RESUMO
The limited success of human myoblast transplantation has been related to immune rejection, poor survival, and limited spread of injected myoblasts after transplantation. An important issue that has received little attention, but is nevertheless of fundamental importance in myoblast transplantation protocols, is the proliferative capacity of human satellite cells. Previous studies from our laboratory have demonstrated that the maximum number of divisions that a population of satellite cells can make decreases with age during the first two decades of life then stabilizes in adulthood. These observations indicate that when satellite cells are used as vectors in myoblast transplantation protocols it is important to consider donor age and the number of divisions that the cells have made prior to transplantation as limiting factors in obtaining an optimal number of donor derived muscle fibers. In this study, myoblasts derived from donors of different ages (newborn, 17 years old, and 71 years old) were isolated and amplified in culture. Their potential to participate in in vivo muscle regeneration in RAG2(-/-)/gamma(c)/C5 triple immunodeficient hosts after implantation was evaluated at 4 and 8 weeks postimplantation. Our results demonstrate that prolonged amplification in culture and the approach to replicative senescence are both important factors that may condition the success of myoblast transplantation protocols.
Assuntos
Senescência Celular , Células Satélites de Músculo Esquelético/transplante , Adolescente , Idoso , Animais , Divisão Celular , Células Cultivadas , Senescência Celular/fisiologia , Feminino , Imunofluorescência , Rejeição de Enxerto/imunologia , Humanos , Recém-Nascido , Masculino , Camundongos , Fibras Musculares Esqueléticas/citologia , Proteínas Musculares/análise , Proteínas Musculares/imunologia , Músculo Esquelético/química , Músculo Esquelético/citologia , RegeneraçãoRESUMO
The cat peroneal muscles have been used in numerous investigations dealing with the physiological properties of motor units, muscle spindles, and Golgi tendon organs. This report presents a study of the organization of peroneal motor pools in the cat spinal cord by means of retrograde axonal transport of horseradish peroxidase from individual muscles to the corresponding motoneurons. The motor nuclei of peroneus longus (PL), peroneus brevis (PB), and peroneus tertius (PT) muscles formed thin columns in the lateral part of the ventral horn in spinal segments L6-S1. In the transverse plane, the PT and PL nuclei occupied, respectively, dorsolateral and ventromedial positions, with PB nucleus in an intermediate position overlapping with the other two nuclei. Measurements of cell body diameters allowed identification of alpha and gamma subgroups in peroneal motoneuron populations. The average numbers of motoneurons were about 96 alpha and 60 gamma in PL, 75 alpha and 54 gamma in PB, and 34 alpha and 23 gamma in PT. Comparison with data from electrophysiological studies indicated that whole populations of motoneurons were labeled in each motor nucleus. The proportions of gamma motoneurons were the same, and cell bodies of gamma motoneurons had similar sizes in the three peroneal populations. In contrast, alpha motoneurons were significantly smaller in PB than in the two other pools, in keeping with the fact that PB contains a proportion of slow motor units larger than the two other muscles. In large samples of homonymous motoneurons, the numbers of first-order dendrites correlated linearly with motoneuron sizes.
Assuntos
Gatos/anatomia & histologia , Neurônios Motores/citologia , Músculos/inervação , Nervo Fibular/anatomia & histologia , Medula Espinal/anatomia & histologia , Animais , Axônios/classificação , Contagem de Células , Dendritos/classificação , Neurônios Motores/classificaçãoRESUMO
The aim of the present study was to investigate whether ultrastructural features can be used as a guide to identify alpha- and gamma-motoneurons among the intermediate-size neurons of the peroneal motor nuclei. The peroneus brevis and peroneus tertius muscles of adult cats were injected with horseradish peroxidase, and motoneurons labeled by retrograde axonal transport were examined by electron microscopy. In both nuclei, the distributions of cell-body diameters, measured in the light microscope, were bimodal covering the range of 28-84 microns, with a trough around 50 microns. The sample of 25 motoneurons selected for the ultrastructural study included not only large (presumed alpha) and small (presumed gamma) neurons but also intermediate-size cell bodies with diameters in the 40-60 microns range. For each motoneuron, 2-5 profiles were reconstructed from ultrathin sections taken at 6-8 microns intervals. Synaptic boutons were counted and their lengths of apposition were measured. On the basis of three criteria, namely: (1) bouton types present on the membrane, (2) percentage of membrane length covered by synapses, and (3) the aspect of the nucleolus, all the examined motoneurons, including those with intermediate sizes, fell into one of two categories. Fourteen motoneurons, with cell-body diameters in a range of 55-84 microns, were contacted by all types of boutons (mainly S-type with spherical vesicles, F-type with flattened vesicles, and C-type with subsynaptic cistern); the synaptic covering of the somatic membrane was over 40% and the nucleus contained a vacuolated nucleolus. These were considered alpha-motoneurons. Eleven motoneurons, with only S and F boutons, a synaptic covering under 30%, a compact nucleolus and a cell-body diameter ranging between 28 and 50 microns, were considered gamma-motoneurons. No other combination of the three criteria was observed. These results show that unequivocal distinction of alpha- and gamma-motoneurons is possible in the peroneal nuclei, on the basis of morphological differences independent of cell-body size.
Assuntos
Neurônios Motores gama/ultraestrutura , Neurônios Motores/ultraestrutura , Nervo Fibular/ultraestrutura , Medula Espinal/ultraestrutura , Animais , Gatos , Nucléolo Celular/ultraestrutura , Feminino , Peroxidase do Rábano Silvestre , Sinapses/ultraestruturaRESUMO
Motoneurons innervating the peroneus brevis muscle of 1 week- and 3 week-old kittens were retrogradely labelled by HRP and examined by electron microscopy. At 1 week the distribution of mean cell body diameters was unimodal. Consequently alpha- and gamma-motoneurons could not be identified by their size. The aim of this study was to see whether the alpha- and gamma-motoneurons of kittens could be identified using the combination of ultrastructural criteria previously defined in the adult cat. Using these three criteria it was not possible to distinguish all the motoneurons as either alpha- or gamma in the kitten and a fourth criterion (frequency of F bouton profiles) was added to aid identification. However, with these four criteria, at 1 week six of 21 motoneurons and at 3 weeks two of 18 could still not be clearly identified as alpha or gamma (four were tentatively considered to be gamma, and four could not be identified). The maturation of alpha-motoneurons between 1 week and the adult was accompanied by an increase in somatic membrane area and a significant decrease in the somatic packing density of F boutons. On gamma-motoneurons there was a decrease in the somatic packing density of F boutons between 1 and 3 weeks. However, the numbers of F and S boutons remained stable for both motoneuron types. Age-related changes in apposition and active zone lengths of F and S boutons characterize the synaptic rearrangements which are occurring during the postnatal development of motoneurons.
Assuntos
Neurônios Motores/citologia , Nervos Periféricos/crescimento & desenvolvimento , Sinapses/ultraestrutura , Fatores Etários , Animais , Animais Recém-Nascidos , Gatos , Divisão Celular/fisiologia , Fíbula , Peroxidase do Rábano Silvestre , Microscopia Eletrônica , Neurônios Motores/ultraestrutura , Músculo Esquelético/inervaçãoRESUMO
The aim of this ultrastructural study was to analyse quantitatively the distribution of gamma-aminobutyric acid (GABA)-like immunoreactivity in axon terminals apposed to somatic and proximal dendritic membranes of cat motoneurons in lumbar column 2. Preembedding immunocytochemistry was used to count the GABAergic terminals contacting profiles of eighteen alpha-and six gamma-motoneurons. Of the 1293 terminals counted on the somatic and proximal dendritic compartments of alpha-motoneurons, 197 were GABAergic. In contrast, a total number of only 62 terminals were counted on gamma-motoneurons, of which 8 were GABAergic. These populations of GABAergic terminals were less numerous than the population of glycinergic terminals observed in a previous study. The morphometric characteristics of GABAergic synapses were analyzed using postembedding immunocytochemistry. Most of the GABAergic terminals contained pleomorphic vesicles (F-type boutons, flattened or pleomorphic vesicles). All terminals presynaptic (P boutons) to large terminals containing sphericle vesicles (M-type boutons, characteristic of alpha-motoneurons), were GABA-immunopositive. These results suggest that there are different distributions of the GABAergic control of excitability on gamma- and alpha-motoneurons. GABA appears to be strongly involved in post-synaptic inhibition of alpha-motoneurons, whereas gamma-motoneurons receive very few GABAergic inhibitory inputs. Morphological correlates of GABAergic presynaptic inhibition were seen on alpha- but not on gamma-motoneurons.
Assuntos
Neurônios Motores/fisiologia , Terminações Pré-Sinápticas/fisiologia , Medula Espinal/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Gatos , Dendritos/ultraestrutura , Imuno-Histoquímica , Neurônios Motores/ultraestrutura , Neurônios Motores gama/fisiologia , Neurônios Motores gama/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura , Inclusão do Tecido , Ácido gama-Aminobutírico/metabolismoRESUMO
The distribution of glycine-like immunoreactivity on cat lumbar motoneurons was examined in electron microscopy, using pre-embedding immunocytochemistry. In the dorsolateral portion of the ventral horn, numerous labeled axon terminals were presynaptic to somatic and dendritic profiles of alpha-motoneurons. Most of the glycinergic boutons contained pleomorphic vesicles and showed symmetrical contacts. On the somatic and proximal dendritic compartments, glycinergic terminals accounted for, respectively, 24.6 and 26.6% of the total number of terminals. There were very few glycinergic terminals on gamma-motoneurons. Immunoreactive axons, dendrites and cell bodies were also observed near the motoneurons. These results support the view that glycine plays a major role in the inhibition of alpha-motoneurons and suggest that inhibitory mechanisms occur on the soma as well as on dendrites.
Assuntos
Glicina/análise , Neurônios Motores/química , Terminações Nervosas/química , Animais , Gatos , Técnicas Imunoenzimáticas , Região Lombossacral , Microscopia Eletrônica , Neurônios Motores/ultraestrutura , Terminações Nervosas/ultraestruturaRESUMO
In 1- to 72-day-old kittens, motoneurons of the 3 peroneal muscle nuclei were labeled by retrograde axonal transport of horseradish peroxidase from individual muscles. At birth, the locations of peroneal nuclei were similar to those of the adult cat. Counts of motoneurons at different ages indicated that postnatal cell death does not occur in peroneal motor nuclei. Primary dendrites were as numerous in motoneurons of newborn kittens as in adult motoneurons but they were thinner, shorter and poorly ramified. The number of recurrent axon collaterals was higher in the first postnatal week than at later stages. The growth of motoneurons followed similar rates in the 3 peroneal nuclei. Distributions of cell body diameters and volumes were unimodal at birth and became bimodal between 15 and 20 days postnatal. The separation of peroneal motoneurons in two size subgroups, presumably corresponding to alpha and gamma populations, was followed by an increase in growth rate which became faster for alpha than for gamma motoneurons.
Assuntos
Envelhecimento/fisiologia , Neurônios Motores/fisiologia , Nervo Fibular/crescimento & desenvolvimento , Animais , Gatos , Contagem de Células , Nervo Fibular/citologiaRESUMO
In adult cats, the fibre population of the retractor bulbi muscle (RB) was analysed, using the histochemical reactions of ATPases. The muscle was found to contain type-2 fibres only, of which 70% were 2a and 30% 2b. Such ATPase profiles, corresponding to fast-twitch fibers, are in agreement with the mechanical properties of the muscle. Both types, 2a and 2b, included fibres in which the oxidative enzyme content was high and fibres in which it was low. The glycogen content of all fibers in the RB was uniformly low.
Assuntos
Músculos Oculomotores/citologia , Adenosina Trifosfatases/metabolismo , Animais , Gatos , Glicogênio/metabolismo , Músculos Oculomotores/enzimologia , Coloração e RotulagemRESUMO
The ependymal junction pattern in the spinal cord of postmetamorphic ribbed newts has been studied, using transmission electron microscopy of ultrathin sections of normal animals and of animals perfused through the IVth ventricle with lanthanum. Contrary to what has been observed in mammalian CNS, the ependyma of the urodelan spinal cord is furnished with tight junctions that seal the luminal border of the terminal bars. These occludens junctions are made up of two to seven punctate fusions of the plasma membranes. Lanthanum tracer remains restricted inside the lumen of the central canal, being stopped at the first punctate fusion on its way through the intercellular clefts. Beyond this point, the extracellular space contains no tracer material. Besides tight junctions, intermediate, desmosomal and gap junctions are also present. Gap junctions and desmosomes are not present in CSF-contacting neurons. It is suggested that ependyma with occluding junctions (special ependyma) overlay the regions of the CNS where the ependymal cells significantly modify the composition of both intercellular and cerebrospinal fluids, through secretory, transporting and permeability control activities.
Assuntos
Epêndima/ultraestrutura , Junções Intercelulares/ultraestrutura , Salamandridae/anatomia & histologia , Medula Espinal/ultraestrutura , Animais , Desmossomos/ultraestrutura , Espaço Extracelular , Lantânio , Masculino , Microscopia EletrônicaRESUMO
An anterograde biocytin and a retrograde WGA-colloidal gold study in the rat can provide information about reciprocal communication pathways between the red nucleus and the trigeminal sensory complex. No terminals were found within the trigeminal motor nucleus, in contrast with the facial motor nucleus. A dense terminal field was observed in the parvicellular reticular formation ventrally to the trigeminal motor nucleus. The parvicellular area may be important for the control of jaw movements by rubrotrigeminal inputs. On the other hand, the contralateral rostral parvicellular part of the red nucleus receives terminals from the same zone in the rostral part of the trigeminal sensory complex, where retrogradely labelled neurones were found after tracer injections into the red nucleus. Such relationships could be part of a control loop for somatosensory information from the orofacial area.
Assuntos
Núcleo Rubro/fisiologia , Núcleos do Trigêmeo/fisiologia , Animais , Transporte Biológico/fisiologia , Transporte Biológico Ativo/fisiologia , Mapeamento Encefálico , Coloide de Ouro/farmacocinética , Lisina/análogos & derivados , Lisina/farmacocinética , Masculino , Vias Neurais/metabolismo , Vias Neurais/fisiologia , Ratos , Ratos Sprague-Dawley , Núcleo Rubro/metabolismo , Núcleos do Trigêmeo/metabolismo , Aglutininas do Germe de Trigo/farmacocinéticaRESUMO
Many survivors of poliomyelitis, several decades after the acute phase of the disease, develop a set of new muscle symptoms called post-polio syndrome. The persistence of poliovirus (PV) in the central nervous system (CNS) may be involved in the aetiology of this syndrome. By using a mouse model, we have shown that PV persists in the CNS of paralysed mice for over a year after the acute disease. Detection of PV plus- and minus-strand RNAs in the spinal cord of paralysed mice suggested continuous PV RNA replication in the CNS. However, infectious PV particles could not be recovered from homogenates of CNS from paralysed mice beyond 20 days post-paralysis, indicating that PV replication was restricted. In an attempt to identify the molecular mechanism by which PV replication was limited, PV plus- and minus-strand RNA levels were estimated in the CNS of persistently infected mice by a semi-quantitative RT-nested PCR method. Results revealed that RNA replication was inhibited at the level of plus-strand RNA synthesis during persistent infection. Similar results were obtained in neuroblastoma IMR-32 cell cultures persistently infected with PV Restriction of PV RNA synthesis could be involved in persistence by limiting PV replication.
Assuntos
Sistema Nervoso Central/virologia , Poliovirus/genética , Poliovirus/fisiologia , Síndrome Pós-Poliomielite/virologia , RNA Viral/biossíntese , Animais , Sistema Nervoso Central/patologia , Feminino , Humanos , Camundongos , Neurônios Motores/ultraestrutura , Neurônios Motores/virologia , Medula Espinal/virologia , Replicação ViralRESUMO
Tubular aggregates (TAs) which have been recently observed in a few mouse myopathies are identical to those described in human diseases. In this study we show that TAs are also found in the skeletal muscle of almost all normal inbred mice strains. In these inbred strains of mice the presence of TAs is shown to be related to both age and sex. Nine different muscles were stained with the modified Gomori trichrome method to reveal the general morphology of the muscles. Anti-SERCA1 ATPase was used to confirm that the TAs were in fact accumulations of sarcoplasmic reticulum and anti-MyHC IIB to demonstrate that these accumulations were found exclusively in the type IIB muscle fibers. An ultrastructural study confirmed the observations revealed by light microscopy that the TAs were derived from the sarcoplasmic reticulum. TAs were never observed in female inbred mice and were only found in type IIB glycolytic muscle fibers of male inbred mice. Therefore when analyzing the effect of genetic knock out and knock in experiments on the muscle phenotype of transgenic mice one should be aware that the presence of these aggregates is a non-specific phenomenon induced by inbreeding.
Assuntos
Envelhecimento/patologia , Endogamia , Corpos de Inclusão/patologia , Músculo Esquelético/patologia , Retículo Sarcoplasmático/patologia , Animais , Anticorpos , ATPases Transportadoras de Cálcio/análise , ATPases Transportadoras de Cálcio/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Rápida/ultraestrutura , Retículo Sarcoplasmático/enzimologia , Retículo Sarcoplasmático/ultraestrutura , ATPases Transportadoras de Cálcio do Retículo SarcoplasmáticoRESUMO
Electrophysiological experiments using averaging techniques, as well as anatomical experiments using horseradish peroxidase staining, have provided further evidence of afferent axons in lumbosacral ventral roots of cats. Recording from dorsal root filaments in L7, S1 or S2, following stimulation of the companion ventral root close to the dura, often shows action potentials of slow conduction velocity belonging to the A delta or C group. Stimulation applied to the proximal part of the ventral root failed to evoke such responses. Recording from multiple sites along a centrally cut ventral root filament shows responses of two types: action potentials of long latency to peripheral nerve stimulation which are seen at all recording locations and which are not seen following dorsal root stimulation. These appear to be afferent fibres which enter the cord via the ventral root; action potentials which follow dorsal root stimulation and which are usually seen only at the most distal ventral root recording site. Some of these were also activated by stimulation of some skin or muscle nerves. At appropriate intervals collision of impulses from dorsal root or peripheral nerve can be demonstrated. Such axons appear to have a recurrent course in the ventral root. Section of the spinal nerve at points progressively closer to the dorsal root ganglion abolishes the dorsal to ventral root continuity of most recurrent type axons at 2 mm distal to the ganglion. Following application of horseradish peroxidase to crushed ends of distal stumps of cut dorsal roots, thin fibres marked by the enzyme are observed in the distal part of companion ventral roots. U-turns of axons have been observed in the distal part of ventral roots and in the spinal nerve near the pole of the ganglion.
Assuntos
Fibras Nervosas/fisiologia , Neurônios Aferentes/fisiologia , Raízes Nervosas Espinhais/fisiologia , Potenciais de Ação , Animais , Axônios/citologia , Axônios/fisiologia , Gatos , Feminino , Masculino , Raízes Nervosas Espinhais/citologia , Fatores de TempoRESUMO
Poliovirus (PV) is the causal agent of paralytic poliomyelitis. Many survivors of the acute disease, after decades of clinical stability, develop new muscular symptoms called postpolio syndrome. It has been hypothesized that the persistence of PV in the spinal cord is involved in the etiology of this syndrome. To investigate the ability of PV to persist in the spinal cord after the onset of paralysis, we exploited a mouse model in which most animals inoculated with a mouse-adapted mutant survived after the onset of paralysis. Light microscopy and ultrastructural immunohistochemical studies and reverse transcription followed by nested PCR performed on spinal cord from paralyzed mice demonstrated that PV persisted in the mouse spinal cord for at least 12 months after the onset of paralysis. This mouse model provides a new tool for studying poliomyelitis evolution after the onset of paralysis.
Assuntos
Modelos Animais de Doenças , Neurônios Motores/virologia , Poliomielite/virologia , Poliovirus/isolamento & purificação , Animais , Camundongos , Poliomielite/fisiopatologia , Síndrome Pós-Poliomielite/virologia , Medula Espinal/virologiaRESUMO
In nembutal anesthetized adult cats, intracellular stimulation of single abducens motoneurones was used to elicit glycogen depletion of their muscle units. Stimulation by short trains (13 pulses at 40 Hz) delivered once a second, was applied for 20 to 110 min. The activation of the motor unit was monitored by intracellular recording of motoneurone action potentials and by EMG. After the end of stimulation, the muscle was excised and frozen to be cut in serial sections that were processed for demonstration of either glycogen, ATPases or SDH. In two experiments, a motor unit could be histochemically identified because 10-15 fibres showed zones of complete glycogen depletion measuring about 5 mm in length. All the depleted fibres had the same histochemical profile: ATPases reactions gave dark staining with alkaline preincubation and light staining with acid preincubation whereas SDH activity was low. In other experiments, prolonged stimulation produced either no depletion at all or very limited zones of partial depletion in a few muscle fibres.
Assuntos
Glicogênio/deficiência , Músculos/metabolismo , Potenciais de Ação , Adenosina Trifosfatases/análise , Animais , Gatos , Estimulação Elétrica , Eletromiografia , Histocitoquímica , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Neurônios Motores/fisiologia , Contração Muscular , Músculos/citologia , Músculos/fisiologia , Succinato Desidrogenase/análise , Fatores de TempoRESUMO
Here we examine the expression of transcription factors GATA-2 and GATA-3 during early stages of embryonic development in the central nervous system (CNS) of the mouse. GATA-2 is expressed as early as 9 dpc in the hindbrain, in ventral rhombomere 4, and transiently in ventral rhombomere 2 (r2). From 9.5 to 11.5 dpc, activation of the gene spreads to many sites of early neuronal differentiation, such as the olfactory bulbs, the pretectum, and the oculomotor nucleus in the midbrain, a thin stripe of cells lining the floor plate from the mesencephalon to the cervical spinal cord and a ventral column of cells spanning the neural tube from rostral hindbrain and including motor neuron as well as ventral interneuron precursors. GATA-3 is expressed in a pattern very similar to that of GATA-2. Distinguishing features are the lack of expression in r2 at 9 dpc and a slight delay in its activation. In addition, GATA-2 is activated in both the ventricular and the subventricular zones of the neural tube, whereas GATA-3 is restricted mainly to the subventricular zone. Expression analyses performed on GATA-2 -/- mouse embryos between E9.5 and 10.5 dpc established that: (i) the expression of GATA-3 in the developing CNS of the mouse embryo is dependent on the presence of GATA-2 and (ii) loss of GATA-2 leads to severe defects in neurogenesis, which strongly suggests that GATA-2 is involved, as in hematopoiesis, in the maintenance of the pool of ventral neuronal progenitors.
Assuntos
Encéfalo/embriologia , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Medula Espinal/embriologia , Transativadores/genética , Fatores de Transcrição/genética , Animais , Axônios/fisiologia , Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA2 , Fator de Transcrição GATA3 , Idade Gestacional , Interneurônios/citologia , Interneurônios/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Medula Espinal/citologia , Medula Espinal/metabolismo , Células-Tronco/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Dedos de ZincoRESUMO
Poliovirus (PV) is the etiological agent of human paralytic poliomyelitis. Paralysis results from the destruction of motoneurons, a consequence of PV replication. However, the PV-induced process leading to the death of motoneurons is not well known. We investigated whether PV-induced central nervous system (CNS) injury is associated with apoptosis by using mice as animal models. Transgenic mice expressing the human PV receptor were infected intracerebrally with either the neurovirulent PV-1 Mahoney strain or a paralytogenic dose of the attenuated PV-1 Sabin strain. Nontransgenic mice were infected with a mouse-adapted PV-1 Mahoney mutant. DNA fragmentation was demonstrated in CNS tissue from paralyzed mice by visualization of DNA oligonucleosomal laddering and by enzyme-linked immunosorbent assay. Viral antigens and DNA fragmentation detected by the in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling technique were colocalized in neurons of spinal cords from paralyzed mice. In addition, morphological changes characteristic of cells undergoing apoptosis were observed in motoneurons by electron microscopy. Thus, we show that PV multiplication and CNS injury during paralytic poliomyelitis are associated with apoptosis.
Assuntos
Apoptose , Sistema Nervoso Central/virologia , Proteínas de Membrana , Poliovirus/fisiologia , Animais , Antígenos Virais/análise , Sistema Nervoso Central/patologia , Fragmentação do DNA , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Neurônios Motores/virologia , Poliovirus/crescimento & desenvolvimento , Poliovirus/imunologia , Receptores Virais/genética , Medula Espinal/patologia , Fatores de Tempo , Células Tumorais Cultivadas , Carga ViralRESUMO
Following our previous studies related to brachial plexus injury and repair, the present experimentation was designed to examine the ultrastructural features of those motoneurons of the locally injured cervical spinal cord of adult rats that were seen to regenerate into peripheral nerve (PN) bridges and to reinnervate nearby skeletal muscles. Here, the peripheral connection of the PN bridge was made with the biceps brachii (BB) muscle. Three months postsurgery, the spinal motoneurons labelled by retrograde axonal transport of horseradish peroxidase (HRP), after its injection into the BB, were selected on thick sections, using light microscopy, for the presence of dark amorphous granules of the HRP reaction product. Serial ultrathin sections were then made from the selected material. For the 10 labelled neurons studied, we examined the synaptic boutons present on the membrane of the neuronal soma. For five of them, we could observe three of the six types of synaptic boutons described for the alpha-motoneurons of the cat (S-type with spherical vesicles, F-types with flattened vesicles, and C-type with subsynaptic cistern). The largest boutons (type C) are specific to alpha-motoneurons. In comparison to normal material, we noticed a decrease in the number of boutons and an increase in the number of glial processes. After a transient phase of trophic changes, the reinnervated BB muscles showed a return of their fibers to nearly normal diameters as well as evidence of fiber type grouping. Simultaneous staining with silver and cholinesterase also revealed the presence of new motor endplates frequently contacted by several motoneurons. The present study indicates that, after a local spinal injury, typical alpha-motoneurons can reinnervate a skeletal muscle by regenerating axons into the permissive microenvironment provided by a PN graft. These data offer prospects for clinical reconstruction of the brachial plexus after avulsion of one or several nerve roots.
Assuntos
Transplante de Tecido Encefálico/métodos , Neurônios Motores/metabolismo , Músculo Esquelético/inervação , Regeneração Nervosa/fisiologia , Nervos Periféricos/transplante , Traumatismos da Medula Espinal/cirurgia , Medula Espinal/cirurgia , Acetilcolinesterase/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Braço/inervação , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Vértebras Cervicais , Denervação/efeitos adversos , Denervação/métodos , Feminino , Peroxidase do Rábano Silvestre/farmacocinética , Microscopia Eletrônica , Neurônios Motores/ultraestrutura , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Nervos Periféricos/metabolismo , Nervos Periféricos/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Confocal microscopy was used to detect GABA-immunoreactive axo-axonic appositions, indicating possible synaptic contacts, on Ib fiber terminals in the lumbosacral spinal cord. A Ib fiber from posterior biceps-semitendinosus muscles was labeled by intra-axonal ejection of tetramethylrhodamine dextran (red), and serial sections of S1-L7 spinal cord segments were processed for GABA immunocytochemistry revealed by fluorescein isothiocynate (green). Appositions between GABA-immunoreactive structures and the labeled fiber appeared as yellow spots because of the presence of both fluorochromes in small volumes (0.3 * 0.3 * 0.5 micrometer(3)) of tissue. These spots were identified as probable axo-axonic contacts when: (1) they were observed in two to four serial confocal planes, indicating that they did not occur by chance; and (2) their sizes, shapes, and locations were similar to those of axo-axonic contacts found on Ia terminals, known to bear presynaptic boutons, and resembled the axo-axonic synapses described in electron microscope studies of Ib boutons in Clarke's column. A total of 59 presumed axo-axonic contacts was observed on two Ib collaterals, representing an estimated 20% of the total complement. In a three-dimensional reconstruction of one collateral, they were mostly located in terminal positions, and some branches bore more contacts than others. Such differential distribution could not result from chance appositions between GABAergic structures and Ib arborization and further supported the identification of axo-axonic contacts. Segmental Ib collaterals bear axo-axonic synapses that might ensure differential funneling of information toward different targets.
Assuntos
Axônios/química , Neurônios Motores/química , Neurônios Aferentes/química , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/imunologia , Animais , Gatos , Tamanho Celular/fisiologia , Cerebelo/citologia , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Microscopia Confocal , Neurônios Motores/ultraestrutura , Neurônios Aferentes/ultraestrutura , Rodaminas , Medula Espinal/citologiaRESUMO
In this communication, we will review the problems caused by cell-mediated gene therapy, taking skeletal muscle as a physiological model. In particular we have utilised vectors transferring telomerase under the control of retroviral promoters into human satellite cells. The set of results presented here has several implications regarding gene therapy trials. Nevertheless, more experiments will be required to fully validate this cellular model and to use telomerase to safely extend the lifespan of putative gene therapy vectors.