Orlando Torres-Fernández

Immunohistochemical Overexpression of MAP-2 in the Cerebral Cortex of Rabies-Infected Mice

La proteina asociada a microtubulos MAP-2 es una parte integral del citoesqueleto y juega un papel importante en la morfogenesis neuronal. Esta proteina es un componente esencial del citoesqueleto de las dendritas, especialmente en el cerebro adulto, y su expresion puede ser alterada en condiciones experimentales o patologicas. El proposito de este estudio fue evaluar el efecto de la infeccion con el virus de la rabia sobre la inmunorreactividad de MAP-2 en la corteza cerebral de ratones. Ratones inoculados con el virus de la rabia fueron sacrificados cuando la enfermedad alcanzo su fase avanzada, junto con animales no infectados de la misma edad. Los cerebros se extrajeron despues de que los animales fueron tratados con paraformaldehido mediante perfusion intracardiaca. En un vibratomo se obtuvieron cortes coronales y estos se procesaron mediante inmunohistoquimica para revelar la presencia de la proteina MAP-2 en las neuronas de la zona motora de la corteza cerebral. Los ratones infectados con rabia mostraron un aumento en la inmunorreactividad de los somas y dendritas apicales en las neuronas piramidales de la corteza motora. Este es un resultado inesperado, ya que previamente se ha demostrado patologia dendritica en rabia, y algunos estudios sobre los trastornos neurologicos asocian las alteraciones dendriticas con perdida de expresion de la proteina MAP-2. Por lo tanto, cualquiera que sea la alteracion en la expresion de esta proteina, disminucion o aumento, podria ser la causa de un desequilibrio bioquimico en la integridad y estabilidad del citoesqueleto neuronal.

Overexpression of MAP2 and NF-H Associated with Dendritic Pathology in the Spinal Cord of Mice Infected with Rabies Virus

Rabies is a viral infection that targets the nervous system, specifically neurons. The clinical manifestations of the disease are dramatic and their outcome fatal; paradoxically, conventional histopathological descriptions reveal only subtle changes in the affected nervous tissue. Some researchers have considered that the pathophysiology of rabies is based more on biochemical changes than on structural alterations, as is the case with some psychiatric diseases. However, we believe that it has been necessary to resort to other methods that allow us to analyze the effect of the infection on neurons. The Golgi technique is the gold standard for studying the morphology of all the components of a neuron and the cytoskeletal proteins are the structural support of dendrites and axons. We have previously shown, in the mouse cerebral cortex and now with this work in spinal cord, that rabies virus generates remarkable alterations in the morphological pattern of the neurons and that this effect is associated with the increase in the expression of two cytoskeletal proteins (MAP2 and NF-H). It is necessary to deepen the investigation of the pathogenesis of rabies in order to find therapeutic alternatives to a disease to which the World Health Organization classifies as a neglected disease.

Entry of Rabies Virus in the Olfactory Bulb of Mice and Effect of Infection on Cell Markers of Neurons and Astrocytes

There are few studies of infection by rabies virus in the olfactory bulb (OB). This work was carried out with the purpose of establishing the time required to detect rabies antigens in the OB of mouse, after the intramuscular inoculation of the virus and to evaluate the effect of the infection on the expression of three proteins: calbindin (CB), parvalbumin (PV) and the glial fib rillary acidic protein (GFAP). Mice were inoculated with rabies virus intramuscularly in the hind limbs. Every 8 hours, after 72 hours postino culation (p.i.), animals were sacrificed by perfusion with paraformaldehyde and coronal sections of OB were obtained for immunohistochem ical study. These cuts were used to reveal the entry and spread of viral antigens. Tissue sections obtained in the terminal phase of the disease (144 hours p.i.), and controls of the same age were also processed for immunohistochemistry of CB, PV and GFAP. Rabies virus an tigens were initially detected at 80 hours p.i. in a few mitral cells. At 88 hours p.i. the antigens had spread through most of these neurons but until the terminal phase of the disease there was little dispersion of the virus towards other cellular layers of the OB. The C B protein was expressed in cells of the glomerular stratum, the PV in cells of the outer plexiform layer and the GFAP was expressed in all th e layers of the OB. Viral infection generated loss of CB expression and increase of PV expression. Immunoreactivity to GFAP was increased i n the outer plexiform layer of the OB as a response to infection.

Evidencia neuroanatómica del transporte del virus de la rabia por la vía propioespinal de la médula espinal de ratones

Introduction: Information about the neuroanatomical details of the ascendant transport of the rabies virus through the spinal cord is scarce. Objective: To identify the neuroanatomical route of dissemination of the rabies virus at each of the levels of the spinal cord of mice after being inoculated intramuscularly. Materials and methods: Mice were inoculated with the rabies virus in the hamstrings. After 24 hours post-inoculation, every eight hours, five animals were sacrificed by perfusion with paraformaldehyde. Then, the spinal cord was removed, and transverse cuts were made at the lumbosacral, thoracic, and cervical levels. These were processed by immunohistochemistry for the detection of viral antigens. Results: The first antigens of rabies were observed as aggregated particles in the lumbar spinal cord at 24 hours post-inoculation, within the ventral horn in the same side of the inoculated limb. At 32 hours post inoculation the first motoneurons immunoreactive to the virus became visible. At 40 hours postinoculation the first immunoreactive neurons were revealed in the thoracic level, located on lamina 8 and at 48 hours post-inoculation in the cervical cord, also on lamina 8. At 56 hours post-inoculation the virus had spread throughout the spinal cord, but the animals still did not show signs of the disease. Conclusion: In the mouse model we used, the rabies virus entered the spinal cord through the motoneurons and probably used the descending propriospinal pathway for its retrograde axonal transport to the encephalus.

Differential Effect of the Route of Inoculation of Rabies Virus on NeuN Immunoreactivity in the Cerebral Cortex of Mice

La rabia es una enfermedad mortal causada por un virus neurotropico que produce discretos cambios morfologicos dificilmente observables con la histopatologia convencional. El desenlace fatal causado por la rabia puede atribuirse a cambios bioquimicos especificos que afectan gravemente la funcion neuronal. La proteina nuclear neuronal (NeuN) es un marcador ampliamente utilizado para la investigacion y el diagnostico histopatologico de enfermedades del sistema nervioso. Este trabajo se realizo con el proposito de evaluar la distribucion de la proteina NeuN en la corteza motora de ratones normales y ratones infectados con virus de la rabia. Ratones ICR adultos fueron inoculados con virus de la rabia por via intramuscular o por via intracerebral. Los animales infectados con rabia fueron sacrificados en la etapa terminal de la enfermedad. Ratones de la misma edad no inoculados con el virus (controles) fueron sacrificados simultaneamente. Se extrajeron los cerebros y se obtuvieron cortes coronales en un vibratomo. Mediante inmunohistoquimica se estudio la expresion de NeuN en el area motora de la corteza cerebral. Se realizaron conteos neuronales, densitometria optica celular y mediciones del diametro de los perfiles neuronales para analizar la inmunorreactividad de la proteina. En los ratones inoculados por via intracerebral hubo disminucion significativa de la inmunorreactividad de NeuN manifestada en los diferentes parametros evaluados. En contraste, estos cambios no fueron estadisticamente significativos en los cerebros de ratones inoculados por la ruta intramuscular. La inmunorreactividad de NeuN o su expresion es afectada por la presencia del virus de la rabia en la corteza cerebral pero dependiendo de la via de inoculacion. Estos resultados contribuyen al conocimiento de las dinamicas de infeccion celular en la patogenesis de la rabia.

Efecto de la degradación post mórtem sobre la detección inmunohistoquímica de antígenos en el cerebro de ratón

Si bien lo ideal es llevar a cabo la preservacion de los tejidos en el menor tiempo posible luego de la muerte de un animal objeto de un estudio neurohistoquimico, con frecuencia es inevitable trabajar con tejido nervioso obtenido varias horas post mortem.

Protocol for the combination of neurohistological techniques on vibratome obtained sections

INTRODUCTION The histological study of the nervous system requires the use of special techniques. Currently, no methods are available to visualize simultaneously all the cellular constituents of nervous tissue. OBJECTIVES A protocol was adapted for staining nervous tissue by modification of a formerly difficult procedure. MATERIALS AND METHODS Slices of brain and spinal cord, 4 mm thick, were taken from adult mice, previously fixed by intracardiac perfusion with 4% paraformaldehyde. Vibratome sections were obtained with thickness of 15-25 µm. These were mounted on glass slides prepared with gelatin as an adhesive. The preparations were subjected to staining protocol Luxol Fast Blue-PAS-hematoxylin (LPH) combined with silver staining method (LPH-Holmes). RESULTS LPH technique yielded an excellent differentiation of gray and white matter in all regions of the nervous system. A panoramic view of the gray matter was colored pink in contrast to the myelinated nerve fibers and tracts which were light blue. The combination LPH-Holmes retained the staining characteristics but significantly improved the demarcation of axons and tracts. CONCLUSIONS A protocol was standardized for the LPH and LPH-Holmes nervous tissue stains applied in combination to tissue slices obtained with a vibratome. The method was shorter, less wasteful and less expensive than the original and also better preserved the integrity of nervous tissue.