A Method to Expose the Inner Wall of Schlemm’s Canal in Mice

Abstract

Purpose : The outflow region in mice exhibits remarkable morphological homology to that in humans, including structural similarities in Schlemm’s canal endothelia (SCE) pores and giant vacuoles (GV). Due to size constraints, SCE study is hampered by lack of a reliable method to expose the tissue. We describe a protocol to dissect the inner wall (IW) in mice for scanning electron microscopy (SEM) analysis.

Methods : A pair of mouse whole globes were fixed in 4% paraformaldehyde, hemisected, the lens was removed and the anterior pole was cut into eight wedges. Each sample, with inner surface upward, was firmly affixed with superglue to a small piece of porcine sclera for support. The outflow region was dissected and SC opened by cutting along its anterior margin towards the posterior, until the tissue detached. These presumptive IW samples were mounted on a second piece of porcine sclera and processed for SEM, examined and assessed for morphological quality, and the quantity of IW area exposed (Fig 1).

Results : We were able expose IW in 11 of 16 (69%) samples dissected. Each wedge had at least 0.0025 mm² of continuous, undamaged SCE visible, amounting to 0.014 mm² per eye, representing 0.59% of the total IW area. By comparison studies in human eyes have sampled 1.8% of the total IW area (Johnson et al., IOVS 2002). The SCE of mice possessed normal appearing GV and pores, similar to those observed in human eyes (Fig 2).

Conclusions : The ability to expose a significant fraction of mouse IW is an important tool for studying aqueous humor dynamics in the mouse. The procedure can be accomplished with high quality, readily available microdissection instruments and needs no specialized equipment. However, the technique requires excellent hand-eye coordination, microdissection skills and extensive practice to achieve the level of success reported here.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Figure 1: Protocol: (A) Anterior half of eye cut into 8 wedges (B) wedge turned on edge, depicting blade plane of section (C) wedge affixed to porcine scleral support, opening up SC with razor blade chip (D) wedge inverted after dissection, affixed to scleral support, showing exposed IW region (E) wedges, dissected and prepared for SEM (F) Fig 2 sample

 

Figure 2: SEM of the inner wall of SC: (A) Human and (B) mouse. Note similar features including elongated SCE cells, I pores (arrowhead), B pores (arrow) and giant vacuoles, some of which are collapsed (_*).