Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.     

Electron microscopic and radioautographic study of the bronchi in chronic inflammation as affected by helium-neon laser treatment

Structural-metabolic changes were studied in 98 biopsies of large bronchial mucosa from 39 male patients with chronic suppurative lung diseases. It was shown (by the level of DNA and RNA synthesis) that the proliferative and metabolic processes were induced by endobronchial treatment of the damaged epithelium with helium-neon laser which passing through a number of transitional stage recovers its structure and differentiation to ciliary and goblet cells with normal ultrastructure. In lamina propria hyperemia, intensive leukodiapedesis, leukocyte infiltrates and granulations develop and proliferative and metabolic activity of endotheliocytes and interstitial cells increases, which results in the formation of delicate fibrous connective tissue. Simultaneous reorganization of the epithelium and underlying connective tissue is interpreted in terms of parenchymatous-stromal interactions.     

Histopathology of the digestive gland of the bivalve mollusk Crenomytilus grayanus (Dunker, 1853) from southwestern Peter the Great Bay, Sea of Japan

The histomorphology of the digestive gland of the bivalve mollusk Crenomytilus grayanus from Sivuchya Bay, which is located in the southwest of Peter the Great Bay and subjected to the effect of polluted waters of Tumannaya River, was studied. Pathological changes of the digestive tubules, channels, and connective tissue of the gland were recorded in all the mussels studied. The epithelium of the tubules and channels was characteristic with erosive disturbances and by heavy vacuolization of digestive cells; connective tissue of the gland was specified by cells with lipofuscin (granulocytomes) and by foci of cells necrosis and lysis. Nervous fibers running in the gland were swollen in some mollusks. Strongly basophilic spherical formations, presumably one of the development stages of a parasitic plasmodium, were found in the granulocytomes and among vesicular cells of connective tissue of all the mussels. It was concluded that pathological changes in digestive gland of Gray’s mussel might be caused by chronic pollution of the bay and by parasitic invasion.     

Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: a biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.     

Regeneration of radiation-damaged digestive tissues in juvenile Pacific oysters (Crassostrea gigas)

Juvenile Pacific oysters, Crassostrea gigas, were irradiated with 16 and 40 krad and their tissues examined histologically. Degenerative syndromes and tissue regeneration processes were determined for the stomach, gut, collecting ducts, and digestive tubules. Following degeneration, tissue regeneration was observed in the digestive tissues of most oysters exposed to 16 krad and in a limited number exposed to 40 krad. Regeneration was first observed in the digestive tubules and subsequently in the stomach, gut, and collecting ducts. Cellular repopulation of the digestive tubules involved epithelialization with large, undifferentiated crypt cells which then differentiated into functional secretory and absorptive cells. Regeneration in the stomach, gut, and collecting ducts was initiated by proliferative islands of small basophilic cells. Mitotic division of those cells and their subsequent differentiation into functional epithelial cells resulted in the rapid restoration and apparent recovery of the affected tissues. The results of these studies indicate that radioresistance of juvenile C. gigas may in part be due to the remarkably efficient regenerative mechanisms involved in replacing injured or lost digestive tissues.     

A method of isolating viable chondrocytes with proliferative capacity from cryopreserved human articular cartilage

This study aimed to optimise methods of cryopreserving human articular cartilage (AC) tissue for the isolation of late chondrocytes. Human AC specimens from osteoarthritis patients who had undergone total knee replacement were used to optimise the chondrocyte isolation process and the choice of cryoprotective agent (CPA). For AC tissue cryopreservation, intact cored cartilage discs (5 mm diameter) and diced cartilage (0.2–1 mm cubes) from the same sized discs were step cooled and stored in liquid nitrogen for up to 48 h before chondrocyte isolation and in vitro assay of cell viability and proliferative potential. The results showed that 10 % dimethyl sulphoxide in 90 % foetal bovine serum was a successful CPA for chondrocyte cryopreservation. Compared with intact cored discs, dicing of AC tissue into 0.2–1 mm cubes significantly increased the viability and proliferative capacity of surviving chondrocytes after cryopreservation. In situ cross-section imaging using focused ion beam microscopy revealed that dicing of cored AC discs into small cubes reduced the cryo-damage to cartilage tissue matrix. In conclusion, modification of appropriate factors, such as the size of the tissue, cryoprotective agent, and isolation protocol, can allow successful isolation of viable chondrocytes with high proliferative capacity from cryopreserved human articular cartilage tissue. Further studies are required to determine whether these cells may retain cartilage differentiation capacity and provide sufficient chondrocytes for use as implants in clinical applications.     

Comparative study of histological and kinetic variations of the digestive mucosa and pancreatic parenchyma after hypophysectomy in the rat. Light and electron microscopic study

Variations of the pancreatic parenchyma, the gastric mucosa and the intestinal mucosa were studied in adult male Wistar rats on day 8 and 15 after hypophysectomy. All results were compared with those obtained in pair-fed control rats. Hypophysectomy affected small intestine as well as gastric mucosa. Hypotrophy was observed on day 8 as most of the morphological parameters reached the maximal decrease. By contrast, hypoplasy occurred on day 15, when the labeling index (LI) decreased significantly. In the intestine, however, a decrease of the LI was observed only for the upper proliferative cells of the crypts. In the gastric mucosa, the LI was reduced only in the proliferative zone containing progenitor cells (isthmic region). Consequently, the cell differentiation is not similarly affected on all levels of the digestive tract.     

栉孔扇贝消化系统γ-氨基丁酸的免疫组织化学定位

采用抗生物素-生物素-过氧化物酶法(ABC法)的免疫组织化学技术,对γ-氨基丁酸(GABA)在栉孔扇贝(Chlamys farreri)消化系统各组织器官中的分布进行了定位研究。结果表明,栉孔扇贝的唇瓣、口唇、肠道的上皮细胞均呈GABA阳性反应,阳性反应物质呈颗粒状分布且多集中于上皮细胞游离端;胃上皮中未见GABA阳性反应;肝胰腺的上皮细胞及部分结缔组织中呈现GABA阳性反应,其中部分上皮细胞中的阳性反应物质呈团块状分布。GABA在栉孔扇贝消化系统除胃以外的各器官均有分布,推测其可能参与消化功能的调节作用。     

NADPH-diaphorase activity in the digestive system of gastropod molluscs <Emphasis Type="Italic">Achatina fulica</Emphasis> and <Emphasis Type="Italic">Littorina littorea</Emphasis>

Distribution and morphological peculiarities of nitroxidergic elements throughout the entire length of digestive tract was studied for the first time in gastropod molluscs Littorina littorea (Prosobranchia) and Achatina fulica (Pulmonata) using histochemical detection of NADPH-diaphorase (NADPHd). NO-ergic cells and fibers were revealed in all parts of the mollusc digestive system beginning from esophagus. Intensive NADPHd activity is found in a great number of intraepithelial cells of the open type and their processes in the intraand subepithelial nerve plexuses, subepithelial neurons, granular connective tissue cells, and multiple nervous fibers distributed among muscular elements of digestive tract as well as those in nerves innervating the tract. NADPHd was also revealed in receptor cells in the oral area and in the A. fulica CNS ganglia innervating the digestive tract. A. fulica has a more complicated organization of A. fulica nitroxidergic system of the digestive tract. A system of glomerular structures formed by thin NADPHd-positive neural fibers coming from epithelium is found directly beneath the epithelium in esophagus, stomach, and midgut of the mollusc. More superficially under the main groups of muscular elements there are revealed small clusters of NADPHd-positive neurons that can be classified as primitive, non-structured microganglia. The distribution pattern and a possible functional role of nitroxidergic elements in digestive tract of molluscs as compared with other invertebrate and vertebrate animals are discussed.     

Control of cell migration in atrioventricular pads during chick early heart development: Analysis of cushion tissue migration in vitro

The formation of the valvular and septal primordia of the embryonic heart depends upon the migration of endocardial cushion tissue mesenchyme (CT) to populate the cardiac jelly (CJ) in specific heart regions (e.g., atrioventricular (AV) pads). It has been proposed that the migration of CT may be directed by macromolecules of the CJ. In this study, [³H]thymidine-labeled endocardial (EC) and CT cells were transplanted onto intact pre- and postmigratory AV pads in vitro to test whether the compositional or structural changes known to occur in the cardiac jelly during development influence the migration of cushion tissue cells. After transplantation of labeled donor cells, host AV pads were fixed, embedded, and sectioned, and autoradiography was performed to determine the distribution of labeled donor cells within the host CJ. The experiments indicate that transplanted mural EC cells remain primarily at the AV pad surface, while grafted CT cells of all developmental ages rapidly invade both developmentally young and older AV pads. Furthermore, CT cells readily migrate in a direction opposite to that of cells in vivo when transplanted to inverted AV pads from which the myocardium has been removed. It is concluded that the CJ matrix, which is clearly a suitable framework for CT cell migration, provides no direct cues to determining the polarity or extent of migration.     

Autoradiographic study of human scirrhous gastric carcinoma during cultivation in diffusion chambers]

The growth of the epithelial and connective tissue cells was noted in cultivation of human schirrous carcinoma of the stomach in diffuse chambers. In difference to the initial tissue of the tumour in vitro in which no incorporation of thymidine-H3 into the connective tissue cells was noted, these cells proved to be labeled under conditions of growth in the chambers. One hour after the administration of thymidine-H3 the percentage of cells with labeled nuclei averaged 25.1% this considerably exceeding the value of the label index determined under conditions of incubation in vitro of the initial tumour tissue (6.6%). Under conditions of cultivation there was revealed a rapidly proliferating subpopulation of cells with the mitotic cycle duration of 14.8 hours.     

A scanning electron microscopic study of the luteo-follicular complex

Summary As observed by SEM, the repair of an ovulated mammalian follicle is accompanied by a sequence of morphogenetic processes. In the initial phase, a mass of cells and coagulated fluids forms at the site of rupture. Shortly thereafter, connective cells, recruited from the adjacent and subjacent connective tissue stroma begin to proliferate and to migrate over this mass such that in the rabbit, the entire site of disruption is covered by a layer of connective cells by approximately 2 days following ovulation. Coincident with the migration of the connective tissue, superficial cells from undisturbed lateral and basal areas of an ovulated follicle also proliferate and begin to migrate over the newly established connective tissue matrix. By approximately 4 days following ovulation in the rabbit, the surface of an ovulated follicle is repopulated by elements of the superficial epithelium. The formation of the underlying corpus luteum (corpora luted) involves characteristic morphological changes as granulosa cells transform into steroid secreting luteal cells. The luteal cells become organized into cords of cells which usually surround capillary vessels. When examined by SEM, the smooth-surfaced endoplasmic reticulum of the luteal cell is quite apparent and is observed to form a three-dimension network of anastomosing tubules which are continuous with the nuclear membrane. Variations in the appearance of the surface of the ovary which directly overlies corpora lutea were observed when the mouse, rat and rabbit were compared. The regression of corpora lutea involves the infiltration of the luteal mass by connective tissue and both degeneration and vacuolization of the luteal cells. The regressing corpus luteum is a honey-comb-like structure in which each space is occupied by a degenerating luteal cell.This work was supported by grants from the National Institutes of Health, Public Health Service (to J.V.B., no. HD-04274), and from Consiglio Nationale delle Ricerche (C.N.R., contracts nos. CT 76.01288.04 and CT 77.01921.4)     

The connective tissue index of <Emphasis Type="Italic">Helix aspersa</Emphasis> as a metal biomarker

The digestive gland of adult land snails, Helix aspersa, sampled from four different sites in São Miguel island (Azores) was submitted to chemical analyses, autometallography and haemalum/eosin staining, in order to quantify the relative abundance of heavy metals, calcium cells and connective tissue cells. Metals were visualized, through light microscopy, as black silver deposits mostly in the connective tissue cells. Metal levels, essentially of Cu and Fe, were related to the relative volumetric density of connective tissue cells but not to the relative volumetric density of calcium cells from the digestive gland epithelium. Thus, the connective tissue index presented herein is suggested as a biomarker of Cu exposure in terrestrial mollusks.     

Structure of the Digestive Tube in the Holothurian Eupentacta fraudatrix (Holothuroidea: Dendrochirota)

In the holothurian Eupentacta fraudatrix,the gut wall exhibits trilaminar organization. It consists of an inner digestive epithelium, a middle layer of connective tissue, and an outer mesothelium (coelomic epithelium). The pharynx, esophagus, and stomach are lined with a cuticular epithelium composed of T-shaped cells. The lining epithelium of the intestine and cloaca lacks a cuticle and consists of columnar vesicular enterocytes. Mucocytes are also encountered in the digestive epithelium. The connective tissue layer is composed of a ground substance, which houses collagen fibers, amoebocytes, morula cells, and fibroblasts. The gut mesothelium is a pseudostratified epithelium, which is dominated by peritoneal and myoepithelial cells and also includes the perikarya and processes of the neurons of the hyponeural plexus and vacuolated cells.     

Microscopic anatomy of the cardiac ganglion of Limulus polyphemus

The morphology of the cardiac ganglion of Limulus polyphemus (L) was examined by reconstructions from stained serial sections. This ganglion is composed of two distinct parts: a fiber tract extending the entire length of the heart and a cellular portion underlying the fiber tract. The cellular portion extends continuously from the third pair of ostia to the posterior terminus of the heart. The mean number of ganglion cell bodies is 231. Most of the ganglion cells are located among the glial elements of the cellular portion. The greatest density of cells is found in segments 5 and 6. Six cell types are recognized: (1) large pigmented unipolar cells approximately 120 μ in diameter with distinct connective tissue capsules around them; (2) large pigmented bipolar cells approximately 120 μ in length which are also encapsulated; (3) pigmented multipolar cells approximately 80 μ in diameter which are free of capsules; (4) small pigmented bipolar cells approximately 40 μ in length which are encapsulated but which are found exclusively within the fiber tract; (5) non-pigmented multipolar cells approximately 30 μ in diameter which are found scattered among the connective tissue elements of the cellular portion; and (6) small non-pigmented cells approximately 10 μ in diameter which are found within the unipolar cell capsule and scattered among the connective tissue elements of the ganglion. The variability in cell numbers and the random location of cells points toward non-specific anatomical connectivity between elements of this ganglion.     

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D₂₈, and s-100. Cultured cells or tissues were incubated with 10?μM BODIPY forskolin for 30?min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D₂₈ (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.     

Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.     

Automated detection of connective tissue by tissue counter analysis and classification and regression trees.

OBJECTIVE: To evaluate the feasibility of the CART (Classification and Regression Tree) procedure for the recognition of microscopic structures in tissue counter analysis. METHODS: Digital microscopic images of H & E; stained slides of normal human skin and of primary malignant melanoma were overlayed with regularly distributed square measuring masks (elements) and grey value, texture and colour features within each mask were recorded. In the learning set, elements were interactively labeled as representing either connective tissue of the reticular dermis, other tissue components or background. Subsequently, CART models were based on these data sets. RESULTS: Implementation of the CART classification rules into the image analysis program showed that in an independent test set 94.1% of elements classified as connective tissue of the reticular dermis were correctly labeled. Automated measurements of the total amount of tissue and of the amount of connective tissue within a slide showed high reproducibility (r=0.97 and r=0.94, respectively; p<0.001). CONCLUSIONS: CART procedure in tissue counter analysis yields simple and reproducible classification rules for tissue elements.