Induced oxidative stress in ex vivo porcine corneas downregulates mRNA expression

Abstract

Purpose : Oxidative stress plays a crucial role in ocular injuries or diseases. Hence, the effect of oxidative stress on the cornea was investigated using ex vivo porcine tissue.

Methods : Pig eyes were obtained from the local abattoir. Explanted corneas were treated for 30 min or 3 h with either 100 mM or 200 mM H₂O₂ to induce oxidative stress. Untreated corneas were used as controls. All five groups were cultured for 24 h. Afterwards, RT-qPCR was performed to analyze inflammation (IL1B, IL6, IL8), angiogenesis (VEGF, VEGFB), tight junctions (TJP1), and aldehyde dehydrogenase (ALDH3A1; n=7/group). Also, histological sections were stained for H&E and ZO-1 and staining area was evaluated (n=6/group).

Results : After 24 h of cultivation, IL1B expression was significantly decreased in the 3 h H₂O₂ groups (100 mM: p=0.005; 200 mM: p=0.001). No changes were noted regarding IL6 and IL10 independent of H₂O₂ concentration and incubation time. 30 min incubation with 100 mM (p=0.031) or 200 mM H₂O₂ (p=0.005) lead to a decreased IL8 mRNA expression. This was also seen with 100 mM H₂O₂ for 3 h (p=0.003). VEGF expression decreased trough H₂O₂, significant effects were seen for 30 min 200 mM H₂O₂ (p=0.014) and 3 h 100 mM H₂O₂ (p=0.010). VEGFB and TJP1 expression was comparable in all five groups. Regarding ALDH3A1, a lower expression was noted with 3 h 100 mM (p=0.017) or 200 mM (p=0.001) H₂O₂. With increasing dose and duration of H₂O₂, more tissue damage was noted in H&E stained sections. ZO-1 area was significantly decreased in the endothelial and epithelial region (p<0.050), but not in the stroma.

Conclusions : Acute oxidative stress decreased the expression of inflammatory, angiogenesis, tight junction, and aldehyde dehydrogenase markers in ex vivo corneas. A damage of corneal tissue was also noted via (immuno-)histology. Hence, this ex vivo model is suitable to mimic oxidative stress. It further can be used to gain further insights into cornea disease pathomechanisms occurring in corneal injuries, like chemical damage, as well as in ocular diseases, including dry eye disease or Fuchs' endothelial dystrophy.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.