RESUMO
Facial nerve injury can cause severe long-term physical and psychological morbidity. There are limited repair options for an acutely transected facial nerve not amenable to primary neurorrhaphy. We hypothesize that a peptide amphiphile nanofiber neurograft may provide the nanostructure necessary to guide organized neural regeneration. Five experimental groups were compared, animals with (1) an intact nerve, (2) following resection of a nerve segment, and following resection and immediate repair with either a (3) autograft (using the resected nerve segment), (4) neurograft, or (5) empty conduit. The buccal branch of the rat facial nerve was directly stimulated with charge balanced biphasic electrical current pulses at different current amplitudes whereas nerve compound action potentials (nCAPs) and electromygraphic responses were recorded. After 8 weeks, the proximal buccal branch was surgically reexposed and electrically evoked nCAPs were recorded for groups 1-5. As expected, the intact nerves required significantly lower current amplitudes to evoke an nCAP than those repaired with the neurograft and autograft nerves. For other electrophysiologic parameters such as latency and maximum nCAP, there was no significant difference between the intact, autograft, and neurograft groups. The resected group had variable responses to electrical stimulation, and the empty tube group was electrically silent. Immunohistochemical analysis and transmission electron microscopy confirmed myelinated neural regeneration. This study demonstrates that the neuroregenerative capability of peptide amphiphile nanofiber neurografts is similar to the current clinical gold standard method of repair and holds potential as an off-the-shelf solution for facial reanimation and potentially peripheral nerve repair.
Assuntos
Fenômenos Eletrofisiológicos , Nervo Facial/fisiopatologia , Nanofibras/química , Regeneração Nervosa/efeitos dos fármacos , Peptídeos/farmacologia , Tensoativos/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Estimulação Elétrica , Eletromiografia , Nervo Facial/efeitos dos fármacos , Nervo Facial/cirurgia , Nervo Facial/ultraestrutura , Feminino , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/ultraestrutura , Nanofibras/ultraestrutura , Ratos Sprague-DawleyRESUMO
One obstacle in neural repair is facilitating axon growth long enough to reach denervated targets. Recent studies show that axonal growth is accelerated by applying tension to bundles of neurites, and additional studies show that mechanical tension is critical to all neurite growth. However, no studies yet describe how individual neurons respond to tensile forces applied to cell bodies and neurites simultaneously; neither do any test motor neurons, a phenotype critical to neural repair. Here we examine the growth of dissociated motor neurons on stretchable substrates. E15 spinal motor neurons were cultured on poly-lactide-co-glycolide films stretched at 4.8, 9.6, or 14.3 mm day(-1). Morphological analysis revealed that substrate stretching has profound effects on developing motor neurons. Stretching increases major neurite length; it also forces neuritogenesis to occur nearest poles of the cell closest to the sources of tension. Stretching also reduces the number of neurites per neuron. These data show that substrate stretching affects neuronal morphology by specifying locations on the cell where neuritogenesis occurs and favoring major neurite growth at the expense of minor neurites. These results serve as a building block for development of new techniques to control and improve the growth of neurons for nerve repair purposes.
Assuntos
Materiais Biocompatíveis/química , Ácido Láctico/química , Neurônios Motores/citologia , Neuritos/metabolismo , Neurogênese , Ácido Poliglicólico/química , Animais , Células Cultivadas , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Neuritos/ultraestrutura , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos Sprague-Dawley , Estresse MecânicoRESUMO
The release of transmitter at neuromuscular junctions (NMJ) of the opener muscle in crayfish is quantal in nature. This NMJ offers the advantage of being able to record quantal events at specific visually identified release sites, thus allowing measurement of the physiological parameters of vesicle release and its response to be directly correlated with synaptic structure. These experiments take advantage of areas between the varicosities on the nerve terminal that we define as "stems." Stems were chosen as the region to study because of their low synaptic output due to fewer synaptic sites. Through 3D reconstruction from hundreds of serial sections, obtained by transmission electron microscopy (TEM), at a site in which focal macropatch recordings were obtained, the number of synapses and AZs are revealed. Thus, physiological profiles with various stimulation conditions can be assessed in regards to direct synaptic structure. Here, we used the properties of the quantal shape to determine if distinct subsets of quantal signatures existed and if differences in the distributions are present depending on the frequency of stimulation. Such a quantal signature could come about by parameters of area, rise time, peak amplitude, latency, and tau decay. In this study, it is shown that even at defined sites on the stem, with few active zones, synaptic transmission is still complex and the quantal responses appear to be variable even for a given synapse over time. In this study, we could not identify a quantal signature for the conditions utilized.
Assuntos
Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Astacoidea , Microscopia Eletrônica de Transmissão , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
Peripheral nerve researchers frequently use the rat sciatic nerve crush as a model for axonotmesis. Unfortunately, studies from various research groups report results from different crush techniques and by using a variety of evaluation tools, making comparisons between studies difficult. The purpose of this investigation was to determine the sequence of functional and morphologic changes after an acute sciatic nerve crush injury with a non-serrated clamp, giving a final standardized pressure of p = 9 MPa. Functional recovery was evaluated using the sciatic functional index (SFI), the extensor postural thrust (EPT) and the withdrawal reflex latency (WRL), before injury, and then at weekly intervals until week 8 postoperatively. The rats were also evaluated preoperatively and at weeks 2, 4, and 8 by ankle kinematics, toe out angle (TOA), and gait-stance duration. In addition, the motor nerve conduction velocity (MNCV) and the gastrocnemius-soleus weight parameters were measured just before euthanasia. Finally, structural, ultrastructural and histomorphometric analyses were carried out on regenerated nerve fibers. At 8 weeks after the crush injury, a full functional recovery was predicted by SFI, EPT, TOA, and gait-stance duration, while all the other parameters were still recovering their original values. On the other hand, only two of the histomorphometric parameters of regenerated nerve fibers, namely myelin thickness/axon diameter ratio and fiber/axon diameter ratio, returned to normal values while all other parameters were significantly different from normal values. The employment of traditional methods of functional evaluation in conjunction with the modern techniques of computerized analysis of gait and histomorphometric analysis should thus be recommended for an overall assessment of recovery in the rat sciatic nerve crush model.
Assuntos
Compressão Nervosa/instrumentação , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Neuropatia Ciática/patologia , Degeneração Walleriana/patologia , Animais , Axônios/patologia , Axotomia/instrumentação , Axotomia/métodos , Modelos Animais de Doenças , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/patologia , Transtornos Neurológicos da Marcha/fisiopatologia , Masculino , Microscopia Eletrônica de Transmissão , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Bainha de Mielina/patologia , Ratos , Ratos Wistar , Reflexo Anormal/fisiologia , Nervo Isquiático/fisiopatologia , Neuropatia Ciática/fisiopatologia , Degeneração Walleriana/etiologia , Degeneração Walleriana/fisiopatologiaRESUMO
A detailed examination of the histopathological features of the clip compression injury in mice was performed to understand the relationships between neurological function and existing pathology of the spinal cord. Adult, female CD1 mice underwent three grades of extradural clip compression injury (3-g, 8-g, and 24-g FEJOTA mouse clips), transection, and sham injury at T3-4. Quantitative behavioural assessments were performed for 4 weeks following SCI. After 4 weeks, Fluoro-Gold was introduced caudal to the SCI site, at T9, and was retrogradely transported for 5 days to the origin of spared axons through the injury site. Counts of retrogradely labeled neurons in the brain-stem, midbrain, and sensory-motor cortex indicated that the number of intact descending axons that traversed the lesion decreased with increasing injury severity (F > 28; df = 4; p < 0.0001; one-way ANOVA). Independent linear correlation analyses were performed between indices of neurological recovery (BBB and IP test), counts of retrogradely labeled neurons and morphometric assessments of normal residual tissue at the injury epicenter. The BBB test correlated strongly with the amount of residual tissue at the injury epicenter (R = 0.945, df = 28, p < 0.0001). Counts of neurons retrogradely labeled with Fluoro-Gold were also strongly correlated with the BBB scores. The extrapyramidal (raphespinal, reticulospinal, vestibulospinal, and rubrospinal) tracts had Pearson correlation coefficients (R) of 0.814, 0.812, 0.813, and 0.747, respectively (df = 28, p < 0.0001). The pyramidal (corticospinal) tract had a correlation of R = 0.747, df = 28, p < 0.0001 with the BBB scores. The IP scores also correlated strongly with the persistence of extrapyramidal (raphespinal, reticulospinal, vestibulospinal and rubrospinal) tracts with correlation coefficients of 0.801, 0.782, 0.790, and 0.836, respectively (df = 28, p < 0.0001). These data indicate that the counts of retrogradely labeled neurons at the origin of distinct descending motor pathways are predictors of the variance of the functional recovery measured by the BBB and IP tests following spinal cord injury. In addition, we provide a detailed neuroanatomical study of clip compression injury in mice that can be used to study the molecular mechanisms of SCI in knockout and transgenic mice.
Assuntos
Modelos Animais de Doenças , Camundongos , Compressão da Medula Espinal/patologia , Compressão da Medula Espinal/fisiopatologia , Instrumentos Cirúrgicos , Animais , Animais não Endogâmicos , Axônios/patologia , Contagem de Células , Feminino , Camundongos Knockout , Atividade Motora , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Recuperação de Função Fisiológica , Índice de Gravidade de Doença , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Compressão da Medula Espinal/mortalidadeRESUMO
The complexity, shape, and branching modes of the dendrites of spinal motoneurons were compared in cat, rat, and frog using topological analysis and growth models. The complexity of motoneuronal dendrites, measured as the mean number of terminal segments, varied significantly among samples and was related to contractile properties of innervated motor units. Despite this variation, all mature motoneurons having a mean number of terminal segments per dendrite greater than ten (up to 24.3) exhibited a narrow range of values of coefficients describing the symmetry of tree shapes (0.42-0.47). This implies low variability in the topological shape of motoneuronal dendrites of different animals. This similarity of tree shapes proved to be a result of the similarity of growth rules. The growth of the dendrites could be described to a first approximation by a two-parameter (Q and S) model called the QS model and by a multitype Markovian model. The estimation of parameters of the QS model, in which parameter Q is related to the probability of branching of intermediate segments, revealed that Q was equal or close to 0, implying that branching of dendrites is restricted to terminal segments. The estimates of the parameter S, which describes whether the probability of branching increases (S < 0) or decreases (S > 0) exponentially with segment order, were positive. This was in agreement with the results of estimation of probabilities of branching provided by the Markovian model, which showed that the branching probabilities decreased with segment order in an exponential manner in most of the neurons studied. The QS and Markovian models involve different assumptions about the sequence and timing of branching events, and selection of the best model can provide insight into details of dendritic outgrowth. Extensive simulation of tree outgrowth using a Markovian model revealed significant differences between stimulated trees and real dendrites, particularly with regard to variability of the number of terminals and to symmetry. In contrast, the QS model provided a good fit to the mean values and standard deviations of basic topological parameters. This model is adequate to describe the shape of mature motoneuronal dendrites. It implies that dendritic branches have many opportunities to bifurcate during the whole time of development and that bifurcating potency of a branch is a function of the number and position of other branches of that dendrite. Combined with analysis of metrical properties such as lengths of segments, the QS model can assist in a quantitative analysis of development and plasticity.
Assuntos
Dendritos/fisiologia , Neurônios Motores/fisiologia , Animais , Gatos , Cobalto , Dendritos/ultraestrutura , Peroxidase do Rábano Silvestre , Lisina/análogos & derivados , Cadeias de Markov , Modelos Biológicos , Neurônios Motores/ultraestrutura , Fatores de Crescimento Neural/farmacologia , Rana esculenta , Rana ridibunda , Especificidade da EspécieRESUMO
The spatial distribution of muscle fibers belonging to a motor unit was studied in the soleus (SOL) and tibialis anterior (TA) of adult cats to provide a detailed description of the spatial patterns which exist within the territory of a motor unit. Glycogen depletion of the motor unit was achieved through repetitive stimulation of either the intracellularly identified motoneuron or the functionally isolated motor axon. Muscle fibers belonging to the stimulated unit were identified in serial cross-sections, and in the cross-section which contained the most depleted fibers the centroid of each depleted fiber was determined. Subsequently, three spatial analyses, ie, a quadrat analysis, a point-to nearest neighbor analysis and an interfiber distance analysis, were used to determine if motor unit fibers were distributed randomly throughout the territory of the unit. Motor unit fibers tended to be localized within the muscle cross-section and were not evenly or homogeneously distributed throughout the territory. In general, the analyses suggested that motor unit fibers may be arranged in clusters or subgroups of varying size. The data demonstrate three different quantitative analyses for studying the organization of muscle fibers of normal motor units, which can be used for objective assessment and diagnosis of neuromuscular diseases.