1：Experimental Design and Method Selection:

The study used a real-time imaging method with Sytox Orange (SO) to assess retinal ganglion cell (RGC) death in rats after N-methyl-D-aspartate (NMDA) injury. This method was based on previous work in mice with optic nerve crush. The design included intravitreal injections of SO, NMDA or PBS (control), and neuroprotective agents like SNJ-1945, followed by imaging and histological analysis.

2：Sample Selection and Data Sources:

Male Brown Norway rats (7-14 weeks old) were used. Sample sizes varied by experiment (e.g., n=6 or n=10 per group). Data were collected from in vivo imaging and ex vivo retinal flat mounts.

3：List of Experimental Equipment and Materials:

Equipment included a confocal scanning laser ophthalmoscope (cSLO, F-10 model from Nidek), fluorescence microscope (Axiovert 200 from Carl Zeiss), Hamilton syringes with 32-gauge needles, and various chemicals such as SO, NMDA, Annexin V, Fluorogold, SNJ-1945, and carboxymethyl cellulose (CMC).

4：Experimental Procedures and Operational Workflow:

Rats were anesthetized, and intravitreal injections of NMDA or PBS were administered to induce injury or serve as controls. SO was injected 10 minutes before imaging with cSLO to visualize damaged cells. For histological confirmation, retinas were flat-mounted and stained with Fluorogold and Annexin V. Neuroprotective effects were tested by administering SNJ-1945 or CMC intraperitoneally or orally before and after NMDA injury, followed by RGC counting.

5：Data Analysis Methods:

Data were analyzed using two-way ANOVA with Bonferroni's post-hoc test and Student's t-test. Statistical significance was set at P<0.05. Software used was JMP Pro version 12.2.0.