This is the first report of the establishment of an OH model in ferrets. There are known difficulties in elevating IOP in this animal and it was a big challenge to establish the OH model in ferrets. We tried previously established methods to increase IOP, such as episcleral vein occlusion10,29,30,31 and trabecular photocoagulation32,33,34, with no success. In addition, injection of latex beads35,36, viscoelastic materials13 and silicone oil37 was tried, but these injected materials were all extracted under the conjunctiva within a few days and IOP did not elevate. Next, we tried angle photocoagulation after intensive flattening of the anterior chamber by aspiration of aqueous fluid, similar to the method for OH mouse models6. However, in ferret eyes, the anterior chamber was immediately recovered and angle closure failed, probably due to the excessive production of aqueous fluid.

Finally, in reference to a report showing epithelial ingrowth leading to secondary glaucoma28, we tried an injection of a conjunctival cell suspension cultured in vitro and artificial angle closure was successful. Histologically, the angle was covered with proliferated cells as indicated in Figure 2c. One of the drawbacks of this method is that the fate and the mechanism of angle occlusion were not investigated in detail. To confirm the role of transplanted cells, we plan to transplant the allogeneic conjunctival fibroblasts with specific markers such as fluorescent protein.

There are only a few reports that measure IOP in ferret eyes38,39,40,41. IOP was measured by applanation tonometry (TonoPen® and TonoVet®) in conscious38,39,40 and anesthetized ferrets41. IOP varied from 14.50 ± 3.27 mmHg38 to 22.8 ± 5.5 mmHg39.

In this study, the IOP of control eyes was 17.36 ± 2.93 mmHg measured using a TonoLab® (Tiolat, Helsinki, Finland). Since IOP was determined in anesthetized ferrets, pharmacological interference of IOP may have occurred. Besides the potential influence of anesthetic procedures, several other factors could be responsible for IOP variations between studies including strain of ferrets, methods of physical restraint and version of the instrument measuring IOP40. Therefore, comparison of the present IOP values with previous values is not possible. However, the TonoLab® may be a better tool to measure IOP in ferrets because of the smaller standard deviation in the measured IOP value than those of previous reports.

In treated eyes, IOP significantly elevated to 31–71 mmHg, the eyeballs were expanded and optic disc cupping was enlarged (Figure 2d). In addition, 14/15 eyes showed over 100% IOP increase compared to untreated eyes. This rate was significantly higher than any other previously reported OH-inducing method in any animal species. Thus, this new method to induce OH may be applied to other animal species to increase IOP effectively and may achieve a higher success rate.

The IOP increase in this ferret OH model was sufficient to damage optic disc and axons in the eye. Optic nerve damage was characterized as degeneration of axons and thickening of the surrounding glial tissues in formed optic nerve bundles8. In this model, the area of optic nerve bundles and intake of fluorescein dye obviously decreased as indicated in Figure 3.

In addition to optic nerve degeneration, one advantage of the ferret OH model is that it may be suitable to investigate secondary degeneration of the optic tract accompanied with glaucomatous optic nerve degeneration in animals with binocular vision. As shown in monkey and human eyes, OH induced secondary degeneration of the LGN4,42,43,44. In this study, anterograde axonal transport was easily visualized by injection of CTB and this showed apparent damage in the optic tract of the right (OH) eye and the damage was also statistically apparent in the optic tract of the left (untreated) eye. This result is coincident with the report that unilateral OH damage induces bilateral brain damage44. In the future, we plan to histological examine the secondary degeneration of LGN, SC and visual cortex in this ferret OH model as a representative small mammal with binocular vision.

There are some disadvantages of our ferret OH model. First, injection of conjunctival cells elicited intracameral inflammation, which may affect retinal degeneration. Second, the proliferative conjunctival cells prevented direct observation of the optic nerve and the retina. Thus, continuous in vivo imaging techniques cannot be applied for this OH model. Third, it was hard to regulate IOP to establish a mild glaucoma model such as human open angle glaucoma. The IOP values were similar to angle closure glaucoma and the IOP was so high that it could lead to ischemic optic neuropathy. However, high IOP inducing ischemia may be compensated by the expansion of eye globes as observed in buphthalmos of congenital glaucoma. In general, it has been a big challenge to establish an ideal glaucoma model similar to open angle glaucoma with only a mild increase in IOP without any inflammation or invasive tissue damage. Even the most popular OH mouse model, the DBA/2J strain, has inevitable degeneration of the anterior segment, cataract and some other ocular deficiencies45,46. In the future, we need to overcome these disadvantages of OH animal models.

In conclusion, injection of a cultured conjunctival cell suspension significantly increased IOP, induced optic disc cupping and degeneration of optic nerve axons in ferret eyes. Additionally, LGN and SC areas projected from the right (OH) eye were macroscopically damaged. In future studies, we plan to analyze the damaged area in the LGN or SC and to investigate the differences in the feasibility of axon damage among the subtypes of retinal ganglion cells, which is expected in the human eye with glaucoma.