Exposure to Green Light-emitting Diodes Alleviates Endometriosis-associated Pain in Mice with Induced Deep Endometriosis

Extensive preclinical and clinical studies have shown that exposure to green light-emitting diodes (gLED) exerts analgesic effect. We hypothesized that exposure to gLED alleviates endometriosis-associated pain. To test this, we induced deep endometriosis in 16 Balb/C female mice. Four weeks post-induction, the mice were randomized into gLED and white LED (wLED) groups (n = 8 each), exposed daily to gLED (520–525 nm in wavelength) and wLED (500–700 nm), respectively, for 8 h over 2 weeks, and were then sacrificed. Endometriotic lesions were weighed and analyzed via immunohistochemistry for E-cadherin, α-Sma, phosphorylated Creb (p-Creb), adrenergic receptor β2 (Adrb2), α7 nicotinic acetylcholine receptor (α7nAChR), κ- and µ-opioid receptors (Kor and Mor), and cannabinoid receptor 1 and 2 (Cb1 and Cb2), alongside Masson trichrome staining. Pain and depression-like behavior was assessed via hotplate tests, tail suspension tests and sucrose preference tests, and serum noradrenaline levels were quantified. Exposed to gLED significantly reduced lesion weight, shortened tail suspension immobility time and increased hotplate latency compared to wLED. Lesions from gLED mice exhibited significantly reduced α-Sma staining, marginally reduced fibrosis, and elevated systemic noradrenaline. IHC analysis revealed significantly lower staining of p-Creb, Adrb2, Cb1 and Cb2 but increased α7nAChR and Mor staining in gLED lesions. These results demonstrate that exposure to gLED significantly alleviates endometriosis-induced pain while decelerates lesional progression in mice with induced deep endometriosis, possibly through the suppression of the Creb-Adrb2-Cb2 signaling pathway as well as increased systemic noradrenaline levels. This raises the possibility of using gLED as a stand-alone or an adjuvant non-pharmacological therapy to alleviate endometriosis-associated pain. Similar content being viewed by others Data Availability The data presented in this study are available upon written request from the corresponding author explaining the use and purposes.

Giudice LC, Kao LC. Endometriosis. Lancet. 2004;364:1789–99. https://doi.org/10.1016/S0140-6736(04)17403-5. Vercellini P, Buggio L, Frattaruolo MP, Borghi A, Dridi D, Somigliana E. Medical treatment of endometriosis-related pain. Best Pract Res Clin Obstet Gynaecol. 2018. https://doi.org/10.1016/j.bpobgyn.2018.01.015. doi:S1521-6934(18)30033-6 [pii]. Burla L, Kalaitzopoulos DR, Metzler JM, Scheiner D, Imesch P. Popularity of endocrine endometriosis drugs and limited alternatives in the present and foreseeable future: A survey among 1420 affected women. Eur J Obstet Gynecol Reprod Biol. 2021;262:232–8. https://doi.org/10.1016/j.ejogrb.2021.05.040. Guo SW, Groothuis PG. Is it time for a paradigm shift in drug research and development in endometriosis/adenomyosis? Hum Reprod Update. 2018;24:577–98. https://doi.org/10.1093/humupd/dmy020. Pellicer A, Taylor HS, Alberich-Bayarri A, Liu Y, Gamborg M, Barletta KE, Pinton P, Heiser PW, Bagger YZ. Quinagolide vaginal ring for reduction of endometriotic lesions: Results from the QLARITY trial. Eur J Obstet Gynecol Reprod Biol. 2025;310:113946. https://doi.org/10.1016/j.ejogrb.2025.113946. De Hoyos G, Ramos-Sostre D, Torres-Reveron A, Barros-Cartagena B, Lopez-Rodriguez V, Nieves-Vazquez C, Santiago-Saavedra F, Appleyard CB, Castro EM, Flores I. Efficacy of an environmental enrichment intervention for endometriosis: a pilot study. Front Psychol. 2023;14:1225790. https://doi.org/10.3389/fpsyg.2023.1225790. Samami E, Shahhosseini Z, Khani S, Elyasi F. Pain-focused psychological interventions in women with endometriosis: A systematic review. Neuropsychopharmacol Rep. 2023;43:310–9. https://doi.org/10.1002/npr2.12348. Kemper KJ. Let there be light. Research on phototherapy, light therapy, and photobiomodulation for healing - Alternative therapy becomes mainstream. Complement Ther Med. 2018;41:A1–6. https://doi.org/10.1016/j.ctim.2018.10.007. Melrose S. Seasonal affective disorder: an overview of assessment and treatment approaches. Depress Res Treat. 2015;2015(178564). https://doi.org/10.1155/2015/178564. Oren DA, Brainard GC, Johnston SH, Joseph-Vanderpool JR, Sorek E, Rosenthal NE. Treatment of seasonal affective disorder with green light and red light. Am J Psychiatry. 1991;148:509–11. https://doi.org/10.1176/ajp.148.4.509. van Maanen A, Meijer AM, van der Heijden KB, Oort FJ. The effects of light therapy on sleep problems: A systematic review and meta-analysis. Sleep Med Rev. 2016;29:52–62. https://doi.org/10.1016/j.smrv.2015.08.009. Iaccarino HF, Singer AC, Martorell AJ, Rudenko A, Gao F, Gillingham TZ, Mathys H, Seo J, Kritskiy O, Abdurrob F, et al. Gamma frequency entrainment attenuates amyloid load and modifies microglia. Nature. 2016;540:230–5. https://doi.org/10.1038/nature20587. Ibrahim MM, Patwardhan A, Gilbraith KB, Moutal A, Yang X, Chew LA, Largent-Milnes T, Malan TP, Vanderah TW, Porreca F, et al. Long-lasting antinociceptive effects of green light in acute and chronic pain in rats. Pain. 2017;158:347–60. https://doi.org/10.1097/j.pain.0000000000000767. Noseda R, Bernstein CA, Nir RR, Lee AJ, Fulton AB, Bertisch SM, Hovaguimian A, Cestari DM, Saavedra-Walker R, Borsook D, et al. Migraine photophobia originating in cone-driven retinal pathways. Brain. 2016;139:1971–86. https://doi.org/10.1093/brain/aww119. Martin LF, Patwardhan AM, Jain SV, Salloum MM, Freeman J, Khanna R, Gannala P, Goel V, Jones-MacFarland FN, Killgore WD, et al. Evaluation of green light exposure on headache frequency and quality of life in migraine patients: A preliminary one-way cross-over clinical trial. Cephalalgia. 2021;41:135–47. https://doi.org/10.1177/0333102420956711. Martin L, Porreca F, Mata EI, Salloum M, Goel V, Gunnala P, Killgore WDS, Jain S, Jones-MacFarland FN, Khanna R, et al. Green light exposure improves pain and quality of life in fibromyalgia patients: a preliminary one-way crossover clinical trial. Pain Med. 2021;22:118–30. https://doi.org/10.1093/pm/pnaa329. Ventura L, do Espirito-Santo RF, Keaser M, Zhang Y, Ro JY, Da Silva JT. Green light exposure reduces primary hyperalgesia and proinflammatory cytokines in a rodent model of knee osteoarthritis: shedding light on sex differences. Biomedicines. 2024;12(9). https://doi.org/10.3390/biomedicines12092005 Cheng KML, Slepian MJ, Patwardhan AM, Ibrahim MM. Mechanisms and pathways of pain photobiomodulation: a narrative review. J Pain. 2021;22(7):763–77. https://doi.org/10.1016/j.jpain.2021.02.005. Khanna R, Patwardhan A, Yang X, Li W, Cai S, Ji Y, Chew LA, Dorame A, Bellampalli SS, Schmoll RW, Gordon J, Moutal A, Vanderah TW, Porreca F, Ibrahim MM. Development and characterization of an injury-free model of functional pain in rats by exposure to red light. J Pain. 2019;20(11):1293–306. https://doi.org/10.1016/j.jpain.2019.04.008. Martin LF, Moutal A, Cheng K, Washington SM, Calligaro H, Goel V, Kranz T, Largent-Milnes TM, Khanna R, Patwardhan A, et al. Green light antinociceptive and reversal of thermal and mechanical hypersensitivity effects rely on endogenous opioid system stimulation. J Pain. 2021;22:1646–56. https://doi.org/10.1016/j.jpain.2021.05.006. Tang YL, Liu AL, Lv SS, Zhou ZR, Cao H, Weng SJ, Zhang YQ. Green light analgesia in mice is mediated by visual activation of enkephalinergic neurons in the ventrolateral geniculate nucleus. Sci Transl Med. 2022;14:eabq6474. https://doi.org/10.1126/scitranslmed.abq6474. Wu XQ, Tan B, Du Y, Yang L, Hu TT, Ding YL, Qiu XY, Moutal A, Khanna R, Yu J, et al. Glutamatergic and GABAergic neurons in the vLGN mediate the nociceptive effects of green and red light on neuropathic pain. Neurobiol Dis. 2023;183:106164. https://doi.org/10.1016/j.nbd.2023.106164. Cao P, Zhang M, Ni Z, Song XJ, Yang CL, Mao Y, Zhou W, Dong WY, Peng X, Zheng C, et al. Green light induces antinociception via visual-somatosensory circuits. Cell Rep. 2023;42:112290. https://doi.org/10.1016/j.celrep.2023.112290. Yan D, Liu X, Guo SW. The establishment of a mouse model of deep endometriosis. Hum Reprod. 2019;34:235–47. https://doi.org/10.1093/humrep/dey361. National Research Council of the National Academies. Guide for the care and use of laboratory animals (8th edition). Washington, DC: The National Academies Press. 2011. Crowley JJ, Blendy JA, Lucki I. Strain-dependent antidepressant-like effects of citalopram in the mouse tail suspension test. Psychopharmacology. 2025;183:257 – 64. https://doi.org/10.1007/s00213-005-0166-5 Primo MJ, Fonseca-Rodrigues D, Almeida A, Teixeira PM, Pinto-Ribeiro F. Sucrose preference test: A systematic review of protocols for the assessment of anhedonia in rodents. Eur Neuropsychopharmacol. 2023;77:80–92. https://doi.org/10.1016/j.euroneuro.2023.08.496. Long Q, Liu X, Qi Q, Guo SW. Chronic stress accelerates the development of endometriosis in mouse through adrenergic receptor beta2. Hum Reprod. 2016;31:2506–19. https://doi.org/10.1093/humrep/dew237. Hao M, Liu X, Guo SW. Activation of alpha7 nicotinic acetylcholine receptor retards the development of endometriosis. Reprod Biol Endocrinol. 2022;20:85. https://doi.org/10.1186/s12958-022-00955-w. Allam S, Paris E, Lazcano I, Bitterman P, Basu S, O’Donnell J, Barua A. Detection of cannabinoid receptor expression by endometriotic lesions in women with endometriosis as an alternative to opioid-based pain medication. J Immunol Res. 2022;2022(4323259). https://doi.org/10.1155/2022/4323259. Liu X, Zhang Q, Guo SW. Histological and immunohistochemical characterization of the similarity and difference between ovarian endometriomas and deep infiltrating endometriosis. Reprod Sci. 2018;25:329–40. https://doi.org/10.1177/1933719117718275. R Core Team. R: A language and environment for statistical computing. Austria: R Foundation for Statistical Computing. Vienna; 2021. Catao MH, Costa RO, Nonaka CF, Junior RL, Costa IR. Green LED light has anti-inflammatory effects on burns in rats. Burns. 2016;42:392–6. https://doi.org/10.1016/j.burns.2015.07.003. Schubert MM, Eduardo FP, Guthrie KA, Franquin J-C, Bensadoun R-JJ, Migliorati CA, Lloid CME, Eduardo CP, Walter N-F, Marques MM, et al. A phase III randomized double-blind placebo-controlled clinical trial to determine the efficacy of low level laser therapy for the prevention of oral mucositis in patients undergoing hematopoietic cell transplantation. Support Care Cancer. 2007;15(10):1145–54. https://doi.org/10.1007/s00520-007-0238-7. Uzunbajakava NETD, Botchkareva NV, Dierickx C, Bjerring P, Town G. Highlighting nuances of blue light phototherapy: Mechanisms and safety considerations. J Biophotonics. 2023;16:e202200257. https://doi.org/10.1002/jbio.202200257. Tantiyavarong J, Charoensuksira S, Meephansan J, Hanvivattanakul S, Rayanasukha Y, Boonkoom T, Tantisantisom K. Red and green LED light therapy: a comparative study in androgenetic alopecia. Photodermatol Photoimmunol Photomed. 2024;40(6):e13004. https://doi.org/10.1111/phpp.13004. Ismail KA, Curfman V, Park J, Louis EM, Korah H, Washington SM, Ibrahim MM, Martin LF. Optimizing photoneuromodulation techniques to evaluate the role of green light-emitting diodes in pain management. J Vis Exp. 2025. https://doi.org/10.3791/67821. Martin LF, Cheng K, Washington SM, Denton M, Goel V, Khandekar M, Largent-Milnes TM, Patwardhan A, Ibrahim MM. Green light exposure elicits anti-inflammation, endogenous opioid release and dampens synaptic potentiation to relieve post-surgical pain. J Pain. 2023;24:509–29. https://doi.org/10.1016/j.jpain.2022.10.011. O’Brien MS, Richter E, Woodward T, Bradshaw HB, McDougall JJ. Visual exposure to green light therapy reduces knee joint pain and alters the lipidome in osteoarthritic rats. Pain. 2025;166:1274–84. https://doi.org/10.1097/j.pain.0000000000003458. Guo SW, Zhang Q, Liu X. Social psychogenic stress promotes the development of endometriosis in mouse. Reprod Biomed Online. 2017;34:225–39. https://doi.org/10.1016/j.rbmo.2016.11.012. Bilgic E, Guzel E, Kose S, Aydin MC, Karaismailoglu E, Akar I, Usubutun A, Korkusuz P. Endocannabinoids modulate apoptosis in endometriosis and adenomyosis. Acta Histochem. 2017;119:523–32. https://doi.org/10.1016/j.acthis.2017.05.005. Pertovaara A. Noradrenergic pain modulation. Prog Neurobiol. 2006;80:53–83. https://doi.org/10.1016/j.pneurobio.2006.08.001. Pertovaara A. The noradrenergic pain regulation system: a potential target for pain therapy. Eur J Pharmacol. 2013;716:2–7. https://doi.org/10.1016/j.ejphar.2013.01.067. Yoo YC, Oh JH, Kwon TD, Lee YK, Bai SJ. Analgesic mechanism of electroacupuncture in an arthritic pain model of rats: a neurotransmitter study. Yonsei Med J. 2011;52:1016–21. https://doi.org/10.3349/ymj.2011.52.6.1016. Obata H. Analgesic mechanisms of antidepressants for neuropathic pain. Int J Mol Sci. 2017;18. https://doi.org/10.3390/ijms18112483. Jasmin L, Narasaiah M, Tien D. Noradrenaline is necessary for the hedonic properties of addictive drugs. Vascul Pharmacol. 2006;45:243–50. https://doi.org/10.1016/j.vph.2005.08.030. Espay AJ, LeWitt PA, Kaufmann H. Norepinephrine deficiency in Parkinson’s disease: the case for noradrenergic enhancement. Mov Disord. 2014;29:1710–9. https://doi.org/10.1002/mds.26048. Jana B, Calka J. Role of beta-adrenergic receptor subtypes in pig uterus contractility with inflammation. Sci Rep. 2021;11:11512. https://doi.org/10.1038/s41598-021-91184-5. Jana B, Calka J, Bulc M. Roles of alpha-2-adrenergic receptor isoforms in inflamed pig uterus contractility in vitro. Theriogenology. 2022;183:41–52. https://doi.org/10.1016/j.theriogenology.2022.01.025. Guo SW, Mao X, Ma Q, Liu X. Dysmenorrhea and its severity are associated with increased uterine contractility and overexpression of oxytocin receptor (OTR) in women with symptomatic adenomyosis. Fertil Steril. 2013;99:231–40. https://doi.org/10.1016/j.fertnstert.2012.08.038. Bulletti C, Rossi S, Albonetti A, Polli VV, De Ziegler D, Massoneau M, Flamigni C. Uterine contractility in patients with endometriosis. J Am Assoc Gynecol Laparosc. 1996;3. https://doi.org/10.1016/s1074-3804(96)80143-5 Leyendecker G, Kunz G, Wildt L, Beil D, Deininger H. Uterine hyperperistalsis and dysperistalsis as dysfunctions of the mechanism of rapid sperm transport in patients with endometriosis and infertility. Hum Reprod (Oxford England). 1996;11:1542–51. https://doi.org/10.1093/oxfordjournals.humrep.a019435. Abobeleira JP, Neto AC, Mauersberger J, Salazar M, Botelho M, Fernandes AS, Martinho M, Serrao MP, Rodrigues AR, Almeida H, et al. Evidence of browning and inflammation features in visceral adipose tissue of women with endometriosis. Arch Med Res. 2024;55:103064. https://doi.org/10.1016/j.arcmed.2024.103064. Willner P. Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology. 2005;52:90–110. https://doi.org/10.1159/000087097. Slattery DA, Cryan JF. Using the rat forced swim test to assess antidepressant-like activity in rodents. Nat Protoc. 2012;7:1009–14. https://doi.org/10.1038/nprot.2012.044. Yin B, Liu X, Guo SW. Caloric restriction dramatically stalls lesion growth in mice with induced endometriosis. Reprod Sci. 2018;25:1024–36. https://doi.org/10.1177/1933719118756755. Gurses G, Omeroglu Akkoc FI, Akti A, Korez MK. Effectiveness of wearing glasses with green lenses on dental anxiety for third-molar surgery: A randomized clinical trial. J Am Dent Assoc. 2024;155(e1):496–503. https://doi.org/10.1016/j.adaj.2024.02.006. Thurnherr N, Burla L, Metzler JM, File B, Imesch P. Attitudes and perceptions of affected women towards endocrine endometriosis therapy: an international survey based on free-word association networks. Hum Reprod. 2024;39:83–92. https://doi.org/10.1093/humrep/dead221. Nelli AWM, Gulur P. Green light-based analgesia - novel nonpharmacological approach to fibromyalgia pain: a pilot study. Pain Physician. 2023;26(4):403–10. Chen J, He Q, Xie H, Gu B, Zhou L, Jiang D, Xie H, Liang L, Zhou Z, Zhang H. Autophagy modulation by hADSCs and green light therapy alleviates inflammation and promotes functional recovery after spinal cord injury. Stem Cell Res Ther. 2025;16(1):256. https://doi.org/10.1186/s13287-025-04367-6. You Y, Huo K, He L, Wang T, Zhao L, Li R, Cheng X, Ma X, Yue Z, Siwko S, Wang N, Liao L, Liu M, Luo J. GnIH secreted by green light exposure, regulates bone mass through the activation of Gpr147. Bone Res. 2025;13(1). https://doi.org/10.1038/s41413-024-00389-7. Huang Q, Liu X, Guo SW. Changing prostaglandin E2 (PGE(2)) signaling during lesional progression and exacerbation of endometriosis by inhibition of PGE(2) receptor EP2 and EP4. Reprod Med Biol. 2022;21:e12426. https://doi.org/10.1002/rmb2.12426.

This research was supported in part by grant 82071623 (SWG) from the National Natural Science Foundation of China. We thank the two anonymous reviewers for their constructive and expert comments and suggestions on an earlier version of this manuscript. Funding This research was funded by grant 82071623 (SWG) from the National Natural Science Foundation of China. Author information Authors and Affiliations Contributions J.D.: Execution and implementation, data acquisition, initial data analysis, manuscript drafting, review and approval. X.L.: Study design, project supervision, manuscript writing, review and approval. Y.W.: Randomization test analysis, manuscript review and revision, and approval. S.-W.G.: Study conception and design, funding, data analysis, manuscript drafting, revision and final approval. Corresponding author Ethics declarations Ethics Approval and Consent to Participate The experiment was performed under the US National Research Council’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Experimental Animals Review Committee of Shanghai OB/GYN Hospital, Fudan University. Conflict of interest J.D., Y.W. and X.L. have no conflict of interest. S.W.G. is a member of the Scientific Advisory Board of Heranova BioSciences, FimmCyte A.G., E3A Healthcare, Foraviset, and Maipl Therapeutics and has provided consultancy advice to these companies, and received a travel support from Ziwig, but these activities had no bearing on this work. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Dong, J., Liu, X. & Guo, SW. Exposure to Green Light-emitting Diodes Alleviates Endometriosis-associated Pain in Mice with Induced Deep Endometriosis. Reprod. Sci. 33, 729–741 (2026). https://doi.org/10.1007/s43032-026-02084-w Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-026-02084-w