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Alucinaciones/psicología , Trastornos de la Percepción/psicología , Anciano , Encéfalo/patología , Electroencefalografía , Femenino , Hemianopsia/etiología , Humanos , Imagen por Resonancia Magnética , Odorantes , Olfato/fisiología , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/terapia , Síndrome , SinestesiaRESUMEN
BACKGROUND: Neuromodulation is used to restore neural function in disorders that stem from an imbalance in the activity of specific neural networks when they prove refractory to pharmacological therapy. The Kir2.1 gene contributes to stabilizing the resting potential below the threshold of activation of voltage-gated sodium channels and action potentials. Therefore, the delivery of the Kir2.1 gene to neuronal cells could reduce the probability of action potential generation, inhibiting excessive neural activity. OBJECTIVE: To address the hypothesis that overexpression of the inwardly rectifying potassium channel 2.1 (Kir2.1) gene could inhibit motor neuron activity and therefore be therapeutically used in gene-based neuromodulation. METHODS: To induce expression of Kir2.1, the inducible RheoSwitch promoter was used and controlled by ligand. In vivo gene expression was accomplished by an adenoviral vector to deliver unilaterally into the lumbar spinal cord of rats. RESULTS: Behavioral assays demonstrated that neuromuscular inhibition was exclusive to rats that received the ligand. Histological analysis also showed evidence of some motor neuron loss in these animals. Behavioral effects of Kir2.1 expression were completely reversible, arguing that the behavioral effect did not result from motor neuron death. CONCLUSION: Delivery of the gene for Kir2.1 inhibits neurons by resisting depolarization to the action potential threshold. Regulated neuronal expression of Kir2.1 may provide an elegant means for neuromodulation in a selected neuronal population.
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Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Neuronas/fisiología , Canales de Potasio de Rectificación Interna/biosíntesis , Médula Espinal/fisiología , Animales , Línea Celular Tumoral , Regulación de la Expresión Génica/fisiología , Humanos , Fármacos Neuromusculares Despolarizantes/farmacología , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/fisiología , Distribución Aleatoria , Ratas , Médula Espinal/citologíaRESUMEN
INTRODUCTION: Carotid sacrifice remains a valuable tool in the treatment of select vascular lesions. Neurointerventionalists have relied on coil embolization as their primary means of carotid sacrifice, a procedure that can be lengthy and expensive with long fluoroscopy times. We investigated a novel technique for carotid sacrifice in a swine model using temporary balloon occlusion to achieve proximal flow arrest in the carotid artery while embolizing the vessel with a liquid embolic agent. METHODS: A total of 10 common carotid artery sacrifices were performed in pigs under fluoroscopic guidance. Various balloons were employed to achieve near total proximal flow arrest to allow an Onyx cast to accumulate in the target vessel. RESULTS: The technique for sacrifice was modified during the experiment with the final procedures yielding successful sacrifice using Onyx through a dimethylsulfoxide-tolerant catheter (Echelon 14) with the assistance of two fibered coils and a 5 mm × 30 mm Hyperglide balloon resulting in a 2.5 cm long cast. CONCLUSION: Carotid artery sacrifice using commercially available non-adhesive liquid embolic agents is feasible with balloon assistance, allowing for reduced radiation and material costs. Coils may be beneficial in providing an anchor point for liquid embolic deposition, as well as reducing the volume of liquid embolysate required to achieve vessel occlusion.
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Oclusión con Balón/métodos , Arteria Carótida Común/diagnóstico por imagen , Dimetilsulfóxido/administración & dosificación , Polivinilos/administración & dosificación , Animales , Oclusión con Balón/instrumentación , Radiografía , PorcinosRESUMEN
S100B, established as prevalent protein of the central nervous system, is a peripheral biomarker for blood-brain barrier disruption and often also a marker of brain injury. However, reports of extracranial sources of S100B, especially from adipose tissue, may confound its interpretation in the clinical setting. The objective of this study was to characterize the tissue specificity of S100B and assess how extracranial sources of S100B affect serum levels. The extracranial sources of S100B were determined by analyzing nine different types of human tissues by ELISA and Western blot. In addition, brain and adipose tissue were further analyzed by mass spectrometry. A study of 200 subjects was undertaken to determine the relationship between body mass index (BMI) and S100B serum levels. We also measured the levels of S100B homo- and heterodimers in serum quantitatively after blood-brain barrier disruption. Analysis of human tissues by ELISA and Western blot revealed variable levels of S100B expression. By ELISA, brain tissue expressed the highest S100B levels. Similarly, Western blot measurements revealed that brain tissue expressed high levels of S100B but comparable levels were found in skeletal muscle. Mass spectrometry of brain and adipose tissue confirmed the presence of S100B but also revealed the presence of S100A1. The analysis of 200 subjects revealed no statistically significant relationship between BMI and S100B levels. The main species of S100B released from the brain was the B-B homodimer. Our results show that extracranial sources of S100B do not affect serum levels. Thus, the diagnostic value of S100B and its negative predictive value in neurological diseases in intact subjects (without traumatic brain or bodily injury from accident or surgery) are not compromised in the clinical setting.
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Tejido Adiposo/metabolismo , Encéfalo/metabolismo , Músculo Esquelético/metabolismo , Proteínas S100/sangre , Tejido Adiposo/química , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Barrera Hematoencefálica/metabolismo , Índice de Masa Corporal , Química Encefálica , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/química , Proteínas S100/análisis , Adulto JovenRESUMEN
It has long been held that chronic seizures cause blood-brain barrier (BBB) damage. Recent studies have also demonstrated that BBB damage triggers seizures. We have used the BBB osmotic disruption procedure (BBBD) to examine the correlation between BBB opening, pattern of white blood cell (WBCs) entry into the brain and seizure occurrence. These findings were compared to results from resected epileptic brain tissue from temporal lobe epilepsy (TLE) patients. We confirmed that a successful BBB osmotic opening (BBBD) leads to the occurrence of acute epileptiform discharges. Electroencephalography (EEG) and time-joint frequency analysis reveal EEG slowing followed by an increase in the 10-20Hz frequency range. Using green fluorescent protein (GFP)-labeled WBCs (GFP-WBCs) suspended in Evans Blue we found that, at time of BBB-induced epileptiform discharges, WBCs populated the perivascular space of a leaky BBB. Similar results were obtained at time of pilocarpine seizure. No frank WBCs extravasation in the brain parenchyma was observed. In TLE brain specimens, CD45-positive leukocytes were detected only in the vascular and perivascular spaces while albumin and IgG extravasates were parenchymal. The pattern was similar to those observed in rats. Our data suggest that neither acute-induced nor chronic seizures correlate with WBC brain parenchymal migration while albumin and IgG brain leakage is a hallmark of acute and chronic seizures.
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Barrera Hematoencefálica/fisiopatología , Epilepsia del Lóbulo Temporal/patología , Leucocitos/patología , Convulsiones/patología , Adolescente , Adulto , Albúminas/farmacocinética , Animales , Preescolar , Modelos Animales de Enfermedad , Electroencefalografía/métodos , Epilepsia del Lóbulo Temporal/fisiopatología , Azul de Evans , Femenino , Proteínas Fluorescentes Verdes/genética , Humanos , Lactante , Antígenos Comunes de Leucocito/metabolismo , Leucocitos/metabolismo , Masculino , Pilocarpina , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Transfección/métodosRESUMEN
BACKGROUND: Stem cells or immune cells targeting the central nervous system (CNS) bear significant promises for patients affected by CNS disorders. Brain or spinal cord delivery of therapeutic cells is limited by the blood-brain barrier (BBB) which remains one of the recognized rate-limiting steps. Osmotic BBB disruption (BBBD) has been shown to improve small molecule chemotherapy for brain tumors, but successful delivery of cells in conjunction with BBBD has never been reported.We have used a clinically relevant model (pig) of BBBD to attempt brain delivery of TALL-104, a human leukemic T cell line. TALL-104 cells are potent tumor killers and have demonstrated potential for systemic tumor therapy. The pig model used is analogous to the clinical BBBD procedure. Cells were injected in the carotid artery after labeling with the MRI T1 contrast agent GdHPDO3A. Contrast CT scans were used to quantify BBBD and MRI was used to detect Gd++-loaded cells in the brain. Transcranial Doppler was used to monitor cerebral blood flow. EEG recordings were used to detect seizures. Immunocytochemical detection (Cresyl Violet, anti-human CD8 for TALL-104, Evans Blue for BBB damage, GFAP and NEUN) was performed. RESULTS: At the concentration used TALL-104 cells were tolerated. Incomplete BBBD did not allow cell entry into the brain. MRI scans at 24 and 48 hours post-injection allowed visualization of topographically segregated cells in the hemisphere that underwent successful BBBD. Perivascular location of TALL-104 was confirmed in the BBBD hemisphere by Cresyl violet and CD8 immunocytochemistry. No significant alteration in CBF or EEG activity was recorded during cell injections. CONCLUSIONS: Our data show that targeted CNS cell therapy requires blood-brain barrier disruption. MRI-detectable cytotoxic anti-neoplastic cells can be forced to transverse the BBB and accumulate in the perivascular space. The virtual absence of toxicity, the high anti-tumor activity of TALL-104, and the clinical feasibility of human osmotic BBBD suggest that this approach may be adopted to treat brain or spinal cord tumors. In addition, BBBD may favor CNS entry of other cells that normally lack CNS tropism.
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Barrera Hematoencefálica/fisiología , Encéfalo , Movimiento Celular , Endotelio Vascular/fisiología , Linfocitos T Citotóxicos/trasplante , Animales , Velocidad del Flujo Sanguíneo , Permeabilidad Capilar , Arterias Carótidas , Línea Celular Tumoral , Circulación Cerebrovascular , Humanos , Imagen por Resonancia Magnética , Porcinos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/fisiología , Factores de TiempoRESUMEN
BACKGROUND: Efficient adenovirus (AdV)-mediated gene transfer is possible only in immature muscle or regenerating muscle, suggesting that a developmentally regulated event plays a major role in limiting AdV uptake in mature skeletal muscle. Previously, we showed that the expression of the primary coxsackie and adenovirus receptor (CAR) is severely down-regulated during muscle maturation and that, in muscle-specific CAR transgenic mice, there is significant enhancement of AdV-mediated gene transfer to mature skeletal muscle. METHODS: To evaluate whether increasing CAR expression can also augment gene transfer to dystrophic muscle that has many regenerating fibers, we crossed CAR transgenics with dystrophin-deficient mice (mdx/CAR). We also tested a two-step protocol in which CAR levels were increased in the target muscle, prior to administration of AdV, through the use of recombinant adeno-associated virus (AAV2) expressing CAR. Lastly, we assessed the effect of histone deacetylase inhibitors on CAR and AdV transduction efficiency in myoblasts and mdx muscle. RESULTS: Although somewhat higher rates of transduction can be achieved in adult mdx mice than in normal mice as a result of ongoing muscle regeneration in these animals, CAR expression in the mdx background (mdx/CAR transgenics) still markedly improved the susceptibility of mature muscle to AdV-mediated gene transfer of dystrophin. Prior administration of AAV2-CAR to normal muscle led to significantly increased transduction by subsequent injection of AdV. The histone deacetylase inhibitor valproate increased CAR transcript and protein levels in myoblasts and mdx muscle, and also increased AdV-mediated gene transfer. CONCLUSIONS: We have developed a method of increasing CAR levels in both normal and regenerating muscle.
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Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/genética , Receptores Virales/genética , Transducción Genética/métodos , Adenoviridae , Animales , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Distrofina/genética , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Fibras Musculares Esqueléticas/efectos de los fármacos , Regeneración , Transcripción Genética/efectos de los fármacos , Ácido Valproico/farmacologíaRESUMEN
OBJECTIVE: The current series represents a preclinical safety validation study for direct parenchymal microinjection of cellular grafts into the ventral horn of the porcine cervical spinal cord. METHODS: Twenty-four 30- to 40-kg female Yorkshire farm pigs immunosuppressed with cyclosporine underwent a cervical laminectomy and ventral horn human neural progenitor cell injection. Cell transplantation in groups 1 to 3 (n = 6 pigs each) was undertaken with the intent of assessing the safety of varied injection volumes: 10, 25, and 50 microL injected at 1, 2.5, and 5 microL/min, respectively. Groups 4 and 5 (n = 3 pigs each) received prolonged immunosuppressant pretreatment in an attempt to demonstrate graft viability. The latter was undertaken in an alternate species (mini-pig versus Yorkshire pig). RESULTS: Neurological morbidity was observed in 1 animal and was attributable to the presence of a resolving epidural hematoma noted at necropsy. Although instances of ventral horn targeting were achieved in all injection groups with a coordinate-based approach, opportunities exist for improvement in accuracy and precision. A relationship between injection volume and graft site cross-sectional area suggested limited reflux. Only animals from group 5 achieved graft survival at a survival end point (t = 1 week). CONCLUSION: This series demonstrated the functional safety of targeted ventral horn microinjection despite evidence for graft site immune rejection. Improvements in graft delivery may be augmented with an adapter to improve control of the cannula entry angle, intraoperative imaging, or larger graft volumes. Finally, demonstration of long-term graft viability in future preclinical toxicity studies may require tailored immunosuppressive therapies, an allograft construct, or tailored choice of host species.
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Microinyecciones/instrumentación , Enfermedades de la Médula Espinal/cirugía , Médula Espinal/citología , Médula Espinal/cirugía , Trasplante de Células Madre/instrumentación , Células Madre/citología , Jeringas/normas , Animales , Células del Asta Anterior/citología , Células del Asta Anterior/fisiología , Células del Asta Anterior/trasplante , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Vértebras Cervicales/anatomía & histología , Vértebras Cervicales/cirugía , Femenino , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/fisiología , Hematoma Espinal Epidural/etiología , Hematoma Espinal Epidural/patología , Hematoma Espinal Epidural/fisiopatología , Humanos , Inmunosupresores/uso terapéutico , Bombas de Infusión , Laminectomía , Microinyecciones/efectos adversos , Microinyecciones/métodos , Neurogénesis/fisiología , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/fisiopatología , Complicaciones Posoperatorias/prevención & control , Médula Espinal/fisiología , Trasplante de Células Madre/efectos adversos , Trasplante de Células Madre/métodos , Células Madre/fisiología , Técnicas Estereotáxicas , Sus scrofa , Jeringas/efectos adversos , Trasplante Heterólogo/efectos adversos , Trasplante Heterólogo/instrumentación , Trasplante Heterólogo/métodos , Resultado del TratamientoRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating disease that is characterized by the progressive loss of motor neurons. Patients with ALS usually die from respiratory failure due to respiratory muscle paralysis. Consequently, therapies aimed at preserving segmental function of the respiratory motor neurons could extend life for these patients. Insulin-like growth factor-I (IGF-I) is known to be a potent survival factor for motor neurons. In this study we induced high levels of IGF-I expression in the cervical spinal cord of hSOD1(G93A) rats with intraspinal cord (ISC) injections of an adeno-associated virus serotype 2 vector (CERE-130). This approach reduced the extent of motor neuron loss in the treated segments of the spinal cord. However, a corresponding preservation of motor function was observed in male, but not female, hSOD1(G93A) rats. We conclude that ISC injection of CERE-130 has the potential to protect motor neurons and preserve neuromuscular function in ALS.
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Esclerosis Amiotrófica Lateral/terapia , Dependovirus/genética , Terapia Genética , Factor I del Crecimiento Similar a la Insulina/genética , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Vectores Genéticos , Inyecciones Espinales , Masculino , Actividad Motora , Neuronas Motoras/fisiología , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Factores Sexuales , Análisis de Supervivencia , Transducción GenéticaRESUMEN
OBJECTIVE: Apoptosis has been shown to play an important role in motor neuron (MN) degeneration in both neurodegenerative disease and peripheral neuropathy. Bcl-xL, an antiapoptotic protein, is down-regulated in these etiologies [corrected] The carboxyl-terminal domain of the tetanus toxin heavy chain (Hc) has high affinity for axon terminal binding and uptake into motor and dorsal root ganglion (DRG) neurons. We report the development of a fusion protein between Hc and Bcl-xL to enhance uptake of Bcl-xL by MNs as a strategy for inhibiting peripheral neuronal apoptosis. METHODS: The genes for Hc, Bcl-xL, and green fluorescent protein were cloned into an Escherichia coli expression system in 2 different arrangements. Fusion proteins were purified through chromatography. Cultured E15 rat spinal cord MNs and DRG cells were used to demonstrate neuron-specific uptake and retrograde transport of the fusion proteins mediated by Hc. Finally, glutamate-induced apoptosis was used as an in vitro model to measure the antiapoptotic effects of the fusion proteins. RESULTS: Bcl-xL fusion proteins were found to bind specifically and undergo uptake into cultured rat spinal MNs. The fusion proteins were also taken up by DRG axonal terminals and transported back to the cell bodies in Campenot compartmentalized chambers (Tyler Research Corp., Edmonton, Canada). Finally, fusion protein application improved cell survival and decreased apoptosis in glutamate-mediated excitotoxicity of the SH-SY5Y neuronal cells. CONCLUSION: Hc can be applied as a universal carrier for therapeutic cargo delivery specifically to MNs or DRGs. The fusion proteins between Bcl-xL and Hc constructed in this study might bear applications to the treatment of MN disease, neuropathy, or nerve injury through nerve or intramuscular injection.
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Apoptosis/efectos de los fármacos , Fragmentos de Péptidos/administración & dosificación , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/fisiología , Toxina Tetánica/administración & dosificación , Proteína bcl-X/administración & dosificación , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fármacos Neuroprotectores/administración & dosificación , Fragmentos de Péptidos/genética , Células del Asta Posterior/citología , Ratas , Proteínas Recombinantes de Fusión/administración & dosificación , Toxina Tetánica/genética , Proteína bcl-X/genéticaRESUMEN
BACKGROUND/AIMS: No validated delivery technique exists for accurate, reproducible delivery of biological therapies to discrete spinal cord targets. To address this unmet need, we have constructed a stabilized platform capable of supporting physiologic mapping, through microelectrode recording, and cellular or viral payload delivery to the ventral horn. METHODS: A porcine animal model (n = 7) has been chosen based upon the inherent morphologic similarities between the human and porcine spine. Animals underwent physiologic mapping and subsequent microinjection of a green-fluorescent-protein-labeled cell suspension. Sacrifice (t = 3 h) was performed immediately following behavioral assessment. RESULTS: Histologic analysis has supported our ability to achieve localization to the ipsilateral ventral horn in the spinal cord. Complications included death due to malignant hyperthermia (n = 1), hindlimb dysfunction attributable to epidural hematoma (n = 1), and hindlimb dysfunction attributable to cord penetration (n = 2). CONCLUSIONS: These results indicate an ability to achieve accurate targeting, but the elevated incidence of neurologic morbidity will require further studies with longer follow-ups that incorporate procedural and equipment modifications that will allow for a reduced number of cord penetrations and will account for observed cardiorespiratory-associated cord movement. These initial results reinforce the challenges of translating biological restorative therapies from small to large animal models and ultimately to humans.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Microinyecciones/métodos , Médula Espinal/patología , Médula Espinal/fisiología , Técnicas Estereotáxicas/instrumentación , Animales , Células del Asta Anterior/patología , Células del Asta Anterior/fisiología , Conducta Animal/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos/instrumentación , Corteza Cerebral/patología , Potenciales Evocados Motores/fisiología , Femenino , Microelectrodos , Microinyecciones/efectos adversos , Microinyecciones/instrumentación , Modelos Animales , Ratas , Reproducibilidad de los Resultados , Técnicas Estereotáxicas/efectos adversos , PorcinosRESUMEN
Clostridial light chain (LC) inhibits synaptic transmission by digesting a vesicle-docking protein, synaptobrevin, without killing neurons. We here report the feasibility of creating a rat hemiparkinsonism model through LC gene expression in the substantia nigra (SN), inhibiting nigrostriatal transmission. 40 adult Sprague Dawley rats were divided into four groups for SN injections of PBS, 6-hydroxydopamine (6-OHDA), or adenoviral vectors for the expression of LC (AdLC), or GFP (AdGFP). Amphetamine and apomorphine induced rotations were assessed before and after SN injection, revealing significant rotational alterations at 8 or 10 days after injection in both AdLC and 6-OHDA but not PBS and AdGFP groups. Induced rotation recovered by one month in AdLC rats but persisted in 6-OHDA rats. Histological analysis of the SN revealed LC and GFP expression with corresponding synaptobrevin depletion in the LC, but not the GFP groups. Tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry (IHC) showed markedly decreased staining in ipsilateral SN and striatum in 6-OHDA but not AdLC or AdGFP rats. Similarly, compared with contralateral, ipsilateral striatal dopamine level only decreased in 6-OHDA but not AdLC, AdGFP, or PBS treated rats. Thus, LC expression induces nigral synaptobrevin depletion with resulting inhibition of nigrostriatal synaptic transmission. Unlike 6-OHDA, LC expression inhibits synaptic activity without killing neurons. This approach, therefore, represents a potentially reversible means of nigrostriatal pathway inhibition as a model for Parkinson's disease. Such a model might facilitate transient and controlled nigral inhibition for studying striatal recovery, dopaminergic re-innervation, and normalization of striatal receptors following the recovery of nigrostriatal transmission.
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Adenoviridae/genética , Clostridium/metabolismo , Metaloendopeptidasas/biosíntesis , Trastornos Parkinsonianos/fisiopatología , Transducción de Señal/fisiología , Sustancia Negra/fisiología , Toxina Tetánica/biosíntesis , Animales , Modelos Animales de Enfermedad , Dopamina/análisis , Masculino , Metaloendopeptidasas/genética , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Proteínas R-SNARE/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Conducta Estereotipada , Transmisión Sináptica , Toxina Tetánica/genéticaRESUMEN
OBJECTIVE: Delivery of biological therapeutics to motor and dorsal root ganglion neurons remains a major hurdle in the development of treatments for a variety of neurological processes, including peripheral nerve injury, pain, and motor neuron diseases. Because nerve cell bodies are important in initiating and controlling axonal regeneration, targeted delivery is an appealing strategy to deliver therapeutic proteins after peripheral nerve injury. METHODS: Tet1 is a 12-aa peptide, isolated through phage display that is selected for tetanus toxin C fragment-like binding properties. In this study, we surveyed its uptake and retrograde transport using compartmented cultures and sciatic nerve injections. We then characterized the time course of this delivery. Finally, to confirm the retrograde transport involvement, a colchicine pretreatment was performed. We also performed competitive binding studies between Tet1 and a recombinant tetanus toxin C fragment using recombinant tetanus toxin C fragment enzyme-linked immunosorbent assay. RESULTS: We were able to demonstrate efficient uptake and retrograde axonal transport of the Tet1 peptide in vitro and in vivo. Intraneural colchicine pretreatment partially blocked fluorescence detection in the spinal cord, revealing a retrograde axonal transport mechanism. Finally, a competitive enzyme-linked immunosorbent assay experiment revealed Tet1-specific binding to the recombinant tetanus toxin C fragment axon terminal trisialogangliosides receptor. CONCLUSION: These properties of Tet1 can be applied to the development of therapeutic viral vectors and fusion proteins for neuronal targeting and enhanced spinal cord delivery in the treatment of nerve regeneration, neuroprotection, analgesia, and spasticity. Small peptides can be easily fused to larger proteins without significantly modifying their function and can be used to alter the binding and uptake properties of these proteins.
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Axones/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Sistema Nervioso Periférico/lesiones , Sistema Nervioso Periférico/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Transporte Axonal/efectos de los fármacos , Transporte Axonal/fisiología , Axones/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/metabolismo , Sistema Nervioso Periférico/efectos de los fármacos , Preparaciones Farmacéuticas/administración & dosificación , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Toxina Tetánica/administración & dosificación , Toxina Tetánica/metabolismoRESUMEN
Spasticity is a condition resulting from excess motor neuron excitation, leading to involuntary muscle contraction in response to increased velocity of movement, for which there is currently no cure. Existing symptomatic therapies face a variety of limitations. The extent of relief that can be delivered by ablative techniques such as rhizotomy is limited by the potential for sensory denervation. Pharmacological approaches, including intrathecal baclofen, can be undermined by tolerance. One potential new approach to the treatment of spasticity is the control of neuromuscular overactivity through the delivery of genes capable of inducing synaptic inhibition. A variety of experiments in cell culture and animal models have demonstrated the ability of neural gene transfer to inhibit neuronal activity and suppress transmission. Similarly, enthusiasm for the application of gene therapy to neurodegenerative diseases of motor neurons has led to the development of a variety of strategies for motor neuron gene delivery. In this review, we discuss the limitations of existing spasticity therapies, the feasibility of motor neuron inhibition as a gene-based treatment for spasticity, potential inhibitory transgene candidates, strategies for control of transgene expression, and applicable motor neuron gene targeting strategies. Finally, we discuss future directions and the potential for gene-based motor neuron inhibition in therapeutic clinical trials to serve as an effective treatment modality for spasticity, either in conjunction with or as a replacement for presently available therapies.
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Marcación de Gen/métodos , Terapia Genética/métodos , Neuronas Motoras/fisiología , Espasticidad Muscular/terapia , Transmisión Sináptica/fisiología , Humanos , Neuronas Motoras/efectos de los fármacos , Espasticidad Muscular/etiología , Espasticidad Muscular/fisiopatología , Transmisión Sináptica/efectos de los fármacos , Toxina Tetánica/farmacologíaRESUMEN
Restraining excitatory neurotransmission within a seizure focus provides a nondestructive treatment strategy for intractable neocortical epilepsy. Clostridial toxin light chain (LC) inhibits synaptic transmission by digesting a critical vesicle-docking protein, synaptobrevin, without directly altering neuronal health. This study tests the treatment efficacy of adenoviral vector delivered LC (AdLC) on a model of seizures in rats induced by motor cortex penicillin (PCN) injection. LC expression significantly reduced electroencephalogram (EEG) frequency, amplitude, duration, and latency compared to control groups injected with either an adenoviral vector bearing green fluorescent protein (AdGFP) or phosphate buffered solution (PBS). Correspondingly, LC gene expression improved behavioral manifestations including seizure severity and latency. There was no statistical difference in motor function before and after vector administration between treatment and control groups. Histological analysis revealed spatially discrete LC expression with corresponding synaptobrevin depletion in the cortex surrounding the injection site. Thus, vector-mediated LC gene delivery is capable of improving both the EEG and behavioral manifestations of PCN-induced focal neocortical seizures through synaptobrevin depletion.
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ADN Viral/genética , Penicilinas/toxicidad , Convulsiones/genética , Convulsiones/terapia , Animales , Conducta Animal , Peso Corporal/efectos de los fármacos , Electroencefalografía , Expresión Génica , Terapia Genética , Masculino , Corteza Motora/efectos de los fármacos , Penicilinas/administración & dosificación , Proteínas R-SNARE/metabolismo , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Transgenes/genéticaRESUMEN
Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. We designed a two-tiered biopanning strategy initially selecting for ganglioside binding and subsequently selecting for binding to PC12 cells. At the completion of biopanning, 54.8% of phage clones bore the identical peptide (Tet.C7C.1). Immunofluorescence confirmed selective binding of this clone to differentiated PC12 cells. Tet.C7C.1 was synthesized and fluorescein conjugated. The synthetic peptide binds neuronal cell lines (SH-SY5Y, NSC-34 and PC12 cells) and tissue (DRG and spinal cord). The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. The neuronal binding properties of this peptide may be applied in the development of neurotropic viral vectors.
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Neuronas/metabolismo , Biblioteca de Péptidos , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Células PC12 , Péptidos/química , Péptidos/genética , Unión Proteica , RatasRESUMEN
OBJECTIVE: The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of programmed cell death. In the current experiment, we examine the potential of adenoviral XIAP gene delivery to protect neurons of the peripheral nervous system using in vitro models of amyotrophic lateral sclerosis (ALS) and diabetic neuropathy. METHODS: XIAP complementary deoxyribonucleic acid was fused in frame with the green fluorescent protein sequence and cloned into a first generation adenoviral vector. The impact of XIAP gene expression on glutamate-induced apoptosis was measured in the neuronal SH-SY5Y cell line with immunohistochemistry for active caspase-3 and with cell density assays. Next, the effect of XIAP expressing neurons on the survival of uninfected neighboring neurons was measured. Finally, the impact of XIAP gene expression on glutamate-induced apoptosis was assessed in embryonic motor neuron and dorsal root ganglion cultures. RESULTS: XIAP gene expression reduced the percentage of active caspase-3 positive SH-SY5Y neurons and preserved cell density after glutamate exposure. In heterogeneously infected cultures, cells infected with XIAP were protected, but uninfected neighboring cells were not. In primary E15 models, inhibition of proapoptotic effects was demonstrated after glutamate insult in motor neurons and glucose insult in dorsal root ganglion cells. CONCLUSION: XIAP gene delivery through the neurosurgical delivery of viral vectors may provide a means for neuroprotection in ALS and diabetic neuropathy.
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Esclerosis Amiotrófica Lateral/patología , Neuropatías Diabéticas/patología , Técnicas de Transferencia de Gen , Fármacos Neuroprotectores , Sistema Nervioso Periférico/patología , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Esclerosis Amiotrófica Lateral/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , Recuento de Células , Línea Celular , Supervivencia Celular/efectos de los fármacos , Neuropatías Diabéticas/metabolismo , Estudios de Factibilidad , Ganglios Espinales/efectos de los fármacos , Expresión Génica , Ácido Glutámico/envenenamiento , Proteínas Fluorescentes Verdes/genética , Humanos , Neuronas Motoras/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Sistema Nervioso Periférico/efectos de los fármacos , Sistema Nervioso Periférico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/farmacologíaRESUMEN
BACKGROUND: Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Polyamine concentrations are elevated in colorectal cancer. Depletion of polyamine content in colorectal cancer by chemotherapy is related to tumor regression and impaired tumorigenicity. The current study evaluates the therapeutic effects of antisense ODC and AdoMetDC sequences on colorectal cancer in vitro and in vivo. METHODS: Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). The human colon cancer cell lines, HT-29 and Caco-2, were infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were performed in order to assess properties of tumor growth and invasiveness. Furthermore, the antitumor effects of Ad-ODC-AdoMetDCas were also evaluated in vivo in a nude mouse tumor model. RESULTS: Our study demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression inhibits tumor cell growth through a blockade of the polyamine synthesis pathway. This inhibitory effect cannot be reversed by the administration of putrescine. Tumor cells were arrested at the G1 phase of the cell cycle after gene transfer and had reduced invasiveness. The adenovirus also induced tumor regression in established tumors in nude mice. CONCLUSIONS: Our study suggests that Ad-ODC-AdoMetDCas has antitumor activity and therapeutic potential for the treatment of colorectal cancer.
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Adenosilmetionina Descarboxilasa/metabolismo , Neoplasias Colorrectales/genética , Vectores Genéticos/genética , Oligonucleótidos Antisentido/farmacología , Ornitina Descarboxilasa/metabolismo , Adenosilmetionina Descarboxilasa/química , Adenosilmetionina Descarboxilasa/genética , Animales , Animales Recién Nacidos , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Citomegalovirus/genética , Dependovirus/clasificación , Dependovirus/genética , Estudios de Evaluación como Asunto , Factor IX/genética , Femenino , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/química , Humanos , Técnicas In Vitro , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Luciferasas/genética , Mediciones Luminiscentes , Masculino , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Oligonucleótidos Antisentido/uso terapéutico , Ornitina Descarboxilasa/química , Ornitina Descarboxilasa/genética , Poliaminas/química , Poliaminas/metabolismo , Serotipificación , Factores Sexuales , Factores de Tiempo , Distribución Tisular , Transgenes , beta-Galactosidasa/metabolismoRESUMEN
AIM: To construct a recombinant adenovirus that can simultaneously express both antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase ( AdoMetDC ) and detect its inhibitory effect on the intracellular polyamine pool and colorectal cancer cell growth. METHODS: A 205-bp cDNA of AdoMetDC was reverse-inserted into recombinant pAdTrack-ODCas vectors and recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the packaging cell HEK293 after they were linearized by PacI. Green fluorescent protein expression was used to monitor the process of adenovirus packaging. The ODC and AdoMetDC protein levels were identified by western blotting, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. A viable cell count was used to determine the growth of HT-29 cells with or without exogenous polyamine. RESULTS: Sequencing confirmed that AdoMetDC cDNA was successfully ligated into the pAdTrack-ODCas vector. GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting demonstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas, and consequently 3 kinds of polyamine (putrescine, spermidine and spermine) were reduced to very low levels. HT-29 cell growth was significantly inhibited as compared with control conditions, and growth arrest was not reversed by exogenous putrescine. CONCLUSION: The successfully constructed recombinant adenovirus, Ad-ODC-AdoMetDCas, blocked polyamine synthesis and has therapeutic potential for treating colorectal cancer in vitro.
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Adenosilmetionina Descarboxilasa/metabolismo , Células HT29/patología , Oligodesoxirribonucleótidos Antisentido , Ornitina Descarboxilasa/metabolismo , Poliaminas/metabolismo , Adenosilmetionina Descarboxilasa/genética , Adenoviridae/genética , Línea Celular , Proliferación Celular , Embrión de Mamíferos , Vectores Genéticos , Células HT29/metabolismo , Humanos , Riñón/citología , Ornitina Descarboxilasa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Recent work implicates excitotoxicity-induced apoptosis as the mechanism triggering motor neuron death in amyotrophic lateral sclerosis (ALS). Our laboratory has previously utilized glutamate excitotoxicity in vitro to study this process. The present experiment tests whether overexpression of the gene for Bcl-xL can inhibit excitotoxicity in this model system. To track Bcl-xL expression, the gene for green fluorescent protein (GFP) was inserted in-frame, upstream of the Bcl-xL gene. The GFP-Bcl-xL gene was then cloned into an adeno-associated viral (AAV2) vector. GFP expression in both SH-SY5Y and embryonic day 15 (E15) motor neurons (MNs) peaked 48 hours after infection. Bcl-xL expression in SH-SY5Y cells significantly reduced terminal deoxy-UTP nick-end labeling (TUNEL)-positive cells and maintained cell density after glutamate exposure. Similarly, Bcl-xL expression inhibited the development of TUNEL staining in E15 MNs and supported cell density after glutamate exposure. These findings suggest that AAV-mediated expression of genes for antiapoptotic proteins may provide a means for ALS gene therapy.