Basement membrane regeneration and TGF-β1 expression in rabbits with corneal perforating injury

Purpose: To evaluate the relationship between basement membrane (BM) regeneration and the spatiotemporal expression of TGF-beta 1 during wound healing in rabbits with corneal perforating injury. Methods: Forty-two rabbits were randomly allocated into 7 experimental groups, with 6 rabbits per group at each time point. The central cornea of the left eye was injured with 2.0 mm trephine to establish the perforating injury model. Six rabbits that received no treatment were used as controls. The cornea was evaluated at 3 days, 1-3 weeks, and 1-3 months after injury with a slit lamp for haze levels. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed to quantify the relative expression of TGF-beta 1 and alpha- SMA mRNA. Immunofluorescence (IF) was used to assess TGF-beta 1 and alpha-smooth actin (alpha- SMA) expression and localization. BM regeneration was assessed using transmission electron microscopy (TEM). Results: After injury, dense haze appeared at 1 month and then gradually faded. The relative expression of TGF-beta 1 mRNA peaked at 1 week and then decreased until 2 months. The relative alpha-SMA mRNA expression reached its peak at 1 week, then reached a small peak again at 1 month. IF results showed that TGF-beta 1 was initially detected in the fibrin clot at 3 days and then in the entire repairing stroma at 1 week. TGF-beta 1 localization gradually diminished from the anterior region to the posterior region at 2 weeks to 1 month, and it was nearly absent at 2 months. The myofibroblast marker alpha-SMA was observed in the entire healing stroma at 2 weeks. Localization of alpha-SMA gradually disappeared from the anterior region at 3 weeks to 1 month, remaining only in the posterior region at 2 months and disappearing at 3 months. Defective epithelial basement membrane (EBM) was first detected at 3 weeks after injury, then gradually repaired, and was nearly regenerated at 3 months. A thin and uneven Descemet's membrane (DM) was initially detected at 2 months after injury, then gradually regenerated to some extent, but remained abnormal at 3 months. Conclusions: In the rabbit corneal perforating injury model, EBM regeneration was observed earlier than DM. At 3 months, complete EBM regeneration was observed, while the regenerated DM was still defective. TGF-beta 1 was distributed throughout the entire wound area in the early stages and then decreased from the anterior to the posterior region. alpha-SMA exhibited a similar temporospatial expression to TGF-beta 1. EBM regeneration may play a key role in low expression of TGF-beta 1 and alpha-SMA in the anterior stroma. Meanwhile, incomplete DM regeneration may contribute to the sustained expression of TGF-beta 1 and a-SMA in the posterior stroma.