RESUMEN
Pruriceptive itch originates following activation of peripheral sensory nerve terminals when pruritogens come in contact with the skin. The ability of botulinum neurotoxins (BoNTs) to attenuate transmitter release from afferent terminals provides a rationale for studying its effect on pruritus. This study investigated the effects of BoNT/A1 and BoNT/B1 on mast cell dependent (Compound 48/80:48/80) and independent (Chloroquine:CQ) scratching. C57Bl/6 male mice received intradermal injection of 1.5 U of BoNT/A1, BoNT/B1 or saline 2, 7, 14 and 21 days prior to ipsilateral 48/80 or CQ at the nape of the neck. Ipsilateral hind paw scratching was determined using an automated recording device. The effect of BoNTs on 48/80 mediated mast cell degranulation was analyzed in human and murine mast cells and the presence of SNAREs was determined using qPCR, immunostaining and Western blot. Pre-treatment with BoNT/A1 and BoNT/B1 reduced 48/80 and CQ induced scratching behavior starting on day 2 with reversal by day 21. Both serotypes inhibited 48/80 induced mast cell degranulation. qPCR and immunostaining detected SNAP-25 mRNA and protein, respectively, in mast cells, however, Western blots did not. This study demonstrates the long-lasting anti-pruritic effects of two BoNT serotypes, in a murine pruritus model using two different mechanistically driven pruritogens. These data also indicate that BoNTs may have a direct effect upon mast cell degranulation.
Asunto(s)
Toxinas Botulínicas Tipo A/farmacología , Mastocitos/efectos de los fármacos , Animales , Toxinas Botulínicas Tipo A/uso terapéutico , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Cloroquina , Humanos , Masculino , Mastocitos/fisiología , Ratones Endogámicos C57BL , Prurito/inducido químicamente , Prurito/tratamiento farmacológico , p-Metoxi-N-metilfenetilaminaRESUMEN
Increasing evidence suggests that botulinum neurotoxins (BoNTs) delivered into the skin and muscle in certain human and animal pain states may exert antinociceptive efficacy though their uptake and transport to central afferent terminals. Cleavage of soluble N-methylaleimide-sensitive attachment protein receptor by BoNTs can impede vesicular mediated neurotransmitter release as well as transport/insertion of channel/receptor subunits into plasma membranes, an effect that can reduce activity-evoked facilitation. Here, we explored the effects of intraplantar botulinum toxin- B (BoNT-B) on peripheral inflammation and spinal nociceptive processing in an inflammatory model of pain. C57BL/6 mice (male) received unilateral intraplantar BoNT (1 U, 30 µL) or saline prior to intraplantar carrageenan (20 µL, 2%) or intrathecal N-methyl-D-aspartate (NMDA), substance P or saline (5 µL). Intraplantar carrageenan resulted in edema and mechanical allodynia in the injected paw and increased phosphorylation of a glutamate subunit (pGluA1ser845) and a serine/threonine-specific protein kinase (pAktser473) in spinal dorsal horn along with an increased incidence of spinal c-Fos positive cells. Pre-treatment with intraplantar BoNT-B reduced carrageenan evoked: (i) allodynia, but not edema; (ii) pGluA1 and pAkt and (iii) c-Fos expression. Further, intrathecal NMDA and substance P each increased dorsal horn levels of pGluA1 and pAkt. Intraplantar BoNT-B inhibited NMDA, but not substance P evoked phosphorylation of GluA1 and Akt. These results suggest that intraplantar toxin is transported centrally to block spinal activation and prevent phosphorylation of a glutamate receptor subunit and a kinase, which otherwise contribute to facilitated states.
Asunto(s)
Analgésicos/farmacología , Toxinas Botulínicas Tipo A/farmacología , Nocicepción , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores AMPA/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Analgésicos/administración & dosificación , Analgésicos/uso terapéutico , Animales , Toxinas Botulínicas Tipo A/administración & dosificación , Toxinas Botulínicas Tipo A/uso terapéutico , Carragenina/toxicidad , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , N-Metilaspartato/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Asta Dorsal de la Médula Espinal/fisiología , Sustancia P/farmacologíaRESUMEN
BACKGROUND: Mononeuropathies (MNs: nerve ligation) and polyneuropathies (PNs: cisplatin) produce unilateral and bilateral tactile allodynia, respectively. We examined the effects of intraplantar (IPLT) and intrathecal (IT) botulinum toxin B (BoNT-B) on this allodynia. METHODS: Mice (male c57Bl/6) were prepared with an L5 nerve ligation. Others received cisplatin (IP 2.3 mg/kg/d, every other day for 6 injections). Saline and BoNT-B were administered through the IPLT or IT route. We examined mechanical allodynia (von Frey hairs) before and at intervals after BoNT. As a control, we injected IPLT BoNT-B treated with dithiothreitol to cleave heavy chain from light chain. We measured motor function using acute thermal escape and sensorimotor tests. RESULTS: MN and PN mice showed a persistent ipsilateral and bilateral allodynia, respectively. IPLT BoNT-B resulted in an ipsilateral dorsal horn reduction in the synaptic protein target of BoNT-B (vesicle-associated membrane protein) and a long-lasting (up to approximately 17 days) reversal of allodynia in PN and MN models. The predominant effect after IPLT delivery was ipsilateral to IPLT BoNT. The effects of IPLT BoNT-B in MN mice were blocked by prior reduction of BoNT-B with dithiothreitol. IT BoNT-B in mice with PN resulted in a bilateral reversal of allodynia. With these dosing parameters, hind paw placing and stepping reflexes were unaltered, and there were no changes in thermal escape latencies. After cisplatin, dorsal root ganglions displayed increases in activation transcription factor 3, which were reduced by IT, but not IPLT BoNT-B. CONCLUSIONS: BoNT-B given IPLT and IT yields a long-lasting attenuation of the allodynia in mice displaying MN and PN allodynia.
Asunto(s)
Analgésicos/administración & dosificación , Toxinas Botulínicas Tipo A/administración & dosificación , Hiperalgesia/tratamiento farmacológico , Mononeuropatías/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Umbral del Dolor/efectos de los fármacos , Polineuropatías/tratamiento farmacológico , Factor de Transcripción Activador 3/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Inyecciones Espinales , Inyecciones Subcutáneas , Masculino , Ratones Endogámicos C57BL , Mononeuropatías/metabolismo , Mononeuropatías/fisiopatología , Mononeuropatías/psicología , Actividad Motora/efectos de los fármacos , Neuralgia/metabolismo , Neuralgia/fisiopatología , Neuralgia/psicología , Dimensión del Dolor , Estimulación Física , Polineuropatías/metabolismo , Polineuropatías/fisiopatología , Polineuropatías/psicología , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Tiempo de Reacción/efectos de los fármacos , Factores de Tiempo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
We addressed the hypothesis that intraplantar botulinum toxin B (rimabotulinumtoxin B: BoNT-B) has an early local effect upon peripheral afferent terminal releasing function and, over time, will be transported to the central terminals of the primary afferent. Once in the terminals it will cleave synaptic protein, block spinal afferent transmitter release, and thereby prevent spinal nociceptive excitation and behavior. In mice, C57Bl/6 males, intraplantar BoNT-B (1 U) given unilaterally into the hind paw had no effect upon survival or motor function, but ipsilaterally decreased: (1) intraplantar formalin-evoked flinching; (2) intraplantar capsaicin-evoked plasma extravasation in the hind paw measured by Evans blue in the paw; (3) intraplantar formalin-evoked dorsal horn substance P (SP) release (neurokinin 1 [NK1] receptor internalization); (4) intraplantar formalin-evoked dorsal horn neuronal activation (c-fos); (5) ipsilateral dorsal root ganglion (DRG) vesicle-associated membrane protein (VAMP); (6) ipsilateral SP release otherwise evoked bilaterally by intrathecal capsaicin; (7) ipsilateral activation of c-fos otherwise evoked bilaterally by intrathecal SP. These results indicate that BoNT-B, after unilateral intraplantar delivery, is taken up by the peripheral terminal, is locally active (blocking plasma extravasation), is transported to the ipsilateral DRG to cleave VAMP, and is acting presynaptically to block release from the spinal peptidergic terminal. The observations following intrathecal SP offer evidence for a possible transsynaptic effect of intraplantar BoNT. These results provide robust evidence that peripheral BoNT-B can alter peripheral and central terminal release from a nociceptor and attenuate downstream nociceptive processing via a presynaptic effect, with further evidence suggesting a possible postsynaptic effect.