Optic neuropathy and increased retinal glial fibrillary acidic protein due to microbead-induced ocular hypertension in the rabbit

Abstract

Aim: To characterize whether a glaucoma model with chronic elevation of the intraocular pressure (IOP) was able to be induced by anterior chamber injection of microbeads in rabbits.

Methods: In order to screen the optimal dose of microbead injection, IOP was measured every 3d for 4wk using handheld applanation tonometer after a single intracameral injection of 10 µL, 25 µL, 50 µL or 100 µL microbeads (5×10⁶ beads/mL; n=6/group) in New Zealand White rabbits. To prolong IOP elevation, two intracameral injections of 50 µL microbeads or phosphate buffer saline (PBS) were made respectively at days 0 and 21 (n=24/group). The fellow eye was not treated. At 5wk after the second injection of microbeads or PBS, bright-field microscopy and transmission electron microscopy (TEM) were used to assess the changes in the retina. The expression of glial fibrillary acidic protein (GFAP) in the retina was evaluated by immunofluorescence, quantitative real-time polymerase chain reaction and Western blot at 5wk after the second injection of microbeads.

Results: Following a single intracameral injection of 10 µL, 25 µL, 50 µL or 100 µL microbead, IOP levels showed a gradual increase and a later decrease over a 4wk period after a single injection of microbead into the anterior chamber of rabbits. A peak IOP was observed at day 15 after injection. No significant difference in peak value of IOP was found between 10 µL and 25 µL groups (17.13±1.25 mm Hg vs 17.63±0.74 mm Hg; P=0.346). The peak value of IOP from 50 µL group (23.25±1.16 mm Hg) was significantly higher than 10 µL and 25 µL groups (all P<0.05). Administration of 100 µL microbead solution (23.00±0.93 mm Hg) did not lead to a significant increase in IOP compared to the 50 µL group (P=0.64). A prolonged elevated IOP duration up to 8wk was achieved by administering two injections of 50 µL microbeads (20.48±1.21 mm Hg vs 13.60±0.90 mm Hg in PBS-injected group; P<0.05). The bright-field and TEM were used to assess the changes of retinal ganglion cells (RGCs). Compared with PBS-injected group, the extended IOP elevation was associated with the degeneration of optic nerve, the reduction of RGC axons (47.16%, P<0.05) and the increased GFAP expression in the retina (4.74±1.10 vs 1.00±0.46, P<0.05).

Conclusion: Two injections of microbeads into the ocular anterior chamber of rabbits lead to a prolonged IOP elevation which results in structural abnormality as well as loss in RGCs and their axons without observable ocular structural damage or inflammatory response. We have therefore established a novel and practical model of experimental glaucoma in rabbits.

Keywords: glial fibrillary acidic protein; microbead; ocular hypertension; optic neuropathy; rabbit.

Figure 1. Distribution of microbeads in the anterior chamber following an intracameral injection

A: The representative image on scattering microbeads taken immediately after injection of 50 µL microbeads (orange). White arrowheads indicate microbeads; B: The representative image of corneal endothelium was taken at 1wk after injection of 50 µL microbeads; C, D: Accumulation and localization of FITC-labelled microbeads (a) in the anterior chamber angle at 8wk after the first injection. Scale bars: 50 µm.

Figure 2. Effect of the IOP levels after the intracameral injection of microbeads

The elevation of IOP was assessed after a single injection of microbeads with different volumes (10 µL, 25 µL, 50 µL or 100 µL) into the ocular anterior chamber of rabbit (n=6/group).

Figure 3. Assessment of IOP elevation following two injections of microbeads

The elevation of IOP was assessed after two injections of microbeads (circle) or PBS (square) into the ocular anterior chamber of rabbit (n=24/group). Arrowhead indicates the repeat injection. The IOP levels after the single injection of 50 µL microbeads (triangle).

Figure 4. Optic nerve abnormality associated with microbead-induced OHT

A: The H&E staining and the TEM images of cross-sections through the optic nerve after two intracameral injections of PBS or microbeads; B: Mean axon density; C: Cross-sectional area of optic nerve from rabbits receiving two intracameral injections of PBS or microbeads (n=6/group; ^aP<0.05 vs PBS-injected group).

Figure 5. Retinal degeneration associated with microbead-induced OHT

The cross-sections of retina obtained from the rabbits receiving two intracameral injections of PBS or microbeads with H&E staining. GCL: Ganglion cell layer; IPL: Inner plexiform layer; INL: Inner nuclear layer; OPL: Outer plexiform layer; ONL: Outer nuclear layer; OLL: Outer limiting membrane layer; RCL: Rods and cones layer. Scale bars: 20 µm.

Figure 6. Increased expression of GFAP following the repeated injection of microbeads

A: Immunofluorescence image illustrated the expression of GFAP (green) in the retina from the rabbits receiving two injections of PBS or microbeads. Sections counter stained with DAPI (blue). Scale bars: 50 µm; B: The representative blot shows Western blot analysis from retina. The protein level of GFAP evaluated by western blot (n=6/group); C: The mRNA level of GFAP evaluated by qPCR (n=6/group). ^aP<0.05.