RESUMEN
Creutzfeldt-Jakob disease (CJD) is a rare and fatal neurodegenerative disorder with a worldwide incidence of 1-1.5 per million. As in other countries, a CJD surveillance unit with a clinical and neuropathological approach was established in Goettingen (Germany) in 1993. Here we report the epidemiological data from a prospective 12-year surveillance. Since 1993, there has been an increasing incidence of CJD, from 0.7 in 1993 to 1.6 in 2005 with a quite stable level since 1998. During this period, the proportion of patients with MV and VV codon 129 genotype rose, possibly because of better identification of atypical subtypes. Six percent of all patients had a PRNP mutation, mainly D178N-129M (FFI), E200K and V210I. Iatrogenic CJD was a rare phenomenon. No patient infected by cadaveric growth hormone extracts was reported. Furthermore, no variant CJD patient has yet been identified in Germany. Differential diagnoses revealed a variety of neurodegenerative diseases, with Alzheimer's disease in the lead. One-third of the non-CJD patients included in this study suffered from a potentially treatable disorder such as metabolic or inflammatory diseases. The incidence and mortality rates in Germany are similar to those in other European countries. In contrast, however, acquired forms, such as iatrogenic and variant CJD are still rare in Germany or have not yet been identified.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/epidemiología , Adulto , Distribución por Edad , Anciano , Anciano de 80 o más Años , Síndrome de Creutzfeldt-Jakob/diagnóstico , Diagnóstico Diferencial , Genotipo , Alemania/epidemiología , Humanos , Incidencia , Persona de Mediana Edad , Polimorfismo Genético , Vigilancia de la Población/métodos , Proteínas PrPSc/genética , Estudios ProspectivosRESUMEN
DNA extracted from formalin-fixed and paraffin-embedded brain tissue is known to contain as yet ill-characterized inhibitors of the PCR process. As part of a project that aims to clarify the role of mitochondrial DNA sequence variation in human neurodegenerative diseases using DNA from various ethnic backgrounds, we have investigated factors that influence the preservation of archival DNA and its suitability for PCR. In this study, neuropathological tissue samples were analysed that had been routinely processed in 18 international centres on four continents. Following DNA extraction, PCR amplification of mitochondrial and nuclear DNA sequences was performed with and without additional purification of the template DNA. In addition, the DNA used for PCR was analysed by HPLC. Phosphate-buffered formalin proved to be a superior fixative compared with unbuffered aldehyde: DNA extraction resulted in greater yields, the molecular weight of the isolated DNA was higher and PCR was more successful. PCR inhibitors were identified as (1) high concentrations of small (<300 bp) DNA fragments that competitively compete with template DNA and (2) contaminants of the DNA template solution including denatured protein that cannot be completely removed by phenolic extraction. HPLC analysis did not reveal significant qualitative differences between DNA isolated from fresh-frozen tissue samples and DNA recovered from formalin-fixed, paraffin-embedded brain tissue. The fact that DNA could be amplified from the majority of tissue specimens in this study suggests that rare diseases and diseases where ethnic background plays an important role can be sampled for genetic polymorphism analysis on a global scale using archival neuropathological collections.
Asunto(s)
Química Encefálica , Encéfalo/patología , ADN Mitocondrial/aislamiento & purificación , ADN/aislamiento & purificación , Variación Genética , Laboratorios/normas , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Cromatografía Líquida de Alta Presión , ADN/genética , ADN Mitocondrial/genética , Síndrome de Down/genética , Síndrome de Down/patología , Humanos , Enfermedad por Cuerpos de Lewy/genética , Enfermedad por Cuerpos de Lewy/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Reacción en Cadena de la Polimerasa , Manejo de Especímenes/métodos , Conservación de Tejido/métodosRESUMEN
We have sequenced all mitochondrial complex I and tRNA genes in five pairs of monozygotic twins with a longitudinal diagnosis of idiopathic Parkinson disease (PD). At the time of molecular genetic analysis, four of the pairs were discordant for PD. Five novel homoplasmic sequence variants, including two missense mutations (ND2 4924 G/A, ND3 10192 C/T), were detected in mitochondrial genes of complex I in four of the pairs. In addition, a total of 20 known polymorphisms affecting both complex I and tRNA genes was found. Importantly, mitochondrial DNA sequences were identical in diseased and non-affected siblings of each pair. Our results demonstrate that missense mutations of mitochondrial complex I may occur in clinically discordant parkinsonian twins, questioning the direct pathogenic relevance of at least some of these mutations.
Asunto(s)
ADN Mitocondrial/genética , Enfermedades en Gemelos/genética , Variación Genética , Mutación Missense , Enfermedad de Parkinson/genética , Gemelos Monocigóticos , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , NAD(P)H Deshidrogenasa (Quinona)/química , NAD(P)H Deshidrogenasa (Quinona)/genética , Subunidades de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Microglia and brain macrophages represent a substantial fraction of the cells present in astrocytic gliomas. Yet, the functional role of microglia in these tumors has remained enigmatic. We have compared rat microglial cells and thymocytes with regard to their ability to present purified CNS proteins, MBP and S100beta, as well as C6 glioma cells to specific T lymphocytes. In addition, a new cytotoxicity assay based on fluorescence activated cell sorting of tumor cells carrying the green fluorescent protein was established. This assay was used to determine the influence of microglial population density and activational state on C6 glioma cell survival in vitro. Microglia were consistently found to present MBP and S100beta less efficiently than thymocytes and appeared to be unable to present C6 glioma cells to cytotoxic T lymphocytes. In addition, high concentrations of microglial cells attenuated the cytotoxic effects of these T cells on C6 glioma cells whereas thymocytes significantly supported their specific killing. It is suggested that defense functions of microglial cells against C6 glioma are severely compromised and that the observed deficiency in antigen presentation may play an important role for astrocytoma growth in vivo.
Asunto(s)
Antígenos de Neoplasias/análisis , Neoplasias Encefálicas/inmunología , Glioma/inmunología , Microglía/inmunología , Linfocitos T Citotóxicos , Animales , Animales Modificados Genéticamente , Células Presentadoras de Antígenos , Separación Celular , Células Cultivadas , Citometría de Flujo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Ratas , Timo/citología , Timo/inmunología , Células Tumorales CultivadasRESUMEN
BACKGROUND: The morphology of retinal transplants is believed to depend on the extent of mechanical disruption of the donor tissue during the surgical procedure and on local factors of the host environment. We hypothesized that oxidative stress during donor tissue preparation and implantation further affects transplant development and investigated the effects of CuZn superoxide dismutase (SOD) overexpression on the survival and morphological development of mouse embryonic retinal transplants. METHODS: Retinae and livers from embryonic day 14-15 SOD overexpressing transgenic mice and CBA control mice were harvested under sterile conditions. In order to identify transgenic mouse embryos, the embryonic livers were analyzed via nondenaturing gel-electrophoresis for the presence of the human SOD protein. Neural retinae were transplanted as fragmented tissue into the subretinal space of albino BALB/c mice. At 4-8 weeks following transplantation, the grafted eyes were fixed in Bouin's solution and processed for histological analysis. RESULTS: Both SOD transgenic and control retinal transplants had developed all retinal layers except for a ganglion cell layer and exhibited a similar extent of rosette formation. Computer-assisted, quantitative assessment of retinal graft volumes revealed a significant, around 58% increase in size of SOD transgenic transplants compared with controls. CONCLUSIONS: Enhanced intracellular SOD levels do not seem to influence retinal transplant morphology as detected by light microscopy. However, volumes of the SOD transgenic transplants were found to be increased compared to control grafts.
Asunto(s)
Trasplante de Tejido Fetal , Retina/enzimología , Retina/trasplante , Superóxido Dismutasa/genética , Animales , Biomarcadores , Estudios de Seguimiento , Supervivencia de Injerto , Líquido Intracelular/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Estrés Oxidativo , Retina/patología , Retina/cirugía , Superóxido Dismutasa/metabolismoRESUMEN
Two polymorphisms of the MnSOD gene, Ile58Thr and Ala9Val, have been associated with Parkinson disease (PD). The Ile58Thr amino acid exchange affects the stability at the tetrameric interface of the enzyme and reduces the enzymatic activity of MnSOD while the Ala/Val substitution at position -9 of the mitochondrial targeting sequence (MTS) may lead to misdirected intracellular trafficking. We have analyzed 63 German Caucasian PD patients for possible sequence variation in the MTS as well as in exon 3 of the MnSOD gene. All 63 PD patients analyzed exhibited a T at nucleotide position 5777 in exon 3 of the MnSOD gene corresponding to ATA, or Ile at the peptide level, and no other sequence variants were found. In addition, both alleles of the Ala9Val polymorphism in the MTS of MnSOD were equally distributed between German PD patients and controls excluding this gene variant as a risk factor for PD in Caucasian subjects.
Asunto(s)
Mitocondrias/enzimología , Enfermedad de Parkinson/enzimología , Polimorfismo Genético/genética , Superóxido Dismutasa/genética , Anciano , Alelos , Cartilla de ADN/genética , Alemania , Humanos , Enfermedad de Parkinson/genética , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Superóxido Dismutasa/metabolismo , Población BlancaRESUMEN
An increased frequency of the A-allele of the alpha-antichymotrypsin (ACT) gene has been recently described in Japanese patients suffering from Parkinson disease (PD). In the present study, we have analyzed 62 German PD patients with regard to their ACT and APOE genotypes and compared them to 53 controls without clinical or pathological evidence of neurodegenerative disease. The A-allele frequency was 47% in PD patients compared to 54% in control cases excluding ACT as a major susceptibility factor for PD in the Caucasian population. Yet, ACT-A allele frequencies were significantly different (p < 0.001) between Japanese and German controls. Therefore, although our data do not suggest that the alpha1-ACT polymorphism is a significant risk factor for the development of PD, a consideration of differences in genetic background seems warranted when evaluating susceptibility factors for neurodegenerative disease.
Asunto(s)
Apolipoproteínas E/genética , Enfermedad de Parkinson/genética , Polimorfismo Genético/genética , alfa 1-Antiquimotripsina/genética , Anciano , Alelos , Femenino , Alemania/epidemiología , Humanos , Modelos Logísticos , Masculino , Persona de Mediana EdadRESUMEN
Mutations in mitochondrially encoded tRNA genes have been described in a variety of neurological disorders. One such mutation, the A to G transition at nucleotide position 4336 of the mitochondrial tRNA(Gln) gene, has been associated with both Alzheimer and Parkinson disease. We have now performed a complete sequence analysis of all 22 mitochondrially encoded tRNA genes in 20 cases of histologically proven idiopathic Parkinson disease. Genomic DNA extracted from the substantia nigra of frozen or formalin-fixed and paraffin-embedded brains was used for amplification by polymerase chain reaction followed by automated sequencing. Two new homoplasmic point mutations were detected in the genes for tRNA(Thr) (15950 G/A) and tRNA(Pro) (15965 T/C) in 1 patient each. Restriction enzyme digestion revealed absence of the 15950 G/A mutation in 96 controls and in 40 cases of neuropathologically confirmed Alzheimer disease. The 15965 T/C mutation was shown to be absent from 100 control subjects and 47 Alzheimer cases. In addition to the two novel mutations, six known sequence variants were detected in a total of 6 different patients in the genes for tRNA(Asp) (G7521A, 1), tRNA(Arg) (T10463C, 1), tRNA(LeuCUN) (A12308G, 2), and tRNA(Thr) (A15924G, 1; G15928A, 2), including 1 patient carrying the tRNA(Gln) (A4336G) mutation. The G15950A transition affects position 70 of the aminoacyl acceptor stem of tRNA(Thr), which has been implicated as a recognition element for threonyl-tRNA synthetase and, at least in some tRNAs, in the processing of primary mitochondrial transcripts. The T15965C point mutation in the mitochondrial tRNA(Pro) gene alters position 64 of the TpsiC stem. The corresponding nucleotide in bacterial aminoacyl-tRNAs is involved in the interaction with elongation factor Tu. Thus, the two novel mutations are likely to be of functional relevance and could contribute to dopaminergic nerve cell death in affected individuals.
Asunto(s)
Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Mutación Puntual , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Prolina/genética , ARN de Transferencia de Treonina/genética , ARN/genética , Sustancia Negra/patología , Anciano , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN Mitocondrial , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia de Prolina/química , ARN de Transferencia de Treonina/química , Valores de ReferenciaRESUMEN
Recent in vitro experiments suggest that neurotoxicity of the prion protein is dependent on the presence of microglia. We have studied 11 cases of Creutzfeldt-Jakob disease (CJD) using immunocytochemistry in combination with computerized image analysis to clarify the relationship between spongiform change and microglial activation. MHC class II-positive microglia were almost exclusively confined to cortical gray matter where the neuropil area occupied by these cells exceeded that of controls more than 350-fold. In cortical regions with a bimodal distribution of spongiform degeneration, the presence of class II-positive microglia correlated well with the presence of vacuolation in layer V, but significantly less with spongiform change in layers II and III. In areas where spongiform degeneration affected the entire depth of the cortex, activated microglia were predominantly located in the inner one-half of the cortex or were evenly distributed throughout all cortical laminae. Here, microglia exhibited atypical, tortuous cell processes and occasionally intracytoplasmic vacuoles, suggesting that microglia themselves may become a disease target. Taken together, our results provide indirect evidence against an early causative involvement of microglia in the development of spongiform change. At later stages, however, diseased microglia could produce harmful factors which mediate both astrogliosis and neuronal injury.
Asunto(s)
Encéfalo/patología , Síndrome de Creutzfeldt-Jakob/patología , Proteínas de Unión al ADN , Microglía/patología , Anciano , Anciano de 80 o más Años , Encéfalo/metabolismo , Proteínas de Unión al Calcio/metabolismo , Síndrome de Creutzfeldt-Jakob/etiología , Síndrome de Creutzfeldt-Jakob/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunoquímica , Masculino , Proteínas de Microfilamentos , Microglía/metabolismo , Persona de Mediana EdadAsunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Encéfalo/patología , Enfermedades del Sistema Nervioso/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Encéfalo/metabolismo , Genotipo , Humanos , Enfermedades del Sistema Nervioso/genética , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Estudios RetrospectivosRESUMEN
Complete sequence analysis of all mitochondrial complex I genes was performed in 22 cases of neuropathologically confirmed idiopathic Parkinson disease (PD). DNA from the substantia nigra was used as a template for polymerase chain reaction-based genomic sequencing. Seven novel mutations causing the exchange of amino acids were detected in subunit genes ND1 (3992 C/ T, 4024 A/G), ND4 (11253 T/C, 12084 C/T), ND5 (13711 G/A, 13768 T/C), and ND6 (14582 T/C). In addition, five known missense mutations affecting the ND1 (3335 T/C, 3338 T/C), ND2 (5460 G/A), ND3 (10398 A/G), and ND5 (13966 A/G) genes as well as three secondary LHON mutations (4216 T/C, 4917 A/ G, 13708 G/A) were found in the PD group. Among the novel mutations, the 11253 T/C transition which changes a conserved isoleucine residue into threonine is most likely to be of functional relevance. Furthermore, 43 synonymous polymorphisms were detected in PD brains, including 20 novel sequence variants. Haplogroup analysis revealed that most unique missense mutations were found in PD cases belonging to the D(c) haplogroup. Our data are in line with the view that PD is not a single disease entity but comprises a genetically heterogeneous group of disorders. The results of our study further suggest that 90% or more of all idiopathic PD cases are not due to sequence variation of mitochondrial complex I, but that mitochondrial mutations may play a pathogenic role in a subset of PD patients.
Asunto(s)
ADN Mitocondrial/genética , Enfermedad de Parkinson/genética , Anciano , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Análisis Mutacional de ADN , ADN Mitocondrial/química , Femenino , Frecuencia de los Genes , Haplotipos , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Mutación Puntual , Polimorfismo Genético , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Intrastriatal grafting of embryonic dopamine-containing neurons is a promising approach for treating clinical and experimental Parkinson's disease. However, neuropathological analyses of grafted patients and transplanted rats have demonstrated that the survival of grafted dopamine neurons is relatively poor. In the present study, we pursued a strategy of transferring a potentially neuroprotective gene into rat embryonic mesencephalic rat cells in vitro, before grafting them into the denervated striatum of 6-hydroxydopamine-lesioned rats. We performed intrastriatal grafts of embryonic day 14 mesencephalic cells infected with replication-defective adenoviruses bearing either the human copper-zinc superoxide dismutase gene or, as a control, the E. coli lac Z marker gene. The transgenes were expressed in the grafts four days after transplantation and the expression persisted for at least five weeks thereafter. After five weeks postgrafting, there was more extensive functional recovery in the superoxide dismutase group as compared to the control (uninfected cells) and beta-galactosidase groups. The functional recovery was significantly correlated with the number of tyrosine hydroxylase-positive cells in the grafts, although the clear trend to increased survival of the dopamine neurons in the superoxide dismutase grafts did not reach statistical significance. Only a moderate inflammatory reaction was revealed by OX-42 immunostaining in all groups, suggesting that ex vivo gene transfer using adenoviral vectors is a promising method for delivering functional proteins into brain grafts.
Asunto(s)
Trasplante de Tejido Encefálico/fisiología , Trasplante de Células/fisiología , Trasplante de Tejido Fetal/fisiología , Neostriado/trasplante , Neuronas/metabolismo , Neuronas/fisiología , Superóxido Dismutasa/metabolismo , Animales , Virus del Sarcoma Aviar/genética , Dopamina/fisiología , Femenino , Vectores Genéticos , Supervivencia de Injerto , Humanos , Inmunohistoquímica , Factores de Lactosa/genética , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
We explored the effects of congeners of nitrogen monoxide (NO) on cultured mesencephalic neurons. Sodium nitroprusside (SNP) was used as a donor of NO, the congeners of which have been found to exert either neurotoxic or neuroprotective effects depending on the surrounding redox milieu. In contrast to a previous report that suggests that the nitrosonium ion (NO+) is neuroprotective to cultured cortical neurons, we found that the nitrosonium ion reduces the survival of cultured dopamine neurons to 32% of control. There was a trend for further impairment of dopamine neuron survival, to only 7% of untreated control, when the cultures were treated with SNP plus ascorbate, i.e. when the nitric oxide radical (NO.) had presumably been formed. We also evaluated the effects of an inhibitor of lipid peroxidation, the lazaroid U-83836E, against SNP toxicity. U-83836E exerted marked neuroprotective effects in both insult models. More than twice as many dopamine neurons (75% of control) survived when the lazaroid was added to SNP-treated cultures and the survival was increased eight-fold (to 55% of control) when U-83836E was added to cultures treated with SNP plus ascorbate. We conclude that the congeners of NO released by SNP are toxic to mesencephalic neurons in vitro and that the lazaroid U-83836E significantly increases the survival of dopamine neurons in situations where congeners of NO are generated.
Asunto(s)
Muerte Celular/efectos de los fármacos , Cromanos/farmacología , Depuradores de Radicales Libres/farmacología , Mesencéfalo/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/farmacología , Nitroprusiato/toxicidad , Piperazinas/farmacología , Animales , Células Cultivadas , Desarrollo Embrionario y Fetal/efectos de los fármacos , Mesencéfalo/embriología , Mesencéfalo/patología , Neuronas/efectos de los fármacos , Oxidación-Reducción , Ratas , Ratas Sprague-DawleyRESUMEN
Areas of striatal grafts which contain neurons that are characteristic of the striatum are called P-zones. We have investigated whether the paucity of P-zones in human xenografts of lateral ganglionic eminence (LGE) tissue in a rat model of Huntington's disease is due (i) to an absence of the appropriate target cells of LGE neurons or (ii) to the persistence of an immature morphology. Striatal tissue from human embryos of varying sizes (21, 24, and 30 mm in crown-to-rump length) was grafted into the ibotenate-lesioned striatum of immunosuppressed rats, which were killed after 15-17 weeks. In most cases, tissue from the LGE and medial ganglionic eminence (MGE) was transplanted together, whereas some rats received grafts of only LGE tissue. Both types of grafts exhibited positive immunostaining for PCNA (proliferating cells), Vimentin (immature astrocytes), and GAP-43 (outgrowing fibers), which indicates that graft maturation is still ongoing up to 4 months after grafting. Graft survival seemed better when MGE was cografted with LGE, suggesting that the MGE may provide trophic support for LGE neurons and can affect the overall survival of human striatal xenografts. However, the extent of P-zone formation was not increased in MIXED, i.e., LGE plus MGE, grafts.
Asunto(s)
Cuerpo Estriado/trasplante , Enfermedad de Huntington/cirugía , Fosfoproteínas , Trasplante Heterólogo , Acetilcolinesterasa/metabolismo , Animales , Benzoxazinas , Colorantes , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Fosfoproteína 32 Regulada por Dopamina y AMPc , Proteína GAP-43 , Supervivencia de Injerto , Histocitoquímica , Humanos , Inmunohistoquímica , Antígeno de Macrófago-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Oxazinas , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Vimentina/metabolismoRESUMEN
Reactive oxygen species are believed to play a crucial role in situations where dopamine neurons die, such as in Parkinson's disease or during intracerebral transplantation of embryonic mesencephalic tissue. The present study was designed to address the question whether, and to what extent, the glutathione redox system is important for the viability of rat embryonic dopamine neurons in vitro. Furthermore, we studied whether the lazaroid U-83836E, a 2-methylaminochroman that inhibits lipid peroxidation, affects the survival of cultured mesencephalic neurons subjected to experimentally induced glutathione depletion. Glutathione depletion was achieved by exposing dissociated mesencephalic cell cultures to L-buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, at four different concentrations (1, 10,100, and 1,000 microM). Dopamine neuron survival was significantly reduced by 65-94% in a concentration-dependent manner by 10-1,000 microM BSO. The neurotoxic effects of BSO were almost completely prevented by supplementing the culture medium with 0.3 microM U-83836E. As assessed by HPLC analysis, BSO treatment was associated with a marked reduction of cellular glutathione content, and this depletion was not altered by the presence of U-83836E. We conclude that in the present insult model of severe glutathione depletion, the lazaroid can afford efficient neuroprotection that does not seem to be mediated by a direct interaction with BSO or glutathione, but rather via an independent pathway.
Asunto(s)
Butionina Sulfoximina/farmacología , Muerte Celular/efectos de los fármacos , Cromanos/farmacología , Depuradores de Radicales Libres/farmacología , Glutatión/metabolismo , Mesencéfalo/citología , Neuronas/citología , Neurotoxinas/farmacología , Piperazinas/farmacología , Análisis de Varianza , Animales , Células Cultivadas , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Mesencéfalo/embriología , Mesencéfalo/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Grafts of striatal tissue comprise two different types of tissue: regions with (P-zones) and without (NP-zones) neurons that express markers characteristic of the striatum, such as dopamine- and cyclic AMP-regulated phosphoprotein with a mol. wt of 32,000 (DARPP-32). It remains unclear whether P-zones alone play a crucial role in functional effects of striatal grafts in an animal model of Huntington's disease. The present study has been performed to determine: (i) the yield of DARPP-32-positive neurons in grafts of lateral ganglionic eminence; (ii) whether treatment of graft tissue with the spin-trapping agent alpha-phenyl-tert-butyl nitrone enhances the survival of implanted DARPP-32-positive neurons; and (iii) the relationship between the number of DARPP-32-positive neurons in the grafts and functional effects of the grafts on paw-reaching ability in rats with unilateral quinolinic acid lesions of the striatum. Dissociated tissue derived from the lateral ganglionic eminence of rat embryos (embryonic day 14), with or without addition of alpha-phenyl-tert-butyl nitrone (3 mM), was implanted into the quinolinic acid-lesioned striatum. Compared to unlesioned normal animals, rats with striatal lesions showed substantial impairment in paw-reaching ability, particularly on the side contralateral to the lesion, as judged from the number of pellets retrieved by each paw. Intrastriatal grafts gave rise to a significant improvement in paw-reaching ability. The mean total number of surviving DARPP-32-positive cells in grafts without alpha-phenyl-tert-butyl nitrone treatment was estimated at 115 x 10(3), which did not significantly differ from that in alpha-phenyl-tert-butyl nitrone-treated grafts. The paw-reaching scores were significantly correlated with the volumes of P-zones and the number of DARPP-32-positive neurons, but with neither the volumes of NP-zones nor the total graft volume. The results suggest that P-zones in striatal grafts mediate graft-derived functional recovery in a complex task such as skilled forelimb use. Although the antioxidant treatment with alpha-phenyl-tert-butyl nitrone failed to promote graft survival, the positive correlation between the yield of DARPP-32-positive cells in the graft and the extent of the functional recovery highly warrants further attempts to increase the yield of the striatal component in the graft.
Asunto(s)
Trasplante de Tejido Encefálico/fisiología , Trasplante de Tejido Fetal/fisiología , Enfermedad de Huntington/fisiopatología , Destreza Motora/fisiología , Neostriado/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas , Acetilcolinesterasa/metabolismo , Animales , Óxidos N-Cíclicos , Fosfoproteína 32 Regulada por Dopamina y AMPc , Femenino , Supervivencia de Injerto , Enfermedad de Huntington/enzimología , Neostriado/enzimología , Neostriado/trasplante , Óxidos de Nitrógeno/farmacología , Ácido Quinolínico/toxicidad , Ratas , Ratas Sprague-Dawley , Marcadores de SpinRESUMEN
Basic parameters which are crucial for the survival of human embryonic striatal grafts need to be investigated before initiating clinical trials in Huntington's disease. In order to define the dissection of human striatal-donor tissue which gives rise to the largest amount of striatal neurons after intrastriatal transplantation, we studied the lateral and medial ganglionic eminences of embryonic striatal primordia obtained from human embryos sized 17-30 mm in crown-to-rump length (corresponding to Carnegie stages 18-23). Anatomical landmarks that demarcated the lateral and medial ganglionic eminences from each other were present only in embryos with 20 mm crown-to-rump length or larger. In monolayer cultures, the lateral ganglionic eminence gave rise to a six-fold higher yield of dopamine- and cyclic AMP-regulated phosphoprotein 32-immunoreactive striatal neurons as compared to the medial ganglionic eminence. We also xenografted the lateral and medial ganglionic eminences from five embryos sized 21-30 mm in crown-to-rump length to the ibotenate lesioned striatum of immunosuppressed rats. The grafts were evaluated with respect to general morphology, survival and integration using (immuno-) histochemical stains for acetylcholinesterase/Cresyl Violet, nicotinamide adenine dinucleotide phosphate-diaphorase, dopamine- and cyclic AMP-regulated phosphoprotein-32, tyrosine hydroxylase and calbindin-D28KD. As assessed 9-25 weeks after implantation, 13 out of 16 and 8 out of 13 grafts, in the groups grafted with the medial and lateral ganglionic eminences, respectively, had survived. Previous studies with rat donor tissue have indicated that the functional efficacy of striatal grafts is related to the development of striatal-specific P-zone regions and that these are enriched in transplants derived from the lateral as opposed to the medial ganglionic eminence. Also in the human striatal xenografts of the present study, P-zones appeared more abundant when the donor tissue was derived from the lateral ganglionic eminence. However, the proportion of graft tissue that expressed P-zone properties was always very low (at most 30%) and never approached the 80-90% previously observed in transplants of rat lateral ganglionic eminence. We conclude that the relative yield of striatal neurons in grafts of the human embryonic striatal primordium has to be improved before neural transplantation should be applied in patients with Huntington's disease.
Asunto(s)
Trasplante de Tejido Encefálico/fisiología , Trasplante de Células/fisiología , Neostriado/embriología , Fosfoproteínas , Trasplante Heterólogo/fisiología , Acetilcolinesterasa/metabolismo , Animales , Calbindinas , Células Cultivadas , Fosfoproteína 32 Regulada por Dopamina y AMPc , Femenino , Ganglios/citología , Ganglios/trasplante , Supervivencia de Injerto , Humanos , Inmunohistoquímica , NADPH Deshidrogenasa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100/metabolismo , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
It has been suggested that oxidative stress plays an important role in mediating excitotoxic neuronal death. We have therefore investigated the protective effects of antioxidants against excitotoxic injury in the rat on striatal neurons both in vitro and in vivo. In the first part of the study, we determined whether two different types of antioxidants, the spin trapping agent, alpha-phenyl-tert-butyl nitrone and an inhibitor of lipid peroxidation, U-83836E, could protect cultured striatal neurons against either hypoglycemic injury or N-methyl-D-aspartate-induced excitotoxicity. Dopamine- and cyclic AMP-regulated phosphoprotein, which is enriched in medium-sized spiny neurons, was chosen as a marker for striatal neurons. alpha-Phenyl-t-butyl nitrone and U-83836E both significantly reduced cell death induced by these insults as indicated by an increased number of surviving dopamine- and cyclic AMP-regulated phospho-protein-positive neurons. The two antioxidants also promoted the survival of cultured striatal neurons grown at low cell density under serum-free culture conditions. In an in vivo experiment systemically administered alpha-phenyl-t-butyl nitrone exerted neuroprotective effects in the rat striatum following injection of the excitotoxin quinolinic acid. Apomorphine-induced rotation tests revealed that alpha-phenyl-t-butyl nitrone-treated animals were significantly less asymmetric in their motor behavior than control rats. Treatment with alpha-phenyl-t-butyl nitrone significantly reduced the size of the quinolinic acid-induced striatal lesions, as assessed by the degree of sparing of dopamine- and cyclic AMP-regulated phospho-protein-positive and nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons, and of microtubule-associated protein-2-immunorective areas. Furthermore, lesion-induced morphological changes in the substantia nigra pars reticulate, i.e. loss of dopamine- and cyclic AMP-regulated phosphoprotein-positive afferent fibers and atrophic changes due to transsynaptic degeneration, were also less extensive in the alpha-phenyl-t-butyl nitrone-treated animals. The results support the hypothesis that oxygen-free radicals contribute to excitotoxic neuronal injury. The in vivo cytoprotective effects of alpha-phenyl-t-butyl nitrone against striatal excitotoxic lesions suggest that antioxidants could be used as potential neuroprotective agents in Huntington's disease, which has been suggested to involve excitotoxicity.
Asunto(s)
Antioxidantes/farmacología , Neostriado/citología , Neuronas/efectos de los fármacos , Neurotoxinas/antagonistas & inhibidores , Fosfoproteínas , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Óxidos N-Cíclicos , Fosfoproteína 32 Regulada por Dopamina y AMPc , Femenino , Hipoglucemia/patología , Inmunohistoquímica , NADPH Deshidrogenasa/metabolismo , Neostriado/efectos de los fármacos , Degeneración Nerviosa/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neurotoxinas/toxicidad , Óxidos de Nitrógeno/farmacología , Ácido Quinolínico/antagonistas & inhibidores , Ácido Quinolínico/toxicidad , Ratas , Ratas Sprague-Dawley , Marcadores de Spin , Conducta Estereotipada/efectos de los fármacos , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacosRESUMEN
We studied the effects of high-dose methylprednisolone on the survival of intrastriatal neural xenografts and the host responses against them. Dissociated mesencephalic tissue from inbred mouse (CBA-strain) embryos was transplanted to the intact striatum of adult Sprague-Dawley rats. The rats received either daily injections of methylprednisolone (30 mg/kg), or cyclosporin A (10 mg/kg), or no immunosuppressive treatment. Two or six weeks after transplantation, there was good survival of xenografts in both the methylprednisolone- and cyclosporin A-treated rats. In contrast, the xenografts in untreated control rats were all rejected by six weeks. There was no marked difference in the degree of expression of MHC class I and II antigens and the accumulation of activated astrocytes and microglial cells/macrophages between the three groups. However, both methylprednisolone and cyclosporin A reduced infiltration of T lymphocytes to the transplantation sites. The expression of pro-inflammatory cytokines (interferon-gamma, tumour necrosis factor-alpha, interleukin-6) in and around the grafts was lower in the methylprednisolone- and cyclosporin A-treated groups than in untreated control rats. Although high-dose methylprednisolone caused significant body weight loss, we conclude that this treatment can prevent rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in the adult.