Procedure for early corneal basement membrane repair and regeneration in corneal penetrating injury in rabbits

Objective

To describe the procedure for early corneal epithelial basement membrane(EBM) repair and regeneration in rabbits with corneal penetrating injury.

Methods

Forty-two New Zealand white rabbits were divided into modeling 1-, 3-, 5-, 7-, 14-, 21-, and 30-day groups using a random number table method, with 6 rabbits in each group; the right eyes were selected as the experimental eyes.Another 6 New Zealand white rabbits without any treatment were taken as the normal control group.A 2.0-mm trephine was used to ablate a full-thickness button of the central corneal tissue of each rabbit.The corneas were observed by slit lamp biomicroscopy at the respective time points after the trephined injury.Corneal epithelial fluorescein staining was used to evaluate re-epithelialization with Image J software and haze grading was evaluated with the Fantes classification.Hematoxylin-eosin staining was used to observe the healing process of the cornea.Transmission electron microscopy was conducted to assess the regeneration of the EBM and the reconstruction of the cornea.The study protocol was approved by the Ethics Committee of Guangxi Medical University (No.201811031). The use and care of the experimental animals complied with the Statement for the Use of Animals in Ophthalmic and Vision Research.

Results

The corneal epithelial fluorescein areas in modeling 1-, 3-, 5-, 7-, and 14-day group were (4.00±0.10), (3.11±0.10), (2.00±0.06), (0.90±0.04) and (0.67±0.03)mm², respectively, with a significant difference among them (F=3 398.88, P<0.01). With the increasing of time after modeling, the corneal epithelium fluorescein area was gradually reduced, showing significant differences between any two groups (all at P<0.05), and the staining was disappeared at 21 and 30 days after modeling.The corneal haze grades were 3.44±0.53, 0.67±0.25, 1.33±0.50, 2.11±0.60, 2.44±0.53, 3.22±0.44 and 3.78±0.44 in modeling 1-, 3-, 5-, 7-, 14-, 21-, and 30-day group, respectively. The corneal opacity score gradually decreased during 1-5 days after modeling and gradually increased during 5-30 days after modeling, with a significant difference among them (F=51.182, P<0.01). Hematoxylin-eosin staining revealed that a fibrin clot formed in the wound area, and a single layer of epithelium covered the area at initial 3 days after modeling, and a large number of fibroblasts and some extracellular matrix were found at 5 days after modeling.At 21 and 30 days after modeling, the collagen fibers were tightly arranged in the anterior stroma.Transmission electron microscopy showed that the wound was filled with irregular collagen fibers and myofibroblasts.The stroma was remodeled at 21 days after modeling, and defective regeneration of the EBM was detected at 21 and 30 days after modeling.

Conclusions

Corneal fibrosis initiates after corneal penetrating injury in rabbits and gradually aggravates, the EBM regenerates defectively.