Histochemical and Molecular Characterization of Spongiosal Cells in Native Tissue, Two- and Three-Dimensional Cultures of Rat Aortic Valve

The histocytochemical and molecular analysis of cells that constitute the aortic valve (AV) of the rat heart was done in this study. We have focussed on the identity of cells in the spongiosal layer of the valve by immunofluorescence studies using lineage specific markers and cytochemical staining. We have established two-dimensional (2D) cultures of cells from isolated rat AV leaflets and maintained endothelial and interstitial valvular cells (IVC) over a period of six to eight weeks. Using “passage 0” cells from 2D valvular cultures, we could reconstruct the three-dimensional (3D) valvular tissue in collagen gels that showed very similar cellular organization and marker expression profile, as that of the native tissue. Lineage specific markers in the native tissue and cell cultures were studied by Reverse Transcriptase-PCR and immunofluorescence for VCAM-I, α-SMA, collagen I, CD71, collagen II, and E-cadherin markers. This is the first report on the identification of cell lineages in the spongiosal layer of AV and the successful reconstruction of 3D valvular tissue from primary cell cultures of AV.

Interspecific Variations in Duration of Tail Regression in Two Tropical Anurans

Anurans breed in an array of habitats and hence employ a variety of evolutionary strategies to adapt to the variable conditions. Particularly, since they undergo a larval phase they develop mechanisms to overcome unfavourable conditions like desiccation, extreme temperatures, and so forth. The anurans, Polypedates maculatus and Duttaphrynus melanostictus, show noticeable variation in the duration of larval period and tadpole tail regression. D. melanostictus breeds throughout the year and hence is subjected to different environmental conditions as compared to P. maculatus which breeds only during the rainy season. Thus, the tadpoles of D. melanostictus have selected to undergo a shorter larval period and duration of tail regression to suit their breeding habits. The present study correlates the interspecific difference in the duration of tail regression with the morphological variations in the tails of the two species. D. melanostictus shortens the duration of larval tail regression by having comparatively larger and more number of melanocytes and a thinner notochord than P. maculatus.

Evaluation and Validation of a Semiautomatic Image Analysis Method: Quantification of Nares Cell Proliferation of Rhinella arenarum (Anura) Detected with the Avidin-Biotin Complex/Peroxidase, DAB

The current report presents the development and application of a novel methodological approach for computer-based methods of processing and analysis of proliferative tissues labeled by ABC-peroxidase method using 3, 3′-diaminobenzidine (DAB) as chromogen. This semiautomatic method is proposed to replace the classical manual approach, widely accepted as gold standard. Our method is based on a visual analysis of the microscopy image features from which a computational model is built to generate synthetic images which are used to evaluate and validate the methods of image processing and analysis. The evaluation allows knowing whether the computational methods applied are affected by the change of the image characteristics. Validation allows determining the method’s reliability and analyzing the concordance between the proposed method and a gold standard one. Additional strongness of this new approach is that it may be a framework adaptable to other studies made on any kind of microscopy.

The Ultrastructure of Secretory Cells of the Islets of Langerhans in South American Catfish Rhamdia quelen

The present work shows that a detailed description of the ultrastructure of the secretory cells of the South American catfish Rhamdia quelen pancreatic islets is presented. Evidence is offered to support the contention that the α-granules consist of a central and an outer portion of different electron densities and solubilities, that the δ-cells are most probably morphologically altered but viable α-cells, and that the β-granules possibly possess a repeating substructure and may therefore represent an intracellular crystalline storage form of insulin.

Tissue Expression of Low and High Molecular Weight Cytokeratins in Lung Carcinoma Sections: Its Correlation with Some Clinic-Pathological Features

The tissue expression of low (LMW) and high (HMW) molecular weight cytokeratins and Ber-EP4 antigen in both small (SCLC) and non-small (NSCLC) cell lung carcinomas, as well as its correlation with a variety of clinic-pathological features, was evaluated. In general, 43/52 (82.7%) of NSCLC sections showed the expression of at least one type of cytokeratin while only 7/16 (43.7%) of SCLC were stained with both LMW cytokeratin and pan-cytokeratins antibodies. Remarkably, 18/52 (34.6%) of NSCLC were positive to both types of cytokeratins. However, none of SCLC showed this pattern of expression. In NSCLC patients, the increasing levels of HMW cytokeratins expression, as shown by 34βE12 antibody, correlated with the occurrence of disease recurrence (P=0.0057; Fisher’s exact test). Consequently, the expression of HMW cytokeratins was found to be associated with a poor 4-year overall survival of NSCLC patients (P=0.0315; Log rank test), not taking into account the histopathological classification of tumors. Similar results were obtained when 8-year overall survival was assessed (P=0.0103; Log rank test). Our results could suggest the assessment of HMW cytokeratins in a larger series of NSCLC samples in order to confirm the potential prognostic value of them.

Comparative Histomorphological and Histochemical Studies on the Oesophagus of Nile Tilapia Oreochromis niloticus and African Catfish Clarias gariepinus

The present work aimed to describe and compare both gross and microscopic structure of the oesophagus of Nile tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus). For this purpose, 60 specimens of oesophagus of Nile tilapia (omnivorous fish) and African catfish (carnivorous fish) were collected and processed. Anatomically, the oesophagus of both species appeared as a short tube with longitudinal mucosal folds. Using scanning electron microscope, the epithelial surface of the esophagus showed primary and secondary mucosal folds in both species while tertiary folds were observed in that of tilapia only. Histologically, the oesophagus consisted of four distinct layers: mucosa, submucosa, muscularis, and serosa. The oesophageal mucosa consisted of stratified epithelium with few mucous secreting cells in catfish and many mucous secreting cells in tilapia. Two types of mucous secreting cells reacted positively with both periodic acid shiff (PAS) and alcian blue (AB); rounded and elongated cells that were recognized in the esophageal epithelium of tilapia and only elongated oval cells were observed in that of catfish. In conclusion, the obtained histomorphological differences in esophagus of both fish species may be attributed to their different feeding habits and type of food.

Histochemical and Immunohistochemical Analysis of the Stomach of Rhinella icterica (Anura, Bufonidae)

The stomach of Rhinella icterica was analyzed at light microscopy, employing histochemical techniques, lectin histochemistry, and immunohistochemistry for identifying enteroendocrine cells (EC). Although the stomach was composed of fundic and pyloric regions, its wall is formed by mucosa, submucosa, muscularis, and serosa. The mucosa was lined by a simple columnar mucous epithelium, supported by loose connective tissue. Several tubular, simple glands were composed of mucous neck cells, containing oxynticopeptic cells and EC cells. The mucous neck cells were rich in neutral glycoconjugates. The oxynticopeptic cells were predominant in fundic glands, exhibiting weaker alcianophilic reaction at their apical cytoplasm. Serotonin (5-HT) immunoreactive (IR) cells occurred throughout the entire stomach, preferentially located among mucous cells at upper part of the fundic glands. The muscularis mucosae, formed of smooth muscle, separated the mucosal layer from the submucosa, both of which were constituted by loose connective tissue, but without glands. Lymphoid modules occurred in the mucosa at the boundary at the stomach and the gut. In addition, the muscularis was constituted by two sublayers, the circular internal and the longitudinal external, being recovered by the connective tissue of the serosa.

Alterations in the Gill Filaments and Secondary Lamellae of Cirrhinus mrigala Exposed to “Nuvan,” an Organophosphorus Insecticide

The alterations in the epithelium of the gill filaments and the secondary lamellae of the gills of Cirrhinus mrigala, on exposure to “Nuvan,” have been explored in the present investigation using light and scanning electron microscopy. The fishes were exposed to two sublethal concentrations, 5 mg/L and 15 mg/L, of “Nuvan.” The changes are more rapid and intensive at higher concentration than at lower concentration, suggesting that the changes are dose dependent. Increase in thickness of epithelium covering secondary lamellae, merger of epithelium of gill filaments and adjacent secondary lamellae, and aneurysm is considered to reduce efficiency of gills for gaseous exchange. A significant decline in the density and area of the mucous goblet cells in the epithelium of the gill filaments and the secondary lamellae of C. mrigala exposed to “Nuvan” could be correlated with excessive loss of the secretory contents of these cells, uncompensated by their production in sufficient quantities. The histopathological changes, in general, take longer time to recover in the fishes exposed to 15 mg/L than those exposed to 5 mg/L indicating that the changes in fishes exposed to higher concentration are more severe than those at lower concentration of the insecticide.

The Histochemical Characterization of the Glycoconjugates in the Epidermal Mucous Cells of the Red Californian Earthworm, Eisenia foetida

The aim of this study was to characterize the nature and regional distribution of the glycoconjugates secreted by epidermal mucous cells in Eisenia foetida (Annelida). Specimens were divided into six regions from anterior to posterior. The histochemistry was carried out by using standard histochemical methods. Histochemical staining properties of glycoconjugates in epidermal mucous cells were determined regionally. The epidermis of all regions contained strong to stronger PAS (+) cells in various degrees. The epidermis of the first, fourth, fifth, and sixth regions had strong to stronger AB pH 2.5 (+) cells. On the contrary, all regions contained weak to moderate AB pH 0.5 and AB pH 1.0 (+) cells. Most of mucous cells in epidermis of the first region contained both PAS (+) and AB (+) mucosubstances. All regions included weaker to weak AF (+) cells. All regions featured KOH/PAS (+) cells, with a slight reduction in reaction intensity in the epidermis of the last three regions. In this context, the different staining patterns observed in epidermal mucous cells hinted at their functional roles with respect to production of mucus with different physical properties. This study provided comprehensive information about the regional distribution patterns of the glycoconjugates and an opportunity to compare their distributional patterns in other annelids.

Histology and Histometric Anatomy of the Male Reproductive System of Bat (Eidolon helvum)

The male reproductive system of fruit bat (Eidolon helvum) was studied histologically using light microscope. Thirty males (17 adults and 13 juveniles) were captured using net, weighed, aged using relative ossification of the wing bone, and dissected and reproductive tissue was processed for histomorphometry. On the basis of histological sections, the structures of a pair of testis containing the seminiferous tubules of adults were compacted in organization with spermatogenic cells. The epididymis has a thinner muscular region than the vas deferens with longitudinal folds on the mucosal lining. Two portions were observed in the prostate gland, while seminal vesicle has numerous trabeculae and bulbourethral gland was observed to have multiacini. There was increase in thickness of muscular region, epithelial height, and luminal diameter of epididymis and vas deferens between adults and juveniles. This work has documented the histology of the male reproductive system in bats, and ultrastructure and histochemistry are recommended for further insight into the reproductive biology.