Development of Animal Models for Lens and Corneal Diseases Using N-Methyl-N-Nitrosourea

Abstract

PURPOSE. N-methyl-N-nitrosourea (MNU) is an alkylating toxicant with potent mutagenic ability. This study was designed to induce apoptosis in lens epithelial cells (LECs) and corneal endothelial cells (CECs) via MNU administration. We sought to build ocular disease models of cataract and corneal endothelial decompensation. METHODS. MNU was delivered into the intraperitoneal cavities of neonatal rats and the anterior chambers of adult rabbits. The MNU-treated animals were then subjected to a series of functional and morphological analyses at various time points. RESULTS. MNU treatment induced pervasive apoptosis of LECs and CECs. These effects were dose and time dependent. Mature cataracts were found in neonatal rats 3 weeks after MNU treatment. Histological analysis revealed that MNU toxicity induced swelling, vacuolation, and liquefaction in lens fibers of MNU-treated rats. Pentacam examination showed that the average density of rat lens increased significantly after MNU administration. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) analysis showed pervasive apoptotic staining in the lenses of MNU-treated rats. In rabbit eyes, intracameral treatment with MNU induced corneal edema and significantly increased central corneal thickness, which peaked at P14. Morphological and immunohistochemical analysis showed that CECs were effectively ablated in the MNU-treated rabbits. The expression of 8-OHdG increased significantly in the cornea of MNU-treated rabbits, compared with vehicle-treated controls. CONCLUSIONS. MNU is sufficient to induce ocular cell apoptosis in animal models. These models of MNU-induced cataract and corneal endothelial decompensation represent valuable tools for efforts to develop relevant therapies.

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway

“…Our team established a mammalian corneal endothelial decompensation model induced by anterior chamber injection of MNU for the first time to simulate the damage of CECs induced by phacoemulsification or TASS, and confirmed its effectiveness and safety. 11 For economic and ethical reasons, New Zealand rabbits were still used as experimental animals for treating MNU-induced corneal endothelial injury with H 2 . The dosage of MNU was 3.0 mg/kg, which could cause corneal endothelial decompensation, DM injury, and severe corneal edema.…”

“…The high concentration of MNU we selected reduced the interference brought by the powerful regeneration ability of rabbit CECs to the experimental results. 11 , 31 Using IVCM, SEM, and section staining, the corneal endothelium was damaged on the first day after MNU injection into the rabbit's anterior chamber and irreparably worsened over the following 28 days. The use of HRS allowed CECs to regenerate around the 14th day after the intervention, and basically returned to normal at day 28, and protected the DM throughout the observation.…”

“…1 ). 11 , 28 The CEC injury model was constructed by dissolving MNU with phosphate buffered saline (PBS) in the dark to make it an MNU solution with a concentration of 80 mmol/L, adding MNU solution into the culture medium containing cells that had been starved for 24 hours, and diluting MNU to a concentration of 6 mmol/L.…”

“…Previously, our team has constructed an N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation model. 11 MNU is represented by the molecular formula C 2 H 5 N 3 O 2 . As an alkylating agent that directly acts on DNA, it is widely believed that it can induce apoptosis of cells, [12][13][14] including eye cells.…”

“…Previously, our team has constructed an N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation model. 11 …”