Effects of Intracameral Drugs and Dyes on Corneal Endothelial Cell Apoptosis in a Rat Model: An In Vivo and In Vitro Analysis

Abstract

Objectives: To evaluate the effects of intracameral drugs and dyes on rat corneal endothelial apoptosis and cell morphology.

Materials and methods: The right eyes of 72 rats were injected intracamerally with 1% lidocaine, 0.01% adrenaline, triamcinolone acetonide (TA) 4 mg/mL, 1% trypan blue (TB), 0.5% indocyanine green (ICG), and fortified balanced salt solution as control. Corneal samples were taken 1 day and 1 week post-injection. Corneal endothelial apoptosis was assessed by the TUNEL technique, and the ratio of apoptotic cells in each group was compared with the control. Corneal endothelial cell morphology was evaluated in each specimen by transmission electron microscopy.

Results: The mean apoptotic endothelial cell ratio was significantly higher at 1 day and 1 week after intracameral adrenaline injection when compared to controls (p=0.03 and 0.021, respectively). TB caused a significantly higher apoptotic cell ratio when compared to controls at 1 week after injection (p=0.043). Lidocaine caused a higher apoptotic cell ratio compared to TA and ICG at 1 week, although not statistically significant (p=0.058, 0.09, 0.69, respectively). In all experimental specimens, transmission electron microscopy showed morphological changes associated with apoptosis.

Conclusion: This study showed that intracameral adrenaline, TB, and lidocaine injections may have toxic effects on corneal tissue, as indicated by ultrastructural and histopathological alterations. Therefore, these agents should be used with caution in intraocular surgery.

Keywords: Intracameral injection; TUNEL assay; apoptosis; corneal endothelium; morphology.

Figure 1

The anterior chamber was entered via the superotemporal corneal quadrant with an MVR knife (A) and 0.05 mL of aqueous humour was drained with a 30-gauge cannula (B) before injection of an intracameral agent

Figure 2

Intracameral injection of 1% preservative-free lidocaine (A), 0.01% adrenaline (B), triamcinolone acetonide 4 mg/mL (C), 0.5% indocyanine green (25 mg/0.5 mL aqueous solvent in 4.5 mL BSS) (D), and 1% trypan blue (E) into the rat anterior chamber

Figure 3

The effect of intracameral agents on endothelial cell apoptosis along the corneal folds was demonstrated by TUNEL technique. Cells with chromatin condensation were labelled and proportioned to the total number of cells. The rate of apoptosis was scored as Grade 0 (0%), Grade 1 (1-5%) (A), Grade 2 (5-25%) (B), Grade 3 (25-50%) (C), and Grade 4 (<50%) (D)

Figure 4

High-magnification transmission electron micrograph of rat corneal endothelium. The normal endothelium (En) is adherent to Descemet’s membrane and the nucleus (Nu) appears long and undulated within the cell (A). The cells in the mid-phase of apoptosis show mitochondrial swelling, cytoplasmic vacuolization (arrows), and chromatin clustering (B). In the late apoptotic phase, the surface cell membrane is disrupted and chromatin condensation, mitochondrial swelling, and nucleus shrinkage are observed