Extracellular matrix remodeling at the early stages of liver regeneration in the rat

Previous studies have shown that activity of urokinase-type plasminogen activator (u-PA) increases very rapidly (within 1 minute) after partial hepatectomy. In view of the well-recognized roles of u-PA as one of the major initiators of the matrix proteolysis cascade and as an activator of plasminogen and hepatocyte growth factor (HGF), we studied matrix degradation in liver shortly after partial hepatectomy. The activation of plasminogen to plasmin following partial hepatectomy was examined by Western blot analysis, and a small increase in plasmin at approximately 15 minutes followed by a large elevation at approximately 3 to 6 hours after partial hepatectomy was detected. In addition, we found that fibrinogen, the major substrate for plasmin, begins to be degraded at approximately 15 to 30 minutes following partial hepatectomy. Using immunohistochemical staining, we detected that the distribution of fibrinogen in normal liver is localized to the perisinusoidal space surrounding the periportal region. A decreased distribution of fibrinogen in the periportal region was found by 15 minutes and continued through 24 hours following partial hepatectomy. In addition, the distribution of fibronectin in normal liver was localized to the perisinusoidal space surrounding the periportal and the pericentral regions. A strikingly decreased distribution of fibronectin in the periportal region was found at 5 minutes after partial hepatectomy. Furthermore, we observed that the protein levels of laminin, entactin, and fibronectin in an extracellular matrix (ECM)-enriched preparation decreased shortly after partial hepatectomy, and were restored later. No changes were observed with either vitronectin or the integrin chain alpha(v). In contrast to the protein levels of the ECM components, the messenger RNA (mRNA) levels of fibronectin, integrin chain beta1, and integrin chain alpha(v) gradually increased over 18 hours and then decreased thereafter. Taken together, these results suggest that rapid reorganization of selected ECM components are important for hepatocyte proliferation at the early stages of liver regeneration.     

Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis

Interferon gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or 3 weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin-positive/bromodeoxyuridine (BrdU)-positive cells) and activated (alpha-smooth-muscle actin [alpha-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by alpha-SMA-positive cells observed in DMN + IFN-gamma-treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation.     

Changes in matrix metalloproteinases during the evolution of interstitial renal fibrosis in a rat experimental model

The aim of the present study was to analyze the matrix metalloproteinase (MMP) activity during the evolution of interstitial renal fibrosis in a rat experimental model of unilateral ureteral obstruction. The interstitial type I collagenase and the gelatinolytic activities were analyzed by radiolabeled substrate degradation. Interstitial collagenase activity was low at all times while gelatinolytic activity increased on day 6 of evolution, with a decrease in activity from this point. The use of organomercurials revealed the presence of latent enzyme in all cases. Normal kidney samples contained MMP-9 in both active and proenzyme forms as revealed by zymography. On day 3 MMP-9 dimers appeared, and increased activity was observed until day 6. A decrease in the gelatinolytic activity was detected from days 9-15 of evolution. This observation was confirmed by Western blot analysis that revealed the presence of proMMP-9 mainly from days 6-12. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was also detected alone and in combination with proMMP-1 and MMP-1, particularly from days 6-15 of evolution. The presence of MMP-9 and MMP-1 was detected in the cytoplasm of cortical tubular cells by immunohistochemistry, with no difference between the experimental and the normal kidneys. There was also an increase in collagen concentration from day 3 after surgery that increased during the entire evolution of the experimental model. This work reveals that the decrease in the MMP-9 and MMP-1 enzymatic activity, due to their interaction with TIMP-1 and to the lack of activation of the latent forms, may participate in the excessive collagen deposit during the evolution of experimental interstitial renal fibrosis.     

Differential expression of flectin in the extracellular matrix and left-right asymmetry in mouse embryonic heart during looping stages

The characteristics of six different indicators of response distortion on the Personality Assessment Inventory (PAI; Morey, 1991) were evaluated by having college students complete the PAI under positive impression management, malingering, and honest responding conditions. The six indicators were the PAI Positive Impression (PIM) and Negative Impression (NIM) scales, the Malingering and Defensiveness Indexes, and two discriminant functions, one developed by Cashel and the other by Rogers. Protocols of students asked to malinger were compared with those of actual clinical patients, while protocols of students asked to manage their impression in a positive direction were compared with those of students asked to respond honestly. Comparisons between groups were accomplished through the examination of effect sizes and receiver operating characteristic (ROC) curves. All six indicators demonstrated the ability to distinguish between actual and feigned responding. The Rogers function was particularly effective in identifying malingering. The Cashel function was less effective than other measures in identifying positive impression management, although it appears to also have promise as an indicator of malingering.     

Extracellular matrix organization in various regions of rat brain grey matter

Previous studies revealed the concentration of extracellular matrix proteoglycans in the so-called perineuronal nets on the one hand and in certain zones of the neuropil on the other. This nonhomogeneous distribution suggested a non-random chemical and spatial heterogeneity of the extracellular space. In the present investigation, regions dominated by one of both distribution patterns, i.e. piriform and parietal cortex, reticular thalamic nucleus, medial septum/diagonal band complex and cerebellar nuclei, were selected for correlative light and electron microscopic analysis. The labelling was performed by the use of the N-acetylgalactosamine-binding plant lectin Wisteria floribunda agglutinin visualized by peroxidase staining and additionally by photoconversion of red carbocyanine fluorescence labelling for electron microscopy. The intense labelling of the neuropil of a superficial piriform region, presumably identical with sublayer Ia, was confined to a fine meshwork spreading over the extracellular space between non-myelinated axons, dendrites and glial profiles. In the reticular thalamic nucleus the neuronal cell bodies were embedded in zones of labelled neuropil. In contrast to these patterns, the labelled extracellular matrix in different cortical layers and in the other subcortical regions was concentrated in perineuronal nets as large accumulations at surface areas of the neuronal perikarya and dendrites and the attached presynaptic boutons. Astrocytic processes usually were separated from the neuronal surface by the interposed extracellular material. Despite a great variability, the width of the extracellular space containing the labelled matrix components in all perineuronal nets appeared to be considerably larger than that in the labelled zones of neuropil and the non-labelled microenvironment of other neurons. Our results support the view that differences expressed in topographical and spatial peculiarities of the extracellular matrix constituents are related to neuron-type and system-specific functional properties.     

Establishment of product morphology during the initial stages of wustite reduction

The product morphologies obtained on the reduction of wustite in CO/CO₂ gas mixtures between 1073 and 1373 K are reported. Three types of product morphology are identified, namely, type A (porous iron), type B (porous wustite covered with dense iron), and type C (dense wustite covered with dense iron). The reactions which occur during the reduction of wustite in CO/CO₂ and H₂/H₂O systems both before and after iron nucleation are examined. The product morphologies obtained on reduction are explained qualitatively in terms of the relative rates of the chemical reaction with the gas and the mass transport processes both in and on the solid. Formerly Postdoctoral Fellow at the Department of Mining and Metallurgical Engineering, University of Queensland, St. Lucia, Brisbane, Australia An erratum to this article is available at .     

In vitro cultivation of mouse embryos during the initial stages of organogenesis

Optimal conditions for in vitro cultivation of mouse embryos were sought. The embryos at 8--9.5 days of development were explanted into the culture in rotating tubes. A number of nutritional media were studied: the human umbilical blood serum; the rat blood serum, as well as its mixture (1 : 1) with Eagle's medium. It was demonstrated that the optimal medium for cultivation of 8-day-old embryos was the mixture of the rat blood serum with Eagle's medium, and for 8.5--9.5-day-old embryos--the rat blood serum, as well. Comparing in vitro and in vivo embryonic development, it was evident that in the cultivated embryos the parameters characterizing their growth decreased significantly. Possible causes of this phenomenon are discussed.     

Differential expression of dystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astrocytes

We have identified isoforms of dystrophin and utrophin, a dystrophin homologue, expressed in astrocytes and examined their expression patterns during dibutyryl-cAMP (dBcAMP)-induced morphological differentiation of astrocytes. Immunoblot and immunocytochemical analyses showed that full-length-type dystrophin (427 kDa), utrophin (395 kDa), and Dp71 (75 kDa), a small-type dystrophin isoform, were coexpressed in cultured nondifferentiated rat brain astrocytes and were found to be located in the cell membrane. During morphological differentiation of the astrocytes induced by 1 mM dBcAMP, the amount of Dp71 markedly increased, whereas that of dystrophin and utrophin decreased. Northern blot analyses revealed that dBcAMP regulates the mRNA levels of Dp71 and dystrophin but not that of utrophin. dBcAMP slightly increased the amount of the beta-dystroglycan responsible for anchoring dystrophin isoforms and utrophin to the cell membrane. Immunocytochemical analyses showed that most utrophin was observed in the cytoplasmic area during astrocyte differentiation, whereas Dp71 was found along the cell membrane of the differentiated astrocytes. These findings suggest that most of the dystrophin/utrophin-dystroglycan complex on cell membrane in cultured astrocytes was replaced by the Dp71-dystroglycan complex during morphological differentiation. The cell biological roles of Dp71 are discussed.     

Simultaneous expression of brain-derived neurotrophic factor and neurotrophin-3 in Cajal-Retzius, subplate and ventricular progenitor cells during early development stages of the rat cerebral cortex

To identify production sites and action targets of neurotrophins during neurogenesis, we investigated immunoreactivities of neurotrophins and their tyrosine kinase receptors in the cerebral cortex of rat embryos. Two sets of ligand-receptor systems, brain-derived neurotrophic factor/TrkB and neurotrophin-3/TrkC, were expressed simultaneously in Cajal-Retzius, subplate neurons and ventricular multipotent stem cells at embryonic days 13 and 15. Intraventricular administration of brain-derived neurotrophic factor or neurotrophin-3 at embryonic day 16 markedly modulated microtubule-associated protein II and/or Hu protein expression in different ways in the cortical plate cells by embryonic day 20. These observations indicate the involvement of autocrine and/or local paracrine action of brain-derived neurotrophic factor and/or neurotrophin-3 during formation of the cerebral cortex.     

Regulation of differentiation and keratin 10 expression by all-trans retinoic acid during the estrous cycle in the rat vaginal epithelium

In rodents, the vaginal epithelium shows cyclic changes with an alternating pattern of keratinization under estrogen control and mucification under progesterone control. Retinoids are powerful regulators of cell differentiation, an excess of retinoids suppressing the keratinizing differentiation of keratinocytes. Here, we have examined the vaginal epithelium during the estrous cycle and compare the effects of retinoids on both types of hormonally induced differentiation, i.e. keratinization and mucification. All-trans retinoic acid was administered either by daily injections during the estrous cycle or by a single injection before the estrogen rise; these two protocols gave similar results. Retinoic acid suppressed estrogen-induced vaginal keratinization and cytokeratin K10 expression (a biochemical marker of terminal differentiation). Progesterone-induced mucification was not impaired; however, retinoic acid impeded mucous cell desquamation, suggesting an effect of retinoic acid on cell adhesiveness. Retinoic acid induced the appearance of apoptotic-like cells, as revealed by immunocytochemical staining of DNA fragmentation.     

A secondary ion mass spectroscopic study of the elemental composition pattern in rat incisor dental enamel during different stages of ameloblast differentiation

In most earlier studies on the elemental composition pattern of dental enamel, a picture is presented which describes a limited region. In this study, estimates of the incorporation of some critical elements into enamel were correlated with the differentiation stages of the ameloblasts through out the whole tooth. Elemental analyses of rat incisor dental enamel during the secretory, transitional and maturation phases were performed using two different modes of secondary ion mass spectrometry (SIMS). The results were presented as ion images and three-dimensional spatial resolution graphs. In the elemental images of 23Na, 26CN, 35Cl and 39K, counts were detected during the secretory and maturation phases of amelogenesis. Variations were interpreted as resulting from secretion of elements during the secretory phase and resorption during the maturation phase. In line scans the ion yield from enamel during different stages of differentiation of the ameloblasts was analysed. The elements investigated were 12C, 19F, 23Na, 31P, 39K and 77CaCl. As seen in the images, most elements exhibited a higher ion yield during the earlier stages of secretion, and lower yields during the maturation-phase resorption. Cl, together with P, increased during the phases of maturation. In the most apical portions of the teeth, corresponding to a presecretory phase, an inverse pattern was seen for most of the elements. If the surface yield was high at the onset of the secretory phase, the presecretory yields were lower, and vice versa.     

Transient CD15 expression reflects stages of differentiation and maturation in the human subcortical central auditory pathway

The expression of the terminal saccharide determinant CD15 (3[a1-3]-fucosyl-N-acetyl-lactosamine) was evaluated in the central auditory system of the human developing brain by using monoclonal antibodies against this epitope. CD15 immunoreactivity was first observed in the ventral cochlear nucleus at 10 weeks of gestation, whereas the dorsal cochlear nucleus became positive from 13 weeks of gestation. In both nuclei, the intensity of immunoreactivity increased until 16 weeks of gestation and lasted until 25 weeks of gestation. In the inferior colliculi, CD15 was poorly expressed in the central nucleus from 13 to 23 weeks of gestation and later with moderate levels until birth. Within the medial geniculate nucleus, a biphasic pattern of expression was observed with peaks around 14-17 and 21-24 weeks of gestation. Heterogeneous expression in the medial geniculate nucleus, which was associated either with neurons or the neuropil, allowed distinction of subnuclei. In many of the auditory pathway structures (e.g., ventral cochlear nucleus and central nucleus of the inferior colliculus), a heterogeneous pattern of CD15 expression in the form of repeating parallel bands, possibly related to tonotopic organization, became transiently apparent around 23 weeks of gestation, whereas in the magnocellular part of the medial geniculate nucleus, a striking modular or compartmental arrangement of immunoreactive structures (which could also be associated with tonotopic organization) was also noted at about 23 weeks of gestation. We propose that the initiation of CD15 expression in each nucleus heralds the appearance of functional contacts and that high levels of neuropil labeling are related to the formation of nonstabilized synaptic contacts. Thus, transient CD15 expression in the central auditory system is possibly correlated with phases of functional plasticity in this pathway.     

Hydrogen peroxide production by monoamine oxidase during ischemia-reperfusion in the rat brain

Monoamine oxidase (MAO) as a source of hydrogen peroxide (H2O2) was evaluated during ischemia-reperfusion in vivo in the rat brain. H2O2 production was assessed with and without inhibition of MAO during and after 15 min of ischemia. Metabolism of H2O2 by catalase during ischemia and reperfusion was measured in forebrain homogenates using aminotriazole (ATZ), an irreversible H2O2-dependent inhibitor of catalase. Catecholamine and glutathione concentrations in forebrain were measured with and without MAO inhibitors. During ischemia, forebrain blood flow was reduced to 8% of baseline and H2O2 production decreased as measured at the microperoxisome. During reperfusion, a rapid increase in H2O2 generation occurred within 5 min as measured by a threefold increase in oxidized glutathione (GSSG). The H2O2-dependent rates of ATZ inactivation of catalase between control and ischemia-reperfusion were similar, indicating that H2O2 was more available to glutathione peroxidase than to catalase in this model. MAO inhibitors eliminated the biochemical indications of increased H2O2 production and increased the catecholamine concentrations. Mortality was 67% at 48 h after ischemia-reperfusion, and there was no improvement in survival after inhibition of MAO. We conclude that MAO is an important source of H2O2 generation early in brain reperfusion, but inhibition of the enzyme does not improve survival in this model despite ablating H2O2 production.     

Brief review of the fibronexus and its significance for myofibroblastic differentiation and tumor diagnosis

This brief review details the structure, nature, and distribution of the fibronexus, and discusses its significance for myofibroblastic differentiation and tumor diagnosis. The fibronexus is a cell surface specialization consisting of intracellular actin filaments and extracellular fibronectin filaments associated with subplasmalemmal plaque material. The fibronexus represents an intercellular junction between myofibroblasts, but in particular is a device for providing contact between myofibroblasts and matrix that mediates continuity between intracellular contractile filaments and extracellular matrix proteins. Immunoelectron microscopy in particular has shown that the intracellular filaments contain actin. The extracellular filaments contain fibronectin and collectively form the fibronectin fibril. The plaque probably contains such proteins as vinculin, talin, alpha-actinin, and integrin. Under appropriate biologic development and fixation conditions, the fibronectin fibril of the fibronexus is characterized by and distinguished from lamina by enhanced density, a rigid appearance, failure to adhere closely to the contours of the cell surface (except focally near the plaque material), and a longitudinally filamentous substructure. Confirmation of the presence of a fibronectin fibril may be obtained by the finding of intense cell surface staining with an antifibronectin antibody. Problems in identifying the fibronexus may be encountered, however, due to poor development and fixation, in which case the filamentous substructure may be inapparent. The fibronexus is such a typical feature of and is often so conspicuous in myofibroblasts that it can be regarded as perhaps essential for the interpretation of myofibroblastic differentiation. Structures with a similar appearance have been documented in fundamentally nonmyofibroblastic cells; these include aortic and scleral spur smooth muscle cells and endothelium. Uncertainties remain in the protein composition of the fibronexus, the nature of its contact with the matrix, and its relationship to similar structures seen in nonmyofibroblastic cells. Immunoelectron microscopy provides a potential means of clarifying some of these questions.