RESUMEN
Microglia/astrocyte and B cell neuroimmune responses are major contributors to the neurological deficits after traumatic spinal cord injury (SCI). Bruton tyrosine kinase (BTK) activation mechanistically links these neuroimmune mechanisms. Our objective is to use Ibrutinib, an FDA-approved BTK inhibitor, to inhibit the neuroimmune cascade thereby improving locomotor recovery after SCI. Rat models of contusive SCI, Western blot, immunofluorescence staining imaging, flow cytometry analysis, histological staining, and behavioral assessment were used to evaluate BTK activity, neuroimmune cascades, and functional outcomes. Both BTK expression and phosphorylation were increased at the lesion site at 2, 7, 14, and 28 days after SCI. Ibrutinib treatment (6 mg/kg/day, IP, starting 3 h post-injury for 7 or 14 days) reduced BTK activation and total BTK levels, attenuated the injury-induced elevations in Iba1, GFAP, CD138, and IgG at 7 or 14 days post-injury without reduction in CD45RA B cells, improved locomotor function (BBB scores), and resulted in a significant reduction in lesion volume and significant improvement in tissue-sparing 11 weeks post-injury. These results indicate that Ibrutinib exhibits neuroprotective effects by blocking excessive neuroimmune responses through BTK-mediated microglia/astroglial activation and B cell/antibody response in rat models of SCI. These data identify BTK as a potential therapeutic target for SCI.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Neuroinmunomodulación , Recuperación de la Función , Traumatismos de la Médula Espinal/inmunología , Adenina/análogos & derivados , Adenina/farmacología , Adenina/uso terapéutico , Agammaglobulinemia Tirosina Quinasa/metabolismo , Animales , Formación de Anticuerpos/efectos de los fármacos , Astrocitos/patología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Peso Corporal/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunoglobulina G/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Microglía/patología , Actividad Motora/efectos de los fármacos , Neuroinmunomodulación/efectos de los fármacos , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , Piperidinas/uso terapéutico , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/metabolismo , Ratas , Recuperación de la Función/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Bazo/patología , Sindecano-1/metabolismo , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) are implicated in the pathophysiology of spinal cord injury (SCI). However, the specific functions of individual ERK isoforms in neurodegeneration are largely unknown. We investigated the hypothesis that ERK2 activation may contribute to pathological and functional deficits following SCI and that ERK2 knockdown using RNA interference may provide a novel therapeutic strategy for SCI. Lentiviral ERK2 shRNA and siRNA were utilized to knockdown ERK2 expression in the spinal cord following SCI. Pre-injury intrathecal administration of ERK2 siRNA significantly reduced excitotoxic injury-induced activation of ERK2 (p < 0.001) and caspase 3 (p < 0.01) in spinal cord. Intraspinal administration of lentiviral ERK2 shRNA significantly reduced ERK2 expression in the spinal cord (p < 0.05), but did not alter ERK1 expression. Administration of the lentiviral ERK2 shRNA vector 1 week prior to severe spinal cord contusion injury resulted in a significant improvement in locomotor function (p < 0.05), total tissue sparing (p < 0.05), white matter sparing (p < 0.05), and gray matter sparing (p < 0.05) 6 weeks following severe contusive SCI. Our results suggest that ERK2 signaling is a novel target associated with the deleterious consequences of spinal injury.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Traumatismos de la Médula Espinal/enzimología , Animales , Modelos Animales de Enfermedad , Femenino , Inyecciones Espinales/métodos , Laminectomía/métodos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/genética , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Células PC12 , Ácido Quiscuálico , ARN Interferente Pequeño/farmacología , Ratas , Ratas Long-Evans , Traumatismos de la Médula Espinal/etiología , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Transfección/métodosRESUMEN
Small molecule inhibitors of calcium-dependent proteases, calpains (CAPNs), protect against neurodegeneration induced by a variety of insults including excitotoxicity and spinal cord injury (SCI). Many of these compounds, however, also inhibit other proteases, which has made it difficult to evaluate the contribution of calpains to neurodegeneration. Calpastatin is a highly specific endogenous inhibitor of classical calpains, including CAPN1 and CAPN2. In the present study, we utilized transgenic mice that overexpress human calpastatin under the prion promoter (PrP-hCAST) to evaluate the hypothesis that calpastatin overexpression protects against excitotoxic hippocampal injury and contusive SCI. The PrP-hCAST organotypic hippocampal slice cultures showed reduced neuronal death and reduced calpain-dependent proteolysis (α-spectrin breakdown production, 145 kDa) at 24 h after N-methyl-D-aspartate (NMDA) injury compared with the wild-type (WT) cultures (n = 5, p < 0.05). The PrP-hCAST mice (n = 13) displayed a significant improvement in locomotor function at one and three weeks after contusive SCI compared with the WT controls (n = 9, p < 0.05). Histological assessment of lesion volume and tissue sparing, performed on the same animals used for behavioral analysis, revealed that calpastatin overexpression resulted in a 30% decrease in lesion volume (p < 0.05) and significant increases in tissue sparing, white matter sparing, and gray matter sparing at four weeks post-injury compared with WT animals. Calpastatin overexpression reduced α-spectrin breakdown by 51% at 24 h post-injury, compared with WT controls (p < 0.05, n = 3/group). These results provide support for the hypothesis that sustained calpain-dependent proteolysis contributes to pathological deficits after excitotoxic injury and traumatic SCI.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Hipocampo/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Animales , Hipocampo/patología , Humanos , Locomoción/fisiología , Ratones , Ratones TransgénicosRESUMEN
We previously reported the serendipitous observation that fenbendazole, a benzimidazole anthelmintic, improved functional and pathological outcomes following thoracic spinal cord contusion injury in mice when administered pre-injury. Fenbendazole is widely used in veterinary medicine. However, it is not approved for human use and it was uncertain if only post-injury administration would offer similar benefits. In the present study we evaluated post-injury administration of a closely related, human anthelmintic drug, flubendazole, using a rat spinal cord contusion injury model. Flubendazole, administered i.p. 5 or 10 mg/kg day, beginning 3 h post-injury and daily thereafter for 2 or 4 weeks, resulted in improved locomotor function after contusion spinal cord injury (SCI) compared with vehicle-treated controls. Histological analysis of spinal cord sections showed that such treatment with flubendazole also reduced lesion volume and improved total tissue sparing, white matter sparing, and gray matter sparing. Flubendazole inhibited the activation of glial fibrillary acidic protein (GFAP); suppressed cyclin B1 expression and Bruton tyrosine kinase activation, markers of B cell activation/proliferation and inflammation; and reduced B cell autoimmune response. Together, these results suggest the use of the benzimidazole anthelmintic flubendazole as a potential therapeutic for SCI.
Asunto(s)
Mebendazol/análogos & derivados , Fármacos Neuroprotectores/farmacología , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal/patología , Médula Espinal/efectos de los fármacos , Animales , Antinematodos/farmacología , Reposicionamiento de Medicamentos , Femenino , Mebendazol/farmacología , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Although calpain (calcium-activated cysteine protease) inhibition represents a rational therapeutic target for spinal cord injury (SCI), few studies have reported improved functional outcomes with post-injury administration of calpain inhibitors. This reflects the weak potency and limited aqueous solubility of current calpain inhibitors. Previously, we demonstrated that intraspinal microinjection of the calpain inhibitor MDL28170 resulted in greater inhibition of calpain activity as compared to systemic administration of the same compound. In the present study, we evaluated the ability of intraspinal MDL28170 microinjection to spare spinal tissue and locomotor dysfunction following SCI. Contusion SCI was produced in female Long-Evans rats using the Infinite Horizon impactor at the 200-kdyn force setting. Open-field locomotion was evaluated until 6 weeks post-injury. Histological assessment of tissue sparing was performed at 6 weeks after SCI. The results demonstrate that MDL28170, administered with a single post-injury intraspinal microinjection (50 nmoles), significantly improves both locomotor function and pathological outcome measures following SCI.
Asunto(s)
Calpaína/antagonistas & inhibidores , Dipéptidos/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Animales , Calpaína/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Modelos Animales de Enfermedad , Femenino , Microinyecciones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Trastornos del Movimiento/tratamiento farmacológico , Trastornos del Movimiento/enzimología , Trastornos del Movimiento/fisiopatología , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/enzimología , Fibras Nerviosas Mielínicas/patología , Ratas , Ratas Long-Evans , Médula Espinal/enzimología , Médula Espinal/patología , Traumatismos de la Médula Espinal/enzimología , Traumatismos de la Médula Espinal/patología , Resultado del TratamientoRESUMEN
Selenium is an essential element required for activity of several antioxidant enzymes, including glutathione peroxidase. Because of the critical role of the antioxidant system in responding to traumatic events, we hypothesized that dietary selenium supplementation would enhance neuroprotection in a rodent model of spinal cord injury. Rats were maintained on either a control or selenium-enriched diet prior to, and following, injury. Dietary selenium supplementation, provided as selenized yeast added to normal rat chow, resulted in a doubling of selenium levels in the spinal cord. Dietary selenium reduced the time required for recovery of bladder function following thoracic spinal cord injury. However, this was not accompanied by improvement in locomotor function or tissue sparing.
Asunto(s)
Suplementos Dietéticos , Locomoción/efectos de los fármacos , Compuestos de Organoselenio/uso terapéutico , Traumatismos de la Médula Espinal/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacos , Animales , Suplementos Dietéticos/análisis , Femenino , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Vejiga Urinaria/fisiopatologíaRESUMEN
PURPOSE: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. METHODS: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. RESULTS: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. CONCLUSIONS: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.
Asunto(s)
Calpaína/genética , Regulación Neoplásica de la Expresión Génica , Células Fotorreceptoras/metabolismo , ARN Neoplásico/genética , Neoplasias de la Retina/genética , Retinoblastoma/genética , Sinapsis/metabolismo , Animales , Western Blotting , Calpaína/biosíntesis , Bovinos , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Neoplasias Experimentales , Células Fotorreceptoras/patología , Retina/metabolismo , Retina/patología , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/metabolismo , Retinoblastoma/patología , Células Tumorales CultivadasRESUMEN
The role of the ERK1/2 signal transduction pathway and related transcription factors in the regulation of gene expression and pain behavior following excitotoxic spinal cord injury (SCI) was examined. Specifically, phosphorylation of ERK1/2, activation of transcription factors NF-kB, ELK-1, and CREB, and gene expression of the neurokinin-1 receptor and NMDA receptor subunits NR1 and NR-2A were investigated. Excitotoxic injury was produced by intraspinal injection of quisqualic acid (QUIS) in male Sprague-Dawley rats. Western blots were used to evaluate phosphorylation and activation of ERK1/2 and transcription factors using phospho-specific or total antibodies. Real-time PCR was used to evaluate gene expression of NK-1R, NR-1, and NR-2A. Assessment of excessive grooming behavior was used to evaluate the presence of spontaneous pain behavior. Excitotoxic spinal injury resulted in: (1) increased phosphorylation of ERK1/2; (2) increased activation of NF-kB and phosphorylation of ELK-1; and (3) increased gene expression for the NK-1 receptor and NR1 and NR-2A subunits of the NMDA receptor. Blockade of the ERK cascade with the MEK inhibitor PD98059 inhibited phosphorylation of ELK-1, activation of NF-kB and gene expression of NR1, NR-2A and NK-1R, and prevented the development of excessive grooming behavior. The results have shown that excitotoxic spinal injury leads to the injury-induced activation of the ERK-->ELK-1 and NF-kB signaling cascades and transcriptional regulation of receptors important in the development of chronic pain. Blockade of this intracellular kinase cascade prevented the onset of injury-induced pain behavior.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Degeneración Nerviosa/enzimología , Traumatismos de la Médula Espinal/enzimología , Médula Espinal/enzimología , Animales , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Genes Reguladores/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/fisiopatología , Dolor/enzimología , Dolor/genética , Dolor/fisiopatología , Ácido Quiscuálico , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Neuroquinina-1/metabolismo , Médula Espinal/efectos de los fármacos , Traumatismos de la Médula Espinal/inducido químicamente , Traumatismos de la Médula Espinal/fisiopatología , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Although high-resolution MR imaging is a valuable diagnostic tool, in vivo MR imaging has not yet been compared with in vitro MR imaging and histologic techniques following experimental spinal cord injury (SCI). The goal of the present study was to evaluate the feasibility of using in vivo MR imaging, in vitro MR imaging, and histologic techniques to study pathologic changes associated with excitotoxic SCI at a single time point. These results are important for future research using in vivo MR imaging to study the temporal profile of pathologic changes following SCI. METHODS: Rats received intraspinal injections of quisqualic acid at the T12-L2 spinal level. In vivo T1- and T2-weighted and dynamic contrast-enhanced MR images were collected 17-24 days postinjury. Once completed, spinal cords were removed and in vitro MR microscopy and histologic assessment were performed. MR images were collected using 4.7-T (in vivo) and 14.1-T magnets (in vitro). RESULTS: Pathologic changes--including hemorrhage, neuronal loss, cavities, and central canal expansion--were visible in T2-weighted in vivo MR images. Evaluation of the blood-spinal cord barrier after injury with contrast agent enhancement showed no disruption at the time points evaluated. In vitro MR images and histologic evaluation confirmed pathologic details observed in vivo. CONCLUSION: Results show that high-resolution in vivo MR imaging has the potential to be used in studying the progression of pathologic changes at multiple time points following SCI. This strategy may provide a way of studying structure-function relationships between therapeutic interventions and different pathologic characteristics of the injured spinal cord.
Asunto(s)
Agonistas de Aminoácidos Excitadores , Imagen por Resonancia Magnética , Ácido Quiscuálico , Enfermedades de la Médula Espinal/inducido químicamente , Enfermedades de la Médula Espinal/diagnóstico , Médula Espinal/patología , Animales , Permeabilidad Capilar , Agonistas de Aminoácidos Excitadores/administración & dosificación , Estudios de Factibilidad , Hemorragia/inducido químicamente , Hemorragia/patología , Inyecciones Espinales , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética , Masculino , Ácido Quiscuálico/administración & dosificación , Ratas , Ratas Sprague-Dawley , Médula Espinal/irrigación sanguínea , Enfermedades de la Médula Espinal/metabolismo , Enfermedades de la Médula Espinal/patologíaRESUMEN
Intraspinal injection of the AMPA/metabotropic receptor agonist quisqualic acid (QUIS) results in a pathophysiology that leads to excessive grooming behavior, which has been proposed as a model of spontaneous at-level pain after spinal cord injury (SCI). To further characterize the onset and progression of this behavior we evaluated the effects of 3 drugs, agmatine (Agm), interleukin-10 (IL-10), and cyclosporin A (CsA), on different characteristics of this behavior. In these experiments rats were given saline, Agm, CsA10, or CsA20 once daily for 14 days (or a single injection of IL-10) starting either 30 minutes post-QUIS (group 1) or 10 to 18 days post-QUIS when excessive grooming behavior had been established (group 2). In the first group of animals agmatine, IL-10, CsA10, or CsA20 reduced the longitudinal extent of neuronal loss in the spinal cord compared to QUIS-injected animals treated with saline. The behavioral consequences of this effect included the delayed onset of excessive grooming behavior, reduction in the area of skin targeted for excessive grooming, and reduced grooming severity. Animals treated at the time of excessive grooming onset showed significantly reduced grooming area, grooming severity, and neuronal loss in the spinal cord compared to QUIS animals treated with saline. In conclusion, systemic administration of Agm, IL-10, or CsA significantly delayed the onset and reduced the severity of a spontaneous pain-like behavior. These effects are believed to be due, in part, to the neuroprotective properties of these drugs against QUIS-induced excitotoxicity. The effective treatment of excessive grooming behavior suggests that Agm, IL-10, and CsA modulate ongoing cellular events responsible for the progression of this behavior.
Asunto(s)
Agmatina/farmacología , Ciclosporina/farmacología , Modelos Animales de Enfermedad , Aseo Animal/efectos de los fármacos , Inmunosupresores/farmacología , Interleucina-10/farmacología , Dolor/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Agonistas de Aminoácidos Excitadores , Masculino , Dolor/psicología , Ácido Quiscuálico , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/inducido químicamente , Factores de TiempoRESUMEN
Using a conjugate of substance P and the ribosome-inactivating protein saporin, neurons expressing the neurokinin-1 receptor in lamina I of the spinal cord were targeted to determine their role in the expression of a spontaneous pain behavior following intraspinal injections of quisqualic acid in the rat. Treatment was carried out at the time of injury in order to prevent the onset of the behavior, and following onset in order to evaluate the potential clinical utility of this intervention. Treatment at the time of injury resulted in significant decreases in onset-time and severity of pain behavior, while treatment at the time of onset led to a significant reduction of the spontaneous self-directed behavior. The results suggest that the substrate for at-level pain following spinal cord injury includes a population of spinal neurons expressing the neurokinin-1 receptor in the superficial laminae of the spinal cord.
Asunto(s)
Neurotoxinas/farmacología , Dolor/etiología , Dolor/fisiopatología , Células del Asta Posterior/metabolismo , Receptores de Neuroquinina-1/metabolismo , Traumatismos de la Médula Espinal/complicaciones , Médula Espinal/fisiopatología , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Modelos Animales de Enfermedad , Aseo Animal/efectos de los fármacos , Aseo Animal/fisiología , Inmunohistoquímica , Inmunotoxinas/farmacología , Inmunotoxinas/uso terapéutico , N-Glicosil Hidrolasas/farmacología , N-Glicosil Hidrolasas/uso terapéutico , Neurotoxinas/uso terapéutico , Dolor/tratamiento farmacológico , Proteínas de Plantas/farmacología , Proteínas de Plantas/uso terapéutico , Células del Asta Posterior/citología , Células del Asta Posterior/efectos de los fármacos , Ratas , Receptores de Neuroquinina-1/efectos de los fármacos , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Automutilación/tratamiento farmacológico , Automutilación/metabolismo , Automutilación/fisiopatología , Piel/inervación , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Sustancia P/farmacología , Sustancia P/uso terapéuticoRESUMEN
The role of NMDA receptor expression and post-translational modification in the pathological and behavioral consequences of injury were examined in rats receiving spinal injections of quisqualate. Spinal cords were removed 3 days following the development of excessive grooming behavior or, if the spontaneous pain-like behavior was not observed, 13 days following injections. Western blots from the spinal tissue demonstrated that non-grooming animals had elevated protein levels of the NR1 subunit of the NMDA receptor. These subunits did not demonstrate an enhanced level of phosphorylation. NR1 protein in grooming rats was not elevated, but there was a significant increase in NR1 serine phosphorylation. These findings suggest that excitotoxic lesions of the spinal cord induce both NR1 expression and NR1 serine phosphorylation. However, the injury-induced excessive grooming behavior is only associated with phosphorylation of the NR1 subunit.
Asunto(s)
Neuronas/metabolismo , Neurotoxinas/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Animales , Modelos Animales de Enfermedad , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Aseo Animal/efectos de los fármacos , Aseo Animal/fisiología , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ácido Quiscuálico/farmacología , Ratas , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Serina/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
To evaluate the hypothesis that calpain 1 knockdown would reduce pathological damage and functional deficits after spinal cord injury (SCI), we developed lentiviral vectors encoding calpain 1 shRNA and eGFP as a reporter (LV-CAPN1 shRNA). The ability of LV-CAPN1 shRNA to knockdown calpain 1 was confirmed in rat NRK cells using Northern and Western blot analysis. To investigate the effects on spinal cord injury, LV-CAPN1shRNA or LV-mismatch control shRNA (LV-control shRNA) were administered by convection enhanced diffusion at spinal cord level T10 in Long-Evans female rats (200-250 g) 1 week before contusion SCI, 180 kdyn force, or sham surgery at the same thoracic level. Intraspinal administration of the lentiviral particles resulted in transgene expression, visualized by eGFP, in spinal tissue at 2 weeks after infection. Calpain 1 protein levels were reduced by 54% at T10 2 weeks after shRNA-mediated knockdown (p<0.05, compared with the LV-control group, n=3 per group) while calpain 2 levels were unchanged. Intraspinal administration of LV-CAPN1shRNA 1 week before contusion SCI resulted in a significant improvement in locomotor function over 6 weeks postinjury, compared with LV-control administration (p<0.05, n=10 per group). Histological analysis of spinal cord sections indicated that pre-injury intraspinal administration of LV-CAPN1shRNA significantly reduced spinal lesion volume and improved total tissue sparing, white matter sparing, and gray matter sparing (p<0.05, n=10 per group). Together, results support the hypothesis that calpain 1 activation contributes to the tissue damage and impaired locomotor function after SCI, and that calpain1 represents a potential therapeutic target.
Asunto(s)
Calpaína/deficiencia , Calpaína/genética , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Animales , Calpaína/fisiología , Línea Celular , Femenino , Técnicas de Silenciamiento del Gen/métodos , Actividad Motora/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Long-Evans , Traumatismos de la Médula Espinal/fisiopatología , Supervivencia Tisular/genética , Resultado del TratamientoRESUMEN
Following contusive spinal cord injury (SCI), calpain activity is dramatically increased and remains elevated for days to weeks. Although calpain inhibition has previously been demonstrated to be neuroprotective following spinal cord injury, most studies administered the calpain inhibitor at a single time point. We hypothesized that sustained calpain inhibition would improve functional and pathological outcomes, as compared to the results obtained with a single postinjury administration of the calpain inhibitor. Contusion SCI was produced in female Long-Evans rats using the Infinite Horizon spinal cord injury impactor at the 200 kdyn force setting. Open-field locomotor function was evaluated until 6 weeks postinjury. Histological assessment of lesion volume and tissue sparing was performed at 6 weeks after SCI. Calpain inhibitor MDL28170 administered as a single postinjury i.v. bolus (20 mg/kg) or as a daily i.p. dose (1 mg/kg) improved locomotor function, but did not increase tissue sparing. Combined i.v. and daily i.p. MDL28170 administration resulted in significant improvement in both functional and pathological outcome measures, supporting the calpain theory of SCI proposed by Dr. Banik and colleagues.