Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

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.......

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.......

Materials

Name	Company	Catalog Number	Comments
10 mL syringe	Zhejiang KDL Medical Devices Co., Ltd. China
5-0 Suture Needle	Suzhou 66 Vision Co., Ltd. China
Absolute ethanol	Shanghai Sinopharm Chemical Reagent Co., Ltd., China
Alexa Fluor 488, Donkey anti mouse IgG	Invitrogen	811493
Alexa Fluor 594, Donkey anti rabbit IgG	Invitrogen	A21207
Anti-Keratin 12 antibody EPR17882	Abcam	ab185627
Anti-PAX6 antibody	Abcam	ab5790
Autoclave	Hirayama. Japan
C57BL/6 mouse	Shanghai Slack Laboratory Animal Co., Ltd.
Centrifuge at room temperature	Eppendorf. Germany
ddH2O	Shanghai Bioengineering Co., Ltd. China
Electronic balances	Shanghai Auhaus Biotech Co., Ltd. China
Fluorescence inverted phase contrast micrography system	TE-2000U,Nikon. Japan
Freeze the cassette	Jiangsu Shitai Experimental Equipment Co., Ltd. China
Freeze the slicing blade	Xiamen Taijing Biotechnology Co., Ltd. China
H-1200 with DAPI mounting medium	Xiamen Juin Biotechnology Co., Ltd. China		mounting medium containing DAPI
H-5000 Tablet Mountant	Vector. USA		mounting medium
HCl	Shanghai Sinopharm Chemical Reagent Co., Ltd., China
Hematoxylin-eosin stain kit	Auragene. USA
Iodine
Ki-67 antibody	Abcam	ab16667
liquid nitrogen	Xiamen Yidong Gas Co., Ltd. China
Microscopic needle holder	Suzhou 66 Vision Co., Ltd. China
Microscopic toothed forceps	Suzhou 66 Vision Co., Ltd. China
Microscopic toothless forceps	Suzhou 66 Vision Co., Ltd. China
Model 3050 frozen slicer	Leica, Deerfield, IL. USA
OCT	Shanghai Maokang Biotechnology Co., Ltd. China
Ofloxacin ointment
Ophthalmic sodium fluorescein test strips	Tianjin Jingming New Technology Development Co., Ltd. China
Paraformaldehyde powder	Sigma. USA
PBS
Phenol red cotton thread
Rapid sterilizer sterilizer	Suzhou 66 Vision Co., Ltd. China
Recombinant Human SPRR1b protein	Abcam	ab167925
Refrigerated tabletop centrifuge	Eppendorf. Germany
Slide holders
Slit lamp	Topcon. Japan
Specimen box	Lambolide (Fuzhou) Biotechnology Co., Ltd. China
Tert-Amyl alcohol	Shanghai. Macklin Biochemical, China	A800283	500 mL
Tribromoethanol
Triton X-100	Sigma. USA
Ultra-low temperature freezer	Thermo Fisher Scientific. USA
Upright fluorescence micrograph	Leica DM2500. USA
Upright normal biomicrography system	Eclipse 50i, Nikon. Japan
Xylene	Shanghai Sinopharm Chemical Reagent Co., Ltd., China
Zeiss Surgical Microscope	VISU150, Carl ZEISS. Germany