Morphological tools to evaluate the digestory apparatus in rocky cavy (Kerodon rupestris)

This research describes for the first time the complete morphology of the digestive apparatus of rock cavies. Dissection, light microscopy, and scanning electron microscopy were performed. The oral cavity has: the hard palate without palatine wrinkles and the soft palate; the tongue composed by striated musculature, with presence of vallate, foliated, and fungiform papillae with taste buds and filiform papillae with mechanical function; and, 20 teeth of the hypsodonts type. Esophagus, stomach, small intestine (duodenum, jejunum, and ileum), and the large intestine (cecum, colon, and rectum) are found. The anus is present at the end of the alimentary channel. Organs of digestive tube are composed by four tunics: mucosa, submucosa, muscular, and serosa. The duodenum, jejunum and ileum have villi. Jejunum, ileum and cecum present Lieberkühn crypts. The cecum has mucous glands. Colon and rectum are folded and have goblet cells. Anus presents sebaceous glands. As associated glands it is found the liver with six lobes and gallbladder; a lobulated pancreas; and a pair of each major salivary gland (parotid, mandibular, and sublingual). Parotid glands have serous acini and mandibular and sublingual glands have mucous acini. Pancreas has adenomers. The liver has hepatocytes and portal vein, hepatic artery, and bile duct (portal triad), separated by sinusoids. It is concluded that the digestive apparatus of the rock cavy has variations in the dentition, lingual papillae, and acini of the salivary glands when compared to other rodents. Other variations refer to the well‐developed cecum characteristic of herbivorous behavior.     

Confocal microscopy with double immunofluorescence staining reveals the functional transient receptor potential vanilloid subtype 1 expressed in myoepithelial cells of human submandibular glands

Myoepithelial cells (MECs) mainly surround acini and intercalated ducts in the human salivary glands. The contraction of MECs provides the expulsive force to promote salivation. We previously found functional transient receptor potential vanilloid subtype 1 (TRPV1) was expressed in rabbit and human submandibular glands and increased saliva secretion. However, it was unknown whether TRPV1 was expressed in MECs of submandibular glands. In this study, we observed the immunoflourescence of TRPV1 was not only located in serous acini and ducts but also surround the basal layer of the acinus and intercalated ducts of human submandibular glands. Double immunofluorescence staining revealed colocalization of TRPV1 with calponin, vimentin, and α-smooth muscle actin, which indicated the myoepithelial expression of TRPV1. Treating submandibular gland tissues with capsaicin, an agonist of TRPV1, substantially increased the phosphorylation of the 20-kDa regulatory light-chain subunit of myosin (MLC(20) ), a crucial molecule for contraction of smooth muscle cells, in MECs. Pretreatment with capsazepine, a specific TRPV1 inhibitor, blocked capsaicin-induced MLC(20) phosphorylation. These results suggest that TRPV1 is expressed in MECs of the human submandibular gland and mediates myoepithelial contraction via a mechanism involving MLC(20) phosphorylation.     

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

In a previous paper, we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently‐stained 12.5‐day old mouse embryos. Here, we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show subcellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal discs are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.     

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.     

Comparative evaluation of vegetable matter involved lesions with oral parasitic infections in the oral cavity

The current research aims to perform a comparative evaluation of vegetable matter involved lesions with oral parasitic infections found in oral mucosa, presenting histochemical methods to differentiate their microscopic similarities. Eight cases were selected out of a sample of 1.975 reports from a single Oral and Maxillofacial Pathology Service of the author's institution from 2012 to 2019. Specimens were examined by hematoxylin and eosin staining (HE), periodic acid-Schiff (PAS) staining, Gomori–Grocott staining, Ziehl–Neelsen staining, Giemsa, and mucicarmine staining. Microscopic analysis included fluorescence, polarized light, and confocal microscopy. Microscopically, in HE coloration, hookworm eggs showed as eosinophilic. Inflammatory multinucleated giant cells and lymphocytes, were usually related to the nematode eggs, forming an intense inflammatory infiltrate. Biofluorescent properties of eggs and larvae revealed to be sensitive in the detection of parasitic structures contrasting with the inflamed connective tissue. Vegetable presence was confirmed by polarized light microscopy and it was found to be associated with microbial biofilms. Confocal microscopy has showed to be an excellent method for morphotype differentiation of parasitic eggs. Parasitic infection and vegetable matter displayed similarities in the inflammatory response, but the latter can rot and agglomerate biofilms. The microscopic diagnosis of such infections requires the interpretation of challenging morphological features since the parasites are usually sectioned and mixed with an inflammatory reaction. These histochemical approaches proved to be excellent to distinguish both lesions.     

Staining tissue with methacrylate casts for light microscopy

Scanning electron microscopy (SEM) of methyl methacrylate casts and light microscopy (LM) of tissue are well-established methods for studying the microcirculation. The two are complimentary, but methacrylate is transparent and thus its presence is often not appreciated by LM. Histologic stains applied to methyl methacrylate in tissue sections would better identify by LM and allow the relationships with the SEM view of cast vasculature. We sought to test different stains on cast tissue to find one that would accent the cast. Surgically removed and autopsied human lungs were cast with methacry late and processed by routine light microscopic methods. They were stained with the hematoxylin and eosin, Masson trichome, elastic-van Gieson, Grocottmethenamine silver, Brown-Brennan, and Ziehl-Neelsen methods. The Ziehl-Neelsen procedure stained the methacry late best, giving it a red color. This procedure also worked well without heating. We conclude that (1) cast methacry late lung can be processed for routine LM with excellent results; (2) methacry late stains well with the Ziehl-Neelsen technique; (3) the acid-fast stained cast lung shows capillaries and cells in both normal and diseased lung better than the routine hematoxylin and eosin stain; (4) this technique can be used to assess filling and correlate findings on the same tissue with the two different microscopic methods.     

Characterization of the primo vascular system in rabbit vagina

The primo vascular system (PVS) is observed in different parts of the body under different physiological and disease conditions. Previously, the PVS was not observed in the vagina. The vaginal samples of this study were collected from the female genitalia of healthy New Zealand white rabbits from the animal house, Faculty of Medicine, Assiut University. The vaginal samples were fixed in Bouin's solution. The sections were stained with hematoxylin and eosin and Crossmon's trichrome. Additionally, the sections were immunohistochemically stained with neuron-specific enolase (NSE) and vascular endothelial growth factor (VEGF). A primo node was observed on the lymph vessel of the vagina and has several characteristics that resemble those of the previously discovered primo nodes. The primo node in this study was surrounded by mesothelial cells that provide positive immunoreactivity to NSE and VEGF. Sinuses of different sizes, floating cells, telocyte-like cell, and primo microcells were observed as the main constituents of the primo node. Additionally, migratory cells were detected, which passed from the primo node to the enclosing lymph vessel.     

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM)

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland‐related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d ‐glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2‐ to 3‐µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in‐solution ASEM for histology and to study vesicle secretion systems. Further, the high‐throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine‐related diseases.     

Effects of anti-CD3 monoclonal antibody in salivary glands of spontaneously diabetic mice

Background: Diabetes mellitus results in many complications, also compromising the salivary glands. The current treatment for this condition should be a substituting method to exogenous insulin. In this aspect, the immunotherapy has been tested, but, it can be inefficient as an agent for the control of damage caused by diabetes. Thus, the aim of this study was to evaluate the anti‐CD3 monoclonal antibody as alternative immunotherapy in the recovery of salivary glands of spontaneously diabetic NOD (nonobese diabetic) mice. Methods: NOD mice were divided into two groups of 10 animals: group I (untreated diabetic mice) and group II (anti‐CD3‐treated diabetic mice). After treatment, the samples of salivary glands were collected for histological examination under both transmitted and polarized light microscopy. Results: Alterations in tissue architecture; increase in extracellular matrix and presence of inflammatory process were observed in untreated animals. Recovery of the salivary acinar cells occurred in treated animals. The parotid glands demonstrated a smaller amount of collagen fibers and were not observed severe inflammatory processes. Conclusion: These results indicate that immunotherapy contributed to reestablishment of tissue damaged by the hyperglycemic condition, demonstrating that the immunomodulation plays an important role in the recovery of salivary glands. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Staining of DNA-phosphate groups with a mixture of azure A and acridine yellow.

This paper deals with staining of DNA-phosphate groups with a mixture of an equal parts of aqueous solution of azure A and acridine yellow in a 1:1 proportion and also embodies a study of the absorption properties of the stained nuclei. It also embodies results of sequential staining of nuclei stained first with azure A followed by staining with acridine yellow and vice versa, after extraction of RNA with cold phosphoric acid. The results indicate that the absorption peaks of nuclei differ from those of nuclei stained for DNA-aldehyde molecules with azure A-SO2 or acridine yellow-SO2. The in vitro absorption characteristics of an aqueous solution of azure A and those of an aqueous solution of acridine yellow are also presented herein. The conclusion obtained from this study is that all the phosphate groups of DNA do not take part in the staining process when staining is carried out with azure A or acridine yellow alone after after RNA has been extracted. This is because the nuclei stained with these dyes sequentially show the presence of acridine yellow-DNA and azure A-DNA complex.     

Blue molybdenum oxides: A stain for light and electron microscopy

Blue molybdenum oxides (molybdenum blues) have been prepared from aqueous phosphomolybdic acid solutions and applied to thin and semi-thin sections of glutaraldehyde-fixed, epoxy-embedded tissues. A light blue colour and high electron opacity were found in mast cell granules, the secretion content of goblet cells, and cytoplasmic granules in Drosophila salivary glands. The possibility that binding of blue molybdenum oxides to polyhydroxylic components accounts for the staining and contrasting reactions in some cell structures is briefly discussed.     

Fine structural aspects of secretion and extrinsic innervation in the olfactory mucosa.

The mucus at the surface of the olfactory mucosa constitutes the milieu in which perireceptor events associated with olfactory transduction occur. In this review, the ultrastructure of olfactory mucus and of the secretory cells that synthesize and secrete olfactory mucus in the vertebrate olfactory mucosa is described. Bowman's glands are present in the olfactory mucosa of all vertebrates except fish. They consist of acini, which may contain mucous or serous cells or both, and ducts that traverse the olfactory epithelium to deliver secretions to the epithelial surface. Sustentacular cells are present in the olfactory epithelium of all vertebrates. In fish, amphibia, reptiles, and birds, they are secretory; in mammals, they generally are considered to be "non-secretory," although they may participate in the regulation of the mucous composition through micropinocytotic secretion and uptake. Goblet cells occur in the olfactory epithelium of fish and secrete a mucous product. Secretion from Bowman's glands and vasomotor activity in the olfactory mucosa are regulated by neural elements extrinsic to the primary olfactory neurons. Nerve fibers described in early anatomical studies and characterized by immunohistochemical studies contain a variety of neuroactive peptides and have several targets within the olfactory mucosa. Ultrastructural studies of nerve terminals in the olfactory mucosa have demonstrated the presence of adrenergic, cholinergic and peptidergic input to glands, blood vessels, and melanocytes in the lamina propria and of peptidergic terminals in the olfactory epithelium. The neural origins of the extrinsic nerve fibers and terminals are the trigeminal, terminal, and autonomic systems.     

Modification of Cunningham's method for the detection of proteases in tissue sections.

A modification of Cunningham's method to show the protease activity in fresh tissue sections on gelatin film is proposed. The diazotization of the gelatin film in two successive baths of Fast blue B salt sharply increases the colour intensity of the film, improving the contrast between the intact zones and the enzymatic lysis zones of the substrate. The various washings of the stained film have been simplified. After incubation, the histological staining of the sections by solid nuclear red is an improvement over the much more tricky Feulgen reaction, combined with PAS, used by Cunningham.     

Effect of black tea on histological and immunohistochemical changes in pancreatic tissues of normal and streptozotocin‐induced diabetic mice (Mus musculus)

The aim of the present study is to evaluate the effect of hot water extract of black tea in regenerating β cells in streptozotocin‐induced diabetic mice. Light microscopic examination of pancreatic sections of streptozotocin‐induced diabetic mice showed the acinar cells to be small, shrunken, and with deteriorated β cells. The dose of streptozotocin not only altered the function of β cells but also damaged the acinar region. The changes in acinar cells were coarsening of endoplasmic reticulation suggesting alteration in their secretory function. The control pancreatic tissue showed well‐defined granulated islets and dark β cells when stained with chrome hematoxylin and phloxine. Interestingly, pancreatic sections of diabetic mice fed with black‐tea extract showed regeneration of β cells and acinar region appeared normal with increased numbers of β cells. To understand the probable mechanism of action of black‐tea extract, we analyzed inducible nitric oxide synthase (iNOS) expression by immunohistochemistry and the results showed an increased iNOS levels in streptozotocin‐induced diabetic pancreas, and such high iNOS levels were inhibited in black‐tea extract treated mice. According to histological results obtained, it can be concluded that the black‐tea extract helps in regeneration of damaged pancreas and protects pancreatic β cells by its antioxidant action against nitrosative stress in streptozotocin‐induced diabetes. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Pure optical contrast in scattering-type scanning near‐field microscopy

A simple contrast enhancement method is presented for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution. The sections were treated with an acidified potassium permanganate oxidizing solution followed by uranyl acetate and lead citrate staining. The method, designated KMnO₄–UA/Pb staining, provided a much greater contrast in electron microscopy than conventional UA/Pb staining. In detail, the visibility of plasma membrane was especially improved and the nuclear heterochromatin, mitochondria and cytoplasmic ribosomes showed an adequate increase in electron density. In the mucous cells of rat Brunner's glands, the Golgi cisternae were well defined with the KMnO₄–UA/Pb staining. Interestingly, the membranes of the intermediate compartments were moderately reactive to the KMnO₄–UA/Pb staining, whereas the cis and the trans compartments were only faintly stained. It should be emphasized that the KMnO₄ oxidation following colloidal gold labelling did not cause a remarkable reduction of immunogold labelling and the enhanced contrast helped us to examine the gold particles with high accuracy. This contrast enhancement method is highly promising, with the potential to become a useful tool for histochemical investigation, including immunocytochemistry with the Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution techniques.     

Immunolocalization of estrogen and progesterone receptors in ewe mammary glands

Immunopresence of estrogen receptor α (ERα), β (ERβ) and progesterone receptor (PR) were examined in the ewe mammary gland from prepubertal stage to involution. Immunolocalization of ERα revealed a strong positive staining in nuclei of cells composing terminal ductular units (TDUs) in prepubertal ewes. A mild immunoreactivity was identified in early lactating gland. During late lactation immunoreactive product to ERα was observed in the cytoplasm of glandular epithelial cells in all alveoli. In mammary glands at involution ERα positivity was clearly nuclear, with weak to moderate cytoplasmic staining. Cytoplasmic strong immunostaining for ERβ was detected in cells of TDUs, whereas some stromal cells exhibited nuclear staining. A nuclear ERβ immunostaining was observed at early lactation, instead during late lactation, the positivity for ERβ showed only a moderate cytoplasmic distribution. At involution, ERβ positivity was very moderate and detected just in the cytoplasm of shrunken alveoli. Scattered nuclear staining of PR was observed just in mammary glands at early lactation. These results showed that in the mammary glands of sheep both estrogen receptor isoforms were displayed during lactation cycle and that PR appeared just at early lactation, reflecting their regulatory role in alveolar cells. Microsc. Res. Tech. 76:955–962, 2013. © 2013 Wiley Periodicals, Inc.     

The innovative safe fixative for histology,histopathology, and immunohistochemistry techniques: “Pilot study using shellac alcoholic solution fixative”

The concerns over health and workplace hazards of formalin fixative, joined to its cross‐linking of molecular groups that results in suboptimal immunohistochemistry, led us to search for an innovative safe fixative. Shellac is a natural material which is used as a preservative in foods and pharmaceutical industries. This study was undertaken to evaluate the fixation adequacy and staining quality of histopathological specimens fixed in the “shellac alcoholic solution” (SAS), and also to determine the validity of immunohistochemical staining of SAS‐fixed material in comparison to those fixed in formalin. Fresh samples from 26 cases from various human tissues were collected at the frozen section room of King Abdulaziz University Hospital, and fixed in SAS fixative or in neutral buffered formaldehyde (NBF) for 12, 18, 24, and 48 h, and processed for paraffin sectioning. Deparaffinized sections were stained with hematoxylin and eosin (H&E) and immunostained for different antigens. The tissues fixed in SAS for >18 h showed best staining quality of H&E comparable to NBF‐fixed tissues. Comparison of the immunohistochemical staining of different tissues yielded nearly equivalent readings with good positive nuclear staining quality in both fixatives. These findings support the fixation and preservation adequacy of SAS. Furthermore, it was concluded that the good staining quality obtained with SAS‐fixed tissues, which was more or less comparable with the quality obtained with the formalin fixed tissues, supports the validity of this new solution as a good innovative fixative. Microsc. Res. Tech. 77:385–393, 2014. © 2014 Wiley Periodicals, Inc.     

Staining of DNA-phosphate groups and DNA-aldehyde molecules with dyes of the azine group

The investigation reports on the use of safranine-SO2 and phenosafranine-SO2, prepared with N HCl or oxalic acid plus potassium metabisulphite, for staining rat liver sections following Feulgen procedure. It has been found that optimum staining of DNA-aldehyde molecules is possible with safranine-SO2 and phenosafranine-SO2, prepared with N HCl and potassium metabisulphite, upto a duration of one week after the preparation of the dye-reagents. Thereafter, staining intensity of the nuclei produced by the dye-reagents is gradually diminished. Staining of acid-hydrolysed sections is also possible with aqueous solutions of these dyes. Moreover, DNA-phosphate groups can also be stained with aqueous solutions of these dyes after selective extraction of RNA with cold phosphoric acid. The in situ absorption spectra of nuclei, stained for DNA-aldehyde molecules with safranine-SO2, phenosafranine-SO2 and aqueous solutions of these dyes, have been presented in this paper. Also presented herein are absorption data of nuclei stained with these dyes after selective extraction of RNA. It has been found that absorption-peaks of nuclei stained differently are different from one another. The implications of these findings have been discussed.