. The expression of c-Fos, a marker for neuronal activity following noxious stimulation, was increased in the dorsal horn of the spinal cord on the light irradiated side. DS-1971a, a selective NaV1.7 inhibitor, was effective in attenuating neuropathic pain in all light-responsive pain mice. In conclusion, optogenetics helps elucidate the specific functions of sodium channel subtypes in pain signaling, thereby advancing our understanding and paving the way for the development of further effective treatments for pain disorders in the future.

 http://dx.doi.org/10.13039/501100001691 Japan Society for the Promotion of Science 21K08925 https://orcid.org/0000-0002-2660-2179 MarutaToyoaki http://dx.doi.org/10.13039/501100001691 Japan Society for the Promotion of Science 18K08859 https://orcid.org/0000-0002-2660-2179 MarutaToyoaki http://dx.doi.org/10.13039/100019890 University of Miyazaki Hospital No number KourokiSatoshi This research was supported by Japan Society for the Promotion of Science (JSPS): KAKENHI Grant Numbers 18K08859, 21K08925, and 16H06276 (Advanced Animal Model Support), and a Grant-in-Aid for Clinical Research from Miyazaki University Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability All relevant data are within the manuscript and its Supporting Information files. Introduction

Neuropathic pain, which affects 7–8% of the population, is considered a social problem as it results in decreased productivity [1]. However, the pathogenesis of neuropathic pain has not been fully elucidated; thus, no treatment has been established for managing neuropathic pain [1,2]. Multiple factors, which are modifiable over time, contribute to the onset of neuropathic pain. Thus, unraveling the complex pathogenesis of neuropathic pain is challenging. The most widely employed models of neuropathic pain attributed to peripheral neuropathy in rodents are physical injury models of peripheral nerves, including the spared nerve injury (SNI), chronic constriction injury (CCI), partial sciatic nerve ligation (PSL), partial sciatic nerve ligation (PSL), and spinal nerve ligation (SNL) (Chung model) models [3]. Although these models produce allodynia, they can cause motor and/or non-pain sensory neuropathies. Therefore, despite the development of various animal models of neuropathic pain in the field of pain research, the creation of models by stimulating only the nociceptors and not the other sensory receptors or motor nerves is desirable.

The NaV1.7, NaV1.8, and NaV1.9, which are voltage-gated Na+ channel subtypes, are predominantly expressed in the spinal dorsal root ganglion (DRG) neurons and are involved in pain signaling and neuropathic pain development [4]. In the voltage-gated Na+ channels, which consist of NaV1.1-1.9 subtypes, each subtype possesses different kinetics and expression patterns, which are reflected in the functional groupings of the peripheral sensory neurons [5]. Large DRG neurons with myelinated Aβ and Aδ fibers express mainly tetrodotoxin-sensitive (TTX-S) NaV1.1, NaV1.6 and NaV1.7. A subset of these neurons also express tetrodotoxin-resistant (TTX-R) NaV1.8, which may correspond to Aβ nociceptors. In contrast, small diameter nociceptive neurons with unmyelinated C fibers express high levels of TTX-R NaV1.8 and NaV1.9, and TTX-S NaV1.7 and NaV1.6. These differences in the Na+ channel expression are reflected in the differences in the morphology of the action potential waveform: NaV1.7 contributes to the rising phase of the action potential and amplifies subthreshold stimulation, while NaV1.8 contributes mainly to the rising phase and NaV1.9 amplifies subthreshold stimuli. Furthermore, NaV1.7, NaV1.8, and NaV1.9 have been reported to cause abnormal pain or painlessness in humans owing to gain-of-function or loss-of-function mutations [5].

Recently, optogenetics using light-responsive ion channels known as opsins has facilitated selective activation and inhibition of the target neurons in vivo [6]. This technique of optogenetics has become widely used in neuroscience and has proven useful in elucidating complex pain pathways in the field of pain research [7–12]. We have developed NaV1.7−channelrhodopsin-2 (ChR2), NaV1.8−ChR2, and NaV1.9−ChR2 mice and applied optogenetics to specifically excite neurons that exhibit expression of each Na+ channel [13,14]. When the plantar of these light-responsive pain mice were irradiated with blue light, nociceptive pain was produced in each mouse at varying light intensities [13]. Previously, Daou et al. reported the occurrence of transient neuropathic pain following long-term exposure to blue light in NaV1.8−ChR2 mice [12]. Such optogenetics-based neuropathic pain models are superior to conventional animal models as they are able to target only the sensory nerves responsible for pain. In this study, we compared the neuropathic pain produced by long-term and continuous exposure of NaV1.7−ChR2, NaV1.8−ChR2, and NaV1.9−ChR2 mice to blue light.

 Materiavls and methods Animals

Wild-type (WT) C57BL/6J mice, commonly known as B6J mice, and Ai32 mice (C57BL/6 background) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All the mice were individually housed in a temperature and humidity-controlled environment with a 12-h light-dark cycle, and were permitted free access to food and water. This study was conducted in strict accordance with the guidelines for the Proper Conduct of Animal Experiments (Science Council of Japan) and approved by the Experimental Animal Care and Use Committee of the University of Miyazaki (Permit Number: 2024-511). Male mice between 2 and 6 months old were used in the experiments. All efforts were made to minimize the number of animals used and their suffering. Because all mice recover to a normal, pain-free state, euthanasia need not be considered. Mice in each group were randomly selected, and the experimenter was blinded to the mouse genotype and drug treatment.

 Production of genetically modified mice

NaV1.x−ChR2 mice were created as described previously [13–15]. We produced the bicistronic founder generation (F0) of NaV1.7−iCre (NaV1.7iCre/+), NaV1.8–iCre (NaV1.8iCre/+), and NaV1.9–iCre (NaV1.9iCre/+) knock-in mice using the CRISPR/Cas9 system, with financial and technical assistance from Advanced Animal Model Support (AdAMS). For each subtype, we targeted the 5′-gaaagcaggaaatagagctt-3′, 5′-cctggacctcagtgaagacactc-3′, or 5′-cttggatgtgcccaagatca-3′ sequence corresponding to the Scn9a, Scn10a, or Scn11a gene, respectively. Following DNA confirmation of the desired genetic modification, NaV1.7–ChR2 (NaV1.7iCre/+;Ai32/+), NaV1.8–ChR2 (NaV1.8iCre/+;Ai32/+), and NaV1.9–ChR2 (NaV1.9iCre/+;Ai32/+) mice were generated by breeding homozygous NaV1.7–iCre (NaV1.7iCre/iCre), NaV1.8–iCre (NaV1.8iCre/iCre), or NaV1.9–iCre (NaV1.9iCre/iCre) mice with homozygous Ai32 mice, carrying the ChR2(H134R)-EYFP gene in the Gt(ROSA)26Sor locus. The ChR2(H134R)-EYFP gene was capable of expression through its CAG promoter by eliminating the loxP-flanked transcriptional STOP cassette after breeding with iCre mice.

 von Frey test for determining mechanical sensitivity

Mechanical sensitivity was examined by determining the paw withdrawal threshold using an electronic von Frey esthesiometer (IITC Life Science Inc., Woodland Hills, CA, USA) fitted with a polypropylene tip. Each adult mouse was placed in a 10 cm × 10 cm suspended chamber with a metallic mesh floor. After acclimation of the mice for 30 min, a polypropylene tip was applied perpendicularly to the plantar surface of the right and left hind paws with sufficient force for 3–4 s. Brisk withdrawal or paw flinching was considered a positive response. The pain threshold was calculated as the mean of three measurements.

In this study, the left and right hindpaws of NaV1.x–ChR2 mice were tested for sensitivity to mechanical stimuli before and after (1, 3, 6, 24, 36, and 48 h) a prolonged (30 min), suprathreshold (5 and 7.5 mW for NaV1.7–ChR2, 1.2 and 1.8 mW for NaV1.8–ChR2, and 1.5 and 2.25 mW for NaV1.9–ChR2) blue-light stimulation of the ipsilateral hindpaw. The blue-light intensity employed in this experiment was the intensity at which the paw withdrawal frequency was nearly 100% in the previously reported blue-light plantar irradiation test and 1.5 times that intensity [13]. The light intensity was determined using a light power meter (LPM-100TM; Bioresearch Center Inc., Aichi, Japan). The non-stimulated contralateral hindpaw was employed as an internal control. Mice were anesthetized with 3.0% sevoflurane during the 30-min stimulation. Laser was pulsed at 2 Hz with 100 ms pulse duration. The settings for these continuous irradiations were determined by a preliminary reported study [12].

To determine the analgesic effects of DS-1971a, a selective NaV1.7 inhibitor, on the long-term optogenetic stimulation-induced neuropathic pain, von Frey test was performed. One side of the hindpaw of NaV1.x –ChR2 mice was tested for sensitivity to mechanical stimuli before and 1 hour after a prolonged (30 min), suprathreshold (7.5 mW for NaV1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2) blue-light stimulation of the ipsilateral hindpaw. DS-1971a (10 mg/kg and 100 mg/kg, Daiichi Sankyo, Tokyo, Japan) in 0.5% methylcellulose or vehicle (0.5% methylcellulose) was subsequently administered orally. The pain threshold in the ipsilateral hindpaw was measured 2 hours after DS-1971a administration. The dose levels of DS-1971a administration were determined by a preliminary reported study [16], in which DS-1971a at 10 mg/kg and 100 mg/kg successfully reduced pain thresholds over a long period of time in the spinal nerve ligation and partial sciatic nerve ligation mouse models.

 Light irradiation test to determine the light-responsive hypersensitivity due to long-term optogenetic stimulation

Light irradiation test was performed before and 1 hour after a prolonged (30 min), suprathreshold (7.5 mW for NaV1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2) light stimulation. Mice were habituated for 1 h in transparent cubicles (10 cm × 6.5 cm × 6.5 cm) set atop a 5-mm-thick glass floor and separated from each other by opaque dividers. Acute nocifensive behaviors were elicited by light from a pulsing light-emitting diode (LED) (465 nm blue light at 10 Hz; Doric Lenses Inc., Quebec, Canada) set at different intensities and aimed at the plantar surface of the hind paw. The light intensity was determined using a light power meter (LPM-100TM). Since the power meter measures light intensity in milliwatts (mW), the light density was calculated in mW/mm2 by dividing the light intensity by the illuminated area in square millimeters (48 mm2). The mice underwent five trials of 1 s each, with 5-s intervals between the trials. The percentage of trials demonstrating hind paw withdrawal or paw licking was recorded.

 Immunohistochemistry

Mice were confirmed hypersensitive by von Frey test 1 hour after a prolonged (30 min), suprathreshold (7.5 mW for Nav1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2) light stimulation. The mice were subsequently anesthetized with sevoflurane and intracardially perfused with 50 mL perfusion buffer, followed by 100 mL 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) (pH 7.4) at room temperature for 30 min. The L3-5 lumbar spinal cord was dissected and post-fixed in 4% PFA for 2 h at 4 °C, cryoprotected in 30% sucrose in PBS, and incubated overnight at 4 °C; 16-μm thick sections were cut from the freeze-fixed spinal cord with the temperature maintained at –20 °C using a cryostat (Leica Biosystems, Nussloch, Germany). Samples of spinal cord were placed directly on slides. The spinal cord sections were incubated in 0.1% Triton X-100 and 5% goat serum in PBS at room temperature for 4 h, followed by incubation with rabbit anti-c-Fos recombinant monoclonal antibody (1:500; catalog #ab222699, Abcam, Cambridge, MA, UK) at 4 °C with overnight agitation. Thereafter, the sections were washed thrice with PBS, followed by incubation with goat anti-rabbit IgG (H+L) (Alexa Fluor™ Plus 594, 1:300; catalog #A32740, Invitrogen, Waltham, MA, USA) for 1 h at room temperature. These sections were washed, air-dried, and mounted with a coverslip using an antifade mounting medium (MowiolTM 4-88, catalog #81381, Sigma-Aldrich, St Louis, MO, USA). The fluorescence of the transgenic ChR2–EYFP was sufficient for visualization without immunostaining. The prepared slides were stored at 4 °C until further examination. The morphology of the different tissues was analyzed using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan). Images were processed using ImageJ software (NIH, Bethesda, MD, USA) to optimize brightness and contrast [17].

 Statistical analysis

Each behavioral experiment evaluated n ≥ 10 animals, whereas the examinations of c-Fos expression evaluated n = 6 animals. For the behavioral experiments, data were analyzed using paired t-test or one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc analysis. The results are presented as mean ± standard deviation (SD). Statistical significance was set at P < 0.05. The statistical software JMP Pro 17 (SAS Institute, Inc., Cary, NC, USA) for Macintosh was used for the statistical analyses.

 Results Mechanical sensitization induced by long-term optogenetic stimulation

To examine whether long-term optogenetic stimulation induces mechanical sensitization, prolonged suprathreshold blue-light irradiation (30 min, 2 Hz, two types of intensity) to the hindpaw plantar surface of anesthetized NaV1.x–ChR2 mice was performed. As shown in Fig 1a,b, and c, compared with the contralateral hindpaw, significant mechanical hypersensitivity was observed in the ipsilateral hindpaw of NaV1.x–ChR2 mice. This mechanical hypersensitivity was reversible and persisted for longer periods at higher light intensities.

10.1371/journal.pone.0323628.g001Paw withdrawal test (von Frey test) in Na<sub>V</sub>1.7–ChR2 (a), Na<sub>V</sub>1.8–ChR2 (b), and Na<sub>V</sub>1.9–ChR2 mice.

The von Frey test was performed with (a) NaV1.7–ChR2, (b) NaV1.8–ChR2, and (c) NaV1.9–ChR2 mice before (Pre) and after (1, 3, 6, 24, 36, and 48 h) light stimulation. Prolonged, suprathreshold blue-light exposure (30 min, 2 Hz, two types of intensity) to the ipsilateral hindpaw of anesthetized NaV1.x –ChR2 mice produces long-term mechanical hypersensitivity lasting up to 24 h and 36 h after stimulation, respectively. The hind paw withdrawal data were analyzed using paired t-test (ipsilateral compared with contralateral) or one-way ANOVA followed by Bonferroni post-hoc analysis (each time compared with Pre). All the results are calculated as mean ± SD of 10 or more animals. *P < 0.05, compared with contralateral. #P < 0.05, compared with before optogenetic stimulation (Pre).

 Light-responsive hypersensitivity due to long-term optogenetic stimulation

To confirm peripheral sensitization by prolonged optogenetic stimulation, we examined the blue LED light irradiation hind paw withdrawal test before and 1 hour after prolonged (30 min) optogenetic stimulation. The leftward shift of the light power-withdrawal curve before and after prolonged optogenetic stimulation indicated that prolonged optogenetic stimulation hypersensitized the response to nociceptive pain induced by light irradiation in the NaV1.x–ChR2 mice (Fig 2).

10.1371/journal.pone.0323628.g002 Light irradiation hind paw withdrawal test before and after long-term optogenetic stimulation. The blue light irradiation hind paw withdrawal test was performed before (Pre) and 1 hour after (Post) a prolonged (30 min), suprathreshold (7.5 mW for Na<sub>V</sub>1.7–ChR2, 1.8 mW for Na<sub>V</sub>1.8–ChR2, and 2.25 mW for Na<sub>V</sub>1.9–ChR2) light stimulation in (a) Na<sub>V</sub>1.7–ChR2, (b) Na<sub>V</sub>1.8–ChR2, (c) Na<sub>V</sub>1.9–ChR2 mice. The data were analyzed using paired <italic>t</italic>-test. All the results are calculated as mean ± SD of 10 or more animals. *<italic>P</italic> < 0.05, compared with Pre. c-Fos expression in the spinal cord dorsal horn following long-term optogenetic stimulation

To investigate whether light irradiation of the hindpaw plantar is transmitted from the peripheral nerve to the central nervous system as a nociceptive signal, we investigated c-Fos expression in the secondary sensory neurons in the dorsal horn of the spinal cord. Mice that demonstrated hypersensitivity using the von Frey test were fixed for immunofluorescence analysis. We observed c-Fos expression in the ipsilateral and contralateral dorsal horn of the lumber spinal cord after long term optogenetic stimulation (Fig 3). In the spinal cord dorsal horn of NaV1.x–ChR2, compared to the contralateral side, c-Fos expression was increased in the ipsilateral side.

10.1371/journal.pone.0323628.g003c-Fos expression in the lumber spinal cord dorsal horn.

c-Fos expression in the ipsilateral and contralateral dorsal horn of the lumber spinal cord after long term (30 min) optogenetic stimulation (7.5 mW for NaV1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2) are shown in (a) NaV1.7–ChR2, (b) NaV1.8–ChR2, (c) NaV1.9–ChR2 mice. Scale bar, 50 μm. Quantification of c-Fos-positive neurons in laminae I–III of the L3–5 lumber spinal segments from optogenetically stimulated NaV1.x–ChR2 mice are shown in (d). The data were analyzed using paired t-test. All results are calculated as mean ± SD of 6 animals. *P < 0.01, compared with the contralateral.

 Analgesic effect of DS-1971a on long-term optogenetic stimulation-induced neuropathic pain

Clinical trials of selective Na+ channel inhibitors are underway for the treatment of acute postoperative pain and neuropathic pain [18]. We have examined the analgesic effect of 10 and 100 mg/kg DS-1971a, which is a selective NaV1.7 inhibitor [16], on long-term optogenetic stimulation-induced neuropathic pain in NaV1.x–ChR2 mice. DS-1971a at both 10 mg/kg and 100 mg/kg almost completely suppressed long-term optogenetic stimulation-induced neuropathic pain in the NaV1.7-ChR2, NaV1.8-ChR2 and NaV1.9-ChR2 mice (Fig 4).

10.1371/journal.pone.0323628.g004Analgesic effect of DS-1971a on long-term optogenetic stimulation-induced neuropathic pain.

von Frey tests were performed in (a) NaV1.7–ChR2, (b) NaV1.8–ChR2, (c) NaV1.9–ChR2 mice before optogenetic stimulation (7.5 mW for NaV1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2), 1 hour after stimulation, and 3 hours after stimulation (2 hours after DS-1971a or vehicle administration). The data were analyzed using one-way ANOVA followed by Bonferroni post-hoc analysis. All data are calculated as mean ± SD of 10 animals. *P < 0.05, compared with pre-stimulation (time 0). †P < 0.01, compared with post-stimulation (1 hour after stimulation).

 Discussion

In this study, we demonstrated that a reversible neuropathic pain state was established persisting for a minimum of 24 hours when light-responsive pain mice (NaV1.x-ChR2 mice) was irradiated with light of an intensity that consistently elicited pain. Furthermore, the mice also showed hypersensitivity to light irradiation and mechanical stimulation. The c-Fos expression increased in the dorsal horn of the spinal cord on the light irradiated side. DS-1971a, a selective NaV1.7 inhibitor, was effective in attenuating neuropathic pain in all light-responsive pain mice.

In our previous studies [13,14], we generated bicistronic NaV1.7−, NaV1.8−, and NaV1.9−iCre recombinase-expressing iCre recombinase under the endogenous NaV1.x gene promoter without disruption of NaV1.7, NaV1.8, and NaV1.9 by CRISPR/Cas9-mediated homologous recombination. Furthermore, by crossing these lines with homozygous Ai32 mice via the Cre-LoxP system, transgenic NaV1.x−ChR2 mouse lines (NaV1.xiCre/+;Ai32/+) were generated in which ChR2 was expressed only in NaV1.x-expressing sensory neurons. The mice expressing ChR2 in the NaV1.7, NaV1.8, and NaV1.9 channels demonstrated a nociceptive response to blue light. Differences in this light sensitivity were observed between NaV1.x−ChR2 mice, indicating that this may depend on the expression and distribution of ChR2 in the DRG, or that light-sensitivity may reflect inherent disparities associated with the varying roles of each Na+ channel subtype in pain transmission [13,14]. Daou et al. reported that prolonged light exposure in NaV1.8−ChR2 mice resulted in an increase in c-Fos expression in the dorsal horn of the spinal cord, which in turn resulted in persistent hypersensitivity to mechanical and thermal stimuli [12]. Similarly, as previously reported by Daou et al., we employed our light-responsive pain mice to demonstrate that prolonged light irradiation induces reversible pain hypersensitivity to mechanical stimuli in NaV1.x−ChR2 mice (Fig 1). This is considered as a “windup phenomenon”; moreover, the fact that not only mechanical stimuli but also light irradiation caused pain hypersensitivity (Fig 2), and increased expression of c-Fos, a marker for neuronal activity following noxious stimulation, in the dorsal horn of the spinal cord on the light-irradiated side (Fig 3) collectively suggest that continuous light irradiation may repeatedly input nociceptive signals from peripheral nerves to the central nervous system, thereby leading to central sensitization [19]. In this study, the selective NaV1.7 inhibitor DS-1971a for long-term optogenetic stimulation-induced neuropathic pain completely suppressed pain in the NaV1.x-ChR2 mice. The fact that selective NaV1.7 inhibitors are effective not only in NaV1.7-ChR2 mice but also in NaV1.8-ChR2 and NaV1.9-ChR2 mice may be attributed to the global expression of NaV1.7. Therefore, we demonstrated that selective NaV1.7 inhibitors are effective in the management of pure neuropathic pain resulting from the stimulation of the nerves responsible for pain. In clinical practice, the use of Na+ channel inhibitors that selectively act on peripheral nerves in the treatment of neuropathic pain may reduce adverse effects, such as arrhythmias and local anesthetic toxicity.

In the present study, we only observed the short-term analgesic effects of DS-1971a; however, successful initial treatment of neuropathic pain may lead to long-term analgesic effects. Furthermore, to understand the role of NaV1.8 in neuropathic pain, it is important to clarify how selective NaV1.8 inhibitors (although no selective NaV1.9 inhibitors currently exist) act in the respective NaV1.x-ChR2 mice. These limitations of the present study and issues should be addressed in future studies.

A multitude of animal models of neuropathic pain have been documented, and a substantial proportion of the knowledge pertaining to the biological alterations associated with neuropathic pain has been derived from these animal models [3,20,21]. Nevertheless, it is challenging to fully extrapolate findings from animal models to humans, as these models may not accurately recapitulate the clinical manifestations of the disease [20]. Several animal models of neuropathic pain have been developed through surgical damage to the sciatic nerve or adjacent nerves [3,21]. For example, the Chung model is typically created by ligating the fifth and sixth spinal nerves of the fourth through sixth lumbar spinal nerves that form the sciatic nerve. However, it is not possible to damage only nerve fibers expressing a specific voltage-gated Na+ channel subtype with this method of creating animal models of neuropathic pain. The light-responsive pain mice can be employed to generate noninvasively neuropathic pain models through the specific stimulation of nerve fibers expressing particular voltage-gated Na+ channel subtypes via prolonged light irradiation. Increased or hyperfunctional Na+ channels not only enhance pain electrical signals but also alter intracellular signaling molecules, which are implicated in pain plasticity [22]. The use of light-responsive pain mice helps elucidate the exact intracellular pathogenesis of neuropathic pain, which involves an increase or hyperfunction of specific voltage-gated Na+ channels. Furthermore, although pain persisted for up to 24–36 hours in this study, more chronic neuropathic pain may occur with higher light intensity or longer exposure duration, which may contribute to the understanding of chronic pain.

In conclusion, our study demonstrated that it is possible to create a reversible neuropathic pain model using light-responsive pain mice, NaV1.7−, NaV1.8−, and NaV1.9−ChR2 mice, by prolonged light irradiation and repeated excitation of peripheral nociceptors. Optogenetics may further contribute to the elucidation of the specific function of sodium channel subtypes in pain signaling and facilitate the acquisition of more in-depth knowledge and the establishment of effective treatments for pain disorders in the future.

 Supporting information Actual values in the respective graphs.

Actual values (mean ± SD) in Figs 1,2,3d, and 4 were shown.

(XLSX)

This study was conducted in the Department of Anesthesiology, Faculty of Medicine, University of Miyazaki. The authors would like to extend their gratitude to Seiya Mizuno and Satoru Takahashi (Laboratory Animal Resource Center in Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan) for the generation of genetically modified mice; Daiichi Sankyo Co., Ltd. for gifting DS-1971a; Noriko Hidaka and Kaori Kaji for their technical and secretarial assistance in this study; and Editage (www.editage.jp) for English language editing.

 References Bouhassira D. Neuropathic pain: definition, assessment and epidemiology. Rev Neurol (Paris). 2019;175(1–2):16–25. doi: 10.1016/j.neurol.2018.09.016 30385075 Baron R, Binder A, Wasner G. Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol. 2010;9(8):807–19. doi: 10.1016/S1474-4422(10)70143-5 20650402 Jaggi AS, Jain V, Singh N. Animal models of neuropathic pain. Fundam Clin Pharmacol. 2011;25(1):1–28. doi: 10.1111/j.1472-8206.2009.00801.x 20030738 Wada A. Roles of voltage-dependent sodium channels in neuronal development, pain, and neurodegeneration. J Pharmacol Sci. 2006;102(3):253–68. doi: 10.1254/jphs.crj06012x 17072104 Bennett DL, Clark AJ, Huang J, Waxman SG, Dib-Hajj SD. The role of voltage-gated sodium channels in pain signaling. Physiol Rev. 2019;99(2):1079–151. doi: 10.1152/physrev.00052.2017 30672368 Deisseroth K. Optogenetics. Nat Methods. 2011;8(1):26–9. doi: 10.1038/nmeth.f.324 21191368 Jarrin S, Finn DP. Optogenetics and its application in pain and anxiety research. Neurosci Biobehav Rev. 2019;105:200–11. doi: 10.1016/j.neubiorev.2019.08.007 31421140 Tsuda M. New approach for investigating neuropathic allodynia by optogenetics. Pain. 2019;160 Suppl 1:S53–8. doi: 10.1097/j.pain.0000000000001506 31008850 St John Smith E. Advances in understanding nociception and neuropathic pain. J Neurol. 2018;265(2):231–8. doi: 10.1007/s00415-017-8641-6 29032407 Uhelski ML, Bruce DJ, Séguéla P, Wilcox GL, Simone DA. In vivo optogenetic activation of Nav1.8+ cutaneous nociceptors and their responses to natural stimuli. J Neurophysiol. 2017;117(6):2218–23. doi: 10.1152/jn.00083.2017 28298301 Carr FB, Zachariou V. Nociception and pain: lessons from optogenetics. Front Behav Neurosci. 2014;8:69. doi: 10.3389/fnbeh.2014.00069 24723861 Daou I, Tuttle AH, Longo G, Wieskopf JS, Bonin RP, Ase AR, et al. Remote optogenetic activation and sensitization of pain pathways in freely moving mice. J Neurosci. 2013;33(47):18631–40. doi: 10.1523/JNEUROSCI.2424-13.2013 24259584 Maruta T, Kouroki S, Kurogi M, Hidaka K, Koshida T, Miura A, et al. Comparison of nocifensive behavior in NaV1.7-, NaV1.8-, and NaV1.9-channelrhodopsin-2 mice by selective optogenetic activation of targeted sodium channel subtype-expressing afferents. J Neurosci Res. 2024;102(10):e25386. doi: 10.1002/jnr.25386 39364619 Maruta T, Hidaka K, Kouroki S, Koshida T, Kurogi M, Kage Y, et al. Selective optogenetic activation of NaV1.7-expressing afferents in NaV1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli. PLoS One. 2022;17(10):e0275751. doi: 10.1371/journal.pone.0275751 36201719 Mizuno-Iijima S, Ayabe S, Kato K, Matoba S, Ikeda Y, Dinh TTH, et al. Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes. Methods. 2021;191:23–31. doi: 10.1016/j.ymeth.2020.04.007 32334080 Shinozuka T, Kobayashi H, Suzuki S, Tanaka K, Karanjule N, Hayashi N, et al. Discovery of DS-1971a, a potent, selective NaV1.7 inhibitor. J Med Chem. 2020;63(18):10204–20. doi: 10.1021/acs.jmedchem.0c00259 32392056 Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5. doi: 10.1038/nmeth.2089 22930834 Jones J, Correll DJ, Lechner SM, Jazic I, Miao X, Shaw D, et al. Selective inhibition of NaV1.8 with VX-548 for acute pain. N Engl J Med. 2023;389(5):393–405. doi: 10.1056/NEJMoa2209870 37530822 Latremoliere A, Woolf CJ. Central sensitization: a generator of pain hypersensitivity by central neural plasticity. J Pain. 2009;10(9):895–926. doi: 10.1016/j.jpain.2009.06.012 19712899 Colvin LA, Dougherty PM. Peripheral neuropathic pain: signs, symptoms, mechanisms, and causes: are they linked? Br J Anaesth. 2015;114(3):361–3. doi: 10.1093/bja/aeu323 25253232 Finnerup NB, Kuner R, Jensen TS. Neuropathic pain: from mechanisms to treatment. Physiol Rev. 2021;101(1):259–301. doi: 10.1152/physrev.00045.2019 32584191 Nemoto T, Miyazaki S, Kanai T, Maruta T, Satoh S, Yoshikawa N, et al. Nav1.7-Ca2+ influx-induced increased phosphorylations of extracellular signal-regulated kinase (ERK) and p38 attenuate tau phosphorylation via glycogen synthase kinase-3beta: priming of Nav1.7 gating by ERK and p38. Eur J Pharmacol. 2010;640(1–3):20–8. doi: 10.1016/j.ejphar.2010.04.048 20470771 10.1371/journal.pone.0323628.r001 Author response to Decision Letter 0 Submission Version 0

2 Jan 2025

 10.1371/journal.pone.0323628.r002 Decision Letter 0 Cheron Julian Academic Editor 2025 Julian Cheron This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

18 Feb 2025

PONE-D-24-58742Reversible neuropathic pain model created by long-term optogenetic nociceptor stimulation using light-responsive pain micePLOS ONE

Dear Dr. Maruta,

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Reviewer #1: Experiments are well executed.

There is one important missing information whether they did a double blind approach for pharmacological treatments in the transgenic mice.

A more precise description of the clinical relevance of their model and additional experiments are needed to establish better comparisons with existing animal models and fully support their conclusions.

Reviewer #2: In the current manuscript the authors use optogenetic models to understand whether activation of distinct Na+ channels changes pain response upon further mechanical stimulation. In summary, the authors showed that opto stimulation generated a prolonged yet reversible pain response.

The experiments in the manuscript are simple and straightforward, convincingly showing that opto stimulation of the three types of Na+ channels does elicit changes in pain response.

The use of an inhibitor further proves the claim that this changes are indeed due to the optogenetic stimulation in this animal model.

Increased c-fos labeling upon opto genetic stimulation shows further proof that the model is indeed working and operating changes at a central level, and yields an interesting observation where neurons in the dorsal horn respond in a manner dependent on their ipsilateral/contralateral location.

Overall, this manuscript is well thought out and does give enough proof to sustain the claims.

Personally, I would just edit some parts of the text, both in sentence construction as well as in some details that seem to be less precise. As an example, the authors claim cfos to be a "a marker of pain perception" and although it might be true in this particular neuronal population/context, cfos is generally used as a proxy for neuronal firing/activation rather than a marker for pain perception.

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Reviewer #1: No

Reviewer #2: Yes:  Rui de Oliveira Beleza

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Submitted filename: reviewing Kouroki et al.pdf

 10.1371/journal.pone.0323628.r003 Author response to Decision Letter 1 Submission Version 1

1 Mar 2025

Dear Dr. Julian Cheron,

Thank you for giving me the opportunity to submit a revised draft of the manuscript titled “Reversible neuropathic pain model created by long-term optogenetic nociceptor stimulation using light-responsive pain mice.” to PLOS ONE (Manuscript ID: PONE-D-24-58742).

We greatly appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback on our manuscript. We are grateful to the reviewers for their insightful comments on this paper.

We have extensively revised our paper to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript in red font on marked version (Revised manuscript with Track Changes). Here is a point-by-point response to the reviewers’ comments and concerns.

Reviewer: 1

Major comments:

1) The use of “neuropathic pain” in the description of their model is confusing. Indeed, neuropathy is large family of pain types which can be acute, a chronic state resulting from neuronal lesion with sensory alterations, combined to inflammation or metabolic alterations... In the discussion, they compare the advantage of their acute model of nociception over sciatic nerve injury that leads to chronic neuropathy, a very different clinical picture. A more precise description of the proposed model in respect to clinical relevance therefore needs to be clarified to draw the right conclusions.

In this line, the authors should give examples and references about the other animal models of neuropathic pain they are comparing theirs to (introduction, line 53) to clarify latter the potential added value and limitations of their model. Also, they refer to a previous work of theirs introducing this optogenetic-based model (introduction, line 61-65) and mention that “nociceptive pain was produced”, not neuropathic pain….

Response: Thank you for your valuable comment. We have already provided examples and references about the other animal models of neuropathic pain, which we are comparing our model to, to clarify latter the potential added value of our model in the discussion section. Kindly see Discussion, Pages 27–29: lines 311–327, “A multitude of animal models of neuropathic pain…specific voltage-gated Na+ channels”.

Our light-responsive pain mice (NaV1.x−ChR2) induce nociceptive pain on exposure to specific wavelengths, similar to the light-responsive pain mice (NaV1.8−ChR2) developed by Daou et al. Moreover, repeated nociceptive pain can lead to neuropathic pain due to central sensitization [16]. Daou et al. and we have also demonstrated that prolonged light exposure can cause neuropathic pain. We have already mentioned these points in the discussion section (Page 26: lines 294-301, “Daou et al. reported that…thereby leading to central sensitization [16]”).

2) To comprehend the importance of each sodium channel they investigate, it is also important to describe better in the introduction their expression pattern in the nociceptive and sensory fiber types, their difference in terms of electrophysiology (for instance their different sensitivity to TTX), and the state of the art knowledge about their implication in neuropathic pain as their involvement is not utterly understood. Their role also needs to be described in line with the type of pain elicited by this optogenetic-induced nociceptor stimulation.

Response: Thank you for your valuable comment. As you have accurately pointed out, in order for the reader to understand the importance of the Na+ channels we are studying, the expression pattern of each Na+ channel, their electrophysiological differences and the latest findings on their involvement in neuropathic pain should be explained. However, these findings are lengthy and would better be summarized in a review article, and cannot be explained in the introduction; we have already mentioned the role of Na+ channels, citing the review by Wada et al. [3] (Pages 5–6: lines 55–57), and further we have added text on Na channels (Pages 6–7: lines 57–70) with an additional reference [4]. Readers of this paper will probably also read the preceding paper [12]. As the role of the Na channel is also described in that preceding paper, we feel that the description of the Na+ channels in this paper is sufficient.

Changes in the text:

“In the voltage-gated Na+ channels, which consist of NaV1.1-1.9 subtypes, each subtype possesses different kinetics and expression patterns, which are reflected in the functional groupings of the peripheral sensory neurons [4]. Large DRG neurons with myelinated Aβ and Aδ fibers express mainly tetrodotoxin-sensitive (TTX-S) NaV1.1, NaV1.6, and NaV1.7. A subset of these neurons also express tetrodotoxin-resistant (TTX-R) NaV1.8, which may correspond to Aβ nociceptors. In contrast, small diameter nociceptive neurons with unmyelinated C fibers express high levels of TTX-R NaV1.8 and NaV1.9, and TTX-S NaV1.7 and NaV1.6. These differences in the Na+ channel expression are reflected in the differences in the morphology of the action potential waveform: NaV1.7 contributes to the rising phase of the action potential and amplifies subthreshold stimulation, while NaV1.8 contributes mainly to the rising phase and NaV1.9 amplifies subthreshold stimuli. Furthermore, NaV1.7, NaV1.8, and NaV1.9 have been reported to cause abnormal pain or painlessness in humans owing to gain-of-function or loss-of-function mutations [4].” (Pages 6–7: lines 57–70)

3) Description of the results should be better written.

Replace “Fig. X shows” throughout the section. For instance, on page 20 you should state “we investigated/examined c-Fos expression” instead of “we observed c-Fos expression” and then “we observed” or “this revealed” instead of “Fig.3 shows”.

Response: Thank you for your valuable comment. We have revised the text in the manuscript accordingly

Changes in the text:

“To confirm peripheral sensitization by prolonged optogenetic stimulation, we examined the blue LED light irradiation hind paw withdrawal test …in the NaV1.x–ChR2 mice (Fig 2).” (Pages 19–20: lines 219–224)

“we investigated c-Fos expression in the secondary sensory neurons in the dorsal horn of the spinal cord. We observed c-Fos expression in the ipsilateral and contralateral dorsal horn of the lumber spinal cord after long term optogenetic stimulation (Fig 3).” (Page 21: lines 237–240)

“We examined the analgesic effect of 10 and 100 mg/kg DS-1971a, which is a selective NaV1.7 inhibitor [15], … in the NaV1.7-ChR2, NaV1.8-ChR2 and NaV1.9-ChR2 mice (Fig 4).” (Page 23: lines 256–261)

4) On page 21, there is no introduction about the aim of the experiment. Why studying DS-1971a effect? Why focusing on NaV1.7 and not investigating the others? Why looking at the responses of the NaV1.8 and NaV1.9 mice after DS-1971a treatment if NaV1.7 is “globally expressed”? This also goes back to better describing the expression and role of these three sodium channels in the introduction. Your description of the results (lines 241-243) is a title of a figure legend but definitively not a result description/explanation. This must be rephrased.

Additional experiments should also be done to fully support your conclusions mentioned in the discussion. It is important to examine the long-lasting effect of the NaV1.7 inhibitor using the same treatment paradigm. Given your model elicits allodynia for 24 h, does NaV1.7 inhibition prevent hypersensitivity appearance when looking at 24 h post-optogenetic stimulation or does it only block the acute phase you investigated here and simply delays sensory alterations?

In this line, NaV expressions are altered in neuropathic pain, it would be of interest to assess whether this is the case in your model 3 h, 24 h, and 48 h after light simulation during the acute phase when DS-1971a is effective, the subacute phase (to link it with the (lack of) effect of DS-1971a at that time point), and when normal sensory response is restored, respectively.

Additionally, given DS-1971a is efficient in all mouse genotypes, you would gain crucial information i/ by examining the influence of DS-1971a in hyperalgia tests in addition to allodynia and ii/ by specifically discussing the role of A-beta sensory fibres, and C vs A-delta nociceptive fibers as the three NaV channels have different expression patterns.

In this line, it would be important to test the impact of low and high TTX doses to discriminate A fibre (low dose) from A+C fibre (high dose) implications in nociceptive and sensory tests.

Also, does blocking NaV1.8 using VX-548 or LTGO-33 for instance lead to the same result in all three genotypes in different nociceptive and sensory tests?

This is crucial for the description of your model, for the understanding of the role of each sodium channel, thereby for proposing specific therapeutic targets for specific pain types.

Response: Thank you for your valuable comment. The purpose of the experiments with DS-1971a has been added. The description of the results has also been revised.

NaV1.7 is abundantly distributed in DRG and is strongly associated with pain generation, and selective NaV1.7 inhibitors have often been studied for their analgesic effects in animal models of neuropathic pain. Fortunately, we were able to use DS-1971a in this study because Daiichi Sankyo gifted us DS-1971a.

Although DS-1971a suppressed neuropathic pain in NaV1.7-ChR2 mice as expected, it was surprising that DS-1971a also suppressed neuropathic pain in NaV1.8-ChR2 and NaV1.9-ChR2 mice. As described in Discussion, since NaV1.7 is globally expressed in various types of DRG, we hypothesize that NaV1.7 is also expressed in DRG expressing NaV1.8 and NaV1.9 and that DS-1971a also suppresses these DRG.

As the analgesic effect of DS-1971a on SNL and PSL mice in previous studies was around 2-4 h [15], in the present study, we believe that DS-1971a also temporarily alleviates neuropathic pain in our model. So, we did not observe any long-term effects of DS-1971a. However, as reviewer 1 has indicated, it is possible that NaV expression is increased in our model of neuropathic pain, and initial administration of DS-1971a may provide long-term pain relief by suppressing it. To elucidate these issues, analysis of changes in NaV expression should also be performed. Therefore, we have not included these matters in this study and would like to leave them for future research.

Selective Na+ channel inhibitors against NaV1.7 and NaV1.8 exist, but there are currently no selective inhibitors against NaV1.9. As reviewer 1 has accurately pointed out, additional experiments with selective inhibitors against NaV1.8, such as VX-548, or with TTX would provide more insight, but due to cost and time constraints, we would like to limit the current report to experiments with DS-1971a. However, the fact that we were able to demonstrate the analgesic effect of DS-1971a on neuropathic pain is of great significance for future clinical practice.

Changes in the text:

“Clinical trials of selective Na+ channel inhibitors are underway for the treatment of acute postoperative pain and neuropathic pain [17]. We have examined the analgesic effect of 10 and 100 mg/kg DS-1971a, which is a selective NaV1.7 inhibitor [15], on long-term optogenetic stimulation-induced neuropathic pain in NaV1.x–ChR2 mice. DS-1971a at both 10 mg/kg and 100 mg/kg almost completely suppressed long-term optogenetic stimulation-induced neuropathic pain in the NaV1.7-ChR2, NaV1.8-ChR2 and NaV1.9-ChR2 mice (Fig 4).” (Page 23: lines 255–261)

Minor comments

1) c-Fos expression is not a pain perception marker as stated several times in the manuscript. It reflects neuronal activity. In this case linked to nociceptor stimulation yet its expression is not specific to pain. This has therefore to be modified.

Response: Thank you for your valuable comment. We have modified the text as per your suggestion.

Changes in the text:

“c-Fos, a marker for neuronal activity following noxious stimulation,” (Page 3: line 36) (Page 26: line 298)

2) Below-mentioned sentences in introduction (lines 65-67) belong to the discussion or need to be rephrased:

“Previously, Daou et al. generated NaV1.8−ChR2 mice using the NaV1.8−Cre recombinase knock-in mouse line and reported results similar to ours [10]. They also reported the occurrence of transient neuropathic pain following long-term exposure to blue light.”

Response: Thank you for your valuable comment. We have rephrased the text as per your suggestion.

Changes in the text:

“Previously, Daou et al. reported the occurrence of transient neuropathic pain following long-term exposure to blue light in NaV1.8−ChR2 mice [11].” (Pages 7–8: lines 78–80)

3) Materials & Methods, line 83. The authors mention that the experimenters are blinded to the mouse genotypes. Are they also blinded to the treatments (DS-1971a vs vehicle) used as this is also crucial?

Response: Thank you for your valuable comment. We have modified the text as per your suggestion.

Changes in the text:

“Mice in each group were randomly selected, and the experimenter was blinded to the mouse genotype and drug treatment.” (Page 9: lines 95–97)

4) Materials & Methods, lines 149-151. The below-mentioned sentence goes into the results section:

“Mice were confirmed hypersensitive by von Frey test 1 hour after a prolonged (30 min), suprathreshold (7.5 mW for NaV1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2) light stimulation.”

Response: Thank you for your valuable comment. However, we believe that this text should be in the Materials & Methods section. Instead, the irradiation times have been added to figure legend.

Changes in the text:

“c-Fos expression in the ipsilateral and contralateral dorsal horn of the lumber spinal cord after long term (30 min) optogenetic stimulation (7.5 mW for NaV1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2) are shown in (a) NaV1.7–ChR2, (b) NaV1.8–ChR2, (c) NaV1.9–ChR2 mice.” (Page 21: line 245)

5) Results. Line 208. “Continuous” should be replaced by “prolonged”.

Response: Thank you for your valuable comment. We have modified the text as per your suggestion.

Changes in the text:

“To confirm peripheral sensitization by prolonged optogenetic stimulation, we examined the blue LED light irradiation hind paw withdrawal test before and 1 hour after prolonged (30 min) optogenetic stimulation.” (Pages 19-20: lines 219-221)

6) Discussion is lacking an introductory a summary of your findings.

Response: Thank you for your valuable comment. We have added the text as per your suggestion.

Changes in the text:

“In this study, we demonstrated that a reversible neuropathic pain state was established persisting for a minimum of 24 hours when light-responsive pain mice (NaV1.x-ChR2 mice) was irradiated with light of an intensity that consistently elicited pain. Furthermore, the mice also showed hypersensitivity to light irradiation and mechanical stimulation. The c-Fos expression increased in the dorsal horn of the spinal cord on the light irradiated side. DS-1971a, a selective NaV1.7 inhibitor, was effective in attenuating neuropathic pain in all the light-responsive pain mice.” (Page 24: lines 273–279)

7) Discussion (lines 276-279). This belongs to the result section.

Response: Thank you for your valuable comment. We have added and revised the texts as per your suggestion.

Changes in the text:

“DS-1971a at both 10 mg/kg and 100 mg/kg almost completely suppressed long-term optogenetic stimulation-induced neuropathic pain in the NaV1.7-ChR2, NaV1.8-ChR2 and NaV1.9-ChR2 mice (Fig 4).” (Page 23: lines 258–261)

“In this study, the selective NaV1.7 inhibitor DS-1971a for long-term optogenetic stimulation-induced neuropathic pain completely suppressed pain in the NaV1.x-ChR2 mice.” (Pages 26–27: lines 301–303)

8) Discussion (line 286). Please give references.

Response: Thank you for your valuable comment. We have added a reference.

Changes in the text:

“…from these animal models [19].” (Page 27: line 313)

Reviewer: 2

1) Overall, this manuscript is well thought out and does give enough proof to sustain the claims.

Personally, I would just edit some parts of the text, both in sentence construction as well as in some

Submitted filename: Response to Reviewers.docx

 10.1371/journal.pone.0323628.r004 Decision Letter 1 Cheron Julian Academic Editor 2025 Julian Cheron This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

16 Mar 2025

PONE-D-24-58742R1Reversible neuropathic pain model created by long-term optogenetic nociceptor stimulation using light-responsive pain micePLOS ONE

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Reviewers' comments:

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Submitted filename: Reviewing R1_PLoS One.docx

 10.1371/journal.pone.0323628.r005 Author response to Decision Letter 2 Submission Version 2

4 Apr 2025

Dear Dr. Julian Cheron,

Thank you for giving me the opportunity to submit a revised draft of the manuscript titled “Reversible neuropathic pain model created by long-term optogenetic nociceptor stimulation using light-responsive pain mice.” to PLoS ONE (Manuscript ID: PONE-D-24-58742).

We greatly appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback on our manuscript. We are grateful to the reviewers for their insightful comments on this paper.

We have extensively revised our paper to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript in red font in the marked version (Revised manuscript with Track Changes). Here is a point-by-point response to the reviewers’ comments and concerns.

Reviewer: 1

Major comments:

1) The use of “neuropathic pain” in the description of their model is confusing. Indeed, neuropathy is large family of pain types which can be acute, a chronic state resulting from neuronal lesion with sensory alterations, combined to inflammation or metabolic alterations... In the discussion, they compare the advantage of their acute model of nociception over sciatic nerve injury that leads to chronic neuropathy, a very different clinical picture. A more precise description of the proposed model in respect to clinical relevance therefore needs to be clarified to draw the right conclusions.

In this line, the authors should give examples and references about the other animal models of neuropathic pain they are comparing theirs to (introduction, line 53) to clarify latter the potential added value and limitations of their model. Also, they refer to a previous work of theirs introducing this optogenetic-based model (introduction, line 61-65) and mention that “nociceptive pain was produced”, not neuropathic pain….

Response: Thank you for your valuable comment. We have already provided examples and references about the other animal models of neuropathic pain, which we are comparing our model to, to clarify latter the potential added value of our model in the discussion section. Kindly see Discussion, Pages 27–29: lines 311–327, “A multitude of animal models of neuropathic pain…specific voltage-gated Na+ channels”.

The authors did not include any example nor references of other models of neuropathic pain in the introduction (as previously asked) to later compare the supposed advantages of their model.

Indeed, in the discussion, they did not get into the specificities of their model vs others in respect to the fact that existing models “may not accurately recapitulate the clinical manifestations of the disease”. I still do not see how their model addresses this question. I understand their model produces central sensitization (which may lead to neuropathic pain) and that the model can be of importance for studying the initial mechanisms of central sensitization, but there is no data supporting that this is a clinically relevant model of neuropathic pain, notably because neuropathic pain is a chronic state (for instance in the Chung model which is the only one they discuss) while sensory alterations are reversible after 24 hours in their model of acute neuropathy.

Our light-responsive pain mice (NaV1.x−ChR2) induce nociceptive pain on exposure to specific wavelengths, similar to the light-responsive pain mice (NaV1.8−ChR2) developed by Daou et al. Moreover, repeated nociceptive pain can lead to neuropathic pain due to central sensitization [16]. Daou et al. and we have also demonstrated that prolonged light exposure can cause neuropathic pain. We have already mentioned these points in the discussion section (Page 26: lines 294-301, “Daou et al. reported that…thereby leading to central sensitization [16]”).

Response 2: Thank you for your valuable comment. Conventional animal models of neuropathic pain in which peripheral nerves are physically damaged may result in motor neuropathy and sensory neuropathy other than pain. Optogenetics-based neuropathic pain models are superior to conventional animal models in that they can precisely target pain nerves. Furthermore, by targeting only the pain nerves, they may more faithfully reproduce the clinical manifestations of neuropathic pain. The text has been added in light of the above.

Changes in the text:

“The most widely employed models of neuropathic pain attributed to peripheral neuropathy in rodents are physical injury models of peripheral nerves, including the spared nerve injury (SNI), chronic constriction injury (CCI), partial sciatic nerve ligation (PSL), partial sciatic nerve ligation (PSL), and spinal nerve ligation (SNL) (Chung model) models [3]. Although these models produce allodynia, they can cause motor and/or non-pain sensory neuropathies.” (Page 5: lines 52–57)

“Such optogenetics-based neuropathic pain models are superior to conventional animal models in that they can target only the sensory nerves responsible for pain.” (Page 8: lines 85–87)

“Furthermore, although pain persisted for up to 24–36 hours in this study, more chronic neuropathic pain may occur with higher light intensity or longer exposure duration, which may contribute to the understanding of chronic pain.” (Page 30: lines 342–344)

4) On page 21, there is no introduction about the aim of the experiment. Why studying DS-1971a effect? Why focusing on NaV1.7 and not investigating the others? Why looking at the responses of the NaV1.8 and NaV1.9 mice after DS-1971a treatment if NaV1.7 is “globally expressed”? This also goes back to better describing the expression and role of these three sodium channels in the introduction. Your description of the results (lines 241-243) is a title of a figure legend but definitively not a result description/explanation. This must be rephrased.

Additional experiments should also be done to fully support your conclusions mentioned in the discussion. It is important to examine the long-lasting effect of the NaV1.7 inhibitor using the same treatment paradigm. Given your model elicits allodynia for 24 h, does NaV1.7 inhibition prevent hypersensitivity appearance when looking at 24 h post-optogenetic stimulation or does it only block the acute phase you investigated here and simply delays sensory alterations?

In this line, NaV expressions are altered in neuropathic pain, it would be of interest to assess whether this is the case in your model 3 h, 24 h, and 48 h after light simulation during the acute phase when DS-1971a is effective, the subacute phase (to link it with the (lack of) effect of DS-1971a at that time point), and when normal sensory response is restored, respectively.

Additionally, given DS-1971a is efficient in all mouse genotypes, you would gain crucial information i/ by examining the influence of DS-1971a in hyperalgia tests in addition to allodynia and ii/ by specifically discussing the role of A-beta sensory fibres, and C vs A-delta nociceptive fibers as the three NaV channels have different expression patterns.

In this line, it would be important to test the impact of low and high TTX doses to discriminate A fibre (low dose) from A+C fibre (high dose) implications in nociceptive and sensory tests.

Also, does blocking NaV1.8 using VX-548 or LTGO-33 for instance lead to the same result in all three genotypes in different nociceptive and sensory tests?

This is crucial for the description of your model, for the understanding of the role of each sodium channel, thereby for proposing specific therapeutic targets for specific pain types.

Response: Thank you for your valuable comment. The purpose of the experiments with DS-1971a has been added. The description of the results has also been revised.

NaV1.7 is abundantly distributed in DRG and is strongly associated with pain generation, and selective NaV1.7 inhibitors have often been studied for their analgesic effects in animal models of neuropathic pain. Fortunately, we were able to use DS-1971a in this study because Daiichi Sankyo gifted us DS-1971a.

Although DS-1971a suppressed neuropathic pain in NaV1.7-ChR2 mice as expected, it was surprising that DS-1971a also suppressed neuropathic pain in NaV1.8-ChR2 and NaV1.9-ChR2 mice. As described in Discussion, since NaV1.7 is globally expressed in various types of DRG, we hypothesize that NaV1.7 is also expressed in DRG expressing NaV1.8 and NaV1.9 and that DS-1971a also suppresses these DRG.

As the analgesic effect of DS-1971a on SNL and PSL mice in previous studies was around 2-4 h [15], in the present study, we believe that DS-1971a also temporarily alleviates neuropathic pain in our model. So, we did not observe any long-term effects of DS-1971a.

They authors did not observe or they did not examine? I still think it is an important experiment to see whether blocking the initial phase of nociception is sufficient to prevent central sensitization.

However, as reviewer 1 has indicated, it is possible that NaV expression is increased in our model of neuropathic pain, and initial administration of DS-1971a may provide long-term pain relief by suppressing it. To elucidate these issues, analysis of changes in NaV expression should also be performed. Therefore, we have not included these matters in this study and would like to leave them for future research.

Again DS-1971a long-term effect should be studied and depending on the results examining NaV expression changes may or may not be of importance.

Selective Na+ channel inhibitors against NaV1.7 and NaV1.8 exist, but there are currently no selective inhibitors against NaV1.9. As reviewer 1 has accurately pointed out, additional experiments with selective inhibitors against NaV1.8, such as VX-548, or with TTX would provide more insight, but due to cost and time constraints, we would like to limit the current report to experiments with DS-1971a. However, the fact that we were able to demonstrate the analgesic effect of DS-1971a on neuropathic pain is of great significance for future clinical practice.

I would have agreed if the authors had focused on NaV1.7 only but they examined the importance of several channels that could have different impact on the establishment of central sensitization. In the end they do not examine deep enough the importance of NaV1.7 nor do they investigate the importance of at least NaV1.8 (as NaV1.9 has no selective inhibitor).

As they point out DS-1971a could be of importance for clinical practice, but they cite a reference supporting this already, thus mellowing the importance of their results, and we circle back to a better understanding of their model in respect to clinical picture.

Experiments 1 and 3 are mandatory and experiment 2 remains optional depending on the result of experiment 1.

Changes in the text:

“Clinical trials of selective Na+ channel inhibitors are underway for the treatment of acute postoperative pain and neuropathic pain [17]. We have examined the analgesic effect of 10 and 100 mg/kg DS-1971a, which is a selective NaV1.7 inhibitor [15], on long-term optogenetic stimulation-induced neuropathic pain in NaV1.x–ChR2 mice. DS-1971a at both 10 mg/kg and 100 mg/kg almost completely suppressed long-term optogenetic stimulation-induced neuropathic pain in the NaV1.7-ChR2, NaV1.8-ChR2 and NaV1.9-ChR2 mice (Fig 4).” (Page 23: lines 255–261)

Response 2: Thank you for your valuable comment. As neuropathic pain in light-responsive pain mice is reversible, over time it becomes impossible to determine whether the analgesia is due to DS-1971a or to a natural process. Furthermore, the effects of DS-1971a last for 2–4 hours. Therefore, I have only observed the effects of DS-1971a for a short time immediately after the onset of neuropathic pain in this study. And unfortunately, I do not currently have temporary possession of the NaV1.x-ChR2 mice due to a change of laboratory in April this year. It is also expected that it will take some time to set up the new laboratory. Therefore, while we fully appreciate that the additional experiments recommended by reviewer would make our study more meaningful, additional experiments could not be performed. Thus, I hope that additional experiments on the long-term effects of DS-1971a and selective NaV1.8 inhibitors could be left for future research. These have been added to the Discussion section as limitations to this study.

Changes in the text:

“In the present study, we only observed the short-term analgesic effects of DS-1971a; however, successful initial treatment of neuropathic pain may lead to long-term analgesic effects. Furthermore, to understand the role of NaV1.8 in neuropathic pain, it is important to clarify how selective NaV1.8 inhibitors (although no selective NaV1.9 inhibitors currently exist) act in the respective NaV1.x-ChR2 mice. These limitations of the present study and issues should be addressed in future studies.” (Page 28: lines 319–324)

Minor comments

4) Materials & Methods, lines 149-151. The below-mentioned sentence goes into the results section:

“Mice were confirmed hypersensitive by von Frey test 1 hour after a prolonged (30 min), suprathreshold (7.5 mW for NaV1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2) light stimulation.”

Response: Thank you for your valuable comment. However, we believe that this text should be in the Materials & Methods section. Instead, the irradiation times have been added to figure legend.

Changes in the text:

“c-Fos expression in the ipsilateral and contralateral dorsal horn of the lumber spinal cord after long term (30 min) optogenetic stimulation (7.5 mW for NaV1.7–ChR2, 1.8 mW for NaV1.8–ChR2, and 2.25 mW for NaV1.9–ChR2) are shown in (a) NaV1.7–ChR2, (b) NaV1.8–ChR2, (c) NaV1.9–ChR2 mice.” (Page 21: line 245)

This is still not a description of the immunofluorescence protocol, but as they included the details in the figure legend, they can simplify by stating that mice that showed hypersensivity using the von Frey test were then fixed for immunofluorescence analysis.

Response 2: Thank you for your valuable comment. We have added the text in the Result section as per your suggestion.

Changes in the text:

“Mice that demonstrated hypersensitivity using the von Frey test were fixed for immunofluorescence analysis.” (Page 22: lines 245–247)

Submitted filename: Response_to_Reviewers_auresp_2.docx

 10.1371/journal.pone.0323628.r006 Decision Letter 2 Cheron Julian Academic Editor 2025 Julian Cheron This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 2

11 Apr 2025

Reversible neuropathic pain model created by long-term optogenetic nociceptor stimulation using light-responsive pain mice

PONE-D-24-58742R2

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Kind regards,

Julian Cheron

Academic Editor

PLOS ONE

 10.1371/journal.pone.0323628.r007 Acceptance letter Cheron Julian Academic Editor 2025 Julian Cheron This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PONE-D-24-58742R2

PLOS ONE

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