Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

Department of Anesthesiology Pain Medicine University of California San Diego La Jolla CA 92037 USA

Department of Anesthesiology University of Ryukyus Okinawa Japan

Department of Biophysics Institute of Experimental Physics Slovak Academy of Sciences Kosice Slovakia

Department of Neurosurgery University of California San Diego La Jolla CA 92037 USA

Departments of Sensory Signaling and Molecular Biophysics Bogomoletz Institute of Physiology Kyiv Ukraine

Departments of Sensory Signaling and Molecular Biophysics Bogomoletz Institute of Physiology Kyiv Ukraine; Kyiv Academic University Kyiv Ukraine

Gene Expression Laboratory and the Howard Hughes Medical Institute Salk Institute for Biological Studies La Jolla CA 92037 USA

Institute of Animal Physiology and Genetics Czech Academy of Sciences Rumburská 89 277 21 Liběchov Czech Republic

Institute of Macromolecular Chemistry Czech Academy of Sciences Department of Biomaterials and Bioanalogous Systems Heyrovsky Square 2 162 06 Prague 6 Czech Republic

Institute of Neurobiology Biomedical Research Center Slovak Academy of Sciences Kosice Slovakia

National Primate Research Center of Thailand Chulalongkorn University Kaengkhoi District Saraburi 18110 Thailand

Neuroregeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla CA 92037 USA

Neuroregeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla CA 92037 USA; Department of Anesthesiology University of Ryukyus Okinawa Japan; Neurgain Technologies 9620 Towne Centre Drive Suite 100 San Diego CA 92121 USA

Neuroregeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla CA 92037 USA; Institute of Neurobiology Biomedical Research Center Slovak Academy of Sciences Kosice Slovakia

Neuroregeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla CA 92037 USA; Neurgain Technologies 9620 Towne Centre Drive Suite 100 San Diego CA 92121 USA

Zobrazit více v PubMed

Widerstrom-Noga E. Neuropathic pain and spinal cord injury: phenotypes and pharmacological management. Drugs. 2017;77:967–984. doi: 10.1007/s40265-017-0747-8. PubMed DOI

Warner F., Cragg J.J., Jutzeler C., Finnerup N., Werhagen L., Weidner N., Maier D., Kalke Y.B., Curt A., Kramer J. The progression of neuropathic pain after acute spinal cord injury: a meta-analysis and framework for clinical trials. J Neurotrauma. 2018;36:1461–1468. doi: 10.1089/neu.2018.5960. PubMed DOI

Alles S.R.A., Smith P.A. Etiology and pharmacology of neuropathic pain. Pharmacol. Rev. 2018;70:315–347. doi: 10.1124/pr.117.014399. PubMed DOI

Felix E.R. Chronic neuropathic pain in SCI. Phys. Med. Rehabil. Clin. N. Am. 2014;25:545–571. doi: 10.1016/j.pmr.2014.04.007. viii. PubMed DOI

Jensen T.S., Madsen C.S., Finnerup N.B. Pharmacology and treatment of neuropathic pains. Curr. Opin. Neurol. 2009;22:467–474. doi: 10.1097/WCO.0b013e3283311e13. PubMed DOI

DeFrates S., Cook A.M. Pharmacologic treatment of neuropathic pain following spinal cord injury. Orthopedics. 2011;34:203–207. doi: 10.3928/01477447-20110124-19. PubMed DOI

Koulousakis A., Kuchta J., Bayarassou A., Sturm V. Intrathecal opioids for intractable pain syndromes. Acta Neurochir Suppl. 2007;97:43–48. doi: 10.1007/978-3-211-33079-1_5. PubMed DOI

Siniscalco D., de Novellis V., Rossi F., Maione S. Neuropathic pain: is the end of suffering starting in the gene therapy? Curr. Drug Targets. 2005;6:75–80. doi: 10.2174/1389450053344966. PubMed DOI

Lee B., Kim J., Kim S.J., Lee H., Chang J.W. Constitutive GABA expression via a recombinant adeno-associated virus consistently attenuates neuropathic pain. Biochem. Biophys. Res. Commun. 2007;357:971–976. doi: 10.1016/j.bbrc.2007.04.061. PubMed DOI

Yang F.R., Chen J., Yi H., Peng L.Y., Hu X.L., Guo Q.L. MicroRNA-7a ameliorates neuropathic pain in a rat model of spinal nerve ligationviathe neurofilament light polypeptide-dependent signal transducer and activator of transcription signaling pathway. Mol. Pain. 2019;15 doi: 10.1177/1744806919842464. 174480691984246. PubMed DOI PMC

Samad O.A., Tan A.M., Cheng X., Foster E., Dib-Hajj S.D., Waxman S.G. Virus-mediated shRNA knockdown of Na(v)1.3 in rat dorsal root ganglion attenuates nerve injury-induced neuropathic pain. Mol. Ther. 2013;21:49–56. doi: 10.1038/mt.2012.169. PubMed DOI PMC

Chen L., Huang J., Zhao P., Persson A.K., Dib-Hajj F.B., Cheng X., Tan A., Waxman S.G., Dib-Hajj S.D. Conditional knockout of NaV1.6 in adult mice ameliorates neuropathic pain. Sci. Rep. 2018;8:3845. doi: 10.1038/s41598-018-22216-w. PubMed DOI PMC

Hirai T., Enomoto M., Kaburagi H., Sotome S., Yoshida-Tanaka K., Ukegawa M., Kuwahara H., Yamamoto M., Tajiri M., Miyata H., et al. Intrathecal AAV serotype 9-mediated delivery of shRNA against TRPV1 attenuates thermal hyperalgesia in a mouse model of peripheral nerve injury. Mol. Ther. 2014;22:409–419. doi: 10.1038/mt.2013.247. PubMed DOI PMC

Fischer G., Pan B., Vilceanu D., Hogan Q.H., Yu H. Sustained relief of neuropathic pain by AAV-targeted expression of CBD3 peptide in rat dorsal root ganglion. Gene Ther. 2014;21:44–51. doi: 10.1038/gt.2013.56. PubMed DOI PMC

Towne C., Pertin M., Beggah A.T., Aebischer P., Decosterd I. Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery. Mol. Pain. 2009;5:52. doi: 10.1186/1744-8069-5-52. PubMed DOI PMC

Miyanohara A., Kamizato K., Juhas S., Juhasova J., Navarro M., Marsala S., Lukacova N., Hruska-Plochan M., Curtis E., Gabel B., et al. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs. Mol. Ther. Methods Clin. Dev. 2016;3:16046. doi: 10.1038/mtm.2016.46. PubMed DOI PMC

Vulchanova L., Schuster D.J., Belur L.R., Riedl M.S., Podetz-Pedersen K.M., Kitto K.F., Wilcox G.L., McIvor R.S., Fairbanks C.A. Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. Mol. Pain. 2010;6:31. doi: 10.1186/1744-8069-6-31. PubMed DOI PMC

Yu H., Fischer G., Hogan Q.H. AAV-mediated gene transfer to dorsal root ganglion. Methods Mol. Biol. 2016;1382:251–261. doi: 10.1007/978-1-4939-3271-9_18. PubMed DOI PMC

Wu H., Jin Y., Buddhala C., Osterhaus G., Cohen E., Jin H., Wei J., Davis K., Obata K., Wu J.Y. Role of glutamate decarboxylase (GAD) isoform, GAD65, in GABA synthesis and transport into synaptic vesicles-Evidence from GAD65-knockout mice studies. Brain Res. 2007;1154:80–83. doi: 10.1016/j.brainres.2007.04.008. PubMed DOI

Saito K., Kakizaki T., Hayashi R., Nishimaru H., Furukawa T., Nakazato Y., Takamori S., Ebihara S., Uematsu M., Mishina M., et al. The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice. Mol. Brain. 2010;3:40. doi: 10.1186/1756-6606-3-40. PubMed DOI PMC

Wojcik S.M., Katsurabayashi S., Guillemin I., Friauf E., Rosenmund C., Brose N., Rhee J.S. A shared vesicular carrier allows synaptic corelease of GABA and glycine. Neuron. 2006;50:575–587. doi: 10.1016/j.neuron.2006.04.016. PubMed DOI

Tadokoro T., Miyanohara A., Navarro M., Kamizato K., Juhas S., Juhasova J., Marsala S., Platoshyn O., Curtis E., Gabel B., et al. Subpial adeno-associated virus 9 (AAV9) vector delivery in adult mice. J. Vis. Exp. 2017;125:55770. doi: 10.3791/55770. PubMed DOI PMC

Bravo-Hernandez M., Tadokoro T., Navarro M.R., Platoshyn O., Kobayashi Y., Marsala S., Miyanohara A., Juhas S., Juhasova J., Skalnikova H., et al. Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS. Nat. Med. 2020;26:118–130. doi: 10.1038/s41591-019-0674-1. PubMed DOI PMC

Bravo-Hernandez M., Tadokoro T., Marsala M. Subpial AAV delivery for spinal parenchymal gene regulation in adult mammals. Methods Mol. Biol. 2019;1950:209–233. doi: 10.1007/978-1-4939-9139-6_12. PubMed DOI

Seltzer Z., Dubner R., Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain. 1990;43:205–218. doi: 10.1016/0304-3959(90)91074-s. PubMed DOI

Kakinohana O., Hefferan M.P., Nakamura S., Kakinohana M., Galik J., Tomori Z., Marsala J., Yaksh T.L., Marsala M. Development of GABA-sensitive spasticity and rigidity in rats after transient spinal cord ischemia: a qualitative and quantitative electrophysiological and histopathological study. Neuroscience. 2006;141:1569–1583. doi: 10.1016/j.neuroscience.2006.04.083. PubMed DOI

Luz L.L., Szucs P., Safronov B.V. Peripherally driven low-threshold inhibitory inputs to lamina I local-circuit and projection neurones: a new circuit for gating pain responses. J. Physiol. 2014;592:1519–1534. doi: 10.1113/jphysiol.2013.269472. PubMed DOI PMC

Todd A.J. Neuronal circuitry for pain processing in the dorsal horn. Nat. Rev. Neurosci. 2010;11:823–836. doi: 10.1038/nrn2947. PubMed DOI PMC

Todd A.J., Puskar Z., Spike R.C., Hughes C., Watt C., Forrest L. Projection neurons in lamina I of rat spinal cord with the neurokinin 1 receptor are selectively innervated by substance p-containing afferents and respond to noxious stimulation. J. Neurosci. 2002;22:4103–4113. doi: 10.1523/jneurosci.22-10-04103.2002. PubMed DOI PMC

Marshall G.E., Shehab S.A., Spike R.C., Todd A.J. Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat. Neuroscience. 1996;72:255–263. doi: 10.1016/0306-4522(95)00558-7. PubMed DOI

Hains B.C., Waxman S.G. Activated microglia contribute to the maintenance of chronic pain after spinal cord injury. J. Neurosci. 2006;26:4308–4317. doi: 10.1523/JNEUROSCI.0003-06.2006. PubMed DOI PMC

Shibata K., Sugawara T., Fujishita K., Shinozaki Y., Matsukawa T., Suzuki T., Koizumi S. The astrocyte-targeted therapy by Bushi for the neuropathic pain in mice. PLoS One. 2011;6:e23510. doi: 10.1371/journal.pone.0023510. PubMed DOI PMC

Stuesse S.L., Cruce W.L., Lovell J.A., McBurney D.L., Crisp T. Microglial proliferation in the spinal cord of aged rats with a sciatic nerve injury. Neurosci. Lett. 2000;287:121–124. doi: 10.1016/s0304-3940(00)01142-3. PubMed DOI

Calvo M., Zhu N., Grist J., Ma Z., Loeb J.A., Bennett D.L.H. Following nerve injury neuregulin-1 drives microglial proliferation and neuropathic pain via the MEK/ERK pathway. Glia. 2011;59:554–568. doi: 10.1002/glia.21124. PubMed DOI PMC

Root D.H., Zhang S., Barker D.J., Miranda-Barrientos J., Liu B., Wang H.L., Morales M. Selective brain distribution and distinctive synaptic architecture of dual glutamatergic-GABAergic neurons. Cell Rep. 2018;23:3465–3479. doi: 10.1016/j.celrep.2018.05.063. PubMed DOI PMC

Tritsch N.X., Granger A.J., Sabatini B.L. Mechanisms and functions of GABA co-release. Nat. Rev. Neurosci. 2016;17:139–145. doi: 10.1038/nrn.2015.21. PubMed DOI PMC

Zhang J.M., An J. Cytokines, inflammation, and pain. Int. Anesthesiol. Clin. 2007;45:27–37. doi: 10.1097/AIA.0b013e318034194e. PubMed DOI PMC

Peng J., Gu N., Zhou L., B Eyo U., Murugan M., Gan W.B., Wu L.J. Microglia and monocytes synergistically promote the transition from acute to chronic pain after nerve injury. Nat. Commun. 2016;7:12029. doi: 10.1038/ncomms12029. PubMed DOI PMC

Liu T., Gao Y.J., Ji R.R. Emerging role of Toll-like receptors in the control of pain and itch. Neurosci. Bull. 2012;28:131–144. doi: 10.1007/s12264-012-1219-5. PubMed DOI PMC

Stokes J.A., Corr M., Yaksh T.L. Spinal Toll-like receptor signaling and nociceptive processing: regulatory balance between TIRAP and TRIF cascades mediated by TNF and IFNβ. Pain. 2013;154:733–742. doi: 10.1016/j.pain.2013.01.012. PubMed DOI PMC

Svensson C.I., Marsala M., Westerlund A., Calcutt N.A., Campana W.M., Freshwater J.D., Catalano R., Feng Y., Protter A.A., Scott B., Yaksh T.L. Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing. J. Neurochem. 2003;86:1534–1544. doi: 10.1046/j.1471-4159.2003.01969.x. PubMed DOI

Yang L.C., Marsala M., Yaksh T.L. Characterization of time course of spinal amino acids, citrulline and PGE2 release after carrageenan/kaolin-induced knee joint inflammation: a chronic microdialysis study. Pain. 1996;67:345–354. doi: 10.1016/0304-3959(96)03106-5. PubMed DOI

Schafers M., Marziniak M., Sorkin L.S., Yaksh T.L., Sommer C. Cyclooxygenase inhibition in nerve-injury- and TNF-induced hyperalgesia in the rat. Exp. Neurol. 2004;185:160–168. doi: 10.1016/j.expneurol.2003.09.015. PubMed DOI

Saito O., Svensson C.I., Buczynski M.W., Wegner K., Hua X.Y., Codeluppi S., Schaloske R.H., Deems R.A., Dennis E.A., Yaksh T.L. Spinal glial TLR4-mediated nociception and production of prostaglandin E(2) and TNF. Br. J. Pharmacol. 2010;160:1754–1764. doi: 10.1111/j.1476-5381.2010.00811.x. PubMed DOI PMC

Schnutgen F., Doerflinger N., Calleja C., Wendling O., Chambon P., Ghyselinck N.B. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat. Biotechnol. 2003;21:562–565. doi: 10.1038/nbt811. PubMed DOI

Saunders A., Sabatini B.L. Cre activated and inactivated recombinant adeno-associated viral vectors for neuronal anatomical tracing or activity manipulation. Curr. Protoc. Neurosci. 2015;72:24.1–1.24.15. doi: 10.1002/0471142301.ns0124s72. PubMed DOI PMC

Lee G., Saito I. Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene. 1998;216:55–65. doi: 10.1016/s0378-1119(98)00325-4. PubMed DOI

Fang H., Lai N.C., Gao M.H., Miyanohara A., Roth D.M., Tang T., Hammond H.K. Comparison of adeno-associated virus serotypes and delivery methods for cardiac gene transfer. Hum. Gene Ther. Methods. 2012;23:234–241. doi: 10.1089/hgtb.2012.105. PubMed DOI PMC

Xu Q., Chou B., Fitzsimmons B., Miyanohara A., Shubayev V., Santucci C., Hefferan M., Marsala M., Hua X.Y. In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery. PLoS One. 2012;7:e32581. doi: 10.1371/journal.pone.0032581. PubMed DOI PMC

Chaplan S.R., Bach F.W., Pogrel J.W., Chung J.M., Yaksh T.L. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods. 1994;53:55–63. doi: 10.1016/0165-0270(94)90144-9. PubMed DOI

Cheng L., Duan B., Huang T., Zhang Y., Chen Y., Britz O., Garcia-Campmany L., Ren X., Vong L., Lowell B.B., et al. Identification of spinal circuits involved in touch-evoked dynamic mechanical pain. Nat. Neurosci. 2017;20:804–814. doi: 10.1038/nn.4549. PubMed DOI PMC

Hargreaves K., Dubner R., Brown F., Flores C., Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988;32:77–88. doi: 10.1016/0304-3959(88)90026-7. PubMed DOI

Pinto V., Szucs P., Lima D., Safronov B.V. Multisegmental A - and C-fiber input to neurons in lamina I and the lateral spinal nucleus. J. Neurosci. 2010;30:2384–2395. doi: 10.1523/JNEUROSCI.3445-09.2010. PubMed DOI PMC

Safronov B.V., Pinto V., Derkach V.A. High-resolution single-cell imaging for functional studies in the whole brain and spinal cord and thick tissue blocks using light-emitting diode illumination. J. Neurosci. Methods. 2007;164:292–298. doi: 10.1016/j.jneumeth.2007.05.010. PubMed DOI PMC

Szucs P., Pinto V., Safronov B.V. Advanced technique of infrared LED imaging of unstained cells and intracellular structures in isolated spinal cord, brainstem, ganglia and cerebellum. J. Neurosci. Methods. 2009;177:369–380. doi: 10.1016/j.jneumeth.2008.10.024. PubMed DOI

Krotov V., Tokhtamysh A., Kopach O., Dromaretsky A., Sheremet Y., Belan P., Voitenko N. Functional characterization of lamina X neurons in ex-vivo spinal cord preparation. Front. Cell Neurosci. 2017;11:342. doi: 10.3389/fncel.2017.00342. PubMed DOI PMC

Kopach O., Krotov V., Belan P., Voitenko N. Inflammatory-induced changes in synaptic drive and postsynaptic AMPARs in lamina II dorsal horn neurons are cell-type specific. Pain. 2015;156:428–438. doi: 10.1097/01.j.pain.0000460318.65734.00. PubMed DOI

Epp J.R., Niibori Y., Liz Hsiang H.L., Mercaldo V., Deisseroth K., Josselyn S.A., Frankland P.W. Optimization of CLARITY for clearing whole-brain and other intact organs. eNeuro. 2015;2 doi: 10.1523/ENEURO.0022-15.2015. PubMed DOI PMC

Bria A., Iannello G. TeraStitcher - a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images. BMC Bioinformatics. 2012;13:316. doi: 10.1186/1471-2105-13-316. PubMed DOI PMC

Liou W., Geuze H.J., Slot J.W. Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 1996;106:41–58. doi: 10.1007/bf02473201. PubMed DOI

Tokuyasu K.T. Immunochemistry on ultrathin frozen sections. Histochem. J. 1980;12:381–403. doi: 10.1007/bf01011956. PubMed DOI

Richardson K.C., Jarett L., Finke E.H. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 1960;35:313–323. doi: 10.3109/10520296009114754. PubMed DOI