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Antihyperalgesic effect of joint mobilization requires Cav3.2 calcium channels

Antihyperalgesic effect of joint mobilization requires Cav3.2 calcium channels The present study was undertaken to explore the relative contributions of Cav3.2 T‑type channels to mediating the antihyperalgesic activity of joint manipulation (JM) therapy. We used the chronic constriction injury model (CCI) to induce peripheral neuropathy and chronic pain in male mice, followed by JM. We demonstrate that JM produces long‑lasting mechanical anti‑hyperalgesia that is abolished in Cav3.2 null mice. Moreover, we found that JM displays a similar analgesic profile as the fatty acid amide hydrolase inhibitor URB597, suggesting a possible converging mechanism of action involving endocannabinoids. Overall, our findings advance our understanding of the mecha‑ nisms through which JM produces analgesia. Keywords Joint mobilization therapy, Cav3.2 channel, Mechanical hyperalgesia, Analgesia CB1 and peripheral CB2 receptors appears to play an Main text important role in this antihyperalgesic action of JM [5]. Joint mobilization (JM) is a common non-allopathic Cannabinoid molecules have emerged as potential thera- treatment approach used by healthcare professionals for peutics for pain management [6]. Natural and synthetic the management of different types of pain such as that cannabinoids, as well as endogenous cannabinoids such arising from inflammation and neuropathies. Mobiliza - as anandamide and 2-AG reduce pain in animal models tion-induced analgesia has been demonstrated in human and humans [7–11]. Outside the brain, many of these pain patients [1], and in animal models [2–4]. In recent molecules produce analgesia by inhibiting Cav3.2 T-type years, evidence has emerged demonstrating that JM may calcium channels expressed in the spinal cord and dor- exert its antihyperalgesic action via endocannabinoid sal root ganglia, rather than just acting via cannabinoid signaling. Indeed, the activation of the spinal cannabinoid receptors [8–10, 12–14]. Thus, we set out to test whether JM mediated analgesia may involve the Cav3.2/endocan- *Correspondence: nabinoid axis. Gerald W. Zamponi All animal care and experimental procedures were car- zamponi@ucalgary.ca 1 ried out in accordance with the National Institutes of Departments of Clinical Neurosciences, and Physiology & Pharmacology, Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, Health’s Animal Care Guidelines (NIH publications No. University of Calgary, Calgary, AB T2N 4N1, Canada 80-23) and conducted following approval of animal pro- Experimental Neuroscience Laboratory (LaNEx), Postgraduate Program tocols by the Ethics and Institutional Animal Care and in Health Sciences, University of Southern Santa Catarina, Palhoça, SC, Brazil Use committees. Ten-week-old male C57BL/6J (wild- Programa de Pós‑Graduação em Neurociências, Centro de Ciências type) and male Cav3.2 knockout mice (25–30  g) were Biológicas, Universidade Federal de Santa Catarina, Campus Universitário‑ purchased from Jackson Laboratories. Stock aliquots Trindade, Florianópolis, SC, Brazil Department of Physical Therapy, Rehabilitation Science Program, of drugs were dissolved in dimethylsulfoxide (DMSO) University of Alabama at Birmingham, Birmingham, AL, USA with a final DMSO concentration of no more than 5% in © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Martins et al. Molecular Brain (2023) 16:60 Page 2 of 4 injection solutions. Control animals received the same Mice were tested for withdrawal thresholds to mechan- vehicle used to dilute the compounds. Drugs were deliv- ical stimuli using von Frey filaments (Stoelting, Chicago, ered by the intraperitoneal (i.p.) route at a volume of USA) applied to the plantar side of the hindpaw and 10  mL/kg of body weight. We used a sciatic nerve con- scored before CCI and 30 min after JM treatment on the striction model (CCI) to induce chronic neuropathic 7th day after surgeries. Animals were kept individually pain. Briefly, mice were anaesthetized (isoflurane 5% in transparent plastic glass boxes set on an elevated wire induction, 2.5% maintenance) and the right sciatic nerve grid platform that allows access to their site of testing on was exposed at the level of the thigh by blunt dissection the hind paw. Each right hind paw was stimulated for 1 s through the biceps femoris. Proximal to the sciatic nerve using a 0.4  g von Frey filament. The percentage of with - trifurcation, about 10 mm of nerve was freed of adhering drawal to 10 stimulations was considered as the nocicep- tissue and three ligatures (silk suture 6–0) were loosely tive response. All experimenters were male. tied around it with about 1–2  mm spacing so that the As shown in Fig.  1a, the CCI model manifested itself epineural circulation was preserved. In sham-operated as long-lasting mechanical hypersensitivity to von Frey mice, the nerve was exposed but not injured. stimulation of the ipsilateral paw. We then assessed to Fig. 1 Joint mobilization reverses mechanical hypersensitivity induced by CCI. a Time course of the effect of a 9‑min of acute ankle joint mobilization on mechanical hypersensitivity in neuropathic mice. Each point represents the mean of 5–7 animals, error bars denote S.E.M. Data are representative of 2 independent sets of experiments. Statistical analyses were performed by two‑ way ANOVA followed by a Tukey’s test. Asterisks denote a significant difference of ***P < 0.001 when compared with the control group, and hashtags denote P < 0.001 for comparison with the sham‑ operated group. b Time‑ course of the effect of 9‑min joint mobilization treatment in either Cav3.2 null mice or wild‑type mice subjected to CCI. Each data point represents the mean of 5–6 mice and data are representative of 2 independent sets of experiments. This particular experiment was performed with the experimenter being blinded to genotype. Statistical analyses were performed by two‑ way ANOVA followed by a Tukey’s test. Asterisks denote a significant difference of ***P < 0.001 when compared with the control group. c Time‑ course and (d) bar graph representing the effect of URB597 (10 mg/kg, i.p.) on nocifensive responses of neuropathic wild type and Cav3.2 null mice, expressed as area under the curve (A.U.C.). Each point or bar represents the mean of 5–6 mice, error bars are SEM. Data are representative of 2 independent sets of experiments. Statistical analyses were performed by two‑ way ANOVA followed by a Tukey’s test. Symbols denote a significant difference of **P < 0.01, ***P < 0.001, or ****P < 0.0001 when compared with the control group M artins et al. Molecular Brain (2023) 16:60 Page 3 of 4 what extent JM could alter this pain response 7 days after between JM mediated anandamide production and an the establishment of neuropathy. Under light isoflurane ensuing inhibition of Cav3.2 channels. anesthesia, the knee joint was stabilized, and the ankle In summary, this study represents the first direct dem - joint was rhythmically flexed and extended to the end of onstration of the role of Cav 3.2 T-type calcium channels the range of movement (animals that did not receive JM in the antihyperalgesic effect of JM. were subjected to the same isoflurane anesthesia). The treatment group received three applications of mobili- Abbreviations zation which each lasted three minutes and which were JM Joint mobilization separated by 30 s of rest. As seen in Fig. 1a, such a 9-min MT Manual therapy CCI Chronic constriction injury JM treatment induced a transient relief from mechanical AEA Anandamide hypersensitivity that abated after one hour. To determine DMSO Dimethylsulfoxide whether this effect involved Cav3.2 T-type calcium chan - nels, we performed analogous experiments in Cav3.2 null Supplementary Information mice which are known to have compensatory mecha- The online version contains supplementary material available at https:// doi. nisms that allow them to develop some types of chronic org/ 10. 1186/ s13041‑ 023‑ 01049‑3. pain (see Fig.  1b). In this batch of mice, wild type ani- Additional file 1: Figure S1 (a) Time course of mibefradil mediated mals exhibited robust JM-induced relief from mechani- reversal of mechanical hypersensitivity in mice with a chronic constriction cal hypersensitivity, whereas Cav3.2 null mice did not injury of the sciatic nerve. Statistical analyses were performed by two‑ way respond to JM. The involvement of Cav3.2 channels in ANOVA followed by Tukey’s test. Asterisks denote a significant difference of **P < 0.01 and ***P < 0.001 when compared with the control group CCI was confirmed further by systemic (intraperitoneal) (n = 5–7). Hashtags denote P < 0.001 for comparison with the sham‑ delivery of 10  mg/kg of the non-selective T-type chan- operated group. (b) Mibefradil at 10 mg/kg inhibits pain responses in nel blocker mibefradil, whereas mibefradil failed to act in CCI operated wild type mice, but not in Cav3.2 null mice *P < 0.05 and ***P < 0.001 (n = 5–6). Cav3.2 null mice (Additional file  1: Fig. S1a, b). In con- trast with 9-min JM treatment, a shorter duration of JM Acknowledgements (3 min) did not mediate pain relief. Not available. A blinded, randomized controlled trial of 31 healthy human subjects measured anandamide (AEA) levels pre- Author contributions DFM, VMG, LMM performed experiments. DFM, VMG designed the experi‑ and post- manipulative treatment (MT) including JM. ments. ARSS and GWZ conceived and supervised the study. DFM, VMG, VS, In subjects receiving MT, serum levels of AEA obtained WLR analysed the data. VMG and GWZ wrote the manuscript. after MT more than doubled while there was no change Funding in control subjects [15]. Because Cav3.2 channels are This work was supported by grants to GWZ from Alberta Innovates and the inhibited by the endocannabinoid anandamide, it is thus Canadian Institutes of Health Research. GWZ holds a Canada Research Chair. possible that anandamide might participate in the actions Availability of data and materials observed in our mouse cohorts. To determine whether All data generated or analysed during this study are included in this published AEA is capable of mediating analgesia in the CCI model, article. we systemically delivered 10  mg/kg URB597. This com - pound is a selective and irreversible inhibitor of fatty acid Declarations amide hydrolase, a key enzyme responsible for the break- Ethics approval and consent to participate down of AEA. As shown in Fig. 1c, d, URB597 mediated This study was approved by the University of Calgary’s and UNISUL’s animal a transient relief from mechanical hypersensitivity in care and ethics committees. wild type mice, but not in Cav3.2 null mice. These data Consent for publication fit with the idea that endogenous AEA is capable of medi - Not applicable. ating pain relief via Cav3.2 channels. Given that JM was equally blunted in mice lacking Cav3.2 and the fact that Competing interests The authors declare no competing interest. endocannabinoids have been previously linked to JM [5, 15], it is thus conceivable that JM induces pain relief by triggering increased production of endocannabinoids Received: 11 June 2023 Accepted: 12 July 2023 such as AEA, which then blocks Cav3.2 channels. How- ever, we did not directly measure AEA levels in our CCI mice, and we did not determine whether degradation of endogenous anandamide blunts the effects of JM. Hence References 1. Ali SS, Ahmed SI, Khan M, Soomro RR. Comparing the effects of manual additional work will be needed to establish a causal link therapy versus electrophysical agents in the management of knee osteo‑ arthritis. Pak J Pharm Sci. 2014;27:1103–6. Martins et al. Molecular Brain (2023) 16:60 Page 4 of 4 2. Skyba DA, Radhakrishnan R, Rohlwing JJ, Wright A, Sluka KA. Joint manip‑ ulation reduces hyperalgesia by activation of monoamine receptors but not opioid or GABA receptors in the spinal cord. Pain. 2003;106:159–68. 3. Martins DF, Mazzardo‑Martins L, Gadotti VM, Nascimento FP, Lima DA, Speckhann B, et al. Ankle joint mobilization reduces axonotmesis‑ induced neuropathic pain and glial activation in the spinal cord and enhances nerve regeneration in rats. Pain. 2011;152:2653–61. 4. Lima CR, Martins DF, Reed WR. Physiological responses induced by manual therapy in animal models: a scoping review. Front Neurosci. 2020;14:430. 5. Martins DF, Mazzardo‑Martins L, Cidral‑Filho FJ, Gadotti VM, Santos ARS. Peripheral and spinal activation of cannabinoid receptors by joint mobilization alleviates postoperative pain in mice. Neuroscience. 2013;255:110–21. 6. Gadotti VM, You H, Petrov RR, Berger ND, Diaz P, Zamponi GW. Analgesic effect of a mixed T ‑type channel inhibitor/CB2 receptor agonist. Mol Pain. 2013;9:32. 7. Casey SL, Mitchell VA, Sokolaj EE, Winters BL, Vaughan CW. Intrathe‑ cal actions of the cannabis constituents Δ(9)‑ Tetrahydrocannabinol and cannabidiol in a mouse neuropathic pain model. Int J Mol Sci. 2022;23(15):8649. 8. Harding EK, Souza IA, Gandini MA, Gadotti VM, Ali MY, Huang S, Antunes FTT, Trang T, Zamponi GW. Differential regulation of Cav 3.2 and Cav 2.2 calcium channels by CB1 receptors and cannabidiol. Brit J Pharmacol. 2023;180:1616–33. 9. Chemin J, Monteil A, Perez‑Reyes E, Nargeot J, Lory P. Direct inhibition of T‑type calcium channels by the endogenous cannabinoid anandamide. EMBO J. 2001;20:7033–40. 10. Barbara G, Alloui A, Nargeot J, Lory P, Eschalier A, Bourinet E, Chemin J. T‑type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids. J Neurosci. 2009;29:13106–14. 11. Wilsey B, Marcotte T, Deutsch R, Gouaux B, Sakai S, Donaghe H. Low‑ dose vaporized cannabis significantly improves neuropathic pain. J Pain. 2013;14:136–48. 12. Gadotti VM, Antunes FTT, Zamponi GW. Analgesia by intrathecal delta‑ 9‑tetrahydrocannabinol is dependent on Cav3.2 calcium channels. Mol Brain. 2023;16:47. 13. Fischbach T, Greffrath W, Nawrath H, Treede RD. Eec ff ts of anandamide and noxious heat on intracellular calcium concentration in nociceptive DRG neurons of rats. J Neurophysiol. 2007;98:929–38. 14. Ross HR, Napier I, Connor M. Inhibition of recombinant human T‑type calcium channels by Delta9‑tetrahydrocannabinol and cannabidiol. J Biol Chem. 2008;283:16124–34. 15. McPartland JM, Giuffrida A, King J, Skinner E, Scotter J, Musty RE. Cannabi‑ mimetic effects of osteopathic manipulative treatment. J Am Osteopath Assoc. 2005;105:283–91. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Brain Springer Journals

Antihyperalgesic effect of joint mobilization requires Cav3.2 calcium channels

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Abstract

The present study was undertaken to explore the relative contributions of Cav3.2 T‑type channels to mediating the antihyperalgesic activity of joint manipulation (JM) therapy. We used the chronic constriction injury model (CCI) to induce peripheral neuropathy and chronic pain in male mice, followed by JM. We demonstrate that JM produces long‑lasting mechanical anti‑hyperalgesia that is abolished in Cav3.2 null mice. Moreover, we found that JM displays a similar analgesic profile as the fatty acid amide hydrolase inhibitor URB597, suggesting a possible converging mechanism of action involving endocannabinoids. Overall, our findings advance our understanding of the mecha‑ nisms through which JM produces analgesia. Keywords Joint mobilization therapy, Cav3.2 channel, Mechanical hyperalgesia, Analgesia CB1 and peripheral CB2 receptors appears to play an Main text important role in this antihyperalgesic action of JM [5]. Joint mobilization (JM) is a common non-allopathic Cannabinoid molecules have emerged as potential thera- treatment approach used by healthcare professionals for peutics for pain management [6]. Natural and synthetic the management of different types of pain such as that cannabinoids, as well as endogenous cannabinoids such arising from inflammation and neuropathies. Mobiliza - as anandamide and 2-AG reduce pain in animal models tion-induced analgesia has been demonstrated in human and humans [7–11]. Outside the brain, many of these pain patients [1], and in animal models [2–4]. In recent molecules produce analgesia by inhibiting Cav3.2 T-type years, evidence has emerged demonstrating that JM may calcium channels expressed in the spinal cord and dor- exert its antihyperalgesic action via endocannabinoid sal root ganglia, rather than just acting via cannabinoid signaling. Indeed, the activation of the spinal cannabinoid receptors [8–10, 12–14]. Thus, we set out to test whether JM mediated analgesia may involve the Cav3.2/endocan- *Correspondence: nabinoid axis. Gerald W. Zamponi All animal care and experimental procedures were car- zamponi@ucalgary.ca 1 ried out in accordance with the National Institutes of Departments of Clinical Neurosciences, and Physiology & Pharmacology, Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, Health’s Animal Care Guidelines (NIH publications No. University of Calgary, Calgary, AB T2N 4N1, Canada 80-23) and conducted following approval of animal pro- Experimental Neuroscience Laboratory (LaNEx), Postgraduate Program tocols by the Ethics and Institutional Animal Care and in Health Sciences, University of Southern Santa Catarina, Palhoça, SC, Brazil Use committees. Ten-week-old male C57BL/6J (wild- Programa de Pós‑Graduação em Neurociências, Centro de Ciências type) and male Cav3.2 knockout mice (25–30  g) were Biológicas, Universidade Federal de Santa Catarina, Campus Universitário‑ purchased from Jackson Laboratories. Stock aliquots Trindade, Florianópolis, SC, Brazil Department of Physical Therapy, Rehabilitation Science Program, of drugs were dissolved in dimethylsulfoxide (DMSO) University of Alabama at Birmingham, Birmingham, AL, USA with a final DMSO concentration of no more than 5% in © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Martins et al. Molecular Brain (2023) 16:60 Page 2 of 4 injection solutions. Control animals received the same Mice were tested for withdrawal thresholds to mechan- vehicle used to dilute the compounds. Drugs were deliv- ical stimuli using von Frey filaments (Stoelting, Chicago, ered by the intraperitoneal (i.p.) route at a volume of USA) applied to the plantar side of the hindpaw and 10  mL/kg of body weight. We used a sciatic nerve con- scored before CCI and 30 min after JM treatment on the striction model (CCI) to induce chronic neuropathic 7th day after surgeries. Animals were kept individually pain. Briefly, mice were anaesthetized (isoflurane 5% in transparent plastic glass boxes set on an elevated wire induction, 2.5% maintenance) and the right sciatic nerve grid platform that allows access to their site of testing on was exposed at the level of the thigh by blunt dissection the hind paw. Each right hind paw was stimulated for 1 s through the biceps femoris. Proximal to the sciatic nerve using a 0.4  g von Frey filament. The percentage of with - trifurcation, about 10 mm of nerve was freed of adhering drawal to 10 stimulations was considered as the nocicep- tissue and three ligatures (silk suture 6–0) were loosely tive response. All experimenters were male. tied around it with about 1–2  mm spacing so that the As shown in Fig.  1a, the CCI model manifested itself epineural circulation was preserved. In sham-operated as long-lasting mechanical hypersensitivity to von Frey mice, the nerve was exposed but not injured. stimulation of the ipsilateral paw. We then assessed to Fig. 1 Joint mobilization reverses mechanical hypersensitivity induced by CCI. a Time course of the effect of a 9‑min of acute ankle joint mobilization on mechanical hypersensitivity in neuropathic mice. Each point represents the mean of 5–7 animals, error bars denote S.E.M. Data are representative of 2 independent sets of experiments. Statistical analyses were performed by two‑ way ANOVA followed by a Tukey’s test. Asterisks denote a significant difference of ***P < 0.001 when compared with the control group, and hashtags denote P < 0.001 for comparison with the sham‑ operated group. b Time‑ course of the effect of 9‑min joint mobilization treatment in either Cav3.2 null mice or wild‑type mice subjected to CCI. Each data point represents the mean of 5–6 mice and data are representative of 2 independent sets of experiments. This particular experiment was performed with the experimenter being blinded to genotype. Statistical analyses were performed by two‑ way ANOVA followed by a Tukey’s test. Asterisks denote a significant difference of ***P < 0.001 when compared with the control group. c Time‑ course and (d) bar graph representing the effect of URB597 (10 mg/kg, i.p.) on nocifensive responses of neuropathic wild type and Cav3.2 null mice, expressed as area under the curve (A.U.C.). Each point or bar represents the mean of 5–6 mice, error bars are SEM. Data are representative of 2 independent sets of experiments. Statistical analyses were performed by two‑ way ANOVA followed by a Tukey’s test. Symbols denote a significant difference of **P < 0.01, ***P < 0.001, or ****P < 0.0001 when compared with the control group M artins et al. Molecular Brain (2023) 16:60 Page 3 of 4 what extent JM could alter this pain response 7 days after between JM mediated anandamide production and an the establishment of neuropathy. Under light isoflurane ensuing inhibition of Cav3.2 channels. anesthesia, the knee joint was stabilized, and the ankle In summary, this study represents the first direct dem - joint was rhythmically flexed and extended to the end of onstration of the role of Cav 3.2 T-type calcium channels the range of movement (animals that did not receive JM in the antihyperalgesic effect of JM. were subjected to the same isoflurane anesthesia). The treatment group received three applications of mobili- Abbreviations zation which each lasted three minutes and which were JM Joint mobilization separated by 30 s of rest. As seen in Fig. 1a, such a 9-min MT Manual therapy CCI Chronic constriction injury JM treatment induced a transient relief from mechanical AEA Anandamide hypersensitivity that abated after one hour. To determine DMSO Dimethylsulfoxide whether this effect involved Cav3.2 T-type calcium chan - nels, we performed analogous experiments in Cav3.2 null Supplementary Information mice which are known to have compensatory mecha- The online version contains supplementary material available at https:// doi. nisms that allow them to develop some types of chronic org/ 10. 1186/ s13041‑ 023‑ 01049‑3. pain (see Fig.  1b). In this batch of mice, wild type ani- Additional file 1: Figure S1 (a) Time course of mibefradil mediated mals exhibited robust JM-induced relief from mechani- reversal of mechanical hypersensitivity in mice with a chronic constriction cal hypersensitivity, whereas Cav3.2 null mice did not injury of the sciatic nerve. Statistical analyses were performed by two‑ way respond to JM. The involvement of Cav3.2 channels in ANOVA followed by Tukey’s test. Asterisks denote a significant difference of **P < 0.01 and ***P < 0.001 when compared with the control group CCI was confirmed further by systemic (intraperitoneal) (n = 5–7). Hashtags denote P < 0.001 for comparison with the sham‑ delivery of 10  mg/kg of the non-selective T-type chan- operated group. (b) Mibefradil at 10 mg/kg inhibits pain responses in nel blocker mibefradil, whereas mibefradil failed to act in CCI operated wild type mice, but not in Cav3.2 null mice *P < 0.05 and ***P < 0.001 (n = 5–6). Cav3.2 null mice (Additional file  1: Fig. S1a, b). In con- trast with 9-min JM treatment, a shorter duration of JM Acknowledgements (3 min) did not mediate pain relief. Not available. A blinded, randomized controlled trial of 31 healthy human subjects measured anandamide (AEA) levels pre- Author contributions DFM, VMG, LMM performed experiments. DFM, VMG designed the experi‑ and post- manipulative treatment (MT) including JM. ments. ARSS and GWZ conceived and supervised the study. DFM, VMG, VS, In subjects receiving MT, serum levels of AEA obtained WLR analysed the data. VMG and GWZ wrote the manuscript. after MT more than doubled while there was no change Funding in control subjects [15]. Because Cav3.2 channels are This work was supported by grants to GWZ from Alberta Innovates and the inhibited by the endocannabinoid anandamide, it is thus Canadian Institutes of Health Research. GWZ holds a Canada Research Chair. possible that anandamide might participate in the actions Availability of data and materials observed in our mouse cohorts. To determine whether All data generated or analysed during this study are included in this published AEA is capable of mediating analgesia in the CCI model, article. we systemically delivered 10  mg/kg URB597. This com - pound is a selective and irreversible inhibitor of fatty acid Declarations amide hydrolase, a key enzyme responsible for the break- Ethics approval and consent to participate down of AEA. As shown in Fig. 1c, d, URB597 mediated This study was approved by the University of Calgary’s and UNISUL’s animal a transient relief from mechanical hypersensitivity in care and ethics committees. wild type mice, but not in Cav3.2 null mice. These data Consent for publication fit with the idea that endogenous AEA is capable of medi - Not applicable. ating pain relief via Cav3.2 channels. Given that JM was equally blunted in mice lacking Cav3.2 and the fact that Competing interests The authors declare no competing interest. endocannabinoids have been previously linked to JM [5, 15], it is thus conceivable that JM induces pain relief by triggering increased production of endocannabinoids Received: 11 June 2023 Accepted: 12 July 2023 such as AEA, which then blocks Cav3.2 channels. How- ever, we did not directly measure AEA levels in our CCI mice, and we did not determine whether degradation of endogenous anandamide blunts the effects of JM. Hence References 1. Ali SS, Ahmed SI, Khan M, Soomro RR. Comparing the effects of manual additional work will be needed to establish a causal link therapy versus electrophysical agents in the management of knee osteo‑ arthritis. Pak J Pharm Sci. 2014;27:1103–6. Martins et al. Molecular Brain (2023) 16:60 Page 4 of 4 2. Skyba DA, Radhakrishnan R, Rohlwing JJ, Wright A, Sluka KA. Joint manip‑ ulation reduces hyperalgesia by activation of monoamine receptors but not opioid or GABA receptors in the spinal cord. Pain. 2003;106:159–68. 3. Martins DF, Mazzardo‑Martins L, Gadotti VM, Nascimento FP, Lima DA, Speckhann B, et al. Ankle joint mobilization reduces axonotmesis‑ induced neuropathic pain and glial activation in the spinal cord and enhances nerve regeneration in rats. Pain. 2011;152:2653–61. 4. Lima CR, Martins DF, Reed WR. Physiological responses induced by manual therapy in animal models: a scoping review. Front Neurosci. 2020;14:430. 5. Martins DF, Mazzardo‑Martins L, Cidral‑Filho FJ, Gadotti VM, Santos ARS. Peripheral and spinal activation of cannabinoid receptors by joint mobilization alleviates postoperative pain in mice. Neuroscience. 2013;255:110–21. 6. Gadotti VM, You H, Petrov RR, Berger ND, Diaz P, Zamponi GW. Analgesic effect of a mixed T ‑type channel inhibitor/CB2 receptor agonist. Mol Pain. 2013;9:32. 7. Casey SL, Mitchell VA, Sokolaj EE, Winters BL, Vaughan CW. Intrathe‑ cal actions of the cannabis constituents Δ(9)‑ Tetrahydrocannabinol and cannabidiol in a mouse neuropathic pain model. Int J Mol Sci. 2022;23(15):8649. 8. Harding EK, Souza IA, Gandini MA, Gadotti VM, Ali MY, Huang S, Antunes FTT, Trang T, Zamponi GW. Differential regulation of Cav 3.2 and Cav 2.2 calcium channels by CB1 receptors and cannabidiol. Brit J Pharmacol. 2023;180:1616–33. 9. Chemin J, Monteil A, Perez‑Reyes E, Nargeot J, Lory P. Direct inhibition of T‑type calcium channels by the endogenous cannabinoid anandamide. EMBO J. 2001;20:7033–40. 10. Barbara G, Alloui A, Nargeot J, Lory P, Eschalier A, Bourinet E, Chemin J. T‑type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids. J Neurosci. 2009;29:13106–14. 11. Wilsey B, Marcotte T, Deutsch R, Gouaux B, Sakai S, Donaghe H. Low‑ dose vaporized cannabis significantly improves neuropathic pain. J Pain. 2013;14:136–48. 12. Gadotti VM, Antunes FTT, Zamponi GW. Analgesia by intrathecal delta‑ 9‑tetrahydrocannabinol is dependent on Cav3.2 calcium channels. Mol Brain. 2023;16:47. 13. Fischbach T, Greffrath W, Nawrath H, Treede RD. Eec ff ts of anandamide and noxious heat on intracellular calcium concentration in nociceptive DRG neurons of rats. J Neurophysiol. 2007;98:929–38. 14. Ross HR, Napier I, Connor M. Inhibition of recombinant human T‑type calcium channels by Delta9‑tetrahydrocannabinol and cannabidiol. J Biol Chem. 2008;283:16124–34. 15. McPartland JM, Giuffrida A, King J, Skinner E, Scotter J, Musty RE. Cannabi‑ mimetic effects of osteopathic manipulative treatment. J Am Osteopath Assoc. 2005;105:283–91. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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Molecular Brain – Springer Journals

Published: Jul 18, 2023

Keywords: Joint mobilization therapy; Cav3.2 channel; Mechanical hyperalgesia; Analgesia

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