RESUMEN
INTRODUCTION: Rodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations. METHODS: This study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single-cell ratiometric calcium imaging, with the activation of neurons to the following pruritogenic and algogenic substances: histamine, chloroquine, canine protease-activated receptor 2 (PAR2) activating peptide (SLIGKT), compound 48/80, 5-hydroxytryptamine receptor agonist (5-HT), bovine adrenal medulla peptide (BAM8-22), substance P, allyl isothiocyanate (AITC), and capsaicin. RESULTS: This study demonstrates a simple dissection and rapid processing of DRG collected from canine cadavers used to create viable primary sensory neuron cultures to measure responses to pruritogens and algogens. CONCLUSION: Ratiometric calcium imaging demonstrated that small-diameter canine sensory neurons can be activated by multiple stimuli, and a single neuron can react to both a pruritogenic stimulation and an algogenic stimulation.
Asunto(s)
Imagen Molecular/métodos , Prurito/fisiopatología , Células Receptoras Sensoriales/fisiología , Animales , Cadáver , Radioisótopos de Calcio , Capsaicina/farmacología , Bovinos , Células Cultivadas , Cloroquina/farmacología , Perros , Ganglios Espinales/fisiopatología , Histamina/farmacología , Humanos , Laminectomía , Neurotransmisores/farmacología , Dolor/fisiopatología , Fragmentos de Péptidos/farmacología , Prurito/inducido químicamente , Radiofármacos , Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Sustancia P/farmacologíaAsunto(s)
Antipruriginosos/farmacología , Inhibidores de las Cinasas Janus/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Animales , Antipruriginosos/química , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Femenino , Humanos , Inhibidores de las Cinasas Janus/química , Ratones , Modelos Moleculares , Conformación Molecular , Imagen Molecular/métodos , Unión Proteica , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Relación Estructura-Actividad , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/metabolismoRESUMEN
The janus kinase-inhibitor oclacitinib is licensed for the control of pruritus associated with allergic skin diseases in dogs. Strikingly, it has been clinically reported that abrupt withdrawal of oclacitinib leads to a rebound pruritus in dogs. Therefore, the primary objective of this study was to mimic the rebound phenomenon of oclacitinib using a chronic pruritic mouse model of allergic contact dermatitis. Chronic allergic contact dermatitis was induced by repetitive toluene-2,4-diisocyanate (TDI) challenge in BALB/c mice. Oclacitinib was orally administered twice daily at 45mg/kg for 7 days, with concurrent TDI challenge, and then treatment of oclacitinib was abruptly discontinued. Scratching bouts following TDI challenge were evaluated to day 15. Additionally, dorsal root ganglia (DRG) and affected skin were isolated from mice receiving oclacitinib and from mice 24h after oclacitinib withdrawal and were used to determine pruritogen induced Ca2+ signals in sensory neurons, the number of activated dendritic cells (DCs) within DRG, and the cytokine profiles of affected skin. Mice treated with oclacitinib showed a significant decrease in scratching bouts during treatment, then following abrupt withdrawal scratching bouts were significantly increased. Furthermore, following abrupt withdrawal more DRG neurons were activated by pruritogenic cytokines, TNFα positive DCs were significantly increased, and affected skin revealed a significant increase of TNFα and TSLP. In conclusion, while oclacitinib significantly reduced itch during treatment the abrupt withdrawal led to a rapid rebound phenomenon which can be explained by an increase in pruritogenic cytokines and fast peripheral sensitization.