significant decrease in choroidal density (Ramrattan et al. 1994). Patients with geographic atrophy demonstrate a lower choroidal blood flow (Grunwald et al. 1998). Experimental and clinical evidence suggests that abnormal interaction between the RPE and choroid is associated with the loss of choroidal density (Klein et al. 1997; McLeod et al. 2002; Sarks et al. 1988; Sarks 1976; Sunness 1999; Zarbin 2004). Abnormal choroidal development is also associated with the loss of retinal integrity in experimental mice (Marneros et al. 2005). Mouse is the only mammal that allows precise genomic manipulation. Therefore, in vivo studies with genetically altered mice are likely to provide more mechanistic insights about the pathogenic mechanisms of the disease. To establish an efficient and quantitative procedure measuring choroidal density in mice for studies related to choroidal development and degeneration, we developed a computer-assisted semi-quantitative method for choroidal density using immunostained mouse choroid. We herein describe the experimental procedure.

40.2 Methods

40.2.1 Immunohistochemial Staining of Choroidal Endothelia

Eyes from albino mice were enucleated and immersed in 4% paraformaldehyde in PBS for 2 h. The choroid/RPE layer was carefully dissected. The RPE cells were then carefully brushed off with a hair loop. The remaining choroidal vessels were washed three times with PBS, blocked with 5% serum containing 1% Triton X-100 in PBS for 1 h, and incubated with a polyclonal anti-CD31 antibody (BD Pharmingen, San Diego, CA) at 37◦C for 2 h. The anti-CD-31 antibody stained choroidal vessels were incubated with FITC-conjugated secondary antibody (Chemicon, Temecula, CA), flat-mounted, and imaged with either a fluorescent microscope or confocal microscope.

40.2.2 Analysis of Choriodal Density with Photoshop 8.0

To quantify choroidal density, fluorescent images of the immunostained choroidal layer were obtained with confocal microscope or fluorescent microscope. Adobe Photoshop 8.0 software was used to calculate choroidal density. The threshold tool (under the adjustment in image) of the software was selected to convert a fluorescent image (Fig. 40.1a) to a black-white image (Fig. 40.1b), in which white area represented choroidal vessels. The threshold was then adjusted to make white area appeared as painted choroidal vessels (Fig. 40.1b), similarly to that in color or grayscale images. The percentage of white area in the adjusted image was calculated with the histogram tool of the software and was used to represent relative choroidal density (Fig. 40.1b). Comparison of a particular region among animals was achieved based on relative choroidal density obtained from identical image window.