Pomegranate extract in polyphosphate-fluoride mouthwash reduces enamel demineralization.

OBJECTIVES: To evaluate the anti-demineralizing effect of a mouthwash comprising pomegranate peel extract (PPE 3%), sodium trimetaphosphate (TMP 0.3%), and fluoride (F 225 ppm) in an in situ study, and to assess its irritation potential in an ex vivo study. METHODS: This double-blind crossover study was conducted in four phases with 7 days each. Twelve volunteers used palatal appliances containing enamel blocks, which were subjected to cariogenic challenges. The ETF formulation (PPE + TMP + F, pH 7.0), TF formulation (TMP + F, pH 7.0), deionized water (W, pH 7.0), and essential oil commercial mouthwash (CM, 220 ppm F, pH 4.3) were dropped onto the enamel twice daily. The percentage of surface hardness loss, integrated loss of subsurface hardness, calcium, phosphorus, and fluoride in enamel and biofilms were determined. In addition, alkali-soluble extracellular polysaccharide concentrations were analyzed in the biofilms. The irritation potential was evaluated using the hen's egg chorioallantoic membrane test through the vascular effect produced during 300-s of exposure. RESULTS: ETF was the most efficacious in preventing demineralization. It also showed the highest concentrations of calcium and phosphorus in the enamel and in the biofilm, as well as the lowest amount of extracellular polysaccharides in the biofilm. In the eggs, ETF produced light reddening, whereas CM led to hyperemia and hemorrhage. CONCLUSIONS: The addition of PPE to formulations containing TMP and F increased its anti-demineralizing property, and this formulation presented a lower irritation potential than the CM. CLINICAL RELEVANCE: ETF can be a promising alternative alcohol-free mouthwash in patients at high risk of caries.

Critical Evaluation of Embedding Media for Histological Studies of Early Stages of Chick Embryo Development.

A histological examination is an important tool in embryology, developmental biology, and correlated areas. Despite the amount of information available about tissue embedding and different media, there is a lack of information regarding best practices for embryonic tissues. Embryonic tissues are considered fragile structures, usually small in size, and frequently challenging to position correctly in media for the subsequent histological steps. Here, we discuss the embedding media and procedures that provided us with appropriate preservation of tissue and easier orientation of embryos at early development. Fertilized Gallus gallus eggs were incubated for 72 h, collected, fixed, processed, and embedded with paraplast, polyethylene glycol (PEG), or historesin. These resins were compared by the precision of tissue orientation, the preview of the embryos in the blocks, microtomy, contrast in staining, preservation, average time, and cost. Paraplast and PEG did not allow correct embryo orientation, even with agar-gelatin pre-embedded samples. Additionally, structural maintenance was hindered and did not allow detailed morphological assessment, presenting tissue shrinkage and disruption. Historesin provided precise tissue orientation and excellent preservation of structures. Assessing the performance of the embedding media contributes significantly to future developmental research, optimizing the processing of embryo specimens and improving results.

Cell Fate of Retinal Progenitor Cells: In Ovo UbC-StarTrack Analysis.

Clonal cell analysis outlines the ontogenic potential of single progenitor cells, allowing the elucidation of the neural heterogeneity among different cell types and their lineages. In this work, we analyze the potency of retinal stem/progenitor cells through development using the chick embryo as a model. We implemented in ovo the clonal genetic tracing strategy UbC-StarTrack for tracking retinal cell lineages derived from individual progenitors of the ciliary margin at E3.5 (HH21-22). The clonal assignment of the derived-cell progeny was performed in the neural retina at E11.5-12 (HH38) through the identification of sibling cells as cells expressing the same combination of fluorophores. Moreover, cell types were assessed based on their cellular morphology and laminar location. Ciliary margin derived-cell progenies are organized in columnar associations distributed along the peripheral retina with a limited tangential dispersion. The analysis revealed that, at the early stages of development, this region harbors multipotent and committed progenitor cells.

The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis.

The mechanisms involved in the development of skeletal muscle fibers have been studied in the last 70 years and yet many aspects of this process are still not completely understood. A myriad of in vivo and in vitro invertebrate and vertebrate animal models has been used for dissecting the molecular and cellular events involved in muscle formation. Among the most used animal models for the study of myogenesis are the rodents rat and mouse, the fruit fly Drosophila, and the birds chicken and quail. Here, we describe the robustness and advantages of the chick primary muscle culture model for the study of skeletal myogenesis. In the myoblast culture obtained from embryonic chick pectoralis muscle it is possible to analyze all the steps involved in skeletal myogenesis, such as myoblast proliferation, withdrawal from cell cycle, cell elongation and migration, myoblast alignment and fusion, the assembly of striated myofibrils, and the formation of multinucleated myotubes. The fact that in vitro chick myotubes can harbor hundreds of nuclei, whereas myotubes from cell lines have only a dozen nuclei demonstrates the high level of differentiation of the autonomous chick myogenic program. This striking differentiation is independent of serum withdrawal, which points to the power of the model. We also review the major pro-myogenic and anti-myogenic molecules and signaling pathways involved in chick myogenesis, in addition to providing a detailed protocol for the preparation of embryonic chick myogenic cultures. Moreover, we performed a bibliometric analysis of the articles that used this model to evaluate which were the main explored topics of interest and their contributors. We expect that by describing the major findings, and their advantages, of the studies using the embryonic chick myogenic model we will foster new studies on the molecular and cellular process involved in muscle proliferation and differentiation that are more similar to the actual in vivo condition than the muscle cell lines.

Characterization of embryonic surface ectoderm cell protrusions.

BACKGROUND: During embryonic development, complex changes in cell behavior generate the final form of the tissues. Extension of cell protrusions have been described as an important component in this process. Cellular protrusions have been associated with generation of traction, intercellular communication or establishment of signaling gradients. Here, we describe and compare in detail from live imaging data the dynamics of protrusions in the surface ectoderm of chick and mouse embryos. In particular, we explore the differences between cells surrounding the lens placode and other regions of the head. RESULTS: Our results showed that protrusions from the eye region in mouse embryos are longer than those in chick embryos. In addition, protrusions from regions where there are no significant changes in tissue shape are longer and more stable than protrusions that surround the invaginating lens placode. We did not find a clear directionality to the protrusions in any region. Finally, we observed intercellular trafficking of membrane puncta in the protrusions of both embryos in all the regions analyzed. CONCLUSIONS: In summary, the results presented here suggest that the dynamics of these protrusions adapt to their surroundings and possibly contribute to intercellular communication in embryonic cephalic epithelia.

Functional Analyses of Embryonic Cerebrospinal Fluid Proteins.

The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development.

MARCKS phosphorylation by PKC strongly impairs cell polarity in the chick neural plate.

Neurulation involves a complex coordination of cellular movements that are in great part based on the modulation of the actin cytoskeleton. MARCKS, an F-actin-binding protein and the major substrate for PKC, is necessary for gastrulation and neurulation morphogenetic movements in mice, frogs, and fish. We previously showed that this protein accumulates at the apical region of the closing neural plate in chick embryos, and here further explore its role in this process and how it is regulated by PKC phosphorylation. PKC activation by PMA caused extensive neural tube closure defects in cultured chick embryos, together with MARCKS phosphorylation and redistribution to the cytoplasm. This was concomitant with an evident disruption of neural plate cell polarity and extensive apical cell extrusion. This effect was not due to actomyosin hypercontractility, but it was reproduced upon MARCKS knockdown. Interestingly, the overexpression of a nonphosphorylatable form of MARCKS was able to revert the cellular defects observed in the neural plate after PKC activation. Altogether, these results suggest that MARCKS function during neurulation would be to maintain neuroepithelial polarity through the stabilization of subapical F-actin, a function that appears to be counteracted by PKC activation.

Chick Embryo Model for Homing and Host Interactions of Tissue Engineering-Purposed Human Dental Stem Cells.

Human dental stem cells (hDSC) have a potential for regenerative therapies and could differentiate in vitro into many tissues, such as dentin, nerve, and vascular endothelium. Gallus gallus domesticus developing fertilized egg or chick embryo is an experimental model absent of xenografts rejection, largely employed in replacement of mammal species in scientific research and preclinical studies to evaluate angiogenesis and vasculogenesis, tissue differentiation, and embryonic development. This multiscale research deals with the homing and cell signaling effects of a standardized hDSC toward the receptor tissues of G. gallus domesticus in ovo. The hDSC were obtained from the explantation from third molars, characterized by cell cytometry, and employed without any further purification procedure. Four experimental groups were studied, according to the kind of cell tracing strategy, named: Control, mCherry-labeled hDSC, QTracker-labeled hDSC, and QTracker-exposed controls. The eggs were kept in an incubator temperature of 37.6°C and humidity 86%, and the embryos were euthanized after 10 days of incubation. In vivo fluorescence and histological analysis were conducted. The fluorescence of the embryos inoculated with mCherry hDSC or the QTracker hDSC was associated with the bones and the beak tooth, and labeled cell islands could be localized in part of the samples. The inoculation of the QTracker probe resulted in proliferating bone tissue labeling. The hDSC inoculated groups presented cartilage plate hypertrophy and atypical morphology, meanwhile Control eggs were negative. The results demonstrated that hDSC can migrate to the cartilaginous tissues of the chick embryos, survive in this environment, implant, and interfere with the growth of developing bone.

Chick embryo model for homing and host interactions of tissue engineering-purposed human dental stem cells

Human dental stem cells (hDSC) have a potential for regenerative therapies and could differentiate in vitro into many tissues, such as dentin, nerve, and vascular endothelium. Gallus gallus domesticus developing fertilized egg or chick embryo is an experimental model absent of xenografts rejection, largely employed in replacement of mammal species in scientific research and preclinical studies to evaluate angiogenesis and vasculogenesis, tissue differentiation, and embryonic development. This multiscale research deals with the homing and cell signaling effects of a standardized hDSC toward the receptor tissues of G. gallus domesticus in ovo. The hDSC were obtained from the explantation from third molars, characterized by cell cytometry, and employed without any further purification procedure. Four experimental groups were studied, according to the kind of cell tracing strategy, named: Control, mCherry-labeled hDSC, QTracker-labeled hDSC, and QTracker-exposed controls. The eggs were kept in an incubator temperature of 37.6 degrees C and humidity 86%, and the embryos were euthanized after 10 days of incubation. In vivo fluorescence and histological analysis were conducted. The fluorescence of the embryos inoculated with mCherry hDSC or the QTracker hDSC was associated with the bones and the beak tooth, and labeled cell islands could be localized in part of the samples. The inoculation of the QTracker probe resulted in proliferating bone tissue labeling. The hDSC inoculated groups presented cartilage plate hypertrophy and atypical morphology, meanwhile Control eggs were negative. The results demonstrated that hDSC can migrate to the cartilaginous tissues of the chick embryos, survive in this environment, implant, and interfere with the growth of developing bone.

Tissue damage and embryonic malformation induced by aqueous extract of Pteridium aquilinum on chorioallantoic membrane of chick embryo (CAM) / Danos teciduais e mal formações morfológicas induzidas pelo extrato aquoso de Pteridium aquilinum sobre a membrana corioalantóica (CAM) e o embrião de galinha

The aim of this study was evaluate the effects of Bracken fern (BF) (Pteridium aquilinum (L.) Kuhn.) on biological systems. When consumed by animals can cause acute intoxication, hematuria, biochemistry alterations and cancer. To humans the toxicity is associated with its intake on contaminated ground water or milk and inhalation of its spores. In order to check the BF aqueous extract (AEB) deleterious effects on animals blood vessels system, chick embryo chorioallantoic membrane (CAM) was used. It were applying on CAM 0.1, 0.5, 1, 5, 10 e 15 µg/mL of AEB and saline as control. The angiogenesis was analyzed and the vascular density index (VDI) calculated. The CAM samples were prepared and stained with H&E to evaluation of microvessels, Massons trichrome to characterize collagen and fibrin deposition and Picro-sirius used to evaluate collagen using polarized light. Also the morphological aspects of embryos were analysed. We observe on the results of neovascularization that AEB did not change significantly the number of vessels/mm², however, membranes treated with AEB (5 or 10 µg/ mL) exhibit opacity and tissue fibrosis, both signs of inflammation. Histological analysis with Massons trichrome and picro-sirius on tissues exposed to AEB respectively has shown increased collagen fibers and presence of fibrilar collagen. The embryos exposed to concentrations of 5 or 10 μg/mL AEB, showed changes as poor face formation and poor closing of abdominal wall. The highest concentration of AEB (15 μg/mL) was lethal to embryos. Although significant effects on the CAMs vasculature has not observed, tissue aggression was detected, a desmoplasia (an extensive inflammatory signal triggered by tissue injury), changes caused on embryos as well as the presence of toxic substances in the AEB show us an important and deleterious pathway of this bracken fern extract on its intoxicants effects on humans and animals, and even cancer or the death of animals.(AU)

O foco principal desse estudo foi avaliar os efeitos do extrato aquoso de Samambaia (EAS) (Pteridium aquilinum (L.) Kuhn) sobre sistemas biológicos. A ingestão das folhas de Samambaia pode provocar intoxicação aguda, hematúria, alterações bioquímicas e câncer se consumida por animais. Em humanos também pode provocar intoxicação ao ser ingerida com água ou leite contaminados e pela inalação de seus esporos. Considerando os efeitos danosos provocados pelo consumo dessa planta esse estudo analisou a ação do EAS sobre um sistema de vasos sanguíneos, a membrana corioalantóide (MCA) de embrião de galinha. Sobre a MCA foram aplicados 0,1, 0,5, 1, 5, 10 e 15 µg/mL de EAS, salina como controle. A angiogênese foi analisada e o índice de densidade vascular (IDV) calculado. Amostras da CAM foram coradas com H&E para avaliação de microvasos, Tricrômico de Masson para caracterização de colágeno e deposição de fibrina e Picro-sirius para analisar o colágeno através de luz polarizada. Os aspectos morfológicos dos embriões também foram analisados. Os resultados da neovascularização mostraram que não houve mudança significativa do número de vasos/mm². Entretanto, as membranas tratadas com 5 ou 10 µg/mL EAS exibiram uma opacidade junto com um tecido fibroso, ambos sinais de inflamação. Corroborando, as análises com Tricrômico Masson e Picro Sirius, respectivamente, apontaram para um aumento das fibras colágenas e presença de colágeno fibrilar. Embriões expostos às concentrações de 5 e 10 μg/mL do EAS sofreram malformações na face e na parede tóraco-abdominal. A concentração de 15 μg/mL foi letal para os embriões. Embora efeitos significativos na vasculatura da MCA não tenham ocorrido, observou-se desmoplasia (um grande sinal de inflamação). Esse fato, juntamente com as malformações no embrião e a presença de compostos tóxicos no EAS inferem, se a planta for ingerida, uma via importante para explicar a intoxicação de animais e humanos, e até mesmo o câncer ou a morte de animais.(AU)

Tissue damage and embryonic malformation induced by aqueous extract of Pteridium aquilinum on chorioallantoic membrane of chick embryo (CAM) / Danos teciduais e mal formações morfológicas induzidas pelo extrato aquoso de Pteridium aquilinum sobre a membrana corioalantóica (CAM) e o embrião de galinha

The aim of this study was evaluate the effects of Bracken fern (BF) (Pteridium aquilinum (L.) Kuhn.) on biological systems. When consumed by animals can cause acute intoxication, hematuria, biochemistry alterations and cancer. To humans the toxicity is associated with its intake on contaminated ground water or milk and inhalation of its spores. In order to check the BF aqueous extract (AEB) deleterious effects on animals blood vessels system, chick embryo chorioallantoic membrane (CAM) was used. It were applying on CAM 0.1, 0.5, 1, 5, 10 e 15 µg/mL of AEB and saline as control. The angiogenesis was analyzed and the vascular density index (VDI) calculated. The CAM samples were prepared and stained with H&E to evaluation of microvessels, Masson’s trichrome to characterize collagen and fibrin deposition and Picro-sirius used to evaluate collagen using polarized light. Also the morphological aspects of embryos were analysed. We observe on the results of neovascularization that AEB did not change significantly the number of vessels/mm², however, membranes treated with AEB (5 or 10 µg/ mL) exhibit opacity and tissue fibrosis, both signs of inflammation. Histological analysis with Masson’s trichrome and picro-sirius on tissues exposed to AEB respectively has shown increased collagen fibers and presence of fibrilar collagen. The embryos exposed to concentrations of 5 or 10 μg/mL AEB, showed changes as poor face formation and poor closing of abdominal wall. The highest concentration of AEB (15 μg/mL) was lethal to embryos. Although significant effects on the CAM’s vasculature has not observed, tissue aggression was detected, a desmoplasia (an extensive inflammatory signal triggered by tissue injury), changes caused on embryos as well as the presence of toxic substances in the AEB show us an important and deleterious pathway of this bracken fern extract on its intoxicants effects on humans and animals, and even cancer or the death of animals.

O foco principal desse estudo foi avaliar os efeitos do extrato aquoso de Samambaia (EAS) (Pteridium aquilinum (L.) Kuhn) sobre sistemas biológicos. A ingestão das folhas de Samambaia pode provocar intoxicação aguda, hematúria, alterações bioquímicas e câncer se consumida por animais. Em humanos também pode provocar intoxicação ao ser ingerida com água ou leite contaminados e pela inalação de seus esporos. Considerando os efeitos danosos provocados pelo consumo dessa planta esse estudo analisou a ação do EAS sobre um sistema de vasos sanguíneos, a membrana corioalantóide (MCA) de embrião de galinha. Sobre a MCA foram aplicados 0,1, 0,5, 1, 5, 10 e 15 µg/mL de EAS, salina como controle. A angiogênese foi analisada e o índice de densidade vascular (IDV) calculado. Amostras da CAM foram coradas com H&E para avaliação de microvasos, Tricrômico de Masson para caracterização de colágeno e deposição de fibrina e Picro-sirius para analisar o colágeno através de luz polarizada. Os aspectos morfológicos dos embriões também foram analisados. Os resultados da neovascularização mostraram que não houve mudança significativa do número de vasos/mm². Entretanto, as membranas tratadas com 5 ou 10 µg/mL EAS exibiram uma opacidade junto com um tecido fibroso, ambos sinais de inflamação. Corroborando, as análises com Tricrômico Masson e Picro Sirius, respectivamente, apontaram para um aumento das fibras colágenas e presença de colágeno fibrilar. Embriões expostos às concentrações de 5 e 10 μg/mL do EAS sofreram malformações na face e na parede tóraco-abdominal. A concentração de 15 μg/mL foi letal para os embriões. Embora efeitos significativos na vasculatura da MCA não tenham ocorrido, observou-se desmoplasia (um grande sinal de inflamação). Esse fato, juntamente com as malformações no embrião e a presença de compostos tóxicos no EAS inferem, se a planta for ingerida, uma via importante para explicar a intoxicação de animais e humanos, e até mesmo o câncer ou a morte de animais.

In vitro study of morphological changes of the cultured otocyst isolated from the chick embryo / Estudio in vitro de los cambios morfológicos del otocisto cultivado aislado del embrión de pollo

The aim of this study was to observe morphological changes of the cultured otocysts isolated from various stages of the chick embryo. Isolated otocysts were dissected from embryonic day, E2.5-4.5 of incubation (HH stage 16-26) according to stages of developing inner ear. Morphology of the chick otocyst exhibited an ovoid shape. The width and height of the otocyst were 0.2 mm and 0.3 mm, respectively. Elongation of a tube-like structure, the endolymphatic duct, was found at the dorsal aspect of the otocyst. The cultured otocyst is lined by the otic epithelium and surrounding periotic mesenchymal cells started to migrate outwards the lateral aspect of such epithelium. Notably, the acoustic-vestibular ganglion (AVG) was observed at the ventrolateral aspect of the otocyst. Appearance of AVG in vitro can be applied for studying chemical-induced ototoxicity and sensorineural hearing loss. It was concluded that the organ-cultured otocyst of the chick embryo could be used as a model to study sensory organ development of avian inner ear.

El objetivo de este estudio fue observar los cambios morfológicos de otocistos cultivados aislados en las diversas etapas del desarrollo del embrión de pollo. Otocistos aislados fueron obtenidos de embriones día, E2.5-4.5 de incubación (HH etapa 16-26) de acuerdo a las etapas de desarrollo del oído interno. El otocisto de pollo presentó una morfología ovoide. El ancho y la altura del otocisto fue de 0,2 mm y 0,3 mm, respectivamente. En la cara dorsal del otocisto se visualizó el alargamiento de una estructura similar a un tubo, el conducto endolinfático. El otocisto cultivado está revestido por epitelio ótico y células mesenquimatosas perióticas que comienzan a migrar hacia el exterior de la cara lateral en búsqueda del epitelio. En particular, el ganglio acústico-vestibular (GAV) fue observado en la parte ventrolateral del otocisto. La aparición de GAV in vitro puede ser aplicado para el estudio de la ototoxicidad inducida por productos químicos y la pérdida de audición neurosensorial. Se concluyó que el otocisto cultivado de embrión de pollo podría ser utilizado como un modelo para estudiar el desarrollo de órganos sensoriales del oído interno aviar.

Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells.

Human adipose-derived stromal cells (hADSC) are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1) regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

Interaction between SCO-spondin and low density lipoproteins from embryonic cerebrospinal fluid modulates their roles in early neurogenesis.

During early stages of development, encephalic vesicles are composed by a layer of neuroepithelial cells surrounding a central cavity filled with embryonic cerebrospinal fluid (eCSF). This fluid contains several morphogens that regulate proliferation and differentiation of neuroepithelial cells. One of these neurogenic factors is SCO-spondin, a giant protein secreted to the eCSF from early stages of development. Inhibition of this protein in vivo or in vitro drastically decreases the neurodifferentiation process. Other important neurogenic factors of the eCSF are low density lipoproteins (LDL), the depletion of which generates a 60% decrease in mesencephalic explant neurodifferentiation. The presence of several LDL receptor class A (LDLrA) domains (responsible for LDL binding in other proteins) in the SCO-spondin sequence suggests a possible interaction between both molecules. This possibility was analyzed using three different experimental approaches: (1) Bioinformatics analyses of the SCO-spondin region, that contains eight LDLrA domains in tandem, and of comparisons with the LDL receptor consensus sequence; (2) Analysis of the physical interactions of both molecules through immunohistochemical colocalization in embryonic chick brains and through the immunoprecipitation of LDL with anti-SCO-spondin antibodies; and (3) Analysis of functional interactions during the neurodifferentiation process when these molecules were added to a culture medium of mesencephalic explants. The results revealed that LDL and SCO-spondin interact to form a complex that diminishes the neurogenic capacities that both molecules have separately. Our work suggests that the eCSF is an active signaling center with a complex regulation system that allows for correct brain development.

Complementary expression of EphA7 and SCO-spondin during posterior commissure development.

Bilaterally symmetric organisms need to exchange information between the two sides of their bodies in order to integrate sensory inputs and coordinate motor control. This exchange occurs through commissures formed by neurons that project axons across the midline to the contralateral side of the central nervous system. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. It is located in the dorsal portion of the prosomere 1, at the caudal diencephalon. The axons of the posterior commissure principally come from neurons of ventrolateral and dorsolateral pretectal nuclei (parvocellular and magnocellular nucleus of the posterior commissure, respectively) that extend their axons toward the dorsal region. The trajectory of these axons can be divided into the following three stages: (1) dorsal axon extension towards the lateral roof plate; (2) fasciculation in the lateral roof plate; and (3) midline decision of turning to the ipsilateral side or continuing to the opposite side. The mechanisms and molecules that guide the axons during these steps are unknown. In the present work, immunohistochemical and in situ hybridization analyses were performed, with results suggesting the participation of EphA7 in guiding axons from the ventral to the dorsal region of the prosomere 1 through the generation of an axonal corridor limited by repulsive EphA7 walls. At the lateral roof plate, the axons became fasciculated in presence of SCO-spondin until reaching the midline. Finally, EphA7 expression was observed in the diencephalic midline roof plate, specifically in the region where some axons turn to the ipsilateral side, suggesting its participation in this decision. In summary, the present work proposes a mechanism of posterior commissure formation orchestrated by the complementary expression of the axon guidance cues SCO-spondin and EphA7.

The involvement of heparin in retinal infection by Toxoplasma gondii in a chick model revealed an ontogenetic-dependent pattern.

This work aimed to test the influence of heparin on the susceptibility of retinal cells to Toxoplasma gondii infection. Primary cultures of retinas from chick embryos of 8 (E8) or 11 (E11) days and fibroblasts (control) were used. To determine the influence of heparin in T. gondii infection, tachyzoites of the RH strain were treated with heparin before addition in the culture. A monoclonal anti-heparin antibody was used to analyze the heparin distribution on fibroblast and retinal cell surfaces. Our results showed that retinal cells (E8 and E11) had a higher infection rate than fibroblasts (91% and 24% versus 13%, respectively). Pre-treatment of T. gondii with heparin decreased infection of E8 retinal cells when compared with non-treated parasites (45% versus 91%, respectively), but not of E11 cells (35% versus 48%). In accordance, retinal cells presented an intense heparin staining by immunofluorescence assay. In conclusion, retinal cells from chick embryos were more susceptible to infection by T. gondii compared to fibroblasts and, pre-treatment of tachyzoites with heparin decreased the number of infected cells and parasite burden particularly for E8 retinal cells.

Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

Biological effects produced by intense pulsed light (Xe-Cl) on the cartilage of the tongue chick embryo using various filters / Efectos biológicos producidos por la luz pulsada intensa (Xe-Cl) sobre el cartílago lingual del embrión de pollo usando diversos filtros

The Laser used correctly in the medical practice offers clear advantages compared with traditional therapies. The improvement and even the elimination of many significant skin lesions can be achieved with reduced risks to patients. However, it is important to keep security measures and understand the possible effects on an experimental model. The chick embryo is a good model to evaluate the direct effects of non-ionizing radiation for its easy handling and availability. The purpose of this communication is to show our histological findings in organs of the chick embryo with and without protective barrier to be subjected to radiation excimer. We used the following issuers: intense pulsed light (excimer Xe-Cl laser of 308 nm wavelength). It was irradiated embryos through an open window on eggshells. Aseptically the eggs were kept for 24 hours in an incubator. The protective barriers were used with and without colored glass, latex, cellophane, paper, polycarbonate of different colors and thicknesses. The most outstanding results, with no barrier and barriers with transparent and green were intense marked congestion in capillaries, edema and focus the necrosis. We concluded that the tissue changes observed are consistent with possible side effects of these radiations fototérmicos we warned about possible side effects when they are applied indiscriminately. We believe it is important to explore different means to safeguard the safety of operators and patients.

El láser utilizado correctamente en la práctica médica ofrece claras ventajas cuando se compara con las terapias tradicionales. La mejoría e incluso la eliminación significativa de muchas lesiones cutáneas se pueden lograr con riesgos reducidos para los pacientes. Sin embargo, es importante guardar medidas de seguridad y conocer los posibles efectos en un modelo experimental. El embrión de pollo es un buen modelo para evaluar los efectos directos de radiaciones no ionizantes por su fácil manipulación y disponibilidad. El objetivo del presente trabajo es comunicar los cambios histopatológicos en órganos del embrión de pollo con y sin barrera de protección al ser sometido a radiación excimer. Se utilizó el siguiente elemento emisor: luz pulsada intensa (Xe-Cl excimer laser de 308 nm de longitud de onda. Se irradiaron los embriones a través de una ventana abierta en la cáscara del huevo. Los huevos fueron mantenidos asépticamente por 24 hs en una incubadora. Las barreras de protección utilizadas fueron vidrio con y sin color, latex, celofán, papel, policarbonato de diferentes colores y espesores. Los resultados más sobresalientes, sin barrera y con barreras transparentes y de color verde fueron: intensa vasocongestión, edema y focosde necrosis. Se concluye que las modificaciones tisulares observadas son compatibles con posibles efectos fototérmicos colaterales de estas radiaciones los que nos advierten sobre posibles efectos adversos cuando las mismas se aplican indiscriminadamente. Creemos que es de importancia estudiar los diferentes medios que permitan resguardar la seguridad de los pacientes y operadores.

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.

Excretion Products of Shigella dysenteriae and Apoptotic Cell Death on Chick Embryo Muscle Tissue / Producto de Excreción de Shigella dysenteriae y la Muerte Celular por Apoptosis en Tejido Muscular de Embrión de Pollo

The aim of this study was to evaluate the acute cell injury of excretion products present in culture filtrate from Shigella dysenteriae in both whole lower limb of chick embryo ex vivo and myoblasts cells developed in hanging-drop cultures in vitro. Three controls were defined: a) Tyrode's solution b) brain-heart broth infusion (CCC) and c) supernatant not toxigenic of E.coli 0157:H7. Shigella dysenteriae were culture for 24 hours and the excretion products were obtained after centrifugation of the culture. After lh of treatment, the morphologic changes in limbs treated with raw filtrate were evaluated through histopathological examination of sections stained with hematoxylin-eosin (H&E-stain) and Gomori's trichrome by image analysis techniques. Quantification of apoptotic cells was measured by an enzyme-linked immunoassay TUNEL. The morphological feature of apoptosis were evaluated in culture myoblasts. In contrast with controls, the longitudinal section on treated thigh of chick embryo limb-buds show atrophy muscle tissue, detachment of few fibers, 57,14% decrease in the number of cells, and loss of collagen substrate. Apoptotic index percent increase and mitotic index decrease in response to excretion products were observed, but were not significant. Membrane blebbing, vacuolation, small aggregates of chromatin around the nucleus and loss of cell adhesion were observed. Culture filtrate from Shigella dysenteriae produced cytotoxic effect on cell of muscle fibers with acute cell injuries suspected to be related to the apoptotic cell death.

El objetivo del presente trabajo ha sido evaluar el daño celular agudo generado por el producto de excreción de Shigella dysentenae sobre el tejido muscular del miembro inferior de embrión de pollo, así como sobre los mioblastos obtenidos a partir del cultivo de explantes de tejido muscular desarrollado en gota pendiente. Tres controles fueron definidos: a) solución de tiroide b) caldo de infusión cerebro-corazón (CCC), c) producto de excreción bacteriano no toxigénico de E. coli 0157:H7. El producto de excreción fue obtenido a partir de la centrifugación y filtrado del medio de cultivo de Shigella dysentenae con 24 horas de crecimiento. Luego de una hora de tratamiento, los cambios morfológicos del tejido muscular fueron evaluados a través del examen histopatológico, con técnicas de análisis de imágenes sobre cortes teñidos con hematoxilina-eosina (H&E) y Tricrómico de Gomori. El ensayo enzimático TÚNEL fue utilizado para evaluar el índice de apoptosis. Señales morfológicas de muerte celular fueron evaluadas en mioblastos en cultivo En contraste con los controles, el tejido muscular presentó signos de atrofia, con fibras desconectadas y pérdida del 57,14% del número total de células así como pérdida de la matriz de colágeno. Un incremento en el índice apoptótico y una reducción de índice mitótico fueron registrados. Esto último fue no significativo. Los mioblastos en cultivo presentaron signos morfológicos de apoptosis tales como protuberancias en la membrana, vacuolización, agregación de cromatina alrededor del núcleo y pérdida de la adhesión celular. Se concluye que el producto de excreción afecta al tejido muscular esquelético del miembro inferior de embrión de pollo e induce lesiones de toxicidad relacionadas con la muerte celular por apoptosis.