RESUMEN
Following injury, dorsal root ganglion (DRG) neurons undergo transcriptional changes so as to adopt phenotypic changes that promote cell survival and axonal regeneration. Here we used a microarray approach to profile changes in a population of small noncoding RNAs known as microRNAs (miRNAs) in the L4 and L5 DRG following sciatic nerve transection. Results showed that 20 miRNA transcripts displayed a significant change in expression levels, with 8 miRNAs transcripts being altered by more than 1.5-fold. Using quantitative reverse transcription PCR, we demonstrated that one of these miRNAs, miR-21, was upregulated by 7-fold in the DRG at 7 days post-axotomy. In dissociated adult rat DRG neurons lentiviral vector-mediated overexpression of miR-21 promoted neurite outgrowth on a reduced laminin substrate. miR-21 directly downregulated expression of Sprouty2 protein, as confirmed by Western blot analysis and 3' untranslated region (UTR) luciferase assays. Our data show that miR-21 is an axotomy-induced miRNA that enhances axon growth, and suggest that miRNAs are important players in regulating growth pathways following peripheral nerve injury.
Asunto(s)
Envejecimiento/metabolismo , Axones/metabolismo , Axotomía , Ganglios Espinales/metabolismo , MicroARNs/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Neuritas/patología , Proteínas Serina-Treonina Quinasas , Ratas , Ratas Wistar , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Nervio Ciático/patología , Regulación hacia Arriba/genéticaRESUMEN
Voltage-gated sodium channels play an essential role in regulating the excitability of nociceptive primary afferent neurones. In particular the tetrodotoxin-sensitive (TTX-S) Na(V)1.7 and the tetrodotoxin-resistant (TTX-R) Na(V)1.8 and Na(V)1.9 channels have been suggested to play a role in inflammatory pain. Previous work has revealed acute administration of inflammatory mediators, such as Freund's complete adjuvant (FCA) or carrageenan caused an upregulation in the levels of Na(V)1.7 and Na(V)1.8 protein in DRG (dorsal root ganglia) tissue up to 4 days post-insult. In the present study, the expression of Na(V)1.7, Na(V)1.8 and Na(V)1.9 was examined over a 28 day timecourse during a rat model of FCA-induced chronic inflammatory joint pain. Using the retrograde tracer Fast Blue (FB) and specific Na(V)1.7, Na(V)1.8 and Na(V)1.9 sodium channel antibodies, immunohistochemical staining techniques were used to study sodium channel expression in a distinct population of L3-L5 knee joint afferent DRGs. In the ganglia, counts were made of positively labelled cells in the FB population. The results demonstrate that, following FCA injection, Na(V)1.9 expression is upregulated at days 14, 21 and 28 post-FCA, with Na(V)1.7 and Na(V)1.8 showing increased channel expression at days 14 and 28. These observations are accompanied by a unilateral joint hypersensitivity in the FCA-injected knee indicated by a behavioural shift in weight distribution measured using an incapacitance tester. The increased presence of these channels suggests that Na(V)1.7, Na(V)1.8 and Na(V)1.9 play a role, at least in part, in the maintenance of chronic inflammatory pain several weeks after the initial insult.
Asunto(s)
Artritis Experimental/fisiopatología , Ganglios Espinales/metabolismo , Hiperalgesia/fisiopatología , Articulación de la Rodilla/inervación , Proteínas del Tejido Nervioso/fisiología , Neuronas Aferentes/metabolismo , Neuropéptidos/fisiología , Canales de Sodio/fisiología , Vías Aferentes/fisiopatología , Animales , Artritis Experimental/complicaciones , Artritis Experimental/genética , Carragenina/toxicidad , Enfermedad Crónica , Adyuvante de Freund/toxicidad , Regulación de la Expresión Génica , Hiperalgesia/etiología , Hiperalgesia/genética , Cojera Animal/etiología , Vértebras Lumbares , Masculino , Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuropéptidos/biosíntesis , Neuropéptidos/genética , Ratas , Ratas Wistar , Canales de Sodio/biosíntesis , Canales de Sodio/genéticaRESUMEN
Chronic joint pain affects physical well being and can lead to severe psychological and social problems, therefore successful long-term management is highly sought-after. No current behavioural measures of pain used in pre-clinical models mimic the clinical dolorimeter, which provides an objective measure of joint hypersensitivity. In this study we aim to use a novel behavioural readout alongside an established measure to mimic the multifactorial measurements taken in the clinic. Using the pressure application measurement (PAM) device a gradually increasing squeeze was applied across the knee joint of rats until the animal gave an indication of pain or discomfort. PAM and the incapacitance tester were used to detect joint hypersensitivity in a well-established rodent model of adjuvant-induced arthritis. Subsequently, the analgesic effects of prednisolone (1, 3 or 10 mg kg(-1)), morphine (3 mg kg(-1)) and celecoxib (15 mg kg(-1)) were assessed. Both PAM and the incapacitance tester detected a reversal of hypersensitivity 1h post-drug administration. Furthermore, the two readouts were highly correlated, and power analysis indicated that PAM was highly reproducible. In conclusion, PAM provides a novel, accurate behavioural tool for detecting a primary mechanical hypersensitivity in a rat model of chronic inflammatory joint pain.
