The ultrastructural effects of beta-amyloid peptide on cultured PC12 cells: changes in cytoplasmic and intramembranous features

SummaryThe fine structural features of cultured PC12 cells were investigated after treatment for 1, 3, or 5 days with different concentrations of the vascular form of - 1–40 (-AP). PC12 cells treated with -AP showed time- and concentration-dependent lysosomal system activation and cell toxicity. We observed increases in the number and size of cytoplasmic lysosomes as indicated by increased acid phosphatase reactivity. Some lysosomes were in the form of multivesicular bodies or large residual bodies that appeared to arise by autophagia or by endocytotic uptake. Double-sided plasma membrane invaginations were observed to give rise to increasingly extensive intracytoplasmic vacuolization that was correlated with duration of -AP treatment. Freeze-fracture studies of the intramembranous particle (IMP) population in the plasma membrane P-face showed that both control and -AP treated cells had two major P-face IMP populations, small-diameter (4–8 nm) IMPs, and large-diameter ( 9nm) IMPs. The larger category of IMPs was found to possess a greater average diameter in the -AP treated cells than in the control cells. These IMPs could represent modifications to existing transmembranous receptors, channels, or transducing molecules by the -AP. These results demonstrate that -AP can induce time- and concentration-dependent ultrastructural changes in PC12 cell membranes.     

Glucocorticoid effects on rabbit fetal lung maturation in vivo: An ultrastructural morphometric study

Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animal were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant-associated protein, SP-A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body crosssectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissué cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell defferentiation, and a concomitant increase in epithelial-mesenchymal interactions.     

Glucocorticoid effects on rabbit fetal lung maturation in vivo: an ultrastructural morphometric study.

Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animals were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant-associated protein, SP-A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body cross-sectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissue cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell differentiation, and a concomitant increase in epithelial-mesenchymal interactions.     

Collagenous colitis: Histologic,morphometric, immunohistochemical and ultrastructural studies. Report of 21 cases

Summary We examined 129 colonic biopsies from 21 patients with collagenous colitis, most of whom presented with diarrhoea. Morphometric measurements gave a mean thickness of the subepithelial collagen deposit of 19.5 µ ± 5.1. The trapped fusiform and/or stellate cells within the deposits were identified immunohistochemically as myoid cells, being positive with antibody against smooth muscle cell alpha-actin. Ultrastructurally, these cells have all the characteristic features of myofibroblasts. Similar cells are also present along the crypts, where they were formerly referred to as pericryptal fibroblasts. Although there is still much debate as to the pathogenesis of this condition, we would like to suggest that collagenous colitis is a disease of pericryptal myofibroblasts. During their migration and maturation into the subepithelial region they may synthesize an excess of collagen, under some yet unknown or undefined stimulus/stimuli.Presented in part at the Pathologists' Meeting Sillon Rhône-Alpes, Grenoble, 18 June 1987, and at the Annual Meeting of the Swiss Society of Pathology, Feldkirch, 6-7 November 1987     

Anthracycline effects on actin and actin-containing thin filaments in cultured neonatal rat myocardial cells

Adriamycin (ADR, Doxorubicin) effects on actin and other proteins in cultured neonatal rat cardiac myocytes were investigated. Heart cells were exposed to ADR in doses of 10(-8) M to 10(-5) M for 24 hours. Cells were harvested in 2 mM of Tris buffer containing Triton X-100, homogenized and centrifuged in a microfuge. Parallel dishes of cultured cardiac myocytes were washed in buffered saline and were fixed at 4 degrees C in Karnovsky's fixative. The supernatant solutions were dialyzed and then incubated with pancreatic DNAase I to quantify actin by enzyme inhibition. In parallel studies, both cell supernatant solutions and pellets were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and the stained polypeptide bands were quantified by densitometry. Results showed that heart cells exposed to 10(-6) M of ADR for 24 hours had unpolymerized actin levels reduced to 7.7 micrograms/10(6) cells (as measured by DNAase I inhibition or by sodium dodecyl sulfate polyacrylamide gel electrophoresis along with densitometry) compared to 11.0 micrograms/10(6) cells in untreated culture heart cells. When ADR concentration was 10(-7) or 10(-8) M, unpolymerized actin levels were similar to the levels of untreated heart cells. Protein content of extract solutions of untreated and ADR-treated myocytes were 1.2 mg/ml and 0.8 mg/ml, respectively. Gel densitometry of electrophoretograms showed actin to account for 12 to 16% of total density of bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Comparative densitometry of ADR-treated cells treated with 10(-6) M of ADR show depolymerized actin to account for 77% of total actin. Ultrastructural results show a large clear cytoplasmic zone of disorganized 12 to 14-nm filaments in cultured myocytes exposed to 10(-6) M ADR. Little change in myocyte ultrastructure was seen at 10(-7) M or 10(-8) M ADR exposure. Data support ADR as a cellular disruptor with toxic effects on cardiac cytoplasmic and contractile proteins and filaments. This ADR effect on heart cells in culture is dose-related.     

Non-Hodgkin's lymphoma classification: ultrastructural morphometric studies for the quantification of nuclear compartments in situ

The condensed chromatin distribution in the nuclei of lymphocytes in non-Hodgkin's lymphoma (NHL) is a key element, along with nuclear size and shape, in the classification of this disease for therapeutic and prognostic purposes. This report describes the ultrastructural comparative quantification of the condensed chromatin and the interchromatinic (nuclear matrix or euchromatin) region in the nuclei of mitogen-stimulated human peripheral T lymphocytes and mouse spleen B lymphocytes, human germinal center lymphocytes, and lymphocytes in ten cases of NHL of a variety of subtypes. The sequential morphologic nuclear changes induced in lymphocytes by mitogens are reflected in human germinal center lymphocyte populations. The common features include the changes in the distribution and volume of condensed chromatin aggregates, as well as the fact that the major increments in nuclear volume during lymphocyte transformation result from increases in the volume of the interchromatinic region. In all subtypes of NHL analyzed morphometrically, subpopulations of lymphocytes were identified in which mean nuclear, condensed chromatin, and interchromatinic volumes were more or less equivalent to those of normal lymphocyte subsets in germinal centers in reactive hyperplasia. However, in NHL the abnormal cytologic characteristics of the nucleus result, at least in part, from a complex interplay of condensed chromatin distribution and amount, and the size of the interchromatinic region. Further complexity is introduced by the fact that in NHL these two nuclear compartments can independently be normal, increased, or reduced in size. Morphometric quantification of lymphocytes in NHL indicates that the interchromatinic (matrix) region of the nucleus is the key element in establishing the nuclear volume of neoplastic lymphocytes. The structural and functional, ribonucleoprotein interchromatinic region of the nucleus was visualized in normal and neoplastic lymphocytes by regressive uranyl-EDTA staining. Quantitative morphometric analysis indicates that the cytologic appearance of neoplastic lymphocytes, even within subtypes of NHL, is heterogeneous and that condensed chromatin quantity and distribution may be more critical than nuclear size in distinguishing between certain subtypes of NHL. Improvements in the classification of NHL will occur only with understanding of the alterations in the biologic mechanisms controlling gross nuclear organization and the morphologic events of the various differentiation pathways available to antigen-stimulated lymphocytes.     

Pregnancy and dexamethasone: effects on morphometric parameters of gonadotropic cells in rats

The effects of pregnancy and multiple dexamethasone (Dx) treatment on morphometric parameters of adenohypophyseal gonadotropic cells that produce follicle stimulating hormone (FSH) and luteinizing hormone (LH) were investigated in female Wistar rats. The rats in the experimental group received injections of 1.0, 0.5 and 0.5mg Dx/kg b.w. on days 16-18 of pregnancy, while the control group received equal volumes of saline. There was also an age-matched adult virgin control group. The experimental and control animals were sacrificed 24 and 72h after the last injection. Using the peroxidase-anti-peroxidase immunohistochemical labeling procedure, morphometric analyses showed that cell volume and volume density of FSH and LH cells on day 19 of pregnancy, as well as the number of LH cells, were significantly decreased compared to the virgin control values. On day 21 of gestation, the volume of FSH and LH cells remained smaller than in the virgin controls. Moreover, FSH and LH cell volume was significantly decreased 24h after multiple Dx treatment in comparison with the pregnant controls. Thus, during the last days of pregnancy, the morphometric parameters of gonadotropic cells decreased in comparison with the control virgin rats, but Dx treatment of pregnant rats had an inhibitory influence on FSH and LH cell size only 24h after the last dose.     

Immunohistochemical and ultrastructural studies of the effects of prednisolone on transformation of fibroblast to regenerated mesothelial cells

We have proposed in the past that chest wall fibroblasts are transformed to regenerated mesothelial cells. This study was conducted to investigate the effects of prednisolone on the differentiation and migration of fibroblasts in their transformation to mesothelial cells. Rat fibroblasts harvested from intercostal thoracic wall specimens were cultured in culture medium until cell spheroids were formed. An experimental cell spheroid group to whose culture medium prednisolone had been added and a control spheroid group with no addition of prednisolone were then subjected to immunohistochemical and ultrastructural studies of the changes in the fibroblasts with the passage of time. On days 1 and 2 of culture, the fibroblasts in each group were cytokeratin negative. However, on day 3 the control group became cytokeratin positive, and ultrastructural observations revealed formation of macula adherens and microvilli. In contrast, the experimental group fibroblasts remained cytokeratin negative even on day 3, but became cytokeratin positive on day 5 of culture. Macula adherens and microvilli also manifested on day 5. Prednisolone inhibited the differentiation and migration of fibroblasts, but it was surmised that fibroblasts that have resisted from the effects of prednisolone finally differentiate into mesothelial cells which have formed macula adherens.This study was presented at the 33rd annual meeting of the Clinical Electron Microscopy Society of Japan, Nagasaki, September 27, 2001     

Hepatoblastoma cells producing alpha-fetoprotein: morphometric, immunohistochemical and electron microscopic studies

Morphometric, immunohistochemical, and electron-microscopic studies were undertaken in an attempt to identify the types of hepatoblastoma cellular elements responsible for the synthesis of alpha-fetoprotein (AFP), and to see how they may relate to serum AFP levels and the metastatic spread and prognosis of the hepatoblastoma. Morphometric studies of 21 hepatoblastomas with statistical treatment of the results revealed a moderately strong reliable correlation of the AFP serum titer with the volume ratio of embryonal tumor cells and with that of those tumor elements of endodermal hepatic diverticulum which are similar to the latter cells with regard to degree of differentiation. Also, a consistent, reliable negative correlation was demonstrated between serum AFP titer and the volume of fetal hepatoblastoma cells. The volume ratio of stromal elements was found to be subject to chance variations and not to correlate with serum AFP level. Immunohistochemical and electron-microscopic studies confirmed the morphometric findings and showed AFP synthesis to be effected by poorly differentiated hepatoblastoma cells--by endodermal hepatic diverticulum elements at first and by embryonal and intermediate tumor cells later--and to decrease as the liver tumor cells differentiate further. It is concluded that a high serum AFP level is, generally, an indication that the hepatoblastoma is an extensive one and consists of poorly differentiated cells so that the prognosis is unfavorable.     

An ultrastructural evaluation of toluene toxicity using cultured mammalian cells

Animal cells were cultured in media containing toluene and then examined by electron microscopy to determine changes in subcellular architecture. Cellular viability and cloning studies indicated toluene toxicity at concentrations of 50-500 ppm. At all dosage levels, toluene accelerated cell death compared to comparable control cells that had not been exposed to toluene. However, media composition and, particularly, the presence or absence of fetal bovine serum (FBS), markedly affected toluene toxicity. In media without FBS reversible changes in filopodia and cell shape were identified after 15 min of exposure to as little as 50 ppm toluene, and cell death occurred within 5 min at concentrations of 500 ppm toluene. In media with FBS, no filopodial changes were observed at 50 ppm toluene, and cell death, although accelerated, was clearly evident only after 24-48 h of exposure to 200-500 ppm toluene. Except for the cell-surface changes, chronic toluene exposures (50-100 ppm without FBS or 200-500 ppm with FBS in the growth medium) produced no unusual intracellular changes until 24-48 h of toluene exposure. With toluene exposures of 24 h or longer, cellular changes included condensation of heterochromatin in nuclei, formation of bulbous protuberances at the cell surface, loss of polyribosomes (but not ribosomes) and, ultimately, degeneration of organelles. These late changes were irreversible and the prelude to cell death.     

High incidence of Langerhans cells in lymph nodes of athymic nude mice: an ultrastructural and morphometric study

Cervical and axillary lymph nodes of athymic nude mice were compared morphometrically and ultrastructurally with those of normal littermates. The lymph nodes of the nude mice were larger and the volumes of the follicle, paracortex, and medulla of the lymph nodes were all larger. The paracortex of nude mice had few lymphocytes and a large number of interdigitating cells (IDC) and Langerhans cells (Lc). The results suggest that the high incidence of Lc and IDC in the lymph nodes of nude mice is due to cell accumulation and not lymphocyte depletion.     

Cytotoxicity effects of amiodarone on cultured cells

Amiodarone is a potent anti-arrhythmic drug used for the treatment of cardiac arrhythmias. Although, the effects of amiodarone are well characterized on post-ischemic heart and cardiomyocytes, its toxicity on extra-cardiac tissues is still poorly understood. To this aim, we have monitored the cytotoxicity effects of this drug on three cultured cell lines including hepatocytes (HepG2), epithelial cells (EAhy 926) and renal cells (Vero). We have investigated the effects of amiodarone on (i) cell viabilities, (ii) heat shock protein expressions (Hsp 70) as a parameter of protective and adaptive response and (iii) oxidative damage.Our results clearly showed that amiodarone inhibits cell proliferation, induces an over-expression of Hsp 70 and generates significant amount of reactive oxygen species as measured by lipid peroxidation occurrence. However, toxicity of amiodarone was significantly higher in renal and epithelial cells than in hepatocytes. Vitamin E supplement restores the major part of cell mortalities induced by amiodarone showing that oxidative damage is the predominant toxic effect of the drug.Except its toxicity for the cardiac system, our findings demonstrated that amiodarone can target other tissues. Therefore, kidneys present a high sensibility to this drug which may limit its use with subjects suffering from renal disorders.     

An ultrastructural study of the effects of asbestos fibres on cultured peritoneal macrophages

The effects of crocidolite and chrysotile fibres on lavaged peritoneal macrophages have been studied by both scanning (SEM) and transmission (TEM) electron microscopy. SEM provided little information (as the surface topography did not reflect the underlying cytoplasmic organization) except that it showed that individual macrophages often partially engulfed many long fibres in a random fashion. TEM revealed the fibres in and protruding from membrane-bound vacuoles, free in the cytoplasm and penetrating the nucleus. The cellular distribution of the fibres is discussed in terms of the cytotoxic nature of the fibres and their ability to produce a selective release of enzymes from the macrophages.     

Injurious effects of ethanol on rat Kupffer cells

The effects of ethanol on rat Kupffer cells were studied functionally and morphologically. Eight g ethanol per kg body weight per day was intragastrically administered to rats for 7 days. An isocaloric glucose solution was administered to control rats. The phagocytic activity of the reticuloendothelial system was measured by the carbon clearance method (57 mg carbon particles per kg body weight) on the 7th day. Kupffer cells having phagocytized carbon particles were counted under the light microscope. Kupffer cells were also observed by scanning electron microscopy. Both the carbon clearance and Kupffer cell number were lower in ethanol-administered rats (32 +/- 8 X 10(-4) mg/ml; 0.6 +/- 0.3/0.01 mm2 liver lobule) as compared to control rats (63 +/- 15; 3.1 +/- 1.0). Microvilli and filopodia of Kupffer cells were fewer in ethanol-administered rats than in control rats. Carbon clearance correlated with Kupffer cell number per 0.01 mm2 liver lobule and liver weight. These results suggest that the decrease in carbon clearance induced by ethanol is due mainly to the decrease in Kupffer cell number and partly to the decrease in Kupffer cell activity as demonstrated by the disappearance of microvilli and filopodia.     

Ultrastructural morphometric analysis of cultured neonatal and adult rat ventricular cardiac muscle cells

Neonatal and adult rat ventricular cardiac muscle cells cultured on laininin differed from similar myocytes grown on plastic in the amount and distribution of their mitochondria and transverse tubules. Point-count morphometry was used at the electron microscopic level to quantify these differences. Adult myocytes grown on laminin contained more mitochondria per unit volume than adult myocytes grown on plastic. No significant differences were observed in the volume percent of myofibrils in either adult or neonatal ventricular myocytes when grown on laminin and compared to those grown on plastic. The transverse tubule system in neonatal and adult myocytes was reduced significantly when both groups were cultured on laminin. Furthermore, neonatal and adult myocytes cultured on laminin were flatter than those cultured on plastic. This may indicate a relationship between the surface/volume ratio and transverse tubule development in cultured myocytes. These studies establish that point-count morphometry can be used to quantify changes in the organelle volume densities of cultured cardiac muscle cells.     

Circadian ultrastructural changes in rat gastric parietal cells under altered feeding regimens: A morphometric study

The ultrastructure of rat gastric parietal cells was studied at six timepoints of the 24-hour day. The rats, maintained on a 12 hour: 12 hour light-dark regimen, had been subjected to either a 40-hour fast or to a 4-hour mid-light restricted feeding period. At each time point, the volume density (Vv) of secretory canaliculi, surface density (Sv) of microvesicles and RER, and the numerical density (Nv) of multivesicular bodies were determined in cells of the neck and base of glands. Circadian variation of the four variables was suggested in both experiments. Canalicular and microvesicular measurements suggested that a rhythm in gastric acid secretion may persist during fasting; a peak and trough, respectively, occurred in the late dark phase, as in our previous report on ad libitum-fed rats. Restriction of feeding to that which is normally the rat's resting phase caused an apparent 180 degree phase-shift in the rhythm. The data suggested, however, that additional factors may have influenced the cellular activity pattern. At all timepoints in both experiments, cells of the neck of glands had higher RER and canalicular values than did cells of the base of glands. This suggests that parietal cells in glandular necks may be more active than those farther removed from the stomach lumen. There was no correlation between the Nv of multivesicular bodies and glandular location of the cells.     

An ultrastructural study on HeLa cells cultured in roller bottles forming aggregates

We will describe here the ultrastructure of HeLa cells cultured in either roller bottles for 8 days or 10 days or in Petri dishes for 7 days. Junctional complexes could be seen between adjacent cells in aggregates formed in roller bottles. The junctional complexes were serially located especially near the free surface of the cells which occupied the outer layer of the aggregates. Two elements of the junctional complex, i.e., tight junctions and intermediate junctions, were observed between adjacent cells. The density and the diameter of microvilli in the intercellular space between cells cultured in roller bottles were denser and narrower than those of microvilli on the free surface of the cells. Junctional complexes and dense microvilli formed intercellular secretory canaliculus-like structure observed in the intercellular space of certain secretory glands.