Aquaporin 1 (AQP1) expression in experimentally induced osteoarthritic knee menisci: an in vivo and in vitro study.

Osteoarthritis (OA) of the knee is a major problem in our society. The development of new treatment options for OA is limited, because the pathophysiological mechanisms are not clearly understood, especially on the molecular level. Aquaporin 1 (AQP1) is a specific protein channels for water transport; it is expressed in articular chondrocytes, human synovitis, in chondrocytes of patients with rheumatoid arthritis or OA and in chondrocyte-like cells of human intervertebral disc. The aim of this study was to investigate the expression of AQP1, through immunohistochemistry, immunocytochemistry and Western blot, in experimentally induced OA knee menisci. AQP1 was studied in vivo in knee OA menisci from 36 rats that underwent medial or lateral meniscectomy, and in vitro on fibrochondrocytes derived from knee OA menisci rats. OA in rats was experimentally induced and tested by histomorphometric analysis. Histological results demonstrated structural alterations in OA menisci accompanied by a very strong AQP1 immunohistochemical and immunocytochemical staining. The Western blot analysis confirmed a strong expression of AQP1 in OA fibrochondrocytes cells. The results of the present research suggest that an activation of AQP1, induced by the OA process, may represent an endogenous mechanism, which can be used to control the tissue degeneration within OA articular joints.

Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold.

Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease.

OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model.

Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular relevance to the use of OA cells and tissue engineering in the therapeutic approach to patients.

Expression profile of ErbB receptor's family in human alveolar type 2-like cell line A549 exposed to hexavalent chromium.

Occupational exposure to hexavalent chromium (Cr (VI)) compounds is associated with increased risk of pulmonary disease. In the present study we have investigated temporal expression of ErbB's receptors family in A549 cells after exposure to Cr (VI). Treatment with 10 microM or 300 microM of Na2CrO4 induced apoptotic cell death within 24h. Based on data obtained by ELISA cell death detection method and fluorescence microscopy, the concentration of 10 microM was chosen to study the expression of ErbB receptors family. Such concentration reflects a condition of acute toxicity in which cells survived up to 24h. Real time quantitative PCR has been performed to analyze the expression profiles of ErbB family genes following chromium toxicity. The expression of EGFR and ErbB2 receptors was significantly reduced after 1h and 4h of treatment while ErbB2 receptor was significantly increased and EGFR receptor returned to basal value after 24h. Instead, ErbB3 receptor was overexpressed after 1h, returned to basal level after 4h and increased its level after 24h. Exposure to chromium did not change expression level of ErbB4 receptor in A549 cell line. The present data suggests that expression changes in ErbB receptors might have a role in the carcinogenic effects induced by this pneumotoxic agent.

Fluoro-edenite fibres induce lung cell apoptosis: an in vivo study.

We previously showed that apoptosis in the lungs of sheep exposed to fluoro-edenite fibres is induced via the receptor pathway. The present study was performed to gain further insights into the mechanisms of activation of programmed cell death induced by the fibres. Fluoro-edenite fibres are similar in size and morphology to some amphibolic asbestos fibres. They have been found in benmoreitic lavas, in the local stone quarry, in building materials and in road paving at Biancavilla, a town in eastern Sicily (Italy), where epidemiological surveys revealed a cluster of mortality from pleural mesothelioma. Inhalation of asbestos fibres can cause chronic inflammation and carcinogenesis. Since fluoro-edenite has been shown to activate the apoptotic process, we set out to characterise the expression of apoptosis-regulating proteins in fluoro-edenite-exposed lung disease and sought to determine if apoptosis results from fluoro-edenite exposure. Lung tissue from apparently healthy sheep habitually grazing near Biancavilla was processed for immunohistochemical localisation of bcl-2 and bax. Results showed epithelial and interstitial bax overexpression, especially in cells directly in contact with the fibres, and negative bcl-2 immunoexpression. TUNEL-positive cells were detected in alveoli and connective tissue. The integrity of alveolar epithelium and alveolar apoptosis are critical determinants in the pathways that initiate fibrogenesis in the lung and fibroblastic foci are usually found close to abnormal or denuded alveolar epithelium. Our results are consistent with the hypothesis that apoptosis is an important mechanism for removing cells with irreparable fluoro-edenite-induced genetic changes that predispose them to a neoplastic evolution.

Effects of exposure to fluoro-edenite fibre pollution on the respiratory system: an in vivo model.

An increased standardised rate of mortality from pleural mesothelioma among the population of Biancavilla (Sicily, Italy) has been attributed to exposure to fluoro-edenite fibres. Our aim was to establish whether and how these fibres may induce pathological effects using an in vivo model. Lung tissue collected from 60 healthy sheep selected from six flocks habitually grazing near Biancavilla and from 10 control sheep was fixed formalin and paraffin-embedded; sections were stained with haematoxylin-eosin, Masson trichrome and Gomori argentic impregnation. Histochemical studies and immunohistochemical analysis for the localisation of TRAIL, DR5 and MMP13 were also performed. The lungs of exposed sheep exhibited fibrosis and loss of alveolar architecture with honeycombing of alveolar cavities. Fluoro-edenite fibres were detected close to the alveolar epithelium and interstitia. The parenchyma showed hyaline degeneration and strong PAS-positivity in the interstitium, proteoglycan alterations, reflecting a damaged basal membrane and an involvement of the interstitial matrix. MMP-13 was overexpressed, mainly in fibroblasts and epithelial cells, while positivity for TRAIL and DR5 was detected on alveolar surfaces and in the vascular stroma. The initial pathological event seems to involve first the alveoli and subsequently the interstitium, giving rise to classic honeycombing. The triggering event at the level of type I pneumocytes would damage the cytoplasmic membrane resulting in loss of cell elements and exposure of underlying capillaries, and eventually in a series of reactions including macrophage activation, possible release of growth factors and metaplasic reconstruction of lung alveoli. Immunopositivity for TRAIL and MMP-13 receptor suggests that apoptotic processes may also be activated by fluoro-edenite.

Effects of norepinephrine depletion in rats during cerebral post-ischemic reperfusion.

The present paper reports the effects of norepinephrine depletion in rats, after treatment with N-(2-chloroethyl)-N-ethyl 2-bromobenzylamine (DSP-4) neurotoxin, on partial cerebral ischemia and reperfusion. Histological observations made under experimental conditions of noradrenergic (NA)-depletion demonstrated that neuronal lesions were not exacerbated; in fact, in DSP-4-treated ischemic animals, a minor number of neurons appeared damaged. Our results suggest that neuronal recovery after post-ischemic reperfusion is not affected by NA-depletion. DSP-4 neurotoxin does not induce 5-hydroxy-triptamine (5-HT) depletion.

Expression of bone morphogenetic protein-6 and transforming growth factor-beta1 in the rat brain after a mild and reversible ischemic damage.

We have examined the distribution of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-6 (BMP-6) in the brain of rats subjected to a mild and reversible ischemic damage produced by a 20-min occlusion of both carotid arteries without occlusion of the vertebral arteries. We have selected this model to study how the expression of trophic factor of the TGF-beta superfamily changes in neurons that recover from a transient insult. Immunocytochemical analysis showed a loss of TGF-beta1 in neurons of all hippocampal subfields immediately after the ischemic period, followed by a recovery of immunoreactivity in CA1 and CA3 neurons after reperfusion. BMP-6 immunoreactivity was also lost in most hippocampal neurons, but immunostaining became particularly intense in the interstitial space after both ischemia and reperfusion. An interstitial localization of BMP-6 was also observed in the cerebral cortex, particularly after reperfusion. Mild ischemia also induced substantial changes in the expression of TGF-beta1 and BMP-6 within the cerebellar cortex. In control animals, these factors appeared to be localized in granule cells (TGF-beta1) and Purkinje cells (both), whereas the molecular layer was not immunopositive. Both TGF-beta1 and BMP-6 were highly expressed in the interstitial spaces of the cerebellar cortex either 20 min after ischemia or 20 min after reperfusion. Taken collectively, these results suggest that a mild and reversible ischemia stimulates the release of BMP-6 from neurons into the interstitial space. We speculate that BMP-6, besides functioning during brain development, may also regulate neuronal resistance to insults of the adult brain.

Fibroblast growth factor-2 and transforming growth factor-beta1 immunostaining in rat brain after cerebral postischemic reperfusion.

Several trophic factors are known to regulate the survival and growth of neurons in brain and peripheral tissues. Several findings suggest that basic fibroblast growth factor-2 (FGF-2) plays an important role in the "self-repair" responses that follow injuries such as trauma and brain ischemia and that FGF-2 contributes to the repair of damaged tissue. Transforming growth factor-beta (TGF-beta) is a potent growth-regulatory protein secreted by virtually all cells. In the present study, we used immunohistochemical techniques to investigate whether FGF-2 and TGF-beta1 participate in the healing of damaged tissue following partial brain ischemia. The profile of the observed immunoreactivities indicated that TGF-beta1 and FGF-2 release varies between the different cerebral areas subjected to ischemic insult. Moreover, the sectorial heterogeneity of immunocytochemical response suggests that, during postischemic reperfusion, neuronal recovery may be due not only to neuron-glia interaction but also to neurochemical conditions involving inhibitory interneurons.

Expression of vimentin intermediate filament in human odontoblast.

BACKGROUND: Vimentin (57 Kda) is a cytoskeletal protein. Odontoblasts contain vimentin and it seems that this protein may function to keep the organelles and the nucleus in a definite place. However little is known about vimentin in the cytoskeleton of odontoblast processes. The purpose of the present study was, therefore to immunolocalize vimentin intermediate filament in odontoblast body and process in order to clarify the distribution of this cytoskeletal element. METHODS: 12 extracted intact premolars, from children, were used in the present study. Each specimen was decalcified in EDTA. Each tissue portion was embedded in paraffin. On sections a monoclonal anti-vimentin antibody was applied. The immunoreaction was visualized by ABC technique. RESULTS: Vimentin was expressed in the cell body and cell process of odontoblasts, however with a different immunolabeling pattern related to the topographical area of observations. In odontoblast cell bodies vimentin showed a perinuclear and cytoplasmatic staining. In the very initial portion of odontoblast process immunoreaction products for vimentin were observed in the core of the process. In the middle zone of dentin vimentin immunoreactions products also showed a granular and cross-bridge arrangements, and also, vimentin was also detected under the plasma membrane, at the periphery of the odontoblast process. Nearby the dentino-enamel junction vimentin immunolabeling was appreciated, mainly under the plasma membrane. CONCLUSIONS: On the basis of vimentin distribution in the odontoblast process it seems plausible to assume that this IF vimentin is important in forming a flexible scaffold essential for structuring cytoplasm.

Effects of glutathione depletors on post-ischemic reperfusion in rat brain.

The present paper reports the effects of GSH depletion (diethylmaleate induced) on partial cerebral ischemia and reperfusion for 7 and 20 days. Our results confirm that there is a paradoxical protective effect of the GSH-depletor and suggest an improved neuronal trophism induced by diethylmaleate treatment.

GFAP, S-100 and vimentin proteins in rat after cerebral post-ischemic reperfusion.

In the present study astrocytes reactivity during cerebral post-ischemic reperfusion was evaluated immunocytochemically by using antibodies to vimentin, glial fibrillary acidic protein (GFAP) and S-100 protein. At the 7th day of post-ischemic reperfusion few GFAP-positive cells were observed in the hippocampus and cerebellum, the number of GFAP-positive cells increased slightly after 20 days of reperfusion. This poor GFAP-positivity may be due to the inhibition of GFAP polymerization by S-100; in fact, S-100 immuno-reactivity was already evident from the 7th day. Vimentin immuno-staining was evident both at the 7th and 20th day of reperfusion in microglial cells and in oligodendrocytes, suggesting that these cells are involved in the recovery of neurons following brain injury.

MAP2, synaptophysin immunostaining in rat brain and behavioral modifications after cerebral postischemic reperfusion.

Plasticity in the central nervous system after cerebral ischemia is a controversial issue; focal cerebral ischemia produces an area of infarction that is surrounded by neurons that may respond to nearby damage by creating new synapses. In the present study the expression of the postsynaptic microtubule-associated protein 2 (MAP2) and the presynaptic marker protein, synaptophysin, was investigated by immunocytochemical techniques in the CA1 sector of hippocampus and in cerebellum of rats made ischemic by bilateral clamping of common carotid arteries and reperfused for 7 and 30 days. In addition, ischemia-induced behavioral alterations were also evaluated after 7 and 30 days of reperfusion. The present study demonstrates a decreased postsynaptic MAP2 immunoreactivity, representative of neuronal loss, particularly in CA1 sector of hippocampus and in cerebellum of ischemic rats reperfused for 7 days. After 30 days of reperfusion, MAP2 immunostaining was similar to control. In the same brain sections an increased presynaptic synaptophysin immunoreactivity has been observed only after 30 days of reperfusion. These data suggest compensatory regenerative changes associated with synaptic remodelling and are supported by behavioral recovery observed under the same experimental conditions.

Neuronal lesions and behavioral modifications in rat following cerebral ischemia and reperfusion.

Neurons of the mammalian CNS differ in their vulnerability to various disease processes and other insults, particularly in their response to total anoxia/ischemia. In this study we have tested the histological and behavioral modifications induced by experimental conditions of partial cerebral ischemia in the rats. The specific morphological and histological alterations, observed in our experimental conditions of reversible partial cerebral ischemia, confirm the selective vulnerability of certain neuronal populations to ischemic injury and are also evidenced by behavioral modifications which may mirror the functional impairment observed in humans after a transitory ischemic attack.

The role of cartilage in osteogenesis in the human fetal vertebral body.

The cartilage canals of the thoracic and lumbar vertebrae of fetuses ranging in crown-rump (C.R.) length from 33 to 180 mm were examined under light microscopy. The activity of the cells within these canals contributed to the anterolateral growth of the vertebral body. Bone formation was seen to commence in the center of the vertebral body. The cells of the connective tissue contained within the cartilage canals appeared to participate in the process of osteogenesis in multiple, discrete foci. In five-month-old fetuses, periosteal bone was noted on the anterior and posterior surfaces of the vertebral body, and calcification in the walls of the cartilage canals gave the histologic appearance of bone spicules.

Ultrastructural study of parietal cells before and after parietal cell vagotomy in patients with duodenal ulcer.

The records of 16 patients of a total of 402 who underwent parietal cell vagotomy without a drainage procedure for duodenal ulcer were reviewed. Biopsies of gastric mucosa were taken before the operation and six months, two years and three years postoperatively. Ultrastructural studies using electron microscopy detail the modifications of the parietal cells which are the main target of the vagal denervation procedure. The parietal cells are greatly modified within six months after parietal cell vagotomy with a significant reduction of the secretory surface of the cells and a sharp increase of tubulovesicular formations regain their preoperative morphologic appearance after two to three years but not their secretory capacity.

[Ultrastructural changes of hypertrophied myocardium in severe experimental hypoxia. Effects of diphosphothiamine and of an antagonist of the vitamin B1. (author's transl)]. / Modificazioni ultrastrutturali del miocardio ipertrofico in corso di ipossia sperimentale. Effetti della difosfotiamina e di una antivitamina B1

The ultrastructural changes occurring in hypertrophic heart cases during severe experimental hypoxia, conducted intermittently for 140 and 250 hours, are reported in the present study. For this purpose, serial right myocardial specimens of control and treated groups of animals were obtained and examined by electron-microscopy. Most areas of the individual myocardial tissue taken during prolonged degrees of exposures to normobaric hypoxia, showed an increased number of mitochondria, their prominent destructive changes, enlargement of cisternae of sarcoplasmic reticulum, reduction and derangement of the cristae and an increase of glycogen and lipif droplets cellular content. Different responses were associated with the various experimental designs which include simple hypoxia, hypoxia with administration of Diphosphothiamine (DPT, 30 mg/hie/Kg body weight) and hypoxia with administration of an antagonist of the vitamin B1, neopyrithiamine (PyTh, 20 mg/die/Kg body weight). Alterations in the fine structure of myocardial cells and cellular organelles could complement the increased glycolitic activity and phospholipid biosynthesis for the compensatory mechanism of the heart to hypoxic stimulus. These changes were most evident in the hypoxic animals treated with PyTh because of a large disturbance of energy production caused by the antivitamin. Most of ultrastructural and glycogen content changes disappeared when DPT was administered to the animals in hypoxia. However, the mitochondria were larger in size and their matrices had higher electron density than in the normal control animals. The significance of the morphological and biochemical findings are discussed.