To determine if bright light exposure induces neuronal damage, retinal tissue from chicks reared under 40,000 and 500 lux, for a period of 7 days, was processed for histology. Following deep anesthesia (5% isoflurane in oxygen), chicks were euthanized and the eyes rapidly enucleated. Eyes were hemisected, with the anterior portion and vitreous body discarded, while the posterior eye cup was fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 4 hours at 4°C. Eye cups were then left overnight in a 30% sucrose solution, dissolved in 0.1 M PBS, for cryoprotection, before being embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Thermo Fischer Scientific, Scoresby, VIC, Australia). Retinal cyrosections 8 μm thick were cut from the nasotemporal plane using a cryostat (CM1850; Leica Microsystems, North Ryde, NSW, Australia). Sections were mounted onto gelatin-coated glass slides and air-dried overnight. Sections were stained for morphological examination using hematoxylin and eosin (H&E; Thermo Fischer Scientific) or TUNEL labeled for analysis of cell death following the protocol of Maslim et al.
21 For TUNEL labeling, slides were immersed in 70% ethanol for 30 minutes before being washed in distilled water. Peroxidase activity was blocked by incubation in 3% H
2O
2 for 10 minutes. Slides were then incubated in terminal deoxynucleotide transferase buffer (3 mM Trizma base, 14 mM sodium cacodylate, and 0.11 mM cobalt chloride) for 10 minutes before incubation in a reaction buffer containing terminal deoxynucleotide transferase (TdT, 0.03 units/μL; Thermo Fischer Scientific), biotinylated deoxyuridine triphosphate (biotin-16-dUTP, 4 μM; Roche, Castle Hill, NSW, Australia) in a TdT buffer for 1 hour at 37°C. The reaction was stopped by incubation in a 300 mM NaCl buffer containing 30 mM sodium citrate. Nonspecific binding was blocked by incubation in a 1% bovine serum albumin solution for 15 minutes. Secondary labeling was achieved through incubation with streptavidin-conjugated Alexa Fluor 594 (Life Technologies, Mulgrave, VIC, Australia) for 1 hour at room temperature. Sections were double stained with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) DNA nuclear stain for visualization of cell layers. As a positive control, retinal tissue from Sprague-Dawley (SD) rats, in which DNA damage and apoptosis had been induced through light damage, was processed simultaneously with the chicken retinal samples. To induce retinal damage, SD rats (90 days of age) were exposed to 1500 lux for a 24-hour period, which is known to induce photoreceptor apoptosis.
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