Corneal Organ Culture Angiogenesis Model

Abstract

 

Purpose

 

Corneal angiogenesis occurs under numerous conditions including corneal injury, infection and hypoxia. These vessels can block light entering the eye and alter vision. Corneal angiogenesis is usually studied in vivo, however, use of animals is expensive and time consuming. An in vitro model that uses limbal endothelial cells and corneal components would be useful for initial screening of angiogenesis stimulators and inhibitors. In this study, an organ culture model of angiogenesis was developed using rat corneas.

 

Methods

 

Whole rat eyes were obtained from PelFreez, washed, treated with Ciprofloxin and the cornea with a 2mm scleral rim was dissected. The corneas were placed over sterile agarose coated glass beads tethered to wells of a 24 well plate. Medium alone or medium containing 100 ng/ml FGF-2 with and without the angiogenesis inhibitor maspin (1µM) and the related non-inhibitor protein ovalbumin (1µM) was added to the level of the limbal region of the cornea. This medium was changed daily and 125 µl dropped on the corneal surface. After seven days the corneas were removed from culture and radial cuts were made in the corneas. The corneas were fixed in acetone, blocked with BSA and stained with phycoerythrin-PECAM (CD31) antibodies. The corneas were flat mounted and the vessels visualized. Vessel lengths were determined using ImageJ. The data was analyzed using an ANOVA and post hoc individual comparisons (Sigma Plot). n=7-12 corneas per group.

 

Results

 

FGF-2 induced the ingrowth of vessels into the cornea relative to medium alone (Fig 1). Seven days in culture was optimal for vessel ingrowth and corneal clarity. The angiogenesis inhibitor, maspin inhibited vessel ingrowth with an average vessel length similar to that of the control. Ovalbumin, a related non-inhibitory protein, did not inhibit vessel ingrowth.

 

Conclusions

 

Corneal angiogenesis can be initially studied using the developed organ culture model. It will be useful for the screening of pro and antiangiogenic molecules.

 

 

Fig 1: Rat organ culture angiogenesis model. Air lifted rat corneas were treated with medium alone (Control, A), FGF-2 (B) and FGF-2 + Maspin (C). The tips of the vessels are outlined by the white line. D. Enlargement of FGF-2 induced vessels. E. The lengths of corneal vessels were averaged for each treatment. n= 7-12 corneas per group. * p < 0.001 relative to control, maspin, FGF-2 + maspin and ovalbumin.

 

Keywords: 480 cornea: basic science • 609 neovascularization