1：Experimental Design and Method Selection:

The study used a rat model of optic nerve crush (ONC) to investigate the neuroprotective effects of carbon monoxide (CO) preconditioning. Rats were divided into groups: CO + crush, crush-only, and sham. CO inhalation at 250 ppm for 1 hour was administered before ONC. Methods included H&E staining, FluoroGold labeling, FVEP analysis, TUNEL staining, and caspase activity assays to assess RGC survival, visual function, and apoptosis.

2：Sample Selection and Data Sources:

108 adult male Sprague-Dawley rats (6 weeks old, 150-200 g) from the experimental animal center of Shanghai Jiao Tong University were used. They were randomly divided into three groups of 36 rats each.

3：List of Experimental Equipment and Materials:

Equipment included a binocular-operating microscope, Sugita Titanium Aneurysm Clip II Applier, Nikon Eclipse E100 microscope, Olympus BX-51 fluorescence microscope, FVEP analyzing instrument (BMLab 4.0), and various kits for staining and assays (e.g., In Situ Cell Death Detection kit, Caspase-9 and Caspase-3 Activity Assay kits). Materials included CO gas (250 ppm from Shanghai Baosteel Gases Co., Ltd.), FluoroGold, hematoxylin, eosin, glutaraldehyde, paraformaldehyde, and anesthetics like chloral hydrate and ketamine/xylazine.

4：0), and various kits for staining and assays (e.g., In Situ Cell Death Detection kit, Caspase-9 and Caspase-3 Activity Assay kits). Materials included CO gas (250 ppm from Shanghai Baosteel Gases Co., Ltd.), FluoroGold, hematoxylin, eosin, glutaraldehyde, paraformaldehyde, and anesthetics like chloral hydrate and ketamine/xylazine. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Rats were preconditioned with CO or air for 1 hour, then anesthetized. ONC was performed on the right eye using the aneurysm clip for 9 seconds. Sham surgery involved exposure without crush. At 1 and 2 weeks post-surgery, rats were euthanized, and retinas were processed for H&E staining, FluoroGold labeling, FVEP measurements, TUNEL staining, and caspase activity assays. Specific steps included fixation, sectioning, staining, imaging, and quantitative analysis.

5：Data Analysis Methods:

Data were analyzed using one-way ANOVA and Tukey's post hoc test. Quantitative data are expressed as mean ± standard deviation. Image Pro Plus software was used for cell counting. Analyzers were blinded to the study groups.