RESUMEN
INTRODUCTION: This study evaluated whether Schwann cells (SCs) from different nerve sources transplanted into cold-preserved acellular nerve grafts (CP-ANGs) would improve functional regeneration compared with nerve isografts. METHODS: SCs isolated and expanded from motor and sensory branches of rat femoral and sciatic nerves were seeded into 14mm CP-ANGs. Growth factor expression, axonal regeneration, and functional recovery were evaluated in a 14-mm rat sciatic injury model and compared with isografts. RESULTS: At 14 days, motor or sensory-derived SCs increased expression of growth factors in CP-ANGs versus isografts. After 42 days, histomorphometric analysis found CP-ANGs with SCs and isografts had similar numbers of regenerating nerve fibers. At 84 days, muscle force generation was similar for CP-ANGs with SCs and isografts. SC source did not affect nerve fiber counts or muscle force generation. CONCLUSIONS: SCs transplanted into CP-ANGs increase functional regeneration to isograft levels; however SC nerve source did not have an effect.
Asunto(s)
Trasplante de Células/métodos , Traumatismos de los Nervios Periféricos/fisiopatología , Traumatismos de los Nervios Periféricos/terapia , Células de Schwann/trasplante , Nervio Ciático/lesiones , Animales , Modelos Animales de Enfermedad , Nervio Femoral/citología , Isoinjertos , Masculino , Factor de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa/fisiología , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Nervio Ciático/citología , Factores de TiempoRESUMEN
INTRODUCTION: We sought to determine whether supplementation of acellular nerve allografts (ANAs) with Schwann cells overexpressing GDNF (G-SCs) would enhance functional recovery after peripheral nerve injury. METHODS: SCs expanded in vitro were infected with a lentiviral vector to induce GDNF overexpression. Wild-type SCs (WT-SCs) and G-SCs were seeded into ANAs used to repair a 14-mm nerve gap defect. Animals were harvested after 6 and 12 weeks for histomorphometric and muscle force analysis. RESULTS: At 6 weeks, histomorphometry revealed that ANAs supplemented with G-SCs promoted similar regeneration compared with isograft at midgraft. However, G-SCs failed to promote regeneration into the distal stump. At 12 weeks, ANAs with G-SCs had lower maximum and specific force production compared with controls. CONCLUSIONS: The combined results suggest that consistent overexpression of GDNF by G-SCs trapped axons in the graft and prevented functional regeneration.
Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Tejido Nervioso/trasplante , Traumatismos de los Nervios Periféricos/cirugía , Recuperación de la Función/fisiología , Células de Schwann/trasplante , Animales , Masculino , Regeneración Nerviosa/fisiología , Tejido Nervioso/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Células de Schwann/metabolismoRESUMEN
Phenotypic differences in Schwann cells (SCs) may help to guide axonal regeneration down motor or sensory specific pathways following peripheral nerve injury. The goal of this study was to identify phenotypic markers for SCs harvested from the cutaneous (sensory) and quadriceps (motor) branches of the rat femoral nerve and to study the effects of expansion culture on the expression patterns of these motor or sensory phenotypic markers. RNA was extracted from SCs harvested from the motor and sensory branches of the rat femoral nerve and analyzed using Affymetrix Gene Chips (Rat Genome 230 v2.0 Array A). Genes that were upregulated in motor SCs compared with the sensory SCs or vice versa were identified, and the results were verified for a subset of genes using quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of the "phenotype-specific" genes were then evaluated in SC expansion cultures at various time points over 30 days by qRT-PCR to determine the effect of expansion on SC phenotype. Expression levels of the phenotype-specific genes were significantly altered after expansion culture for both the motor and the sensory markers compared with fresh nerve tissue. These results indicate that both motor and sensory SC gene expression patterns are disrupted during expansion in vitro and may affect the ability of SCs to express phenotype-specific genes after transplantation.
Asunto(s)
Nervio Femoral/citología , Regulación de la Expresión Génica/fisiología , Células de Schwann/metabolismo , Análisis de Varianza , Animales , Proteínas Portadoras/metabolismo , Citocinas/metabolismo , Perfilación de la Expresión Génica , Masculino , Proteína Básica de Mielina/metabolismo , Proteínas de Neurofilamentos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Técnicas de Cultivo de Órganos , Proteína Quinasa C/metabolismo , Ratas , Ratas Endogámicas Lew , Células de Schwann/clasificación , Factores de TiempoRESUMEN
Schwann cells (SCs) secrete growth factors and extracellular matrix molecules that promote neuronal survival and help guide axons during regeneration. Transplantation of SCs is a promising strategy for enhancing peripheral nerve regeneration. However, we and others have shown that after long-term in vitro expansion, SCs revert to a de-differentiated state similar to the phenotype observed after injury. In vivo, glial cell-line derived neurotrophic factor (GDNF) may guide the differentiation of SCs to remyelinate regenerating axons. Therefore, we hypothesized that exogenous GDNF may guide the differentiation of SCs into their native phenotypes in vitro through stimulation of GDNF family receptor (GFR)α-1. When activated in SCs, GFRα-1 promotes phosphorylation of Fyn, a Src family tyrosine kinase responsible for mediating downstream signaling for differentiation and proliferation. In this study, SCs harvested from the sensory and motor branches of rat femoral nerve were expanded in vitro and then cultured with 50 or 100ng/mL of GDNF. The exogenous GDNF promoted differentiation of sensory and motor-derived SCs back to their native phenotypes, as demonstrated by decreased proliferation after 7days and increased expression of S100Βß and phenotype-specific markers. Furthermore, inhibiting Fyn with Src family kinase inhibitors, PP2 and SU6656, and siRNA-mediated knockdown of Fyn reduced GDNF-stimulated differentiation of sensory and motor-derived SCs. These results demonstrate that activating Fyn is necessary for GDNF-stimulated differentiation of femoral nerve-derived SCs into their native phenotypes in vitro. Therefore GDNF could be incorporated into SC-based therapies to promote differentiation of SCs into their native phenotype to improve functional nerve regeneration.
Asunto(s)
Nervio Femoral/citología , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Neuroglía/efectos de los fármacos , Células de Schwann/citología , Células de Schwann/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/fisiología , Fenotipo , Fosforilación , Ratas , Células de Schwann/fisiología , Nervio Ciático/citología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Peripheral nerve regeneration after injury depends on environmental cues and trophic support. Schwann cells (SCs) secrete trophic factors that promote neuronal survival and help guide axons during regeneration. The addition of SCs to acellular nerve grafts is a promising strategy for enhancing peripheral nerve regeneration; however, inconsistencies in seeding parameters have led to varying results. The current work sought to establish a systematic approach to seeding SCs in cold-preserved acellular nerve grafts. Studies were undertaken to (1) determine the needle gauge for optimal cell survival and minimal epineurial disruption during injection, (2) track the seeded SCs using a commercially available dye, and (3) evaluate the seeding efficiency of SCs in nerve grafts. It was determined that seeding with a 27-gauge needle resulted in the highest viability of SCs with the least damage to the epineurium. In addition, Qtracker(®) dye, a commercially available quantum dot nanocrystal, was used to label SCs prior to transplantation, which allowed visualization of the seeded SCs in nerve grafts. Finally, stereological methods were used to evaluate the seeding efficiency of SCs in nerve grafts immediately after injection and following a 1- or 3-day in vitro incubation in SC growth media. Using a systematic approach, the best needle gauge and a suitable dye for SC visualization in acellular nerve grafts were identified. Seeding efficiency in these grafts was also determined. The findings will lead to improvements ability to assess injection of cells (including SCs) for use with acellular nerve grafts to promote nerve regeneration.