RESUMEN
BACKGROUND: Antibodies to human full-length myelin oligodendrocyte glycoprotein (MOG-IgG) as detected by new-generation cell-based assays have recently been described in patients presenting with acute demyelinating disease of the central nervous system, including patients previously diagnosed with multiple sclerosis (MS). However, only limited data are available on the relevance of MOG-IgG testing in patients with chronic progressive demyelinating disease. It is unclear if patients with primary progressive MS (PPMS) or secondary progressive MS (SPMS) should routinely be tested for MOG-IgG. OBJECTIVE: To evaluate the frequency of MOG-IgG among patients classified as having PPMS or SPMS based on current diagnostic criteria. METHODS: For this purpose, we retrospectively tested serum samples of 200 patients with PPMS or SPMS for MOG-IgG using cell-based assays. In addition, we performed a review of the entire English language literature on MOG-IgG published between 2011 and 2017. RESULTS: None of 139 PPMS and 61 SPMS patients tested was positive for MOG-IgG. Based on a review of the literature, we identified 35 further MOG-IgG tests in patients with PPMS and 55 in patients with SPMS; the only reportedly positive sample was positive just at threshold level and was tested in a non-IgG-specific assay. In total, a single borderline positive result was observed among 290 tests. CONCLUSION: Our data suggest that MOG-IgG is absent or extremely rare among patients with PPMS or SPMS. Routine screening of patients with typical PPMS/SPMS for MOG-IgG seems not to be justified.
Asunto(s)
Inmunoglobulina G/sangre , Esclerosis Múltiple Crónica Progresiva/inmunología , Esclerosis Múltiple Crónica Progresiva/metabolismo , Glicoproteína Mielina-Oligodendrócito/inmunología , Adolescente , Adulto , Anciano , Estudios de Cohortes , Bases de Datos Bibliográficas , Femenino , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Transfección , Adulto JovenRESUMEN
Systemic administration of human umbilical cord blood (HUCB) mononuclear cells (MNC) following middle cerebral artery occlusion (MCAO) in the rat reduces infarct size and, more importantly, restores motor function. The HUCB cell preparation is composed of immature T-cells, B-cells, monocytes and stem cells. In this study we examined whether the beneficial effects of HUCB injection were attributable to one of these cell types. Male Sprague Dawley rats underwent permanent MCAO followed 48 h later by intravenous administration of HUCB MNC preparations depleted of either CD14(+) monocytes, CD133(+) stem cells, CD2(+) T-cells or CD19(+) B cells. Motor function was measured prior to MCAO and 30 days post-stroke. When CD14(+) monocytes were depleted from the HUCB MNC, activity and motor asymmetry were similar to the MCAO only treated animals. Monocyte depletion prevented HUCB cell treatment from reducing infarct size while monocyte enrichment was sufficient to reduce infarct size. Administration of monocyte-depleted HUCB cells did not suppress Iba1 labeling of microglia in the infarcted area relative to treatment with the whole HUCB preparation. These data demonstrate that the HUCB monocytes provide the majority of the efficacy in reducing infarct volume and promoting functional recovery.
Asunto(s)
Sangre Fetal/trasplante , Infarto de la Arteria Cerebral Media/terapia , Monocitos/trasplante , Antígeno AC133 , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Linfocitos B/trasplante , Antígenos CD2/genética , Antígenos CD2/metabolismo , Sangre Fetal/citología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/metabolismo , Masculino , Monocitos/metabolismo , Péptidos/genética , Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Linfocitos T/metabolismo , Linfocitos T/trasplanteRESUMEN
There is currently a significant lack of therapeutic options for acute ischemic stroke, and no drug has been approved for treating patients at delayed time points (≥6h post-stroke). Afobazole, an anxiolytic currently used clinically in Russia, has been shown to reduce neuronal and glial cell injury in vitro following ischemia. Experiments using the permanent middle cerebral artery occlusion (MCAO) rat model were carried out to determine if afobazole can reduce ischemic stroke damage in vivo and expand the therapeutic window for stroke treatment. Post-stroke (24h) application of afobazole (0.3-3mg/kg) significantly decreased infarct volume at 96h post-surgery, as determined by Fluoro-Jade and NeuN staining of brain sections. Moreover, afobazole helped preserve both the levels and normal histological distribution of myelin basic protein, indicating a reduction in white matter injury. A time-dependence study showed that either pre-treatment or treatment started 6 to 48h post-stroke with the drug yields improved outcomes at 96h. The decrease in infarct volume produced by afobazole was blocked by the application of either a σ-1 (BD 1063, 30mg/kg) or a σ-2 (SM-21, 1mg/kg) antagonist, indicating that both receptor subtypes are involved in the effects of afobazole. Treatment with afobazole starting at 24h post-stroke resulted in enhanced survival one month following surgery. Behavioral testing of animals 28-32days post-surgery using the elevated body swing and forelimb grip-strength tests revealed that treatment with afobazole starting 24h post-stroke significantly reduces behavioral deficits caused by ischemic stroke. The increase in survival and improved functional outcomes are accompanied by a reduction in infarct volume, as determined by thionin staining of brain sections. Taken together, our data support the use of afobazole as a post-stroke pharmacological agent to expand the current therapeutic window.
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Bencimidazoles/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Morfolinas/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Bencimidazoles/administración & dosificación , Isquemia Encefálica/patología , Fuerza de la Mano/fisiología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Masculino , Morfolinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores sigma/agonistas , Accidente Cerebrovascular/patología , Resultado del TratamientoRESUMEN
Previous reports have shown that human umbilical cord blood cells (HUCBCs) administered intravenously 48 hr following middle cerebral artery occlusion reduce infarct area and behavioral deficits of rodents. This cellular therapy is potently neuroprotective and antiinflammatory. This study investigates the effect of HUCBC treatment on white matter injury and oligodendrocyte survival in a rat model of ischemia. Intravenous infusion of 10(6) HUCBCs 48 hr poststroke reduced the amount of white matter damage in vivo as seen by quantification of myelin basic protein staining in tissue sections. To determine whether HUCBC treatment was protective via direct effects on oligodendrocytes, cultured oligodendrocytes were studied in an in vitro model of oxygen glucose deprivation. Active caspase 3 immunohistochemistry and the lactate dehydrogenase assay for cytotoxicity were used to determine that HUCBCs provide protection to oligodendrocytes in vitro. Based on these results, it is likely that HUCBC administration directly protects oligodendrocytes and white matter. This effect is likely to contribute to the increased behavioral recovery observed with HUCBC therapy.
Asunto(s)
Isquemia Encefálica/terapia , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal/trasplante , Oligodendroglía/patología , Animales , Caspasa 3/metabolismo , Muerte Celular/fisiología , Sangre Fetal/citología , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratas , Ratas Sprague-DawleyRESUMEN
An environmental assessment of long-chain alcohols (LCOH) has recently been conducted under the OECD SIDS High Production Volume (HPV) Program via the Global International Council of Chemical Associations (ICCA) Aliphatic Alcohols Consortium. LCOH are used primarily as intermediates, as a precursor to alcohol-based surfactants and as alcohol per se in a wide variety of consumer product applications. Global production volume is approximately 1.58 million metric tonnes. The OECD HPV assessment covers linear to slightly branched LCOH ranging from 6 to 22 alkyl carbons (C). LCOH biodegrade exceptionally rapidly in the environment (half-lives on the order of minutes); however, due to continuous use and distribution to wastewater treatment systems, partitioning properties, biodegradation of alcohol-based surfactants, and natural alcohol sources, LCOH are universally detected in wastewater effluents. An environmental risk assessment of LCOH is presented here by focusing on the most prevalent and toxic members of the linear alcohols, specifically, from C(12-15). The assessment includes environmental monitoring data for these chain lengths in final effluents of representative wastewater treatment plants and covers all uses of alcohol (i.e., the use of alcohol as a substance and as an intermediate for the manufacturing of alcohol-based surfactants). The 90th percentile effluent discharge concentration of 1.979microg/L (C(12)-C(15)) was determined for wastewater treatment plants in 7 countries. Chronic aquatic toxicity studies with Daphnia magna demonstrated that between C(13) and C(15) LCOH solubility became a factor and that the structure-activity relationship was characterized by a toxicity maximum between C(13) and C(14). Above C(14) the LCOH was less toxic and become un-testable due to insolubility. Risk quotients based on a toxic units (TU) approach were determined for various scenarios of exposure and effects extrapolation. The global average TU ranged from 0.048 to 0.467 depending on the scenario employed suggesting a low risk to the environment. The fact that environmental exposure calculations include large fractions of naturally derived alcohol from animal, plant, and microbially mediated biotransformations further supports a conclusion of low risk.
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Contaminantes Ambientales/toxicidad , Alcoholes Grasos/toxicidad , Animales , Biodegradación Ambiental , Canadá , Daphnia , Monitoreo del Ambiente , Contaminantes Ambientales/química , Europa (Continente) , Alcoholes Grasos/química , Relación Estructura-Actividad Cuantitativa , Medición de Riesgo , Aguas del Alcantarillado/análisis , Estados Unidos , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/toxicidadRESUMEN
Daphnia magna reproduction tests were performed with C(10), C(12), C(14) and C(15) alcohols to establish a structure-activity relationship of chronic effects of long-chain alcohols. The data generation involved substantial methodological efforts due to the exceptionally rapid biodegradability of the test substances and the need to test as close as possible to their water solubility limits. Test concentrations were determined by GC-MS before and after test solution renewal. Whereas apparent toxicity based on survival and reproduction increased with increasing C-chain lengths up to C(14), observations of toxicity to C(15) alcohol were not in line with lower chain lengths due to the lack of toxicity below the level of water solubility. When omitting C(15), the slope of most (Q)SARs approach -1, being consistent with the expectation of a non-polar narcotic mode of action. Further testing at higher chain lengths is not sensible due to progressively lower solubility, at remaining biodegradability. Effects on mortality and reproduction are not expected below the level of water solubility.
Asunto(s)
Alcoholes Grasos/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Daphnia , Interpretación Estadística de Datos , Monitoreo del Ambiente , Crecimiento/efectos de los fármacos , Relación Estructura-Actividad Cuantitativa , Reproducibilidad de los Resultados , Reproducción , Medición de Riesgo , Soluciones/análisis , Sobrevida , Agua/químicaRESUMEN
Human NT cells derived from the NTera2/D1 cell line express a dopaminergic phenotype making them an attractive vehicle to supply dopamine to the depleted striatum of the Parkinsonian patient. In vitro, hNT neurons express tyrosine hydroxylase (TH), depending on the length of time they are exposed to retinoic acid. This study compared two populations of hNT neurons that exhibit a high yield of TH+ cells, MI-hNT and DA-hNT. The MI-hNT and DA-hNT neurons were intrastriatally transplanted into the 6-OHDA hemiparkinsonian rat. Amelioration in rotational behavior was measured and immunohistochemistry was performed to identify surviving hNT and TH+ hNT neurons. Results indicated that both MI-hNT and DA-hNT neurons can survive in the striatum, however, neither maintained their dopaminergic phenotype in vivo. Other strategies used in conjunction with hNT cell replacement are likely needed to enhance and maintain the dopamine expression in the grafted cells.
Asunto(s)
Trasplante de Células/fisiología , Dopamina/fisiología , Enfermedad de Parkinson Secundaria/fisiopatología , Receptores de Dopamina D1/fisiología , Animales , Apomorfina/toxicidad , Conducta Animal/efectos de los fármacos , Línea Celular , Agonistas de Dopamina/toxicidad , Supervivencia de Injerto , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Oxidopamina , Enfermedad de Parkinson Secundaria/inducido químicamente , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D1/genética , Conducta Estereotipada/efectos de los fármacos , Simpaticolíticos , Tirosina 3-Monooxigenasa/biosíntesis , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.
Asunto(s)
Mesencéfalo/trasplante , Neuronas/trasplante , Células de Sertoli/trasplante , Animales , Ganglios Basales/cirugía , Trasplante de Tejido Encefálico/inmunología , Rechazo de Injerto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Células de Sertoli/metabolismo , Trasplante Heterólogo/fisiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The mitochondrial toxin, 3-nitropropionic acid (3-NP), produces motor dysfunction and striatal atrophy in rats. However, rat strain and method of administration may contribute to variability in the deficits caused by 3-NP toxicity. To evaluate this, changes in nocturnal spontaneous locomotor activity from chronic administration of 3-NP using an osmotic mini pump, were examined in the Lewis rats. Lewis rats were treated with 3-NP or saline for 2 days and behavior was tested daily for a 15 day period. Animals receiving 3-NP displayed significantly less spontaneous activity than animals in the saline group. 3-NP treated animals also weighed significantly less when compared to saline treated animals. These results demonstrate that even though there were no significant alterations in overt anatomical pathology, even short-term exposure to 3-NP produced significant effects. This short-term administration may present a potential paradigm for examination of sub-threshold neurotoxicity.
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Conducta Animal/efectos de los fármacos , Convulsivantes/administración & dosificación , Nitrocompuestos/administración & dosificación , Propionatos/administración & dosificación , Animales , Peso Corporal/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Esquema de Medicación , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Masculino , Actividad Motora/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Factores de TiempoRESUMEN
BACKGROUND AND PURPOSE: Human umbilical cord blood cells (HUCBC) are rich in stem and progenitor cells. In this study we tested whether intravenously infused HUCBC enter brain, survive, differentiate, and improve neurological functional recovery after stroke in rats. In addition, we tested whether ischemic brain tissue extract selectively induces chemotaxis of HUCBC in vitro. METHODS: Adult male Wistar rats were subjected to transient (2-hour) middle cerebral artery occlusion (MCAO). Experimental groups were as follows: group 1, MCAO alone (n=5); group 2, 3x10(6) HUCBC injected into tail vein at 24 hours after MCAO (n=6) (animals of groups 1 and 2 were killed at 14 days after MCAO); group 3, MCAO alone (n=5); group 4, MCAO injected with PBS at 1 day after stroke (n=8); and group 5, 3x10(6) HUCBC injected into tail vein at 7 days after MCAO (n=5). Rats of groups 3, 4, and 5 were killed at 35 days after MCAO. Behavioral tests (rotarod and Modified Neurological Severity Score [mNSS]) were performed. Immunohistochemical staining was used to identify cells derived from HUCBC. Chemotactic activity of ischemia brain tissue extracts toward HUCBC at different time points was evaluated in vitro. RESULTS: Treatment at 24 hours after MCAO with HUCBC significantly improved functional recovery, as evidenced by the rotarod test and mNSS (P<0.05). Treatment at 7 days after MCAO with HUCBC significantly improved function only on the mNSS (P<0.05). Some HUCBC were reactive for the astrocyte marker glial fibrillary acidic protein and the neuronal markers NeuN and microtubule-associated protein 2. In vitro, significant HUCBC migration activity was present at 24 hours after MCAO (P<0.01) compared with normal brain tissue. CONCLUSIONS: Intravenously administered HUCBC enter brain, survive, migrate, and improve functional recovery after stroke. HUCBC transplantation may provide a cell source to treat stroke.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Infarto de la Arteria Cerebral Media/terapia , Accidente Cerebrovascular/terapia , Animales , Conducta Animal , Encéfalo/citología , Isquemia Encefálica/fisiopatología , Diferenciación Celular , Extractos Celulares/farmacología , Supervivencia Celular , Quimiotaxis/efectos de los fármacos , Sangre Fetal/citología , Sangre Fetal/fisiología , Humanos , Infarto de la Arteria Cerebral Media/fisiopatología , Infarto de la Arteria Cerebral Media/psicología , Infusiones Intravenosas , Masculino , Ratas , Ratas Wistar , Accidente Cerebrovascular/fisiopatología , Accidente Cerebrovascular/psicologíaRESUMEN
Huntington's disease is a fatal neurological disorder characterized by chorea and deterioration in cognitive and neuropsychiatric function. Primary pathological changes are found in the striatum, where GABAergic neurons undergo degenerative changes. Local interneurons are relatively spared. Here, we describe the rationale for clinical trials of fetal striatal tissue transplantation for the treatment of Huntington's disease. Specifically, the reasons for utilizing tissue derived from the far lateral aspect of the lateral ventricular eminence as a source of striatal tissue will be discussed.
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Trasplante de Tejido Encefálico , Cuerpo Estriado/trasplante , Trasplante de Tejido Fetal , Enfermedad de Huntington/cirugía , Animales , Ensayos Clínicos como Asunto , HumanosRESUMEN
Cell transplantation therapy for diabetes and Parkinson's disease offers hope for long-term alleviation of symptoms. However, successful protocols remain elusive due to obstacles, including rejection and lack of tropic support for the graft. To enhance engraftment, testis-derived postmitotic Sertoli cells have been cotransplanted with islets in the diabetic rat (Db) and neurons in the Parkinsonian rat (PD). Sertoli cell tropic, regulatory, and nutritive factors that nourish and stimulate germ cells also support isolated neurons and islets in vitro. Likewise, immunosuppressive properties of Sertoli cells, extant in the testis, are expressed by extratesticular Sertoli cells evidenced by allo- and xenograft immunoprotection of grafts in both the CNS (in the PD model) and the periphery (in the Db model). On this basis, we have created Sertoli islet cell aggregates (SICA) and Sertoli neuron aggregated cells (SNAC) using simulated microgravity culture technology developed by NASA. Isolated rat and pig Sertoli cells were cocultured with neonatal pig islets (SICA) and with immortalized N-Terra-2 (NT2) neurons (SNAC) in the HARV biochamber. Formed aggregates were assayed for desirable functional and structural characteristics. Cell viability in SICA and SNAC exceeded 90% and FasL immunopositive Sertoli cells were present in both. Sertoli cells did not interfere with insulin secretion by SICA and promoted differentiation of NT2 cells to the dopaminergic hNT cell type in SNAC. Addition of Matrigel resulted in structural reorganization of the aggregates and enhanced insulin secretion. We conclude that SICA, SNAC, and Matrigel-induced islet- and neuron-filled "Sertoli cell biochambers" are suitable for long-term transplantation treatment of Db and PD.
Asunto(s)
Células de Sertoli , Ingravidez , Animales , Animales Recién Nacidos , Trasplante de Células , Técnicas de Cocultivo , Islotes Pancreáticos/citología , Islotes Pancreáticos/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Ratas , PorcinosRESUMEN
Taurine acts as an antioxidant able to protect neurons from free radical-mediated cellular damage. Moreover, it modulates the immune response of astrocytes that participate in neurodegenerative processes. The objective of this study was to examine whether taurine can prevent or attenuate the host inflammatory response induced by the xenotransplantation of neurons derived from the human teratocarcinoma cell line (hNT neurons). Male Sprague-Dawley rats were treated IP with either saline or taurine. Animals from both groups were perfused on the 4th or 11th day and the saline or taurine was administered from the start of the study until the day prior to sacrifice. The brains were processed immunohistochemically using antibodies against glial fibrillary acidic protein (GFAP), microglia (OX42), and human nuclear matrix antigen (NuMA). In the saline group, NuMA labeling revealed small grafts on the 4th day and no surviving cells on the 11th day. However, in the group that received taurine there were surviving grafts at both time points. Strong immunoreactivity for GFAP and OX42 was detected in the saline group surrounding the transplant. These effects were reduced in animals receiving taurine. Taken together, these results demonstrated that taurine was able to facilitate graft survival and attenuate the immune response generated by the xenograft.
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Neoplasias Encefálicas/tratamiento farmacológico , Cuerpo Estriado/patología , Degeneración Nerviosa/tratamiento farmacológico , Taurina/farmacología , Teratocarcinoma/tratamiento farmacológico , Animales , Antioxidantes/farmacología , Astrocitos/química , Astrocitos/metabolismo , Astrocitos/patología , Neoplasias Encefálicas/patología , Proteínas de Ciclo Celular , Cuerpo Estriado/metabolismo , Cuerpo Estriado/cirugía , Proteína Ácida Fibrilar de la Glía/análisis , Supervivencia de Injerto/efectos de los fármacos , Inmunohistoquímica , Masculino , Microglía/química , Microglía/metabolismo , Microglía/patología , Trasplante de Neoplasias , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/análisis , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Teratocarcinoma/patología , Trasplante HeterólogoRESUMEN
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.
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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
The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.
Asunto(s)
Trasplante de Tejido Encefálico/patología , Mesencéfalo/trasplante , Neuronas/patología , Células de Sertoli/fisiología , Animales , Trasplante de Tejido Encefálico/fisiología , Recuento de Células , Diferenciación Celular , Supervivencia Celular , Técnicas de Cocultivo , Trasplante de Tejido Fetal/patología , Trasplante de Tejido Fetal/fisiología , Humanos , Masculino , Mesencéfalo/citología , Mesencéfalo/enzimología , Neuronas/enzimología , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/cirugía , Ratas , Porcinos , Células Tumorales Cultivadas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.
Asunto(s)
Compuestos Cromogénicos/metabolismo , Galactósidos/metabolismo , Histocitoquímica/métodos , Indoles/metabolismo , Neuronas/trasplante , beta-Galactosidasa/genética , Animales , Artefactos , Encéfalo/metabolismo , Escherichia coli/genética , Fijadores , Expresión Génica , Genes Reporteros , Concentración de Iones de Hidrógeno , Ratones , Neuronas/metabolismo , Ratas , beta-Galactosidasa/metabolismoRESUMEN
The presence and distribution of vagal fibers and terminals throughout esophagus and gastrointestinal tract that could be anterogradely labeled by nodose ganglion tracer injections was quantitatively assessed in capsaicin- and vehicle-pretreated adult rats, in order to identify the capsaicin-resistant population. Up to 90% of the intraganglionic laminar endings (IGLEs), in the myenteric plexus of the esophagus, and 70-90% in the stomach, as well as 57% of the intramuscular endings or arrays (IMAs) in the fundic stomach survived the capsaicin treatment, while in the upper small intestine only few and in the lower small intestine, the cecum and colon, virtually no IGLEs survived capsaicin treatment. Intramucosal terminals were not assessed. Furthermore, gastric balloon distension-induced c-Fos expression in the dorsal vagal complex was not significantly decreased in capsaicin-treated rats. It is concluded that among primary vagal afferents there is a capsaicin-resistant population that primarily innervates the esophagus and upper gastrointestinal tract, and a capsaicin-sensitive population that innervates mainly the lower tract. At least vagal gastric tension-sensitive afferents also seems to be functionally intact in that they may be capable of synaptically activating second-order neurons in the brainstem.
Asunto(s)
Capsaicina/farmacología , Esófago/inervación , Estómago/inervación , Nervio Vago/citología , Animales , Carbocianinas , Cateterismo , Colorantes Fluorescentes , Masculino , Fibras Nerviosas/química , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/metabolismo , Neuronas Aferentes/química , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Ganglio Nudoso/citología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Ratas , Ratas Sprague-Dawley , Núcleo Solitario/química , Núcleo Solitario/citología , Núcleo Solitario/fisiología , Transmisión Sináptica/fisiología , Nervio Vago/anatomía & histologíaRESUMEN
One of the major issues in neural transplantation is the low survival rate (<5%) of transplanted dopamine (DA) neurons [3]. Recently it has been shown that it is possible to enhance the survival of these neurons, which in turn may decrease the amount of tissue that is required for each transplantation patient. The present paper demonstrates a novel approach for enhancing neuronal survival by co-transplantation of neuronal tissue with Testis-derived Sertoli cells (SC). This strategy could improve neuronal survival through the provision of trophic support.
Asunto(s)
Trasplante de Tejido Encefálico/métodos , Dopamina/fisiología , Neuronas/trasplante , Células de Sertoli/trasplante , Animales , Comunicación Celular/fisiología , Trasplante de Células/métodos , Cuerpo Estriado , Desnervación , Supervivencia de Injerto/fisiología , Masculino , Neuronas/citología , Neuronas/enzimología , Oxidopamina , Ratas , Ratas Sprague-Dawley , Células de Sertoli/citología , Simpaticolíticos , Tirosina 3-Monooxigenasa/análisisRESUMEN
Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.
Asunto(s)
Apoptosis/fisiología , Neuronas/citología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , ADN Nucleotidilexotransferasa/análisis , Dopamina/fisiología , Humanos , Neoplasias de Células Germinales y Embrionarias , Neuronas/enzimología , Células Madre/citología , Células Madre/enzimología , Tretinoina/farmacología , Células Tumorales Cultivadas , Tirosina 3-Monooxigenasa/análisisRESUMEN
We investigated the catecholaminergic nature of cultured hNT neurons previously treated either for 4 or 5 weeks with retinoic acid (RA). There were significantly more tyrosine hydroxylase (TH)-positive neurons (60%) in cultures treated for 4 weeks with RA compared to 5 week-treated cultures (=15%). Furthermore, numerous TH-positive hNT cells were also immunoreactive to dopamine transporter (DAT), dopamine receptor (D2) and aldehyde dehydrogenase (AHD-2), an enzyme exclusively expressed by dopaminergic (DA) ventral mesencephalic (VM) precursors. Thus this cell line has all the necessary cellular machinery to produce functional DA neurons and therefore is a good alternative tissue source to fetal VM.