RESUMEN
A growing number of gain-of-function (GOF) BK channelopathies have been identified in patients with epilepsy and movement disorders. Nevertheless, the underlying pathophysiology and corresponding therapeutics remain obscure. Here, we utilized a knock-in mouse model carrying human BK-D434G channelopathy to investigate the neuronal mechanism of BK GOF in the pathogenesis of epilepsy and dyskinesia. The BK-D434G mice manifest the clinical features of absence epilepsy and exhibit severe motor deficits and dyskinesia-like behaviors. The cortical pyramidal neurons and cerebellar Purkinje cells from the BK-D434G mice show hyperexcitability, which likely contributes to the pathogenesis of absence seizures and paroxysmal dyskinesia. A BK channel blocker, paxilline, potently suppresses BK-D434Ginduced hyperexcitability and effectively mitigates absence seizures and locomotor deficits in mice. Our study thus uncovered a neuronal mechanism of BK GOF in absence epilepsy and dyskinesia. Our findings also suggest that BK inhibition is a promising therapeutic strategy for mitigating BK GOF-induced neurological disorders.
Asunto(s)
Canalopatías , Discinesias , Epilepsia Tipo Ausencia , Canales de Potasio de Gran Conductancia Activados por el Calcio , Animales , Discinesias/genética , Epilepsia Tipo Ausencia/tratamiento farmacológico , Epilepsia Tipo Ausencia/genética , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Ratones , Neuronas , ConvulsionesRESUMEN
Alternating Hemiplegia of Childhood (AHC) is a devastating autosomal dominant disorder caused by ATP1A3 mutations, resulting in severe hemiplegia and dystonia spells, ataxia, debilitating disabilities, and premature death. Here, we determine the effects of delivering an extra copy of the normal gene in a mouse model carrying the most common mutation causing AHC in humans, the D801N mutation. We used an adeno-associated virus serotype 9 (AAV9) vector expressing the human ATP1A3 gene under the control of a human Synapsin promoter. We first demonstrated that intracerebroventricular (ICV) injection of this vector in wild-type mice on postnatal day 10 (P10) results in increases in ouabain-sensitive ATPase activity and in expression of reporter genes in targeted brain regions. We then tested this vector in mutant mice. Simultaneous intracisterna magna and bilateral ICV injections of this vector at P10 resulted, at P40, in reduction of inducible hemiplegia spells, improvement in balance beam test performance, and prolonged survival of treated mutant mice up to P70. Our study demonstrates, as a proof of concept, that gene therapy can induce favorable effects in a disease caused by a mutation of the gene of a protein that is, at the same time, an ATPase enzyme, a pump, and a signal transduction factor.
Asunto(s)
Dependovirus , Hemiplejía , Animales , Dependovirus/genética , Dependovirus/metabolismo , Terapia Genética , Hemiplejía/genética , Hemiplejía/terapia , Ratones , Mutación , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
OBJECTIVE: To define the risks and consequences of cardiac abnormalities in ATP1A3-related syndromes. METHODS: Patients meeting clinical diagnostic criteria for rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC), and cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) with ATP1A3 genetic analysis and at least 1 cardiac assessment were included. We evaluated the cardiac phenotype in an Atp1a3 knock-in mouse (Mashl+/-) to determine the sequence of events in seizure-related cardiac death. RESULTS: Ninety-eight patients with AHC, 9 with RDP, and 3 with CAPOS (63 female, mean age 17 years) were included. Resting ECG abnormalities were found in 52 of 87 (60%) with AHC, 2 of 3 (67%) with CAPOS, and 6 of 9 (67%) with RDP. Serial ECGs showed dynamic changes in 10 of 18 patients with AHC. The first Holter ECG was abnormal in 24 of 65 (37%) cases with AHC and RDP with either repolarization or conduction abnormalities. Echocardiography was normal. Cardiac intervention was required in 3 of 98 (≈3%) patients with AHC. In the mouse model, resting ECGs showed intracardiac conduction delay; during induced seizures, heart block or complete sinus arrest led to death. CONCLUSIONS: We found increased prevalence of ECG dynamic abnormalities in all ATP1A3-related syndromes, with a risk of life-threatening cardiac rhythm abnormalities equivalent to that in established cardiac channelopathies (≈3%). Sudden cardiac death due to conduction abnormality emerged as a seizure-related outcome in murine Atp1a3-related disease. ATP1A3-related syndromes are cardiac diseases and neurologic diseases. We provide guidance to identify patients potentially at higher risk of sudden cardiac death who may benefit from insertion of a pacemaker or implantable cardioverter-defibrillator.
Asunto(s)
Ataxia Cerebelosa/genética , Deformidades Congénitas del Pie/genética , Pérdida Auditiva Sensorineural/genética , Hemiplejía/genética , Mutación/genética , Atrofia Óptica/genética , Reflejo Anormal/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Adolescente , Adulto , Ataxia Cerebelosa/metabolismo , Ataxia Cerebelosa/terapia , Niño , Preescolar , Estudios de Cohortes , Femenino , Deformidades Congénitas del Pie/metabolismo , Deformidades Congénitas del Pie/terapia , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/terapia , Hemiplejía/diagnóstico , Hemiplejía/terapia , Humanos , Lactante , Masculino , Persona de Mediana Edad , Atrofia Óptica/metabolismo , Atrofia Óptica/terapia , Fenotipo , Convulsiones/terapia , Adulto JovenRESUMEN
De novo mutations causing dysfunction of the ATP1A3 gene, which encodes the α3 subunit of Na+/K+-ATPase pump expressed in neurons, result in alternating hemiplegia of childhood (AHC). AHC manifests as paroxysmal episodes of hemiplegia, dystonia, behavioral abnormalities, and seizures. The first aim of this study was to characterize a novel knock-in mouse model (Atp1a3E815K+/-, Matoub, Matb+/-) containing the E815K mutation of the Atp1a3 gene recognized as causing the most severe and second most common phenotype of AHC with increased morbidity and mortality as compared to other mutations. The second aim was to investigate the effects of flunarizine, currently the most effective drug used in AHC, to further validate our model and to help address a question with significant clinical implications that has not been addressed in prior studies. Specifically, many E815K patients have clinical decompensation and catastrophic regression after discontinuing flunarizine therapy; however, it is not known whether this is congruent with the natural course of the disease and is a result of withdrawal from an acute beneficial effect, withdrawal from a long-term protective effect or from a detrimental effect of prior flunarizine exposure. Our behavioral and neurophysiological testing demonstrated that Matb+/- mice express a phenotype that bears a strong resemblance to the E815K phenotype in AHC. In addition, these mice developed spontaneous seizures with high incidence of mortality and required fewer electrical stimulations to reach the kindled state as compared to wild-type littermates. Matb+/- mice treated acutely with flunarizine had reduction in hemiplegic attacks as compared with vehicle-treated mice. After withdrawal of flunarizine, Matb+/- mice that had received flunarizine did neither better nor worse, on behavioral tests, than those who had received vehicle. We conclude that: 1) Our mouse model containing the E815K mutation manifests clinical and neurophysiological features of the most severe form of AHC, 2) Flunarizine demonstrated acute anti-hemiplegic effects but not long-term beneficial or detrimental behavioral effects after it was stopped, and 3) The Matb+/- mouse model can be used to investigate the underlying pathophysiology of ATP1A3 dysfunction and the efficacy of potential treatments for AHC.
Asunto(s)
Modelos Animales de Enfermedad , Hemiplejía/genética , Hemiplejía/fisiopatología , Mutación/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Animales , Conducta Exploratoria/fisiología , Femenino , Técnicas de Sustitución del Gen/métodos , Fuerza de la Mano/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiologíaRESUMEN
OBJECTIVE: Na+ /K+ -ATPase dysfunction, primary (mutation) or secondary (energy crisis, neurodegenerative disease) increases neuronal excitability in the brain. To evaluate the mechanisms underlying such increased excitability we studied mice carrying the D801N mutation, the most common mutation causing human disease, specifically alternating hemiplegia of childhood (AHC) including epilepsy. Because the gene is expressed in all neurons, particularly γ-aminobutyric acid (GABA)ergic interneurons, we hypothesized that the pathophysiology would involve both pyramidal cells and interneurons and that fast-spiking interneurons, which have increased firing rates, would be most vulnerable. METHODS: We performed extracellular recordings, as well as whole-cell patch clamp recordings from pyramidal cells and interneurons, in the CA1 region on hippocampal slices. We also performed immunohistochemistry from hippocampal sections to count CA1 pyramidal cells as well as parvalbumin-positive interneurons. In addition, we performed video-electroencephalography (EEG) recordings from the dorsal hippocampal CA1 region. RESULTS: We observed that juvenile knock-in mice carrying the above mutation reproduce the human phenotype of AHC. We then demonstrated in the CA1 region of these mice the following findings as compared to wild type: (1) Increased number of spikes evoked by electrical stimulation of Schaffer collaterals; (2) equalization by bicuculline of the number of spikes induced by Schaffer collateral stimulation; (3) reduced miniature, spontaneous, and evoked inhibitory postsynaptic currents, but no change in excitatory postsynaptic currents; (4) robust action potential frequency adaptation in response to depolarizing current injection in CA1 fast-spiking interneurons; and (5) no change in the number of pyramidal cells, but reduced number of parvalbumin positive interneurons. SIGNIFICANCE: Our data indicate that, in our genetic model of Atp1α3 mutation, there is increased excitability and marked dysfunction in GABAergic inhibition. This supports the performance of further investigations to determine if selective expression of the mutation in GABAergic and or glutamatergic neurons is necessary and sufficient to result in the behavioral phenotype.
Asunto(s)
Modelos Animales de Enfermedad , Epilepsia/fisiopatología , Hipocampo/fisiopatología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Niño , Análisis Mutacional de ADN , Electroencefalografía , Epilepsia/genética , Potenciales Evocados , Tamización de Portadores Genéticos , Hemiplejía/genética , Hemiplejía/fisiopatología , Humanos , Técnicas In Vitro , Interneuronas/fisiología , Ratones , Ratones Mutantes Neurológicos , Técnicas de Placa-Clamp , Células Piramidales/fisiología , ATPasa Intercambiadora de Sodio-Potasio/genética , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Our recent terminal experiments revealed that administration of a single train of repetitive spinal electromagnetic stimulation (sEMS; 35 min) enhanced synaptic plasticity in spinal circuitry following lateral hemisection spinal cord injury. In the current study, we have examined effects of repetitive sEMS applied as a single train and chronically (5 wk, every other day) following thoracic T10 contusion. Chronic studies involved examination of systematic sEMS administration alone and combined with exercise training and transgene delivery of neurotrophin [adeno-associated virus 10-neurotrophin 3 (AAV10-NT3)]. Electrophysiological intracellular/extracellular recordings, immunohistochemistry, behavioral testing, and anatomical tracing were performed to assess effects of treatments. We found that administration of a single sEMS train induced transient facilitation of transmission through preserved lateral white matter to motoneurons and hindlimb muscles in chronically contused rats with effects lasting for at least 2 h. These physiological changes associated with increased immunoreactivity of GluR1 and GluR2/3 glutamate receptors in lumbar neurons. Systematic administration of sEMS alone for 5 wk, however, was unable to induce cumulative improvements of transmission in spinomuscular circuitry or improve impaired motor function following thoracic contusion. Encouragingly, chronic administration of sEMS, followed by exercise training (running in an exercise ball and swimming), induced the following: 1) sustained strengthening of transmission to lumbar motoneurons and hindlimb muscles, 2) better retrograde transport of anatomical tracer, and 3) improved locomotor function. Greatest improvements were seen in the group that received exercise combined with sEMS and AAV-NT3.
Asunto(s)
Terapia por Ejercicio , Vectores Genéticos/uso terapéutico , Magnetoterapia/métodos , Plasticidad Neuronal , Neurotrofina 3/uso terapéutico , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Animales , Terapia Combinada , Dependovirus/fisiología , Potenciales Evocados Motores , Femenino , Miembro Posterior/fisiopatología , Imanes , Actividad Motora , Neuronas Motoras/fisiología , Músculo Esquelético/fisiopatología , Neuronas/metabolismo , Neurotrofina 3/genética , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/metabolismo , Recuperación de la Función , Traumatismos de la Médula Espinal/virología , TransgenesRESUMEN
OBJECTIVES: Mutations in the ATP1α3 subunit of the neuronal Na+/K+-ATPase are thought to be responsible for seizures, hemiplegias, and other symptoms of alternating hemiplegia of childhood (AHC). However, the mechanisms through which ATP1A3 mutations mediate their pathophysiologic consequences are not yet understood. The following hypotheses were investigated: (1) Our novel knock-in mouse carrying the most common heterozygous mutation causing AHC (D801N) will exhibit the manifestations of the human condition and display predisposition to seizures; and (2) the underlying pathophysiology in this mouse model involves increased excitability in response to electrical stimulation of Schaffer collaterals and abnormal predisposition to spreading depression (SD). METHODS: We generated the D801N mutant mouse (Mashlool, Mashl+/-) and compared mutant and wild-type (WT) littermates. Behavioral tests, amygdala kindling, flurothyl-induced seizure threshold, spontaneous recurrent seizures (SRS), and other paroxysmal activities were compared between groups. In vitro electrophysiologic slice experiments on hippocampus were performed to assess predisposition to hyperexcitability and SD. RESULTS: Mutant mice manifested a distinctive phenotype similar to that of humans with AHC. They had abnormal impulsivity, memory, gait, motor coordination, tremor, motor control, endogenous nociceptive response, paroxysmal hemiplegias, diplegias, dystonias, and SRS, as well as predisposition to kindling, to flurothyl-induced seizures, and to sudden unexpected death. Hippocampal slices of mutants, in contrast to WT animals, showed hyperexcitable responses to 1 Hz pulse-trains of electrical stimuli delivered to the Schaffer collaterals and had significantly longer duration of K+-induced SD responses. SIGNIFICANCE: Our model reproduces the major characteristics of human AHC, and indicates that ATP1α3 dysfunction results in abnormal short-term plasticity with increased excitability (potential mechanism for seizures) and a predisposition to more severe SD responses (potential mechanism for hemiplegias). This model of the human condition should help in understanding the molecular pathways underlying these phenotypes and may lead to identification of novel therapeutic strategies of ATP1α3 related disorders and seizures.
Asunto(s)
Conducta Animal , Encéfalo/fisiopatología , Hemiplejía/fisiopatología , Convulsiones/fisiopatología , Amígdala del Cerebelo/fisiopatología , Animales , Convulsivantes , Modelos Animales de Enfermedad , Electroencefalografía , Fenómenos Electrofisiológicos , Flurotilo , Técnicas de Sustitución del Gen , Hemiplejía/genética , Excitación Neurológica/fisiología , Aprendizaje , Locomoción , Memoria , Ratones , Ratones Transgénicos , Convulsiones/inducido químicamente , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Transmission through descending pathways to lumbar motoneurons, although important for voluntary walking in humans and rats, has not been fully understood at the cellular level in contusion models. Major descending pathways innervating lumbar motoneurons include those at corticospinal tract (CST) and ventrolateral funiculus (VLF). We examined transmission and plasticity at synaptic pathways from dorsal (d)CST and VLF to individual motoneurons located in ventral horn and interneurons located in dorsomedial gray matter at lumbar segments after thoracic chronic contusion in adult anesthetized rats. To accomplish this, we used intracellular electrophysiological recordings and performed acute focal spinal lesions during the recordings. We directly demonstrate that after thoracic T10 chronic contusion the disrupted dCST axons spontaneously form new synaptic contacts with individual motoneurons, extending around the contusion cavity, through spared ventrolateral white matter. These detour synaptic connections are very weak, and strengthening these connections in order to improve function may be a target for therapeutic interventions after spinal cord injury (SCI). We found that degradation of scar-related chondroitin sulfate proteoglycans with the enzyme chondroitinase ABC (ChABC) combined with adeno-associated viral (AAV) vector-mediated prolonged delivery of neurotrophin NT-3 (AAV-NT3) strengthened these spontaneously formed connections in contused spinal cord. Moreover, ChABC/AAV-NT3 treatment induced the appearance of additional detour synaptic pathways innervating dorsomedial interneurons. Improved transmission in ChABC/AAV-NT3-treated animals was associated with increased immunoreactivity of 5-HT-positive fibers in lumbar dorsal and ventral horns. Improved locomotor function assessed with automated CatWalk highlights the physiological significance of these novel connections.
Asunto(s)
Condroitina ABC Liasa/metabolismo , Plasticidad Neuronal , Neurotrofina 3/metabolismo , Tractos Piramidales/fisiopatología , Traumatismos de la Médula Espinal/terapia , Animales , Condroitina ABC Liasa/administración & dosificación , Condroitina ABC Liasa/genética , Contusiones/fisiopatología , Contusiones/terapia , Dependovirus/genética , Dependovirus/metabolismo , Femenino , Terapia Genética , Interneuronas/metabolismo , Interneuronas/fisiología , Locomoción , Neuronas Motoras/metabolismo , Neuronas Motoras/fisiología , Neurotrofina 3/administración & dosificación , Neurotrofina 3/genética , Tractos Piramidales/metabolismo , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/fisiopatología , Sinapsis/metabolismo , Sinapsis/fisiología , Traumatismos Torácicos/fisiopatología , Traumatismos Torácicos/terapiaRESUMEN
NG2 belongs to the family of chondroitin sulfate proteoglycans that are upregulated after spinal cord injury (SCI) and are major inhibitory factors restricting the growth of fibers after SCI. Neutralization of NG2's inhibitory effect on axon growth by anti-NG2 monoclonal antibodies (NG2-Ab) has been reported. In addition, recent studies show that exogenous NG2 induces a block of axonal conduction. In this study, we demonstrate that acute intraspinal injections of NG2-Ab prevented an acute block of conduction by NG2. Chronic intrathecal infusion of NG2-Ab improved the following deficits induced by chronic midthoracic lateral hemisection (HX) injury: (1) synaptic transmission to lumbar motoneurons, (2) retrograde transport of fluororuby anatomical tracer from L5 to L1, and (3) locomotor function assessed by automated CatWalk gait analysis. We collected data in an attempt to understand the cellular and molecular mechanisms underlying the NG2-Ab-induced improvement of synaptic transmission in HX-injured spinal cord. These data showed the following: (1) that chronic NG2-Ab infusion improved conduction and axonal excitability in chronically HX-injured rats, (2) that antibody treatment increased the density of serotonergic axons with ventral regions of spinal segments L1-L5, (3) and that NG2-positive processes contact nodes of Ranvier within the nodal gap at the location of nodal Na(+) channels, which are known to be critical for propagation of action potentials along axons. Together, these results demonstrate that treatment with NG2-Ab partially improves both synaptic and anatomical plasticity in damaged spinal cord and promotes functional recovery after HX SCI. Neutralizing antibodies against NG2 may be an excellent way to promote axonal conduction after SCI.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos/inmunología , Actividad Motora/efectos de los fármacos , Proteoglicanos/inmunología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Transmisión Sináptica/efectos de los fármacos , Análisis de Varianza , Animales , Anticuerpos Monoclonales/farmacología , Antígenos/farmacología , Transporte Biológico/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiopatología , Moléculas de Adhesión Celular Neuronal/metabolismo , Dextranos/metabolismo , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Conducta Exploratoria/efectos de los fármacos , Femenino , Lateralidad Funcional , Marcha/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Técnicas de Placa-Clamp , Proteoglicanos/farmacología , Nódulos de Ranvier/efectos de los fármacos , Nódulos de Ranvier/metabolismo , Ratas , Ratas Sprague-Dawley , Rodaminas/metabolismo , Serotonina/metabolismo , Canales de Sodio/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
As we reported previously, propagation of action potentials through surviving axons is impaired dramatically, resulting in reduced transmission to lumbar motoneurons after midthoracic lateral hemisection (HX) in rats. The aim of the present study was to evoke action potentials through the spared fibers using noninvasive electromagnetic stimulation (EMS) over intact T2 vertebrae in an attempt to activate synaptic inputs to lumbar motoneurons and thus to enhance plasticity of spinal neural circuits after HX. We found that EMS was able to activate synaptic inputs to lumbar motoneurons and motor-evoked potentials (MEP) in hindlimb muscles in adult anesthetized rats. Amplitude of MEP was attenuated in parallel with the decline of responses recorded from the motoneuron pool after HX. Repetitive EMS (50 min, 0.2 Hz) facilitated the amplitudes of responses elicited by electric stimulation of lateral white matter or dorsal corticospinal tracts in HX rats. Facilitation sustained for at least 1.5 h after termination of EMS. The N-methyl-d-aspartate (NMDA) receptor blocker MK-801, injected intraspinally close to the recording electrode prior to EMS, did not alter these responses but blocked the EMS-induced facilitation, suggesting that activation of NMDA receptors is required to initiate an EMS-evoked increase. When MK-801 was administered after EMS-induced facilitation was established, it induced depression of these elevated responses. Results suggest that repetitive EMS over intact vertebrae could be used as a therapeutic approach to open a window of synaptic plasticity after incomplete midthoracic injuries, i.e., to activate NMDA receptors in the lumbar motoneuron pool at synaptic inputs and to strengthen transmission in damaged spinal cord.
Asunto(s)
Campos Electromagnéticos , Plasticidad Neuronal/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Sinapsis/fisiología , Animales , Estimulación Eléctrica/métodos , Potenciales Evocados Motores/fisiología , Femenino , Vértebras Lumbares , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/terapia , Transmisión Sináptica/fisiología , Vértebras Torácicas , Factores de TiempoRESUMEN
To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by descending fibers after an intervening lateral thoracic (T10) hemisection (Hx), we treated adult rats with the following agents: (i) anti-Nogo-A antibodies to neutralize the growth-inhibitor Nogo-A; (ii) neurotrophin-3 (NT-3) via engineered fibroblasts to promote neuron survival and plasticity; and (iii) the NMDA-receptor 2d (NR2d) subunit via an HSV-1 amplicon vector to elevate NMDA receptor function by reversing the Mg(2+) block, thereby enhancing synaptic plasticity and promoting the effects of NT-3. Synaptic responses evoked by stimulation of the ventrolateral funiculus ipsilateral and rostral to the Hx were recorded intracellularly from ipsilateral lumbar motoneurons. In uninjured adult rats short-latency (1.7-ms) monosynaptic responses were observed. After Hx these monosynaptic responses were abolished. In the Nogo-Ab + NT-3 + NR2d group, long-latency (approximately 10 ms), probably polysynaptic, responses were recorded and these were not abolished by re-transection of the spinal cord through the Hx area. This suggests that these novel responses resulted from new connections established around the Hx. Anterograde anatomical tracing from the cervical grey matter ipsilateral to the Hx revealed increased numbers of axons re-crossing the midline below the lesion in the Nogo-Ab + NT-3 + NR2d group. The combined treatment resulted in slightly better motor function in the absence of adverse effects (e.g. pain). Together, these results suggest that the combination treatment with Nogo-Ab + NT-3 + NR2d can produce a functional 'detour' around the lesion in a laterally hemisected spinal cord. This novel combination treatment may help to improve function of the damaged spinal cord.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Proteínas de la Mielina/inmunología , Neurotrofina 3/farmacología , Subunidades de Proteína/farmacología , Receptores de N-Metil-D-Aspartato/uso terapéutico , Traumatismos de la Médula Espinal/patología , Médula Espinal/efectos de los fármacos , Animales , Conducta Animal/fisiología , Femenino , Humanos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Neurotrofina 3/uso terapéutico , Proteínas Nogo , Subunidades de Proteína/uso terapéutico , Desempeño Psicomotor , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Previously, we reported a pronounced reduction in transmission through surviving axons contralateral to chronic hemisection (HX) of adult rat spinal cord. To examine the cellular and molecular mechanisms responsible for this diminished transmission, we recorded intracellularly from lumbar lateral white matter axons in deeply anesthetized adult rats in vivo and measured the propagation of action potentials (APs) through rubrospinal/reticulospinal tract (RST/RtST) axons contralateral to chronic HX at T10. We found decreased excitability in these axons, manifested by an increased rheobase to trigger APs and longer latency for AP propagation passing the injury level, without significant differences in axonal resting membrane potential and input resistance. These electrophysiological changes were associated with altered spatial localization of Nav1.6 sodium channels along axons: a subset of axons contralateral to the injury exhibited a diffuse localization (>10 µm spread) of Nav1.6 channels, a pattern characteristic of demyelinated axons (Craner MJ, Newcombe J, Black JA, Hartle C, Cuzner ML, Waxman SG. Proc Natl Acad Sci USA 101: 8168-8173, 2004b). This result was substantiated by ultrastructural changes seen with electron microscopy, in which an increased number of large-caliber, demyelinated RST axons were found contralateral to the chronic HX. Therefore, an increased rheobase, pathological changes in the distribution of Nav1.6 sodium channels, and the demyelination of contralateral RST axons are likely responsible for their decreased conduction chronically after HX and thus may provide novel targets for strategies to improve function following incomplete spinal cord injury.
Asunto(s)
Potenciales de Acción , Axones/metabolismo , Hemiplejía/fisiopatología , Canales de Sodio/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/metabolismo , Animales , Células Cultivadas , Femenino , Canal de Sodio Activado por Voltaje NAV1.6 , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Distribución TisularRESUMEN
Chronic unilateral hemisection (HX) of the adult rat spinal cord diminishes conduction through intact fibers in the ventrolateral funiculus (VLF) contralateral to HX. This is associated with a partial loss of myelination from fibers in the VLF (Arvanian et al., 2009). Here, we again measured conduction through the VLF using electrical stimulation while recording the resulting volley and synaptic potentials in target motoneurons. We found that intraspinal injection of chondroitinase-ABC, known to digest chondroitin sulfate proteoglycans (CSPGs), prevented the decline of axonal conduction through intact VLF fibers across from chronic T10 HX. Chondroitinase treatment was also associated with behavior suggestive of an improvement of locomotor function after chronic HX. To further study the role of CSPGs in axonal conduction, we injected three purified CSPGs, NG2 and neurocan, which increase in the vicinity of a spinal injury, and aggrecan, which decreases, into the lateral column of the uninjured cord at T10 in separate experiments. Intraspinal injection of NG2 acutely depressed axonal conduction through the injected region in a dose-dependent manner. Similar injections of saline, aggrecan, or neurocan had no significant effect. Immunofluorescence staining experiments revealed the presence of endogenous and exogenous NG2 at some nodes of Ranvier. These results identify a novel acute action of CSPGs on axonal conduction in the spinal cord and suggest that antagonism of proteoglycans reverses or prevents the decline of axonal conduction, in addition to stimulating axonal growth.