Inmunorreactividad de neuronas gabaergicas y glutamatergicas en la corteza y el cerebelo de ratones infectados con rabia

La rabia es una enfermedad viral que produce cambios en la función neuronal. Estos cambios, entre otros, inducen alteraciones en el metabolismo de los neurotransmisores. Los signos clínicos de la rabia indican que se podría afectar el sistema GABA-glutamato y, por lo tanto, el balance entre la inhibición y excitación neuronal. El objetivo de este trabajo consistió en evaluar la inmunorreactividad de neuronas GABAérgicas y glutamatérgicas en la corteza y el cerebelo de ratones infectados con rabia, mediante dos vías de inoculación. Se inocularon ratones adultos por vía intramuscular e intracerebral con virus de la rabia CVS; en la etapa terminal de la enfermedad se anestesiaron y se sacrificaron por perfusión con paraformaldehído y glutaraldehído. Se obtuvieron cortes en vibrátomo y se procesaron para inmunohistoquímica con anticuerpos anti-Glu (glutamato), anti-GABA y anti-PV (parvoalbúmina); una proteina buffer de calcio que además sirve como marcador de algunas células GABAérgicas. Se realizaron conteos celulares y análisis densitométricos. En la corteza cerebral motora se demostró aumento significativo en la Glu-ir y pérdida de GABA-ir por las dos vías de inoculación, pero en la corteza somatosensorial los resultados fueron contrarios. En las folias del cerebelo se halló aumento de Glu-ir en la capa granular así como aumento de PV-ir y pérdida de GABA-ir en la capa molecular, sólo después de la inoculación intramuscular. Los resultados fueron similares en los núcleos profundos. La inoculación intracerebral del virus no provocó cambios de inmunorreactividad para GABA, Glu, y PV en ninguna de las áreas del cerebelo. Aunque el estudio de PV no hacía parte de los objetivos permitió realizar diversas interpretaciones entre ellas el establecer que la expresión de la proteína aumenta como respuesta al aumento del glutamato y la pérdida de GABA, por otro lado con este trabajo se ilustra por primera vez que que el virus de la rabia afecta el metabolismo de GABA y el glutamato de manera antagónica y que los efectos varían según la ruta de inoculación y el área encefálica evaluada.Abstract. Rabies is a viral disease that induces changes in neuronal function. These changes induce alterations in the metabolism of neurotransmitters. Clinical signs of rabies indicate that the system could affect GABA-glutamate and, therefore, the balance between inhibition and neuronal excitation. The aim of this study was to evaluate the immunoreactivity of GABAergic and glutamatergic neurons in the cortex and cerebellum of mice infected with rabies through two routes of inoculation. Adult mice were inoculated intramuscularly and intracerebrally with CVS rabies virus, in the terminal stage of disease were anesthetized and sacrificed by perfusion with paraformaldehyde and glutaraldehyde. In vibratome sections were obtained and processed for immunohistochemistry with anti-Glu (glutamate), anti-GABA and anti-PV (parvoalbúmina), a calcium buffer protein also serves as a marker of GABAergic cells. Cell counts were performed and densitometric analysis. In the motor cortex showed significant increase in the Glu-ir and loss of GABA-ir by both routes of inoculation, but in the somatosensory cortex results were contrary. In the cerebellar folia was found increased Glu-ir in the granular layer and increased PV-ir and GABA-ir loss in the molecular layer, only after intramuscular inoculation. The results were similar in the deep nuclei. Intracerebral inoculation of virus did not cause changes in immunoreactivity for GABA, Glu, and PV in any area of the the cerebellum. Although the study of PV was not part of the objectives allowed for different interpretations including establishing that the protein expression increases in response to increased glutamate and GABA loss, on the other side with this work is illustrated for the first time the rabies virus affects the metabolism of GABA and glutamate in an antagonistic manner and that the effects vary by route of inoculation and the brain area evaluated.Maestrí

Disminución del número de neuronas que expresan GABA en la corteza cerebral de ratones infectados con rabia

Introduction. GABAergic neurons synthesize and release gamma-aminobutyric acid, the predominant inhibitory neurotransmitter in the brain. Certain clinical signs of rabies and previous experimental studies have suggested that rabies viral infections affect the host GABAergic system.Objective. The effect of rabies virus infection on the expression of GABA was evaluated in neurons of the mouse cerebral cortex.Materials and methods. Adult mice were inoculated by intramuscular injection with the standard strain of rabies (CVS virus). The animals were sacrificed in the terminal stage of the illness and perfused with 4% paraformaldehyde and 1% glutaraldehyde. Frontal sections were obtained in a Vibratome® and treated with appropriate immunohistochemical reactions for identifying the GABAergic neurons in the cerebral cortex. Counts and comparative quantitative analysis of the GABA+ neurons were compared in samples of infected and normal mice.Results. In the animals infected with rabies virus, the distribution pattern of cortical GABAergic neurons was not changed, but their number diminished significantly. The mean value of GABA+ cells number in 1 μm2 of cerebral cortex was 293±32 in normal samples and 209±13 in infected samples. Despite the loss in GABA+ cell number, the average size of GABA+ cells per unit increased from 104±8 μm2 in normal mice to 122±10 μm2 in infected mice because the cell loss consisted more frequently of smaller neurons. Nevertheless, the rank of GABA+ cell sizes in infected samples was similar to normal samples.Conclusion. This evidence supported the hypothesis that GABA is involved in rabies pathology.Introducción. Algunos signos clínicos de la rabia y estudios experimentales previos sugieren que esta infección viral podría afectar al sistema GABAérgico.Objetivo. Evaluar el efecto de la infección con el virus de la rabia sobre la expresión de GABA en neuronas de la corteza cerebral de ratón.Materiales y métodos. Se inocularon ratones adultos con virus CVS de la rabia por vía intramuscular. Los animales se sacrificaron en la etapa terminal de la enfermedad y se fijaron por perfusión con paraformaldehído al 4% y glutaraldehído al 1%. Se procesaron cortes coronales de cerebro obtenidos en un Vibratome®, mediante inmunohistoquímica para identificar neuronas GABAérgicas en la corteza cerebral. Se realizaron conteos y análisis cuantitativo de las neuronas positivas para GABA en muestras de ratones normales e infectados.Resultados. En los animales infectados con rabia no se alteró el patrón de distribución de las neuronas GABAérgicas corticales pero su número disminuyó significativamente. El promedio de células positivas para GABA en 1 mm2 de corteza fue de 293±32 en los controles y de 209±13 en los infectados. Por otra parte, el valor promedio del área de los perfiles neuronales positivos para GABA aumentó significativamente de 104±8 μm2 en los controles a 122±10 μm2 en las muestras infectadas, debido a que la pérdida de células positivas para GABA fue más evidente en las neuronas de menor tamaño. No obstante, el rango de tamaños de las células inmunopositivas para GABA fue similar en muestras de animales normales e infectados.Conclusiones. Este trabajo aporta nueva evidencia en favor de la hipótesis que propone la participación del GABA en la fisiopatología de la rabia

Nuclei ultrastructural changes of C6/36 cells infected with virus dengue type 2

Introduction: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. Objective: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. Materials and methods: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. Results: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. Conclusions: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism

Glucógeno hepático en dengue severo: análisis histopatológico

5 páginasLiver glycogen in severe dengue: histopathological analysisGlucógeno hepático en dengue severo: análisis histopatológic

Glucógeno hepático en dengue severo: análisis histopatológico

Antecedentes: El virus del dengue afecta distintos órganos, pero se ha determinado que el hígado es el principal blanco de acción y en donde ocurre la mayor severidad del daño. Existen pocos estudios sobre los cambios histológicos durante la infección por dengue. Objetivos: Analizar las alteraciones histopatológicas post-mortem en hígados de pacientes que presentaron la forma grave del dengue. Métodos: Se revisaron los cortes de hígado de 20 pacientes con dengue severo y se realizaron coloraciones y pruebas para glucógeno. Resultados: Encontramos pérdida de glucógeno citoplasmático en todos los casos analizados y la presencia de glucógeno intranuclear en dos de ellos. Conclusiones: En este estudio se reporta por primera vez la presencia de masas de glucógeno intranuclear en hepatocitos de dos niños fallecidos con dengue grave

Morphological and Molecular Changes in the Cortex and Cerebellum of Immunocompetent Mice Infected with Zika Virus

Zika virus (ZIKV) disease continues to be a threat to public health, and it is estimated that millions of people have been infected and that there have been more cases of serious complications than those already reported. Despite many studies on the pathogenesis of ZIKV, several of the genes involved in the malformations associated with viral infection are still unknown. In this work, the morphological and molecular changes in the cortex and cerebellum of mice infected with ZIKV were evaluated. Neonatal BALB/c mice were inoculated with ZIKV intraperitoneally, and the respective controls were inoculated with a solution devoid of the virus. At day 10 postinoculation, the mice were euthanized to measure the expression of the markers involved in cortical and cerebellar neurodevelopment. The infected mice presented morphological changes accompanied by calcifications, as well as a decrease in most of the markers evaluated in the cortex and cerebellum. The modifications found could be predictive of astrocytosis, dendritic pathology, alterations in the regulation systems of neuronal excitation and inhibition, and premature maturation, conditions previously described in other models of ZIKV infection and microcephaly

5'/3' RACE method for sequencing the 5' and 3' untranslated regions of Zika virus

The spread of Zika virus (ZIKV) from the African continent to the Americas promoted its molecular evolution, as reflected by mutations in its RNA genome. Most of the ZIKV genome sequences in the GenBank database have incomplete 5' and 3' UTR sequences, reflecting the deficiency of whole-genome sequencing technologies to resolve the sequences of the genome ends. We modified a protocol for rapid amplification of cDNA ends (RACE) to determine the complete sequences of the 5' and 3' UTRs of a previously reported ZIKV isolate (GenBank no. MH544701.1). This strategy is useful for determining 5' and 3' UTR sequences of ZIKV isolates and will be useful for comparative genomics applications

Genotyping of dengue virus from infected tissue samples embedded in paraffin

Abstract Dengue has become one of the vector-borne diseases that affect humans worldwide. In Latin American countries, Colombia is historically one of the most affected by epidemics of this flavivirus. The underreporting of signs and symptoms of probable cases of dengue, the lack of characterization of the serotypes of the infection, and the few detailed studies of postmortem necropsies of patients are among other conditions that have delayed progress in the knowledge of the pathogenesis of the disease. This study presents the results of fragment sequencing assays on paraffin-embedded tissue samples from fatal DENV cases during the 2010 epidemic in Colombia. We found that the predominant serotype was DENV-2, with the Asian/American genotype of lineages 1 and 2. This work is one of the few reports of the circulating genotypes of dengue during the 2010 epidemic in Colombia, one of the most lethal dates in the country's history

Multisystem Failure in Fatal Dengue: Associations between the Infectious Viral Serotype and Clinical and Histopathological Findings

Dengue is the most important arthropod-borne viral infection of humans. However, its viral pathogenesis is still unknown. The information collected from dengue fatal cases is crucial for understanding the complex interactions between virulence and host factors. This study aimed to establish possible associations between the clinical characteristics, histopathological changes, replication, and tissue location of viral serotypes in dengue fatal cases. Clinical and histopathological characterizations, antigen localization in tissue, and detection of the infecting serotype and replication using real-time polymerase chain reaction were all performed on the dengue fatal cases. The majority of the cases involved people under the age of 20. Bleeding (48.3%), abdominal pain (44.8%), myalgia (52.9%), and headache (48.3%) were the most common clinical manifestations in the cases. There was multiorgan pathology, with histopathological changes primarily in the liver, spleen, and lung. Similarly, the viral antigen was found primarily in these organs; however, there were no associations between tissue changes, viral location, infecting serotypes, and replication processes. Dengue infection should be considered a multiorgan disease, the outcome of which is possibly not associated with the infecting viral serotype