Fig 1.
Experimental Flow Chart of the Image-Guided Laser-Induced CNV Model and Data Collection.
(A) Overview of the procedure for CNV induction involving mouse preparation and followed by experimental treatment, sample preparation and analysis. (B) Representative image of normal fundus (Green check mark). (C) Representative image of anomalous structure (white arrow) in the eye, which is not suitable for laser photocoagulation (Red X). (D) Representative image of normal fundus with 4 laser burns shown as bright white spots. (E) Representative image of a successful laser burn (white arrow) with 3D OCT. (F&G) Representative ocular FFA images at 5 and 10 minutes after the injection of fluorescent dye at day 6 after laser burn. (H) Representative images of flat-mounted choroid with IB4 staining at day 7 after laser photocoagulation. Scale bar: 200 μm. ON, optic nerve. (I) Higher magnification of the laser-induced CNV lesion highlighted in panel H. Scale bar: 50 μm.
Fig 2.
Focus Adjustment Using Micron IV.
(A) Prior to lens contact with the eye, the ON was positioned in the center of the vision by moving the mouse support (a) and adjusting the height through knob b. (B) After lens contact with cornea, the ON was re-positioned in the center of the vision by fine adjustment through knob c and mouse platform (d). (C) Demonstration of representative incorrect alignment showed retinal ON fibers unevenly in the vision with the bottom half (solid arrow) much clearer than the upper half (hollow arrow), indicating the camera axis was not aligned with the eye axis. θ, the intersection angle between the eye and camera axises. (D) Even radial reflection of retinal ON fibers (yellow arrows) in all of the 4 quadrants indicated an ideal alignment (θ = 0) of the eye axis with the camera axis, which is critical to induce consistent and reliable laser photocoagulation.
Fig 3.
Suggested Retinal Positions of Laser Photocoagulation and Indicator of Successful Rupture of Bruch’s Membrane.
(A) Four laser burns per eye were applied at 3, 6, 9 and 12 o’clock (a) or in 4 individual quadrants (c) approximately double the disc diameter of the optic nerve away from it, which was between the 2 circles. Main retinal vessels (solid arrow in b) and choroid vessels (empty arrow in b) should be avoided to prevent severe breeding. The distance between laser burns (yellow arrows in b&d) should be at least double the optic nerve diameter. (B) A successful laser-induced rupture of Bruch’s membrane (BM) was identified by the appearance of a vaporization bubble and haze area around the lesion right after laser photocoagulation (upper panels). If the Bruch’s membrane was not ruptured, vaporization bubble or haze area would not occur (lower panels). (C) The rupture of BM (yellow arrows) induced by laser burn was confirmed by both 2D cross-sectional OCT scan and 3D reconstructed OCT image. NFL: nerve fiber layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium. (D) Cross-sectional OCT scans of the lesion showing the rupture of BM at day 0 (yellow arrow in a), a typical butterfly-like shape of retinal hyper-reflectivity at day 1 (b), choroidal fibro-vascular tissue (marked by red dot line) formation at day 7 (c) and a typical section of laser-induced CNV lesion (yellow arrow in d) stained with hematoxylin and eosin at day 7 after laser photocoagulation. Scale bar: 200 μm.
Fig 4.
Exclusion Criteria for the Laser-Induced Lesions.
(A) Laser-induced choroidal hemorrhages were graded as follows: Grade 0, the major axis of the bleeding area was smaller than the diameter of the laser-induced lesion; Grade 1, the major axis of the bleeding area was bigger than the diameter of the lesion but smaller than 2 times of the lesion diameter (LD); and Grade 2, the major axis of the bleeding area was bigger than 2 LD. Lesions with Grade 0 bleeding were included, lesions with Grade 1 bleeding were excluded and any eyes with Grade 2 bleeding were excluded. (B) Lesions with choroidal damage (yellow circle in bright field image) were excluded. Scale bar: 200 μm. (C) Fused lesions (yellow arrow) were excluded. Scale bar: 200 μm. (D) Outlier lesions (yellow arrow) with more than 5 times larger than the mean area of the lesions in the same eye were excluded. Scale bar: 200 μm.
Table 1.
Percentages of Lesion Types with Different Laser Power Levels.
BM, Bruch’s membrane.
Table 2.
Number of CNV Lesions, Mean Area CNV, SEM, SD and % Lesion Area Relative to Area at 240 mW Laser Power.
Fig 5.
The Area of Lesions Was Positively Correlated to the Power Levels of Laser.
Laser photocoagulation was induced with different levels of laser power in C57BL/6 mice using Micron IV. The area of lesions was quantified in flat-mounted choroids with IB4 staining 7 days after laser injection. n = 10 mice/group. * p < 0.05; *** p < 0.001.
Fig 6.
Gender Had Little Effect on CNV Lesion Area in Younger Mice.
Laser photocoagulation was induced in C57BL/6 mice of both genders at 6–8 or 12–16 weeks age using Micron IV. The area of lesions was quantified in flat-mounted choroids with IB4 staining 7 days after laser injection. n = 10 mice/group. n.s. not significant; *** p < 0.001.
Table 3.
Number of CNV Lesions, Mean Area CNV, SEM, SD and % Lesion Area Relative to Area of 6–8 Week Male Mice.
Fig 7.
Dietary Intake of Omega-3 Polyunsaturated Fatty Acid Reduced CNV.
C57BL/6 mice were fed with omega-6 (ω-6) or omega-3 (ω-3) polyunsaturated fatty acid from 7 days before laser photocoagulation to 7 days after laser injection. The area of lesions was quantified in flat-mounted choroids with IB4 staining 7 days after laser injection. n = 20 mice/group. *** p < 0.001.
Table 4.
Number of CNV Lesions, Mean Area CNV, SEM, SD and % Lesion Area Relative to Area of Mice on ω-6 Feed.
Table 5.
Comparison between Slit Lamp System and Micron IV Platform.
OCT, optical coherence tomography; FFA, fundus fluorescein angiography.