RESUMEN
In the present study, the continuity between the central nervous system (CNS) and the peripheral nervous system (PNS) was restored by mean of a collagen channel in order to reinnervate a skeletal muscle. Three groups of animals were considered. In the first group, one end of the collagen channel was implanted in the cervical spinal cord of adult rats. The other end was connected to a 30-mm autologous peripheral nerve graft (PNG) implanted into the denervated biceps brachii muscle. The gap between the spinal cord and the proximal nerve stump varied from 3 to 7 mm. In the second group of animals, the distal end of the PNG graft was ligatured in order to compare the survival of the growing axons in the presence and in the absence of a muscular target. In the third group of animals, the extraspinal stump of the collagen channel was ligatured. Our study demonstrates that spinal neurons and dorsal root ganglion (DRG) neurons can grow long axons through the collagen channel over a 7-mm gap and reinnervate a denervated skeletal muscle. The results also indicate that the presence of a PNG at the extraspinal stump of the collagen channel is essential for axonal regrowth and that the muscle target contributes to the long-term maintenance of the regenerating axons. These data might be interesting for clinical application when the continuity between the CNS and PNS is interrupted such as in root avulsion.
Asunto(s)
Implantes Absorbibles , Colágeno/farmacología , Conos de Crecimiento/metabolismo , Músculo Esquelético/inervación , Regeneración Nerviosa/fisiología , Radiculopatía/cirugía , Médula Espinal/cirugía , Animales , Trasplante de Tejido Encefálico/métodos , Femenino , Conos de Crecimiento/ultraestructura , Peroxidasa de Rábano Silvestre/farmacocinética , Microscopía Electrónica , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Nervios Periféricos/trasplante , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Médula Espinal/ultraestructura , Raíces Nerviosas Espinales/lesiones , Raíces Nerviosas Espinales/cirugía , Raíces Nerviosas Espinales/ultraestructuraRESUMEN
Scar formation following adult spinal cord (SC) hemisection is accompanied by important remodeling of the surrounding extracellular matrix (ECM). Since ECM molecules provide the substrate for axon growth, these changes in ECM composition are likely to influence the process of axonal regeneration. Here we investigated whether scar formation could be associated with the activation of matrix metalloproteinases (MMPs), a class of proteins implicated in ECM remodeling thought to favor axonal regeneration in the peripheral nervous system. Two members of the MMP family, MMP-2 and MMP-9, were found to be transiently upregulated in the SC wound. In situ fluorescent zymography revealed a MMP-related gelatinase activity (GA) in the wound, which was spatially and temporally correlated with scar formation. The GA formed a striking pattern of interwoven pathways along which neurites were seen to grow. These pathways corresponded to the distribution of other ECM molecules, which are known to have antagonistic effects on axonal regrowth. Our results suggest that neurite ingrowth into the wound may transiently benefit from this ECM remodeling and, in particular, from the upregulation of MMPs.
Asunto(s)
Cicatriz/metabolismo , Matriz Extracelular/metabolismo , Gelatinasas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Regeneración Nerviosa/fisiología , Neuritas/enzimología , Traumatismos de la Médula Espinal/metabolismo , Envejecimiento/fisiología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Axotomía , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Cicatriz/patología , Cicatriz/fisiopatología , Femenino , Proteína GAP-43/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Inmunohistoquímica , Laminina/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neuritas/ultraestructura , Proteínas de Neurofilamentos/metabolismo , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Mediators of extracellular matrix proteins degradation, the matrix metalloproteinases (MMPs), involved in inflammation as well as facilitation of process outgrowth of oligodendrocytes are interesting targets for neural repair. Recent data reported their activation after seizures, cerebral ischemia and spinal cord injury. The present study was designed to localize at cellular level the gelatinase activity by in situ zymography in a rat spinal cord contusion model. The kinetic of gelatinase activation was monitored by in situ zymography on 20 microm cryostat sections. The fluorescein-quenched DQ gelatin digestion yielded cleaved fluorescent peptides enabling the detection of gelatinase activity at cellular level. Twenty four hours and 48 h after injury, a strong gelatinase activity was detected at the lesion site in and around vascular structures and infiltrated cells. A preincubation with either MMP-2 or MMP-9 antibodies significantly decreases the gelatinase activity pattern, suggesting the involvement of at least both MMPs. Our results are consistent with a role for MMPs in the blood spinal barrier disruption, the leukocytes infiltration, the disruption of the extracellular matrix and the clearance of debris.
Asunto(s)
Metaloproteinasas de la Matriz/uso terapéutico , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/enzimología , Animales , Contusiones/tratamiento farmacológico , Matriz Extracelular/metabolismo , Femenino , Gelatinasas/metabolismo , Humanos , Inmunohistoquímica , Hibridación in Situ , Cinética , Leucocitos/metabolismo , Metaloproteinasa 2 de la Matriz/uso terapéutico , Metaloproteinasa 9 de la Matriz/uso terapéutico , Neovascularización Patológica , Perfusión , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Factores de TiempoRESUMEN
In order to compensate the loss of motoneurons resulting from severe spinal cord injury and to reestablish peripheral motor connectivity, solid pieces of fetal spinal cord, taken from embryonic day 14 rat embryos, were transplanted into unilateral aspiration lesions of the cervical spinal cord of adult rats. Concomitantly, one end of a 3.5-cm autologous peripheral nerve graft was put in close contact with the embryonic graft; the other end was sutured to the distal stump of the musculocutaneous nerve which innervate the biceps brachii muscle. The animals were examined 3 and 6 months after surgery. Following intramuscular injection of horseradish peroxidase, retrograde axonal labeling studies indicated that both transplanted and host spinal neurons were able to extend axons all the way through the peripheral nerve graft and nerve stump, up to the reconnected muscles. The labeled cells in the transplant were generally observed close to the intraspinal tip of the peripheral nerve graft. Retrograde axonal tracing, as well as electrophysiological and histological data, demonstrated the sensory and motor reinnervation of the reconnected muscles. This muscular reinnervation was able to reverse the atrophic changes observed in the denervated muscle. In control experiments, the extraspinal end of the peripheral nerve graft was ligatured in order to compare the differentiation of the transplanted neurons and the survival of their growing axons with or without their muscular targets. Six months after both types of surgery, large-size grafted neurons, identified as motoneurons by immunocytochemistry for peripherine and calcitonin gene-related peptide, were only observed in fetal spinal cord transplants which were connected to denervated muscles, thus demonstrating the trophic influence of the muscle target on the survival and differentiation of the transplanted neurons and on the maintenance of the axons they had grown into the peripheral nerve graft.
Asunto(s)
Trasplante de Tejido Fetal , Músculo Esquelético/inervación , Nervios Periféricos/trasplante , Traumatismos de la Médula Espinal/cirugía , Médula Espinal/trasplante , Animales , Axones/fisiología , Axones/ultraestructura , Péptido Relacionado con Gen de Calcitonina/metabolismo , Recuento de Células , Diferenciación Celular/fisiología , Tamaño de la Célula , Desnervación , Modelos Animales de Enfermedad , Estimulación Eléctrica , Femenino , Supervivencia de Injerto/fisiología , Peroxidasa de Rábano Silvestre , Cuello , Neuronas/citología , Neuronas/metabolismo , Neuronas/trasplante , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Médula Espinal/citología , Médula Espinal/embriología , Traumatismos de la Médula Espinal/patología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
This study investigates the effect of insulin-like growth factor-1 (IGF-1) and phorbol 12-myrystate 13-acetate (PMA) on 3H-thymidine, 35SO(4) and 3H -glycine incorporations, adenosine 3':5'-cyclic monophosphate (cAMP) production and protein kinase C (PKC) activation in cultured rat articular chondrocyte monolayers (RACM) derived from animals of different ages. It was found that IGF-1 stimulates all these cellular functions in cultures derived from all age groups in a concentration dependent manner, although the cells from 14-month old animals responded poorly. IGF-1 also induces in cells from 1-month old rats an increase in the expression of mRNAs specific for aggrecan and type II collagen molecules as shown with RT-PCR. These effects are mediated via IGF-1 interaction with specific receptors because the monoclonal antibody against the receptor protein suppresses more than 60% of the ligand-induced DNA synthesis. PMA, a direct PKC activator, potentiated IGF-1-induced effects in all cells but much more strongly in cells from young than in cells from 14-month old animals. The age-related failure of RACM to respond adequately to IGF-1 was correlated with a decrease in IGF-1-induced cAMP production, and IGF-1-induced and PMA-induced PKC activations. These results show that IGF-1 regulates the synthesis of DNA, proteoglycans (PG) and collagen II at the level of transcription and suggest that the reduced response of cell monolayers derived from 14-month old rats to IGF-1 is probably due to a failure of old cells to adequately transduce IGF-1 receptor-generated downstream signaling.
Asunto(s)
Envejecimiento/fisiología , Cartílago Articular/efectos de los fármacos , Proteínas de la Matriz Extracelular , Factor I del Crecimiento Similar a la Insulina/farmacología , Transducción de Señal/fisiología , Agrecanos , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/fisiología , Cartílago Articular/citología , Cartílago Articular/metabolismo , Células Cultivadas , Colágeno/biosíntesis , Colágeno/genética , AMP Cíclico/biosíntesis , AMP Cíclico/genética , ADN/biosíntesis , Expresión Génica/fisiología , Lectinas Tipo C , Masculino , Proteoglicanos/biosíntesis , Proteoglicanos/genética , Ratas , Ratas Wistar , Acetato de Tetradecanoilforbol/farmacología , Timidina/metabolismoRESUMEN
The authors described the reconstruction of a terminal branch of the brachial plexus (the median nerve) by different kinds of peripheral nerve grafts, in rats. Fresh or frozen autografts from Sprague-Dawley rats and fresh or frozen xenografts from Beagle dogs were used. Three, six, nine and twelve months after grafting, rats underwent histological assessment (muscle, nerve and spinal cord) and simple functional assessment by the grasping test. The immune reaction was prevented by the freezing and thawing method that had rendered xenografts acellular. This process allowed a satisfactory reinnervation of the flexor carpi radialis muscle (FCR) and a function recovery about 75% of control value. Nevertheless, the force recovery in rats that received frozen grafts was slower than those received fresh autografts. Probably, the destruction of cellular elements by freezing produced a deficient environment for nerve regeneration. However, this gap was partially compensated at twelve months after surgery by the maturation and the secondary adaptation of regenerated nerve fibers. Theses results showed that the force recovery is directly correlated to the capability of the nerve fibers to reproduce, histologically, a next to normal nerve pattern.
Asunto(s)
Nervio Mediano/trasplante , Animales , Perros , Femenino , Congelación , Masculino , Nervio Mediano/anatomía & histología , Nervio Mediano/cirugía , Músculo Esquelético/inervación , Ratas , Ratas Sprague-Dawley , Recuperación de la FunciónRESUMEN
A lateral surgical approach of the cervical spinal cord and brachial plexus was developed in nine dogs for avulsion and reimplantation of the ventral cervical spinal roots (C). The surgical steps involved in exposing the spinal cord and roots are described. The avulsed rootlets of C6 and C7 were reimplanted in their initial position. As a direct consequence of the avulsion, flaccid paralysis of the shoulder and severe amyotrophy developed within 5-7 weeks on the injured side. In addition, the dogs exhibited clinical signs resulting from damage to long fiber tracts due to the reimplantation procedure. A partial recovery of these deficits was observed during the 6 postoperative months. Retrograde axonal tracing with horseradish peroxidase applied to the distal stump of the musculocutaneous, suprascapular, and subscapular nerves (originating from C5, C6 and C7) revealed the presence of labelled neuronal somata that were located in the ipsilateral ventral horn, close to the tip of the reimplanted rootlets. It is concluded that the dog constitutes a worthwhile animal model for the study of avulsion and reimplantation of brachial plexus root via a lateral surgical approach.
Asunto(s)
Plexo Braquial/cirugía , Reimplantación/métodos , Raíces Nerviosas Espinales/cirugía , Animales , Axones/fisiología , Vértebras Cervicales/cirugía , Perros , Femenino , Histocitoquímica , Peroxidasa de Rábano Silvestre , Laminectomía , Masculino , Músculo Esquelético/inervación , Músculo Esquelético/cirugía , Regeneración Nerviosa , Médula Espinal/patologíaRESUMEN
In an attempt at repairing the injured spinal cord of adult mammals (rat, dog and marmoset) and its damaged muscular connections, we are currently using: 1) peripheral nerve autografts (PNG), containing Schwann cells, to trigger and direct axonal regrowth from host and/or transplanted motoneurons towards denervated muscular targets; 2) foetal spinal cord transplants to replace lost neurons. In adult rats and marmosets, a PNG bridge was used to joint the injured cervical spinal cord to a denervated skeletal muscle (longissimus atlantis [rat] or biceps brachii [rat and marmoset]). The spinal lesion was obtained by the implantation procedure of the PNG. After a post-operative delay ranging from 2 to 22 months, the animals were checked electrophysiologically for functional muscular reconnection and processed for a morphological study including retrograde axonal tracing (HRP, Fast Blue, True Blue), histochemistry (AChE, ATPase), immunocytochemistry (ChAT) and EM. It was thus demonstrated that host motoneurons of the cervical enlargement could extend axons all the way through the PNG bridge as: a) in anaesthetized animals, contraction of the reconnected muscle could be obtained by electrical stimulation of the grafted nerve; b) the retrograde axonal tracing studies indicated that a great number of host cervical neurons extended axons into the PNG bridge up to the muscle; c) many of them were assumed to be motoneurons (double labelling with True Blue and an antibody against ChAT); and even alpha-motoneurons (type C axosomatic synapses in HRP labelled neurons seen in EM in the rat); d) numerous ectopic endplates were seen around the intramuscular tip of the PNG. In larger (cavitation) spinal lesions (rat), foetal motoneurons contained in E14 spinal cord transplants could similarly grow axons through PNG bridges up to the reconnected muscle. Taking all these data into account, it can be concluded that neural transplants are interesting tools for evaluating both the plasticity and the repair capacities of the mammalian spinal cord and of its muscular connections.