Figure 1.
IOP range of normal C57BL/6 mouse population.
IOP was measured on 281 healthy adult C57BL/6 mice under anesthesia. IOP of right (OD) and left eyes (OS) were recorded and analyzed separately. X-axis represents the IOP and y-axis represents the frequency distribution at different IOP levels.
Table 1.
IOP distribution of normal C57BL/6 mice.
Figure 2.
IOP elevation after laser treatment.
IOP of laser treated and fellow untreated eyes was shown at different time points after laser. Values are mean ±SEM. Dashed line represents the 99.5th percentile of normal IOP range as shown in Table 1. The difference between two groups at each time points is statistically significant (p<0.05, paired t-test).
Table 2.
IOP changes at different time points after laser treatment.
Figure 3.
Mouse anterior chamber angle after laser treatment.
A: Mouse eye at 4 weeks after laser treatment showing laser burn locations. B: Plastic section of a normal control eye with anterior chamber angle open (arrowhead). C: Plastic section of a laser-treated eye at 4 weeks with anterior chamber angle open (arrowhead). D–F: Representative images of SD-OCT scans. D: Normal control (IOP = 14 mmHg). E: Laser treated eye at 20 weeks (IOP = 30 mmHg) with anterior chamber angle open. F: Laser treated eye at 20 weeks with partial synechia (pointed by arrows, IOP = 28 mmHg). SD-OCT scans of each eye show the angles with rotations of the eye at 0, 45, 90 and 135 degrees. AC, anterior chamber. TM, trabecular meshwork. SC, Schlemm's canal. IOP, intraocular pressure.
Figure 4.
Photopic negative response (PhNR) of normal and laser-treated mouse eyes.
A: a wave, b wave and PhNR of untreated left control eye (OS) and laser-treated right eye (OD) of step 1 with the stimulus at 1 cd.s/m2. B: Depicts the waves of step 2 with the stimulus at 5 cd.s/m2. C: Represents the waves of step 3 with the stimulus at 7 cd.s/m2. D: Shows the statistical analysis of the amplitude of PhNR at step 2 with the stimulus at 5 cd.s/m2 compared to the laser-treated and untreated control eyes (p<0.0001, paired t-test).
Figure 5.
RGC and optic nerve axonal damage following IOP elevation.
A and B represent images of plastic-embedded retinal sagittal sections peripheral to the optic nerve with hemotoxylin-eosin staining. A: Laser-treated. B: Normal control. C: Statistical analysis of the RGC number counts (p<0.001, paired t-test). D and E show toluidine blue staining of semithin transverse sections of optic nerves. D: Laser treated eye with swollen axons (arrowheads) and shrunken axons (arrows). E: Normal control with more homogeneous axons. D1–E1 are magnified views of the regions encased in black boxes in D–E. F Represents the statistical analysis of the axons (p<0.0001, paired t-test). RNFL, retinal nerve fiber layer; RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.
Figure 6.
SPARC immunolocalization in the TM.
A1–E1: DAPI stains nuclei as blue in the cryosections. A2–E2: SPARC stains the TM and peripheral cornea and sclera green. A3–E3: Bright field images for tissue orientation. A–E: Merged fluorescent images and bright field provide detailed structural orientation. Asterisk (*) shows the location of Schlemm's canal (SC). Arrow points the TM. Anterior chamber (AC) and iris are indicated as well. A: Untreated control. B: 24 hrs after laser treatment. C: 1 week after laser. D: 2 weeks after laser. E: 12 weeks after laser. Bars, 50 µm.
Figure 7.
CD45 and F-actin immunolocalization in the TM wholemounts.
A1–E1: DAPI stains nuclei as blue in the wholemounts. A2–E2: Inflammatory cells positive to CD45 (red) appear at 24 hrs after laser (B2) and decrease thereafter. A3–E3: F-actin (Green) stains the TM cells clearly shown the location of the TM region. A–E: Merged fluorescent images provide detailed structural orientation. The TM and cornea are indicated. A: Untreated control. B: 24 hrs after laser treatment. C: 1 week after laser. D: 2 weeks after laser. E: 12 weeks after laser. Bars, 50 µm.
Figure 8.
TM ultrastructure changes after laser.
The region encased in the black box in the upper row is magnified in the lower row. A and A1: TEM of normal mouse TM revealed aligned collagen beams with three layers of normal TM cells. A giant vacuole (V) was seen. The JCT appeared loose and disorganized. B and B1: 24 hrs after laser, the TM tissue had only 1–2 layers of cells with disorganized beams. C and C1: 12 weeks after laser, the TM had disorganized beams and the collagen fibrils were more compacted compared to the normal structure and the JCT was more compacted with more and disorganized extracellular matrix. Abbreviations: SC, Schlemm's canal; JCT, Juctacanalicular connective tissue; V, giant vacuole.
Figure 9.
TUNEL staining on cryosections.
A1–E1: DAPI stains nuclei as blue. A2–E2: TUNEL assay stains apoptotic cells as red peak at 24 hrs after laser (B2). A3–E3: Bright field for tissue orientation. A–E: Merged fluorescent images and bright field provide detailed structural orientation. Asterisk (*) represents the location of Schlemm's canal (SC). Arrow points the TM. Anterior chamber (AC) and iris are indicated as well. A: Untreated control. B: 24 hrs after laser treatment. C: 1 week after laser. D: 2 weeks after laser. E: 12 weeks after laser. Bars, 50 µm.