RESUMEN
Our letter to the editor addresses important questions regarding the role of the blood-spinal cord barrier in amyotrophic lateral sclerosis. The novel finding of barrier dysfunction in ALS has implications for disease pathogenesis. This discussion should prove of widespread interest to researchers and may help in formulating various new therapeutic strategies to protect barrier function and thus extend functionality and lifespan in ALS patients.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Barrera Hematoencefálica/fisiopatología , Esclerosis Amiotrófica Lateral/genética , HumanosRESUMEN
BACKGROUND/OBJECTIVE: We previously reported that chronic hyperglycemia, but not hypoglycemia, was associated with the reduction of neuronal size in the rat brain. We hypothesized that hyperglycemia-induced changes in neuronal structure would have negative consequences, such as impaired learning and memory. We therefore assessed the effects of hyperglycemia and hypoglycemia on neuronal dendritic structure and cognitive functioning in young rats. DESIGN/METHODS: Experimental manipulations were conducted on male Wistar rats for 8 wk, beginning at 4 wk of age. At the completion of the treatments, all rats were trained in the radial-arm water maze, a spatial (hippocampus-dependent) learning and memory task. Three groups of rats were tested: an untreated control group, a streptozotocin-induced diabetic (STZ-D) group, and an intermittent hypoglycemic group. Following behavioral training, the brains of all animals were examined with histologic and biochemical measurements. RESULTS: Peripheral hyperglycemia was associated with significant increases in brain sorbitol (7.5 +/- 1.6 vs. 5.84 +/- 1.0 microM/mg) and inositol (9.6 +/- 1.4 vs. 7.1 +/- 1.1 microM/mg) and reduced taurine (0.65 +/- 0.1 vs. 1.3 +/- 0.1 mg/mg). Histologic evaluation revealed neurons with reduced dendritic branching and spine density in STZ-D rats but not in control or hypoglycemic animals. In addition, the STZ-D group exhibited impaired performance on the water maze memory test. CONCLUSIONS: Hyperglycemia, but not hypoglycemia, was associated with adverse effects on the brain polyol pathway activity, neuronal structural changes, and impaired long-term spatial memory. This finding suggests that the hyperglycemic component of diabetes mellitus has a greater adverse effect on brain functioning than does intermittent hypoglycemia.
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Dendritas/patología , Diabetes Mellitus Experimental/fisiopatología , Hiperglucemia/complicaciones , Trastornos de la Memoria/etiología , Neuronas/patología , Animales , Química Encefálica/fisiología , Corteza Cerebral/metabolismo , Espinas Dendríticas/patología , Espinas Dendríticas/fisiología , Hipocampo/metabolismo , Hiperglucemia/fisiopatología , Hipoglucemia/complicaciones , Hipoglucemia/fisiopatología , Inositol/metabolismo , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Ratas , Ratas Wistar , Sorbitol/metabolismo , Conducta Espacial/fisiología , Taurina/metabolismoRESUMEN
Stem cell transplantation to replace damaged tissue or correct metabolic disease holds the promise of helping a myriad of human afflictions. Although a great deal of attention has focused on pluripotent stem cells derived from embryos, adult stem cells have been described in a variety of tissues, and they likely will prove to be as beneficial as embryonic stem cells in cell replacement therapy and control of inbred errors of metabolism. We describe methods by which stem cells can be introduced into the nervous system, although the techniques are applicable to any portion of the body to be targeted or any cell that may be used for cell therapy. The first and most straight-forward method is introduction of stem cells directly into the brain parenchyma. The second, which in our hands has proven to be superior in some instances, is introduction of the stem cells into the circulatory system.
Asunto(s)
Trasplante de Células Madre/métodos , Células Madre/metabolismo , Animales , Ratones , Cuidados Posoperatorios , RatasRESUMEN
The 796RMB cell line is a multipotent stem cell line isolated from human fetal midbrain tissues, a region from which dopamine neurons of the substantia nigra develop. It would be useful to increase the dopaminergic characteristics of this cell line to enhance its usefulness as a cell therapy for Parkinson's disease utilizing transplantation protocols. Sertoli cells and its conditioned media isolated from the testis have been previously shown to enhance tyrosine hydroxylase expression in ventral mesencephalon neurons both in vitro and in vivo. Therefore, the present preliminary study investigated the ability of Sertoli cell pre-conditioned medium to enhance differentiation of the 796MB cell line toward the domaminergic phenotype. Results showed that secretory products derived from Sertoli cell conditioned medium increased cell proliferation and enhanced dopaminergic neuronal differentiation of the 796RMB cell line. These findings may lead to alternative therapeutic cell transplantation protocols for the treatment of Parkinson's disease.
Asunto(s)
Medios de Cultivo Condicionados/farmacología , Mesencéfalo/citología , Neuronas/fisiología , Células de Sertoli/química , Células Madre/efectos de los fármacos , Animales , Animales Recién Nacidos , Recuento de Células/métodos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dopamina/metabolismo , Feto , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Ratas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Numerous data support passage of maternal cells into the fetus during pregnancy in both human and animal models. However, functional benefits of maternal microchimerism in utero are unknown. The current study attempted to take advantage of this route for prenatal delivery of alpha-N-acetylglucosaminidase (Naglu) enzyme into the enzyme-deficient mouse model of Sanfilippo syndrome type B (MPS III B). Enzymatically sufficient mononuclear cells from human umbilical cord blood (MNC hUCB) were intravenously administered into heterozygote females modeling MPS III B on the 5th day of pregnancy during blastocyst implantation. The major findings were 1) administered MNC hUCB cells transmigrated and diffused into the embryos (E12.5); 2) some transmigrated cells expressed CD34 and CD117 antigens; 3) transmigrated cells were found in both the maternal and embryonic parts of placentas; 4) transmigrated cells corrected Naglu enzyme activity in all embryos; 5) administered MNC hUCB cells were extensively distributed in the organs and the blood of heterozygote mothers at one week after transplantation. Results indicate that prenatal delivery of Naglu enzyme by MNC hUCB cell administration into mothers of enzyme-deficient embryos is possible and may present a significant opportunity for new biotechnologies to treat many inherited disorders.
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Acetilglucosaminidasa/genética , Trasplante de Células Madre de Sangre del Cordón Umbilical , Terapias Fetales , Leucocitos Mononucleares/trasplante , Intercambio Materno-Fetal , Mucopolisacaridosis III/terapia , Acetilglucosaminidasa/deficiencia , Animales , Antígenos CD34/análisis , Linaje de la Célula , Movimiento Celular , Femenino , Terapias Fetales/métodos , Humanos , Leucocitos Mononucleares/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Mucopolisacaridosis III/embriología , Mucopolisacaridosis III/enzimología , Mucopolisacaridosis III/genética , Placenta/ultraestructura , Embarazo , Proteínas Proto-Oncogénicas c-kit/análisis , Trasplante HeterólogoRESUMEN
The purpose of this study was to determine the ultrastructure of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in G93A SOD1 mice modeling ALS at different stages of disease. Electron microscope examination of brainstem, cervical and lumbar spinal cords was performed in ALS mice at early and late stages of disease. Our results show disorganized mitochondrial cristae and degenerating mitochondria in endothelial cells and neuropil, swollen astrocyte foot processes, swollen and degenerating capillary endothelial cells, astrocytes and motor neurons and extensive extracellular edema. In spite of progressive extracellular edema in neural tissue, capillary endothelial cell tight junctions appeared to remain intact in early and late symptomatic animals. Results show that disruption of BBB and BSCB was evident in areas of motor neuron degeneration in G93A mice at both early and late stages of disease. Capillary rupture was observed in brainstem in early symptomatic G93A mice. Capillary ultrastructure revealed that endothelial cell membrane and/or basement membrane damage occurred, followed by vascular leakage.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Barrera Hematoencefálica/patología , Edema Encefálico/patología , Capilares/patología , Células Endoteliales/patología , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Astrocitos/patología , Astrocitos/ultraestructura , Membrana Basal/patología , Membrana Basal/ultraestructura , Barrera Hematoencefálica/fisiopatología , Barrera Hematoencefálica/ultraestructura , Encéfalo/irrigación sanguínea , Encéfalo/patología , Encéfalo/ultraestructura , Edema Encefálico/etiología , Edema Encefálico/fisiopatología , Capilares/fisiopatología , Capilares/ultraestructura , Modelos Animales de Enfermedad , Células Endoteliales/ultraestructura , Espacio Extracelular/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias/patología , Mitocondrias/ultraestructura , Neuronas Motoras/patología , Neuronas Motoras/ultraestructura , Mutación/genética , Médula Espinal/irrigación sanguínea , Médula Espinal/patología , Médula Espinal/ultraestructura , Superóxido Dismutasa-1 , Uniones Estrechas/patología , Uniones Estrechas/ultraestructuraRESUMEN
Our previous studies demonstrate enhanced neural protective effects of cord blood (CB) cells in comparison to stem cells from adult marrow. To determine further whether mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) possess optimal characteristics for neural therapy, we isolated populations of plastic-adherent CB MSCs. These cells generated CD34-, CD45-, CD11b-, CD3-, CD19- cells in culture and failed to produce CFU-M, CFU-GEMM, or CFU-GM hematopoietic colonies in methylcellulose. However, cultured CB MSCs possessed a remarkable ability to support proliferation as well as differentiation of hematopoietic cells in vitro. In addition, supernatants from cultured CB MSCs promoted survival of NT2 N neural cells and peripheral blood mononuclear cells (MNCs) cultured under conditions designed to induce cell stress and limit protein synthesis. After incubation in neural differentiation medium, CB MSCs expressed the neural cell-surface antigen A2B5, the neurofilament polypeptide NF200, the oligodendrocyte precursor marker 04, intermediate filament proteins characteristic of neural differentiation (nestin and vimentin), as well as the astrocyte marker glial fibrillary acidic protein (GFAP) and the neural progenitor marker TUJ-1. We examined the immunomodulatory effects of the CB MSCs after co-culture with murine splenocytes. Whereas spleen cells from normal C57Bl/6 mice exhibited a prominent immunoglobulin M (IgM) response after immunization with the T cell-dependent antigen sheep red blood cells, this response was significantly decreased after incubation with CB MSCs. These data indicate that CB MSCs possess multiple utilities that may contribute to their therapeutic potency in the treatment of neurological disorders.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Sangre Fetal/citología , Células Madre Mesenquimatosas/citología , Enfermedades del Sistema Nervioso/terapia , Animales , Biomarcadores , Diferenciación Celular , Separación Celular , Supervivencia Celular , Células Cultivadas , Eritrocitos/inmunología , Sustancias de Crecimiento/biosíntesis , Hematopoyesis/fisiología , Humanos , Inmunoglobulina M/inmunología , Enfermedades del Sistema Nervioso/patología , Neuroglía/citología , Neuronas/citología , OvinosRESUMEN
Numerous reports elucidate that tissue-specific stem cells are phenotypically plastic and their differentiation pathways are not strictly delineated. Although the identity of all the epigenetic factors which may trigger stem cells to make a lineage selection are still unknown, the plasticity of adult stem cells opens new approaches for their application in the treatment of various disorders. There is increasing researcher interest in hematopoietic stem cells for treatment of not only blood-related diseases but also various unrelated disorders including neurodegenerative diseases. Human umbilical cord blood (hUCB) cells, due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, including neural cells, may be useful as an alternative cell source for cell-based therapies requiring either the replacement of individual cell types and/or substitution of missing substances. Here we focus on recent findings showing the robustness of adult stem cells derived from hUCB and their potential as a source of transplant cells for the treatment of diseased or injured brains and spinal cords. Depending upon the pathological microenvironment in which the hUCB cells are introduced, neuroprotective and/or trophic effects of these cells, from release of various growth or anti-inflammatory factors to moderation of immune-inflammatory effectors, may be more likely than neural replacement. These protective effects may prove essential to maintaining restored tissue integrity over the course of various diseases or injuries.
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Lesiones Encefálicas/terapia , Tratamiento Basado en Trasplante de Células y Tejidos , Traumatismos de la Médula Espinal/terapia , Esclerosis Amiotrófica Lateral/terapia , Animales , Sangre Fetal/citología , Humanos , Accidente Cerebrovascular/terapiaRESUMEN
Children with diabetes onset before 5 years of age have reduced neurocognitive function. This problem has been attributed to hypoglycemia, a complication of insulin therapy. The eye, kidney, and nerve complications of diabetes (hyperglycemia) have been reduced by intensified insulin therapy which is associated with a 3-fold increase in severe hypoglycemia and therefore is not recommended for children less than 13 years of age. Since hyperglycemia is much more common than intermittent hypoglycemia during early childhood diabetes, it is important to determine if hyperglycemia affects brain growth and development. Rats were exposed to 4 weeks of either continuous hyperglycemia (diabetes) or intermittent (3 h, 3 times/week) hypoglycemia from 4 to 8 weeks of age. The brains of these animals were compared to those of similarly aged normal control animals. The cell number was increased, and the cell size reduced in the cortex of diabetic animals as assessed by DNA/wet weight of brain and protein/DNA content. Reduced amounts of protein, fatty acids, and cholesterol/microgram DNA also indicate smaller cells with reduced myelin content in the cortex of the diabetic animals. Histologic evaluation of these brains confirmed the biochemical findings. These observations require further confirmation and evaluation but indicate that continuous hyperglycemia may be more damaging than intermittent hypoglycemia to the developing brain. This is an important consideration for the management of diabetes mellitus in young children.
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Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/patología , Hiperglucemia/etiología , Animales , Astrocitos/patología , Recuento de Células/métodos , Corteza Cerebral/patología , Diabetes Mellitus Experimental/complicaciones , Glucosa/metabolismo , Hipocampo/patología , Inmunohistoquímica/métodos , Masculino , Factores de Crecimiento Nervioso/metabolismo , Neuronas/patología , Neuronas/fisiología , Ratas , Ratas Wistar , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismo , Factores de TiempoRESUMEN
Sertoli cells (SCs) are testis-derived cells that secrete trophic factors important for the development of germ cells. Both porcine and rat SCs have been used as graft facilitators - neonatal porcine SCs to support islets in diabetes and 15-day-old rat SCs to enhance dopaminergic neuron transplants in Parkinson's disease models. However, there has never been a study examining the optimal SCs preparation to enhance tyrosine hydroxylase expression in the ventral mesencephalon (VM) neuron. The aim of this study was to compare the ability of both rat and porcine SCs to enhance tyrosine hydroxylase expression (TH) and neuronal survival at the same postnatal developmental ages. The SCs were isolated from 1-, 9-, or 15-day-old rat, or neonate (2-5 days), 2-month, or 4-month-old pig, and co-cultured with VM tissue from 13.5-day-old embryos. Our results showed that VM neurons co-cultured with SCs dispersed over the culture plate and had extensive neuritic outgrowth, while VM neurons cultured alone tended to cluster together forming a mass of cells with limited neurite outgrowth. TH expression was significantly increased when VM neurons were co-cultured with 15-day rat SCs or 2-month pig SCs but not when the cells were co-cultured with other ages of SCs. This suggests that secretion of trophic factors by SCs varies according to the developmental age, and it is critical for the success of graft facilitation that SCs from the appropriate age and species be used.
Asunto(s)
Mesencéfalo/citología , Mesencéfalo/enzimología , Neuronas/enzimología , Células de Sertoli/fisiología , Tirosina 3-Monooxigenasa/biosíntesis , Tirosina 3-Monooxigenasa/genética , Animales , Animales Recién Nacidos , División Celular/fisiología , Separación Celular , Supervivencia Celular , Células Cultivadas , Técnicas de Cocultivo , Inmunohistoquímica , Masculino , Mesencéfalo/embriología , Neuritas/fisiología , Neuronas/ultraestructura , Ratas , Células de Sertoli/ultraestructura , PorcinosRESUMEN
Human umbilical cord blood (HUCB) is now considered a valuable source for stem cell-based therapies. HUCB cells are enriched for stem cells that have the potential to initiate and maintain tissue repair. This potential is especially attractive in neural diseases for which no current cure is available. Furthermore, HUCB cells are easily available and less immunogenic compared to other sources for stem cell therapy such as bone marrow. Accordingly, the number of cord blood transplants has doubled in the last year alone, especially in the pediatric population. The therapeutic potential of HUCB cells may be attributed to inherent ability of stem cell populations to replace damaged tissues. Alternatively, various cell types within the graft may promote neural repair by delivering neural protection and secretion of neurotrophic factors. In this review, we evaluate the preclinical studies in which HUCB was applied for treatment of neurodegenerative diseases and for traumatic and ischemic brain damage. We discuss how transplantation of HUCB cells affects these disorders and we present recent clinical studies with promising outcome.
Asunto(s)
Lesiones Encefálicas/terapia , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal/citología , Enfermedades Neurodegenerativas/terapia , Células Madre/metabolismo , Animales , Lesiones Encefálicas/patología , Isquemia Encefálica/terapia , Diferenciación Celular , Ensayos Clínicos como Asunto , Humanos , Enfermedades Neurodegenerativas/patología , Células Madre/citología , Trasplante HeterólogoRESUMEN
Interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) are major inflammatory cytokines produced after spinal cord injury (SCI). This study sought to evaluate the effects of methylprednisolone (MP) on IL-1beta and IL-6 protein in spinal cord tissue following SCI. Halothane-anesthetized, female Sprague-Dawley rats weighing (280-320 g) underwent laminectomy at T7-T8. No lesions were produced in animals in the saline control and MP control groups. SCI was induced by temporary placement of an aneurysm clip at T7-T8, with a closing pressure of 55 g at the spinal level of T7-T8, resulting in spinal cord compression for one minute. Animals with SCI were treated with MP (30 mg/kg sc) or an equal volume of saline. IL-1beta and IL-6 spinal cord protein were measured by enzyme-linked immunosorbent assays (ELISA). Data were summarized as mean +/- SD and compared by two-way analysis of variance (ANOVA). IL-1beta and IL-6 levels were elevated in the SCI + Saline animals (P < 0.01) compared with saline control, MP control, and SCI + MP-treated animals. The rise in IL-1beta and IL-6 levels after SCI was blunted after administration of MP, suggesting an interaction between glucocorticosteroids and the cytokine cascade after spinal cord trauma. Further evaluation of the effects of MP on the cytokine cascade may be important in assessing whether or not the anti-inflammatory effects of glucocorticosteroids confer neuroprotection after SCI.
Asunto(s)
Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Metilprednisolona/farmacología , Fármacos Neuroprotectores/farmacología , Compresión de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Animales , Femenino , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacosRESUMEN
Recently, our laboratory began to characterize the mononuclear cells from human umbilical cord blood (HUCB) both in vitro and in vivo. These cryopreserved human cells are available in unlimited quantities and it is believed that they may represent a source of cells with possible therapeutic and practical value. Our previous molecular and immunocytochemical studies on cultured HUCB cells revealed their ability to respond to nerve growth factor (NGF) by increased expression of neural markers typical for nervous system-derived stem cells. In addition, the DNA microarray detected downregulation of several genes associated with development of blood cell lines. To further explore the survival and phenotypic properties of HUCB cells we transplanted them into the developing rat brain, which is known to provide a conducive environment for development of neural phenotypes. Prior to transplantation, HUCB cells were either cultured with DMEM and fetal bovine serum or were exposed to retinoic acid (RA) and nerve growth factor (NGF). Neonatal pups (1 day old) received unilateral injection of cell suspension into the anterior part of subventricular zone. One month after transplantation animals were perfused, their brains cryosectioned, and immunocytochemistry was performed for identification of neural phenotypes. Our results clearly demonstrated that approximately 20% of transplanted HUCB survived (without immunosuppression) within the neonatal brain. Additionally, double-labeling with cell-type-specific markers revealed that some HUCB-derived cells (recognized by anti-human nuclei labeling) were immunopositive for glial fibrillary acidic protein (GFAP) and few donor cells expressed the neuronal marker TuJ1 (class III beta-tubulin). These findings suggest that at least some of the transplanted HUCB cells differentiated into cells with distinct glial or neuronal phenotypes after being exposed to instructive signals from the developing brain.
Asunto(s)
Diferenciación Celular , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal/citología , Células Madre Multipotentes/trasplante , Neuronas/metabolismo , Prosencéfalo/cirugía , Animales , Animales Recién Nacidos , Biomarcadores , Técnicas de Cultivo de Célula/métodos , Linaje de la Célula , Supervivencia Celular , Células Cultivadas , Trasplante de Tejido Fetal , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/fisiología , Factor de Crecimiento Nervioso/farmacología , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/química , Fenotipo , Prosencéfalo/citología , Ratas , Ratas Sprague-Dawley , Trasplante de Células Madre , Tubulina (Proteína)/metabolismoRESUMEN
Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1-4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT--specific for NT2 cells) and cholera toxin subunit B (CTb--specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD.
Asunto(s)
Neuronas/citología , Células de Sertoli/citología , Ingeniería de Tejidos/métodos , Simulación de Ingravidez , Animales , Agregación Celular/fisiología , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Línea Celular Transformada , Técnicas de Cocultivo/métodos , Técnica del Anticuerpo Fluorescente , Humanos , Uniones Intercelulares/fisiología , Uniones Intercelulares/ultraestructura , Masculino , Microscopía Electrónica de Transmisión , Proteínas Mitocondriales/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuronas/fisiología , Neuronas/ultraestructura , Ratas , Ratas Sprague-Dawley , Células de Sertoli/fisiología , Células de Sertoli/ultraestructuraRESUMEN
Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation.
Asunto(s)
Reactores Biológicos , Diferenciación Celular/fisiología , Neuronas/citología , Células de Sertoli/citología , Células Madre/citología , Animales , Animales Recién Nacidos , Western Blotting/métodos , Células Cultivadas , Técnicas de Cocultivo/métodos , Humanos , Inmunohistoquímica/métodos , Indoles/metabolismo , Masculino , Ratas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
OBJECT: Human neuroteratocarcinoma (hNT)-derived neurons are differentiated postmitotic neurons derived from a human teratocarcinoma cell line following treatment with retinoic acid. In preclinical transplantation studies investigators have demonstrated both their safety as a source of neurons for transplantation and efficacy in treating stroke-related behavioral deficits. The objective of this study was to examine whether hNT neurons transplanted in an area of complete spinal cord contusion would improve electrophysiological measures of spinal cord function. METHODS: Complete spinal cord contusion injury, defined as the complete loss of motor evoked potentials (MEPs), was produced in 30 rats at T-8. Ten rats with contused spinal cords underwent transplantation with hNT neurons within the site of contusion immediately after injury (immediate transplant group). Ten rats underwent hNT neuron transplantation following a 2-week evaluation for loss of MEPs (delayed transplant group). Ten other rats with contusion injury served as a spinal cord injury control group, and 10 rats underwent only a T-8 laminectomy and served as non-injured controls. All rats survived 8 weeks after transplantation. In the delayed transplant group significant functional recovery was observed, as demonstrated by return of MEPs and a modest improvement of motor function. Immunohistochemical analysis showed the survival, integration, and long fiber outgrowth of the grafted hNT neurons. CONCLUSIONS: These findings suggest that the transplantation of the hNT neurons may be an effective means of reestablishing electrical connectivity of the injured spinal cord.
Asunto(s)
Contusiones/fisiopatología , Contusiones/cirugía , Neuronas/trasplante , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/cirugía , Teratocarcinoma/patología , Animales , Electrofisiología , Potenciales Evocados Motores , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Médula Espinal/cirugíaRESUMEN
Problems with immunosuppression and graft survival limit clinical applications of neurotransplantation protocols for neurodegenerative disease. Sertoli cells, testes-derived cells with immunosuppressive and trophic properties, may serve as an alternative cell source for transplantation. Sertoli cells were transplanted into the striatum of rats following two injections of 3-nitropropionic acid (3-NP) to determine whether they could ameliorate abnormalities in a model of early stage Huntington's disease. 3-NP-induced locomotor hyperactivity was significantly reduced in rats receiving Sertoli transplants compared to controls, with some behaviors returning to baseline. Sertoli cells survived in the striatum without systemic immunosuppression and some formed tubule-like structures. These results show that Sertoli transplants are able to ameliorate locomotor abnormalities in a 3-NP model of early HD. Thus, Sertoli cells should be further evaluated as a possible treatment strategy for the early stages of Huntington's disease.
Asunto(s)
Enfermedad de Huntington/inducido químicamente , Enfermedad de Huntington/terapia , Propionatos/toxicidad , Células de Sertoli/trasplante , Animales , Conducta Animal/efectos de los fármacos , Ventrículos Cerebrales/patología , Enfermedad de Huntington/psicología , Inmunohistoquímica , Masculino , Microscopía Fluorescente , Actividad Motora/efectos de los fármacos , Neostriado/citología , Neostriado/fisiología , Nitrocompuestos , Ratas , Ratas Sprague-Dawley , Conducta Estereotipada/efectos de los fármacos , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
An increasing number of in vitro and in vivo studies suggest that anesthesia and surgery could be risk factors for later cognitive impairment in the young and aged brain. General anesthesia has been shown to impair spatial memory in rats and this performance is dependent on hippocampal function and postnatal hippocampal neurogenesis. Anesthetic induced alteration of one or more stages of postnatal hippocampal neurogenesis may in part explain this cognitive impairment following anesthesia. Three different populations of proliferating cells in the dentate gyrus (DG) were labeled with different thymidine analogs (EdU, IdU, and CldU) at 4, 8, and 21 days, respectively, in young (3-month-old) and aged (20-month-old) rats prior to a 3h exposure to isoflurane, control, propofol, or 10% intralipid. 24h following general anesthesia, brains were collected for analysis. The number of cells co-localized with neuronal differentiation and maturation labels with each of the thymidine analogs was quantified. In addition, new cell proliferation 24hr following anesthesia was assessed with anti-Ki67. The effect of anesthesia on astrocytes was also assessed with anti-S100ß. Isoflurane or propofol did not affect new cell proliferation, as assessed by Ki67, in the DG of young or aged rats. However, propofol significantly decreased the number of differentiating neurons and increased the number of astrocytes in the DG of young, but not aged, rats. Isoflurane significantly decreased the number of maturing neurons and increased the number of astrocytes in the DG of aged, but not young, rats. Isoflurane and propofol anesthesia altered postnatal hippocampal neurogenesis in an age and agent dependent matter.
Asunto(s)
Anestésicos por Inhalación/toxicidad , Hipocampo/efectos de los fármacos , Isoflurano/toxicidad , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Propofol/toxicidad , Envejecimiento/fisiología , Anestesia General , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Giro Dentado/efectos de los fármacos , Hipocampo/citología , Masculino , Memoria/efectos de los fármacos , Memoria/fisiología , Neurogénesis/fisiología , Neuronas/citología , Ratas , Ratas Endogámicas F344RESUMEN
There is a growing body of evidence showing that a statistically significant number of people experience long-term changes in cognition after anesthesia. We hypothesize that this cognitive impairment may result from an anesthetic-induced alteration of postnatal hippocampal cell proliferation. To test this hypothesis, we investigated the effects of isoflurane and propofol on new cell proliferation and cognition of young (4 month-old) and aged (21 month-old). All rats were injected intraperitoneally (IP) with 50 mg/kg of 5-bromo-2-deoxyuridine (BrdU) immediately after anesthesia. A novel appetitive olfactory learning test was used to assess learning and memory two days after anesthesia. One week after anesthesia, rats were euthanized and the brains analyzed for new cell proliferation in the dentate gyrus, and proliferation and migration of newly formed cells in the subventricular zone to the olfactory bulb. We found that exposure to either isoflurane (p=0.017) or propofol (p=0.006) decreased hippocampal cell proliferation in young, but not in aged rats. This anesthetic-induced decrease was specific to new cell proliferation in the hippocampus, as new cell proliferation and migration to the olfactory bulb was unaffected. Isoflurane anesthesia produced learning impairment in aged rats (p=0.044), but not in young rats. Conversely, propofol anesthesia resulted in learning impairment in young (p=0.01), but not in aged rats. These results indicate that isoflurane and propofol anesthesia affect postnatal hippocampal cell proliferation and learning in an age dependent manner.
Asunto(s)
Envejecimiento/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Giro Dentado/efectos de los fármacos , Isoflurano/administración & dosificación , Aprendizaje/efectos de los fármacos , Propofol/administración & dosificación , Envejecimiento/fisiología , Anestesia por Inhalación/métodos , Anestesia Intravenosa/métodos , Animales , Giro Dentado/citología , Giro Dentado/fisiología , Aprendizaje/fisiología , Masculino , Distribución Aleatoria , Ratas , Ratas Endogámicas F344RESUMEN
Vascular pathology, including blood-brain/spinal cord barrier (BBB/BSCB) alterations, has recently been recognized as a key factor possibly aggravating motor neuron damage, identifying a neurovascular disease signature for ALS. However, BBB/BSCB competence in sporadic ALS (SALS) is still undetermined. In this study, BBB/BSCB integrity in postmortem gray and white matter of medulla and spinal cord tissue from SALS patients and controls was investigated. Major findings include (1) endothelial cell damage and pericyte degeneration, (2) severe intra- and extracellular edema, (3) reduced CD31 and CD105 expressions in endothelium, (4) significant accumulation of perivascular collagen IV, and fibrin deposits (5) significantly increased microvascular density in lumbar spinal cord, (6) IgG microvascular leakage, (7) reduced tight junction and adhesion protein expressions. Microvascular barrier abnormalities determined in gray and white matter of the medulla, cervical, and lumbar spinal cord of SALS patients are novel findings. Pervasive barrier damage discovered in ALS may have implications for disease pathogenesis and progression, as well as for uncovering novel therapeutic targets.