Figure 1.
Wafer implantation into the rat anterior chamber.
A. A full thickness corneal incision was made parallel to the plane of the iris (arrows). Wafers were introduced to the anterior chamber using toothless forceps (dotted circle), and displaced to its temporal position (dotted arrow) in the anterior chamber. B. Side-view schematic showing the approximate location of the implant. C. The final position of all implants was an average of 0.8 mm from the limbus (dotted line). Ch = cholesterol, 7KCh = 7-ketocholesterol, n = number of wafers implants.
Figure 2.
Angiogenesis in Ch versus 7KCh implants.
Ch (20% w/w) and 7KCh (10% w/w) implants were placed in the anterior chamber of the rat eye as described in Fig. 1. Both implants were imaged 7 days post implantation with and without fluorescein angiography. Arrows mark the locations of the iris, limbus and implants.
Figure 3.
Effects of time and 7KCh concentrations on angiogenesis.
Wafers containing 5, 7, 10 and 15% 7KCh (w/w) were implanted into rats anterior chambers as described in Fig. 1 and representative images are shown at 7, 10, 14 and 21 days. The vessel volume for each panel is shown in Fig. 4b, in mm2.
Figure 4.
Technique used to quantify neovessel formation.
Vessels were imaged using a fluorescent dissecting microscope immediately after IP injection of fluorescein as described in Methods. A. Neovessel area was quantified using Nikon NIS elements hands free tool by delineating the base of neovessels (blue line), the border of the neovessels (red line) and subtracting areas with no vessels (green line). B. Graph showing neovessel area from the experiments described in Fig. 3 in mm2.
Figure 5.
Localization of corneal neovessels with isolectin IB4
. Cryosections of temporal corneas were prepared 14 days after implantation with a 20% 7KCh wafer. The sections were labeled with AlexaFluor 568 Isolectin IB4 (red) and the nuclei stained with DAPI (blue). A. DAPI stained section demonstrating the location of the corneal stroma and epithelium. B. Isolectin IB4 labeling of neovessels, corneal epithelium and iris. C. Combined image. Arrows mark the different structures.
Figure 6.
Histological comparison of Ch and 7KCh implants using isolectin IB4.
Rat eyes were implanted with 20% Ch and 7KCh wafers and cryosections prepared 14 days post implantation. A. Ch implant labeled with isolectin IB4. B. Ch implant dual labeled with DAPI. C. 7KCh implant labeled with isolectin IB4. D. 7KCh implant dual labeled with DAPI.
Figure 7.
Localization of CD68 positive cells and VEGF in Ch and 7KCh implants.
Rat eyes were implanted with 20% Ch and 7KCh wafers and cryosections prepared 14 days post implantation. The sections were labeled with AlexaFluor 488 (green) for anti-CD68 or anti-VEGF and the nuclei stained with DAPI (blue). A. Ch implant, anti-CD68. B. 7KCh implant, anti-CD68. C. Ch implant, anti-VEGF. D. 7KCh implant, anti-VEGF.
Figure 8.
SDS-PAGE and immunoblotting of AH from Ch and 7KCh implants.
AH (5 µl) from control (un-implanted eye), Ch and 7KCh implanted eyes were separated by SDS-PAGE and blotted. A. Ponceau S red stained blot demonstrating the difference in protein content between samples. B. Anti-VEGF labeling of blot.
Table 1.
Protein concentration of aqueous humor.
Figure 9.
Quantification of VEGF, IL-1β and GRO/KC in AH 4 and 7 days after implantation.
AH from eyes of un-implanted (control) and implanted with 20% Ch and 7KCh wafers were analyzed for cytokine levels 4 and 7 days post implantation, as described above. A. VEGF. B. IL-1β. C. GRO/KC.