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In This Article

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

Summary

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Abstract

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Introduction

Dry eye has become the most common ocular surface disease worldwide, with an estimated 5% to 50% of the population suffering from the disease1. Eye dryness and discomfort caused by dry eye, if persistent, can lead to visual disturbances and unstable tear films, further leading to ocular surface damage2. Some patients with dry eye may even suffer from chronic pain2.

According to the 2017 TFOS DEWS II Dry Eye Consensus, dry eye is a complex condition that involves multiple factors and is characterized by the disruption of the natural balance of the tear film. This conditi....

Protocol

Female C57BL/6 (C57) mice, aged 7-8 weeks, were used in this study. All procedures adhered to the ARVO guidelines for the use of animals in ophthalmic and vision research and were approved by the Animal Ethics Committee of Guizhou Medical University (Approval No. 2305193). No ocular surface lesions were observed under a slit lamp examination.

1. Preoperative steps

  1. Sterilize the instruments using a rapid sterilizer: needle holders, a 5-0 suture needle with thread, o.......

Representative Results

To investigate the effects and underlying mechanisms of aqueous deficiency dry eye on the ocular surface, we established a mouse model of aqueous deficiency dry eye by surgically removing both the ELG and ILG (Figure 1A). Following a 2-week period post tear gland resection, tear secretion in the dry eye (after removal of lacrimal glands) mice notably decreased compared to the normal group (Figure 1B,C). Evaluation of the cornea via slit lamp mic.......

Discussion

We successfully established a mouse model of aqueous deficiency dry eye disease by surgically excising both the ELG and ILG, aiming to investigate changes in the ocular surface under conditions of aqueous deficiency dry eye disease. We meticulously documented the morphological and physiological alterations observed in this mouse model of dry eye resulting from the double excision of the ELG and ILG. Two weeks post modeling, we observed and sampled the established mouse model of dry eye.

Consi.......

Acknowledgements

This study was supported in part by the Guizhou Provincial Science and Technology Projects (QKHJC-ZK[2024]ZD043), Fujian Provincial Science Fund for Distinguished Young Scholars (2023J06053 [to S.O]), the Natural Science Foundation of China (No.82101084 [to S.O.] and China Scholarship Council (CSC, 202306310049 [to Y.W.]).

....

Materials

NameCompanyCatalog NumberComments
10 mL syringeZhejiang KDL Medical Devices Co., Ltd. China
5-0 Suture NeedleSuzhou 66 Vision Co., Ltd. China
Absolute ethanolShanghai Sinopharm Chemical Reagent Co., Ltd., China
Alexa Fluor 488, Donkey anti mouse IgGInvitrogen811493
Alexa Fluor 594, Donkey anti rabbit IgGInvitrogenA21207
Anti-Keratin 12 antibody EPR17882Abcamab185627
Anti-PAX6 antibodyAbcamab5790
AutoclaveHirayama. Japan
C57BL/6 mouseShanghai Slack Laboratory Animal Co., Ltd.
Centrifuge at room temperatureEppendorf. Germany
ddH2OShanghai Bioengineering Co., Ltd. China
Electronic balancesShanghai Auhaus Biotech Co., Ltd. China
Fluorescence inverted phase contrast micrography systemTE-2000U,Nikon. Japan
Freeze the cassetteJiangsu Shitai Experimental Equipment Co., Ltd. China
Freeze the slicing bladeXiamen Taijing Biotechnology Co., Ltd. China
H-1200 with DAPI mounting mediumXiamen Juin Biotechnology Co., Ltd. Chinamounting medium containing DAPI
H-5000 Tablet MountantVector. USAmounting medium
HClShanghai Sinopharm Chemical Reagent Co., Ltd., China
Hematoxylin-eosin stain kitAuragene. USA
Iodine
Ki-67 antibodyAbcamab16667
liquid nitrogenXiamen Yidong Gas Co., Ltd. China
Microscopic needle holderSuzhou 66 Vision Co., Ltd. China
Microscopic toothed forcepsSuzhou 66 Vision Co., Ltd. China
Microscopic toothless forcepsSuzhou 66 Vision Co., Ltd. China
Model 3050 frozen slicerLeica, Deerfield, IL. USA
OCTShanghai Maokang Biotechnology Co., Ltd. China
Ofloxacin ointment
Ophthalmic sodium fluorescein test stripsTianjin Jingming New Technology Development Co., Ltd. China
Paraformaldehyde powderSigma. USA
PBS
Phenol red cotton thread
Rapid sterilizer sterilizerSuzhou 66 Vision Co., Ltd. China
Recombinant Human SPRR1b protein Abcamab167925
Refrigerated tabletop centrifugeEppendorf. Germany
Slide holders
Slit lampTopcon. Japan
Specimen boxLambolide (Fuzhou) Biotechnology Co., Ltd. China
Tert-Amyl alcoholShanghai. Macklin Biochemical, ChinaA800283500 mL
Tribromoethanol
Triton X-100Sigma. USA
Ultra-low temperature freezerThermo Fisher Scientific. USA
Upright fluorescence micrographLeica DM2500. USA
Upright normal biomicrography systemEclipse 50i, Nikon. Japan
XyleneShanghai Sinopharm Chemical Reagent Co., Ltd., China
Zeiss Surgical MicroscopeVISU150, Carl ZEISS. Germany

References

  1. Stapleton, F., et al. Tfos dews ii epidemiology report. Ocul Surf. 15 (3), 334-365 (2017).
  2. Galor, A., Levitt, R. C., Felix, E. R., Martin, E. R., Sarantopoulos, C. D. Neuropathic ocular pain: An important yet underevaluated feature of dry eye.<....

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