Optimized intraorbital optic nerve exposure: a translational surgical paradigm for neural regeneration in rat model

To establish a standardized microsurgical protocol for atraumatic exposure of extended intraorbital optic nerve segments in rat models, enabling precision interventions in neural regeneration research.

Aseptic surgical procedures were performed on anesthetized Sprague-Dawley rats, involving three critical steps: (1) precision skin incision with bilateral silk suture retraction, (2) meticulous periorbital adipose tissue dissection, and (3) atraumatic full-length optic nerve exposure. Post-exposure interventions comprised optic nerve crush (ONC) modeling, intrasheath injection, scaffold and hydrogel implantation. Validation encompassed functional (Functional Visual Evoked Potentials, FVEP), vascular (Fluorescein Fundus Angiography, FFA), and cellular (RGC density and axonal integrity via cholera toxin B subunit (CTB) anterograde tracing) assesments.

The approach reliably exposed 5-mm intraorbital optic nerve segments (4.96 ± 0.13 mm) with intact vasculature (FFA-confirmed perfusion). Complete ONC validation was demonstrated through three principal findings: (a) Complete FVEP signal ablation (Ampliude P1 (OS/OD) pre-operation 1.05 ± 0.19, sham 1.00 ± 0.06, ONC 0.03 ± 0.02, *  p < 0.05), (b) Progressive RGC loss quantified by CTB anterograde signal attenuation (Week 1: 1496.7 ± 186.3; Week 2: 146.3 ± 13.0; Week 3: 110.0 ± 12.4;Week 4: 78.75 ± 5.1, * p <  0.05), and (c) Axonal discontinuity confirmed by anterograde CTB truncation. Scaffold implantation permitted CTB-traced axonal regrowth across the lesion site.

This refined surgical protocol provides enhanced operative accessibility for optic nerve interventions, enabling precise modeling of neural injury and repair mechanisms while maintaining microvascular homeostasis. The technical reproducibility and quantifiable outcome measures establish a robust platform for translational research in neuroprotection and regenerative therapies.