Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Summary

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Abstract

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Introduction

Every blink of an eye replenishes the smooth tear film dispersed over the cornea. The ocular surface epithelia facilitate the distribution and correct orientation of the tear film on the ocular surface. Mucins are provided by the cornea and conjunctiva epithelial cells to help position the aqueous part of the tear film coming from the lacrimal glands on the eyes' surface. Finally, MG secretes lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film^1,2,3. In this fashion, the coordinated functions of all the ocular organs protect the ocular surface from invading pathogens or injury and support crystal clear vision without any pain or discomfort.

In a healthy ocular surface, the ocular flowing discharge or eye rheum sweeps away dust, dead epithelial cells, bacteria, mucus, and immune cells. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin and incorporate these components in the eye rheum. AggNETs resolve inflammation by the proteolytic degradation of pro-inflammatory cytokines and chemokines⁴. However, when they become dysfunctional, these aberrant aggNETs drive the pathogenesis of diseases such as vascular occlusions in COVID-19⁵, gallstones⁶, and sialolithiasis⁷. Similarly, aggNETs on the ocular surface play a protective role and contribute to resolving inflammation of the highly exposed surface⁸. Either an exaggerated formation or lack of aggNETs in the ocular surface can impair the tear film stability and/or cause corneal wounds, cicatrizing conjunctivitis, and dry eye disease. For example, the obstruction of MG is a leading cause of dry eye disease⁹. AggNETs are also known to plug the flow of lipid secretion from the ducts of MG and cause Meibomian gland dysfunction (MGD). The congestion of MG orifices by aggNETs causes a lack of fatty fluid enveloping the ocular surface and retrograde bottled-up fluid, resulting in dysfunction of the gland function and acinar damage. This dysfunction can result in tear film evaporation, fibrosis of the margins on the eyelids, eye inflammation, and detrimental damage to the MG^10,11.

Several animal models have been developed over the years to imitate the pathological process of MGD in humans. For example, C57BL/6 mice aged 1 year have helped study age-related effects on dry eye disease (DED) and MGD, reflecting the ocular disease pathology in patients aged 50 years and older^12,13,14. Furthermore, rabbits are appropriate models for investigating the effects of pharmacological interventions. Therefore, inducing MGD in rabbits has been reported by either the topical administration of epinephrine or the systemic introduction of 13-cis-retinoic acid (isotretinoin)^{15,16,17,18,19}.

Although these animal models were adequate for determining the different factors contributing to the pathophysiology of MGD, they were restricted in their utilization. For instance, the murine model of age-related MGD was ideal for deciphering elements in older adults only, and hence, rabbits appeared to be the most suitable animal model to study ocular surface diseases, as they enable the investigation of multiple pathophysiological mechanisms. However, due to the lack of comprehensive analytical tools to detect proteins at the ocular surface and because many parts of the rabbit genome are unannotated, they are limited for investigations^20,21.

In addition, these animal models used to investigate the pathogenesis of dry eye disease did not provide adequate details to analyze the immunological arm of the disorder that instigates the inflammation of the ocular surface. Accordingly, the murine model of MGD developed by Reyes et al. showed an association between allergic eye disease in mice and MGD in humans and highlighted the immune etiology responsible for obstructive MGD²¹. This model associates allergic eye disease with a TH17 response that recruits neutrophils to the conjunctiva and eyelid, causing MGD and chronic ocular inflammation²¹. The induction of MGD and ocular inflammation in this murine model is a valuable tool for investigating upstream events during the development of local inflammation driven by an ongoing immune response²¹. The current protocol describes the ocular surface inflammation accompanied by obstructive MGD. In this method, mice are immunized and, after 2 weeks, challenged on the ocular surface with the immunogen for 7 days. Furthermore, the steps to isolate ocular exudate and the associated ocular organs during acute inflammation and the dissection of the cornea, conjunctiva, and eyelids are described.

Protocol

All procedures involving animals were conducted according to the institutional guidelines on animal welfare and approved by the animal welfare commission of the Friedrich-Alexander-University Erlangen-Nuremberg (FAU) (permit number: 55.2.2-2532-2-1217). Female C57Bl/6 mice, aged 7-9 weeks were used for the present study. The mice were obtained from commercial sources (see Table of Materials) and kept in specific pathogen-free conditions with 12 h day/night cycles.

1. Induction of murine ocular surface inflammation

1. Perform immunogen preparation for immunization.
   1. Prepare the immunogen freshly on the day of immunization, mixing ovalbumin (OVA, 50 mg/mL) and pertussis toxin (100 µg/mL) in saline and the adjuvant aluminum hydroxide (40 mg/mL) (see Table of Materials) in a 1:1 proportion.
      CAUTION: Perform the opening, reconstitution, and immunogen preparation of pertussis toxin in a laminar safety cabinet. Wear protective clothing and avoid any contact with the skin.
   2. Incubate the immunogen and adjuvant mixture at room temperature for 30 min and load 100 µL into a 1 mL syringe.
   3. Perform intraperitoneal injection of the immunogen solution in a non-anesthetized mouse.
      1. Hold the mouse softly by the tail while grasping the cage grid. Hold firmly the skin of the back and the neck region between the thumb and index finger and fix the tail and lower limbs between the ring and little finger against the palm of the hand.
      2. Keep the fixed mouse with its head downward.
      3. Inject 100 µL of the prepared immunogen solution in the right or left quadrant of the lower abdominal cavity.
2. Perform ocular surface challenge 2 weeks after immunization.
   1. Anesthetize the mouse with isoflurane (2.5%).
   2. Apply 5 µL of OVA (50 mg/mL) or saline (0.9 % NaCl) per eye on both eyes and wait until the drop gets absorbed by the eye. This takes ~5 min.
   3. Repeat the procedure 1x daily for 7 days.
      ​NOTE: The topical administration of medications can be done in the same way.

2. Collection of ocular exudates

1. Recover the ocular exudates formed during the challenge phase by applying 50 µL of sterile saline to the eye immediately after the challenge.
2. To obtain a single-cell suspension, treat the collected ocular discharge with recombinant MNase (2 x 10⁶ gel U/mL, see Table of Materials) containing the cofactor calcium (5 mM) at 37 °C for 20 min.
3. Centrifuge at 400 x g for 7 min at room temperature.
4. Isolate the supernatant and measure the cytokines and chemokines using multiplexed ELISA according to the manufacturer's instructions (see Table of Materials).
   ​NOTE: The obtained supernatant can be used for protein analysis and the pellet for functional assays such as immunophenotyping, phagocytosis, degranulation, and gene expression.

3. Excision of ocular surface tissues

1. Dissect the eyelids and eye globe following the steps below.
   1. Euthanize the mouse by CO₂ asphyxiation and cervical dislocation.
   2. Place the mouse on an even surface.
   3. Disinfect the orbital area around the eye with a swab impregnated with 70% ethanol.
   4. Make an incision between the ear and the retro-orbital sinus and along the surface above the squamous bone vertically, extending the incision horizontally below the lower eyelid along the maxillary bone and above the upper eyelid along the frontal bone. This forms an incision around the eye (Figure 1).
   5. Carefully hold the dissected tissue around the eye using curved forceps and pull the tissue and eyeball out.
   6. Place the excised organs in sterile PBS.
   7. Trim excess facial muscle tissue around the excised upper eyelids using a scalpel and under the stereomicroscope.
2. Collect the conjunctiva following the steps below.
   1. Place the upper eyelid on a dry Petri dish under the stereomicroscope.
   2. Using fine tweezers and a scalpel, peel the whitish-mucous layer very gently out of the inner surface of the eyelid.
3. Next, dissect the cornea following the steps below.
   1. Place the eye globe on a new dry Petri dish on top of dry ice for 3 min.
   2. Take the Petri dish onto the bench surface in a stable position.
   3. Make a small incision at the border of the cornea next to the limbus using fine sharp scissors.
   4. Prolong the incision with the scalpel around the eye globe, separating the sclera from the cornea.
   5. Remove remnants of the iris and the lens from the backside of the cornea by generously flushing with saline solution.

4. Documentation of Meibomian gland (MG) obstruction

1. To assess the MG and its orifices, place excised eyelids in an upright position under the stereomicroscope (Figure 2).
2. Capture images with white light epi-illumination according to the camera's exposure time and ISO settings.
   NOTE: Morphometric analysis of these images provides reliable quantification of the plug size at the outlet of the glands. The quantification can be performed by outlining with the wand tool the eye plugs and executing the "Analyze Particles" command of the Image J software (see Table of Materials).

5. Transillumination of eyelids (Meibomian gland morphology)

1. To assess the MG area, place the excised eyelids in a horizontal position and turn on the backlight of the stereomicroscope equipped with an infrared camera (Figure 3).
2. Capture images by adjusting the exposure time according to the camera's (see Table of Materials) ISO.
   NOTE: Infrared imaging of these transilluminated eyelids under the stereomicroscope can help measure the shape and size of each acini. Morphometric analysis of these images provides reliable quantification of the size and number of MG. The quantification can be performed by outlining the MG with the wand tool and executing the "Analyze Particles" command of the Image J software.

Results

The present protocol describes the sequential steps for establishing a murine model of ocular surface inflammation. The protocols aim to show how to apply therapeutics locally, obtain ocular exudates, and excise associated accessory organs such as healthy and inflamed eyelids (Figure 2), the cornea, and the conjunctiva. Attention must be paid when the upper eyelids are dissected for the isolation of the conjunctiva, and it must be stored in 1x PBS during the dissection of the cornea. This wi...

Discussion

The oily secretion of the Meibomian glands is of great importance for a healthy eye²². However, the obstruction of these sebaceous glands by aggregated neutrophil extracellular traps (aggNETs) that line up as parallel strands located on the tarsal plates of both eyelids can disrupt the tear film²³. This disruption results in Meibomian gland dysfunction (MGD)¹ and accelerated tear evaporation and conditions the damage of the ocular surface

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x PBS	Gibco
Aluminium Hydroxide	Imject alum Adjuvant	77161	40 mg/ mL Final Concentration: in vivo: 1 mg/ 100 µL
C57Bl/6 mice, aged 7–9 weeks	Charles River Laboratories
Calcium	Carl roth	CN93.1	1 M Final Concentration: 5 mM
Curved forceps	FST by Dumont SWITZERLAND	5/45 11251-35
Fine sharp scissor	FST Stainless steel, Germany	15001-08
Laminar safety cabinet	Herasafe
Macrophotography Camera	Canon	EOS6D
Macrophotography Camera (without IR filter)	Nikon	D5300
Mnase	New England biolabs	M0247S	2 x 106 gel U/mL
Multi-analyte flow assay kit (Custom mouse 13-plex panel)	Biolegend	CLPX-200421AM-UERLAN
NaCl 0,9% (Saline)	B.Braun
Ovalbumin (OVA)	Endofit, Invivogen	9006-59-1	10 mg/200 µL in saline
Pertussis toxin	ThermoFisher Scientific	PHZ1174	50 µg/ 500 µL in saline Final Concentration: in vivo: 100 µg/ 100 µL
Petridish	Greiner bio-one	628160
Scalpel	Feather disposable scalpel	No. 21	Final Concentration: in vivo:  300 ng/ 100 µL
Stereomicroscope	Zeiss	Stemi508
Syringe (corneal/iris washing)	BD Microlane	27 G x 3/4 - Nr.20 0,4 x 19 mm
Syringe (i.p immunization)	BD Microlane	24 G1"-Nr 17, 055* 25 mm