Histology and histopathology Vol.14, nº 4 (1999)
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- PublicationOpen AccessQuantitative in situ hybridization for the evaluation of gene expression in asynchronous and synchronized cell cultures and in tissue sections(Murcia : F. Hernández, 1999) Barlati, S.; Zoppi, N.; Copeta, A.; Tavian, D.; De Petro, G.; Colombi, M.We describe an image analysis (IA) system that has been applied for the quantitative evaluation of mRNAs evidenced by in situ hybridization (ISH) with radiolabelled probes in cultured cells and in tissue sections. The ISH-IA method was used for the evaluation of cultured cell morphological parameters such as cell and nucleous area (CA and NA, respectively) in parallel with the levels of mRNAs detected as hybridization grains areas (GA). The evaluation of these parameters, together with the analysis of the levels of mRNAs (c-jun, cyclin A) specific for given cell cycle phases (i.e. G1 and S/G2), allowed the identification, in asynchronous cultures of human skin fibroblasts, of cells in G1 and SlG2 phases. The mRNA levels measured by ISH-AI were comparable with those detected by RT-PCR. This method was also applied for the analysis of fibronectin (FN) gene expression in control skin fibroblasts in relationship with the different phases of the cell cycle and in comparison with a tumor cell line (Sk-Hepl), heterogeneous either for morphometric parameters or for the levels of this transcript. Finally, the ISH-AI was applied for the semiquantitative evaluation of the expression, localization and alternative splicing pattern of FN mRNA in normal liver and in hepatocellular carcinoma (HCC) tissue sections.
- PublicationOpen AccessHeart mitochondria in rats submitted to chronic hypoxia(Murcia : F. Hernández, 1999) Cervós-Navarro, J.; Kunas, R.Ch.; Sampaolo, S.; Mansmann, U.The effect of prolonged exposure to normobaric hypoxia on the mitochondria of myocard of rats exposed for several weeks to 8 and 7% O2 has been morphometrically evaluated. Twelve male Wistar rats housed in Nalgene cages (2 per cage) with a batch of six cages placed in plexiglass chambers were maintained in air/N2 mixtures containing different concentrations of 02. Six animals kept in similar cages under normoxia served as controls. When at day 60 the FIOZ was reduced to 8%, the weight increase stagnated and after the 81st test day, on which the hypoxic animals were subdivided into 8% and 7% groups the weight curve showed a decrease in the mean body weight for both groups. The arrest and the following loss of weight beyond the 85th day may be interpreted as the expression of a limit reached in the compensation capacity. In the 8%-group the shape of the mitochondria varied more markedly often with budding and furrowing of the surface. In the 7%-group bizarre shapes and wide variations in size with a decided shift towards larger mitochondria were noteworthy. While rats kept under 8% oxygen exhibited a numerical increase in myocardial mitochondria compared to controls, the mitochondria of the 7%-group were numerically reduced. The results suggest that hypoxia of 8% oxygen is compensatable, if only to some extent, by an increasing surface of mitochondrial membranes, and that further reduction of oxygen causes compensation mechanisms to fail as seen by the severe alterations of the mitochondrial population of the cardiomyocyte in the 7%-group.
- PublicationOpen AccessSodium transport systems in human chondrocytes II. Expression of ENaC, Na+ K+ 2CI- cotransporter and Na+ H+ exchangers in healthy(Murcia : F. Hernández, 1999) Trujillo, E.; Alvarez de la Rosa, D.; Mobasheri, A.; Gonzalez, T.; Canessa, C.M.; Martín Vasallo, P.In this article, the second of two, we continue our studies of sodium-dependent transport systems in human cartilage from healthy individuals and with osteoarthritis (OA) and rheumatoid arthritis (RA). We demonstrate the presence of the epithelia1 sodium channel (ENaC), previously undescribed in chondrocytes. This system is composed of three subunits, a, 13 and y. We have shown that the human chondrocytes express at least the a and the l3 subunit of ENaC. The expression of these subunits is altered in arthritic chondrocytes. In RA samples the quantity of a and B is significantly higher than in control samples. On the other hand, ENaC a and B subunits are absent in the chondrocytes of OA cartilage. Human chondrocytes also possess three isoforms of the Na+/H+ exchanger (NHE), NHE1, NHE2 and NHE3. The NHE system is composed of a single protein and is believed to participate in intracellular pH regulation. Furthermore, our studies indicate that at least one isoform of the electroneutral Naf/K+/2C1- cotransporter (NKCC) is present in human chondrocytes. There are no obvious variations in the relative expression of NHE isoforms or NKCC between healthy and arthritic cartilage. Our data suggests that chondrocytes from arthritic cartilage may adapt to changes in their environmental sodium concentration through variations in ENaC protein levels. ENaC is also likely to serve as a major sodium entry mechanism, a process that, along with cytoskeletal proteins, may be part of mechanotransduction in cartilage.
- PublicationOpen Accesslntraocular neovascularization(Murcia : F. Hernández, 1999) Yoshida, A.; Yoshida, S.; Ishibashi, T.; Inomata, H.An important character of the eye is transparency, so intraocular neovascularization, which is fragile and likely to result in hemorrhage, would cause a functional disorder of the eye and contribute to loss of vision associated with such diseases as retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration. Recently interest in the mechanisms of intraocular neovascularization has increased, and the mechanisms have been gradually elucidated using several in vitro and in vivo angiogenesis models. Blood vessels in the eye are composed of, and surrounded by, various types of cells that produce multiple factors. Neovascularization is regulated by complex interactions among these angiogenic factors, angiostatic factors, and adhesion molecules, and some of these angiogenesis-related molecules have also been suggested as new targets for novel therapeutic agents of intraocular neo-vascularization. This review focuses on in vivo representative angiogenesis models of the corneal pocket model and the model of oxygen-induced retinopathy, and discusses the role of some angiogenesisrelated factors and adhesion molecules in intraocular neovascularization.
- PublicationOpen AccessHeterotopic neogenesis of skeletal muscle induced in the adult rat diaphragmatic peritoneum. Ultrastructural and transplantation studies(Murcia : F. Hernández, 1999) Drakontides, A.B.; Danon, M.J.; Levine, S.During the course of a mild chemical peritonitis, new skeletal muscle fibers develop and persist over a twelve-month interval in the diaphragmatic peritoneum. Light and electron microscopic studies revealed that the ectopic fibers developed from myoblasts and myotubes to fully differentiated muscle cells in the same manner as normally situated skeletal muscle. The ectopic fibers were separated from the intrinsic muscle by dense connective tissue and an elastic lamina. Diaphragms taken from normal rats and transplanted to the omentum of isogeneic recipients also developed skeletal muscle neogenesis in the same ectopic location as in the normal diaphragm. Satellite cells, reactive fibroblasts in the peritoneum, mesenchymal stem cells or blood-borne myoblast precursor cells could be the source of these ectopic muscle fibers. The results of the present studies, however, cannot provide conclusive evidence for the origin of the new muscle fibers. Regardless of their source, the methods employed may represent a unique model for the development and prolonged maintenance of skeletal muscle fibers in a heterotopic location in vivo.