Immunoperoxidase tracing of Junin virus neural route after footpad inoculation.

To determine the pathway adopted by peripherally inoculated Junin virus (JV) to reach the CNS, rat tissues were serially harvested to trace the sequence of viral progression from right hind footpad to brain. Immunoperoxidase (PAP) labeling of viral antigen, concomitantly with infectivity assays and histological examination of each selected sample, were carried out. As from the 2nd week post-infection (pi), neurological disease inducing 100% mortality at 1 month was evident. At day 5 pi, viral antigen was first detected at footpad level in epidermic and dermic cells, as well as in neighbouring myocytes; labeled macrophages infiltrating small nerve branches were also disclosed. As from 10-15 days pi, viral antigen became apparent along ipsilateral sciatic nerve structures and within lumbar spinal ganglion neurons, followed by a fast viral spread throughout CNS neurons that involved spinal cord and brain. Concurrent histopathology featured minimal inflammatory reaction together with generalized astrocytic activation. Hematogenous viral transport was negligible, since JV was isolated much earlier and in higher infectivity titers in neural tissues than in blood. It may be concluded that after viral replication in footpad, JV neural route was demonstrated by its PAP labeling from peripheral nerves to cerebral cortex.

A triple staining procedure to evaluate phagocytic role of differentiated astrocytes.

To determine whether phagocytic activity is affected by a viral infection known to induce astrocyte differentiation, a triple procedure (PAP labeling for GFAP, PAS reaction for added baker's yeast cells and hematoxylin for nuclear staining of the whole monolayer) was applied to Junin virus-inoculated cultures, as well as matched controls. The three-step staining simplified yeast cell count for subsequent statistical analysis, which discerned preferential uptake by differentiated rather than immature astrocytes. Accordingly, greater cell maturation induced by Junin virus was concomitant with early enhancement of phagocytic activity.

Murine acinar pancreatitis preceding necrotizing myocarditis after Coxsackievirus B3 inoculation.

Balb/C weanling mice were inoculated intraperitoneally with a myocarditic variant of coxsackie-virus B3, with the aim of characterizing more fully the cell damage induced in the heart as well as in other organs. During the first week postinfection (pi), all animals developed acinar pancreatitis, followed by focal myocarditis. In accordance with the increasing infectivity titers, such progressive histopathological changes correlated with local viral replication. From day 4 pi, acinar degeneration accompanied by diffuse inflammatory exudate was observed in the pancreas, followed by fatty tissue replacement by day 8. In the heart, focal necrosis rather than inflammatory reaction first appeared at 4 days pi and became widespread by 6-8 days pi. Necrotic foci usually presented calcium deposits, with absence of myofibrils in the affected fibers. The fact that both periodic acid Schiff (PAS) and Best carmine staining remained positive even after diastase treatment ruled out basophilic necrosis. In summary, the pancreas appeared to be the site of primary viral replication leading to viremia.

Astrocytic reaction predominance in chronic encephalitis of Junin virus-infected rats.

Junin virus antigen distribution and astrocytic reaction to prolonged infection were characterized in rat brain by the PAP technique. During the acute stage of neurologic disease following intracerebral inoculation, Junin antigen was detected in 100% of animals, strongly in most neurons but also to a much lesser degree in scattered astrocytes, dropping to 20% of rats at 540 days postinfection. Initially labeled in all brain areas, viral antigen gradually disappeared from hippocampus but persisted irregularly in cerebral cortex, basal ganglia, Purkinje cells, pons, and medulla oblongata. Such a pattern suggests that specific neuronal subpopulations, in spite of apparently unaltered cell morphology, may persistently harbor the virus, leading on occasion to a delayed neurologic syndrome. During both the acute and chronic stages of disease, a mild inflammatory exudate was observed, characterized by the presence of T and B lymphocytes, as well as macrophages and unidentified round cells. GFAP immunostaining showed increased astrocytic reaction as infection lapsed into chronicity. Corpus callosum, hippocampus, and cerebellum exhibited the sharpest reactive astrocytosis, followed by basal ganglia, pons, and medulla oblongata, whereas in cerebral cortex it was considerably less. Astrocyte activation, which failed to correlate with viral antigen presence in neurons, seems to result from a generalized condition, possibly including diffusible brain factors triggered by viral infection. Such widespread astroglial reaction may thus contribute to the outcome of the late neurologic syndrome.

A controlled silver impregnation method to characterize cultured cardiomyocytes.

A morphological characterization of cultured cardiomyocytes was attempted using a modification of a silver impregnation technique originally described for connective tissue. Cardiac cells, obtained from newborn rats and grown as dissociated cultures on plastic surfaces, were fixed in methanol plus 5% glacial acetic acid, treated with potassium permanganate, decolorized in oxalic acid, sensitized with potassium bichromate, impregnated with a silver-ammonium complex, reduced in gelatin-formalin preparation, toned with gold chloride and fixed in sodium thiosulfate. The cultured cardiac cells tended to form a monolayer, although many myocytes remained isolated. Spherical nuclei, sharply stained with silver, were centrally located and surrounded by relatively plentiful cytoplasm packed with well delineated myofibrils. Contaminating fibroblasts were readily distinguished by their spindle-shaped nuclei and the presence of overstained collagen fibers, as well as the absence of myofibrils. In the absence of specific antibody for immunocytochemical identification of cardiomyocytes, morphological characterization of cell type and degree of differentiation by the controlled silver impregnation procedure described here provides a viable alternative, both in short- and long-term studies.

PAP labeling enhancement by osmium tetroxide-potassium ferrocyanide treatment.

We describe a procedure to intensify staining of antigens labeled by the peroxidase-antiperoxidase (PAP) method. Routinely PAP-stained samples were enhanced by application of 5% osmium tetroxide for 30 min followed by freshly prepared 2% potassium ferrocyanide, plus 1% hydrochloric acid for 15 min. The staining color changed from the original golden-brown, through transient gray, to a very dark brown that was almost black. In addition to stronger labeling, antigen locations not apparent in untreated specimens may thus be disclosed. Furthermore, retrospective staining intensification can be performed in stored PAP-labeled samples even years later.

Junín virus persistence in mice.

Newborn mice surviving intracerebral infection with Junín virus (JV) strain XJ showed viral persistence in brain up to 140 days post-infection (p.i.). Mild meningoencephalitis or encephalitis, but not the neutralizing antibody titres (NtAb) correlated with virus presence.

Morphological characterization of brain cell cultures by controlled silver impregnation

Dada la heterogeneidad del tejido nervioso, el uso de cultivos neurales como modelo de estudios neurobiológicos exige una caracterización precisa, tanto de los tipos celulares presentes en las monocapas como de su grado de diferenciación. Si bien la microscopía de contraste de fase ha sido el recurso más empleado en el análisis morfológico de los cultivos neurales, es reconocida la superioridad de la tinción con plata. Con el objeto de lograr una más fina caracterización celular, recurrimos a una técnica que habíamos desarrollado años atrás para la impregnación argéntica de fibras colágenas y reticulares. El procedimiento debió adecuarse a su aplicación a neuronas y astrocitos cultivados y, por tanto, inmaduros o en vías de maduración. Los pasos fundamentales consistieron en el uso de dicromato de potasio como mordiente, en forma previa a la impregnación argéntica, la que era seguida por reducción en solución formólica de gelatina, que actuaba como coloide protector. Los lavados posteriores se realizaron con etanol 95- cuando se pretendía destacar neuronas, o con agua piridinada cuando se trataba de astrocitos. En cultivos derivados de encéfalo de embrión de ratón, la plata precisó los variados tipos neuronales y su grado de maduración morfológica. Asimismo, en cultivos obtenidos de cerebro de ratón recien nacido, se logró caracterizar las sucesivas etapas de diferenciación astrocitaria. En neuronas y astrocitos madurados, la identificación fue confirmada por marcación (método PAP) de enolasa específica de neurona y proteína gliofibrilar ácida, respectivamente. La técnica propuesta, que puede definirse como impregnanción argéntica controlada, parece ser indicada para la caracterización morfológica de células neurales en cultivo, particularmente antes de la expresión de los marcadores específicos en toda la extensión de la monocapa (AU)

Experimental study of the fibroblastic reaction induced by carbon fiber

En años recientes, ha sido propuesto el uso de fibras de carbono en cirugía reparadora de tendones y ligamentos, ya que dichas fibras incitaban una intensa reacción del tejido conjuntivo, con sustitución de las estructuras lesionadas y recuperación de la función afectada. El objeto del trabajo fue el análisis histológico de esa reacción fibroblástica bajo las siguientes condiciones experimentales: a) sistema in vivo: reemplazo quirúrgico total del tendón de Aquiles de ratas adultas por un grueso haz de fibras de carbono operando como tutor, y estudio histológico de la zona operada; b) sistema in vitro: en tubos Leighton, siembra de fibroblastos de embrión de rata acompañados de pequeños segmentos de fibras de carbono, con cosecha a los 1, 2 y 6 días de incubación a 37-C y examen ulterior por microscopías ópticas y electrónica de barrido. El estudio histológico de la zona operada a los 30 días de la intervención, mostró franca reacción tisular, caracterizada por aparición de células gigantes multinucleadas fagocitando pequeños restos de fibras de carbono, acompañadas de gran proliferación de capilares sanguíneos, fibroblastos y fibras colágenas. La reacción fue mucho más acentuada a los 90 y 120 días de la cirugía, observándose que amplios sectores antes ocupados por fibras de carbono habían sido reemplazados por masas de tejido conjuntivo fibroso. En cultivos celulares, la microscopía óptica mostró que los fibroblastos multiplicaban adhiréndose a las fibras de carbono. En muestras análogas, la microscopía electrónica de barrido precisó las características de... (AU)

Experimental study of the fibroblastic reaction induced by carbon fiber

En años recientes, ha sido propuesto el uso de fibras de carbono en cirugía reparadora de tendones y ligamentos, ya que dichas fibras incitaban una intensa reacción del tejido conjuntivo, con sustitución de las estructuras lesionadas y recuperación de la función afectada. El objeto del trabajo fue el análisis histológico de esa reacción fibroblástica bajo las siguientes condiciones experimentales: a) sistema in vivo: reemplazo quirúrgico total del tendón de Aquiles de ratas adultas por un grueso haz de fibras de carbono operando como tutor, y estudio histológico de la zona operada; b) sistema in vitro: en tubos Leighton, siembra de fibroblastos de embrión de rata acompañados de pequeños segmentos de fibras de carbono, con cosecha a los 1, 2 y 6 días de incubación a 37-C y examen ulterior por microscopías ópticas y electrónica de barrido. El estudio histológico de la zona operada a los 30 días de la intervención, mostró franca reacción tisular, caracterizada por aparición de células gigantes multinucleadas fagocitando pequeños restos de fibras de carbono, acompañadas de gran proliferación de capilares sanguíneos, fibroblastos y fibras colágenas. La reacción fue mucho más acentuada a los 90 y 120 días de la cirugía, observándose que amplios sectores antes ocupados por fibras de carbono habían sido reemplazados por masas de tejido conjuntivo fibroso. En cultivos celulares, la microscopía óptica mostró que los fibroblastos multiplicaban adhiréndose a las fibras de carbono. En muestras análogas, la microscopía electrónica de barrido precisó las características de...

Morphological characterization of brain cell cultures by controlled silver impregnation

Dada la heterogeneidad del tejido nervioso, el uso de cultivos neurales como modelo de estudios neurobiológicos exige una caracterización precisa, tanto de los tipos celulares presentes en las monocapas como de su grado de diferenciación. Si bien la microscopía de contraste de fase ha sido el recurso más empleado en el análisis morfológico de los cultivos neurales, es reconocida la superioridad de la tinción con plata. Con el objeto de lograr una más fina caracterización celular, recurrimos a una técnica que habíamos desarrollado años atrás para la impregnación argéntica de fibras colágenas y reticulares. El procedimiento debió adecuarse a su aplicación a neuronas y astrocitos cultivados y, por tanto, inmaduros o en vías de maduración. Los pasos fundamentales consistieron en el uso de dicromato de potasio como mordiente, en forma previa a la impregnación argéntica, la que era seguida por reducción en solución formólica de gelatina, que actuaba como coloide protector. Los lavados posteriores se realizaron con etanol 95- cuando se pretendía destacar neuronas, o con agua piridinada cuando se trataba de astrocitos. En cultivos derivados de encéfalo de embrión de ratón, la plata precisó los variados tipos neuronales y su grado de maduración morfológica. Asimismo, en cultivos obtenidos de cerebro de ratón recien nacido, se logró caracterizar las sucesivas etapas de diferenciación astrocitaria. En neuronas y astrocitos madurados, la identificación fue confirmada por marcación (método PAP) de enolasa específica de neurona y proteína gliofibrilar ácida, respectivamente. La técnica propuesta, que puede definirse como impregnanción argéntica controlada, parece ser indicada para la caracterización morfológica de células neurales en cultivo, particularmente antes de la expresión de los marcadores específicos en toda la extensión de la monocapa

Chronic neurologic disease in Junín virus-infected rats.

The purpose of this study was to determine whether Junín virus persistence in CNS of rats was capable of inducing late neurologic disease. Following intracerebral inoculation of newborn animals with XJ strain, three distinct stages could be discerned: an early phase of acute disease, up to 30 days pi, with 5% mortality; an intermediate one, extending to 280 days pi, without clinical signs but with evident viral persistence; and a final period of chronic illness, featuring clinical neurologic syndrome, severe perivascular inflammatory reaction, PAP-labeled viral antigen in a few cerebral and cerebellar neurons, and virus recovery only by coculture. Late neurologic disease seems associated to the lack of effective clearance of brain virus, leading to viral persistence and long lasting immunologic stimulation. The importance of animal models for pathogenic studies on CNS persistent viral infections leading to late neurologic disease is stressed.

Brain inflammatory exudate in Junin virus-infected rats: its characterization by the immunoperoxidase (PAP) technique.

Morphologic changes in cyclophosphamide (CY)-suppressed vs. control non-suppressed new-born rats infected i.c. with XJC13 strain of Junin virus were compared and the cells involved in CNS lesions were identified by the PAP technique. Fifty per cent of the control rats exhibited widespread cerebral necrosis vs. only 15% of the immunosuppressed animals. The first cells to reach Junin virus-infected CNS in controls were T lymphocytes, which destroyed viral antigen-laden target neurons and astrocytes. B lymphocytes and macrophages, presumably attracted by viral antigen and/or by lymphokines, made their appearance a day or two later. Activated macrophages phagocytosed necrotic cells and perhaps exerted a cytotoxic effect upon target neural cells, whereas the actual role of B lymphocytes requires further explanation. In CY-treated rats, cerebral lesions were smaller and the cellular exudate, though similar, proved much scantier than in controls. A similar extent of cerebellar necrosis was observed in both groups.

Modification of Junin virus neurotropism in mice by selective brain or spinal cord passaging.

The percentage of suckling mice that developed paralysis after intracerebral Junin virus (XJ-JV pathogenic strain) inoculation (13.8%) consistently increased after 5 serial passages of virus-infected brain or spinal cord obtained from paralytic animals, reaching 37.9 and 45.7%, respectively. As expected, all paralytic mice exhibited an identical spinal cord histologic picture, with widespread JV antigen in spinal cord astrocytes and neurons, particularly the large motor neurons of the anterior horn. These findings strongly support the existence of a motor neurotropic viral particle subpopulation in parental XJ-JV stock.

Autonomic nervous system involvement in experimental genital infection by herpes simplex virus type 2.

Peroxidase-antiperoxidase technique and histology were employed to elucidate the peripheral routes involved in HSV-2 progression from vagina towards the central nervous system in mice. 12 week-old female Balb/c mice were intravaginally infected with 5 X 10(5)LD50 of HSV-2. Sixty per cent of animals developed vulvovaginitis, perigenital alopecia and hind-limb paresia. Death occurred at 9-11 days post-infection. Colon dilatation and urinary bladder distention were observed in all cases. Complete transversal sections from vulva to kidneys were obtained of each animal, including the spinal cord in situ. Herpes antigen were regularly detected in vulvovaginal epithelium, intramural, perigenital and perivesical small nerves. Besides, their invariable presence in Auerbach's plexus and sympathetic ganglia, strongly suggests preferential autonomic nervous system involvement in the progression of HSV-2 intravaginal infection towards the spinal cord.

Effect of staggered cyclophosphamide-immunosuppression on resistance to experimental Junin virus infection.

Otherwise resistant adult mice were rendered susceptible to intracerebral Junin virus (JV) infection only when a staggered cyclophosphamide (CY) schedule was used. Forty-five-day old Balb/c mice, intracerebrally JV-infected and immunosuppressed with four 50 mg/kg body weight CY doses at days -1, +1, +4, +6 (day 0: viral infection) developed a lethal disease (86.6 per cent mortality) with high CNS viral titers and brain lesions. Neutralizing antibodies were absent throughout, while immunofluorescent antibody levels were considerably diminished. The transfer of hyperimmune serum conferred partial though significant protection on CY-treated animals but no correlation was found between CNS viral titers and mortality since in both infected CY-treated and untreated mice similar brain viral content was found. This was also confirmed by immune spleen cell transfer at day 0 where the clearance achieved was unable to modify the time course of the disease. Feasible mechanisms explaining recovery from JV infection by means of the protective effect of antibodies and the cell-mediated clearance are discussed.