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Angiogenesis is a dynamic process that requires an interaction of pro-and antiangiogenic factors. It is known that the cytokine leptin stimulates endothelial cell growth and angiogenesis, but further quantitative analysis is necessary to understand leptin angiogenic effects. The quail chorioallantoic membrane ͑CAM͒ assay has been used to study angiogenesis in vivo by focusing on morphometric parameters that quantify vascular complexity and density. We quantify the angiogenic activity of leptin using the CAM assay by digital morphometry and a computer-assisted image analysis to evaluate more precisely vessel length, diameter, branching, and tortuousity. CAM images are obtained from ex ovo cultures of E8-E9 quail embryos. MATLAB® and custom software are used for our analysis. The effects of leptin, vascular endothelial growth factor-165 ͑VEGF 165 ͒, and their corresponding neutralizing antibodies are compared. Our results show that CAM treated with leptin and VEGF 165 has a significant increase in vascular complexity and density. A corresponding decrease is observed using neutralizing antibodies. Notably, leptin induced more significant changes than VEGF in vessel length and tortuousity. Conversely, VEGF induced a greater increase in vessel branching than leptin. These results underscore the importance of using multiparametric quantitative methods to assess several aspects of angiogenesis and enable us to understand the proangiogenic effects of leptin.

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Biological specimens are three-dimensional structures. However, when capturing their images through a microscope, there is only one plane in the field of view that is in focus, and out-of-focus portions of the specimen affect image quality in the in-focus plane. It is well-established that the microscope's point spread function (PSF) can be used for blur quantitation, for the restoration of real images. However, this is an ill-posed problem, with no unique solution and with high computational complexity. In this work, instead of estimating and using the PSF, we studied focus quantitation in multi-spectral image sets. A gradient map we designed was used to evaluate the sharpness degree of each pixel, in order to identify blurred areas not to be considered. Experiments with realistic multi-spectral Pap smear images showed that measurement of their sharp gradients can provide depth information roughly comparable to human perception (through a microscope), while avoiding PSF estimation. Spectrum and morphometrics-based statistical analysis for abnormal cell detection can then be implemented in an image database where the axial structure has been refined.

Angiogenesis is a dynamic process that requires interaction of proangiogenic and antiangiogenic factors. Several methodologies have been used to evaluate this process. The quail chorioallantoic membrane (CAM) is a suitable model to explore angiogenesis in vivo. Recently, the morphometric parameters of fractal dimension (Df) and grid intersection (ρ) are used to quantify angiogenesis in CAM. However, more information is necessary to understand angiogenic mechanisms. New methods to measure additional parameters such as the average blood vessel thickness, tortuousity, total branch length and complexity (junction node number), are described herein. For the analysis, CAM images were obtained from ex ovo culture of E8‐E9 quail embryos. MatLab®(The MathWorks, Natick, MA) software was used for the analyses of branches nodes, thickness and length of the forming branches. Conventional morphometry was done using IPLab (Scanalytics, Fairfax, VA). To validate the new parameters, leptin, VEGF and neutralizing antibodies were evaluated. Leptin and VEGF treated CAM had increased complexity and vascular densities (Df = 1.5 ± 0.005; ρ = 12.3 ± 0.689) and (Df = 1.6 ± 0.006; ρ = 14.3 ± 0.465), respectively, compared to PBS control (Df = 1.3 ± 0.035; ρ = 8.6 ± 0.439). There was a corresponding decrease antileptin and anti‐VEGF antibodies. The node‐structural map applied to CAM images reveled 53% increase in the number of one‐branch nodes and 50% increase in three‐branch nodes with leptin compared to controls. Additionally, the total branch length was 30% greater with leptin along with a decrease in vessel thickness, The analyses allowed quantification of branching, tortuousity, thickness, and length of the new blood vessels. Together with the conventional morphometric parameters, these new analyses will help understand the mechanisms exerted by angiogenesis‐modulating molecule.

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