RESUMEN
There is a lack of experimental methods in genetically tractable mouse models to analyze the developmental period at which newborns mature weight-bearing locomotion. To overcome this deficit, we introduce methods to study l-3,4-dihydroxyphenylalanine (l-DOPA)-induced air-stepping in mice at postnatal day (P)7 and P10. Air-stepping is a stereotypic rhythmic behavior that resembles mouse walking overground locomotion but without constraints imposed by weight bearing, postural adjustments, or sensory feedback. We propose that air-stepping represents the functional organization of early spinal circuits coordinating limb movements. After subcutaneous injection of l-DOPA (0.5 mg/g), we recorded air-stepping movements in all four limbs and electromyographic (EMG) activity from ankle flexor (tibialis anterior, TA) and extensor (lateral gastrocnemius, LG) muscles. Using DeepLabCut pose estimation, we analyzed rhythmicity and limb coordination. We demonstrate steady rhythmic stepping of similar duration from P7 to P10 but with some fine-tuning of interlimb coordination with age. Hindlimb joints undergo a greater range of flexion at older ages, indicating maturation of flexion-extension cycles as the animal starts to walk. EMG recordings of TA and LG show alternation but with more focused activation particularly in the LG from P7 to P10. We discuss similarities to neonatal rat l-DOPA-induced air-stepping and infant assisted walking. We conclude that limb coordination and muscle activations recorded with this method represent basic spinal cord circuitry for limb control in neonates and pave the way for future investigations on the development of rhythmic limb control in genetic or disease models with correctly or erroneously developing motor circuitry.NEW & NOTEWORTHY We present novel methods to study neonatal air-stepping in newborn mice. These methods allow analyses at the onset of limb coordination during the period in which altricial species like rats, mice, and humans "learn" to walk. The methods will be useful to test a large variety of mutations that serve as models of motor disease in newborns or that are used to probe for specific circuit mechanisms that generate coordinated limb motor output.
Asunto(s)
Levodopa , Músculo Esquelético , Recién Nacido , Animales , Ratas , Ratones , Humanos , Animales Recién Nacidos , Levodopa/farmacología , Electromiografía , Músculo Esquelético/fisiología , Movimiento , Locomoción/fisiología , Miembro Posterior/fisiologíaRESUMEN
Limited axon regeneration following peripheral nerve injury may be related to activation of the lysosomal protease, asparaginyl endopeptidase (AEP, δ-secretase) and its degradation of the microtubule associated protein, Tau. Activity of AEP was increased at the site of sciatic nerve transection and repair but blocked in mice treated systemically with a specific AEP inhibitor, compound 11 (CP11). Treatments with CP11 enhanced axon regeneration in vivo. Amplitudes of compound muscle action potentials recorded 4 weeks after nerve transection and repair and 2 weeks after daily treatments with CP11 were double those of vehicle-treated mice. At that time after injury, axons of significantly more motor and sensory neurons had regenerated successfully and reinnervated the tibialis anterior and gastrocnemius muscles in CP11-treated mice than vehicle-treated controls. In cultured adult dorsal root ganglion neurons derived from wild type mice that were treated in vitro for 24 h with CP11, neurites were nearly 50% longer than in vehicle-treated controls and similar to neurite lengths in cultures treated with the TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF). Combined treatment with CP11 and 7,8-DHF did not enhance outgrowth more than treatments with either one alone. Enhanced neurite outgrowth produced by CP11 was found also in the presence of the TrkB inhibitor, ANA-12, indicating that the enhancement was independent of TrkB signalling. Longer neurites were found after CP11 treatment in both TrkB+ and TrkB- neurons. Delta secretase inhibition by CP11 is a treatment for peripheral nerve injury with great potential.
Asunto(s)
Axones , Traumatismos de los Nervios Periféricos , Animales , Ratones , Secretasas de la Proteína Precursora del Amiloide , Traumatismos de los Nervios Periféricos/tratamiento farmacológico , Regeneración Nerviosa , NeuritasRESUMEN
Introduction-Recovery from peripheral nerve injuries is poor even though injured peripheral axons can regenerate. Novel therapeutic approaches are needed. The most successful preclinical experimental treatments have relied on increasing the activity of the regenerating axons, but the approaches taken are not applicable to many nerve-injured patients. Bioluminescent optogenetics (BL-OG) is a novel method of increasing the excitation of neurons that might be similar to that found with activity-dependent experimental therapies. We investigated the use of BL-OG as an approach to promoting axon regeneration following peripheral nerve injury. Methods-BL-OG uses luminopsins, light-sensing ion channels (opsins) fused with a light-emitting luciferase. When exposed to a luciferase substrate, such as coelenterazine (CTZ), luminopsins expressed in neurons generate bioluminescence and produce excitation through their opsin component. Adeno-associated viral vectors encoding either an excitatory luminopsin (eLMO3) or a mutated form (R115A) that can generate bioluminescence but not excite neurons were injected into mouse sciatic nerves. After retrograde transport and viral transduction, nerves were cut and repaired by simple end-to-end anastomosis, and mice were treated with a single dose of CTZ. Results-Four weeks after nerve injury, compound muscle action potentials (M waves) recorded in response to sciatic nerve stimulation were more than fourfold larger in mice expressing the excitatory luminopsin than in controls expressing the mutant luminopsin. The number of motor and sensory neurons retrogradely labeled from reinnervated muscles in mice expressing eLMO3 was significantly greater than the number in mice expressing the R115A luminopsin and not significantly different from those in intact mice. When viral injection was delayed so that luminopsin expression was induced after nerve injury, a clinically relevant scenario, evoked M waves recorded from reinnervated muscles were significantly larger after injury in eLMO3-expressing mice. Conclusions-Treatment of peripheral nerve injuries using BL-OG has significant potential to enhance axon regeneration and promote functional recovery.
Asunto(s)
Axones , Traumatismos de los Nervios Periféricos , Ratones , Animales , Axones/fisiología , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/terapia , Optogenética , Regeneración Nerviosa/genética , Neuronas , Nervio Ciático/lesionesRESUMEN
The common neurotrophin receptor, p75NTR , has been proposed to be an inhibitor of axon regeneration after peripheral nerve injury, but whether this effect is on the regenerating axons, immune cells migrating into the injury site, or cells in the pathway surrounding the axons is not clear. Cut nerves in mice expressing fluorescent proteins in axons were repaired with grafts from non-fluorescent hosts to study axon elongation when p75NTR was eliminated separately from axons and immune cells in the proximal stump of cut nerves, from cells in the regeneration pathway, or both. Two weeks later, axons from wild type mice regenerating into grafts devoid of p75NTR had elongated more than twice as far as axons in grafts from wild type mice. No enhancement of regeneration of axons in p75NTR knockout mice was observed, whether nerves were repaired with grafts from wild type mice or from p75NTR knockout mice. To evaluate whether inhibition of p75NTR could be used to improve regeneration, nerves in wild type mice repaired without grafts were exposed to a specific inhibitor of the p75NTR receptor, LM11A-31, at the time of nerve repair. This local blockade of p75NTR resulted in successful regeneration of axons of nearly three times as many motoneurons and reinnervation of twice as many muscle fibers by regenerating motor axons as untreated controls. Expression of p75NTR surrounding regenerating axons contributes to poor regeneration during the first 2 weeks after peripheral nerve injury. Inhibition of p75NTR might be a therapeutic target for treatments of peripheral nerve injuries.
Asunto(s)
Axones , Traumatismos de los Nervios Periféricos , Animales , Ratones , Ratones Noqueados , Neuronas Motoras , Regeneración NerviosaRESUMEN
Functional recovery after peripheral nerve injury (PNI) is poor, mainly due to the slow and incomplete regeneration of injured axons. Experimental therapies that increase the excitability of the injured axons have proven remarkably successful in promoting regeneration, but their clinical applicability has been limited. Bioluminescent optogenetics (BL-OG) uses luminopsins, fusion proteins of light-generating luciferase and light-sensing ion channels that could be used to increase neuronal excitability if exposed to a suitable substrate. Excitatory luminopsins were expressed in motoneurons of transgenic mice and in wildtype mice transduced with adeno-associated viral vectors. Intraperitoneal administration of coelenterazine (CTZ), a known luciferase substrate, generated intense bioluminescence in peripheral axons. This bioluminescence increased motoneuron excitability. A single administration of CTZ immediately after sciatic nerve transection and repair markedly enhanced motor axon regeneration. Compound muscle action potentials were 3-4 times larger than controls by 4 weeks after injury. The results observed with transgenic mice were comparable to those of mice in which the luminopsin was expressed using viral vectors. Significantly more motoneurons had successfully reinnervated muscle targets four weeks after nerve injury in BL-OG treated mice than in controls. Bioluminescent optogenetics is a promising therapeutic approach to enhancing axon regeneration after PNI.
Asunto(s)
Regeneración Nerviosa/fisiología , Optogenética/métodos , Traumatismos de los Nervios Periféricos/terapia , Animales , Axones/fisiología , Modelos Animales de Enfermedad , Potenciales Evocados Motores , Femenino , Humanos , Imidazoles/administración & dosificación , Sustancias Luminiscentes/administración & dosificación , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/fisiología , Traumatismos de los Nervios Periféricos/fisiopatología , Pirazinas/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Medicina Regenerativa/métodosRESUMEN
Inhibitory luminopsins (iLMO2) integrate opto- and chemo-genetic approaches and allow for cell-type specific inhibition of neuronal activity. When exposed to a Renilla luciferase substrate, Coelenterazine (CTZ), iLMO2 generates bioluminescence-mediated activation of its amino-terminal halorhodopsin, resulting in neuronal inhibition. Moderate daily exercise in the form of interval treadmill-training (IT) applied following a peripheral nerve injury results in enhanced motor axon regeneration and muscle fiber reinnervation in female mice. We hypothesized that iLMO2 mediated inhibition of motoneuron activity during IT would block this enhancement. Unilateral intramuscular injections of Cre-dependent AAV2/9-EF1a-DIO-iLMO2 (â¼8.5 x 1013 vg/ml) were made into the gastrocnemius and tibialis anterior muscles of young female ChAT-IRES-Cre mice, thereby limiting iLMO2 expression specifically to their motoneurons. Four to six weeks were allowed for retrograde viral transduction after which a unilateral sciatic nerve transection (Tx) and repair was performed. Animals were randomized into four groups: IT only, IT + CTZ, CTZ only, and untreated (UT). Three weeks post Tx-repair, the maximal amplitude direct muscle responses (M-max) in both muscles in the IT only group were significantly greater than in UT mice, consistent with the enhancing effects of this exercise regimen. Inhibiting motoneuron activity during exercise by a single injection of CTZ, administered 30 minutes prior to exercise, completely blocked the enhancing effect of exercise. Similar treatments with CTZ in mice without iLMO2 had no effect on regeneration. Neuronal activity is required for successful enhancement of motor axon regeneration by exercise.
Asunto(s)
Actividad Motora , Neuronas Motoras/fisiología , Traumatismos de los Nervios Periféricos/fisiopatología , Recuperación de la Función , Animales , Potenciales Evocados Motores , Femenino , Imidazoles/administración & dosificación , Luciferasas de Renilla/genética , Luciferasas de Renilla/fisiología , Sustancias Luminiscentes/administración & dosificación , Ratones Transgénicos , Regeneración Nerviosa , Optogenética , Traumatismos de los Nervios Periféricos/rehabilitación , Pirazinas/administración & dosificación , Nervio Ciático/fisiopatologíaRESUMEN
Injuries to the peripheral nervous system are major sources of disability and often result in painful neuropathies or the impairment of muscle movement and/or normal sensations. For gaps smaller than 10 mm in rodents, nearly normal functional recovery can be achieved; for longer gaps, however, there are challenges that have remained insurmountable. The current clinical gold standard used to bridge long, nonhealing nerve gaps, the autologous nerve graft (autograft), has several drawbacks. Despite best efforts, engineering an alternative "nerve bridge" for peripheral nerve repair remains elusive; hence, there is a compelling need to design new approaches that match or exceed the performance of autografts across critically sized nerve gaps. Here an immunomodulatory approach to stimulating nerve repair in a nerve-guidance scaffold was used to explore the regenerative effect of reparative monocyte recruitment. Early modulation of the immune environment at the injury site via fractalkine delivery resulted in a dramatic increase in regeneration as evident from histological and electrophysiological analyses. This study suggests that biasing the infiltrating inflammatory/immune cellular milieu after injury toward a proregenerative population creates a permissive environment for repair. This approach is a shift from the current modes of clinical and laboratory methods for nerve repair, which potentially opens an alternative paradigm to stimulate endogenous peripheral nerve repair.
Asunto(s)
Regeneración Nerviosa/inmunología , Traumatismos de los Nervios Periféricos/terapia , Nervio Ciático/fisiología , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Autoinjertos , Quimiocina CX3CL1/farmacología , Traumatismos de los Nervios Periféricos/inmunología , Traumatismos de los Nervios Periféricos/patología , Ratas , Nervio Ciático/trasplanteRESUMEN
Locomotion outcomes after peripheral nerve injury and repair in cats have been described in the literature for the period immediately following the injury (muscle denervation period) and then again for an ensuing period of long-term recovery (at 3 mo and longer) resulting in muscle self-reinnervation. Little is known about the changes in muscle activity and walking mechanics during midrecovery, i.e., the early reinnervation period that takes place between 5 and 10 wk of recovery. Here, we investigated hindlimb mechanics and electromyogram (EMG) activity of ankle extensors in six cats during level and slope walking before and every 2 wk thereafter in a 14-wk period of recovery after the soleus (SO) and lateral gastrocnemius (LG) muscle nerves in one hindlimb were surgically transected and repaired. We found that the continued increase in SO and LG EMG magnitudes and corresponding changes in hindlimb mechanics coincided with the formation of neuromuscular synapses revealed in muscle biopsies. Throughout the recovery period, EMG magnitude of SO and LG during the stance phase and the duration of the stance-related activity were load dependent, similar to those in the intact synergistic medial gastrocnemius and plantaris. These results and the fact that EMG activity of ankle extensors and locomotor mechanics during level and upslope walking recovered 14 wk after nerve transection and repair suggest that loss of the stretch reflex in self-reinnervated muscles may be compensated by the recovered force-dependent feedback in self-reinnervated muscles, by increased central drive, and by increased gain in intermuscular motion-dependent pathways from intact ankle extensors. NEW & NOTEWORTHY This study provides new evidence that the timeline for functional recovery of gait after peripheral nerve injury and repair is consistent with the time required for neuromuscular junctions to form and muscles to reach preoperative tensions. Our findings suggest that a permanent loss of autogenic stretch reflex in self-reinnervated muscles may be compensated by recovered intermuscular force-dependent and oligosynaptic length-dependent feedback and central drive to regain adequate locomotor output capabilities during level and upslope walking.
Asunto(s)
Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , Traumatismos de los Nervios Periféricos/fisiopatología , Recuperación de la Función , Caminata , Animales , Fenómenos Biomecánicos , Gatos , Electromiografía , Articulación de la Cadera/fisiopatología , Articulación de la Rodilla/fisiopatología , Desnervación Muscular , Sinapsis/fisiologíaRESUMEN
Brief neuronal activation in injured peripheral nerves is both necessary and sufficient to enhance motor axon regeneration, and this effect is specific to the activated motoneurons. It is less clear whether sensory neurons respond in a similar manner to neuronal activation following peripheral axotomy. Further, it is unknown to what extent enhancement of axon regeneration with increased neuronal activity relies on a reflexive interaction within the spinal circuitry. We used mouse genetics and optical tools to evaluate the precision and selectivity of system-specific neuronal activation to enhance axon regeneration in a mixed nerve. We evaluated sensory and motor axon regeneration in two different mouse models expressing the light-sensitive cation channel, channelrhodopsin (ChR2). We selectively activated either sensory or motor axons using light stimulation combined with transection and repair of the sciatic nerve. Regardless of genotype, the number of ChR2-positive neurons whose axons had regenerated successfully was greater following system-specific optical treatment, with no effect on the number of ChR2-negative neurons (whether motor or sensory neurons). We conclude that acute system-specific neuronal activation is sufficient to enhance both motor and sensory axon regeneration. This regeneration-enhancing effect is likely cell autonomous.
Asunto(s)
Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/metabolismo , Nervio Ciático/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Axones/fisiología , Axotomía/métodos , Femenino , Masculino , Ratones Transgénicos , Neuronas Motoras/fisiologíaRESUMEN
Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools used to modulate neuronal excitability. We hypothesized that activation of excitatory (Gq) DREADD by its designer ligand, clozapine-N-oxide (CNO), would increase the excitability of neurons whose axons have been transected following peripheral nerve injury, and that this increase will lead to an enhanced functional recovery. The lateral gastrocnemius (LG) muscle of adult female Lewis rats was injected unilaterally with AAV9- hsyn- hM3Dq-mCherry (7.6 × 109 viral genomes/µL) to transduce Gq-DREADD expression in LG neurons. The contralateral LG muscle served as an uninjected control. No significant changes in either spontaneous EMG activity or electrically evoked direct muscle (M) responses were found in either muscle after injection of CNO (1 mg/kg, i.p.). The amplitude of monosynaptic H-reflexes in LG was increased after CNO treatment exclusively in muscles previously injected with virus, suggesting that Gq-DREADD activation increased neuronal excitability. After bilateral sciatic nerve transection and repair, additional rats were treated similarly with CNO for up to three days after injury. Electrophysiological data were collected at 2, 4 and 6 weeks after injury. Evoked EMG responses were observed as early as 2 weeks after injury only in Gq-DREADD expressing virus injected LG muscle. By 4 weeks after injury, both M-response and H-reflex amplitudes were significantly greater in muscles previously injected with viral vector than contralateral, uninjected muscles. Increases in the excitability of injured neurons produced by this novel use of Gq-DREADD were sufficient to promote an enhancement in functional recovery after a sciatic nerve injury.
Asunto(s)
Terapia Genética/métodos , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos/terapia , Animales , Células Cultivadas , Clozapina/análogos & derivados , Clozapina/farmacología , Clozapina/uso terapéutico , Potenciales Evocados Motores , Femenino , Músculo Esquelético/inervación , Músculo Esquelético/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Traumatismos de los Nervios Periféricos/diagnóstico por imagen , Ratas , Ratas Endogámicas Lew , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Nervio Ciático/lesionesRESUMEN
Following peripheral nerve injury, moderate daily exercise conducted on a level treadmill results in enhanced axon regeneration and modest improvements in functional recovery. If the exercise is conducted on an upwardly inclined treadmill, even more motor axons regenerate successfully and reinnervate muscle targets. Whether this increased motor axon regeneration also results in greater improvement in functional recovery from sciatic nerve injury was studied. Axon regeneration and muscle reinnervation were studied in Lewis rats over an 11 wk postinjury period using stimulus evoked electromyographic (EMG) responses in the soleus muscle of awake animals. Motor axon regeneration and muscle reinnervation were enhanced in slope-trained rats. Direct muscle (M) responses reappeared faster in slope-trained animals than in other groups and ultimately were larger than untreated animals. The amplitude of monosynaptic H reflexes recorded from slope-trained rats remained significantly smaller than all other groups of animals for the duration of the study. The restoration of the amplitude and pattern of locomotor EMG activity in soleus and tibialis anterior and of hindblimb kinematics was studied during treadmill walking on different slopes. Slope-trained rats did not recover the ability to modulate the intensity of locomotor EMG activity with slope. Patterned EMG activity in flexor and extensor muscles was not noted in slope-trained rats. Neither hindblimb length nor limb orientation during level, upslope, or downslope walking was restored in slope-trained rats. Slope training enhanced motor axon regeneration but did not improve functional recovery following sciatic nerve transection and repair.
Asunto(s)
Axones/fisiología , Neuronas Motoras/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/fisiopatología , Recuperación de la Función/fisiología , Caminata/fisiología , Animales , Axones/patología , Fenómenos Biomecánicos , Modelos Animales de Enfermedad , Electromiografía , Terapia por Ejercicio , Femenino , Miembro Posterior/inervación , Miembro Posterior/patología , Miembro Posterior/fisiopatología , Neuronas Motoras/patología , Músculo Esquelético/inervación , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Traumatismos de los Nervios Periféricos/patología , Traumatismos de los Nervios Periféricos/rehabilitación , Ratas Endogámicas Lew , Reflejo/fisiología , Nervio Ciático/lesiones , Nervio Ciático/patología , Nervio Ciático/fisiopatologíaRESUMEN
Enhancing the regeneration of axons is often considered to be a therapeutic target for improving functional recovery after peripheral nerve injury. In this review, the evidence for the efficacy of electrical stimulation (ES), daily exercise and their combination in promoting nerve regeneration after peripheral nerve injuries in both animal models and in human patients is explored. The rationale, effectiveness and molecular basis of ES and exercise in accelerating axon outgrowth are reviewed. In comparing the effects of ES and exercise in enhancing axon regeneration, increased neural activity, neurotrophins and androgens are considered to be common requirements. Similarly, there are sex-specific requirements for exercise to enhance axon regeneration in the periphery and for sustaining synaptic inputs onto injured motoneurons. ES promotes nerve regeneration after delayed nerve repair in humans and rats. The effectiveness of exercise is less clear. Although ES, but not exercise, results in a significant misdirection of regenerating motor axons to reinnervate different muscle targets, the loss of neuromuscular specificity encountered has only a very small impact on resulting functional recovery. Both ES and exercise are promising experimental treatments for peripheral nerve injury that seem to be ready to be translated to clinical use.
Asunto(s)
Terapia por Estimulación Eléctrica , Terapia por Ejercicio , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos/terapia , Animales , Humanos , Músculo Esquelético/inervación , Traumatismos de los Nervios Periféricos/fisiopatologíaRESUMEN
Despite significant advances in robotics, commercially advanced prosthetics provide only a small fraction of the functionality of the amputated limb that they are meant to replace. Peripheral nerve interfacing could provide a rich controlling link between the body and these advanced prosthetics in order to increase their overall utility. Here, we report on the development of a fully integrated regenerative microchannel interface with 30 microelectrodes and signal extraction capabilities enabling evaluation in an awake and ambulatory rat animal model. In vitro functional testing validated the capability of the microelectrodes to record neural signals similar in size and nature to those that occur in vivo. In vitro dorsal root ganglia cultures revealed striking cytocompatibility of the microchannel interface. Finally, in vivo, the microchannel interface was successfully used to record a multitude of single-unit action potentials through 63% of the integrated microelectrodes at the early time point of 3 weeks. This marks a significant advance in microchannel interfacing, demonstrating the capability of microchannels to be used for peripheral nerve interfacing.
Asunto(s)
Potenciales de Acción , Electrofisiología/métodos , Vigilia , Amplificadores Electrónicos , Animales , Células Cultivadas , Electrofisiología/instrumentación , Ganglios Espinales/fisiología , Microelectrodos , Nervios Periféricos/fisiología , RatasRESUMEN
Treatments with two-small molecule tropomyosin receptor kinase B (trkB) ligands, 7,8 dihydroxyflavone (7,8 DHF) and deoxygedunin, were evaluated for their ability to promote the regeneration of cut axons in injured peripheral nerves in mice in which sensory and motor axons are marked by YFP. Peripheral nerves were cut and repaired with grafts from strain-matched, nonfluorescent donors and secured in place with fibrin glue. Lengths of profiles of regenerating YFP(+) axons were measured 2 wk later from confocal images. Axon regeneration was enhanced when the fibrin glue contained dilutions of 500-nM solution of either small-molecule trkB agonist. In mice in which the neurotrophin receptor trkB is knocked out selectively in neurons, axon regeneration is very weak, and topical treatment with 7,8 DHF had no effect on axon regeneration. Similar treatments with deoxygedunin had only a modest effect. In conditional BDNF knockout mice, topical treatments with either 7,8 DHF or deoxygedunin resulted in a reversal of the poor regeneration found in controls and produced significant enhancement of regeneration. In WT mice treated with 2 wk of daily i.p. injections of either 7,8 DHF or deoxygedunin (5 mg/kg), regenerating axon profiles were nearly twice as long as in controls. Restoration of direct muscle responses evoked by sciatic nerve stimulation to pretransection levels over an 8-wk survival period was found only in the treated mice. Treatments with either small-molecule trkB agonist enhanced axon regeneration and muscle reinnervation after peripheral nerve injuries.
Asunto(s)
Axones/fisiología , Adhesivo de Tejido de Fibrina/farmacología , Flavanonas/farmacología , Limoninas/farmacología , Regeneración Nerviosa/fisiología , Receptor trkB/agonistas , Análisis de Varianza , Animales , Axones/efectos de los fármacos , Proteínas Bacterianas , Potenciales Evocados/fisiología , Modelos Lineales , Proteínas Luminiscentes , Ratones , Regeneración Nerviosa/efectos de los fármacosRESUMEN
Synaptic contacts onto motoneurons were studied in mice in which the gene for the trkB neurotrophin receptor was knocked out selectively in a subset of spinal motoneurons. The extent of contacts by structures immunoreactive for either of two different vesicular glutamate transporters (VGLUT1 and VGLUT2), the vesicular GABA transporter, or glutamic acid decarboxylase 67 (GAD67) with the somata of motoneurons, was studied in wild type and trkB knockout cells in tamoxifen treated male and female SLICK-trkB(-/-) mice. Selective knockout of the trkB gene resulted in a marked reduction in contacts made by VGLUT2- and GAD67-immunoreactive structures in both sexes and a significant reduction in contacts containing only glycine in male mice. No reduction was found for glycinergic contacts in female mice or for VGLUT1 immunoreactive contacts in either sex. Signaling through postsynaptic trkB receptors is considered to be an essential part of a cellular mechanism for maintaining the contacts of some, but not all, synaptic contacts onto motoneurons.
Asunto(s)
Neuronas Motoras/metabolismo , Receptor trkB/metabolismo , Transducción de Señal/fisiología , Sinapsis/metabolismo , Animales , Femenino , Masculino , Ratones Transgénicos , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Electrical stimulation and exercise are treatments to enhance recovery from peripheral nerve injuries. Brain-derived neurotrophic factor and androgen receptor signaling are requirements for the effectiveness of these treatments. Increased neuronal activity is adequate to promote regeneration in injured nerves, but the dosing of activity and its relationship to neurotrophins and sex steroid hormones is less clear. Translation of these therapies will require principles associated with their cellular mechanisms.
Asunto(s)
Axones , Terapia por Estimulación Eléctrica , Terapia por Ejercicio , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos/terapia , Nervios Periféricos/fisiopatología , Animales , Axones/metabolismo , Axones/patología , Femenino , Hormonas Esteroides Gonadales/metabolismo , Humanos , Masculino , Traumatismos de los Nervios Periféricos/diagnóstico , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/fisiopatología , Nervios Periféricos/metabolismo , Nervios Periféricos/patología , Recuperación de la Función , Transducción de Señal , Resultado del TratamientoRESUMEN
Transection of a peripheral nerve results in withdrawal of synapses from motoneurons. Some of the withdrawn synapses are restored spontaneously, but those containing the vesicular glutamate transporter 1 (VGLUT1), and arising mainly from primary afferent neurons, are withdrawn permanently. If animals are exercised immediately after nerve injury, regeneration of the damaged axons is enhanced and no withdrawal of synapses from injured motoneurons can be detected. We investigated whether delaying the onset of exercise until after synapse withdrawal had occurred would yield similar results. In Lewis rats, the right sciatic nerve was cut and repaired. Reinnervation of the soleus muscle was monitored until a direct muscle (M) response was observed to stimulation of the tibial nerve. At that time, rats began 2 wk of daily treadmill exercise using an interval training protocol. Both M responses and electrically-evoked H reflexes were monitored weekly for an additional seven wk. Contacts made by structures containing VGLUT1 or glutamic acid decarboxylase (GAD67) with motoneurons were studied from confocal images of retrogradely labeled cells. Timing of full muscle reinnervation was similar in both delayed and immediately exercised rats. H reflex amplitude in delayed exercised rats was only half that found in immediately exercised animals. Unlike immediately exercised animals, motoneuron contacts containing VGLUT1 in delayed exercised rats were reduced significantly, relative to intact rats. The therapeutic window for application of exercise as a treatment to promote restoration of synaptic inputs onto motoneurons following peripheral nerve injury is different from that for promoting axon regeneration in the periphery.
Asunto(s)
Axones/patología , Terapia por Ejercicio/métodos , Neuronas Motoras/patología , Plasticidad Neuronal , Traumatismos de los Nervios Periféricos/fisiopatología , Sinapsis/patología , Animales , Ergometría , Femenino , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/terapia , Ratas , Ratas Endogámicas Lew , Transmisión Sináptica , Factores de Tiempo , Resultado del TratamientoRESUMEN
Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of ß-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of ß-actin mRNA, and introducing GFP with the 3'UTR of ß-actin mRNA depletes axons of endogenous ß-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of ß-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.
Asunto(s)
Actinas/genética , Axones/fisiología , Glicoproteínas/metabolismo , Regeneración Nerviosa/fisiología , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Actinas/metabolismo , Animales , Transporte Axonal/genética , Proliferación Celular , Células Cultivadas , Proteína GAP-43/deficiencia , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Conos de Crecimiento/fisiología , Ratones , Ratones Endogámicos C57BL , Transporte de ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismoRESUMEN
This article outlines the novel hypothesis that exercise promotes axon regeneration after peripheral nerve injury through neuronal brain-derived neurotrophic factor (BDNF), and there are three required means of promoting BDNF expression: 1) increased signaling through androgen receptors, 2) increased cAMP-responsive element-binding protein expression, and 3) increased expression of the transcription factor SRY-box containing gene 11.
Asunto(s)
Axones/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/fisiopatología , Animales , Factor Neurotrófico Derivado del Encéfalo/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , Receptores Androgénicos/metabolismo , Factores de Transcripción SOXC/metabolismo , Transducción de SeñalRESUMEN
The withdrawal of synaptic inputs from the somata and proximal dendrites of spinal motoneurons following peripheral nerve injury could contribute to poor functional recovery. Decreased availability of neurotrophins to afferent terminals on axotomized motoneurons has been implicated as one cause of the withdrawal. No reduction in contacts made by synaptic inputs immunoreactive to the vesicular glutamate transporter 1 and glutamic acid decarboxylase 67 is noted on axotomized motoneurons if modest treadmill exercise, which stimulates the production of neurotrophins by spinal motoneurons, is applied after nerve injury. In conditional, neuron-specific brain-derived neurotrophic factor (BDNF) knockout mice, a reduction in synaptic contacts onto motoneurons was noted in intact animals which was similar in magnitude to that observed after nerve transection in wild-type controls. No further reduction in coverage was found if nerves were cut in knockout mice. Two weeks of moderate daily treadmill exercise following nerve injury in these BDNF knockout mice did not affect synaptic inputs onto motoneurons. Treadmill exercise has a profound effect on synaptic inputs to motoneurons after peripheral nerve injury which requires BDNF production by those postsynaptic cells.