Angela María Suburo

Lipopolysaccharide Directly Affects Pancreatic Acinar Cells

We have explored whether lypopolisaccharide (LPS, endotoxin) induces pancreatic injury on pancreatic acinar cells both in vivo and in vitro. Wistar male rats were treated with four intraperitoneal injections of 10 mg/kg LPS, and AR4-2J cells were exposed to increasing doses of LPS. Expression of pancreatitis-associated-protein (PAP) mRNA was strongly induced in AR4-2J cells exposed to LPS, while amylase mRNA was reduced. LPS also induced apoptosis and expression of TNF-α, IL-1β, and IL-8 mRNA in AR4-2J cells. The in vivo effect of LPS showed structural signs of cellular damage, including numerous cytoplasmic vacuoles, severe nuclear alterations, and high expression of PAP mRNA. This study demonstrated that LPS induced pancreatic damage by directly affecting the pancreatic acinar cells. The role of LPS in the pathophysiology of acute pancreatitis may be partly due to the effect LPS has on the acinar cell.

Cell interactions and the regulation of cholinergic enzymes during neural differentiation in vitro

Abstract Seven-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rembencephalon (Ro) were dissociated, and the resulting cell suspensions were allowed to reaggregate in vitro during 3 days either independently or in different binary combinations. Interactions could be detected by the comparison of the activity of the enzymes of the cholinergic system, choline acetyltransferase (CAT) and acetylcholinesterase (ACE), in “pure” and “combined” aggregates. The results clearly show that the activity of both enzymes in embryonic neural cells can be modified selectively by interactions between different cell populations. Thus, combined NR-OL aggregates show an increase in CAT without changes in ACE, NR-T an increase in CAT and a decrease in ACE, T-Ro a decrease in both CAT and ACE, and OL-T no changes at all. Experiments in which NR and OL cells were combined in different proportions indicate that the interactions require the presence of defined numbers of cells from each kind. Isochronous and heterochronous combinations of 7- and 10-day-old NR and OL cells show that the interactive capacities of the cells change with development.

Electron staining of synaptic vesicles using the Champy-Maillet technique

Synaptic vesicles from rat pineal nerves can be stained by Champy‐Maillet mixtures containing osmium tetroxide and a soluble iodide. Two components of the vesicle (the matrix and the dense core) are revealed in a different way depending on at least two factors: the iodide employed and the final pH of the mixture. We have tried the following mixtures: zinc iodide at pH 5·5 (ZIO), cadmium iodide at pH 6·0 (CIO) and potassium iodide at pH 7·2 (KIO‐7) or pH 5·5 (KIO‐5). The matrix is more reactive with ZIO, while the dense core is more reactive with CIO and especially with KIO‐7. With the only difference of a lower pH, KIO‐5 stains the whole matrix of some vesicles and also reveals some tubular structures which are more frequently found in preterminal axons.

Differentiation between 5-Hydroxytryptamine and Catecholamines in Synaptic Vesicles

Publisher Summary The results presented in this chapter show that it is possible to differentiate between catechol and indolamines in synaptic vesicles. Two methods have been successfully assayed in the pineal nerves of the rat, one of them based on the use of current fixation methods combined with pharmacological treatment and the other using a histochemical technique. After both fixation methods, the typical population of clear and granulated vesicles could be observed with minor differences such as a greater density of the intermediate type of vesicles after glutaraldehyde-osmium tetroxide and darker and better defined cores of the small vesicles after osmium tetroxide alone. However, the heterogeneity of monoamine-containing vesicles as revealed by current and special fixation methods indicates that these results cannot be extrapolated to other granulated vesicles without demonstration. The chapter also shows that different fixatives may preserve different components in synaptic vesicles even if they give a similar picture at the electron microscopic level. Not only employed the fixative but also the used precise schedule may involve substantial differences in the preservation of monoamines. Differences in binding may be an important parameter in the localization of monoamines in synaptic vesicles.

Neuronal and glial differentiation in reaggregation cultures

SummaryDissociation and reaggregation cultures from different portions of the chick embryo neural tube were made, and the resulting aggregates were fixed for electron microscopy after 1, 5, 8, 14, 16 and 22 days in vitro. All cultures (pure aggregates of telencephalon, optic lobe or neural retina, and combined aggregates made from mixtures of optic lobe plus neural retina or optic lobe plus telencephalon) show a common timing of neuronal and glial morphological differentiation.During the first week in vitro, some cells developed neuronal characteristics in the absence of morphological evidence of glial differentiation. Numerous axonic processes usually formed fascicles with all the fibers running parallel to each other. Axonic growth cones were abundant and a few immature synapses were also present. The second week in culture was characterized by the disappearance of growth cones and the increase in number and morphological maturation of synapses. Morphologically detectable glial differentiation began by the end of this week, and during the third week almost every neuronal element, including the axonic fascicles, became associated with glial cells showing astrocytic features.

Effect of tyramine on the compartments of the granulated vesicles in rat pineal nerve endings

Abstract Granulated vesicles in rat pineal nerve endings consist of 2 compartments; the core and the matrix. The core can be stained by a variety of techniques including osmium tetroxide alone and a mixture of cosmium tetroxide-zinc iodide (ZIO), and gives histochemical reactions for cathecol and indolamines. The matrix can be stained with ZIO. To determine whether the matrix ZIO-reactive material represents a store of noradrenaline, different amounts of tyramine were adminisered i.p. in 50 mg/kg doses at 15-min intervals. The pineal glands were then studied under the electron microscope using the above techniques. Noradrenaline was determined fluorimetrically. It was observed that after the first dose of tyramine, no significant change in the noradrenaline content occurred although the matrix ZIO-reactive material decreased by 80%; this strongly suggests that the matrix ZIO-reactive material is not a noradrenaline store but represents some other component of the vesicle which is also depleted by tyramine. In contrast, the core components revealed by the techniques used disappeared gradually.

Action ofp-chlorophenylalanine on the synaptic vesicles from rat pineal nerves

Se demuestra que lap-clorofenilalanina depleciona los componentes de las vesiculas sinapticas de los nervios pineales de la rata revelables por la mezcla tetroxido de osmio-yoduro de zinc pero no afecta a los revelables por tetroxido de osmio-yoduro de potasio.Se demuestra que lap-clorofenilalanina depleciona los componentes de las vesículas sinápticas de los nervios pineales de la rata revelables por la mezcla tetróxido de osmio-yoduro de zinc pero no afecta a los revelables por tetróxido de osmio-yoduro de potasio.

Presynaptic tubular structures in photoreceptor cells.

SummaryAfter the application of fixatives including phosphotungstic acid or a mixture of osmium tetroxide and zinc iodide, complex tubular structures are evident in the presynaptic side of the synapses between photoreceptor and bipolar cells of the rats retina. In the first case only the limiting membranes are visualized, while in the second only the content of the tubules is stained. These tubules seem to be related, on a morphological ground, with the formation of synaptic vesicles. These tubular structures are not observed when fixation is done with osmium tetroxide or glutaraldehyde-osmium tetroxide.

Neural differentiation in aggregates containing mixtures of cell types.

Se describe la diferenciación ultraestructural alcanzada en cultivos de reagregación por células neurales embrionarias despues de 8 días in vitro. Se estudiaron agregados «puros» formados por células de lóbulo óptico, retina neural o telencéfalo, y cultivos «combinados» de lóbulo óptico+retina neural (OL+NR), lóbulo óptico+telencéfalo (OL+T), y lóbulo óptico+esbozo de miembro (OL+LB). En los cultivos «puros» las céculas poseen caracterśticas de neuroblastos, pero el proceso de sinaptogénesis está poco desarrollado. No se encuentran diferencias significativas entre la diferenciación alcanzada por los cultivos «puros» y los «combinados» OL+NR y OL+T. Por el contrario, en los «combinados» OL+LB la sinaptogénesis está mucho más avanzada y se encuentran prolongaciones sinápticas semejantes a las del adulto.

Osmium tetroxide-zinc iodide reactive sites in the photoreceptor cells

Abstract In a previous study it had been shown that after osmium tetroxide-zinc iodide (ZIO) fixation there are heavy electron dense deposits in the outer segments of rat photoreceptor cells. A positive reaction can also be observed in synaptic vesicles from the photoreceptor cells. Our observations in the retina of the pigeon seem to indicate that the reaction is confined to the rod outer segments, as no electron dense deposits could be observed in the cone outer segments. ZIO reactive synaptic vesicles can also be observed in the photoreceptor nerve endings of the pigeon. To study the light sensitivity of the ZIO reactive material present in the rod outer segment, we fixed the retina of rats subjected to 96 hr of either continuous illumination or darkness. It was observed that the electron dense deposits are more concentrated after continuous illumination. The reactivity of different substances with the ZIO mixture was studied in vitro . Considerations are made about the possible relationship between ZIO reactive material and the visual pigments.