Robot-Assisted Stromal Injection in Mice: Precision Delivery and Validation in Experimental Ophthalmic Surgery

Abstract

Because the murine cornea is extremely thin, manual corneal intrastromal injection is technically challenging and lacks a unified standard protocol, and no robot-assisted solution has yet been systematically validated for this model. This paper therefore aims to systematically evaluate the feasibility and safety of a robot-assisted microinjection system for corneal intrastromal injection in mice, to investigate its potential as a standardized technical pathway for precise corneal drug delivery in small-animal models, and to report its first application in experimental ophthalmic surgery in the mouse. A portable high-precision ophthalmic surgical robot was employed. Following a gentle epithelial incision made by the ophthalmologist with a 15° knife, a 41G microneedle mounted on the robotic end-effector advanced at 0.05 mm/s to deliver $2\mu $ L of trypan blue into the stromal layer. Surgical success rates and intraoperative complications, primarily corneal perforation, were recorded. Postoperative evaluation included anterior segment optical coherence tomography (AS-OCT) to measure tunnel length and depth, slit-lamp photography and whole-mount imaging to assess dye diffusion, and frozen histological sections for validation. A total of 84 eyes were included: 44 in the robot-assisted group and 40 in the manual group. The success rate was 81.81% for the robot-assisted group, significantly higher than the 20% in the manual group. The mean tunnel length was $389.8~\pm ~44.47~\mu $ m (CV = 11.41%), and the mean depth was $74.19~\pm ~13.01~\mu $ m(CV = 17.53%), indicating high consistency. Imaging confirmed homogeneous stromal dye distribution. AS-OCT revealed no significant changes in central corneal thickness with injection volume, except for mild edema at $3\mu $ L ( $p \lt 0.05$ ). Dye infiltration area expanded significantly with increasing volume ( $3\mu $ L $\gt 2\mu $ L, $p \lt 0.001$ ), without excessive tissue damage. Histology further demonstrated uniform dispersion across stromal lamellae. These findings establish the feasibility of robot-assisted stromal injection in mice, providing a reliable and standardized platform for precise delivery in small-animal ophthalmic models and laying the groundwork for translational ocular therapeutics.