Targeted Corneal Sensory Nerve Depletion via Subconjunctival Injection: A Model for Investigating Bacterial Adhesion and Neuroimmune Interactions

Summary

This study establishes a refined model for corneal sensory nerve depletion using bupivacaine, revealing its impact on corneal nerve and bacterial adhesion. The model simulates neuropathic conditions, providing insights into neuroimmune interactions in ocular infections and offering applications in drug delivery, neuroprotection, immune modulation, and gene modification studies.

Abstract

Corneal sensory nerves play a pivotal role in supporting ocular surface integrity and immune defense mechanisms. Loss of this innervation has been associated with increased vulnerability to microbial invasion, yet the precise contribution of nerve depletion to bacterial adhesion on the cornea remains insufficiently characterized. Here, we present a reproducible and temporally controlled method for selective corneal sensory nerve suppression using bupivacaine, a long-acting sodium channel blocker. By combining subconjunctival and topical delivery routes, this dual-application strategy achieves robust, sustained denervation, allowing for precise investigation of how altered sensory input influences corneal epithelial susceptibility to bacterial colonization.

Using this model, we investigate how sensory denervation influences microbial adhesion dynamics for Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa three clinically relevant pathogens with distinct adhesion mechanisms. Standardized bacterial inoculation via the laboratory wipe blotting method ensures uniform deposition on the corneal surface, followed by quantitative assessment of bacterial adhesion. Bupivacaine-induced nerve depletion correlates with reduced corneal nerve density and increased bacterial adhesion, confirming a functional link between sensory depletion and microbial susceptibility.

By simulating neuropathic conditions such as diabetic neuropathy and neurotrophic keratitis, this approach provides a novel framework for studying neuroimmune interactions in ocular infections. Beyond infection models, this subconjunctival injection strategy offers a versatile platform for investigating ocular drug pharmacokinetics, neuroprotective interventions, and immune modulation. Furthermore, it can be adapted for gene modification studies, including subconjunctival delivery of CRISPR/Cas constructs or viral vectors, broadening its applications in ophthalmic research and therapeutics.

Introduction

Corneal sensory innervation plays a critical role in maintaining ocular surface integrity and immune homeostasis. The cornea is one of the most densely innervated tissues in the body, predominantly supplied by the ophthalmic division of the trigeminal nerve¹. These sensory nerves not only mediate nociception and blink reflexes but also regulate epithelial proliferation, wound healing, and immune surveillance^2,3. Any disruption in sensory innervation can, therefore, compromise the corneal barrier, altering the ocular surface microenvironment and increasing susceptibility to microbial inf....

Protocol

All procedures involving animals were carried out in accordance with the standards established by the Association for Research in Vision and Ophthalmology and were approved by the Institutional Animal Care and Use Committee (IACUC) at New England College of Optometry and adhered to PHS policy on the humane care and use of laboratory animals. Six- to eight-week-old male and female C57BL/6J (Wild-Type; strain no. 000664) mice were obtained from the Jackson Laboratory, and mice were bred under normal circadian rhythms (a 12 h light/dark cycle) and were provided food and water. Maintain sterile technique throughout all procedures.

1. Anim....

Results

Unless otherwise specified, all experiments were independently performed in triplicate, with each replicate including 3-4 mice per group.

Significant reduction of corneal nerve density following bupivacaine treatment
To systematically assess the impact of targeted corneal sensory nerve depletion, corneal nerve density was evaluated using immunolabeling and confocal microscopy. Mice received alternate-day subconjunctival injections of bupivacaine for 15 days, either alone o.......

Discussion

Corneal sensory nerves play a pivotal role in maintaining ocular surface integrity, regulating epithelial homeostasis, and modulating immune responses. Corneal neuritis, characterized by nerve degeneration and inflammation, is commonly observed in systemic conditions such as diabetes mellitus, where chronic hyperglycemia contributes to peripheral neuropathy, including corneal nerve loss. Studies have demonstrated that diabetic neuropathy leads to reduced corneal sensitivity, dry eye, impaired wound healing, and an increa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5% bupivacaine hydrochloride solution	Pfizer INC. USA	NDC 00409-1162-01
30 G needle	Manufacturer: BD Biosciences, Franklin Lakes, NJ, USA	305106
33 G small hub blunt needle (point style 3)	Hamilton Company, Reno, NV, USA	7803-05
4',6-Diamidino-2-Phenylindole, Dihydrochloride	Thermo Fisher Scientific, Waltham, MA, USA	D1306
Bovine serum albumin	Sigma-Aldrich, St. Louis, MO, USA	A7906
C57BL/6J (Wild-Type) mice	Jackson Laboratory	. 000664
Cantrifuge rotator	The Lab Depot, Inc. (Dawsonville, GA, USA)	R2020
Clear Nail Protector	Wet and Wild	N/A
Dissecting microscope (SMZ-160-TLED Stereo (dissecting) Microscope With Trinocular Port)	Stellar Scientific (Baltimore, MD, USA)	1.1002E+12
Ethylenediaminetetraacetic acid	Sigma-Aldrich, St. Louis, MO, USA	E6511
Goat anti-Rabbit IgG Alexa Fluor 488	Thermo Fisher Scientific, Waltham, MA, USA	A-11008
Hamilton 10-μL syringe	Hamilton Company, Reno, NV, USA	7635-01
High Precision Dissecting Micro Scissors	Thermo Fisher Scientific, Waltham, MA, USA	08-953-1B
Isoflurane	Patterson Veterinary, USA	07-890-8115
Isoflurane vaporizer	Kent Scientific, USA	VETFLO1205S
Ketamine Hydrochloride	Dechra Veterinary Products, Overland Park, KS, USA	DP00050
Kimwipe lint free tissue paper	Kimberly-Clark Professional, Roswell, GA, USA	34155
Micro-dissecting forcep curved front (for enucleation)	Sigma-Aldrich, St. Louis, MO, USA	F4142-1EA
Micro-dissecting forceps straight front	Sigma-Aldrich, St. Louis, MO, USA	F4017
Petri Dishes (100 mm)	Thermo Fisher Scientific, Waltham, MA, USA	263991
Petri Dishes (35 mm)	Avantor, via VWR International (Radnor, PA, USA).	25373-041
Phosphate-buffered saline	Thomas Scientific, Swedesboro, NJ, USA	C987D24
ProLong Gold Antifade Mountant	Thermo Fisher Scientific, Waltham, MA, USA	P36930
Rabbit anti-β-Tubulin III	Sigma-Aldrich, St. Louis, MO, USA)	T2200
Round Cover Slip German Glass (8 mm)	Thermo Fisher Scientific, Waltham, MA, USA	50-949-314
Scalpel handles	Sigma-Aldrich, St. Louis, MO, USA)	S2896-1EA
Slides, microscope	Sigma-Aldrich, St. Louis, MO, USA)	S8902-1PAK
Snap Cap Low Retention Microcentrifuge Tubes	Thermo Fisher Scientific, Waltham, MA, USA	3453
Sorvall Instruments RT6000B Benchtop Centrifuge	Thermo Fisher Scientific, Waltham, MA, USA	3367898
Surgical feather blades	World Precision Instruments (Sarasota, FL, USA)	504169
Tryptic soy agar	Sigma-Aldrich, St. Louis, MO, USA)	22091
Xylazine	Sigma-Aldrich, St. Louis, MO, USA	1236-20-8