Fig. 40.1 Semi-quantitative analysis of choroidal density. (a) a grayscale image showing anti-CD31 antibody stained choroidal vessels of a wild-type mouse. (b) a black-white image converted from a. The white area is a representation of choroidal density and its value was 67.3%, calculated by Adobe Photoshop 8.0 software. (c–d) choroidal vessel images from identical region of an RPE-specific VEGF knockout (KO) mouse (c) and wild-type control (d). The calculated choroidal densities in the conditional VEGF KO mouse and wild-type control were 48.6 and 64.5%, respectively. The conditional VEGF KO mouse demonstrated a 24.7% loss of choroidal density, compared with that of the wild-type control (defined as 100%). The size of choroidal vessels was also reduced in the conditional VEGF KO mouse. (e) choroidal vessel image from a pigmented mouse

40.3 Results and Discussion

40.3.1 Analysis Of Choroidal Density

In this study, we utilized Photoshop 8.0 imaging software and converted fluorescent choroidal vessel images to black-white images. As demonstrated, a fluorescent image of immunostained choroidal vessels (Fig. 40.1a) can be converted to a blackwhite image (Fig. 40.1b). Since the white area in this black-white image was a representation of choroidal density, the value of the white area in Fig. 40.1b could be calculated by Adobe Photoshop and many other computer programs. The conversion of fluorescent images into black-white images is critical to the methodology and makes it possible to calculate the area of choroidal vessels, rather than fluorescent intensity, which was difficult to control in experiments. Since the result is not affected by the intensity of fluorescent images, we could use this method to compare images generated from different experiments. In our effort to investigate the function of the RPE-produced vascular endothelial growth factor (VEGF), we used this

method to confirm its role in choroidal development. Figure 40.1c–d demonstrated a 24.7% reduction of choroidal density in an RPE-specific VEGF knockout mouse, as reported previously (Marneros et al. 2005). Since the procedure simplifies a threedimensional structure to two-dimensional, the method may only be considered as semi-quantitative.

40.3.2 Usefulness of the Methodology

Choroidal vasculature provides approximately 70–80% of retinal blood circulation and thus is vital to the function and maintenance of the retina. To study the biology of choroid and outer blood retina barrier, attempts have been made to imaging choroidal vessels. Immunohistostaining of alkaline phosphatase for choroidal vessels is effective with human and primates (McLeod et al. 2002; Otsuji et al. 2002). However, the method does not work well with mice (Dr. G. Lutty, personnel communication). The reproducibility of corrosion cast (Majji et al. 2000) for mouse choroidal vessel is not easy to control, at least not in our hands. The leakage of fluorescin-conjugated large molecular weight dextran is an issue in visualization choroidal vessels with angiography. Taken together, there has not been a uniform and reliable methodology for imaging mouse choroidal vessels. The experimental procedure described in this study allows us to visualize choroidal density within one day, which is relatively efficient. Although images obtained in this study is two dimensional, they gave a similar readout (Fig. 40.1) as that derived from corrosion cast (Majji et al. 2000). There are also technical challenges associated with this method. For pigmented mice, it is more difficult to obtain reproducible results and images are usually less clear (Fig. 40.1e). The choroid is organized as dense vessels in wild-type mice, the method developed in this study may not be suitable for detecting subtle changes in the choroidal vasculature, which is a weakness of all other methods described above. However, a moderate change in choroidal density, as well as the size of choroidal vessels, can be detected and quantified (Fig. 40.1c–d).

40.3.3 Summary

At present, a major challenge in preclinical studies in dry-AMD field is the difficulties to image pathological changes in choroidal vasculature. The procedure developed in this study may be a supplement to existing methods. However, given the difficulties in imaging choroid, a dense vascular network with vessels overlaying closely, a combination of methods including those discussed above and ultrastructural analysis may be required to detect the changes in choroidal vasculature. Since mouse is the only mammal that allows precise genomic manipulation, semiquantitative analyses of choroidal density in genetically altered mice is likely to provide more insights about the pathogenic mechanisms and therapeutic strategies for geographic atrophy.