A novel flow cytometry based assay for quantification of corneal angiogenesis in the mouse model of herpetic stromal keratitis.
In this study a novel flow cytometry based quantitative assay for measuring corneal angiogenesis is demonstrated. Corneas of Balb/c mice were lightly scarified and infected with 5 x 10(5), 5 x 10(4) and 5 x 10(3) pfu Herpes simplex virus 1 (HSV-1) RE virus. Development of corneal angiogenesis was studied on day 15 post infection by direct visualisation of infected cornea with a slit-lamp biomicroscope. Endothelial cells constituting the newly developed blood vessels in cornea were stained with murine anti CD31 antibody on frozen corneal sections and corneal whole mounts at day 15 post infection. Total number of endothelial cells was quantified at day 15 post infection by flow cytometry. Mice infected with different doses of HSV-1 RE developed severe to mild corneal angiogenesis at day 15 post infection. Endothelial cells constituting the newly formed blood vessels expressed CD31 at day 15 post infection. Flow cytometry revealed that, the number of CD31 positive cells isolated from diseased corneas increased with the increase in Neovascularisation index. The flow cytometry analysis used in this present study is a useful, accurate and cost effective method for quantifying corneal angiogenesis.
Biswas
Partha S
PS
Department of Comparative and Experimental Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.
Banerjee
Kaustuv
K
Kim
Bumseok
B
Smith
Jordan
J
Rouse
Barry T
BT
D000818:Animals; D016510:Corneal Neovascularization / Q000150:complications / Q000473:pathology*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000473:pathology; D004730:Endothelium, Vascular / Q000473:pathology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D016849:Keratitis, Herpetic / Q000150:complications / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal / Q000379:methods; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D018139:Simplexvirus / Q000302:isolation & purification; D017154:Stromal Cells / Q000473:pathology; D019562:Viral Load
D019408:Platelet Endothelial Cell Adhesion Molecule-1