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 infections.

Corneal sensory dysfunction is associated with systemic conditions such as diabetes mellitus and contributes to ocular pathologies, including dry eye disease, diabetic keratopathy, neurotrophic keratitis, and post-surgical complications^{3,4,5,6,7,8,9,10}. Sensory nerve impairment leads to reduced tear production, epithelial thinning, and delayed wound healing, all of which facilitate bacterial colonization^11,12. However, the precise mechanistic link between sensory nerve depletion and bacterial adhesion remains inadequately understood. While studies have demonstrated increased bacterial adhesion following corneal denervation, there is a lack of standardized models that allow controlled investigation of this relationship.

To address this gap, we developed a targeted model of corneal sensory nerve depletion using bupivacaine, a long-acting sodium channel blocker that mimics clinical neuropathy without causing permanent nerve damage^13,14,15. This pharmacological approach provides a controlled and reproducible method for studying corneal nerve depletion and its effects on the ocular surface¹⁵. By integrating this model with bacterial adhesion assays, we can systematically evaluate the impact of sensory nerve depletion on microbial colonization, mimicking key aspects of diabetic keratopathy and neurotrophic keratitis.

Microbial adhesion is a critical early event in corneal infections, influenced by tear film composition, epithelial surface properties, and host immune responses. Among the diverse pathogens capable of colonizing the ocular surface, S. aureus, S. epidermidis, and P. aeruginosa represent three clinically significant bacteria with distinct adhesion mechanisms. P. aeruginosa, a highly virulent Gram-negative bacterium, utilizes pili, flagella, and secreted virulence factors to rapidly adhere to and invade corneal epithelial cells, often leading to severe ulcerative keratitis^16,17. In contrast, S. epidermidis, a Gram-positive commensal bacterium, predominantly exploits disrupted epithelial surfaces and tear film alterations to establish biofilms, contributing to persistent and often subclinical infections^18,19. The differences in adhesion strategies between these species highlight the need to understand how corneal nerve depletion alters the ocular microenvironment to favor bacterial colonization.

A key challenge in studying bacterial adhesion is the choice of an appropriate inoculation model. The conventional scratch injury model, frequently used in infection studies, creates epithelial defects to facilitate bacterial adherence. While effective in simulating trauma-induced infections, this approach introduces significant confounding variables, including excessive tissue damage and an exaggerated inflammatory response, which do not accurately represent the early stages of bacterial colonization in neuropathic corneas. Additionally, the variability in mechanical injury makes it difficult to achieve reproducible results. To overcome these limitations, a more controlled and physiologically relevant bacterial inoculation method is required.

The laboratory wipe blotting technique offers a standardized approach to bacterial deposition, ensuring uniform bacterial adherence without disrupting the epithelial barrier²⁰. This method closely mimics real-world susceptibility scenarios, where bacterial adhesion occurs in the absence of overt mechanical trauma but under conditions of altered tear composition and epithelial homeostasis. By eliminating the variability associated with mechanical injury, the laboratory wipe blotting method provides a more accurate assessment of microbial adhesion dynamics in neuropathic corneas. Furthermore, it allows for the controlled evaluation of tear film contributions to bacterial colonization, which is particularly relevant in cases of corneal sensory dysfunction where tear secretion is impaired.

Given the critical role of corneal sensory nerves in regulating tear production and epithelial homeostasis, it is essential to establish a reproducible model for studying the consequences of sensory nerve depletion on microbial adhesion. This study aims to refine a protocol for targeted corneal sensory nerve depletion using bupivacaine, a long-acting local anesthetic, to evaluate its effects on tear secretion and bacterial adhesion. By employing a combination of subconjunctival and topical bupivacaine application, this approach ensures localized and sustained sensory depletion at a defined time point of peak efficacy. Using this model, we investigate how sensory denervation alters the adhesion dynamics of S. aureus, S. epidermidis, and P. aeruginosa, providing valuable insights into the relationship between nerve depletion, tear film alterations, and microbial susceptibility. These findings have broader implications for understanding infection susceptibility in neuropathic conditions such as diabetic keratopathy and neurotrophic keratitis and offer a foundation for future studies on ocular neuroimmune interactions and therapeutic interventions.

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. Animal preparation for subconjunctival injection

1. Induce anesthesia using 2-3% isoflurane in oxygen via a calibrated vaporizer. Monitor anesthesia depth by checking for the absence of reflexive response to a gentle toe pinch.
2. Continuously observe the animal for any signs of severe discomfort or distress.
3. If distress is observed, induce deep anesthesia with 5% isoflurane and confirm unresponsiveness.
4. Perform cervical dislocation to ensure euthanasia.
5. Otherwise, immediately perform enucleation using curved enucleation forceps for downstream analyses.

2. Corneal nerve block using subconjunctival injection

NOTE: Implement a two-step protocol involving subconjunctival injection and topical application of bupivacaine to achieve targeted corneal sensory nerve depletion. Complete subconjunctival injections and topical applications within 15 min to minimize systemic anesthetic exposure.

1. Site preparation and incision
   1. Create a small (~0.5 mm) incision at the superotemporal limbal region using a 30 G needle. Avoid blood vessels and minimize tissue trauma. Use the incision to access the subconjunctival space for controlled drug delivery.
2. Injection of bupivacaine
   1. Prepare a 0.5% bupivacaine hydrochloride solution under sterile conditions.
   2. Load 5 µL of the solution into a Hamilton 10 µL syringe fitted with a 33 G small hub blunt needle (point style 3).
   3. Inject the solution into the subconjunctival space at a 15-20° angle to minimize reflux and ensure uniform distribution.
   4. Observe bleb formation at the injection site; allow it to persist for 5-10 s and disperse within the subconjunctival pocket until fully absorbed.
3. Topical reinforcement of corneal desensitization
   1. Apply an additional 5 µL of 0.5% bupivacaine directly to the central cornea immediately following the subconjunctival injection.
      NOTE: Use this step to enhance surface nerve desensitization and prolong the local anesthetic effect.
4. Control group
   1. Administer 5 µL of phosphate-buffered saline (PBS) via both subconjunctival injection and topical application at identical time points for control animals. Match procedural steps and volume to account for injection-related effects.
5. Treatment duration and end point analysis
   1. Administer bupivacaine treatment every other day for 15 days to achieve sustained sensory nerve depletion while minimizing toxicity.
   2. On day 15, euthanize mice 4 h after the final bupivacaine treatment using 5% isoflurane in oxygen via a calibrated vaporizer, followed by cervical dislocation.
   3. Immediately collect corneas from one group of mice (see step 5.5.) for assessing nerve density alterations using immunohistochemistry (see Figure 1 and Section 6 of the protocol), and from another group for bacterial adhesion analysis (see Figure 1 and Section 5 of the protocol).

3. Animal preparation for bacterial inoculation

1. Administer intraperitoneal ketamine (80-100 mg/kg) and dexmedetomidine (0.25-0.5 mg/kg) to anesthetize mice four hours after the final bupivacaine injection and topical application on day 15.
2. Confirm deep anesthesia by verifying the absence of reflexive responses to toe pinch.
3. Rinse the ocular surface 3x using a pipette with 5 µL of sterile PBS to remove residual tear fluid and debris, ensuring a clean corneal surface for bacterial adhesion studies (see Figure 1)
4. In the designated experimental group, use a laboratory wipe lint-free tissue to blot the corneal surface in a standardized manner.
   1. Fold the tissue paper to ensure a secure grip.
   2. Gently wipe the cornea of the anesthetized mouse 4x, starting at the central cornea and using a rotational motion to the right and left to include the peripheral regions²⁰.
      NOTE: Apply minimal pressure to avoid mechanical injury while effectively removing tear fluid to facilitate bacterial adhesion.

4. Bacterial inoculum preparation and corneal inoculation

1. Bacterial culture and preparation
   1. Culture S. aureus ATCC 25923, S. epidermidis ATCC 12228, and P. aeruginosa O1 on tryptic soy agar (TSA) and incubate at 37 °C with sterile water placed in the incubator to maintain humidity.
   2. Harvest individual colonies and suspend in phosphate-buffered saline (PBS). Centrifuge the suspension at 5,000 × g for 5 min once to remove dead cells and metabolic byproducts.
   3. Resuspend the bacterial pellet in PBS and adjust the concentration to ~10¹¹ CFU/mL to ensure a high bacterial load for adhesion studies.
2. Corneal inoculation protocol
   1. Apply a 5 µL aliquot of the prepared bacterial suspension to the corneal surface once every hour for 4 h under sustained anesthesia.
   2. Position the mice at a slight incline to prevent runoff and ensure consistent exposure to the inoculum.
   3. Repeat the inoculation at hourly intervals to mimic persistent bacterial exposure relevant to clinical infection scenarios.
3. Post inoculation euthanasia and bacterial adhesion assessment
   1. After 4 h, administer an intraperitoneal injection of ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg) to ensure deep anesthesia.
   2. Perform cervical dislocation to euthanize the mice humanely.
   3. Enucleate eyes using sterile, curved-tip forceps while maintaining strict aseptic conditions to prevent contamination.
   4. Rinse the ocular surface 5-6x with sterile 2 mL of PBS in a Petri dish using gentle shaking to thoroughly remove non-adherent bacteria.
   5. Homogenize the enucleated eyes in 1 mL of sterile PBS using a mechanical tissue homogenizer to ensure consistent bacterial recovery and maintain aseptic conditions to prevent contamination.
   6. Perform serial six-fold dilutions of the homogenate in sterile PBS.
   7. Plate aliquots of each dilution on TSA plates and incubate overnight at 37 °C to allow colony growth.
   8. Count colony-forming units (CFUs) and express results as log CFU per cornea for quantitative comparison.

5. Immunohistochemistry

1. Euthanize mice via cervical dislocation on day 15 post treatment.
2. Carefully enucleate eyes and rinse once with PBS at room temperature under gentle rotation.
3. Fix the enucleated eyes in 2% paraformaldehyde (PFA) for 1 h to preserve structural integrity
4. Wash the fixed eyes 3 x 10 min in 1 mL of PBS under gentle rotation at room temperature.
5. Follow the previously described corneal dissection protocol^21,22
   1. Dissect the corneas within a 35 mm wide Petri dish filled with a minimal amount of PBS.
   2. Use straight edge forceps to stabilize the enucleated eye and surgical scissors to create a small incision at the posterior aspect of the globe.
   3. Use the small incision point to carefully cut along the posterior aspect 360°.
   4. Discard the murine retina, optic nerve, lens, iris pigment, and any additional tissue structures that are not the cornea.
   5. Remove any excess tissue using straight-edge forceps or by carefully incising with surgical scissors keeping the cornea intact.
   6. Wash the dissected corneas 3 x 10 min in 1 mL of PBS placed in microcentrifuge tubes under gentle rotation at room temperature.
6. Incubate the corneas in 1 mL of blocking solution (3% BSA + Triton X-100 in PBS) for 1 h at room temperature with continuous rotation.
7. Enhance tissue permeabilization by incubating corneas in 1 mL of 20% EDTA for an additional hour under the same conditions.
8. Incubate the corneas overnight at 4 °C with rabbit anti-β-Tubulin III primary antibody under gentle rotation at room temperature.
9. On the following day, incubate the tissues with Alexa Fluor 488-conjugated Goat anti-Rabbit IgG secondary antibody and DAPI for nuclear staining for 2-3 h at 4 °C in the dark.
10. Wash the corneas 3 x 10 min with PBS under gentle rotation.
11. Follow the Corneal flat mounting protocol described previously^21,22
    1. Flat mount the stained corneas on glass slides by placing cornea at edge of slide to remove excess fluid.
    2. Reposition the cornea in the center of the microscope slide with the epithelial side facing down, in contact with the slide surface.
    3. Make four straight incisions (1-2 mm in length) from the peripheral edge toward the center using a surgical feather blade to create semi-quadrants.
    4. Apply 20 µL of mountant and place an 8 mm glass coverslip over the cornea.
    5. Gently press down on the coverslip to remove any air bubbles and prevent wrinkling of the cornea.
    6. Store the sample at room temperature in the dark for drying.
    7. Seal the edges of the coverslip with clear nail protector to prevent air drying, and store the slides in the dark until ready for imaging.

6. Imaging

1. Perform confocal microscopy using an upright two-photon confocal microscope with a 20x/1.00 NA water-dipping objective.
2. Place the flat-mounted samples on the microscope stage and set up the 20x/1.00 NA water-immersion objective lens.
3. Dim the room lights and cover the microscope with an opaque black cloth to minimize external light interference during imaging.
4. Visualize the corneal nerves using the Alexa Fluor 488 nm laser channel and detect cell nuclei using the DAPI 405 nm laser channel.
5. Set the frame size to 1,536 x 1,536, the pinhole to 1 Airy unit (AU), and the scan speed to 1.02 µs. Enable bidirectional scanning and apply 2x averaging.
6. Adjust the master gain (~750 V) and minimize laser power intensity to prevent photobleaching and phototoxicity while ensuring clear image capture.
7. Perform Z-stack imaging to capture the full corneal thickness. Set the First focal plane at the anterior-most aspect and the Last at the posterior-most aspect of the sample.
8. Acquire Z-stacks at 0.8 µm or 1.0 µm step intervals, capturing approximately 60 - 80 total stacks per sample.
9. Corneal nerve density assessment
   1. Analyze corneal nerve density using Imaris software.
   2. Confirm the full image thickness by checking the volume dimensions.
   3. Enable the volume rendering option to generate a 3D reconstruction.
   4. Create a maximum intensity projection to convert the Z-stack into a 2D view for visualization and quantification.
   5. Grade corneal nerve density on a 0-10 scale by blinded evaluators to ensure objective assessment.

7. Statistical analysis

1. Evaluate the data distribution using the Shapiro-Wilk and Kolmogorov-Smirnov tests.
2. If the majority of the data follow a normal distribution, present the results as the mean ± standard deviation (SD).
3. For comparisons between two groups, apply Student's t-test, and use one-way ANOVA followed by Tukey's multiple comparisons test for analyses involving three or more groups.
4. Consider a p-value < 0.05 statistically significant.

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