In vitro evaluation of the sinus sagittalis superior thrombosis model in the rat using 3D micro- and nanocomputed tomography.

INTRODUCTION: Thrombosis of the cerebral veins and sinus are common causes of stroke. Animal models help us to understand the underlying pathophysiology of this condition. Therefore, the purpose of our study was to evaluate a well-established model for sinus sagittalis (SSS) thrombosis using micro- and nanocomputed tomography (CT) imaging. METHODS: SSS thrombosis was performed in four rats. After contrast perfusion, brains were isolated and scanned using micro-CT at (8 microm)(3) voxel size to generate 3D images of the cerebral vasculature. For more detailed information on vascular perfusion territories, nano-CT imaging was performed to investigate the boundary layer of contrast-enhanced vessels and the occluded veins. The venous and arterial vascular volume fraction and gray scale measurements were obtained in the SSS thrombosis group and compared to controls. The significance of differences in vascular volume fraction and gray scale measurements was tested with analysis of variance. Results were complemented with histology. RESULTS: Micro-CT proved to accurately visualize and differentiate vascular occlusion territories performed in the SSS thrombosis model. Moreover, 3D micro-CT provided quantitative information on arterial and venous vascular volume fraction. Micro-CT imaging enables a total 3D visualization of complications (ventricle rupture) in the SSS thrombosis model. We established gray scale measurements by which focal cerebral ischemia could be radiographically categorized (p < 0.001). CONCLUSIONS: Using nano-CT, the interface of contrast-perfused and occluded veins can be visualized. Micro-CT is feasible for analysis and differentiation of perfusion territories in an animal model of focal cerebral ischemia.

Effects of 3-deazaadenosine on homocysteine and atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: In the past decade, elevated homocysteine concentration has achieved widespread recognition as an independent risk factor in the development of atherosclerosis. 3-Deazaadenosine (c3Ado) is a potent inhibitor and substrate for S-adenosylhomocysteine hydrolase and therefore may reduce homocysteine concentrations. The current study investigated the effect of c3Ado on serum homocysteine, atherosclerotic lesions, and the expression of adhesion molecules in apoE-knockout mice. METHODS AND RESULTS: Animals were placed on an atherogenic diet with or without c3Ado for 12 and 24 weeks. Frozen cross-sections of the aortic sinus and the proximal aorta were analyzed by computer-aided planimetry for fatty plaque formation. Macrophages, VCAM-1 and ICAM-1 were quantified by immunhistochemistry and oligo-cell reverse transcription polymerase chain reaction after laser microdissection. Application of c3Ado resulted in significant reduction of homocysteine levels by 35.9 and 45.3% after 12 and 24 weeks, respectively (P < 0.001). Neointimal area and atherosclerotic plaque formation were significantly reduced in animals treated with c3Ado (P < 0.01). Moreover, monocyte adhesion and concomitant ICAM-1 and VCAM-1 antigen and RNA expression on the endothelial layer were significantly reduced (P < 0.001, P < 0.01). CONCLUSION: Our results demonstrate that c3Ado induces a marked reduction of homocysteine concentrations which might explain in part the anti-atherogenic effect of the drug.

Effects of multi-deficiencies-diet on bone parameters of peripheral bone in ovariectomized mature rat.

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.

Evaluation of angiogenesis using micro-computed tomography in a xenograft mouse model of lung cancer.

Quantitative evaluation of lung tumor angiogenesis using immunohistochemical techniques has been limited by difficulties in generating reproducible data. To analyze intrapulmonary tumor angiogenesis, we used high-resolution micro-computed tomography (micro-CT) of lung tumors of mice inoculated with mouse Lewis lung carcinoma (LLC1) or human adenocarcinoma (A549) cell lines. The lung vasculature was filled with the radiopaque silicone rubber, Microfil, through the jugular vein (in vivo application) or pulmonary artery (ex vivo application). In addition, human adenocarcinoma lung tumor-bearing mice treated site-specifically with humanized monoclonal antibody (bevacizumab) against vascular endothelial growth factor. Quantitative analysis of lung tumor microvessels imaged with micro-CT showed that more vessels (mainly small, <0.02 mm(2)) were filled using the in vivo (5.4%) compared with the ex vivo (2.1%) method. Furthermore, bevacizumab-treated lung tumor-bearing mice showed significantly reduced lung tumor volume and lung tumor angiogenesis compared with untreated mice as assessed by micro-CT. Interestingly, microvascularization of mainly the smaller vessels (<0.02 mm(2)) was reduced after bevacizumab treatment. This observation with micro-CT was nicely correlated with immunohistochemical measurement of microvessels. Therefore, micro-CT is a novel method for investigating lung tumor angiogenesis, and this might be considered as an additional complementary tool for precise quantification of angiogenesis.

Glomerular filtration rate measured by using triphasic helical CT with a two-point Patlak plot technique.

PURPOSE: To determine the accuracy of the two-point Patlak plot in the calculation of glomerular filtration rate (GFR). MATERIALS AND METHODS: Fifty patients without acute renal disorder were included. GFR was calculated by using a two-point Patlak plot technique. The computed tomography (CT) protocol consisted of a plain examination followed by two contrast material-enhanced examinations in the arterial and portovenous phase. Each examination included the entire kidneys and was performed after injection of 120 mL iopromide and 300 mg of iodine per milliliter given per 75 kg of body weight. All examinations were performed with a standard abdominal protocol. Section thickness was 4 x 2.5 mm, and table advance was 12.5 mm. Bolus triggering commenced 10 seconds after the start of contrast medium injection. Twelve dynamic scans were obtained with reduced tube current every 3 seconds to obtain sufficient arterial input function data. Correction for hematocrit level was made by using the unenhanced attenuation of the aorta. As a reference method, plasma clearance of the contrast medium injected for CT was calculated from three iodine plasma concentration measurements obtained 3, 4, and 5 hours after injection. Linear correlation was performed. RESULTS: GFR was calculated from CT data in 48 patients. Two patients were excluded because of breathing errors. Mean GFR was 80 mL/min (range, 17-153 mL/min) as measured with iopromide plasma clearance and 82 mL/min (range, 28-148 mL/min) as measured with CT. Linear correlation between the two methods was r = 0.889; GFR calculated with the two-point Patlak plot was equal to 15 plus 0.83 times GFR (plasma clearance). The mean difference between GFRs as determined with the two methods was -1.2 mL/min (95% CI: -27.1, 24.6). CONCLUSION: Total GFR can be measured accurately with minimally extended triphasic CT in patients without acute renal disorder by using a two-point Patlak plot technique.

Acute rat lung injury: feasibility of assessment with micro-CT.

PURPOSE: To evaluate the feasibility of micro-computed tomography (CT) for analysis of the lung fine structure and its alterations during endotoxin-induced lung injury. MATERIALS AND METHODS: Intravital perfusion-fixed rat lungs with (n = 5) and without (n = 5) endotoxin perfusion were scanned with micro-CT. Three imaging modalities (conventional histology, intravital microscopy, and electron microscopy) were used to document the effect of endotoxin and the in vivo application of contrast agent (a mixture of barium sulfate, gelatin, and thymol). The effect of endotoxin on structural changes of the lung was evaluated with analysis of variance. RESULTS: Intravital microscopy, conventional histology, and electron microscopy demonstrated capillary perfusion of contrast agent, inflated alveoli, and no extravasation of barium sulfate in the extravascular space. Systemic application of endotoxin led to a significant increase in the soft-tissue volume of the lungs (ie, tissue edema) (58.09 microm(3)+/- 4.6 [standard error of the mean] vs 8.31 microm(3)+/- 1.63, P <.001) and significant thickening of the alveolar walls (34.01 microm +/- 4.5 vs 14.83 microm +/- 2.5, P <.001) at micro-CT. Simultaneously, endotoxin-treated rat lungs showed a significant reduction in total air space (49.74 microm(3)+/- 1.72 vs 100.99 microm(3)+/- 1.16, P <.001). CONCLUSION: These findings indicate that micro-CT is feasible for structural evaluation of the lung fine structure and its alterations during endotoxin-induced lung injury.

Atherosclerotic lesions at micro CT: feasibility for analysis of coronary artery wall in autopsy specimens.

PURPOSE: To evaluate the feasibility of micro computed tomography (CT) for analysis of the coronary artery wall. MATERIALS AND METHODS: With micro CT, two-dimensional transverse images were generated from 10 human autopsy specimens of coronary arteries (2.5-3.5 cm long), with section thickness of 6 microm. Vessel wall perimeter, plaque area, calcified lesion area, media area, and lumen area were determined by three experienced radiologists. Results were compared with those obtained from a detailed conventional histomorphometric analysis of corresponding cross sections. Hotelling T(2) test (a multivariate generalization of the univariate Student t test) and Pearson correlation coefficient were used to assess the correlation between micro CT findings and conventional histologic measurements. The significance of differences in gray-scale measurements was tested with analysis of variance. RESULTS: Micro CT provided quantitative information about plaque morphology equivalent to that provided with histomorphometric analysis. Hotelling T(2) test revealed significantly smaller values for vessel wall perimeter and lumen area with histologic sections (P <.001). Gray-scale measurements were established with which lesions could be categorized after histologic classification. CONCLUSION: Micro CT is feasible for analysis of the coronary artery wall.