Anatomical, Histological, and Histochemical Analyses of the Scent Glands of the Scorpion Mud Turtle (Kinosternon scorpioides scorpioides).

Fossil evidence suggests that scent glands are basal features of Testudines. However, we know little about the structure of these glands in the Brazilian Kinosternidae. In this study, we described the macroscopic anatomy, histology, and histochemistry of the scent glands of three males and three females of Kinosternon scorpioides scorpioides from the Marajó mesoregion, Pará State, Brazil. In all of the specimens analyzed, regardless of sex, we found four scent glands, including two axillary and two inguinal glands that were structurally similar to each other. Each gland consisted of a single holocrine secretory lobule, a large lumen surrounded by relatively thin glandular secretory epithelium, an adjacent narrow layer of loose connective tissue, and a thick layer of skeletal striated muscle tissue surrounded by a serous tunic. The secretory epithelium produced a characteristic malodorous yellowish substance that was passed via a single duct through a bone channel in the bridge connecting the carapace to the plastron and excreted through an outer pore in the plate of each respective gland. Histologically, the secretory epithelium presented cells with two types of secretory vacuoles. Type 1 vacuoles stained red were the largest and most frequently found, and stained positively with Periodic acid-Schiff (PAS), suggesting they contained glycoproteic complexes. Type 2 vacuoles were translucent, smaller in size and fewer in number, and negative for PAS staining. Because they are very primitive structures, scent glands must play important roles in the lives of chelonians, but their real function remains unknown. Several hypotheses suggest that they can act as protection against ectoparasites, as a repellent of predators, in addition to attracting mates and eliciting other pheromonal responses. In this study, all animals reacted by exuding malodorous substances when handled, as a form of defense. However, these are just assumptions that need to be clarified with additional studies on animal behavior. Anat Rec, 303:1489-1500, 2020. © 2019 American Association for Anatomy.

Can Paraplegia by Disruption of the Spinal Cord Tissue Be Reversed? The Signs of a New Perspective.

Central nervous system (CNS) trauma is often related to tissue loss, leading to partial or complete disruption of spinal cord function due to neuronal death. Although generally irreversible, traditional therapeutic efforts, such as physical therapy exercises, are generally recommended, but with a poor or reduced improvement of the microenvironment, which in turn stimulates neuroplasticity and neuroregeneration. Mesenchymal stem cells (MSCs) have paracrine, immunomodulatory, and anti-inflammatory effects. Here we use stem cells to see if they can promote not only physical but also the functional regeneration of neuronal tissue in dogs with CNS traumas. Two dogs, one with chronic spinal cord injury and one with subacute spinal cord injury, underwent infusion of autologous MSCs in association with physiotherapy. The two treatments in combination were able to partially or completely recover the dog's walking movement again. The treatment of MSCs in association with physical therapy improved the microenvironment, which could be evidence of a paradigm shift that the CNS is not capable of functional regeneration after aggressive traumas. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1812-1820, 2020. © 2019 American Association for Anatomy.

Morphological description of smell glands in Cyclopes didactylus.

A macroscopic and microscopic study of the mandibular organ of the silky anteater (Cyclopes didactylus) was carried out. The organ extends from below the zygomatic bone line to the middle of the mandible body, between the skin and the masseter muscle, on both sides of the animal. It has an average length of 11.7 mm and a width of 6.3 mm. In the mesoscopic analysis, it was observed that the organ presents in yellowish color due to the high amount of sebaceous content. In the histological analysis, the mandibular organ was observed to be composed of innumerable alveoli of the specialized sebaceous gland, surrounded by a layer of adventitia tunica. Scanning electron microscopy (SEM), revealed an apparent alveolar division with what appeared to be a sulcus at its center. The information here presented regarding the constitution and location of this structure has not been previously explored for other species and differs with respect to other descriptions for anteaters. Based on the present study, it is suggested that the mandibular organ is involved in social interaction in this species.

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes.

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.

Growth arrest and morphological changes triggered by emodin on Trypanosoma cruzi epimastigotes cultivated in axenic medium.

Emodin is an anthraquinone obtained from Rheum palmatum rootstocks. Here we tested the cytotoxic effects of emodin on Trypanosoma cruzi epimastigotes, as well as the morphological changes that were induced by this compound in the parasite. Emodin was permeable and blocked in vitro cell division of T. cruzi epimastigotes in axenic medium, causing growth arrest in a dose-dependent but reversible manner. Emodin-exposed epimastigotes underwent duplication of organelles, such as the nucleus, kinetoplast and flagellum, but were incapable of completing cytokinesis. Neither elongation of the parasite body nor appearance of the regular longitudinal cleavage furrow was displayed, suggesting that emodin is most likely affecting components of the parasite cytoskeleton. Moreover, drug-treated parasites acquired alterations such as protuberances, folds and indentations on their membrane surface. Since emodin has been shown to be a potent protein kinase CK2 inhibitor, and we have previously described an association between tubulin and CK2 in T. cruzi epimastigotes (De Lima et al. Parasitology132, 511-523, 2006), we also measured the indirect effect of the drug on tubulin. Incubation of epimastigotes with axenic medium containing emodin hindered the endogenous phosphorylation of tubulin in whole-cell parasite extracts. All our results suggested that the parasite CK2 may be important for the maintenance of the morphology and for the regulation of mitosis-cytokinesis transition in T. cruzi epimastigotes.

Hypertrophy and neuron loss: structural changes in sheep SCG induced by unilateral sympathectomy.

Recently, superior cervical ganglionectomy has been performed to investigate a variety of scientific topics from regulation of intraocular pressure to suppression of lingual tumour growth. Despite these recent advances in our understanding of the functional mechanisms underlying superior cervical ganglion (SCG) growth and development after surgical ablation, there still exists a need for information concerning the quantitative nature of the relationships between the removed SCG and its remaining contralateral ganglion and between the remaining SCG and its modified innervation territory. To this end, using design-based stereological methods, we have investigated the structural changes induced by unilateral ganglionectomy in sheep at three distinct timepoints (2, 7 and 12 weeks) after surgery. The effects of time, and lateral (left-right) differences, were examined by two-way analyses of variance and paired t-tests. Following removal of the left SCG, the main findings were: (i) the remaining right SCG was bigger at shorter survival times, i.e. 74% at 2 weeks, 55% at 7 weeks and no increase by 12 weeks, (ii) by 7 weeks after surgery, the right SCG contained fewer neurons (no decrease at 2 weeks, 6% fewer by 7 weeks and 17% fewer by 12 weeks) and (iii) by 7 weeks, right SCG neurons were also larger and the magnitude of this increase grew substantially with time (no rise at 2 weeks, 77% by 7 weeks and 215% by 12 weeks). Interaction effects between time and ganglionectomy-induced changes were significant for SCG volume and mean perikaryal volume. These findings show that unilateral superior cervical ganglionectomy has profound effects on the contralateral ganglion. For future investigations, it would be interesting to examine the interaction between SCGs and their innervation targets after ganglionectomy. Is the ganglionectomy-induced imbalance between the sizes of innervation territories the milieu in which morphoquantitative changes, particularly changes in perikaryal volume and neuron number, occur? Mechanistically, how would those changes arise? Are there any grounds for believing in a ganglionectomy-triggered SCG cross-innervation and neuroplasticity?

On the atrophy of the internal carotid artery in capybara.

Capybara might be a useful model for studying changes in cerebral circulation as the natural atrophy of the internal carotid artery (ICA) occurs in this animal at maturation. In this study, confocal and electron microscopy combined with immunohistochemical techniques were applied in order to reveal the changes in morphology and innervation to the proximal part of ICA in young (6-month-old) and mature (12-month-old) capybaras. Some features of the basilar artery (BA) were also revealed. The ICA of young animals degenerated to a ligamentous cord in mature animals. Immunolabelling positive for pan-neuronal marker protein gene product 9.5 but negative for tyrosine hydroxylase was observed in the proximal part of ICA at both ages examined. Axon varicosities positive for synaptophysin were present in the adventitia of ICA of young animals but were absent in the ligamentous cord of mature animals. In the ICA of young animals, adventitial connective tissue invaded the media suggesting that the process of regression of this artery began within the first 6 months of life. An increase in size of the BA was found in mature animals indicating increased blood flow in the vertebro-basilar system, possibly making capybara susceptible to cerebrovascular pathology (e.g. stroke). Capybara may therefore provide a natural model for studying adaptive responses to ICA regression/occlusion.

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi.

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi.

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

Early and late molecular and morphologic changes that occur during the in vitro transformation of Trypanosoma cruzi metacyclic trypomastigotes to amastigotes.

The amastigogenesis primary of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. The in vitro study of the macromolecular changes that occur over several days during the transformation process should provide significant indications of how the parasite adapts to the mammalian host environment. We show here that metacyclic trypomastigotes pre-incubated at 37 degrees C in a protein-rich medium reach a high degree of transformation to amastigotes when re-incubated in the fresh medium. Giemsa-stained smears show that during the pre-incubation phase, the metacyclic trypomastigotes undergo lengthening at the posterior end and a thinning out of the entire body. SDS-PAGE analysis of polypeptides and glycopeptides or Western blot with stage-specific antisera analyses indicate that the in vitro primary amastigogenesis is associated with abrupt changes in protein, glycoprotein, and stage-specific antigens that occur simultaneously during the first 24 hours of pre-incubation. Since the differentiating system consists of a rich media at 37 degrees C, temperature and medium constitution must trigger a macromolecular differentiation to amastigotes that precedes the morphological transformation by several days. This transformation is associated with the rearrangement of stage-specific antigens and takes place when the culture medium is changed.

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37ºC. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37ºC for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90 percent of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts

Producción y purificación de anticuerpos (IgY) a partir de huevos de gallinas inmunizadas con epimastigotas de trypanosoma cruzi / Production and purification of IgY antibodies from eggs laid by hens inmunized with trypanosoma cruzi epimastigotes

No obstante las ventajas de producir anticuerpos en gallinas, no se ha reportado inducción y purificación de anticuerpos contra estadios de Trypanosoma cruzi en el modelo aviario. La inducción de anticuerpos anti-estadios de T. cruzi en gallina puede garantizar la producción de reactivos útiles en diagnóstico y terapéutica de la enfermedad de Chagas. Aquí reportamos la obtención y rápido aislamiento de IGY procedente de yema de huevos de gallinas inmunizadas contra epimastigotas de T. cruzi a los 7, 22 y 61 días post-inmunización. Se compararon tres estrategias de purificación de IgY: el método de la dilución acuosa, el método del polietilén glicol (PEG) y el método del cloroformo-PEG, respecto a un estuche comercial. El método del cloroformo-PEG dio un rendimiento de 6,4 mg/mL de proteínas de yema de huevo de 61 días con una sensibilidad tal que 7 ng de IgY detectaron 1 µg de antígeno (ELISA). Por Western blot, 5 µg/mL de IgY revelaron 11 antígenos diferentes en 8 µg de proteína total de epimastigotas. El análisis por SDS-PAGE demostró que las IgY extraídas contienen dos bandas proteícas dominantes de 70/57 y 37/35 kDa y dos tenues de 81 y 41 kDa, las cuales pueden ser eliminadas por re-precipitación con PEG. Para la extracción de IgY el método del cloroformo-polietilén glicol dio la mejor combinación por facilidad de ejecución y bajo costo. Si bien rindió 50 por ciento menos que el estuche comercial bajo las mismas condiciones, la sensibilidad de los anticuerpos fue 4 veces mayor. Estos resultados evidencian la factibilidad del modelo aviario para producir anticuerpos contra estadios de T. cruzi y muestran la experticia para purificarlos usando una tecnología local de bajo costo

Separación de polipéptidos antigénicos de trypanosoma cruzi usando soluciones de pH diferentes / Separation of antigenic polypeptides of tripanosoma cruzi using defferential solubility in different pHs

Fracciones antigénicas útiles para el diagnóstico de la enfermedad de Chagas son obtenidas utilizando metodologías costosas, por eso es necesario desarrollar tecnologías que reduzcan los costos de producción. Previamente mostramos que los polipéptidos de trypanosoma cruzi se solubilizan diferencialmente en soluciones con valores de pH diferentes. El Propósito en este estudio fue obtener fracciones enriquecidas en polipéptidos de T. cruzi antigénicamente funcionales en base a su solubilidad diferencial. Los perfiles polipeptídicos de los epimastigotas fueron identicados por electroforesis en geles de poliacrilamida con dodecil sulfato de sodio (SDS-PAGE), coloreados por el método de la plata metálica. La antigenicidad de los polipéptidos se estudió mediante Western Bot usando un suero específico anti-epimastigotas. Se encontró que: a) la solubilidad de los polipéptidos no fue afectada por cambios en la fuerza iónica entre 0 y 250 mM NaCl; b) todos los polipéptidos se precipitaron a pH 4,0 mientras que el incremento secuencial del pH de la solución de resuspensión produjo un enriquecimiento diferencial de polipéptidos de mayor peso molecular; c) las fracciones enriquecidas, procedentes de soluciones con valores pH menores que 7,0, mostraron una fuerte actividad proteolítica la cual fue inhibida a pH alcalino y; d) los polipéptidos conservaron su antigenicidad. Estos resultados muestran una metodología sencilla y relativamente económica para producir fracciones antigénicas. Están en progreso experimentos para reducir la actividad proteolítica y obtener polipéptidos útiles para el diagnóstico de la enfermedad de Chagas