Suitability of different staining methods for the identification of isolated and cultured cells from guinea pig (Cavia aperea porcellus) stomach.

Cell suspensions from the guinea pig gastric mucosa were obtained using a pronase/collagenase isolation method, and cultured on Petri dishes in minimum essential medium at 37 degrees C. For proper identification of different gastric cell types in cytospots, cell suspensions or culture, selective staining methods were employed, modified and evaluated. Mucous cells and mucous neck cells were detected by use of lectins. Mucous cells were stained on cytospots and in primary cultures with lectins from peanut, Helix pomatia, Ulex europaeus, wheat germ, and from soybean. Vital chief cells in suspensions but not in culture, were selectively stained by Nile blue sulphate, brilliant cresyl blue or the fluorescence dye dihexyloxacarbocyanine iodide. Pepsinogen granules of isolated and cultured chief cells were detected with a polyclonal antibody against porcine pepsinogen. Isolated parietal cells were identified in cytospots by using acidophilic dyes (aurantia, eosin). In suspensions and in cultures vital parietal cells were identified by enzymatic detection of succinic dehydrogenase or carboanhydrase activity and by the vital stain Janus green. In cultures exclusively, parietal cells were additionally identified by the vital stain rhodamine. Cytochemically, they were identified with phalloidin by binding to actin filaments. Endocrine cells in the suspension were visualised immunocytochemically with antibodies directed against different amines or peptides. Fibroblasts and endothelial cells were identified after isolation and in primary culture with a vimentin antibody. Mast cells in suspension were either visualised by a histamine antibody or by metachromatic staining behaviour to toluidine blue, respectively. Endothelial cells in suspension or culture were distinguished from fibroblasts by endocytosis of acetylated low-density-lipoprotein. In conclusion, the developed methods are highly suitable to identify guinea pig gastric cells after isolation and follow up their fate in primary culture.

Improvement of tracheal autograft revascularization by means of fibroblast growth factor.

Ischemic airway complications after lung transplantation remain a significant problem despite the use of bronchial omentopexy. Clinical observations suggest that enhancement of vascular ingrowth could possibly increase the efficacy of a bronchial omental flap. This study was therefore designed to investigate whether basic fibroblast growth factor can enhance blood supply of an ischemic airway by acceleration of vascular ingrowth in a rabbit autotransplant model. Segments of the trachea were harvested and transplanted into a subcutaneous pouch. The animals were randomly assigned to one of four groups: group I, no omentopexy; group II, omentopexy; group III, omentopexy and fibrin glue; or group IV, omentopexy and fibrin glue enriched with 2.5 micrograms basic fibroblast growth factor. After 14 days the animals were sacrificed. The extent of perfusion was investigated by means of radioactive microspheres. The morphology of the tracheal segments was investigated in a blinded fashion macroscopically, by means of light microscopy, and by means of scanning electron microscopy. The radioactivity measurements revealed a significantly increased perfusion of group IV (77% +/- 42%) as compared with groups I (17% +/- 13%) and III (20% +/- 16%). By macroscopic and light microscopic assessment, the epithelial integrity of group IV was significantly improved compared with groups I and II. At electron microscopy the integrity of group IV was significantly superior to all remaining groups. We conclude that a deposit of basic fibroblast growth factor and fibrin glue appears to increase revascularization of an ischemic airway from omentum and thus results in improved epithelial preservation of a tracheal autograft.

Diversity of cell-cell interactions formed by gastric parietal cells in culture: morphological study on guinea pig cells.

Parietal cells of gastric glands are specialized to produce acid. Tight junctions between the parietal cells and their neighbouring cells (usually chief cells and mucous cells, less commonly parietal cells) avoid acid back-diffusion. Alterations of these junctions are accompanied by a defective epithelial barrier function. The conditions leading to junction formation, e.g. during epithelial restitution and regeneration are entirely unknown. The present study has the purpose to establish an in vitro model which allows studying these junctions. Freshly isolated gastric epithelial cells of guinea pig, moderately enriched with parietal cells, were cocultured for 2 days. Highly specific staining techniques showed the following composition in the near-confluent monolayer: 45% parietal cells (succinic dehydrogenase-positive), 36% mucous cells (lectin-binding granules), 18% chief cells (pepsinogen-positive granules) and 1% subepithelial cells (vimentin-positive). Ultrastructural investigations of sections of these monolayers revealed a high tendency of parietal cells to form cell junctions with the following characteristics: 1) virtually all parietal cells formed junctions with their neighbouring cells; 2) only junctions, but no desmosomes, were observed among neighbouring parietal cells; 3) junctional complexes and desmosomes were regularly present between parietal cells and their neighbouring mucous and chief cells; 4) parietal cells were sometimes integrated into three-dimensional structures, resembling rudimentary gastric glands. In conclusion, parietal cells under standard coculture conditions, generate de novo the same types of cell junctions that are observed in the intact gastric epithelium. The results show that parietal cells in vitro spontaneously make junctions with parietal and non-parietal cells, resembling the junctions in the intact tissue.

Morphology and histogenesis of squamous cell metaplasia of the rat lung after chronic exposure to a pyrolized pitch condensate and/or carbon black, or to combinations of pyrolized pitch condensate, carbon black and irritant gases.

Female Wistar rats were exposed to different concentrations of a pyrolized pitch condensate and/or carbon black particles and/or a combination of irritant gases for 18 hours/day, 5 days/week for 10 months, followed by a clean air period of up to 20 months. Bronchiolo-alveolar hyperplasia and squamous metaplasia were important components of the resulting lesions. Squamous metaplasia and associated hyperplasia was investigated by routine histology, scanning and transmission electron microscopy, and by immunohistochemical detection of various cytokeratins (CKs). Intensely CK positive squamous metaplasia lacking a distinct stratum spinosum was distinguishable from squamous metaplasia with a distinct stratum spinosum that reacted weakly CK positive or CK negative. The CK positive type was histologically characterized by narrow intercellular spaces, the weakly CK positive or CK negative type had markedly enlarged intercellular spaces. Differentiated hyperplastic epithelium and the normal lung parenchyma reacted CK negative. In poorly differentiated hyperplasia of the alveolar type associated with squamous metaplasia scattered cells with characteristics of squamous differentiation were detected. Ultrastructurally these cells showed increased amounts of filament bundles and immunohistochemically a positive reaction with the CK antibody. These cells were regarded as precursor stages of squamous metaplasia of the lung periphery in rats.

Gastric mucosal repair in vitro.

We tested whether cultured gastric mucosal cells would be suitable to study the two major steps of repair: restitution and proliferation. Preparations of freshly isolated epithelial cells of guinea pig gastric mucosa were used for the studies. The cells attached to Petri dishes within 10 h and formed monolayers after 48-72 h. Electron microscopy showed that each cell type was able to form lamellipodia (i.e., cell protrusions) during restitution in vivo. When monolayers were wounded with a razor blade, most cells at the edge of damage died within a few minutes, but some recovered from injury. Later, intact cells migrated from the edge into the denuded zone and restored the monolayer within 24-48 h. An increased number of cells near the edge started to synthesize DNA. In conclusion, this model allows one to study in vitro both aspects of mucosal repair.

Quantification of proliferative lesions in hamster lungs after chronic exposure to cadmium aerosols.

The development of proliferative areas in the lungs of Syrian golden hamsters was studied after chronic inhalation of cadmium oxide, cadmium sulfide, cadmium chloride or cadmium sulfate. Lung tissue from randomly selected animals in each group was evaluated by morphometric histopathologic techniques. Estimation of the volumetric ratio of proliferative areas within the lungs of exposed animals showed significantly different extents of these lesions in dependence on the respective cadmium compound administered. The most severe changes were observed after inhalation of cadmium oxide and cadmium sulfide. Lesions were mainly found in the peribronchial region of the lung. Electron microscopic analysis of these proliferative areas revealed that they were composed of ciliated and Clara cells. From its histophatologic appearance this of lesion was qualitatively comparable in all hamsters which had been treated with the different cadmium compounds.

Ultrastructural observations in hamster and rat lungs after chronic inhalation of cadmium compounds.

Long-term inhalation of CdCl2 at concentrations as low as 12.6 micrograms Cd/m3 causes development of lung tumors in rats (4). No information, however, was available on the chronic carcinogenicity of CdO, CdS and CdSO4 which are especially relevant to the occupational area. In the present joint study of the Fh-ITA and the Fh-IUCT, rats and hamsters were exposed to CdCl2, CdSO4, CdO and CdS in a chronic inhalation carcinogenicity set-up (2, 3). The goal of the ultrastructural investigation was to compare inflammatory reactions and fibrotic lesions, as well as epithelial alterations occurring in the species under study. The present communication focusses especially on observations obtained from male and female hamsters and rats chronically exposed to CdO. In addition, we report preliminary results from a short-term inhalation study with CdO.

An improved embedding method for obtaining semithin and ultrathin sections from critical-point dried trachea specimens.

Critical-point dried trachea specimens were rinsed for 48 h in dimethylsulphoxide before being embedded in Epon 812 to obtain semithin and ultrathin sections. Compared with methods hitherto published, this treatment leads to better resin infiltration of the specimens and better preservation of intracellular structures.

Light and scanning electron microscopic investigation of the laryngeal mucosa of Syrian golden hamsters.

The laryngeal epithelium of Syrian golden hamsters (SGH) at 8, 12.5 and 17 months was studied by scanning electron microscopy (SEM) and light microscopy (LM). Stratified squamous epithelium, covered with shallow microvilli or microplicae, was observed covering the upper two-thirds of the laryngeal epiglottis, the false folds, the vocal cords and the luminal protrusions of the arytenoid cartilages. Pseudostratified respiratory epithelium, characterized by mucus producing cells with microvilli and ciliated cells, covered the base of the epiglottis and the entire subglottis. Transitional zones between squamous and respiratory epithelium were composed of stratified cuboidal epithelium. Towards the base of the epiglottis cuboidal cells with a relatively large surface area were present which displayed short surface microvilli, while cells with a small surface area were covered with long microvilli. Age related changes were not observed. Degenerative changes of submucosal glands or cartilages occurred in almost every animal, but no epithelial lesions were found. The findings confirm a low incidence of spontaneous metaplasia in the laryngeal epithelium of the SGH.

Spontaneous lesions in the respiratory epithelium of the Syrian golden hamster as seen by scanning electron microscopy.

The respiratory epithelium of 8, 12.5 and 17.5-month-old Syrian golden hamsters was investigated by scanning electron microscopy (SEM) and routine light microscopy (LM) of paraplast sections. In selected cases, SEM-specimens were embedded in Epon after SEM evaluation. The samples were cut in semithin sections and examined by light microscopy. The density of ciliated cells in the respiratory epithelium differs from hamster to hamster. Care must, therefore, be taken when diagnosing simple metaplasia by SEM alone unless a sufficient number of damaged cilia are present. Only 1 animal (8 months old) exhibited squamous metaplasia of the tracheal mucosa. However, surface polymorphism resembling squamous metaplasia was seen in almost every specimen. The polymorphism was caused by either submucosal calcifications or by cystic changes of hypertrophic submucosal glands. In addition, variously formed aggregations of mucus were seen protruding from duct openings of hypertrophic submucosal glands. To avoid false positive or negative diagnosis which can occur when screening is done by SEM alone, light-microscopical examination of sections cut from SEM identified lesions appears necessary.

High-energy phosphates, myocardial contractile function and material properties after short periods of oxygen deficiency.

To investigate myocardial performance and diastolic properties after repeated periods of oxygen deficiency auxotonic and isovolumic measurements were performed after three periods (4 min) of asphyxia in Wistar rats (n = 19). Additionally, the response of the peak isovolumic left ventricular pressure to postextrasystolic potentiation was measured. The hemodynamic results were compared to the levels of high-energy phosphates. Already after 15 min of recovery from asphyxia auxotonic measures of systolic function were completely normal compared to the control group (n = 19). Isovolumic measurements after 20 min of postasphyctic recovery, however, demonstrated a considerable reduction of the peak left ventricular pressure (226.5 +/- 7.5 mm Hg vs. 262.6 +/- 3.4 mm Hg in controls, mean +/- SEM (p less than 0.01) indicating persistence of decreased postischemic contractile performance. The relative effect of postextrasystolic potentiation was similar in both groups, but could not compensate for the reduced performance of the postasphyctic hearts: the absolute postextrasystolic peak isovolumic pressure of the postasphyctic hearts was lower than the value of the regular isovolumic peak pressure in the controls. Diastolic properties (pressure/volume and stress/strain relationships) of the postasphyctic myocardium remained unchanged. The total sum of the adenine-nucleotides decreased from 7.2 +/- 0.2 to 5.6 +/- 0.3 mumol/gww (p less than 0.01). ATP was reduced from 4.8 +/- 0.2 to 3.9 +/- 0.3 mumol/gww (p less than 0.01). Phosphocreatine was elevated to 7.0 +/- 0.6 mumol/gww, x +/- SEM (p less than 0.01). Our results demonstrated normal postasphyctic basal hemodynamics and material properties. Thus, the energy supply was sufficient to maintain steady state conditions - in spite of decreased overall adenine-nucleotide levels. Isovolumic measurements and postextrasystolic potentation tests, however, indicated that the contractile performance of the postischemic myocardium was still reduced. This functional limitation cannot be explained by altered material properties and is probably not causally related to the decreased overall ATP content.

Androgen receptor-deficient Tfm cells in the mosaic epididymis of sex-reversed mice heterozygous for Tfm: an autoradiographic study with [3H]-dihydrotestosterone and [3H]-estradiol.

Testicular feminization (Tfm) in the mouse is characterized by androgen insensitivity of the target cells. We describe the presence of androgen-insensitive Tfm cells in the epididymis of mosaic mice produced by converting female carriers of the Tfm mutation (XTfm/X+) to males via the sex reversal factor (Sxr). The mosaic arises by random X-inactivation. In the epididymal duct, flat undifferentiated Tfm cells are interspersed between high columnar wild-type cells. By thaw-mount autoradiography we show that after injection of [3H]dihydrotestosterone, radioactivity is concentrated in the nuclei of high columnar wild-type cells, whereas the nuclei of the low cuboid Tfm cells remain free of nuclear radioactivity. After injection of [3H]estradiol, both Tfm and wild-type cells show nuclear labeling. Our observations demonstrate that Tfm cells in the mosaic epididymis selectively lack nuclear dihydrotestosterone-binding sites, whereas estradiol-binding sites are intact.

Intersex mice composed of androgen insensitive Tfm and wild-type cells analysed by 3H dihydrotestosterone autoradiography.

The X-linked testicular feminization mutation (Tfm) in the mouse is characterized by an androgen receptor defect. Due to random X-chromosome inactivation, XTfm/X+ heterozygotes are mosaics with respect to Tfm. They are composed of androgen receptor deficient XTfm cells and normal X+ wild-type cells. If Tfm heterozygotes are converted to XX males by the sex reversal factor (Sxr) the mosaicism is expressed. Therefore in sex reversed Tfm heterozygotes (XTfm/X+-Sxr) intersexual sex organs develop. In five intersexes with small male accessory glands and hypospadia and one heavily feminized intersex with vagina and caudally dislocated deferent ducts the mosaic is visualized by 3H-DHT-autoradiography. In the epididymis differentiated wild-type cells show nuclear labeling, whereas undifferentiated Tfm cells are unlabeled. Unlabeled Tfm cells are also encountered in the vesicular glands of the heavily feminized animal, demonstrating that Tfm cells can participate in the formation of male sex glands. The urethral glands of the mosaic animals are composed of unlabeled Tfm lobules exhibiting the female phenotype of the glands, and of labeled wild-type lobules exhibiting the male phenotype. Formation of a vagina and deviation of the deferent ducts is correlated with lack of androgen binding sites in the connective tissue.

Embryonic cholinesterase activity during morphogenesis of the mouse genital tract. Light- and electron-microscopic observations.

In the genital tract of male and female mouse embryos cholinesterase activity is described that is independent from innervation. The enzyme activity is localized in the mesenchyme at the junction of Wolffian and Müllerian ducts with the urogenital sinus. During male development prostate buds and vesicular glands grow out into the cholinesterase-active mesenchyme. During female development the active mesenchyme participates in the downgrowth of the vaginal anlage. Ultrastructurally the cholinesterase activity is localized in the perinuclear cisterna and in smooth endoplasmic reticulum of the mesenchymal cells. The enzyme activity disappears with definitive differentiation of the tissue. The embryonic cholinesterase is a component of a primitive muscarinic system. Its relation to the morphogenetic action of testosterone and its possible general functions are discussed.

Autoradiographic binding studies with [3H]oestradiol and [3H]dihydrotestosterone in the autonomic genital ganglion (plexus of Frankenhäuser) of the mouse.

Male, female and Tfm mice (testicular feminization) were injected with [3H]oestradiol or [3H]dihydrotestosterone, and autoradiograms prepared of male accessory sex organs and of the cervico-vaginal portion of the female reproductive tract. After injection of [3H]oestradiol in male, female and Tfm animals a nuclear concentration of radioactivity was found in a subpopulation--about 20-30%--of the neurons of the genital ganglion. No such concentration was seen after [3H]dihydrotestosterone. The results suggest a direct genomic effect of oestradiol on certain neurons of the autonomic genital ganglion in both sexes.

The vagina is formed by downgrowth of Wolffian and Müllerian ducts. Graphical reconstructions from normal and Tfm mouse embryos.

We use the Tfm (testicular feminization) mutation of the mouse to reexamine the role of Wolffian and Müllerian ducts during formation of the vagina. Three dimensional graphical reconstructions of the lower genital tract are prepared from serial sections of male, female, and Tfm embryos from day 15 p.c. until 8 days after birth. The reconstructions show that in female and Tfm animals the caudal segments of Wolffian and Müllerian ducts fuse and migrate caudally, whereas in the male they do not fuse and remain in their original position. Following down-growth, separate Wolffian and Müllerian ducts emerge from the fused caudal tips of the ducts. The Wolffian ducts degenerate, while the Müllerian ducts fuse with each other and form the vagina. Wolffian and Müllerian ducts are connected to the urogenital sinus by the sinus ridges which in later stages are separated from the sinus by lateral furrows. The sinus ridges are replaced by the Müllerian ducts. We conclude that the vagina develops by down-growth of Wolffian and Müllerian ducts along the sinus ridges. Wolffian ducts and sinus ridges regress so that the definitive vagina is formed by the Müllerian ducts. In Tfm embryos the vagina forms as in the female but subsequently degenerates, probably due to the action of AMH. The vaginal pocket in the Tfm is the variable remainder of the vagina at the end of the degeneration process.

Differential distribution of 3H dihydrotestosterone and 3H estradiol nuclear binding sites in mouse male accessory sex organs. An autoradiographic study.

The distribution of specific nuclear binding sites for androgens and estrogens in the male accessory sex organs of the mouse was assessed by autoradiography with 3H dihydrotestosterone (3H DHT) and 3H estradiol (3H E2). With 3H DHT nuclear labeling differed among the epithelia of the organs. It was high in seminal vesicle and ampullary gland, moderate in ventral prostate, urethral gland, prostatic excretory ducts and the ampulla ductus deferentis, low in dorsal prostate and low or absent in coagulation gland. With 3H E2, in contrast, epithelial nuclear labeling was high only in coagulation gland, moderate or low in seminal vesicle, low or absent in ventral and dorsal prostate and absent in ampullary gland and ampulla ductus deferentis. In the lamina propria of all organs nuclear labeling with 3H DHT was generally moderate and existed only in some cells, with the highest number in the ampulla ductus deferentis. With 3H E2, nuclear labeling in the lamina propria showed a high intensity in all organs, except in ventral and dorsal prostate which remained unlabeled. Many labeled cells were found in the deferent duct and its ampulla, while in the other organs only a few cells showed nuclear labeling with 3H E2. In the smooth muscle sheath of all organs, some muscle cells were moderately labeled with 3H DHT, but not with 3H E2. The results indicate the presence of nuclear receptors in male accessory sex organs for both dihydrotestosterone and estradiol. The differential patterns of 3H DHT and 3H E2 nuclear uptake suggest differential sensitivities of the individual organs and their tissue compartments for androgens and estrogens.

Metabolic effects of direct current stimulation on cultured vascular smooth muscle cells.

Vascular smooth muscle cells from rabbit arteries were grown in tissue culture and stimulated by DC impulses (1 mA, 1 V, 10 Hz, 1 ms/imp). Scanning microscopic examination disclosed that in stimulated cultures the cell surface was enlarged by numerous microvilli. This was interpreted as being indicative of an increase in cell activity. Cellular metabolism was characterized by analyzing the incubation medium for glucose, glutamate/glutamine, and lactate. When compared to unstimulated controls, stimulation caused an increase in the uptake of glucose and glutamine as well as an increased lactate production. The enhancing effect on metabolism was prevented when the "calcium antagonist" verapamil was present (5 X 10(-6) M). Although the exact mechanism by which DC stimulation influences the cells remains obscure, this finding indicates an important mediating role of Ca2+ ions.

Connective tissue content and myocardial stiffness in pressure overload hypertrophy. A combined study of morphologic, morphometric, biochemical, and mechanical parameters.

We investigated samples of left ventricular myocardium from Goldblatt II (4 and 8 weeks after operation) and spontaneously hypertensive rats (SHR; 40 and 80 weeks old) by histological and morphometric methods. From the same hearts, the distensibility of the left ventricular papillary muscle was analyzed by means of resting tension curves, and the collagen content of the whole left ventricular wall was determined by means of hydroxyproline concentration. In all groups, myocardial fibrosis was observed to accompany myocardial hypertrophy. The severity of fibrotic lesions increased with the duration of hypertension, and, in late stages, degenerative changes of cardiac myocytes were found. Morphometric determinations and chemical analysis of the hydroxyproline concentration revealed a decrease in myocardial muscle content, which was paralleled by an increase in collagen content when compared to the respective controls. In general, morphometric and chemical findings correlate with increased myocardial stiffness observed during mechanical measurements in isolated papillary muscle preparations from the same hearts. Differences were found, however, between chemical analysis and mechanical measurements in the 40-week-old SHR group, which may result from different patterns of collagen distribution between interstitium, perivascular spaces, and the walls of blood vessels. The comparison between histological, morphometric, chemical, and physiological data shows that (1) cardiac hypertrophy of Goldblatt and SH-rats is accompanied by myocardial fibrosis, and (2) changes in passive elastic properties of myocardium is better reflected in morphometric than in chemical analysis.