Electron microscopic observations of human hair medulla

In the study of human hair, medulla is the less studied structure because it is believed that it has no influence on the fibre properties. The aim of this paper is to contribute to the better understanding of medulla morphology. Using reproducible methods for hair samples preparations allowed observing the inner fibre by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Three medulla subunits were observed in cryofractured samples. In addition, the application of plasma etching on samples allowed accessing chemical differences between them. Two kinds of medulla were identified using stereomicroscopy: thin and thick medulla. They were morphologically differentiated using TEM. These methods can be used to study systematically the effects of medulla on hair properties and to evaluate the efficiency of cosmetic products.     

Immunocytochemical localization of neuropeptides in the adrenal medulla

The distribution of neuropeptides exhibits pronounced interspecies heterogeneity. Neuropeptides may function as hormones secreted from chromaffin cells or as neurotransmitters/neuromodulators released from nerve terminals. However, other possible functions such as trophic or intracellular effects should also be considered. Thus, to understand the role of neuropeptides, it is important to explore their localization in different species. The distribution of enkephalins, neurotensin, neuropeptide Y, calcitonin gene-related peptide, and galanin in the adrenal medulla of rat, cat, hamster, and mouse is presented in detail.     

Ultrastructural demonstration of exocytosis in the intact rat adrenal medulla

Evidence is presented for morphological proof of exocytosis in the rat adrenal medulla in situ. Techniques were modified to allow perfusion of the intact adrenal gland with secretagogues (or electrical stimulation) followed by tannic acid. Unstimulated specimens demonstrated exocytotic (omega-shaped) profiles filled with flocculent material. This flocculation was also seen in the intercellular space. Stimulation of the adrenal medulla also resulted in the appearance of exocytotic profiles and an accumulation of the flocculent mass. This was often most evident in the subendothelial space. This is the first demonstration of exocytosis in the rat adrenal medulla by electron microscopy. The techniques used in this study will be useful for studying the pathway of secretory products of the adrenal chromaffin cell before they enter the vascular system.     

Histological and immunocytochemical localization of serotonin-like immunoreactivity in the brain and optic ganglia of the Indian white shrimp, Fenneropenaeus indicus

Serotonin is one of the important neurotransmitter and neuromodulator so far studied in crustacean models. With its secretory sites well-studied in higher crustaceans, its function in controlling the release of metabolic hormones from their storage and release sites has been well proved. The present study attempts to localize serotonin-like immunoreactivity in Fenneropenaeus indicus, a commercially important shrimp species and a natural inhabitant of the Indian oceans. Histological studies were employed to visualize the different types of neurosecretory cells and their regions of occurrence in brain and optic ganglia on the basis of their size, shape, and tinctorial properties. Immunocytochemical studies were performed in the brain and optic ganglia with specific antisera against serotonin in combination with peroxidase anti-peroxidase to map the serotonin-like immunoreactive cells. Variations in the immunoreactivity were observed on comparing the cells of brain and optic ganglia. Medulla terminalis region had intense serotonin immunoreactivity suggesting it to be the primary source of the neurotransmitter.     

Immunoelectron microscopic localization of neurotransmitters in the cochlea

This paper presents the works and methods of our respective laboratories using electron microscopic immunocytochemistry to identify and localize cochlear neurotransmitters. Antibodies to various prospective neurotransmitters and associated enzymes have been used to study the ultrastructural localization of several candidates for olivocochlear efferent neurotransmitters previously suggested by light microscopic immunocytochemistry. Antibodies against enkephalins label lateral olivocochlear efferent fibers. Antibodies against choline acetyltransferase (ChAT) (an enzyme marker for acetylcholine) label a major population of both lateral and medial efferent fibers and terminals, whereas antibodies to γ-aminobutyric acid (GABA) label what might be a small subpopulation of both the lateral and medial efferent systems. The GABA-like immunostained medial efferent fibers are preferentially located in the upper turns of the guinea pig cochlea, particularly the third turn. Immunoelectron microscopy shows that neither GABA nor ChAT immunolabels all medial efferent terminals, regardless of cochlear turn. All the different types of immunolabeled efferent terminals have been observed to make characteristic synaptic contacts; lateral efferent terminals on afferent dendrites and medial efferent terminals on outer hair cells and occasionally on type II afferent dendrites. Other types of contacts involving GABA-like, and sometimes met-enkephalin-like, immunostained fibers are occasionally seen particularly in the upper turns of the cochlea. Immunoelectron microscopic results suggest that both medial and lateral efferent systems might be further subdivided on the basis of differences in neurotransmitters. Future trends of immunocytochemical research on cochlear neurotransmitters are proposed, particularly colocalization studies, which show a complex pattern of coexistence of neurotransmitters in the lateral efferent system.     

Stem cells in the development and differentiation of the human adrenal glands

There are no studies on stem cells (SCs) and development and differentiation (DD) of the human adrenal glands. The SCs in DD of the adrenal glands were herein investigated histochemically and immunohistochemically in 18 human embryonic adrenal glands at gestational week (GW) 7–40. At 7 GW, the adrenal glands were present, and at 7 GW, numerous embryonic SCs (ESCs) are seen to create the adrenal cortex. The ESCs were composed exclusively of small cells with hyperchromatic nuclei without nucleoli. The ESCs were positive for neural cell adhesion molecule, KIT, neuron‐specific enolase, platelet‐derived growth factor receptor‐α, synaptophysin, and MET. They were negative for other SC antigens, including chromogranin, ErbB2, and bcl‐2. They were also negative for lineage antigens, including cytokeratin (CK)7, CK8, CK18, and CK19, carcinoembryonic antigen, carbohydrate antigen 19‐9, epithelial membrane antigen, HepPar1, mucin core apoprotein (MUC)1, MUC2, MUC5AC, and MUC6, and cluster differentiation (CD)3, CD45, CD20, CD34, and CD31. The Ki‐67 labeling index (LI) was high (Ki‐67 LI = around 20%). α‐Fetoprotein was positive in the ESCs and adrenal cells. The ESC was first seen in the periphery of the adrenal cortex at 7–10 GW. The ESC migrates into the inner part of the adrenal cortex. Huge islands of ESC were present near the adrenal, and they appeared to provide the ESC of the adrenal. At 16 GW, adrenal medulla appeared, and the adrenal ESCs were present in the periphery or the cortex, in the cortical parenchyma, corticomedullary junctions, and in the medulla. The adrenal essential architecture was established around 20 GW; however, there were still ESCs. At term, there are a few ESCs. These data suggest that the adrenal glands were created by ESCs. Microsc. Res. Tech., 78:59–64, 2015. © 2014 Wiley Periodicals, Inc.     

Mitotic cell division in the extraadrenal chromaffin system of various species

Mitotic activity often has been reported in embryonic and fetal sympathetic neuroblasts, principal sympathoblasts, and primitive sympathetic cells in various species at different stages of development. Postnatal adrenal medullary cells also are known to undergo mitosis, but such dividing capabilities rarely have been observed in the true postnatal extraadrenal chromaffin system. Although few in number, this work nevertheless has clearly identified such cells in varying stages of the mitotic cycle in the young dog, Syrian hamster, mouse, rabbit, and rat. The dividing cells were noted in paraaortic chromaffin organs, paraganglia, and within the inferior mesenteric ganglion as well. They displayed the morphological character usually associated with their adrenal medullary catecholaminergic counterparts, including numerous dense-cored vesicles known to be the harbingers of catecholamines and various peptides. Nerve endings were not noticed upon the mitotic cells. The phenomenon of dividing extraadrenal chromaffin cells augments existing data and perhaps suggests that these cells are more endocrine than neural in type and subservient to the adrenal medulla in its classic endocrine function.     

Distribution of neurotrophin and TrK receptor-like immunoreactivity in the adrenal gland of birds

The occurrence and localization of neurotrophins and their specific TrK receptor-like proteins in the adrenal gland of chicken, duck and ostrich were examined by immunohistochemical methods. In all species studied NGF-, TrK A- and TrK C-like immunoreactivity was observed in neurons and fibers of adrenal ganglia. Thin TrK A- and TrK C-like immunoreactive fibers were also observed among chromaffin cells. NT-3-like immunoreactivity was detected in chromaffin cells as revealed by the double immunolabelings NT-3/chromogranin A and NT-3/DbetaH. The interrenal tissue never showed IR to any neurotrophins and TrK tested, and none of the adrenal structures displayed immunoreactivity to BDNF and TrK B. Double immunolabelings NGF/TrK A, NGF/TrK C and TrK A/TrK C showed colocalization in some neurons and fibers in adrenal ganglia. In adrenal glands of the species studied, the distribution of neurotrophins and TrK receptors could suggest an involvement of NT-3 on neuronal populations innervating adrenal ganglia by means of its high affinity receptor TrK C and low affinity receptor TrK A. In addition, NGF could be utilized by neuronal populations of adrenal ganglia through its preferential receptor TrK A by an autocrine or paracrine modality of action.     

Morphological observations of small granule-containing (Chromaffin) cells in the celiac ganglion of the guinea pig,with emphasis on cell contacts

Utilizing electron microscopic observation, several contacts between small, granule-containing cells (SGC) and postganglionic neurons (PGN) in the celiac ganglion of the guinea pig have been observed. A SGC in very close association with a PGN was seen to receive a distinct synaptic contact that contained many vesicles with dense cores. This contact was morphologically unlike cholinergic synapses previously reported on chromaffin cells. Because the SGC and PGN were clearly separated by a thin rim of satellite cell cytoplasm mutual to both cells, it is not known how or if the SGC would possibly exert a synaptic or paracrine effect on the PGN. Also, intraganglion SGC existed as large well-vascularized islands within the celiac ganglion. These intraganlion clusters sometimes contained more than 50 cells and perhaps could be considered to function as localized neuroendocrine components within the ganglion by secreting granule products into the nearby blood vessels for local or distant effects, although this certainly is not known. This work reports a unique synaptic ending upon a single-occurring SGC, which, in turn, closely approximates a ganglion neuron in a soma-somatic relationship. In addition, a very close association (but no actual contact) was observed between granule-containing processes, presumably emanating from the intraganglion clusters, and PGN. Whatever the function of ganglionic SGC may be, the exact relationship between SGC and PGN presumably would be of great interest and potential importance. © 1994 Wiley-Liss, Inc.     

Distribution of NGF and NT-3-like protein immunoreactivity in the teleost kidney

By means of immunochemistry and immunohistochemistry, we investigated in the kidney of freshwater and marine teleostean species for the presence and localization of three neurotrophins: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3. In both species studied, NGF-like and NT-3-like immunoreactivity were present in the kidney with different distribution patterns, while BDNF-like immunoreactivity was never detected. In goldfish, NGF-like and NT-3-like immunoreactivity were identified extensively in cells along part of the arterial branches adjacent to the afferent arterioles. In scorpion fish, NGF-like and NT-3-like immunoreactive cells were observed both on afferent arterioles and on adjacent secondary branches derived from renal arteries. No immunoreactivity was detected in other renal structures. A staining pattern of immunoreactivity similar to that obtained for NGF and NT-3 was detected utilizing S100 antibody as a juxtaglomerular (JG) cell marker. Double immunolabellings NGF/S100 and NT-3/S100 evidenced the coexistence of neurotrophin-like proteins and S100-like protein in the same immunoreactive cells, thus identifying them as juxtaglomerular cells. Western blot analysis revealed the presence of molecules immunoreactive to NGF and NT-3, whose molecular weights were very similar to those of the corresponding mammalian neurotrophins. These findings extend the presence and distribution of NGF-like and NT-3-like IR in the kidney to teleost species, suggesting a probable participation of these proteins in the renal functions of freshwater and marine teleosts.     

Electron microscopic and biochemical study of the effects of rapamycin on glycogen autophagy in the newborn rat liver

The effects of rapamycin on glycogen autophagy in the newborn rat liver were studied using biochemical determinations, electron microscopy, and morphometric analysis. Rapamycin increased the fractional volume of hepatocytic autophagic vacuoles, the liver lysosomal glycogen-hydrolyzing activity of acid glucosidase, the degradation of glycogen inside the autophagic vacuoles, and decreased the activity of acid mannose 6-phosphatase. These findings suggest that rapamycin, a known inhibitor of the mammalian target of rapamycin (mTOR) signaling, induces glycogen autophagy in the newborn rat hepatocytes. mTOR may participate in the regulation of this process.     

A new method for obtaining scanning electron microscopic images of the reorganization process of human dermal microvascular endothelium in vitro

A new method for obtaining scanning electron microscopic images of the reorganization process of endothelial cells has been developed. When covered with a collagen-coated disk, all the cultured endothelial cells reorganized on the collagen of the disk, which was easily taken out from the dish to process for SEM. The reorganization process could be divided into four stages: endothelial cell growth (Stage 1), reticular network formation (Stage 2), tubular structure formation (Stage 3), and cytolysis of the tube (Stage 4). Between Stages 1 and 2 the endothelial cells transformed from a cobblestone to a spindle-shaped pattern and fused each other, forming a board-like structure. Between Stages 2 and 3 break up of parts of the board-like structure and outflow of a necrotic mass from the centre of the structure occur. At Stage 3 a tubular structure is formed following enwrapping of the cleared centre by the surrounding endothelial cells. This method produces a means to study the angiogenesis in a variety of disorders including tumours and wound-healing process using SEM.     

Histological,transmission electron microscopic,and immunohistochemical study of the adrenal gland in the Persian squirrel (Sciurus anomalus)

This research was aimed to present the histological and ultrastructure properties of the adrenal gland in the Persian squirrel. Two male and female animals were included in the study. The adrenal gland was bean-shaped and located on the cranial pole of kidney. The enveloping capsule was dense connective tissue that reacted positively with Periodic-Acid Schiff (PAS) and Masson trichrome stainings. The parenchyma of the gland consisted of two-part, namely cortex and medulla; the cortex had three layers: zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR). The cells of the ZG were mainly spherical and ovoid with circular arrangement and few lipid droplets in TEM micrographs. The cells of the ZF were columnar and spherical that were arranged in cord-like rows. Transmission electron microscopy (TEM) indicated conspicuous lipid droplets and mitochondria in this zone. The cells of the ZR were arranged in a tangled networks and were almost similar to those in the ZF. TEM images showed fewer lipid vesicles in the ZR compared to the ZF and ZG. Chromaffin cells were located in the medulla of the adrenal gland in two layers. TEM images showed that some of them were smaller and contained fewer secretory granules; other cells were larger and contained more electron-dense secretory granules. Immunofluorescence staining showed that steroidogenic factor 1 (SF1) expressed from cortex to the corticomedullary junction (CMJ) and tyrosine hydroxylase (TH) expressed in the medulla. In conclusion, the results indicated both similarities and differences between the adrenal gland of the Persian squirrel and other animals such as mammals and rodents.     

Leptin‐like immunoreactivity in the muscle of juvenile sea bass (Dicentrarchus labrax)

The mammalian hormone, leptin, is mainly synthesized in adipose tissue along with other tissues. Leptin plays a role in numerous processes such as in the control of food intake, homeostasis, immune function and reproduction. In this study, we detected and localized leptin immunoreactivity to the muscle of early juvenile sea bass (Dicentrarchus labrax) by Western blot analysis and immunohistochemistry. A leptin immunopositive band with a molecular weight of ～16 kDa, corresponding to mammalian leptin, was identified in trunk skeletal muscle homogenate. Furthermore, leptin immunopositive cells were detected in the endomysium of skeletal muscular fibers. These cells showed immunostained cytoplasmic granules and roundish and oval nuclei. The most intense immunostaining was observed in the endomysial space among the superficial red muscular fibers of the trunk. These findings suggest that in early juvenile sea bass, leptin is mostly produced by skeletal muscles. Therefore, during the developmental stage lacking adipose tissue, skeletal muscles can be considered an important source of leptin. As already suggested in mammals, we can hypothesize the potential roles of leptin not only in energy expenditure for muscle contraction but also during muscle differentiation and growth. Microsc. Res. Tech. 73:797–802, 2010. © 2010 Wiley‐Liss, Inc.     

Ultrathin cryosections in the plane of cell monolayers: Evaluation of their potential for antibody localization studies of the cytoskeleton

In spite of the fact that the preembedding method is satisfactory for the ultrastructural localization of cytoskeletal proteins, there is a need for a localization method that retains the cells' ground substance, delicate filament arrangements, and membrane-filament interactions and provides a good delineation of ultrastructural detail. Ultracryomicrotomy, a resinless sectioning method, can combine good morphology with optimal antibody labeling. Until now, however, it has not been possible to section cell monolayers parallel to their plane of growth. This is a prerequisite for the localization of proteins along segments of filaments, contained within the section thickness. We describe such a method and give a first appreciation of its potential for antibody localization studies of cytoskeletal proteins. The method consists of seeding cells on a parallel 0.75-mm-thick gelatin substrate that can later be cut and used as a mounting block. An adapted negative staining has yielded a very useful delineation of the well-preserved structures within the cells, even in combination with immunogold labeling. The latter has been in its indirect version less satisfactory in dense microfilament bundles because of penetration problems, and more satisfactory on microtubules. Clearly, the penetration properties of gold probes will have to be improved before this method will become widely applicable. The availability of a sectioning method like this will provide the basis for further progress. There will be many cases which will justify the use of this relatively more difficult approach.     

Application of the disector method in the light microscopic quantification of type II pneumocytes in control and ozone-exposed rats

Alveolar type II cells in control and ozone-exposed rat lungs were counted at the light microscopical level with the ‘disector method’. The type II cells were unequivocally marked by histochemical staining for alkaline phosphatase activity in 2 μm plastic sections. By this counting method, the mean number of type II cells per lung in control rats was of the same magnitude as those reported in the literature, using point counting methods. After exposure of rats to 1.6 mg ozone/m³ for 7 days, a 50% increase in the mean number of type II cells was observed. The use of the disector method at the light microscopical level offers some advantages above a quantification at the electron microscopical level. The procedure is less time-consuming, larger areas can be screened, two parallel countings can be performed in one set of sections and there is no need for an exact knowledge about the diameter of the measured particle.     

Intracellular localization of the antitumour drug adriamycin in living cultured cells: A confocal microscopy study

The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy. In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin-treated M14 cells were allowed to recover in drug-free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug-resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild-type cells. In particular, in resistant human breast carcinoma cells (MCF-7), adriamycin appeared to be exclusively located within the cytoplasm whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P-glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a very useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.     

Hypothalamic regulatory peptides and their receptors: Cytochemical studies of their role in regulation at the adenohypophyseal level

Hypothalamic regulatory peptides bind to specific receptors on target cells in the pituitary and control secretion. They in turn can be regulated at the pituitary level by steroid and peptide modulators. Affinity cytochemical techniques are important tools for the identification of specific target binding sites for these regulatory peptides. This presentation reviews the work in which potent, biotinylated ligands of gonadotropin releasing hormone (bio-GnRH), corticotropin releasing hormone (bio-CRH), and arginine vasopressin (bio-AVP) were applied to study the target cell responses. Bio-GnRH, bio-CRH, and bio-AVP bind to membrane receptors on specific anterior pituitary cells. Dual labeling for either gonadotropin or adrenocorticotropin (ACTH) antigens further identified the target cells. After 1–3 minutes, the label was in patches or capped on the surface. After 3 minutes, it was internalized in small vesicles and sent to receptosomes and vacuoles in the Golgi complex. Eventually the biotinylated peptides, or a metabolite, was found in the lysosomes (multivesicular bodies) and a subpopulation of secretory granules. The route and rate of uptake was similar to that described for the classical receptor-mediated endocytosis process. In contrast, intermediate lobe corticotropes internalized the bio-CRH in less than 1 minute. The route through the Golgi complex appeared to be bypassed. Instead the labeled peptide was in vesicles, on the membranes of scattered vacuoles, and in multivesicular bodies. Modulation of ligand binding by steroids showed that changes in receptor numbers correlated with changes in the number of cells that bound the ligand. In male rats, dihydrotestosterone reduced the percentage of GnRH-bound cells by 50%. Most of the reduction appeared in cells that stored luteinizing hormone (LH) antigens. In diestrous female rats, estradiol increased the percentage of bio-GnRH-bound cells. However, the steroid decreased the percentage of GnRH-bound cells in cells from proestrous rats. Glucocorticoids decreased the percentage of CRH-bound corticotropes in as little as 10 minutes. Potentiation of secretion by these ligands was correlated with increases in the percentage of ligand-bound cells. AVP pretreatment of corticotropes increased the percentage of cells that bound bio-CRH. It also increased the rate of receptor-mediated endocytosis of CRH and changed the route so that the Golgi complex was bypassed. This effect could be mimicked by activation of its second messengers (calcium and protein kinase C). Similarly, CRH pretreatment increased the percentage of corticotropes that bound AVP. Thyrotropin releasing hormone (TRH) pretreatment also increased the percentage of thyrotropes that bound AVP. Finally, calcium or sodium channel blockers altered CRH binding so that fewer cells were labeled. This binding by CRH was not dependent on extracellular calcium and tests with a calcium channel agonist showed that it was related to activation of calcium channels. To summarize, these affinity cytochemical studies have identified specific target cells in the pituitary for GnRH, CRH, and AVP. They have also identified heterogeneity in the population. They have demonstrated new information about the direct modulatory effects of steroids, ion channels, and neuropeptides on neuropeptide binding by subpopulations of these target cells.     

Ultrastructural localization of nucleic acids in plant tissues following the use of malachite green or neutral red in the fixative solution

Malachite green and neutral red, when added to glutaraldehyde for fixation of various tissues, yielded high-contrast images of cell ultrastructure. Malachite green, in acid conditions, appeared to increase contrast of heterochromatin material in the nucleus whereas neutral red gave greater clarity to the nucleolus and to cytoplasmic ribosomes. Control tissue fixed under acid conditions showed little damage but there were ‘crystalline’ areas at the periphery of the nucleolus. RNase did not digest cytoplasmic ribosomes from tissue after neutral red glutaraldehyde fixation. These results suggested that neutral red became bound to RNA in the tissues. Fixation with malachite green, at a pH below 6, did not affect the digestion of RNA by RNase but did protect chromatin against the bleaching action of the chelating agent EDTA. The addition of malachite green (pH < 6) or neutral red to glutaraldehyde are useful techniques for the investigation of the ultrastructure of nuclear material and cytoplasmic ribosomes.     

Effects of microwave irradiation and chemical fixation on the localization of perisinusoidal cells in rat liver by gold impregnation

Modified gold impregnation is one of the methods that are used in light microscopical demonstration of hepatic perisinusoidal cells. This method has some disadvantages, such as restriction of fixation time to 16 h, which allows limited time for processing the tissues, especially when dealing with a large amount of material, and a long impregnation time (16–24 h). We investigated the effect of prolonged fixation on the staining of sections, to shorten the time needed for gold impregnation by using microwave irradiation. Liver specimens were fixed in Baker's calcium–formalin for different periods of time. After fixation, frozen sections were impregnated in gold chloride solution either at room temperature or in a microwave oven. The staining quality of the sections which had been impregnated in the microwave oven for a much shorter time were equal to or even superior to the ones impregnated at room temperature. Prolonging the fixation time up to 7 days did not affect the staining results by microwave irradiation, whereas satisfactory results were not obtained from sections stained at room temperature and fixed for more than 3 days. We conclude that microwave irradiation can be used to shorten the impregnation time in gold chloride solution and the duration of fixation can be prolonged up to 3 days in the original method and up to 7 days when microwave irradiation is used during impregnation.