Establishment of an experimental glaucoma animal model: A comparison of microbead injection with or without hydroxypropyl methylcellulose

Abstract

The present study aimed to compare microbead injection with and without hydroxypropyl methylcellulose (HPM) in order to establish an experimental animal model of glaucoma. This model was established in C57BL/6 mice and transgenic mice expressing cyan fluorescent protein (CFP) under the control of the Thy1 promoter in retinal ganglion cells (RGCs). C57BL/6 mice aged between 12 and 20 weeks old were randomly separated into three groups, which received different injections into the anterior chamber of the eye. Group A (microbead) received 2 µl microbeads (10×106 beads/ml) and 1 µl air. Group B (microbeads + HPM) received 2 µl microbeads and 1 µl HPM. Group C (control group) received 2 µl PBS and 1 µl air. The intraocular pressure (IOP) was measured with a tonometer under topical anesthesia daily for 1 month. A single injection of microbeads, with or without HPM, induced consistent IOP elevation when compared with the control group. Thy1-CFP mice received an injection of 2 µl microbeads and 1 µl HPM into the anterior chamber of the eyes, and the number of CFP+ RGCs was subsequently assessed in vivo by confocal scanning laser microscopy in the same area of the retina weekly for 6 weeks. The results from in vivo imaging of Thy1-CFP mice were comparable with the immunohistochemical staining results from the C57BL/6 mice. The combined injection of microbeads and HPM induced longer and higher peaks of IOP elevation when compared with the microbeads alone. The rate of RGC loss following the administration of microbeads alone was 25.0±1.3% 6 weeks after the initial IOP elevation, while it was 33.2±1.9% following the administration of microbeads + HPM. These results indicate that the injection of microbeads + HPM is a more effective method of establishing a mouse model with chronic elevation of IOP. In addition, the in vivo imaging that can be used with this technique provides an effective and noninvasive approach for monitoring the progress of RGC loss.

Keywords: animal model; glaucoma; hydroxypropyl methylcellulose; intraocular pressure; microbead; retinal ganglion cell.

Figure 1.

Distribution of microbeads in the anterior chamber of the eye following injection. (A) Microbead injection group. (B) Microbeads + HPM injection group. More microbeads entered the Schlemm's canal in the microbeads + HPM injection group. (C) Accumulation of microbeads in the anterior chamber angle and Schlemm's canal (arrows) in the microbeads + HPM group. Magnification, ×200; scale bar=50 µm. HPM, hydroxypropyl methylcellulose.

Figure 2.

Assessment of IOP elevation following the injection of microbeads or microbeads + HPM. Microbeads and microbeads + HPM injection induced IOP elevation, but the microbeads + HPM injections induced a longer and higher peak of IOP elevation compared with microbeads alone. Compared with the control group, an elevation in IOP was induced within 2 days after injection in the microbeads and microbeads + HPM groups. Data are presented as mean ± standard deviation. *P<0.05 vs. microbeads group. ^#P<0.05 vs. microbeads + HPM group. Magnification, ×200. IOP, intraocular pressure; HPM, hydroxypropyl methylcellulose.

Figure 3.

Quantification of RGC loss in mice following the injection of microbeads or microbeads + HPM. Distribution of Brn3b⁺ RGCs in the ganglion cell layer of the retina in the (A) control group 6 weeks after injection, (B) microbeads group 3 weeks after injection, (C) microbeads group 6 weeks after injection, (D) control group 6 weeks after injection, (E) microbeads + HPM group 3 weeks after injection. and (F) microbeads + HPM group 6 weeks after injection. (G) Quantification of RGC loss 3 and 6 weeks after injection. Scale bar=100 µm. RGC death was significantly higher in the microbeads + HPM at 3 weeks and 6 weeks after injection compared with the microbeads group. **P<0.05 vs. microbeads + HPM group. Magnification, ×200. RGC, retinal ganglion cells; HPM, hydroxypropyl methylcellulose.

Figure 4.

In vivo imaging of RGC loss in Thy1-CFP mice following the injection of microbeads + HPM. In vivo imaging of CFP (A) prior to microbeads + HPM injection, (B) 3 weeks after injection and (C) 6 weeks after injection. Scale bar=100 µm. The number of CFP⁺ RGCs in the same area of the retina was assessed in vivo and a progressive decrease in the number of fluorescent points was detected using serial blue-light confocal scanning laser ophthalmoscopy images following IOP elevation. Magnification, ×200. RGC, retinal ganglion cells; CFP, cyan fluorescent protein; HPM, hydroxypropyl methylcellulose; IOP, intraocular pressure.

Figure 5.

Percentage of RGC loss in Thy1-CFP mice measured by in vivo imaging is similar compared with that observed in C57BL/6 mice with in vitro immunohistochemistry following the injection of microbeads + hydroxypropyl methylcellulose. RGC, retinal ganglion cells; CFP, cyan fluorescent protein.