Central Sensitization in the Trigeminal Nucleus Caudalis Produced by a Conjugate of Substance P and the A Subunit of Cholera Toxin


      Individuals with chronic craniofacial pain experience symptoms that are consistent with central sensitization. In fact, central sensitization may constitute the major disease process in these conditions, particularly if the original injury has healed or the condition is idiopathic. To understand central sensitization we have developed a conjugate of substance P and cholera toxin (SP-CTA). SP-CTA is selectively taken up by cells that express neurokinin receptors. Twenty-four hours following intracisternal administration of SP-CTA, wild-type rats and mice demonstrated signs of persistent background nociception, but when tested for facial cold sensitivity, they did not differ from controls. However, treating the SP-CTA–injected animals with naloxone exposed cold hypersensitivity in the face. Mu-opioid receptor knockout mice treated with SP-CTA demonstrated hypersensitivity without naloxone treatment. These findings suggest that central sensitization leads to activation of an endogenous opioid system. The data also demonstrate that the intracisternal administration of SP-CTA in rodents is a useful model for studying central sensitization as a disease process without having to induce a peripheral injury.


      Central sensitization is a concern in many craniofacial pain conditions. In this project, we utilize a conjugate of substance P and the catalytic subunit of cholera toxin to induce central sensitization in the nucleus caudalis of rodents. The data indicate that the injected animals become hypersensitive in the face.

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        • Bozic C.R.
        • Lu B.
        • Hopken U.E.
        • Gerard C.
        • Gerard N.P.
        Neurogenic amplification of immune complex inflammation.
        Science. 1996; 273: 1722-1725
        • Brenner G.J.
        • Ji R.R.
        • Shaffer S.
        • Woolf C.J.
        Peripheral noxious stimulation induces phosphorylation of the NMDA receptor NR1 subunit at the PKC-dependent site, serine-896, in spinal cord dorsal horn neurons.
        Eur J Neurosci. 2004; 20: 375-384
        • Burstein R.
        • Cutrer M.F.
        • Yarnitsky D.
        The development of cutaneous allodynia during a migraine attack clinical evidence for the sequential recruitment of spinal and supraspinal nociceptive neurons in migraine.
        Brain. 2000; 123: 1703-1709
        • Caudle R.M.
        • Mannes A.J.
        • Benoliel R.
        • Eliav E.
        • Iadarola M.J.
        Intrathecally administered cholera toxin blocks allodynia and hyperalgesia in persistent pain models.
        J Pain. 2001; 2: 118-127
        • Caudle R.M.
        • Mannes A.J.
        • Keller J.
        • Perez F.M.
        • Suckow S.K.
        • Neubert J.K.
        Sensitization of Spinal Cord Nociceptive Neurons with a Conjugate of Substance P and Cholera Toxin.
        BMC Neurosci. 2007; 8: 30
        • Chaban V.V.
        • Li J.
        • Ennes H.S.
        • Nie J.
        • Mayer E.A.
        • McRoberts J.A.
        N-methyl-D-aspartate receptors enhance mechanical responses and voltage-dependent Ca2+ channels in rat dorsal root ganglia neurons through protein kinase C.
        Neuroscience. 2004; 128: 347-357
        • Chen L.
        • Huang L.Y.
        Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation.
        Nature. 1992; 356: 521-523
        • Chung K.M.
        • Suh H.W.
        Pretreatment with cholera or pertussis toxin differentially modulates morphine- and beta-endorphin-induced antinociception in the mouse formalin test.
        Neuropeptides. 2001; 35: 197-203
        • Deberry J.
        • Ness T.J.
        • Robbins M.T.
        • Randich A.
        Inflammation-induced enhancement of the visceromotor reflex to urinary bladder distention: Modulation by endogenous opioids and the effects of early-in-life experience with bladder inflammation.
        J Pain. 2007; 8: 914-923
        • Desmeules J.A.
        • Cedraschi C.
        • Rapiti E.
        • Baumgartner E.
        • Finckh A.
        • Cohen P.
        • Dayer P.
        • Vischer T.L.
        Neurophysiologic evidence for a central sensitization in patients with fibromyalgia.
        Arthritis Rheum. 2003; 48: 1420-1429
        • Dodick D.
        • Silberstein S.
        Central sensitization theory of migraine: Clinical implications.
        Headache. 2006; 46: 182-191
        • Dougherty P.M.
        • Palecek J.
        • Paleckova V.
        • Willis W.D.
        Neurokinin 1 and 2 antagonists attenuate the responses and NK1 antagonists prevent the sensitization of primate spinothalamic tract neurons after intradermal capsaicin.
        J Neurophysiol. 1994; 72: 1464-1475
        • Draisci G.
        • Iadarola M.J.
        Temporal analysis of increases in c-fos, preprodynorphin and preproenkephalin mRNAs in rat spinal cord.
        Brain Res Mol Brain Res. 1989; 6: 31-37
        • Dubner R.
        • Ren K.
        Brainstem mechanisms of persistent pain following injury.
        J Orofac Pain. 2004; 18: 299-305
        • Fishman P.H.
        Internalization and degradation of cholera toxin by cultured cells: Relationship to toxin action.
        J Cell Biol. 1982; 93: 860-865
        • Ganguly N.K.
        • Kaur T.
        Mechanism of action of cholera toxin & other toxins.
        Indian J Med Res. 1996; 104: 28-37
        • Hao J.X.
        • Yu W.
        • Xu X.J.
        Evidence that spinal endogenous opioidergic systems control the expression of chronic pain-related behaviors in spinally injured rats.
        Exp Brain Res. 1998; 118: 259-268
        • Ji R.R.
        • Woolf C.J.
        Neuronal plasticity and signal transduction in nociceptive neurons: Implications for the initiation and maintenance of pathological pain.
        Neurobiol Dis. 2001; 8: 1-10
        • Jones S.L.
        • Gebhart G.F.
        Inhibition of spinal nociceptive transmission from the midbrain, pons and medulla in the rat: Activation of descending inhibition by morphine, glutamate and electrical stimulation.
        Brain Res. 1988; 460: 281-296
        • Kawasaki Y.
        • Kohno T.
        • Zhuang Z.Y.
        • Brenner G.J.
        • Wang H.B.
        • Van der Meer C.
        • Befort K.
        • Woolf C.J.
        • Ji R.R.
        Ionotropic and metabotropic receptors, protein kinase A, protein kinase C, and Src contribute to C-fiber-induced ERK activation and cAMP response element-binding protein phosphorylation in dorsal horn neurons, leading to central sensitization.
        J Neurosci. 2004; 24: 8310-8321
        • Klepstad P.
        • Rakvag T.T.
        • Kaasa S.
        • Holthe M.
        • Dale O.
        • Borchgrevink P.C.
        • Baar C.
        • Vikan T.
        • Krokan H.E.
        • Skorpen F.
        The 118 A > G polymorphism in the human mu-opioid receptor gene may increase morphine requirements in patients with pain caused by malignant disease.
        Acta Anaesthesiol Scand. 2004; 48: 1232-1239
        • Latremoliere A.
        • Woolf C.J.
        Central sensitization: A generator of pain hypersensitivity by central neural plasticity.
        J Pain. 2009; 10: 895-926
        • Lee B.H.
        • Park S.H.
        • Won R.
        • Park Y.G.
        • Sohn J.H.
        Antiallodynic effects produced by stimulation of the periaqueductal gray matter in a rat model of neuropathic pain.
        Neurosci Lett. 2000; 291: 29-32
        • Lin Q.
        • Peng Y.B.
        • Willis W.D.
        Possible role of protein kinase C in the sensitization of primate spinothalamic tract neurons.
        J Neurosci. 1996; 16: 3026-3034
        • Lovati C.
        • D'Amico D.
        • Bertora P.
        Allodynia in migraine: Frequent random association or unavoidable consequence?.
        Expert Rev Neurother. 2009; 9: 395-408
        • Madland G.
        • Feinmann C.
        Chronic facial pain: A multidisciplinary problem.
        J Neurol Neurosurg Psychiatry. 2001; 71: 716-719
        • Madland G.
        • Newton-John T.
        • Feinmann C.
        Chronic idiopathic orofacial pain: I: What is the evidence base?.
        Br Dent J. 2001; 191: 22-24
        • Messersmith D.J.
        • Kim D.J.
        • Iadarola M.J.
        Transcription factor regulation of prodynorphin gene expression following rat hindpaw inflammation.
        Brain Res Mol Brain Res. 1998; 53: 260-269
        • Neubert K.
        • King C.
        • Malphurs W.
        • Wong F.
        • Weaver J.P.
        • Jenkins A.C.
        • Rossi H.L.
        • Caudle R.M.
        Characterization of mouse orofacial pain and the effects of lesioning TRPV1-expressing neurons on operant behavior.
        Mol Pain. 2008; 4: 43
        • Neubert J.K.
        • Rossi H.L.
        • Malphurs W.
        • Vierck Jr., C.J.
        • Caudle R.M.
        Differentiation between capsaicin-induced allodynia and hyperalgesia using a thermal operant assay.
        Behav Brain Res. 2006; 170: 308-315
        • Neubert J.K.
        • Rossi H.L.
        • Pogar J.
        • Jenkins A.C.
        • Caudle R.M.
        Effects of mu- and kappa-2 opioid receptor agonists on pain and rearing behaviors.
        Behav Brain Funct. 2007; 3: 49
        • Neubert J.K.
        • Widmer C.G.
        • Malphurs W.
        • Rossi H.L.
        • Vierck Jr., C.J.
        • Caudle R.M.
        Use of a novel thermal operant behavioral assay for characterization of orofacial pain sensitivity.
        Pain. 2005; 116: 386-395
        • Newton-John T.
        • Madland G.
        • Feinmann C.
        Chronic idiopathic orofacial pain: II. What can the general dental practitioner do?.
        Br Dent J. 2001; 191: 72-73
        • Oertel B.G.
        • Kettner M.
        • Scholich K.
        • Renne C.
        • Roskam B.
        • Geisslinger G.
        • Schmidt P.H.
        • Lotsch J.
        A Common Human {micro}-Opioid Receptor Genetic Variant Diminishes the Receptor Signaling Efficacy in Brain Regions Processing the Sensory Information of Pain.
        J Biol Chem. 2009; 284: 6530-6535
        • Pietrobon D.
        Migraine: New molecular mechanisms.
        Neuroscientist. 2005; 11: 373-386
        • Randich A.
        • Mebane H.
        • DeBerry J.J.
        • Ness T.J.
        Rostral ventral medulla modulation of the visceromotor reflex evoked by urinary bladder distension in female rats.
        J Pain. 2008; 9: 920-926
        • Ren K.
        • Dubner R.
        Central nervous system plasticity and persistent pain.
        J Orofac Pain. 1999; 13: 155-163
        • Rossi H.L.
        • Neubert J.K.
        Effects of environmental enrichment on thermal sensitivity in an operant orofacial pain assay.
        Behav Brain Res. 2008; 187: 478-482
        • Rossi H.L.
        • Vierck Jr., C.J.
        • Caudle R.M.
        • Neubert J.K.
        Characterization of cold sensitivity and thermal preference using an operant orofacial assay.
        Mol Pain. 2006; 2: 37
        • Scott D.B.
        • Blanpied T.A.
        • Ehlers M.D.
        Coordinated PKA and PKC phosphorylation suppresses RXR-mediated ER retention and regulates the surface delivery of NMDA receptors.
        Neuropharmacology. 2003; 45: 755-767
        • Sessle B.J.
        Acute and chronic craniofacial pain: Brainstem mechanisms of nociceptive transmission and neuroplasticity, and their clinical correlates.
        Crit Rev Oral Biol Med. 2000; 11: 57-91
        • Shen K.F.
        • Crain S.M.
        Cholera toxin-A subunit blocks opioid excitatory effects on sensory neuron action potentials indicating mediation by Gs-linked opioid receptors.
        Brain Res. 1990; 525: 225-231
        • Silberstein S.D.
        Migraine pathophysiology and its clinical implications.
        Cephalalgia. 2004; 24: 2-7
        • Sluka K.A.
        Activation of the cAMP transduction cascade contributes to the mechanical hyperalgesia and allodynia induced by intradermal injection of capsaicin.
        Br J Pharmacol. 1997; 122: 1165-1173
        • Sluka K.A.
        • Rees H.
        • Chen P.S.
        • Tsuruoka M.
        • Willis W.D.
        Capsaicin-induced sensitization of primate spinothalamic tract cells is prevented by a protein kinase C inhibitor.
        Brain Res. 1997; 772: 82-86
        • Smith P.K.
        • Krohn R.I.
        • Hermanson G.T.
        • Mallia A.K.
        • Gartner F.H.
        • Provenzano M.D.
        • Fujimoto E.K.
        • Goeke N.M.
        • Olson B.J.
        • Klenk D.C.
        Measurement of protein using bicinchoninic acid.
        Anal Biochem. 1985; 150: 76-85
        • Sohn J.H.
        • Lee B.H.
        • Park S.H.
        • Ryu J.W.
        • Kim B.O.
        • Park Y.G.
        Microinjection of opiates into the periaqueductal gray matter attenuates neuropathic pain symptoms in rats.
        Neuroreport. 2000; 11: 1413-1416
        • Srikiatkhachorn A.
        Chronic daily headache: A scientist's perspective.
        Headache. 2002; 42: 532-537
        • Staud R.
        • Bovee C.E.
        • Robinson M.E.
        • Price D.D.
        Cutaneous C-fiber pain abnormalities of fibromyalgia patients are specifically related to temporal summation.
        Pain. 2008; 139: 315-323
        • Staud R.
        • Craggs J.G.
        • Perlstein W.M.
        • Robinson M.E.
        • Price D.D.
        Brain activity associated with slow temporal summation of C-fiber evoked pain in fibromyalgia patients and healthy controls.
        Eur J Pain. 2008; 12: 1078-1089
        • Staud R.
        • Koo E.
        • Robinson M.E.
        • Price D.D.
        Spatial summation of mechanically evoked muscle pain and painful aftersensations in normal subjects and fibromyalgia patients.
        Pain. 2007; 130: 177-187
        • Staud R.
        • Robinson M.E.
        • Price D.D.
        Temporal summation of second pain and its maintenance are useful for characterizing widespread central sensitization of fibromyalgia patients.
        J Pain. 2007; 8: 893-901
        • Tingley W.G.
        • Roche K.W.
        • Thompson A.K.
        • Huganir R.L.
        Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain.
        Nature. 1993; 364: 70-73
        • Vierck C.J.
        • Acosta-Rua A.J.
        • Johnson R.D.
        Bilateral chronic constriction of the sciatic nerve: A model of long-term cold hyperalgesia.
        J Pain. 2005; 6: 507-517
        • Willis W.D.
        Role of neurotransmitters in sensitization of pain responses.
        Ann NY Acad Sci. 2001; 933: 142-156
        • Woolf C.J.
        The pathophysiology of peripheral neuropathic pain–abnormal peripheral input and abnormal central processing.
        Acta Neurochir Suppl (Wien). 1993; 58: 125-130
        • Woolf C.J.
        • Thompson S.W.
        • King A.E.
        Prolonged primary afferent induced alterations in dorsal horn neurones, an intracellular analysis in vivo and in vitro.
        J Physiol (Paris). 1988; 83: 255-266
        • Woolf C.J.
        • Thompson S.W.N.
        The induction and maintenance of central sensitization is dependent on N-methyl-D-aspartic acid receptor activation; Implications for the treatment of postinjury pain hypersensitivity states.
        Pain. 1991; 44: 293-299
        • Zoppi M.
        • Maresca M.
        Symptoms accompanying fibromyalgia.
        Reumatismo. 2008; 60: 217-220
        • Zou X.
        • Lin Q.
        • Willis W.D.
        Role of protein kinase a in phosphorylation of NMDA receptor 1 subunits in dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats.
        Neuroscience. 2002; 115: 775-786
        • Zou X.J.
        • Lin Q.
        • Willis W.D.
        Enhanced phosphorylation of NMDA receptor 1 subunits in spinal cord dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats.
        J Neurosci. 2000; 20: 6989-6997