Light Microscopic Immunohistochemistry Research Articles

Physiology and pathophysiology of the RANKL/RANK system

The bone resorption activity of osteoclasts is regulated at many levels, including differentiation of their monocytes precursors, fusion into multi-nucleated cells, migration to the resorption site, polarization of the mature osteoclasts and assembly of a podosome-based sealing zone. Another function of osteoclasts is relative to the integrity of the actin cytoskeleton, depending on the substratum upon which the osteoclasts are spread. There are two different structures of actin known as podosomes and sealing zone, actived in specialized matrix contacts and delimiting the membrane domain, where the ruffled border is formed. When a dual coculture of murine osteoblasts and murine mononuclear monocytes, in absolute absence of exogenous cytokines and other growth factors, was cultured on glass, the basic architecture of podosomes units and ruffled border was maintained regularly (1). We studied the osteoclast morphology and its behaviour in adhesion and in vesicle traffic by combination of light microscopy immunohistochemistry and transmission electron microscopy (TEM) immunolabeling. The adhesion and the fusion of preosteoclasts were observed by scanner electron microscopy (SEM). The osteoclasts produced by our physiological dual co-culture (without interaction of specific cytokines) are functionally and biologically active TRAP + and multinucleated cells. In fact the role of RANK, expressed by osteoblasts, controls the modulation of OPG bioavailability in the extracellular compartment. The fusion of monocytes is influenced by the presence of osteoblasts, that is based on RANK-RANKL interaction and communication between osteoblasts and preosteoclasts mediated by several molecules (2). Osteoclasts and osteoblasts can make direct contact, allowing membrane-bound ligands and receptors to interact and initiate intracellular signalling. RANKL-RANK complexes are likely internalized via rafts and then degraded in lysosomes. A recent study has shown that membrane-bound RANKL complexes to OPG is internalized by endocytosis process. A potential interaction of RANKL with clathrin components prior to the OPG binding, as shown by the kinetic results, OPG is intracellularly degraded after being internalized: our observation of osteoclast membrane at TEM has shown that immunogold labelled RANKL colocalizes with immunoglod labelled clathrin via the clathrin-coated-pit-mediated (3) pathway and both proteins are degraded by lysosome and proteasome pathway. 1) Nicolin V. et al.,. 2006 J.Mol.Histol.37 :171-177 2) Narducci P., et al.,. Acta Histochem.113(2):73-81. 3) Narducci P., et al., 2010, Eur J Histochem 54(1):e6.

Localization and Expression of Prothrombin in Rodent Osteoclasts and Long Bones

The serum protein prothrombin (PT) is proteolytically converted to thrombin during the coagulation cascade by the cell-associated prothrombinase complex. In vitro, RANKL-differentiated osteoclasts express tissue factor and coagulation factor Xa, which convert PT to thrombin (Karlstrom et al. Biochem Biophys Res Commun 394:593-599, 2010). The present study investigated the localization of PT in bone as well as the expression of PT mRNA in bone and osteoclasts. Herein, immunoblot analysis detected PT and smaller proteolytically cleaved fragments with sizes consistent with the action of prothrombinase in a protein fraction extracted with guanidine-HCl EDTA from mouse tibia. Light microscopic and ultrastructural immunohistochemistry demonstrated the presence of PT in the newly formed bone matrix of the metaphysis. Furthermore, fluorescent immunohistochemistry on metaphyseal trabecular bone showed that PT colocalized with MMP-9-expressing subepiphyseal osteoclasts, whereas cathepsin K-expressing osteoclasts were closely associated with PT of the bone matrix. RT-qPCR analysis revealed that PT mRNA was detected in tibia. Expression of PT mRNA in the tibia was 0.2% of the level in the liver. In addition, PT mRNA expression was increased by RANKL-induced differentiation of bone marrow macrophages to osteoclasts. The results demonstrate that PT is synthesized and proteolytically processed in bone. Furthermore, PT is present mainly in the newly formed bone matrix of the metaphyseal trabecular bone compartment in close association to osteoclasts. In addition, MMP-9-positive osteoclasts contain PT, and PT expression is increased during osteoclastogenesis.

Ultrastructural characterization of tryptophan hydroxylase 2-specific cortical serotonergic fibers and dorsal raphe neuronal cell bodies after MDMA treatment in rat

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a widely used recreational drug known to cause selective long-term serotonergic damage. The aim of this study was to characterize the ultrastructure of serotonergic pericarya and proximal neurites in the dorsal raphe nucleus as well as the ultrastructure of serotonergic axons in the frontal cortex of adolescent Dark Agouti rats 3 days after treatment with 15 mg/kg i.p. MDMA. Light microscopic immunohistochemistry and pre-embedding immunoelectron microscopy with a novel tryptophan hydroxylase-2 (Tph2) specific antibody, as a marker of serotonergic structures. Light microscopic analysis showed reduced serotonergic axon density and aberrant swollen varicosities in the frontal cortex of MDMA-treated animals. According to the electron microscopic analysis, Tph2 exhibited diffuse cytoplasmic immunolocalization in dorsal raphe neuronal cell bodies. The ultrastructural-morphometric analysis of these cell bodies did not indicate pathological changes or significant alteration in the cross-sectional areal density of any examined organelles. Proximal serotonergic neurites in the dorsal raphe exhibited no ultrastructural alteration. However, in the frontal cortex among intact fibers, numerous serotonergic axons with destructed microtubules were found. Most of their mitochondria were intact, albeit some injured axons also contained degenerating mitochondria; moreover, a few of them comprised confluent membrane whorls only. Our treatment protocol does not lead to ultrastructural alteration in the serotonergic dorsal raphe cell bodies and in their proximal neurites but causes impairment in cortical serotonergic axons. In these, the main ultrastructural alteration is the destruction of microtubules although a smaller portion of these axons probably undergo an irreversible damage.

Localization of NK1 receptors and roles of substance-P in subepithelial fibroblasts of rat intestinal villi

Subepithelial fibroblasts of the intestinal villi, which form a contractile cellular network beneath the epithelium, are in close contact with epithelial cells, nerve varicosities, capillaries, smooth muscles and immune cells, and secrete extracellular matrix molecules, growth factors and cytokines, etc. Cultured subepithelial fibroblasts of the rat duodenal villi display various receptors such as endothelins, ATP, substance-P and bradykinin, and release ATP in response to mechanical stimulation. In this study, the presence of functional NK1 receptors (NK1R) was pharmacologically confirmed in primary culture by Ca(2+) measurement, and the effects of substance-P were measured in an acute preparation of epithelium-free duodenal villi from 2- to 3-week-old rats using a two-photon laser microscope. Substance-P elicited an increase in the intracellular Ca(2+) concentration and contraction of the subepithelial fibroblasts in culture and the isolated villi. The localization of NK1R and substance-P in the villi was examined by light and electron microscopic immunohistochemistry. NK1R-like immunoreactivity was intensely localized on the plasma membrane of villous subepithelial fibroblasts in 10-day- to 4-week-old rats and mice and was decreased or absent in adulthood. The pericryptal fibroblasts of the small and large intestine were NK1R immuno-negative. These villous subepithelial fibroblasts form synapse-like structures with both substance-P-immunopositive and -immunonegative nerve varicosities. Here, we propose that the mutual interaction between villous subepithelial fibroblasts and afferent neurons via substance-P and ATP plays important roles in the maturation of the structure and function of the small intestine.

Campylobacter jejuni 81-176 forms distinct microcolonies on in vitro-infected human small intestinal tissue prior to biofilm formation

Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy. Organisms adhered to the apical surface of ileal tissues at all time points in large numbers, in areas where mucus was present and in distinct groups. Microcolony formation was evident at 1-2 h, with bacteria adhering to mucus on the tissue surface and to each other by flagellar interaction. At later time points (3-4 h), biofilm formation on ileal tissue was evident. Flagellar mutants did not form microcolonies or biofilms in tissue. Few organisms were observed in colonic tissue, with organisms present but not as abundant as in the ileal tissue. This study shows that C. jejuni 81-176 can form microcolonies and biofilms on human intestinal tissue and that this may be an essential step in its ability to cause diarrhoea in man.

Serotonin fibre sprouting and increase in serotonin transporter immunoreactivity in the CA1 area of hippocampus in a triple transgenic mouse model of Alzheimer’s disease

Alzheimer's disease (AD) is a neurodegenerative disease that deteriorates cognitive functions and associated brain regions such as the hippocampus, being the primary cause of dementia. Serotonin (5-HT) is widely present in the hippocampus, being an important neurotransmitter involved in learning and memory. Although recent evidence suggests alterations in 5-HT neurotransmission in AD, it is not clear how hippocampal 5-HT innervation is modified. Here, we studied hippocampal 5-HT innervation by analysing: (i) the expression, density and distribution of 5-HT transporter (SERT)-immunoreactive fibres; (ii) the specific morphological characteristics of serotonergic fibres and their relation to amyloid plaques; and (iii) the total number of 5-HT neurons within the raphe nuclei in triple transgenic mouse model of AD. We used quantitative light microscopy immunohistochemistry comparing transgenic and non-transgenic animals of different ages (3, 6, 9, 12 and 18 months). The transgenic animals showed a significant increase in SERT fibres in the hippocampus in a subfield-, strata- and age-specific manner. The increase in SERT fibres was specific to the CA1 stratum lacunosum-moleculare. An increase in SERT fibres in transgenic animals was observed at 3 months (by 61%) and at 18 months (by 74%). No changes, however, were found in the total number of raphe 5-HT neurons at any age. Our results indicate that triple transgenic mice display changes in the expression of SERT and increased SERT fibres sprouting, which may account for imbalanced serotonergic neurotransmission associated with (or linked to) AD cognitive impairment.

Effects of 8 weeks of vibration training at different frequencies (1 or 15 Hz) in senior sportsmen on torque and force development and of 1 year of training on muscle fibers

Objective: To examine the effects of 8 weeks of vibration training at different frequencies (1 and 15 Hz) on maximal isometric torque and force development in senior sportsmen, and of 1 year of heavy-resistance and vibration trainings on muscle fibers.Methods: Seven healthy senior sportsmen (mean age: 69·0 ± 5·4 years) performed an 8 weeks of strength training of knee extensors. Vibrations were applied vertically to the axis of movement during training. One leg of each subject was trained at a frequency of 1 Hz, while the other leg was trained at 15 Hz. Measures of isometric peak torque (at knee-angles of 60, 90 and 120°) and force development were recorded before and after training. Four sportsmen continued a year-long heavy-resistance training adding every second week a session of vibration training. After training, muscle biopsies were harvested from their quadriceps muscles and used for structural analyses. Morphometry of muscle fibers was performed by light microscopy. Immunohistochemistry using anti-MHCemb and anti-N-CAM antibodies was performed to measure potential muscle damage. Data from muscle morphometry were compared to that of a series of vastus lateralis biopsies harvested from 12 young sportsmen and four healthy elderly.Results: Our results showed a significant increase in isometric peak torque at both 1 and 15 Hz vibration frequency in all three measured angles of the knee. There was no significant difference between the two frequencies, but we could find a higher increase in percentage of maximum power after the 1 Hz training. The results of force development showed a slight increase at the 1 Hz training in measured time frames from 0 to 50 and 200 ms, without statistical significance. A trend to significance was found at the 1 Hz training at the time window up to 200 ms. The 15 Hz training showed no significant changes of force development. Muscle biopsies show that the muscles of these well trained senior sportsmen contain muscle fibers which are 35% larger than those of sedentary elderly and, unexpectedly, 10% larger than those of young sportsmen. Despite 1 year of heavy resistance and vibration training, no evidence of muscle damage or denervation/reinnervation could be observed by light microscopy analyses, ATPase histochemistry and immunohistochemistry using anti-N-CAM or anti-MHC-emb antibodies.Discussion: Integration of vibration to conventional strength training in elderly sportsmen induces similar improvement of isometric peak torque and force development independently from the vibration frequency after 8 weeks of training, and long-term results in the surprising evidence of hypertrophic muscle fibers larger than those of young active sportsmen. The observation that the vibration training with low frequency is safe opens the possibility to test these rehabilitation procedures in sedentary elderly.

T-SNARE and v-SNARE in the GABAergic and glutamatergic synapses of the rat cerebellar cortex

Using light microscope immunohistochemistry, the distributions of the t-SNARE, SNAP-25 and syntaxin-1a, and v-SNARE, VAMP-2 (or synaptobrevin-2), have been analyzed in the rat cerebellar cortex and compared with those of GAD-65/67, marker of GABAergic neurons, and vGLUT-1 and vGLUT-2, markers of glutamatergic neurons. SNAP-25 immunoreactivity was detected throughout the cortex, highly diffused in the neuropil. In the molecular and Purkinje layers, the immunoreactivity surrounded dendrite trunks of the Purkinje neurons, and bodies of stellate, basket and Purkinje neurons, which appeared all immunonegative. In the granular layer, the immunoreactivity was less diffuse and formed small grains localized within the protoplasmic islands of Held (synaptic glomeruli). Immunoreactivity was also observed in the white matter. The distribution of syntaxin-1a immunoreactivity appeared superimposable to that of SNAP-25. VAMP-2 immunoreactivity was observed in small dots finely dispersed in the neuropil of the molecular/Purkinje layer; a moderate labelling of the Purkinje neuron bodies was also observed. In the granular layer, VAMP-2 immunoreactivity formed larger deposits, presumably corresponding to mossy fibre terminals. Experiments of double labelling immunohistochemistry revealed no co-localization between t-SNARE (SNAP-25) or v-SNARE (VAMP-2) and GAD-65/67. On the contrary, co-localization between SNAP-25 and VAMP-2, on one side, and v-GLUT-1 or vGLUT-2, on the other side, was frequently revealed. Briefly, in the molecular layer, v-GLUT-1 and vGLUT-2-positive terminals only partly displayed also SNAP-25 immunoreactivity; whereas in the granular layer, all v-GLUT-1 and vGLUT-2-positive terminals displayed this co-localization. The results of this study indicate that t-SNARE and v-SNARE largely characterize the glutamatergic synapses in the rat cerebellar cortex. The same proteins are lacking in the GABAergic synapses.

Expression of transient receptor potential ankyrin 1 (TRPA1) in the rat trigeminal sensory afferents and spinal dorsal horn

Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold and pungent compounds, is implicated in the mediation of nociception, but little is known about the processing of the TRPA1-mediated nociceptive information within the trigeminal sensory nuclei (TSN) and the spinal dorsal horn (DH). To address this issue, we characterized the TRPA1-positive (+) neurons in the trigeminal ganglion (TG) and investigated the distribution of TRPA1(+) afferent fibers and their synaptic connectivity within the rat TSN and DH by using light and electron microscopic immunohistochemistry. In the TG, TRPA1 was expressed in unmyelinated and small myelinated axons and also occasionally in large myelinated axons. Many TRPA1(+) neurons costained for the marker for peptidergic neurons substance P (26.8%) or the marker for nonpeptidergic neurons IB4 (44.5%). In the CNS, small numbers of axons and terminals were immunopositive for TRPA1 throughout the rostral TSN, in contrast to the dense network of positive fibers and terminals in the superficial laminae of the trigeminal caudal nucleus (Vc) and DH. The TRPA1(+) terminals contained clear round vesicles, were presynaptic to one or two dendrites, and rarely participated in axoaxonic contacts, suggesting involvement in relatively simple synaptic circuitry with a small degree of synaptic divergence and little presynaptic modulation. Immunoreactivity for TRPA1 was also occasionally observed in postsynaptic dendrites. These results suggest that TRPA1-dependent orofacial and spinal nociceptive input is processed mainly in the superficial laminae of the Vc and DH in a specific manner and may be processed differently between the rostral TSN and Vc.

Immunohistochemical localization of apyrase during initial differentiation and germination of pea seeds.

Localization of the 49-kDa apyrase (ATP diphosphohydrolase, EC3.6.1.5; DDBJ/EMBL/GenBank BAB40230) was investigated during early stages of germination of pea (Pisum sativum L. var. Alaska) at the organ, tissue, cellular, and sub-cellular level using light-microscopical immunohistochemistry. Whole mount tissues were immuno-reacted with anti-APY1 serum, pre-immune serum or anti-actin antibody for control. Antigen to the anti-APY1 serum was not detected until 16 h after sowing (26 h after start of imbibition), when the antigen was detected throughout the tissue, especially in the epidermis and cortex. At 35 h after sowing, the younger regions including the root tip and the tip of the stele were more strongly stained than the control. Both, epidermal and cortical cells of the epicotyl and root tip were stained. The stain was mainly localized in the cytoplasm and around nuclei in the apical meristem and the root tip, while vacuoles and cell walls were not stained. At 62 h, there was major staining in the plumule, hook, and elongating regions of the epicotyl and in the region between cotyledons and the epicotyl. After 84 h, lateral root primordia were stained. The pre-immune serum showed virtually no staining while the anti-actin antibody reacted solely with the cytoplasm. Since the antigen to the anti-APY1 serum was primarily found in the cytoplasm and around nuclei in elongating and differentiating tissues and labeling declined in mature tissues, it is suggested that apyrases may play a role in growth and development of tissues, for example, lateral roots.

The nematode parasite Onchocerca volvulus generates the transforming growth factor-beta (TGF-beta)

Transforming growth factor-beta (TGF-beta) is a highly conserved cytokine that has a well-known regulatory role in immunity, but also in organ development of most animal species including helminths. Homologous tgf-b genes and mRNA have been detected in the filaria Brugia malayi. The in situ protein expression is unknown for filariae. Therefore, we examined several filariae for the expression and localization of latent (stable) TGF-beta in adult and larval stages. A specific goat anti-human latency associated protein (LAP, TGF-beta 1) antibody, purified by affinity chromatography, was used for light and electron microscopic immunohistochemistry. Adult Onchocerca volvulus, Onchocerca gibsoni, Onchocerca ochengi, Onchocerca armillata, Onchocerca fasciata, Onchocerca flexuosa, Wuchereria bancrofti, Dirofilaria sp., B. malayi, and infective larvae of W. bancrofti reacted with the antibody. Labeling of worm tissues varied between negative and all degrees of positive reactions. Latent TGF-beta was strongly expressed adjacent to the cell membranes of the hypodermis, epithelia, and muscles and adjacent to many nuclei in all organs. TGF-beta was well expressed in worms without Wolbachia endobacteria eliminated by doxycycline treatment. Pleomorphic neoplasms in O. volvulus were also labeled. We conclude that latent TGF-beta protein is expressed by filariae independently of Wolbachia, possibly regulating worm tissue homeostasis.

Immunohistochemical Detection of Prolactin and its Receptors in Human Testis/Immunhistochemische Bestimmung von Prolaktin und seinen Rezeptoren im menschlichen Hoden

Prolactin (PRL) and PRL receptors were localized by light microscopic immunohistochemistry in the human testes on interstitial Leydig-like cells. Similar stainings were obtained with both monoclonal antihuman PRL antibodies and polyclonal antibodies raised against rabbit mammary gland PRL receptors. Moreover, the detection of PRL binding sites by radio-receptor assay on the same samples gave a confirmation of these results. This suggests that PRL might act directly on Leydig cells in the man, in the same way as in the rodent.

Juxtanodin in the rat olfactory epithelium: Specific expression in sustentacular cells and preferential subcellular positioning at the apical junctional belt

In the CNS, juxtanodin (JN) is an actin-binding oligodendroglial protein that functions to promote differentiation of the host cells during postnatal development. In other tissues, JN expression and function remain unknown. We surveyed rat peripheral nerve, skeletal muscle and various epithelial tissues using immunoblotting and light-microscopic immunohistochemistry, and found prominent JN expression only in the olfactory epithelium (OE). Confocal and immunoelectron microscopy further revealed specific JN expression in the glia-like sustentacular cells and in the ductal cells of Bowman's glands. JN immunoreactivity was especially prominent in sustentacular cell apices extending superficially from the zone of terminal webs and associated adherens junctions, through the zone of tight junctions, to the roots of sustentacular microvilli. Moderate JN was also found in the supranuclear regions of sustentacular cells, whereas distal parts of sustentacular microvilli were devoid of JN. Distribution of JN in the OE differed from that of class III β-tubulin or nestin, but partially overlapped with a zone of intense F-actin staining near the OE mucous surface. Together these results identify JN as a marker protein for OE sustentacular cells, and support the glia-like nature of OE sustentacular cells. Functionally, JN in the OE might help in the molecular specialization of distinct compartments of olfactory receptor neurons (ORNs), in the interaction of sustentacular cells with ORNs, and/or in maturation/maintenance of sustentacular cells.

The Expression of Matrix Metalloproteinase according to Hydrostatic Pressure in Varicose Veins

Purpose: The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) caused by hydrostatic pressure stress is important in the development of varicose veins (VVs). This study was performed to analyse the expression of various MMPs and TIMPs according to the hydrostatic stress and the anatomical level of human great saphenous vein (GSV). Methods: Forty-nine vein samples were obtained from 10 patients with VVs (control group), and 34 samples from 7 VV patients after 1-hour hydrostatic stress just before surgery (stress group) at each anatomical site (proximal, Hunter, Dodd, and Boyd perforators) of GSV. Light microscopic examination and immunohistochemistry for MMP-1, -2, -9, -13 and TIMP-1, -2 were performed. Results: Intimal hyperplasia, fragmentation and loss of elastic fibers, infiltration of collagen fibers, and disorganization of medial muscle layers were evident in most vein samples. The degree of vein wall degeneration was not different between the 2 groups, and the anatomical sites of GSV. By immunohistochemistry, the expression of MMPs and TIMPs was not significantly different according to the group and the site. The expression of MMP-9 was more intense than that of other MMPs and TIMPs in all samples. MMP-9 was well localized to endothelial cells, medial muscle layers, and adventitial vasa vasorum. Conclusion: There are no distinct differences in the varicose vein samples after short-term postural blood stasis compared to the resting group. MMP-9 may be the key enzyme of the venous wall remodeling.

Demonstration of autoantibody binding to muscarinic acetylcholine receptors in the salivary gland in primary Sjögren's syndrome

A significant pathogenetic role of antimuscarinic acetylcholine receptor-3 (anti-m3AChR) autoantibodies in primary Sjögren's syndrome (pSS) has been suggested. However, the binding of these antibodies to the receptors in the target tissues has not yet been demonstrated. In this study, the binding characteristics of pSS sera and anti-m3AChR-monospecific sera affinity-purified from pSS patients to labial salivary gland samples from healthy subjects were studied with light- and electron microscopy. Furthermore, the ultrastructural localisation of in vivo deposited antibodies in pSS salivary glands was also investigated. Light microscopic immunohistochemistry revealed the binding of the anti-m3AChR-specific sera to the membrane of acinar cells. Similar reaction end-products were observed in the pSS salivary gland epithelial cell membranes. With electron microscopy, the autoantibody binding was observed to be localised to the junctions of epithelial cell membranes with nerve endings, both in normal and pSS glands. The results indicate that anti-m3AChR antibodies bind to the receptors in the salivary glands.

Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid

The annular ligament across the stapediovestibular joint connects the stapes footplate and the vestibular window and plays an important role in the sound conductive system of the ear. In this study, we investigated the distribution of extracellular matrix components in the ligament by histochemical methods at light and electron microscopic levels. As results, light microscopic immunohistochemistry of fibrillin and 36-kDa microfibril-associated glycoprotein (MAGP-36) showed intense immunoreactivities in the annular ligament between the stapes footplate and vestibular window. In addition, the histochemical localization of hyaluronic acid by using biotinylated hyaluronic acid-binding protein (HABP) clarifi ed the presence of hyaluronic acid in the annular ligament. At the electron microscopic level, the immunogold labeling of fibrillin showed intense labeling on the periphery of the electron-dense mantle. Furthermore, the labeling of fibrillin was preferentially seen on the fibrous components among the electronlucent amorphous substance. The immunogold labeling of MAGP-36 was seen on the electron-dense mantle and scattered on the electron-lucent amorphous substance. The gold labeling with biotinylated HABP clearly showed a distribution of hyaluronic acid throughout the amorphous space in the ligament. The present results provide a histochemical profile of the annular ligament of the rat stapediovestibular joint that may provide clues to elucidation of pathological changes in the ligaments and conductive hearing loss in humans.

Application of light microscopical and ultrastructural immunohistochemistry in the study of goblet cell carcinoid in the appendix

BackgroundGoblet cell carcinoids appear less frequently in the appendix than do other carcinoids. In the presented work a case with a goblet cell carcinoid of the appendix is described.MethodsRoutine histological and histochemical methods were employed, with a combination of histochemistry and immunohistochemistry on one section and light and electron microscopical immunohistochemisty on paraffin-embedded material, were applied to identify the type of the carcinoid and to reveal the fine structure of cell types in the tumour nests of the appendix.ResultsDuring the biopsy of a patient who had undergone appendectomy, an infiltration with clusters of goblet cells in the submucosa of the appendix was found. After a second operation of right-sided hemicolectomy, similar clusters of goblet cells were detected in the muscle layers of the caecum. After 18 months the patient died from cirrhosis and had not developed metastases or any recurrence. Immunohistochemically the serotonin-, somatostatin-, chromogranin A- and synaptophysin-positive endocrine cells were basally attached to mucin-secreting cells. The combined staining revealed simultaneously present endocrine cells (chromogranin-A-positive) and mucin-secreting cells (PAS- or alcian blue-positive). The ultrastructural immunohistochemistry showed that chromogranin A-positive cells had discoid and pleomorphic granules and were located in tumour nests or as single cells in the appendiceal wall.ConclusionThe combined histochemical and immunohistochemical procedure and the ultrastructural immunohistochemistry on archival material could contribute in clarifying the diagnosis of goblet cell carcinoid.

Interstitial Cajal‐like cells in rat mesentery: an ultrastructural and immunohistochemical approach

Interstitial Cajal-like Cells (ICLC) were recently recognized in a plethora of non-digestive organs. Here, we describe a cell type of rat mesentery sharing ultrastructural and immunohistochemical features with ICLC. Mesenteric ICLC were demonstrated by transmission electron microscopy (TEM) and further tested by light microscope immunohistochemistry. The cell described here fulfils the TEM diagnostic criteria accepted for ICLC: location in the connective interstitium; close vicinity to nerves, capillaries and other interstitial cells; characteristic long, moniliform cell processes; specialized cell-to-cell junctions; caveolae; mitochondria at 5–10% of cytoplasmic volume; rough endoplasmic reticulum at about 1–2%; intermediate and thin filaments, microtubules; undetectable thick filaments. The processes of this mesenteric ICLC were particularly long, with a mean length of 24.91 μm (10.27–50.83 μm), and a convolution index of 2.32 (1.37–3.63) was calculated in order to measure their potential length. Mean distances versus main target cells of ICLC–nerve bundles, vessels, adipocytes and macrophages–were 110.69, 115.80, 205.07 and 34.65 nm, respectively. We also tested the expression of CD117/c-kit, CD34, vimentin, α-smooth muscle actin, nestin, NK-1, tryptase and chymase and the antigenic profile of the mesenteric ICLC was comparable if not identical with that recently observed in ICLC from other extra-digestive tissues. Due to the peculiar aspect of the mesenteric ICLC processes it can be hypothesized that these cells form a three-dimensional network within the mesentery that is at the same time resistant and deformable following stretches consequent to intestine movements, mainly avoiding blood vessels closure or controlling blood vessels rheology. It remains, however, to be established if and how such cells are connected with the archetypal enteric ICC.

Expression of calcium channel CaV1.3 in cat spinal cord: Light and electron microscopic immunohistochemical study

In spinal neurons, plateau potentials serve to amplify neuronal input signals. To a large extent, the underlying persistent inward current is mediated by a subtype of the L-type calcium channel (Ca(V)1.3). In the present investigation, we have studied its distribution and cellular localization in the cat spinal cord by light and electron microscopic immunohistochemistry. The results show that Ca(V)1.3-like immunoreactivity is widely distributed in all segments of the spinal cord but that the distribution in the different laminae of the spinal gray matter varies, with the highest density of labeled neurons in lamina IX and the lowest in lamina II. The labeling intensity was highest in neuronal somata, but a certain length of the proximal dendrite was also labeled. Some neuronal groups exhibited a particularly dense labeling; these include the lateral motoneuronal group in the cervical and the lumbar enlargements and the phrenic nucleus in cervical, Clarke's nucleus in lower thoracic and upper lumbar, and Onuf's nucleus in upper sacral segments. At the ultrastructural level, Ca(V)1.3-immunoreactive products were found in neuronal somata and dendrites of different sizes. In the soma, they were predominantly associated with the rough endoplasmic reticulum but some also with the plasma membrane. In dendrites, they were associated with both intracellular organelles, including microtubules and microchondria, and the plasma membrane. These results indicate that significant proportions of the neurons in cat spinal cord, including projection neurons, interneurons, and motoneurons, are endowed with ion channels that subserve persistent inward currents and act to amplify synaptic input signals.

Chitin Nanofibrils Linked to Chitosan Glycolate as Spray, Gel, and Gauze Preparations for Wound Repair

Recent advances in process chemistry have made it possible to make chitosan and chitin nanofibril materials more flexible and useful for the development of new biorelated products. In this study, the effectiveness of three chitin nanofibril/chitosan glycolate-based preparations, a spray (Chit-A), a gel (Chit-B), and a gauze (Chit-C), in healing cutaneous lesions are assessed macroscopically and by light microscopy immunohistochemistry. These evaluations are compared to the results obtained using a laser co-treatment. The wound repair provided by the three preparations is clearly evident even without the synergistic effect of the laser co-treatment. These results confirm the effectiveness of chitin nanofibril/chitosan glycolate-based products in restoring subcutaneous architecture. The spray seems to be most effective in healing superficial lesions, including extensive ones; the gel is more effective in repairing shallow lesions as well as an aesthetic factor while the gauze is effective in slow-healing dermo-epidermal wounds.