2.1. Animal and Tissue Sample Collection

All canines (mixed breed canines) in this experiment were of medium size and they died of non-ophthalmic diseases, with ages ranging from 8 months old to 3 years old. Among them, 9 canines were less than 1 year old, 8 canines were between 1–2 years old, and 6 canines were between 2–3 years old. The death causes of canines were mainly caused by congenital diseases and trauma. The eyeballs were collected within 1 h after the canine died. Afterwards, they were placed in fresh 4% formaldehyde solution for later use; 39 corneas from 23 canines were successfully stained, including 12 male canines and 11 female canines (21 corneas from the left eyeballs and 18 corneas from the right eyeballs).

Eyeballs were collected within 1 h after the death of canines, and were marked in the direction of 12 o’clock before completely removing them. After taking out these eyeballs, the blood was flushed with normal saline first. Subsequently, these eyeballs were dried with the gauze and placed into a jar containing formalin solution (4% formaldehyde solution). Then, the cornea was removed from the fixed eyeball along the corneoscleral limbus, and a short incision was made with ophthalmic scissors in the direction of 12 o’clock for marking. The corneal endodermis and Descemet’s membrane were torn with toothed forceps. Long incisions were made in the direction of 2 o’clock, 4 o’clock, 6 o’clock, 8 o’clock, and 10 o’clock, respectively, so that the cornea was divided into 6 quadrants. In view of the symmetry of both eyeballs, 6 corneal zones of the right eyeball of canine were marked as Ra, Rb, Rc, Rd, Re, and Rf, respectively, in a clockwise direction, while 6 corneal zones of the left eyeball were marked as La, Lb, Lc, Ld, Le, and Lf, in a counterclockwise direction (Figure 1A). Five researchers were invited to independently count the number of nerve roots entering the cornea from the corneal limbus, and finally conduct statistics and analysis.

Before corneal staining, (A) the positions of the six zones (Ra–Rf, La–Lf) made on the isolated canine cornea. The black arrow indicates the corneoscleral limbus. Photoshop software was used to stitch (B) the cornea staining maps in each zone and draw (C) the nerve direction.