RESUMEN
Denervated skeletal muscles show decreased Akt activity and phosphorylation, resulting in atrophy. Akt inhibits downstream transcription of atrophy-associated ubiquitin ligases like muscle ring-finger protein 1 (MuRF-1). In addition, reduced Akt signaling contributes to aberrant protein synthesis in muscles. In ALS mice, we recently found that carboxyl-terminator modulator protein (CTMP) expression is increased and correlated with reduced Akt signaling in atrophic skeletal muscle. CTMP has also been implicated in promoting muscle degeneration and catabolism in an in vitro muscle atrophy model. The present study examined whether sciatic nerve injury (SNI) stimulated CTMP expression in denervated skeletal muscle during muscle atrophy. We hypothesized that CTMP deficiency would reduce neurogenic atrophy and reverse Akt signaling downregulation. Compared to the unaffected contralateral muscle, wild-type (WT) gastrocnemius muscle had a significant increase in CTMP (p < 0.05). Furthermore, denervated CTMP knockout (CTMP-KO) gastrocnemius weighed more than WT muscle (p < 0.05). Denervated CTMP-KO gastrocnemius also showed higher Akt and downstream glycogen synthase kinase 3ß (GSK3ß) phosphorylation compared to WT muscle (p < 0.05) as well as ribosomal proteins S6 and 4E-BP1 phosphorylation (p < 0.001 and p < 0.05, respectively). Moreover, CTMP-KO mice showed significantly lower levels of E3 ubiquitin ligase MuRF-1 and myostatin than WT muscle (p < 0.05). Our findings suggest that CTMP is essential to muscle atrophy after denervation and it may act by reducing Akt signaling, protein synthesis, and increasing myocellular catabolism.
Asunto(s)
Atrofia Muscular , Proteínas Proto-Oncogénicas c-akt , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Atrofia Muscular/metabolismo , Músculo Esquelético/metabolismo , Desnervación , Proteínas Portadoras/metabolismo , Palmitoil-CoA Hidrolasa/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease, with no present cure. The progressive loss of MNs is the hallmark of ALS. We have previously shown the therapeutic effects of the phosphatase and tensin homolog (PTEN) inhibitor, potassium bisperoxo (picolinato) vanadium (bpV[pic]), in models of neurological injury and demonstrated significant neuroprotective effects on MN survival. However, accumulating evidence suggests PTEN is detrimental for MN survival in ALS. Therefore, we hypothesized that treating the mutant superoxide dismutase 1 G93A (mSOD1G93A) mouse model of ALS during motor neuron degeneration and an in vitro model of mSOD1G93A motor neuron injury with bpV(pic) would prevent motor neuron loss. To test our hypothesis, we treated mSOD1G93A mice intraperitoneally daily with 400 µg/kg bpV(pic) from 70 to 90 days of age. Immunolabeled MNs and microglial reactivity were analyzed in lumbar spinal cord tissue, and bpV(pic) treatment significantly ameliorated ventral horn motor neuron loss in mSOD1G93A mice (p = 0.003) while not significantly altering microglial reactivity (p = 0.701). Treatment with bpV(pic) also significantly increased neuromuscular innervation (p = 0.018) but did not affect muscle atrophy. We also cultured motor neuron-like NSC-34 cells transfected with a plasmid to overexpress mutant SOD1G93A and starved them in serum-free medium for 24 h with and without bpV(pic) and downstream inhibitor of Akt signaling, LY294002. In vitro, bpV(pic) improved neuronal viability, and Akt inhibition reversed this protective effect (p < 0.05). In conclusion, our study indicates systemic bpV(pic) treatment could be a valuable neuroprotective therapy for ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Neuronas Motoras/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Compuestos de Vanadio/uso terapéutico , Esclerosis Amiotrófica Lateral/patología , Animales , Células del Asta Anterior/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Medio de Cultivo Libre de Suero/farmacología , Humanos , Ratones Transgénicos , Microglía/efectos de los fármacos , Modelos Animales , Morfolinas/farmacología , Atrofia Muscular/etiología , Atrofia Muscular/patología , Mutación Missense , Unión Neuromuscular/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fosfohidrolasa PTEN/antagonistas & inhibidores , Mutación Puntual , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Superóxido Dismutasa-1/deficiencia , Superóxido Dismutasa-1/genética , Compuestos de Vanadio/farmacologíaRESUMEN
OBJECTIVES: To determine whether functional and anatomical outcomes following suture neurorrhaphy are improved by the addition of electrical stimulation with or without the addition of polyethylene glycol (PEG). METHODS: In a rat model of facial nerve injury, complete facial nerve transection and repair was performed via (a) suture neurorrhaphy alone, (b) neurorrhaphy with the addition of brief (30 minutes) intraoperative electrical stimulation, or (c) neurorrhaphy with the addition electrical stimulation and PEG. Functional recovery was assessed weekly for 16 weeks. At 16 weeks postoperatively, motoneuron survival, amount of regrowth, and specificity of regrowth were assessed by branch labeling and tissue analysis. RESULTS: The addition of brief intraoperative electrical stimulation improved all functional outcomes compared to suturing alone. The addition of PEG to electrical stimulation impaired this benefit. Motoneuron survival, amount of regrowth, and specificity of regrowth were unaltered at 16 weeks postoperative in all treatment groups. CONCLUSION: The addition of brief intraoperative electrical stimulation to neurorrhaphy in this rodent model shows promising neurological benefit in the surgical repair of facial nerve injury. LEVEL OF EVIDENCE: Animal study.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron (MN) disease with no cure. Accumulating evidence indicates ALS involves a complex interaction between central glia and the peripheral immune response and neuromuscular interface. Stem cell secretomes contain various beneficial trophic factors and cytokines, and we recently demonstrated that administration of the secretome of adipose-derived stem cells (ASCs) during early neuromuscular junction (NMJ) denervation in the mutant superoxide dismutase (mSOD1G93A) ALS mouse ameliorated NMJ disruption. In the present study, we hypothesized that administration of dental pulp stem cell secretome in the form of conditioned medium (DPSC-CM) at different stages of disease would promote NMJ innervation, prevent MN loss and extend lifespan. Our findings show that DPSC-CM significantly improved NMJ innervation at postnatal day (PD) 47 compared to vehicle treated mSOD1G93A mice (p < 0.05). During late pre-symptomatic stages (PD70-P91), DPSC-CM significantly increased MN survival (p < 0.01) and NMJ preservation (p < 0.05), while reactive gliosis in the ventral horn remained unaffected. For DPSC-CM treated mSOD1G93A mice beginning at symptom onset, post-onset days of survival as well as overall lifespan was significantly increased compared to vehicle treated mice (p < 0.05). This is the first study to show therapeutic benefits of systemic DPSC secretome in experimental ALS, and establishes a foundation for future research into the treatment effects and mechanistic analyses of DPSC and other stem cell secretome therapies in ALS.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease involving motor neuron death, paralysis and, ultimately, respiratory failure. Motor neuron dysfunction leads to target skeletal muscle atrophy involving dysregulation of downstream cell survival, growth and metabolic signaling. Decreased Akt activity is linked to muscle atrophy in ALS and is associated with increased atrophy gene expression. Unfortunately, the regulating mechanism of Akt activity in atrophic muscle remains unclear. Recent research indicates a role of carboxyl-terminal modulator protein (CTMP) in Akt-signaling related neurologic dysfunction and skeletal muscle metabolism. CTMP is known to bind and reduce Akt phosphorylation and activation. We hypothesized that CTMP expression might progressively increase in ALS skeletal muscle as the disease progresses, downregulating Akt activity. We found that CTMP protein expression significantly increased in hindlimb skeletal muscle in the mSOD1G93A mouse model of ALS in late stages of the disease (P < 0.05), which negatively correlated with Akt phosphorylation over this period (R2 = -0.77). Co-immunoprecipitation of Akt revealed CTMP binding in pre-symptomatic and end-stage skeletal muscle, suggesting a possible direct role in reduced Akt signaling during disease progression. Inflammatory TNFα and downstream cellular degradation process markers for autophagy, lysosome production, and atrophy significantly increased in a pattern corresponding to increased CTMP expression and reduced Akt phosphorylation. In an in vitro model of skeletal muscle atrophy, differentiated C2C12 cells exhibited reduced Akt activity and decreased FOXO1 phosphorylation, a process known to promote transcription of atrophy genes in skeletal muscle. These results corresponded with increased Atrogin-1 expression compared to healthy control cells (P < 0.05). Transfection with CTMP siRNA significantly increased Akt phosphorylation in atrophic C2C12 cells, corresponding to significantly decreased CTMP expression. In conclusion, this is the first study to provide evidence for a link between elevated CTMP expression, downregulated Akt phosphorylation and muscle atrophy in ALS and clearly demonstrates a direct influence of CTMP on Akt phosphorylation in an in vitro muscle cell atrophy model.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Portadoras/metabolismo , Atrofia Muscular/metabolismo , Palmitoil-CoA Hidrolasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Línea Celular , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Técnicas In Vitro , Ratones , Ratones Mutantes , Modelos Biológicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/patología , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Palmitoil-CoA Hidrolasa/antagonistas & inhibidores , Palmitoil-CoA Hidrolasa/genética , Fosforilación , ARN Interferente Pequeño/genética , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Although mechanisms involved in progression of cell death in spinal cord injury (SCI) have been studied extensively, few are clear targets for translation to clinical application. One of the best-understood mechanisms of cell survival in SCI is phosphatidylinositol-3-kinase (PI3K)/Akt and associated downstream signaling. Clear therapeutic efficacy of a phosphatase and tensin homologue (PTEN) inhibitor called bisperoxovanadium (bpV) has been shown in SCI, traumatic brain injury, stroke, and other neurological disease models in both neuroprotection and functional recovery. The present study aimed to elucidate mechanistic influences of bpV activity in neuronal survival in in vitro and in vivo models of SCI. Treatment with 100 nM bpV(pic) reduced cell death in a primary spinal neuron injury model (p < 0.05) in vitro, and upregulated both Akt and ribosomal protein S6 (pS6) activity (p < 0.05) compared with non-treated injured neurons. Pre-treatment of spinal neurons with a PI3K inhibitor, LY294002 or mammalian target of rapamycin (mTOR) inhibitor, rapamycin blocked bpV activation of Akt and ribosomal protein S6 activity, respectively. Treatment with bpV increased extracellular signal-related kinase (Erk) activity after scratch injury in vitro, and rapamycin reduced influence by bpV on Erk phosphorylation. After a cervical hemicontusive SCI, Akt phosphorylation decreased in total tissue via Western blot analysis (p < 0.01) as well as in penumbral ventral horn motor neurons throughout the first week post-injury (p < 0.05). Conversely, PTEN activity appeared to increase over this period. As observed in vitro, bpV also increased Erk activity post-SCI (p < 0.05). Our results suggest that PI3K/Akt signaling is the likely primary mechanism of bpV action in mediating neuroprotection in injured spinal neurons.
Asunto(s)
Fármacos Neuroprotectores/farmacología , Traumatismos de la Médula Espinal/metabolismo , Compuestos de Vanadio/farmacología , Animales , Células Cultivadas , Femenino , Fosfohidrolasa PTEN/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Traumatismos de la Médula Espinal/patología , Serina-Treonina Quinasas TOR/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Spinal cord injury (SCI) afflicts hundreds of thousands of Americans, and most SCI (â¼80%) occurs in males. In experimental animal models, however, many studies used females. Funding agencies like the National Institutes of Health recommend that new proposed studies should include both genders due to variations in gender response to injuries, diseases, and treatments. However, cost and considerations for some animal models, such as SCI, affect investigators in adapting to this recommendation. Research has increased comparing gender effects in various disease and injury models, including SCI. However, most studies use weight-matched animals, which poses issues in comparing results and outcomes. The present study compared histologic and functional outcomes between age-matched male and female Sprague-Dawley rats in a moderate thoracic contusion SCI model. Cresyl violet and eosin staining showed no significant differences in lesion volume between genders after 9 weeks post-SCI (p > 0.05). Luxol fast blue-stained spared myelin was similar between genders, although slightly greater (â¼6%) in spared myelin, compared with cord volume (p = 0.044). Glial reactivity and macrophage labeling in the lesion area was comparable between genders, as well. Basso, Beattie, Bresnahan (BBB) functional scores were not significantly different between genders, and Hargreaves thermal hyperalgesia and Gridwalk sensorimotor analyses also were similar between genders, compared with uninjured gender controls. Analysis of covariance showed weight did not influence functional recovery as assessed through BBB (p = 0.65) or Gridwalk assessment (p = 0.63) in this study. In conclusion, our findings suggest age-matched male and female rats recover similarly in a common clinically relevant SCI model.
Asunto(s)
Contusiones/fisiopatología , Actividad Motora/fisiología , Recuperación de la Función/fisiología , Caracteres Sexuales , Traumatismos de la Médula Espinal/fisiopatología , Factores de Edad , Animales , Peso Corporal/fisiología , Contusiones/patología , Femenino , Masculino , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/patología , Vértebras Torácicas/lesionesRESUMEN
IMPORTANCE: Functional and anatomical outcomes after surgical repair of facial nerve injury may be improved with the addition of polyethylene glycol (PEG) to direct suture neurorrhaphy. The application of PEG has shown promise in treating spinal nerve injuries, but its efficacy has not been evaluated in treatment of cranial nerve injuries. OBJECTIVE: To determine whether PEG in addition to neurorrhaphy can improve functional outcomes and synkinesis after facial nerve injury. DESIGN, SETTING, AND SUBJECTS: In this animal experiment, 36 rats underwent right facial nerve transection and neurorrhaphy with addition of PEG. Weekly behavioral scoring was done for 10 rats for 6 weeks and 14 rats for 16 weeks after the operations. In the 16-week study, the buccal branches were labeled and tissue analysis was performed. In the 6-week study, the mandibular and buccal branches were labeled and tissue analysis was performed. Histologic analysis was performed for 10 rats in a 1-week study to assess the association of PEG with axonal continuity and Wallerian degeneration. Six rats served as the uninjured control group. Data were collected from February 8, 2016, through July 10, 2017. INTERVENTION: Polyethylene glycol applied to the facial nerve after neurorrhaphy. MAIN OUTCOMES AND MEASURES: Functional recovery was assessed weekly for the 16- and 6-week studies, as well as motoneuron survival, amount of regrowth, specificity of regrowth, and aberrant branching. Short-term effects of PEG were assessed in the 1-week study. RESULTS: Among the 40 male rats included in the study, PEG addition to neurorrhaphy showed no functional benefit in eye blink reflex (mean [SEM], 3.57 [0.88] weeks; 95% CI, -2.8 to 1.9 weeks; P = .70) or whisking function (mean [SEM], 4.00 [0.72] weeks; 95% CI, -3.6 to 2.4 weeks; P = .69) compared with suturing alone at 16 weeks. Motoneuron survival was not changed by PEG in the 16-week (mean, 132.1 motoneurons per tissue section; 95% CI, -21.0 to 8.4; P = .13) or 6-week (mean, 131.1 motoneurons per tissue section; 95% CI, -11.0 to 10.0; P = .06) studies. Compared with controls, neither surgical group showed differences in buccal branch regrowth at 16 (36.9 motoneurons per tissue section; 95% CI, -14.5 to 22.0; P = .28) or 6 (36.7 motoneurons per tissue section; 95% CI, -7.8 to 18.5; P = .48) weeks or in the mandibular branch at 6 weeks (25.2 motoneurons per tissue section; 95% CI, -14.5 to 15.5; P = .99). Addition of PEG had no advantage in regrowth specificity compared with suturing alone at 16 weeks (15.3% buccal branch motoneurons with misguided projections; 95% CI, -7.2% to 11.0%; P = .84). After 6 weeks, the number of motoneurons with misguided projections to the mandibular branch showed no advantage of PEG treatment compared with suturing alone (12.1% buccal branch motoneurons with misguided projections; 95% CI, -8.2% to 9.2%; P = .98). In the 1-week study, improved axonal continuity and muscular innervation were not observed in PEG-treated rats. CONCLUSIONS AND RELEVANCE: Although PEG has shown efficacy in treating other nervous system injuries, PEG in addition to neurorraphy was not beneficial in a rat model of facial nerve injury. The addition of PEG to suturing may not be warranted in the surgical repair of facial nerve injury. LEVEL OF EVIDENCE: NA.
Asunto(s)
Traumatismos del Nervio Facial/tratamiento farmacológico , Traumatismos del Nervio Facial/cirugía , Polietilenglicoles/administración & dosificación , Animales , Modelos Animales de Enfermedad , Masculino , Procedimientos Neuroquirúrgicos , Ratas , Ratas Wistar , Recuperación de la Función , Técnicas de SuturaRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is devastating, leading to paralysis and death. Disease onset begins pre-symptomatically through spinal motor neuron (MN) axon die-back from musculature at â¼47 days of age in the mutant superoxide dismutase 1 (mSOD1G93A) transgenic ALS mouse model. This period may be optimal to assess potential therapies. We previously demonstrated that post-symptomatic adipose-derived stem cell conditioned medium (ASC-CM) treatment is neuroprotective in mSOD1G93A mice. We hypothesized that early disease onset treatment could ameliorate neuromuscular junction (NMJ) disruption. OBJECTIVE: To determine whether pre-symptom administration of ASC-CM prevents early NMJ disconnection. METHODS: We confirmed the NMJ denervation time course in mSOD1G93A mice using co-labeling of neurofilament and post-synaptic acetylcholine receptors (AchR) by α-bungarotoxin. We determined whether ASC-CM ameliorates early NMJ loss in mSOD1G93A mice by systemically administering 200µl ASC-CM or vehicle medium daily from post-natal days 35 to 47 and quantifying intact NMJs through co-labeling of neurofilament and synaptophysin with α-bungarotoxin in gastrocnemius muscle. RESULTS: Intact NMJs were significantly decreased in 47 day old mSOD1G93A mice (pâ<â0.05), and daily systemic ASC-CM prevented disease-induced NMJ denervation compared to vehicle treated mice (pâ<â0.05). CONCLUSIONS: Our results lay the foundation for testing the long-term neurological benefits of systemic ASC-CM therapy in the mSOD1G93A mouse model of ALS.
Asunto(s)
Adipocitos/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Medios de Cultivo Condicionados/farmacología , Unión Neuromuscular/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Células Madre/metabolismo , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones Transgénicos , Molibdoferredoxina , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inervación , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Unión Neuromuscular/patología , Unión Neuromuscular/fisiopatología , Distribución Aleatoria , Receptores Colinérgicos/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of lower and upper motor neurons (MN) leading to muscle weakness, paralysis and eventually death. Although a highly varied etiology results in ALS, it broadly manifests itself as sporadic and familial forms that have evident similarities in clinical symptoms and disease progression. There is a tremendous amount of knowledge on molecular mechanisms leading to loss of MNs and neuromuscular junctions (NMJ) as major determinants of disease onset, severity and progression in ALS. Specifically, two main opposing hypotheses, the dying forward and dying back phenomena, exist to account for NMJ denervation. The former hypothesis proposes that the earliest degeneration occurs at the central MNs and proceeds to the NMJ, whereas in the latter, the peripheral NMJ is the site of precipitating degeneration progressing backwards to the MN cell body. A large body of literature strongly indicates a role for the immune system in disease onset and progression via regulatory involvement at the level of both the central and peripheral nervous systems (CNS and PNS). In this review, we discuss the earliest reported immune responses with an emphasis on newly identified immune players in mutant superoxide dismutase 1 (mSOD1) transgenic mice, the gold standard mouse model for ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/inmunología , Análisis Espacio-Temporal , Animales , Humanos , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Unión Neuromuscular/inmunología , Unión Neuromuscular/patología , Células de Schwann/inmunología , Células de Schwann/patologíaRESUMEN
Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A2 (cPLA2), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA2. Inhibition of RhoA, Rho kinase and cPLA2 significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA2 activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA2. The immunofluorescence staining showed that ROCK1 or ROCK2, two isoforms of Rho kinase, was co-localized with cPLA2 in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK1 or ROCK2 bonded directly with cPLA2 and phospho-cPLA2. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA2 activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA2 activation.
Asunto(s)
Fosfolipasas A2 Grupo IV/metabolismo , Neuronas/metabolismo , Quinasas Asociadas a rho/fisiología , Animales , Muerte Celular/fisiología , Femenino , Ratones , Ratones Endogámicos C57BL , Neuronas/patología , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
PURPOSE: Cervical injuries are the most common form of spinal cord injury (SCI), and are often complicated by pathological secondary damage. Therefore, cervical SCI is of great clinical importance for understanding pathology and potential therapies. Here we utilize a weight drop cervical hemi-contusion injury model using a NYU/MASCIS impactor that produced graded anatomical and functional deficits. METHODS: Three groups of rats were established: 1) Sham (laminectomy only) (nâ=â6), 12.5âmm weight drop (nâ=â10), and 25âmm weight drop (nâ=â10) SCI groups. Forelimb functional assessments of grooming ability, cereal manipulation, and forepaw adhesive removal were performed weekly after injury. Using transcranial magnetic motor evoked potentials (tcMMEPs), supraspinal motor stimulations were recorded in both forelimbs and hindlimbs at 5 and 28d post-injury. Lesion volume and myelinated tissue area were assessed through histological analysis. RESULTS: A 12.5âmm weight drop height produced considerable tissue damage compared to Sham animals, while a 25âmm drop induced even greater damage than the 12.5âmm drop (pâ<â0.05). Forelimb functional assessments showed that increased injury severity and tissue damage was correlated to the degree of forelimb functional deficits. Interestingly, the hindlimbs showed little to no motor function loss. Upon tcMMEP stimulation, surprisingly little motor signal was recorded in the hindlimbs despite outward evidence of hindlimb motor recovery. CONCLUSIONS: Our findings highlight a correlation between anatomical damage and functional outcome in a graded cervical hemi-contusion model, and support a loss of descending motor control from supraspinal inputs and intraspinal plasticity that promote spontaneous hindlimb functional recovery in this model.
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Enfermedades Desmielinizantes/etiología , Lateralidad Funcional/fisiología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Estimulación Magnética Transcraneal , Animales , Vértebras Cervicales/patología , Modelos Animales de Enfermedad , Potenciales Evocados Motores/fisiología , Femenino , Miembro Anterior/fisiopatología , Aseo Animal/fisiología , Desempeño Psicomotor/fisiología , Ratas , Ratas Sprague-Dawley , Sustancia Blanca/patologíaRESUMEN
We have established a physiologically relevant mechanism of CD4+ T cell-mediated neuroprotection involving axotomized wildtype (WT) mouse facial motoneurons (FMN) with significance in the treatment of amyotrophic lateral sclerosis (ALS), a fatal MN disease. Use of the transgenic mouse model of ALS involving expression of human mutant superoxide dismutase genes (SOD1(G93A); abbreviated here as mSOD1) has accelerated basic ALS research. Superimposition of facial nerve axotomy (FNA) on the mSOD1 mouse during pre-symptomatic stages indicates that they behave like immunodeficient mice in terms of increased FMN loss and decreased functional recovery, through a mechanism that, paradoxically, is not inherent within the MN itself, but, instead, involves a defect in peripheral immune: CNS glial cell interactions. Our goal is to utilize our WT mouse model of immune-mediated neuroprotection after FNA as a template to elucidate how a malfunctioning peripheral immune system contributes to motoneuron cell loss in the mSOD1 mouse. This review will discuss potential immune defects in ALS, as well as provide an up-to-date understanding of how the CD4+ effector T cells provide neuroprotection to motoneurons through regulation of the central microglial and astrocytic response to injury. We will discuss an IL-10 cascade within the facial nucleus that requires a functional CD4+ T cell trigger for activation. The review will discuss the role of T cells in ALS, and our recent reconstitution experiments utilizing our model of T cell-mediated neuroprotection in WT vs mSOD1 mice after FNA. Identification of defects in neural:immune interactions could provide targets for therapeutic intervention in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/inmunología , Linfocitos T CD4-Positivos/inmunología , Neuronas Motoras/inmunología , Regeneración Nerviosa/inmunología , Neuroprotección/inmunología , Animales , HumanosRESUMEN
Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.
Asunto(s)
Vértebras Cervicales/lesiones , Modelos Animales de Enfermedad , Traumatismos de la Médula Espinal/etiología , Animales , Femenino , Ratas , Ratas Sprague-Dawley , Restricción FísicaRESUMEN
Schwann cells (SCs) hold promise for spinal cord injury (SCI) repair; however, there are limitations for its use as a lone treatment. We showed that acute inhibition of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) by bisperoxovanadium (bpV) was neuroprotective and enhanced function following cervical hemicontusion SCI. We hypothesized that combining acute bpV therapy and delayed SC engraftment would further improve neuroprotection and recovery after cervical SCI. Adult female Sprague-Dawley (SD) rats were randomly sorted into 5 groups: sham, vehicle, bpV, SC transplantation, and bpV+SC transplantation. SCs were isolated from adult green fluorescent protein (GFP)-expressing SD rats (GFP-SCs). 200 µg/kg bpV(pic) was administered intraperitoneally (IP) twice daily for 7 days post-SCI in bpV-treated groups. GFP-SCs (1×10(6) in 5 µl medium) were transplanted into the lesion epicenter at the 8th day post-SCI. Forelimb function was tested for 10 weeks and histology was assessed. bpV alone significantly reduced lesion (by 40%, p<0.05) and cavitation (by 65%, p<0.05) and improved functional recovery (p<0.05) compared to injury alone. The combination promoted similar neuroprotection (p<0.01 vs. injury); however, GFP-SCs alone did not. Both SC-transplanted groups exhibited remarkable long-term SC survival, SMI-31(+) axon ingrowth and RECA-1(+) vasculature presence in the SC graft; however, bpV+SCs promoted an 89% greater axon-to-lesion ratio than SCs only. We concluded that bpV likely contributed largely to the neuroprotective and functional benefits while SCs facilitated considerable host-tissue interaction and modification. The combination of the two shows promise as an attractive strategy to enhance recovery after SCI.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Células de Schwann/fisiología , Células de Schwann/trasplante , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/cirugía , Compuestos de Vanadio/uso terapéutico , Análisis de Varianza , Animales , Células del Asta Anterior/efectos de los fármacos , Células del Asta Anterior/fisiología , Moléculas de Adhesión Celular/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Miembro Anterior/fisiopatología , Trastornos Neurológicos de la Marcha/etiología , Trastornos Neurológicos de la Marcha/terapia , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Traumatismos de la Médula Espinal/complicaciones , Factores de TiempoRESUMEN
Animal models of traumatic brain injury (TBI) are essential for testing novel hypotheses and therapeutic interventions. Unfortunately, due to the broad heterogeneity of TBI in humans, no single model has been able to reproduce the entire spectrum of these injuries. The controlled cortical impact (CCI) model is one of the most commonly used models of contusion TBI. However, behavioral evaluations have revealed transient impairment in motor function after CCI in rats and mice. Here we report a new semicircular CCI (S-CCI) model by increasing the impact tip area to cover both the motor cortex and hippocampal regions in adult mice. Mice were subjected to S-CCI or CCI using an electromagnetic impactor (Impactor One, MyNeuroLab; semicircular tip: 3mm radius; CCI tip diameter: 3mm). We showed that S-CCI, at two injury severities, significantly decreased the neuroscore and produced deficits in performance on a rotarod device for the entire duration of the study. In contrast, the CCI induced motor deficits only at early stages after the injury, suggesting that the S-CCI model produces long-lasting motor deficits. Morris water maze test showed that both CCI and S-CCI produced persisting memory deficits. Furthermore, adhesive removal test showed significant somatosensory and motor deficits only in the S-CCI groups. Histological analysis showed a large extent of cortical contusion lesions, including both the sensory and motor cortex, and hippocampal damage in the S-CCI. These findings collectively suggest that the current model may offer sensitive, reliable, and clinically relevant outcomes for assessments of therapeutic strategies for TBI.
Asunto(s)
Lesiones Encefálicas/patología , Trastornos del Conocimiento/etiología , Hipocampo/lesiones , Modelos Animales , Trastornos del Movimiento/etiología , Corteza Sensoriomotora/lesiones , Heridas no Penetrantes/patología , Animales , Lesiones Encefálicas/psicología , Trastornos del Conocimiento/fisiopatología , Contusiones/etiología , Contusiones/patología , Contusiones/fisiopatología , Contusiones/psicología , Diseño de Equipo , Femenino , Hipocampo/patología , Hipocampo/fisiopatología , Discapacidades para el Aprendizaje/etiología , Discapacidades para el Aprendizaje/fisiopatología , Aprendizaje por Laberinto , Trastornos de la Memoria/etiología , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Endogámicos C57BL , Trastornos del Movimiento/fisiopatología , Desempeño Psicomotor , Tiempo de Reacción , Prueba de Desempeño de Rotación con Aceleración Constante , Corteza Sensoriomotora/patología , Corteza Sensoriomotora/fisiopatología , Índice de Severidad de la Enfermedad , Heridas no Penetrantes/psicologíaRESUMEN
The goal for treatment in acute spinal cord injury (SCI) is to reduce the extent of secondary damage and facilitate neurologic regeneration and functional recovery. Although multiple studies have investigated potential new therapies for the treatment of acute SCI, outcomes and management protocols aimed at ameliorating neurologic injury in patients remain ineffective. More recent clinical and basic science research have shown surgical interventions to be a potentially valuable modality for treatment; however, the role and timing of surgical decompression, in addition to the optimal surgical intervention, remain one of the most controversial topics pertaining to surgical treatment of acute SCI. As an increasing number of potential treatment modalities emerge, animal models are pivotal for investigating its clinical application and translation into human trials. This review critically appraises the available literature for both clinical and basic science studies to highlight the extent of investigation that has occurred, specific therapies considered, and potential areas for future research.
RESUMEN
Cervical spinal cord injury (SCI) damages axons and motor neurons responsible for ipsilateral forelimb function and causes demyelination and oligodendrocyte death. Inhibition of the phosphatase and tensin homologue, PTEN, promotes neural cell survival, neuroprotection and regeneration in vivo and in vitro. PTEN inhibition can also promote oligodendrocyte-mediated myelination of axons in vitro likely through Akt activation. We recently demonstrated that acute treatment with phosphatase PTEN inhibitor, bisperoxovanadium (bpV)-pic reduced tissue damage, neuron death, and promoted functional recovery after cervical hemi-contusion SCI. Evidence suggests bpV can promote myelin stability; however, bpV effects on myelination and oligodendrocytes in contusive SCI models are unclear. We hypothesized that bpV could increase myelin around the injury site through sparing or remyelination, and that bpV treatment may promote increased numbers of oligodendrocytes. Using histological and immunofluorescence labeling, we found that bpV treatment promoted significant spared white matter (30%; p<0.01) and relative Luxol Fast Blue (LFB)(+) myelin area rostral (Veh: 0.56 ± 0.01 vs. bpV: 0.64 ± 0.02; p<0.05) and at the epicenter (Veh: 0.42 ± 0.03 vs. bpV: 0.54 ± 0.03; p<0.05). VLF oligodendrocytes were also significantly greater with bpV therapy (109 ± 5.3 vs. Veh: 77 ± 2.7 mm(-2); p<0.01). In addition, bpV increased mean motor neuron soma area versus vehicle-treatment (1.0 ± 0.02 vs. Veh: 0.77 ± 0.02) relative to Sham neuron size. This study provides key insight into additional cell and tissue effects that could contribute to bpV-mediated functional recovery observed after contusive cervical SCI.