Effects of NO synthase inhibitors on the synovial microcirculation in the mouse knee joint

Production of nitric oxide by the inducible NO synthase (iNOS) is known to be enhanced in chronic joint inflammation and osteoarthritis as well as aseptic loosening of joint prostheses. Initial studies yielded promising results after inhibition of the nitric oxide synthase (NOS). However, the effect of NOS inhibition has not been studied at the site of the primary function of NO, the microcirculation of the synovium in vivo. Using our recently developed model for the in vivo study of synovial microcirculation in the mouse knee joint, the effects of selective versus nonselective inhibition of iNOS were investigated by means of intravital fluorescence microscopy. After resection of the patella tendon, the synovial fatty tissue was exposed for intravital microscopy. Diameter of arterioles, functional capillary density (FCD), diameter of venules, venular red blood cell velocity and leukocyte-endothelial cell interaction were quantitatively analyzed before, and 10 and 60 min after intravenous injection of NOS inhibitors {[}selective iNOS inhibitor N-iminoethyl-L-lysine (L-NIL), and nonselective NOS inhibitor N-G-nitro-L-arginine methyl ester (L-NAME)]. Our results demonstrate that L-NAME causes a significant decrease in the arteriolar diameter and FCD associated with an increase in the leukocyte accumulation in the synovium in vivo. In contrast, L-NIL neither altered the microhemodynamics nor the leukocyte-endothelial cell interaction in the synovium, indicating its potential use for selective inhibition of iNOS in joint inflammation. Using our method, further studies will provide new insights into the unknown effect of NOS inhibition on the synovial microvasculature in inflammatory joint disease in vivo. Copyright (C) 1999 S. Karger AG, Basel

Milieu-adopted in vitro and in vivo differentiation of mesenchymal tissues derived from different adult human CD34-negative progenitor cell clones

Adult mesenchymal stem cells with multilineage differentiation potentially exist in the bone marrow, but have also been isolated from the peripheral blood. The differentiation of stem cells after leaving their niches depends predominately on the local milieu and its new microenvironment, and is facilitated by soluble factors but also by the close cell-cell interaction in a three-dimensional tissue or organ system. We have isolated CD34-negative, mesenchymal stem cell lines from human bone marrow and peripheral blood and generated monoclonal cell populations after immortalization with the SV40 large T-antigen. The cultivation of those adult stem cell clones in an especially designed in vitro environment, including self-constructed glass capillaries with defined growth conditions, leads to the spontaneous establishment of pleomorphic three-dimensional cell aggregates ( spheroids) from the monoclonal cell population, which consist of cells with an osteoblast phenotype and areas of mineralization along with well-vascularized tissue areas. Modifications of the culture conditions favored areas of bone-like calcifications. After the transplantation of the at least partly mineralized human spheroids into different murine soft tissue sites but also a dorsal skinfold chamber, no further bone formation could be observed, but angiogenesis and neovessel formation prevailed instead, enabling the transplanted cells and cell aggregates to survive. This study provides evidence that even monoclonal adult human CD34-negative stem cells from the bone marrow as well as peripheral blood can potentially differentiate into different mesenchymal tissues depending on the local milieu and responding to the needs within the microenvironment. Copyright (C) 2005 S. Karger AG, Basel

Visualization of leukocyte transendothelial and interstitial migration using reflected light oblique transillumination in intravital video microscopy

Dynamic visualization of the intravascular events leading to the extravasation of leukocytes into tissues by intravital microscopy has significantly expanded our understanding of the underlying molecular processes. In contrast, the detailed observation of leukocyte transendothelial and interstitial migration in vivo has been hampered by the poor image contrast of cells within turbid media that is obtainable by conventional brightfield microscopy. Here we present a microscopic method, termed reflected light oblique transillumination microscopy, that makes use of the optical interference phenomena generated by oblique transillumination to visualize subtle gradients of refractive indices within tissues for enhanced image contrast. Using the mouse cremaster muscle, we demonstrate that this technique makes possible the reliable quantification of extravasated leukocytes as well as the characterization of morphological phenomena of leukocyte transendothelial and interstitial migration

Quantitative assessment of angiogenesis in murine antigen-induced arthritis by intravital fluorescence microscopy

Inhibition of angiogenesis might be a therapeutic approach to prevent joint destruction caused by the overgrowing synovial tissue during chronic joint inflammation. The aim of this study was to investigate angiogenesis in the knee joint of mice with antigen-induced arthritis (AIA) by means of intravital microscopy. In 14 mice (C57BL6/129Sv) intravital microscopic assessment was performed on day 8 after AIA induction in two groups (controls, AIA). Synovial tissue was investigated by intravital fluorescence microscopy using FITC-dextran (150 kD). Quantitative assessment of vessel density was performed according to the following categories: functional capillary density (FCD, vessels 10 mum) and FVD of vessels with angiogenic criteria (convoluted vessels, abrupt changes of diameter, vessels which are generated by sprouting and progressively pruned and remodelled). Microvessel count was performed using immunohistochemistry. There was no significant difference in FCD between the control group (337 +/- 9 cm/cm(2); mean +/-SEM) and the AIA group (359 +/- 13 cm/cm(2)). The density of vessels larger than 10 gm diameter was significantly increased in animals with AIA (135 +/- 10 vs. 61 +/- 5 cm/cm(2) in control). The density of blood vessels with angiogenic criteria was enhanced in arthritic animals (79 +/- 17 vs. 12 +/- 2 cm/cm(2) in control). There was a significant increase in the microvessel count in arthritic animals (297 +/- 25 vs. 133 +/- 16 mm(-2) in control). These findings demonstrate that angiogenesis in murine AIA can be assessed quantitatively using intravital microscopy. Further studies will address antiangiogenic strategies in AIA

ROS-mediated TNF-α and MIP-2 gene expression in alveolar macrophages exposed to pine dust

BACKGROUND: Respiratory symptoms, impaired lung function, and asthma have been reported in workers exposed to wood dust in a number of epidemiological studies. The underlying pathomechanisms, however, are not well understood. Here, we studied the effects of dust from pine (PD) and heat-treated pine (HPD) on the release of reactive oxygen species (ROS) and inflammatory mediators in rat alveolar macrophages. METHODS: Tumour necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-2 (MIP-2) protein release, TNF-α and MIP-2 mRNA expression, and generation of ROS were studied as end points after treatment of rat alveolar macrophages with PD or HPD. In a separate series of experiments, the antioxidants glutathione and N-acetyl-L-cysteine were included in combination with wood dust. To determine the endogenous oxidative and antioxidant capacity of wood dusts, electron spin resonance (ESR) spectroscopy was used. RESULTS: After 4 h incubation, both PD and HPD elicited a significantly (p < 0.05) increased mRNA expression of TNF-α and MIP-2 as well as a concentration-dependent release of TNF-α and MIP-2 protein. Interestingly, PD induced a significantly higher TNF-α and MIP-2 production than HPD. Moreover, a significantly increased ROS production was observed in alveolar macrophages exposed to both PD and HPD. In the presence of the antioxidants glutathione and N-acetyl-L-cysteine, the PD- and HPD-induced release of ROS, TNF-α, and MIP-2 was significantly reduced. Finally, electron spin resonance analyses demonstrated a higher endogenous antioxidant capacity of HPD compared to PD. Endotoxin was not present in either dust sample. CONCLUSION: These results indicate that pine dust is able to induce expression of TNF-α and MIP-2 in rat alveolar macrophages by a mechanism that is, at least in part, mediated by ROS

Parasitism of the root-lesion nematode Pratylenchus thornei on chickpea

Pratylenchus thornei-chickpea interactions were investigated under controlled and fluctuating environmental conditions in the growth chamber, greenhouse and shadehouse. Under controlled conditions. P. thornei infected chickpea hnes 12071/10054 and P2245 and cultivars Andoum 1, JG62 and UC 27. Line P 2245 and cv. JG 62 were the most susceptible genotypes on the basis of root damage and nematode reproduction, but nematode infection did not significantly reduce root and shoot weights. Cultivars Andoum 1 and UC27 and line 12071/10054 showed the least root damage and nematode reproduction. Inoculation of cv. Andoum 1 with 2500, 5000 or lOOOO nematodes per plant in pots did not affect shoot weight, regardless ofthe conditions of water stress ofthe plants. However, root weight was significantly reduced by nematode infection in plants grown under water stress and fluctuating temperature conditions in the greenhouse, but was not affected by any other treatment. The nematode reproduction index was not affected by soil water content under shadehouse conditions, but was greater on plants watered to soil water-holding capacity than in water-stressed plants under greenhouse conditions. For both environments, the nematode reproduction index decreased when inoculum density was greater than 5000 nematodes per plant.This research was supported by grant AGF92- 0910-CO2-01 from Comision Interministerial de Ciencia y Technologia (CICYT), We thank H, M, Halila, INRA, Tunisia for providing us with seeds of cultivar Andoum 1, and F, Orgaz, Instituto de Agricultura Sostenible, CSIC, Spain for measuring water potential.Peer reviewe