RESUMEN
BACKGROUND: Both non-traumatic and traumatic spinal cord injuries have in common that a relatively minor structural lesion can cause profound sensorimotor and autonomous dysfunction. Besides treating the cause of the spinal cord injury the main goal is to restore lost function as far as possible. AIM: This article provides an overview of current innovative diagnostic (imaging) and therapeutic approaches (neurorehabilitation and neuroregeneration) aiming for recovery of function after non-traumatic and traumatic spinal cord injuries. MATERIAL AND METHODS: An analysis of the current scientific literature regarding imaging, rehabilitation and rehabilitation strategies in spinal cord disease was carried out. RESULTS: Novel magnetic resonance imaging (MRI) based techniques (e.g. diffusion-weighted MRI and functional MRI) allow visualization of structural reorganization and specific neural activity in the spinal cord. Robotics-driven rehabilitative measures provide training of sensorimotor function in a targeted fashion, which can even be continued in the homecare setting. From a preclinical point of view, defined stem cell transplantation approaches allow for the first time robust structural repair of the injured spinal cord. CONCLUSION: Besides well-established neurological and functional scores, MRI techniques offer the unique opportunity to provide robust and reliable "biomarkers" for restorative therapeutic interventions. Function-oriented robotics-based rehabilitative interventions alone or in combination with stem cell based therapies represent promising approaches to achieve substantial functional recovery, which go beyond current rehabilitative treatment efforts.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Terapia Pasiva Continua de Movimiento/métodos , Robótica/métodos , Enfermedades de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/terapia , Trasplante de Células Madre/métodos , HumanosRESUMEN
Neural stem cells have exhibited efficacy in pre-clinical models of spinal cord injury (SCI) and are on a translational path to human testing. We recently reported that neural stem cells must be driven to a spinal cord fate to optimize host axonal regeneration into sites of implantation in the injured spinal cord, where they subsequently form neural relays across the lesion that support significant functional improvement. We also reported methods of deriving and culturing human spinal cord neural stem cells derived from embryonic stem cells that can be sustained over serial high passage numbers in vitro, providing a potentially optimized cell source for human clinical trials. We now report further optimization of methods for deriving and sustaining cultures of human spinal cord neural stem cell lines that result in improved karyotypic stability while retaining anatomical efficacy in vivo. This development improves prospects for safe human translation.
Asunto(s)
Diferenciación Celular , Células-Madre Neurales , Traumatismos de la Médula Espinal , Médula Espinal , Humanos , Células-Madre Neurales/citología , Médula Espinal/citología , Animales , Traumatismos de la Médula Espinal/terapia , Diferenciación Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Ratones , Trasplante de Células Madre/métodosRESUMEN
The identification of axon growth-promoting genes, and overexpression of these genes in central nervous system (CNS) neurons projecting to the spinal cord, has emerged as one potential approach to enhancing CNS regeneration. Assessment of the regenerative potential of candidate genes usually requires axonal tracing of spinal projections, ideally limited to neurons that express the candidate gene. Alternatively, coexpression of a reporter gene such as enhanced green fluorescent protein (GFP) from an internal ribosomal entry site can be used to identify neurons expressing the candidate gene, but this strategy does not label corticospinal axons in the spinal cord. We therefore developed a dual promoter lentiviral vector in which a potentially therapeutic transgene is expressed from the cytomegalovirus-enhanced chicken beta-actin promoter and the fluorescent protein copGFP is expressed from the elongation factor-1alpha promoter. The vector was constructed to be compatible with the Gateway recombination system for efficient introduction of transgenes through entry shuttle vectors. We show both simultaneous expression of a candidate and reporter gene in corticospinal and red nucleus neurons, and efficient labeling of their axons after lesions in the cervical spinal cord. This expression system is therefore an accurate and efficient means of screening candidate genes in vivo for enhancement of axonal growth.
Asunto(s)
Axones/fisiología , Terapia Genética/métodos , Vectores Genéticos , Lentivirus , Regeneración Nerviosa , Traumatismos de la Médula Espinal/terapia , Actinas/genética , Animales , Femenino , Ratones , Factor 1 de Elongación Peptídica/genética , Regiones Promotoras Genéticas , RatasRESUMEN
Growth and progression of malignant melanoma cells is influenced by a complex network of growth-stimulating and -inhibiting factors produced by both the tumor cells and the local environment. Here we report the purification and molecular cloning of a novel growth regulating protein, designated melanoma inhibitory activity (MIA) and provide a preliminary functional characterization. MIA is translated as a 131-amino acid precursor and processed into a mature 107-amino acid protein after cleavage of a putative secretion signal. A murine complementary DNA was isolated that encoded a MIA-protein with 88% amino acid identity. MIA is secreted into the culture supernatant by several malignant melanoma cell lines as an M(r) 11,000 autocrine growth factor and acts as a potent tumor cell growth inhibitor for malignant melanoma cells and some other neuroectodermal tumors, including gliomas. MIA has no homology to any other known protein and, therefore, represents a novel type of growth-regulatory factor. Furthermore, we describe a molecular approach to express functionally active MIA in Escherichia coli, which might be attractive as a future antitumor therapeutical substance.
Asunto(s)
Melanoma/química , Proteínas de Neoplasias/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/química , ADN Complementario/genética , Proteínas de la Matriz Extracelular , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , ARN Mensajero/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Glial-cell-line--derived neurotrophic factor (GDNF) has been identified as a potent survival and differentiation factor for several neuronal populations in the central nervous system (CNS), but to date, distinct effects of GDNF on motor axon growth and regeneration in the adult have not been demonstrated. In the present study, ex vivo gene delivery was used to directly examine whether GDNF can influence axonal growth, expression of neuronal regeneration-related genes, and sustain the motor neuronal phenotype after adult CNS injury. Adult Fischer 344 rats underwent unilateral transections of the hypoglossal nerve, followed by intramedullary grafts of fibroblasts genetically modified to secrete GDNF. Control animals received lesions and grafts of cells expressing a reporter gene. Two weeks later, GDNF gene delivery (1) robustly promoted the growth of lesioned hypoglossal motor axons, (2) altered the expression and intracellular trafficking of the growth-related protein calcitonin gene-related peptide (CGRP), and (3) significantly sustained the cholinergic phenotype in 84 +/- 6% of hypoglossal neurons compared with 39 +/- 6% in control animals (P < 0.001). This is the first neurotrophic factor identified to increase the in vivo expression of the trophic peptide CGRP and the first report that GDNF promotes motor axonal growth in vivo in the adult CNS. Taken together with previous in vitro studies, these findings serve as the foundation for a model wherein GDNF and CGRP interact in a paracrine manner to regulate neuromuscular development and regeneration.
Asunto(s)
Axones/fisiología , Péptido Relacionado con Gen de Calcitonina/biosíntesis , Sistema Nervioso Central/citología , Terapia Genética , Neuronas Motoras/fisiología , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/genética , Animales , Northern Blotting , Recuento de Células , Femenino , Fibroblastos , Factor Neurotrófico Derivado de la Línea Celular Glial , Inmunohistoquímica , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Sistema Nervioso Parasimpático/citología , Sistema Nervioso Parasimpático/fisiología , Fenotipo , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas F344 , Transducción Genética , Transgenes/genéticaRESUMEN
Neurotrophic factors exert many effects on the intact and lesioned adult central nervous system (CNS). Among these effects are prevention of neuronal death (neurotrophism) and promotion of axonal growth (neurotropism) after injury. To date, however, it has not been established whether survival and axonal growth functions of neurotrophins can be independently modulated in injured adult neurons in vivo. To address this question, the ability of brain-derived neurotrophic factor (BDNF) to influence corticospinal motor neuronal survival and axonal growth was examined in two injury paradigms. In the first paradigm, a survival assay, adult Fischer 344 rats underwent subcortical lesions followed by grafts to the lesion cavity of syngenic fibroblasts genetically modified to secrete high amounts BDNF or, in control subjects, the reporter gene green fluorescent protein. In control subjects, only 36.2 +/- 7.0% of the retrogradely labeled corticospinal neurons survived the lesion, whereas 89.8 +/- 5.9% (P < 0.001) of the corticospinal neurons survived in animals that received BDNF-secreting grafts. However, in an axonal growth assay, BDNF-secreting cell grafts that were placed into either subcortical lesion sites or sites of thoracic spinal cord injury failed to elicit corticospinal axonal growth. Despite this lack of a neurotropic effect on lesioned corticospinal axons, BDNF-secreting cell grafts placed in the injured spinal cord significantly augmented the growth of other types of axons, including local motor, sensory, and coerulospinal axons. Immunolabeling for tyrosine kinase B (trkB) demonstrated that BDNF receptors were present on corticospinal neuronal somata and apical dendrites but were not detected on their projecting axons. Thus, single classes of neurons in the adult CNS appear to exhibit disparate survival and growth sensitivity to neurotrophic factors, potentially attributable at least in part to differential trafficking of neurotrophin receptors. The possibility of tropic/trophic divergence must be considered when designing strategies to promote CNS recovery from injury.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Corteza Cerebral/fisiología , Neuronas/efectos de los fármacos , Médula Espinal/fisiología , Animales , Axones/efectos de los fármacos , Axones/fisiología , Supervivencia Celular/efectos de los fármacos , Trasplante de Células , Corteza Cerebral/citología , Fibroblastos/metabolismo , Fibroblastos/trasplante , Masculino , Vías Nerviosas/citología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/crecimiento & desarrollo , Neuronas Aferentes/efectos de los fármacos , Fenotipo , Ratas , Ratas Endogámicas F344 , Receptor trkB/metabolismo , Médula Espinal/citologíaRESUMEN
Schwann cells contribute to efficient axonal regeneration after peripheral nerve injury and, when grafted to the central nervous system (CNS), also support a modest degree of central axonal regeneration. This study examined (1) whether Schwann cells grafted to the CNS exhibit normal patterns of differentiation and association with spinal axons and what signals putatively modulate these interactions, and (2) whether Schwann cells overexpressing neurotrophic factors enhance axonal regeneration. Thus, primary Schwann cells were transduced to hypersecrete human nerve growth factor (NGF) and were grafted to spinal cord injury sites in adult rats. Comparisons were made to nontransfected Schwann cells. From 3 days to 6 months later, grafted Schwann cells exhibited a phenotypic and temporal course of differentiation that matched patterns normally observed after peripheral nerve injury. Schwann cells spontaneously aligned into regular spatial arrays within the cord, appropriately remyelinated coerulospinal axons that regenerated into grafts, and appropriately ensheathed but did not myelinate sensory axons extending into grafts. Coordinate expression of the cell adhesion molecule L1 on Schwann cells and axons correlated with establishment of appropriate patterns of axon-Schwann cell ensheathment. Transduction of Schwann cells to overexpress NGF robustly increased axonal growth but did not otherwise alter the nature of interactions with growing axons. These findings suggest that signals expressed on Schwann cells that modulate peripheral axonal regeneration and myelination are also recognized in the CNS and that the modification of Schwann cells to overexpress growth factors significantly augments their capacity to support extensive axonal growth in models of CNS injury.
Asunto(s)
Terapia Genética , Glicoproteínas de Membrana/genética , Factor de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa/fisiología , Moléculas de Adhesión de Célula Nerviosa/genética , Células de Schwann/trasplante , Traumatismos de la Médula Espinal/terapia , Animales , Péptido Relacionado con Gen de Calcitonina/análisis , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Expresión Génica/fisiología , Supervivencia de Injerto/fisiología , Complejo de Antígeno L1 de Leucocito , Fibras Nerviosas Mielínicas/química , Fibras Nerviosas Mielínicas/enzimología , Factor de Crecimiento Nervioso/genética , Neuronas Aferentes/citología , Neuronas Aferentes/ultraestructura , Fenotipo , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Células de Schwann/citología , Células de Schwann/metabolismo , Transducción de Señal/fisiología , Médula Espinal/citología , Médula Espinal/cirugía , Tirosina 3-Monooxigenasa/análisisRESUMEN
Previously we reported that grafts of cells genetically modified to produce human nerve growth factor (hNGF) promoted specific and robust sprouting of spinal sensory, motor, and noradrenergic axons. In the present study we extend these investigations to assess NGF effects on corticospinal motor axons and on functional outcomes after spinal cord injury. Fibroblasts from adult rats were transduced to express human NGF; control cells were not genetically modified. Fibroblasts were then grafted to sites of midthoracic spinal cord dorsal hemisection lesions. Three months later, recipients of NGF-secreting grafts showed deficits on conditioned locomotion over a wire mesh that did not differ in extent from control-lesioned animals. On histological examination, NGF-secreting grafts elicited specific sprouting from spinal primary sensory afferent axons, local motor axons, and putative cerulospinal axons as previously reported, but no specific responses from corticospinal axons. Axons responding to NGF robustly penetrated the grafts but did not exit the grafts to extend to normal innervation territories distal to grafts. Grafted cells continued to express NGF protein through the experimental period of the study. These findings indicate that 1) spinal cord axons show directionally sensitive growth responses to neurotrophic factors, 2) growth of axons responding to a neurotrophic factor beyond an injury site and back to their natural target regions will likely require delivery of concentration gradients of neurotrophic factors toward the target, 3) corticospinal axons do not grow toward a cellular source of NGF, and 4) functional impairments are not improved by strictly local sprouting response of nonmotor systems.
Asunto(s)
Fibroblastos/trasplante , Factores de Crecimiento Nervioso/metabolismo , Traumatismos de la Médula Espinal/cirugía , Enfermedad Aguda , Animales , Axones/fisiología , Northern Blotting , Péptido Relacionado con Gen de Calcitonina/análisis , Células Cultivadas , Colina O-Acetiltransferasa/análisis , Dopamina beta-Hidroxilasa/análisis , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/fisiología , Técnicas de Transferencia de Gen , Humanos , Locomoción/fisiología , Sondas Moleculares , Factores de Crecimiento Nervioso/genética , Neuronas/química , Neuronas/enzimología , Tractos Piramidales/citología , Tractos Piramidales/cirugía , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Tirosina 3-Monooxigenasa/análisis , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre ConjugadaRESUMEN
Neurotrophic factors are a large class of molecules that can influence neuronal survival, modulate neuronal function and induce axonal growth. They act on specific receptors in specific subsets of neurons and glia. Since their discovery, neurotrophic factors have received considerable interest as possible means for the treatment of neurodegenerative diseases and central nervous system (CNS) trauma. In vitro experiments, as well as studies in animal models of CNS disease, have supported the concept of neuroprotection as a new treatment strategy in degenerative neurologic disease, using neurotrophic factor delivery to CNS targets. Despite the encouraging results from animal studies, progress in adaptation of this treatment strategy to human disease has been slow and sometimes disappointing. One of the major keys for the successful use of neurotrophic factors in treating neurologic disease is the mode of delivery. Neurotrophic factors need to be specifically targeted and regionally restricted to the area of interest to achieve significant results without widespread, unwanted adverse effects. This article summarizes results from recent studies of neurotrophic factors in models of neurodegenerative diseases and CNS trauma and outlines current knowledge and future perspectives of trophic factor gene therapy in the CNS.
RESUMEN
Dissection of the cavernous nerves eliminates spontaneous erections and may lead to irreversible erectile dysfunction due to degeneration of cavernous tissue. Novel procedures to reconstruct penile innervation include cavernous nerve interposition grafting and neurotrophic treatments to revitalize penile neural input, evaluated thus far in various preclinical models of cavernous nerve injury. Schwann cells crucially contribute to successful axonal regeneration by mechanical and paracrine mechanisms in the injured nerve, and Schwann cells seeded into guidance channels have been successfully employed to support regeneration in animal models of cavernous nerve injury. Gene therapy, tissue engineering, and reconstructive techniques have been combined to deliver neurotrophic factors and recover erectile function.
Asunto(s)
Disfunción Eréctil/cirugía , Regeneración Nerviosa/fisiología , Pene/inervación , Pene/cirugía , Enfermedades del Sistema Nervioso Periférico/cirugía , Células de Schwann/trasplante , Ingeniería de Tejidos/métodos , Implantes Absorbibles , Animales , Disfunción Eréctil/diagnóstico , Disfunción Eréctil/etiología , Disfunción Eréctil/terapia , Terapia Genética/métodos , Humanos , Masculino , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/uso terapéutico , Enfermedades del Sistema Nervioso Periférico/complicaciones , Enfermedades del Sistema Nervioso Periférico/terapia , Resultado del TratamientoRESUMEN
Brain-derived neurotrophic factor (BDNF) has been identified as a potent promoter of neurite growth, a finding that has led to an ongoing exploration of this neurotrophin as a potential treatment for spinal cord injury. BDNF's many effects in the nervous system make it an excellent candidate for neuroprotective strategies as well as for promoting axonal regeneration, plasticity and re-myelination. In addition, neuronal activity and physical exercise can modulate the expression of BDNF, suggesting that non-invasive means to increase BDNF levels might exist. Nonetheless, depending on the location, amount and duration of BDNF delivery, this potent neurotrophin can also have adverse effects, such as modulation of nociceptive pathways or contribution to spasticity. Taken together, the benefits and possible risks require careful assessment when considering this multifaceted neurotrophin as a treatment option for spinal cord injury.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/uso terapéutico , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Traumatismos de la Médula Espinal/patologíaAsunto(s)
Lesiones Encefálicas/terapia , Factores de Crecimiento Nervioso/genética , Enfermedades Neurodegenerativas/terapia , Traumatismos de la Médula Espinal/terapia , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/terapia , Animales , Humanos , Factores de Crecimiento Nervioso/fisiologíaRESUMEN
This is the second part of a review summarizing progress and prospects in gene therapy clinical research. Twenty key diseases/strategies are succinctly described and commented on by leaders in the field. This part includes clinical trials for skin diseases, neurological disorders, HIV/AIDS, ornithine transcarbamylase deficiency, alpha(1)-antitrypsin deficiency, haemophilia and cancer.
Asunto(s)
Terapia Genética/tendencias , Ensayos Clínicos como Asunto , Técnicas de Transferencia de Gen/efectos adversos , Técnicas de Transferencia de Gen/tendencias , Terapia Genética/métodos , Vectores Genéticos , Humanos , Neoplasias/terapia , Trasplante de Células Madre/efectos adversos , Trasplante de Células Madre/tendenciasRESUMEN
OBJECTIVE: This article reviews novel restorative therapies for cavernous nerves that may be used to replace resected cavernous nerves at the time of pelvic surgery. METHODS: A literature-based presentation (Medline search) on current nerve replacement strategies was conducted with emphasis on neurobiological factors contributing to the restoration of erectile function after cavernous nerve injuries. RESULTS: A promising alternative to autologous nerve grafts for extending the length of successful nerve regeneration are artificial nerve guides. The addition of neurotrophic factors, extracellular matrix components and Schwann cells has been shown to promote cavernous nerve regeneration. Neurotrophic factors can be incorporated in the scaffold or can be supplied by cells seeded into the stroma. The regenerative capacity of these cells can be further enhanced by genetic modification with neurotrophic factor encoding genes. CONCLUSIONS: Artificial nerve guides, especially biodegradable ones containing growth-promoting factors or cells, are a promising option for the repair of cavernous nerve lesions.
Asunto(s)
Disfunción Eréctil/cirugía , Regeneración Tisular Dirigida/métodos , Regeneración Nerviosa/fisiología , Erección Peniana/fisiología , Pene/inervación , Células de Schwann/trasplante , Disfunción Eréctil/etiología , Humanos , Masculino , Recuperación de la FunciónRESUMEN
Gene transfer is a potential means of treating chronic neurologic disorders and injury related neural degeneration. One approach for transferring genes to the CNS is to genetically modify cells in vitro and then transplant the cells to the CNS. For example, fibroblasts can be infected with a replication-defective retrovirus expressing a transgene, and can then be transplanted into the brain or spinal cord, thereby providing neurotrophic factors and substrates for axonal growth and elongation. In this review we discuss the grafting of neurotrophic factor secreting autologous fibroblasts in the rat and primate CNS. NGF secreting grafts have been shown to prevent degeneration of cholinergic neurons in the basal forebrain of primates and to induce sprouting of sensory, motor, and noradrenergic neurites after spinal cord injury. These results suggest the potential usefulness of ex vivo gene transfer for the treatment of Alzheimer's disease and spinal cord injury.
Asunto(s)
Enfermedad de Alzheimer/terapia , Terapia Genética , Vectores Genéticos , Retroviridae/genética , Traumatismos de la Médula Espinal/terapia , Animales , Fibroblastos/metabolismo , Fibroblastos/trasplante , Humanos , Factores de Crecimiento Nervioso/metabolismoRESUMEN
Vector systems for the regulated and reversible expression of therapeutic genes are likely to improve the safety and efficacy of gene therapy for medical disease. In the present study, we investigated whether the expression of genes transferred into the central nervous system by ex vivo gene therapy can be regulated in vivo leading to controlled neuronal survival and axonal growth. Primary rat fibroblasts were transfected with a retrovirus containing a tetracycline responsive promoter for the expression of the neurotrophin nerve growth factor (NGF) or green fluorescent protein as a control (GFP). After lesions of basal forebrain cholinergic neurons, NGF-mediated neuronal rescue and axonal growth could be completely controlled over a 2-week period by the addition or removal of the tetracycline modulator doxycycline in the animals' drinking water. Further, continued expression of the reporter gene GFP could be reliably and repeatedly turned on and off in the injured CNS for at least 3 months post-grafting, the longest time point investigated. These data constitute the first report of regulated neuronal rescue and axonal growth by controlled neurotrophin gene delivery and long-term, regulated expression using ex vivo CNS gene therapy.
Asunto(s)
Antibacterianos/uso terapéutico , Enfermedades del Sistema Nervioso Central/terapia , Doxiciclina/uso terapéutico , Terapia Genética/métodos , Polisacáridos/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Análisis de Varianza , Animales , Axones/metabolismo , Femenino , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Vectores Genéticos/administración & dosificación , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Neuronas/metabolismo , Ratas , Ratas Endogámicas F344 , Retroviridae/genética , Médula Espinal , Factores de TiempoRESUMEN
Little spontaneous regeneration of axons occurs after acute and chronic injury to the CNS. Previously we have shown that the continuous local delivery of neurotrophic factors to the acutely injured spinal cord induces robust growth of spinal and supraspinal axons. In the present study we examined whether chronically injured axons also demonstrate significant neurotrophin responsiveness. Adult rats underwent bilateral dorsal hemisection lesions that axotomize descending supraspinal pathways, including the corticospinal, rubrospinal, and cerulospinal tracts, and ascending dorsal spinal sensory projections. One to three months later, injured rats received grafts of syngenic fibroblasts genetically modified to produce nerve growth factor (NGF). Control subjects received unmodified cell grafts or cells transduced to express the reporter gene beta-galactosidase. Three to five months after grafting, animals that received NGF-secreting grafts showed dense growth of putative cerulospinal axons and primary sensory axons of the dorsolateral fasciculus into the grafted lesion site. Growth from corticospinal, raphaespinal, and local motor axons was not detected. Thus, robust growth of defined populations of supraspinal and spinal axons can be elicited in chronic stages after spinal cord injury by localized, continuous transgenic delivery of neurotrophic factors.
Asunto(s)
Axones/fisiología , Terapia Genética/métodos , Supervivencia de Injerto/fisiología , Factores de Crecimiento Nervioso/biosíntesis , Regeneración Nerviosa , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Trasplante Isogénico/métodos , Animales , Transporte Axonal , Axones/ultraestructura , Axotomía , División Celular , Femenino , Fibroblastos/trasplante , Técnicas de Transferencia de Gen , Fibras Nerviosas/fisiología , Fibras Nerviosas/ultraestructura , Factores de Crecimiento Nervioso/fisiología , Ratas , Ratas Endogámicas F344 , Serotonina/análisis , Traumatismos de la Médula Espinal/patología , Trasplante Isogénico/fisiologíaRESUMEN
The injured adult mammalian spinal cord shows little spontaneous recovery after injury. In the present study, the contribution of projections in the dorsal half of the spinal cord to functional loss after adult spinal cord injury was examined, together with the effects of transgenic cellular delivery of neurotrophin-3 (NT-3) on morphological and functional disturbances. Adult rats underwent bilateral dorsal column spinal cord lesions that remove the dorsal corticospinal projections or underwent more extensive resections of the entire dorsal spinal cord bilaterally that remove corticospinal, rubrospinal, and cerulospinal projections. Long-lasting functional deficits were observed on a motor grid task requiring detailed integration of sensorimotor skills, but only in animals with dorsal hemisection lesions as opposed to dorsal column lesions. Syngenic primary rat fibroblasts genetically modified to produce NT-3 were then grafted to acute spinal cord dorsal hemisection lesion cavities. Up to 3 months later, significant partial functional recovery occurred in NT-3-grafted animals together with a significant increase in corticospinal axon growth at and distal to the injury site. These findings indicate that (1) several spinal pathways contribute to loss of motor function after spinal cord injury, (2) NT-3 is a neurotrophic factor for the injured corticospinal projection, and (3) functional deficits are partially ameliorated by local cellular delivery of NT-3. Lesions of the corticospinal projection may be necessary, but insufficient in isolation, to cause sensorimotor dysfunction after spinal cord injury in the rat.
Asunto(s)
Axones/efectos de los fármacos , División Celular/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Neurotrofina 3 , Ratas , Ratas Endogámicas F344RESUMEN
The delivery of neurotrophic factors to the adult nervous system has potential applications for the treatment of neurodegenerative diseases and trauma. In vivo and ex vivo gene therapy offer a means of delivering growth factors and other therapeutic substances to the central nervous system (CNS) in an intraparenchymal, accurately targeted, and regionally restricted manner. Ideally, gene therapy delivery systems should also be regulatable, allowing exogenous control of amount of gene product delivery. In the present experiment, a tetracycline-regulatable gene expression system was generated to determine whether controllable release of nerve growth factor (NGF) and green fluorescent protein (GFP) from primary rat fibroblasts could modulate biological responses (neurite outgrowth) in vitro. Using a tetracycline-repressible construct, it was found that NGF mRNA, NGF protein, and NGF-induced neurite outgrowth could be tightly regulated within a 24 hour period, and in a dose-dependent fashion, by exposure to the tetracycline analog doxycycline. Similarly, levels of green fluorescence could be regulated in GFP-transfected cells. These findings in a neurobiological system lay the framework for future studies using regulated neurotrophin delivery in in vivo models of neurodegenerative diseases and CNS injury.
Asunto(s)
Antibacterianos/farmacología , Expresión Génica/fisiología , Terapia Genética , Vectores Genéticos/metabolismo , Factor de Crecimiento Nervioso/genética , Neuritas/fisiología , Tetraciclina/farmacología , Animales , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Vectores Genéticos/efectos de los fármacos , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Ratas , Ratas Endogámicas F344 , TransfecciónRESUMEN
Transforming growth factor-beta (TGF-beta) is a potent cytokine that has influence upon immunosuppressive as well as proinflammatory processes. In this context we have investigated the role of endogenous TGF-beta in human peripheral blood mononuclear cells (PBMCs) by TGF-beta 1 phosphorothioate antisense oligodeoxynucleotides (TGF-beta 1-S-ODNs). In short-term cultures (up to 3 days), TGF-beta 1-S-ODNs-treated, interleukin 2-activated PBMCs displayed a growth advantage (up to 148%) compared to nonsense or untreated controls as measured by cell counting and [3H]thymidine incorporation. Long-term antagonization of TGF-beta production (up to 12 days) showed no significant differences between both antisense- and nonsense-treated PBMCs. The efficacy and specificity of TGF-beta 1-S-ODNs effects was validated by gene expression studies (Northern blot and ELISA) and analysis of cellular uptake of BrdU-labeled S-ODN (Immunocytochemistry). TGF-beta 1-S-ODNs at the final concentration of 1 microM reduced TGF-beta 1 protein concentration up to 65% in PBMCs cultures without significant changes in TGF-beta 1 mRNA expression. These experiments demonstrate that TGF-beta 1-S-ODNs specifically antagonize TGF-beta 1-mediated autocrine suppression of IL-2-dependent PBMC activation. TGF-beta 1-specific antisense oligonucleotides may represent a promising immunoresponse modifying agent that could be used for T-cell directed immunostimulation.