Decrease in fibrin content of venous thrombi in selectin-deficient mice.

The purpose of this study was to quantify the fibrin content of thrombi produced in a mouse model of venous thrombosis and correlate this to thrombus mass. The role of P-selectin, E-selectin, and IL-10 on thrombus fibrin content was analyzed using knockout (KO) mice. Five groups of mice were evaluated: control (N = 10), P-selectin KO (N = 7), E-selectin KO (N = 5), combined E-/P-selectin KO (N = 12), and IL-10 KO (N = 10). Venous thrombosis was induced by ligation of the infrarenal IVC. Mice were sacrificed on postoperative days (POD) 2 and 6. Thrombus mass was calculated. Sections of IVC were stained with an antibody that cross reacts with mouse fibrin. The distribution of RGB color pixels was generated from digitized micrographs of the thrombus of each animal. The mean pixel value for each group was compiled and analyzed using 2-way ANOVA. Mean pixel value per group was correlated with the mean thrombus mass per group. Color analysis demonstrated significant decreases in the analyzed fibrin content on POD-2 between the control vs E-/P-selectin KO (P < 0.05) and control vs IL-10 KO (P < 0.05) groups. In addition, significantly less fibrin staining was noted on POD-6 between the control vs E-selectin KO (P = 0.03), control vs P-selectin KO (P = 0.01), and control vs E-/P-selectin KO (P < 0.01). There was a strong overall correlation between the mean pixel value for each group and the thrombus mass (R = 0.964; P < 0.01). This study demonstrates a difference in fibrin content of thrombi produced in animals deficient in E-selectin, P-selectin, and IL-10, supporting their importance in thrombus amplification, fibrin formation, and the mass of thrombus formed.

Triplex ultrasound: elasticity imaging to age deep venous thrombosis.

Deep venous thrombosis (DVT), and its sequela, pulmonary embolism (PE), is the leading cause of preventable in-hospital mortality in the USA and other developed countries. After it is detected, acute clots must be differentiated from chronic DVT for appropriate treatment. However, there are no reliable thrombus staging methods presently available in clinical practice. In this study, we tested the hypothesis that blood clots can be detected and staged using a triplex ultrasound (US) test. Triplex US is based on a "gold standard" duplex US technique augmented by US-based reconstructive elasticity imaging. Fibrin-composed blood clots harden with development and organization. By imaging clot elasticity, it may be possible to both detect and differentiate clots and, therefore, provide an urgently needed noninvasive means of DVT staging.

Computed tomographic venography is specific but not sensitive for diagnosis of acute lower-extremity deep venous thrombosis in patients with suspected pulmonary embolus.

PURPOSE: Duplex ultrasound scanning (US) is the accepted standard means of diagnosis for lower-extremity suprageniculate deep venous thrombosis (LE-DVT). Computed tomographic venography (CTV) has been proposed as an alternative modality for diagnosis of LE-DVT in patients with suspected pulmonary embolism (PE). This study compared CTV with US as a means of diagnosing acute LE-DVT. METHODS: A retrospective review of US and CTV scans from 136 patients with suspected PE who underwent both studies to exclude acute LE-DVT at a single institution was performed. Studies were reviewed and coded in a blinded manner. US was considered to be the reference test. Direct costs of each study were determined by using commercial software. RESULTS: The sensitivity and specificity rates of CTV were 71% and 93%, respectively. The positive predictive value, negative predictive value, and accuracy rates of CTV were 53%, 97%, and 90%, respectively. DVT localization was the same in eight of 10 cases in which the results of both US and CTV were positive. CTV costs and charges per study were greater than those of US by $46.88 and $602.00, respectively. CONCLUSION: CTV is specific, but has a lower sensitivity rate and positive predictive value for the diagnosis of acute LE-DVT compared with US. Additionally, CTV is more costly than US scanning. Because of the lower sensitivity rate and positive predictive value and the increased cost of CTV, US remains the screening study of choice in cases of suspected acute LE-DVT.

Nitric oxide inhibition increases matrix metalloproteinase-9 expression by rat aortic smooth muscle cells in vitro.

OBJECTIVE: The hypothesis to be tested was that diminished bioavailable nitric oxide (NO) affects matrix metalloproteinase (MMP) expression and activation in vascular smooth muscle cells (SMCs). METHODS: Cultivated rat aortic SMCs (RA-SMCs) were exposed to increasing concentrations of L-N-monomethyl arginine (L-NMMA), a nonselective inhibitor of NO synthase, in the presence of proinflammatory cytokines (50 ng/mL interleukin [IL]-1beta, 50 ng/mL interferon-gamma, and 30 microg/mL lipopolysaccharide). Nitrite and nitrate, two of the final end products of NO metabolism, were measured in media collected at 48 hours with the use of the Saville assay (n = 4). MMP activity was measured with 1% gelatin zymography (n = 4). In separate experiments in which 2 ng/mL of IL-1beta and L-NMMA was used, MMP protein and messenger RNA (mRNA) levels were determined with Western blot analysis (n = 3) and semiquantitative reverse transcriptase-polymerase chain reaction (n = 3), respectively. Data were analyzed with nonparametric analysis of variance. RESULTS: Increasing concentrations of the NO synthase inhibitor L-NMMA caused a dose-dependent decrease (P <.05) in nitrite and nitrate production by RA-SMCs after cytokine exposure. Zymography documented an early dosedependent increase (P <.05 compared with cytokines alone) in 92-kd MMP activity, with no significant changes in 72-kd MMP activity after treatment with L-NMMA (P >.05 compared with cytokines alone). Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that the addition of L-NMMA to IL-1beta-stimulated RA-SMCs led to significant increases in MMP-9 mRNA (n = 3, P <.01 for 1.0 mmol/L L-NMMA) and MMP-9 protein levels (n = 3, P <.05), respectively. No differences in MMP-2 mRNA or protein levels were demonstrated. CONCLUSIONS: Inhibition of cytokine-induced NO expression in RA-SMCs is associated with a selective, dose-dependent increase in MMP-9 expression and synthesis. These findings suggest that alterations in local NO synthesis may influence MMP-9-dependent vessel wall damage.

Quantitative ultrasonography of the liver in cats during obesity induction and dietary restriction.

A study was designed to evaluate quantitative ultrasonographic characteristics of a diffuse parenchymal disease in an experimental model of subclinical fatty infiltration of the liver using diet-induced obesity and dietary restriction in the cat. Ultrasound images of livers were quantitatively analysed by a video signal analysis technique before, during and at the end of obesity induction, and then during and following dietary restriction. Attenuation and brightness (backscatter coefficient) were correlated with hepatic lipid content obtained from the livers by surgical and ultrasound-guided percutaneous biopsy. Attenuation and backscatter increased as hepatic lipid content increased. Both attenuation and backscatter significantly correlated with the hepatic lipid content (P=0.002 and P=0.02 respectively). This model of subclinical fatty infiltration of the liver in the cat demonstrates that hepatic lipid content increases as a consequence of obesity and of severe dietary restriction. Infiltration of the liver with fat may be evaluated non-invasively using quantitative ultrasonography by the video signal analysis technique. This method of image analysis may prove useful for the evaluation of diffuse parenchymal organ disease.

Attenuation and backscatter estimation using video signal analysis applied to B-mode images.

Most methods for in vivo quantitation of ultrasound attenuation and backscatter are not available clinically because they rely upon acquiring and analyzing radio frequency (rf) echo signals. This paper describes a technique to estimate ultrasound attenuation and backscatter from B-mode image data. The video signal analysis (VSA) technique utilizes images of a reference phantom, taken using the same instrument settings used to record images from the patient or sample, to account for effects of the transducer beam, system gain and signal processing on image data. A 'gray-scale look-up table' is derived to convert image pixel value data within a region of interest to echo signal amplitudes relative to echo signals from the same depth in the reference phantom. These relative echo levels enable estimates of attenuation and backscatter in the region of interest. VSA was used to quantify acoustic properties of test phantoms using 3 different clinical scanners and various transducers. The level of agreement between results obtained with different ultrasound imaging systems was very good. VSA attenuation and backscatter levels also compare favorably with attenuation coefficients and backscatter coefficients obtained using rf analysis.