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 mm2) 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 mm2) 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

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

Biochemical markers reflect the bone metabolism.

<p>(A–D) Opn, BGP, RANKL, and PTH concentration in serum was higher in the 3 M OVX+Diet than the 3 M Sham. (D -F) lower concentrations of TRAP5b and leptin were seen in the OVX+Diet at 3 M compared to the Sham group. (* = (p≤0.05), Mann Whitney U with bonferroni correction, n = 8 per group).</p

Qualitative analysis of trabecular bone shows inferior bone quality resulting from multi-deficiencies diet combined with bilateral ovariectomy in rats after 3

<p> <b>M.</b> (A) BV/TV showed how bone tissue is affected within the total volume; these results suggest that at 3 M the treatment results in less mineralized tissue in OVX+Diet when compared to the 3 M Sham. (B) Structure Model Index (SMI) indicated the trabecular shape change at 3 M in the OVX+Diet group. (C) Tb. N was lower in the 3 M OVX+Diet group compared to the Sham group. (D) Higher trabecular separation in the OVX+Diet group when compared to the Sham after 3 M of treatment. (* = p≤0.05, one-way ANOVA with bonferroni correction, 0 M, n = 8; 1 M, n = 3 per group; 3 M n = 8 per group).</p

µCT analysis of cortical bone parameters show affected porosity and thickness in the OVX+Diet treatment after 3 months.

<p>(A) Porosity measurement showed that both groups; the 0 M control and the 3 M Sham have significantly lower porosity than the 3 MOVX+Diet. (B) Cortical thickness was significantly higher in the 3 M Sham group when compared to either 3 M OVX+Diet or 0 M control group. (* = p≤0.05, Mann Whitney U with bonferroni correction, n = 8 per group).</p

A chart depicting the work plan and experimental design.

<p>Female Sprague-Dawley rats were utilized for this experiment, 10 of which were sacrificed, analysis on these animals were carried out to obtain an initial bone status. Eleven animals were then ovariectomized (OVX), and another eleven rats were Sham operated (Sham) and animals were left to recover for two weeks. After recovery OVX animals were given a multi-deficiencies-diet where Sham animals received a standard diet. One month (1 M) and three months (3 M) after dietary treatment begun, 3 and 8 animals were sacrificed, respectively. Rats were scanned by DEXA at every time point and DEXA results were reported elsewhere. Besides DEXA, at 1 M only µCT analysis was performed on the left tibia. At both 0 M and 3 M Left tibia was analyzed in µCT before performing undecalcified histology; right femur and right tibia were tested biomechanically, and right femur was used for decalcified histology.</p

Biomechanical testing of tibia and femur of the ovariectomized rat after three months of multi-deficient diet treatment.

<p>(A) Ultimate torque at failure shows no significance between the groups at 3 M. (B) Tibia of treated rats showed lower torsional stiffness compared with the Sham operated rats. (C) Breaking load of the femur shows an increased needed load to break the femur of treated animals at 3 M. (D) Bending stiffness of the femur showed no significant difference between the treatment group and the Sham group. (* = p≤0.05, Mann Whitney U with bonferroni correction, n = 8 per group).</p