Fabián Nishida

Effects of Solanum glaucophyllum toxicity on cell proliferation and apoptosis in the small and large intestine of rabbits

Abstract Vitamin D regulates mineral homeostases and enterocyte proliferation and differentiation. Hypervitaminosis D generates changes in cell proliferation, differentiation and apoptosis in several organs. We analysed morphometric parameters and proliferative and apoptotic indices in the intestinal epithelium of rabbits with hypervitaminosis D induced by the chronic treatment with the calcinogenic plant Solanum glaucophyllum. Rabbits were treated for 15 or 30days. A group was treated for 15days and led to possible recovery for 30days. Another group was nutritionally restricted for 30days. Morphological, morphometric, proliferative and apoptotic changes were found in the treated animals. Mild atrophy and reduced proliferation was found in the jejunum and ileum. Apoptosis increased in the crypts of the ileum and in the superficial epithelium and crypts of the rectum. Most of the alterations were partially recovered. The possible involvement in these changes of the hypervitaminosis D-like state induced by S. glaucophyllum is discussed.

Increased Number of Neurons in the Cervical Spinal Cord of Aged Female Rats

In the brain, specific signaling pathways localized in highly organized regions called niches allow the persistence of a pool of stem and progenitor cells that generate new neurons in adulthood. Much less is known about the spinal cord where a sustained adult neurogenesis is not observed. Moreover, there is scarce information concerning cell proliferation in the adult mammalian spinal cord and virtually none in aging animals or humans. We performed a comparative morphometric and immunofluorescence study of the entire cervical region (C1-C8) in young (5 mo.) and aged (30 mo.) female rats. Serum prolactin (PRL), a neurogenic hormone, was also measured. Gross anatomy showed a significant age-related increase in size of all of the cervical segments. Morphometric analysis of cresyl violet stained segments also showed a significant increase in the area occupied by the gray matter of some cervical segments of aged rats. The most interesting finding was that both the total area occupied by neurons and the number of neurons increased significantly with age, the latter increase ranging from 16% (C6) to 34% (C2). Taking the total number of cervical neurons the age-related increase ranged from 19% (C6) to 51% (C3), C3 being the segment that grew most in length in the aged animals. Some bromodeoxyuridine positive-neuron specific enolase negative (BrdU+-NSE−) cells were observed and, occasionally, double positive (BrdU+-NSE+) cells were detected in some cervical segments of both young and aged rats groups. As expected, serum PRL increased markedly with age. We propose that in the cervical spinal cord of female rats, both maturation of pre-existing neuroblasts and/or possible neurogenesis occur during the entire life span, in a process in which PRL may play a role.

Neurons of the rat cervical spinal cord express vimentin and neurofilament after intraparenchymal injection of kainic acid

Intermediate filaments (IF) can be altered under disorders such as neurodegenerative diseases. Kainic acid (KA) induce behavioral changes and histopathological alterations of the spinal cord of injected rats. Our goal was to evaluate the IF expression in neurons during this injury model. Animals were injected with KA at the C5 segment of the cervical spinal cord and euthanized at 1, 3 and 7 post injection (pi) days. Neuronal cell counting showed a significant loss of neurons at the injection site when compared with those of sham and non-operated animals. Immunohistochemistry for vimentin and neurofilament showed positive labeling of perikarya in sham and KA-injected animals since day 1 pi that lasted for the remaining experimental days. Colocalization analysis between enolase and vimentin or neurofilament confirmed a high index of colocalization in both experimental groups at day 1 pi. This index decreased in sham animals by day 3 pi whereas that of KA-injected animals remained high throughout the experiment. These results may suggest that perikarya initiate an unconventional IF expression, which may respond to the neuronal damage induced by the mechanical injury and the excitotoxic effect of KA. It seems that vimentin and neurofilament expression may be a necessary change to promote recovery of the damaged tissue.

Effects of equid herpesvirus 1 (EHV-1) AR8 and HH1 strains on BALB-c mice

Here, we used a murine model to describe and compare the pathogenic potential of the Argentinean equid herpesvirus 1 (EHV-1) AR8 strain with the Japanese HH1 reference strain. In AR8-inoculated animals, clinical signs began earlier, but the viremic phase was shorter. Virus isolation and DNA detection in the lungs, liver and spleen were positive for both strains at different times postinfection (pi). Infection foci produced by both strains were immunohistochemically detected in lungs from day 1 to day 4 pi. We conclude that whichever EHV-1 strain is selected to experimentally reproduce the disease, it needs appropriate standardization in order to provide valid conclusions.

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Abstract Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.

Effects of an Intraparenchymal Injection of Lidocaine in the Rat Cervical Spinal Cord

Lidocaine effects in the spinal cord have been extensively investigated over the years. Although the intrathecal route is usually used to treat insults occurring in the spinal cord, the local delivery drug via intraparenchymal infusions has gained increasing favor for the treatment of some neurodegenerative disorders. The aim of the present study was to evaluate the behavioral and tissue effects of the intraparenchymal injection of different concentrations of lidocaine into the rat cervical spinal cord. Young male Sprague–Dawley rats were intraparenchymally injected with 0.5%, 1% or 2% lidocaine at the C5 segment of the spinal cord. Other rats were injected with saline solution (sham group). Hot plate test was determined at 0, 1, 2, 3, 7 and 14 post-injection (pi) days. Rats of each experimental group were euthanized either at 1, 2, 3, 7 or 14 pi days. Intact animals were used as controls. Sections of the C5 segment were used for histological, immunohistochemical or immunofluorescence analysis. Injection of 0.5% lidocaine did not affect neuronal counting, did not evoke an inflammatory reaction, nor induce astrocyte activation. Therefore, a concentration of 0.5% lidocaine is suggested to promote anti-inflammatory effects after injury.

Association between Degree of Anaplasia and Degree of Inflammation with the Expression of COX-2 in Feline Injection Site Sarcomas

Feline injection site sarcomas (FISSs) are mesenchymal neoplasms that develop at the sites of delivery of vaccines or other injectable products. Vaccine adjuvants can trigger an intense and persistent inflammatory response that may lead to neoplastic transformation. The proinflammatory role of cyclo-oxygenase (COX)-2 is well known and its overexpression has prognostic value in multiple neoplastic processes. One hundred and seventeen FISSs were evaluated for the degree of inflammation and anaplasia. Immunohistochemistry was used to determine the expression of COX-2 in these sarcomas. There was a significant association between the degree of inflammation and the expression of COX-2 by neoplastic cells. COX-2 expression was lower in tumours with higher degrees of anaplasia. These findings may be useful in predicting the sensitivity of FISSs to treatment with COX-2 inhibitors. The potential therapeutic use of such agents could then be restricted to tumours with lower degrees of anaplasia.

Uso de la técnica CLARITY para la identificación de marcadores fluorescentes en cortes gruesos de la médula espinal

espanolEl estudio de las interconexiones que establecen las neuronas entre si o con las celulas de la glia es uno de los mayores desafios de la neurociencia. Los anticuerpos fluorescentes permiten la identificacion y localizacion de diferentes estructuras tisulares. Sin embargo, su uso eficiente requiere de la utilizacion de cortes delgados de muestras, que constituyen una limitacion para el estudio de las interconexiones en el sistema nervioso. Asimismo, un mayor espesor de la muestra limita la penetracion de la luz emitida por el microscopio, mientras que la opacidad propia del tejido nervioso debida a su alto contenido lipidico dificulta la adquisicion de las imagenes al restringir la resolucion de los objetos. La tecnica CLARITY (del ingles, Clear, Lipidexchanged, Acrylamidehybridized Rigid, Imaging/Immunostaining/In situ hybridizationcompatible, Tissue hYdrogel) permite subsanar estos inconvenientes. Esta tecnica fue adaptada en nuestro laboratorio para el estudio de la medula espinal de rata. De acuerdo con el procedimiento realizado, pudimos obtener un organo completamente translucido, estructuralmente intacto y que permitio su procesamiento mediante tecnicas de inmunofluorescencia y de lectinhistoquimica sin mayores dificultades. A partir de muestras de mas de 1 mm de espesor se obtuvieron imagenes confocales de gran resolucion y de mayor penetrabilidad que las que se obtienen utilizando las tecnicas de procesamiento convencionales. La implementacion de esta tecnica en nuestro laboratorio permitira optimizar la informacion obtenida a partir de muestras de interes. EnglishThe study of the interconnections established between neurons themselves and with glial cells is one of the major challenges of neuroscience. Fluorescent antibodies allow identification and localization of different tissue structures. However, their efficient employment requires the use of thin sections of samples, which constitute a limitation for the study of nervous system interconnections. Likewise, increased thickness of samples limits penetration of the light emitted by the microscope, whereas the opacity of the nervous tissue due to its high lipid content limits the resolution of objects, making the acquisition of images difficult. The CLARITY (Clear, Lipidexchanged, Acrylamidehybridized Rigid, Imaging/Immunostaining/In situ hybridizationcompatible, Tissue hYdrogel) technique makes possible to remedy these disadvantages. This technique was adapted in our laboratory for the study of the rat spinal cord. According to the described procedure we obtained a completely translucid and structurally intact organ that allowed processing through immunofluorescence and lectinhistochemistry techniques without major drawbacks. Confocal images of higher resolution and greater penetrability in comparison with those captured using conventionalprocessing techniques were obtained from more than 1 mm thick samples. The implementation of this technique in our laboratory will improve the information obtained from our samples of interest.

Characterization of paneth cells in alpacas (Vicugna pacos, Mammalia, Camelidae).

Abstract Paneth cells are secretory epithelial cells of the innate immune system of the intestine of several mammals, including alpacas. Little is known about the latter; thus, in the present study we described the morphology and histochemical characteristics of Paneth cells in healthy fetuses, and young and adult alpacas. For this purpose, samples of duodenum, jejunum and ileum were taken from 6 fetuses at different days of pregnancy (between days 221–330), 66 offsprings (between 0 and 45-days-old) and 5 adult alpacas (>2-years-old). Samples were fixed in 10% buffered formalin and processed for histological and morphometrical analysis using HE and Masson Trichomićs technique. Immunohistochemistry was used to identify Paneth cells using anti-lysozyme antibody. In addition, the lectinhistochemichal binding-pattern of Paneth celĺs granules was evaluated. Lyzozyme was immunohistochemically detected in the granules of Paneth cells from day 283 of pregnancy in all the small intestinal sections of the studied fetuses. In newborn alpacas Paneth cells were initially found in the duodenum, but the following days (days 18–21 after birth) they were also found in the ileum. Their size gradually increased after birth, but then no significant differences were found. In adult alpacas the number was lower than offsprings. We suggest that Paneth cells early differentiate in the small intestine of alpacas, and the increase in their number during the first two weeks of life strongly support their possible involvement in the intestinal defensive functions against the enteric diseases that occur during the lactancy stage.

Microvascular lesions and changes in cell proliferation and death, and cytokine expression in the placentas of mice experimentally infected with Equid Herpesvirus 1

This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.