AIM: To test the expression of erythropoietin (Epo) and its receptor EpoR in normal and neovascularized murine corneas induced by alkali burns, and to investigate whether Epo/EpoR is involved in the process of corneal angiogenesis.METHODS: The expression of Epo/EpoR was tested in normal and neovascularized murine corneas induced by alkali burns through immunohistochemistry of corneal frozen sections. Epo cloning, expression, and purification were carried out. Then Epo protein (6μL, 1μg) and control (6μ L of vector control or saline) were injected into the corneal stroma respectively, and the corneas were checked at the 14th day after injection to see whether corneal neovascuarization occurred.RESULTS: Epo/EpoR was expressed in epithelial cells, endothelial cells and stromal cells in normal and neovascularized corneas induced by alkaline burns, and also expressed strongly in neovascularized cornea. They were expressed at the same time in stromal inflammatory cells and new vessels. Corneal neovascularization was induced by Epo intrastromal injection in 5 out of 6 eyes ,but no new vessels were observed in all controls (n = 6) at day 14 after vector control or saline intrastromal injection in normal corneas.CONCLUSION: This paper first reported the expression of Epo and its receptor in normal and neovascularized cornea. Injection of Epo into the corneal stroma may induce the corneal neovascularization. Epo/EpoR is associated with the formation of corneal neovascularization.

BACKGROUNDIn vivo quantification of choroidal neovascularization (CNV) based on noninvasive optical coherence tomography (OCT) examination and in vitro choroidal flatmount immunohistochemistry stained of CNV currently were used to evaluate the process and severity of age-related macular degeneration (AMD) both in human and animal studies. This study aimed to investigate the correlation between these two methods in murine CNV models induced by subretinal injection.

METHODSCNV was developed in 20 C57BL6/j mice by subretinal injection of adeno-associated viral delivery of a short hairpin RNA targeting sFLT-1 (AAV.shRNA.sFLT-1), as reported previously. After 4 weeks, CNV was imaged by OCT and fluorescence angiography. The scaling factors for each dimension, x, y, and z (μm/pixel) were recorded, and the corneal curvature standard was adjusted from human (7.7) to mice (1.4). The volume of each OCT image stack was calculated and then normalized by multiplying the number of voxels by the scaling factors for each dimension in Seg3D software (University of Utah Scientific Computing and Imaging Institute, available at http://www.sci.utah.edu/cibc-software/seg3d.html). Eighteen mice were prepared for choroidal flatmounts and stained by CD31. The CNV volumes were calculated using scanning laser confocal microscopy after immunohistochemistry staining. Two mice were stained by Hematoxylin and Eosin for observing the CNV morphology.

RESULTSThe CNV volume calculated using OCT was, on average, 2.6 times larger than the volume calculated using the laser confocal microscopy. The correlation statistical analysis showed OCT measuring of CNV correlated significantly with the in vitro method (R 2 =0.448, P = 0.001, n = 18). The correlation coefficient for CNV quantification using OCT and confocal microscopy was 0.693 (n = 18, P = 0.001).

CONCLUSIONSThere is a fair linear correlation on CNV volumes between in vivo and in vitro methods in CNV models induced by subretinal injection. The result might provide a useful evaluation of CNV both for the studies using CNV models induced by subretinal injection and human AMD studies.

Animals ; Choroidal Neovascularization ; pathology ; physiopathology ; Disease Models, Animal ; Fluorescein Angiography ; Humans ; Mice ; Mice, Inbred C57BL ; Tomography, Optical Coherence