RESUMO
SUMMARY: This study investigated the role and mechanism of aspirin combined with rehabilitation training in the nerve injury repair and Schwann cell changes in rats with sciatic nerve injury. Totally, 120 male healthy SD rats were randomly divided into sham, model, aspirin, and aspirin + rehabilitation groups, with 30 rats in each group. The sciatic nerve function index (SFI), photothermal pain tolerance threshold and inclined plane test results at 4, 6, and 8 weeks after operation were compared. The distance of sensory nerve regeneration and the expression of S100B protein in Schwann cells were analyzed. Compared with the sham group, the SFI of the model, aspirin, and aspirin+rehabilitation groups were significantly lower at 4, 6, and 8 weeks after operation. However, the aspirin and aspirin+rehabilitation groups had significantly higher SFI than the model group. The SFI at 6 and 8 weeks after operation was higher in the aspirin+rehabilitation group than that in the aspirin group (P<0.05). The photothermal pain tolerance threshold of the sham, aspirin, and aspirin+rehabilitation groups were significantly higher than those of the model group at 4, 6, and 8 weeks after operation (P<0.05). The inclination angles of the model, aspirin, and aspirin+rehabilitation groups were significantly lower than those of the sham group at 4, 6, and 8 weeks after operation, and the inclination angle of the aspirin+rehabilitation group was significantly higher than that of the model and aspirin groups (P<0.05). The sensory nerve regeneration distance in aspirin and aspirin+rehabilitation groups was higher than that in the sham and model groups (P<0.05). The expression of S100B protein in the aspirin and aspirin+rehabilitation groups was higher than that in the model group (P<0.05). Aspirin combined with rehabilitation training can promote the functional recovery of sciatic nerve injury, and the mechanism may be related to the increase of the expression of S100B protein in Schwann cells.
En este estudio se investigó el papel y el mecanismo que desempeña la aspirina combinada, con el entrenamiento de rehabilitación en la reparación de lesiones nerviosas y los cambios en los schwannocitos en ratas con lesiones en el nervio ciático. En total, 120 ratas SD macho sanas se dividieron aleatoriamente en cuatro grupos de 30 ratas en cada uno: simulación, modelo, aspirina y aspirina + rehabilitación. Se compararon el índice de función del nervio ciático (SFI), el umbral de tolerancia al dolor fototérmico y los resultados de la prueba del plano inclinado a las 4, 6 y 8 semanas después de la operación. Se analizó la distancia de regeneración del nervio sensorial y la expresión de la proteína S100B en los schwannocitos. En comparación con el grupo simulado, el SFI de los grupos modelo, aspirina y aspirina+rehabilitación fue significativamente menor a las 4, 6 y 8 semanas después de la operación. Sin embargo, los grupos de aspirina y aspirina + rehabilitación tuvieron un SFI significativamente más alto que el grupo modelo. El SFI a las 6 y 8 semanas después de la operación fue mayor en el grupo de aspirina + rehabilitación que en el grupo de aspirina (P<0,05). El umbral de tolerancia al dolor fototérmico de los grupos simulado, aspirina y aspirina+rehabilitación fue significativamente mayor que el del grupo modelo a las 4, 6 y 8 semanas después de la operación (P<0,05). Los ángulos de inclinación de los grupos modelo, aspirina y aspirina+rehabilitación fueron significativamente menores que los del grupo simulado a las 4, 6 y 8 semanas después de la operación, y el ángulo de inclinación del grupo aspirina+rehabilitación fue significativamente mayor que el de los grupos modelo y aspirina (P<0.05). La distancia de regeneración del nervio sensorial en los grupos de aspirina y aspirina+rehabilitación fue mayor que en los grupos simulado y modelo (P<0,05). La expresión de la proteína S100B en los grupos de aspirina y aspirina+rehabilitación fue mayor que en el grupo modelo (P<0,05). La aspirina combinada con el entrenamiento de rehabilitación puede promover la recuperación funcional de la lesión del nervio ciático, y el mecanismo puede estar relacionado con el aumento de la expresión de la proteína S100B en los schwannocitos.
Assuntos
Animais , Ratos , Nervo Isquiático/citologia , Exercício Físico , Aspirina/uso terapêutico , Neuropatia Ciática/reabilitação , Células de Schwann , Imuno-Histoquímica , Limiar da Dor , Terapia Combinada , Neuropatia Ciática/fisiopatologia , Modelos Animais de DoençasRESUMO
A promising alternative to conventional nerve grafting is the use of artificial grafts made from biodegradable and biocompatible materials and support cells. The aim of this study has been to produce a biodegradable nerve conduit and investigate the cytocompatibility with stem cells and its regeneration promoting properties in a rat animal model. A poly (lactic-co-glycolic acid) (PLGA) conduit of aligned nanofibers was produced by the electrospinning method, functionalized with gelatin and seeded either with mouse embryonic stem cells (mESCs) or with human mesenchymal stem cells (SHED). The cell proliferation and viability were analyzed in vitro. The conduits were implanted in a rat model of sciatic nerve lesion by transection. The functional recovery was monitored for 8 weeks using the Sciatic Functional Index (SFI) and histological analyses were used to assess the nerve regeneration. Scaffolds of aligned PLGA fibers with an average diameter of 0.90 ± 0.36 µm and an alignment coefficient of 0.817 ± 0.07 were produced. The treatment with gelatin increased the fiber diameter to 1.05 ± 0.32 µm, reduced the alignment coefficient to 0.655 ± 0.045 and made the scaffold very hydrophilic. The cell viability and Live/dead assay showed that the stem cells remained viable and proliferated after 7 days in culture. Confocal images of phalloidin/DAPI staining showed that the cells adhered and proliferated widely, in fully adaptation with the biomaterial. The SFI values of the group that received the conduit were similar to the values of the control lesioned group. In conclusion, conduits composed of PLGA-gelatin nanofibers were produced and promoted a very good interaction with the stem cells. Although in vitro studies have shown this biomaterial to be a promising biomaterial for the regeneration of nerve tissue, in vivo studies of this graft have not shown significant improvements in nerve regeneration.
Assuntos
Gelatina/química , Nanofibras/química , Regeneração Nervosa/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/fisiologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Ácido Poliglicólico/química , Ratos , Células de Schwann/citologia , Nervo Isquiático/citologia , Células-Tronco/citologia , Alicerces Teciduais/químicaRESUMO
INTRODUCTION: Treatments based on cell biology need reliable and precise carriers for reaching the desired targets. For that reason, a PDO-based cell carrier was idealized, with the purpose of carrying stem cells to distant sites at room temperature. MATERIALS AND METHODS: Three modalities of the same carrier were evaluated: one containing undifferentiated human dental pulp stem cells (DPSCs); one loaded with stem cells induced to neurogenic differentiation (DPSCNs); and one without cells (Blank). The carriers were implanted in sciatic nerve gaps in 48 Wistar rats that were divided in three groups. Two other rats were included in a SHAM control group. Immunohistochemical, histological and clinical analyses were performed in two, four, six and eight weeks of time. RESULTS: Efficacy of human stem cell transportation at room temperature to rats was attested. Moreover, it was possible to confirm that those cells show tropism for inflamed environments and are also prone to induction of neurogenesis in the first two weeks, vanishing after that period. CONCLUSION: Clinical evaluation of the animals' gait recovery shows a promising perspective of success with the inclusion of stem cell-loaded PDO tubes in nerve gaps, which may be positively compared to previously published studies. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors - www.springer.com/00266.
Assuntos
Tecido Adiposo/citologia , Movimento Celular/fisiologia , Polpa Dentária/citologia , Nervo Isquiático/citologia , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Animais , Biópsia por Agulha , Diferenciação Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Sensibilidade e EspecificidadeRESUMO
Peripheral nerve injury is an important cause of incapability and has limited available treatment. Autologous donor nerve implant is the golden standard treatment, however, may cause secondary deficits. Stem cells show positive results in preclinical settings, preserving tissue and function. We tested the efficacy of stem cells derived from human exfoliated deciduous teeth seeded in poly (lactide-co-glycolide) scaffolds in sciatic nerve transection model. Seventy-two adult male Wistar rats had 7-mm nerve gap bridge using scaffolds with (or without) stem cells. Animals were randomly divided into: sham-operated; sham-operated without scaffold; sham-operatedâ¯+â¯scaffoldâ¯+â¯stem cells; sciatic transectionâ¯+â¯no treatment; sciatic transectionâ¯+â¯acellular scaffolds; sciatic transectionâ¯+â¯scaffoldâ¯+â¯stem cells. Sciatic Functional Index and Ladder Rung Walking tests were performed before (-1), 14 and 28â¯days after surgery. Morphometric nerve measurement and muscle weights were assessed. Scaffolds with stem cells improved function in Sciatic Functional Index. Acellular scaffold was effective, promoting functional recovery and nerve regeneration following nerve injury. Scaffolds provide better nerve regeneration and functional recovery after sciatic transection. Despite cell therapy promoting faster recovery after sciatic transection in the Sciatic Index Score, stem cells did not improve functional and morphological recovery after nerve injury. This is the first study testing the potential use of scaffolds combined with stem cells in the early stages after injury. Scaffolds with stem cells could accelerate nerve recovery and favor adjuvant therapies, evidencing the need for further studies to increase the knowledge about stem cells' mechanisms.
Assuntos
Regeneração Nervosa/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Alicerces Teciduais , Animais , Humanos , Masculino , Ratos , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/citologia , Nervo Isquiático/fisiologia , Transplante de Células-Tronco , Caminhada/fisiologiaRESUMO
BACKGROUND: Despite the regenerative potential of the peripheral nervous system, severe nerve lesions lead to loss of target-organ innervation, making complete functional recovery a challenge. Few studies have given attention to combining different approaches in order to accelerate the regenerative process. OBJECTIVE: Test the effectiveness of combining Schwann-cells transplantation into a biodegradable conduit, with treadmill training as a therapeutic strategy to improve the outcome of repair after mouse nerve injury. METHODS: Sciatic nerve transection was performed in adult C57BL/6 mice; the proximal and distal stumps of the nerve were sutured into the conduit. Four groups were analyzed: acellular grafts (DMEM group), Schwann cell grafts (3×105/2 µL; SC group), treadmill training (TMT group), and treadmill training and Schwann cell grafts (TMT + SC group). Locomotor function was assessed weekly by Sciatic Function Index and Global Mobility Test. Animals were anesthetized after eight weeks and dissected for morphological analysis. RESULTS: Combined therapies improved nerve regeneration, and increased the number of myelinated fibers and myelin area compared to the DMEM group. Motor recovery was accelerated in the TMT + SC group, which showed significantly better values in sciatic function index and in global mobility test than in the other groups. The TMT + SC group showed increased levels of trophic-factor expression compared to DMEM, contributing to the better functional outcome observed in the former group. The number of neurons in L4 segments was significantly higher in the SC and TMT + SC groups when compared to DMEM group. Counts of dorsal root ganglion sensory neurons revealed that TMT group had a significant increased number of neurons compared to DMEM group, while the SC and TMT + SC groups had a slight but not significant increase in the total number of motor neurons. CONCLUSION: These data provide evidence that this combination of therapeutic strategies can significantly improve functional and morphological recovery after sciatic injury.
Assuntos
Transplante de Células , Regeneração Nervosa , Condicionamento Físico Animal , Células de Schwann/citologia , Nervo Isquiático/citologia , Nervo Isquiático/fisiologia , Animais , Axônios/fisiologia , Sobrevivência Celular , Modelos Animais de Doenças , Masculino , Camundongos , Neurônios Motores/fisiologia , Fatores de Crescimento Neural/metabolismo , Junção Neuromuscular , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Traumatismos dos Nervos Periféricos/terapia , Poliésteres/metabolismo , Recuperação de Função Fisiológica , Nervo Isquiático/ultraestruturaRESUMO
Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.
Assuntos
Transporte Axonal/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Axônios/metabolismo , Transporte Biológico , Células Cultivadas , Hipocampo/citologia , Membranas Intracelulares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nervo Isquiático/citologia , Sinaptossomos/metabolismoRESUMO
Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFbeta isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFbeta1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.
Assuntos
Anticorpos Heterófilos/imunologia , Galinhas/imunologia , Imunoglobulinas/imunologia , Neuregulina-1/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Anticorpos Heterófilos/biossíntese , Anticorpos Heterófilos/isolamento & purificação , Especificidade de Anticorpos , Células Cultivadas , Meios de Cultivo Condicionados , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Immunoblotting , Imunoglobulinas/biossíntese , Imunoglobulinas/isolamento & purificação , Neuregulina-1/análise , Fragmentos de Peptídeos/síntese química , Isoformas de Proteínas/análise , Isoformas de Proteínas/imunologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/imunologia , Células de Schwann/imunologia , Células de Schwann/metabolismo , Nervo Isquiático/citologiaRESUMO
Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.
Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.
Assuntos
Animais , Feminino , Ratos , Anticorpos Heterófilos/imunologia , Galinhas/imunologia , Imunoglobulinas/imunologia , Neuregulina-1/imunologia , Fragmentos de Peptídeos/imunologia , Especificidade de Anticorpos , Anticorpos Heterófilos/biossíntese , Anticorpos Heterófilos/isolamento & purificação , Células Cultivadas , Meios de Cultivo Condicionados , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Immunoblotting , Imunoglobulinas/biossíntese , Imunoglobulinas/isolamento & purificação , Neuregulina-1/análise , Fragmentos de Peptídeos/síntese química , Isoformas de Proteínas/análise , Isoformas de Proteínas/imunologia , Ratos Sprague-Dawley , Proteínas Recombinantes/imunologia , Células de Schwann/imunologia , Células de Schwann/metabolismo , Nervo Isquiático/citologiaRESUMO
Compression of spinal roots is an important medical problem, which may arise from intervertebral disc herniation, tumor growth or as a result of high energy accidents. Differently from avulsion, root crushing maintains the central/peripheral nervous system (CNS/PNS) connection, although the axons are axotomized and motoneurons degenerate. Such neuronal death may decrease and delay motor function recovery. In the present study we have investigated the neuroprotective effects of mesenchymal stem cell (MSC) therapy following such proximal lesions. Motor recovery and synaptic stabilization were analyzed by the use of morphological and functional approaches. For that, crushing the ventral roots at L4, L5 and L6 was unilaterally performed in Lewis rats. Four weeks after injury, an increased motoneuron survival was observed in the MSC-treated group, coupled with a smaller decrease of inputs at the motoneuron surface and nearby neuropil, seen by synaptophysin and synapsin immunolabeling and decreased astrogliosis, seen by GFAP immunolabeling. In this sense, MSC-treated group displayed a significant preservation of GABAergic terminals, indicating a possible neuroprotection to glutamate excitotoxicity. Motor function recovery was acutely improved in MSC-treated group as compared to Dulbeco's modified eagle medium (DMEM)-treated. Overall, we provide evidence that ventral root crushing (VRC), although milder than avulsion, results in significant loss of motoneurons (~51%) that can be reduced by MSC administration within the spinal cord. Such treatment also improves the number of synapses immunoreactive against molecules present in inhibitory inputs. Also, an increased number of regenerated axons was obtained in the MSC-treated group, in comparison to the DMEM-treated control. Overall, MSC therapy acutely improved limb strength and gait coordination, indicating a possible clinical application of such treatment following proximal lesions at the CNS/PNS interface.
Assuntos
Axotomia , Transplante de Células-Tronco Mesenquimais , Neurônios Motores/fisiologia , Regeneração Nervosa/fisiologia , Medula Espinal/citologia , Raízes Nervosas Espinhais/fisiologia , Sinapses/fisiologia , Animais , Sobrevivência Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Marcha/fisiologia , Imuno-Histoquímica , Força Muscular , Compressão Nervosa , Neuroglia/fisiologia , Ratos , Ratos Endogâmicos Lew , Recuperação de Função Fisiológica , Nervo Isquiático/citologia , Nervo Isquiático/fisiologiaRESUMO
To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.