Optic Nerve Injury Enhanced Mitochondrial Fission and Increased Mitochondrial Density without Altering the Uniform Mitochondrial Distribution in the Unmyelinated Axons of Retinal Ganglion Cells in a Mouse Model

**Background:** Mitochondrial dysfunction is implicated in neurodegenerative diseases and optic neuropathies, including glaucoma. Retinal ganglion cells (RGCs) have high energy demands, and their unmyelinated axons contain uniformly distributed mitochondria. The pathophysiological significance of this distribution pattern in injury is unknown. This study investigated how optic nerve crush (ONC) alters mitochondrial distribution, density, and size in RGC axons using Thy1-mito-YFP mice, both in vitro (flat-mounted retinas) and in vivo (confocal scanning laser ophthalmoscope, cSLO).

**Methods:** Transgenic mice expressing YFP in RGC mitochondria were subjected to ONC on the right eye; the left eye received sham surgery. At 1 and 7 days post-ONC, flat-mounted retinas were immunostained for Brn3 (RGC marker) and imaged by confocal microscopy. Mitochondrial distribution was quantified using the Iδ index (uniform if approaching 1), density (mitochondria/μm), and area (μm²). In vivo, cSLO fundus imaging measured total fluorescence intensity and single-axon mitochondrial distribution and density. Statistical comparisons used Mann–Whitney U tests or repeated-measures ANOVA with Tukey’s post hoc test.

**Key Results:** Brn3-positive RGC counts did not differ at 1 day post-ONC (157.6 ± 11.2 vs. 129.7 ± 14.6/1.9 mm², p=0.39) but decreased significantly by 7 days (29.8 ± 6.0 vs. 155.9 ± 7.3/1.9 mm², p=0.0079). Mitochondrial distribution remained uniform at both time points (Iδ index sigmoid curves approaching 1). Mitochondrial density was unchanged at 1 day (1.26 ± 0.08 vs. 1.16 ± 0.04/μm, p=0.69) but increased at 7 days (1.50 ± 0.05 vs. 1.24 ± 0.04/μm, p=0.0003). Mitochondrial area was similar at 1 day (4.36 ± 0.19 vs. 4.29 ± 0.22 μm², p=0.39) but significantly smaller at 7 days (2.13 ± 0.07 vs. 3.46 ± 0.14 μm², p<0.0001), with a shift toward smaller mitochondria (kurtosis 23.5 vs. 6.0). In vivo cSLO imaging confirmed increased mitochondrial density at 7 days and uniform distribution, and total fundus fluorescence decreased over time (p<0.0001 at 7 days vs. baseline). Comparison of in vivo and in vitro images of the same axons showed consistent mitochondrial distribution.

**Clinical Implications:** Optic nerve injury induces mitochondrial fission and increased density without disrupting the uniform distribution pattern in unmyelinated RGC axons. This suggests that fission may be a compensatory response to maintain local ATP supply and axonal integrity. The preservation of uniform distribution may be critical for axonal survival. In vivo cSLO imaging of mitochondrial dynamics could serve as a non-invasive biomarker for optic neuropathy severity and progression, and mitochondrial fission/fusion pathways represent potential therapeutic targets for glaucoma and traumatic optic neuropathy.