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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 film1,2,3. In this fashion, the coordinated functions of all the ocular organs prote....

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. Ind.......

Representative 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

Acknowledgements

This work was partially supported by the German Research Foundation (DFG) 2886 PANDORA Project-No.B3; SCHA 2040/1-1; MU 4240/2-1; CRC1181(C03); TRR241(B04), H2020-FETOPEN-2018-2020 Project 861878, and by the Volkswagen-Stiftung (Grant 97744) to MH.

....

Materials

NameCompanyCatalog NumberComments
1x PBSGibco
Aluminium HydroxideImject alum Adjuvant7716140 mg/ mL
Final Concentration: in vivo: 1 mg/ 100 µL
C57Bl/6 mice, aged 7–9 weeksCharles River Laboratories 
CalciumCarl rothCN93.11 M
Final Concentration: 5 mM
Curved forcepsFST by Dumont SWITZERLAND5/45 11251-35
Fine sharp scissorFST Stainless steel, Germany15001-08
Laminar safety cabinetHerasafe
Macrophotography CameraCanonEOS6D
Macrophotography Camera (without IR filter)NikonD5300
MnaseNew England biolabsM0247S2 x 106 gel U/mL
Multi-analyte flow assay kit (Custom mouse 13-plex panel)BiolegendCLPX-200421AM-UERLAN
NaCl 0,9% (Saline)B.Braun
Ovalbumin (OVA)Endofit, Invivogen9006-59-110 mg/200 µL in saline
Pertussis toxin ThermoFisher Scientific PHZ117450 µg/ 500 µL in saline
Final Concentration: in vivo: 100 µg/ 100 µL
PetridishGreiner bio-one628160
ScalpelFeather disposable scalpelNo. 21 Final Concentration: in vivo:  300 ng/ 100 µL
StereomicroscopeZaissStemi508
Syringe (corneal/iris washing)BD Microlane27 G x 3/4 - Nr.20 0,4 x 19 mm
Syringe (i.p immunization)BD Microlane24 G1"-Nr 17, 055* 25 mm

References

  1. Gilbard, J. P., Rossi, S. R., Heyda, K. G. Tear film and ocular surface changes after closure of the meibomian gland orifices in the rabbit. Ophthalmology. 96 (8), 1180-1186 (1989).
  2. Mishima, S., Maurice, D. M.

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Ocular Surface InflammationMeibomian Gland DysfunctionNeutrophil Extracellular TrapsImmunogenOvalbuminOcular ExudatesOcular Surface TissuesConjunctivaCornea

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