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

Rifampin and Triclosan but not Silver is Effective in Preventing Bacterial Infection of Vascular Dacron Graft Material

AbstractObjectives. To evaluate the efficacy of silver- or Triclosan-coated prosthetic material compared to Rifampin bonded Dacron concerning their resistance to infection following subcutaneous implantation and contamination with Staphylococcus aureus.Design. Animal experimental study in mice.Material and methods. Thirty-six C3H/HcN mice (Charles River Lab., Sulzfeld, Germany) with a weight between 24 and 27 g were randomised into six groups counting six animals each. Group I: control, gel-sealed dacron graft, group II: gel-sealed dacron graft and local contamination, group III: Intergard®-Silver-prosthesis and contamination, group IV: silver/gel-sealed dacron prosthesis (test graft) and contamination, group V: Rifampin-bonded gel-sealed graft and contamination, group VI: Triclosan/collagen-coated dacron graft and contamination. Dacron graft material 0.8×1 cm was subcutaneously implanted in mice. Local contamination with 2×107/0.2 ml S. aureus ATCC 25923 was carried out in groups II to VI. On day 14 the animals were killed and the grafts were explanted. The microscopic, histologic and microbiological evaluation of the graft material and the perigraft tissue was performed.Results. In control group I no case of infection was detected. In group II, 6 of 6 animals showed infection. In group III (Intergard®-Silver) and group IV (silver/gel-test graft) were 6 of 6, in group V (Rifampin) only 1 of 6 grafts and in group VI (Triclosan) 4 of 6 grafts were infected. The difference between the low rate of infection in group V (Rifampin) in comparison to the completely infected groups III and IV (Silver) as well as the control group II was significant. Treatment of grafts with Triclosan could prevent infection only in 1/3 of the cases in group IV.Conclusion. Silver coating failed to prevent graft infection material. A potential antimicrobial property was evident for Triclosan whereas Rifampin-bonded grafts exhibit a significantly reduced infection rate. Thus, silver-coated vascular grafts cannot ensure protection from vascular graft infection

Platelet-Associated CD40/CD154 Mediates Remote Tissue Damage after Mesenteric Ischemia/Reperfusion Injury

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage

Orthogonal polarisation spectral imaging as a new tool for the assessment of antivascular tumour treatment in vivo: a validation study

Tumour angiogenesis plays a key role in tumour growth, formation of metastasis, detection and treatment of malignant tumours. Recent investigations provided increasing evidence that quantitative analysis of tumour angiogenesis is an indispensable prerequisite for developing novel treatment strategies such as anti-angiogenic and antivascular treatment options. Therefore, it was our aim to establish and validate a new and versatile imaging technique, that is orthogonal polarisation spectral™ imaging, allowing for non-invasive quantitative imaging of tumour angiogenesis in vivo. Experiments were performed in amelanotic melanoma A-MEL 3 implanted in a transparent dorsal skinfold chamber of the hamster. Starting at day 0 after tumour cell implantation, animals were treated daily with the anti-angiogenic compound SU5416 (25 mg kg bw−1) or vehicle (control) only. Functional vessel density, diameter of microvessels and red blood cell velocity were visualised by both orthogonal polarisation spectral™ imaging and fluorescence microscopy and analysed using a digital image system. The morphological and functional properties of the tumour microvasculature could be clearly identified by orthogonal polarisation spectral™ imaging. Data for functional vessel density correlated excellently with data obtained by fluroescence microscopy (y=0.99x+0.48, r2=0.97, RS=0.98, precision: 8.22 cm−1 and bias: −0.32 cm−1). Correlation parameters for diameter of microvessels and red blood cell velocity were similar (r2=0.97, RS=0.99 and r2=0.93, RS=0.94 for diameter of microvessels and red blood cell velocity, respectively). Treatment with SU5416 reduced tumour angiogenesis. At day 3 and 6 after tumour cell implantation, respectively, functional vessel density was 4.8±2.1 and 87.2±10.2 cm−1 compared to values of control animals of 66.6±10.1 and 147.4±13.2 cm−1, respectively. In addition to the inhibition of tumour angiogenesis, tumour growth and the development of metastasis was strongly reduced in SU5416 treated animals. This new approach enables non-invasive, repeated and quantitative assessment of tumour vascular network and the effects of antiangiogenic treatment on tumour vasculature in vivo. Thus, quantification of tumour angiogenesis can be used to more accurately classify and monitor tumour biologic characteristics, and to explore aggressiveness of tumours