Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Summary

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

Abstract

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover was used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3^rd day after modeling, the rats' corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7^th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

Introduction

Limbal stem cell dysfunction (LSCD) is characterized by persistent epithelial defects, corneal vascularization, chronic inflammation, scarring, and conjunctivalization of cornea^1,2. It may arise from the depletion or dysfunction of limbal stem cells after severe ocular surface diseases, such as chemical burns, thermal burns, Stevens-Johnson syndrome, and iatrogenic injury caused by ocular surgeries^2,3,4. The pathogenic mechanisms of LSCD mainly involve alterations in stem cells and disturbances in the stem cell microe....

Protocol

All procedures conformed to the ARVO guidelines for using animals in ophthalmic and vision research and were approved by the Animal Ethics Committee of Guizhou Medical University (Approval No. 2305192). The rats were housed at the Animal Center of Guizhou Medical University, adhering to relevant animal management regulations.

1. Animal selection

1. Use female Sprague-Dawley (SD) rats aged 7-8 weeks. Ensure that there are no ocular surface lesions under slit lamp exami.......

Discussion

Inflammation plays a key role in the development of limbal stem cell deficiency¹⁸, but its specific mechanisms and effects require further exploration. To address this issue, we induced LSCD by scraping the central corneal epithelium and suturing the central corneal stroma to cause severe corneal inflammation. We observed the evolution of corneal inflammation and the status of the limbal stem cells on days 1, 3, and 7 post-modeling. On the third day post-modeling, significant edema of the rat limb.......

Materials

Name	Company	Catalog Number	Comments
10-0 suture line	Alcon, Inc., USA	8065698001
2 mm corneal trephine	Suzhou XVI Vision Company, China	http://www.66vision.com/
20 μ L, 100 μ L, 1 mL pipette gun heads, cell culture plate culture plates, centrifuge tubes	Guangzhou Jiete Biological Co., Ltd., China	https://www.jetbiofil.com/index.html
-20/-80 °C fridge	Qingdao Haier Company, China	https://www.haier.com/cn/
A 10-mL syringe	Zhejiang Condelai Medical Device Co., Ltd., China	https://en.kdlchina.com/zhejiang/25.html
Adhere to the slide	ShiTai Company	188105
Amylene alcohol	Shanghai. Macklin Biochemical, China	http://www.macklin.cn/products/
Anti-Cytokertin	Abcam, United Kingdom	ab76318
Anti-Keratin 12 antibody EPR17882	Abcam, United Kingdom	ab185627
Anti-PAX6 antibody	Abcam, United Kingdom	ab195045
Anti-stripping slides, cover slips	Jiangsu Shitai Experimental Equipment Co., Ltd., China	https://cn.citotest.com/
Autoclave	Hirayama, Japan	https://hirayama.com.cn/about/
Avertin	Shanghai Aladdin Biochemical Technology, China	https://www.aladdin-e.com/zh_cn/A111225.html
Chloroform	Shanghai Biological Engineering Co., Ltd., China	https://www.siobp.com/web
CO2 Oven incubator, ultrasonic crusher, centrifuge	Thermo Fisher Technologies Inc., USA	https://www.thermofisher.cn/cn/zh/home.html
Confocal microscope	Shanghai Qinxiang Scientific Instrument Co., Ltd., China	https://www.clinx.cn/about
Corneal epithelial forceps	Suzhou XVI Vision Company, China	53418D
DAPI	Vector. Inc., USA	H-1000
DEPC water	Shanghai Biological Engineering Co., Ltd., China	https://www.siobp.com/web
Dicuro eye cream (ofloxacin eye cream)	Santian Pharmaceutical Corporation, Japan	https://www.santen.com/asia
D-KSFM, culture medium, double antibody, and PBS	Thermo Fisher Scientific, Inc., USA	https://www.thermofisher.cn/cn/zh/home.html
ED1	AbD Serote c, United Kingdom	MCA341GA
Electronic balance	Shanghai Ohaus Biotechnology Company, China	http://shbio.com/company
Enclosure of the membrane	Parafilm, Inc., USA	https://www.sigmaaldrich.cn
Fluorescein sodium test strip was used in ophthalmology	Tianjin Jingming New Technology Development Co., Ltd., China	https://www.eworldtrade.com/c/jingmingtechnological/
Fluorescent inverted phase-contrast micrographic system	TE-2000U, Nikon, Japan	https://www.microscopyu.com/museum/eclipse-te2000-inverted-microscope
Frozen table-top centrifuge	Eppendorf, Germany	https://www.eppendorf.sh.cn
Glass sheet frame	Xiamen Tianjing Biotechnology Co., Ltd., China	http://www.tagene.net/
H-1200 with DAPI sealagent	Xiamen Juin Biotechnology Co., Ltd., China	https://www.bosonbio.com.cn/
HE staining kit	Auragene, China	http://jushengwu.com/a048/
Hematoxylin-eosin dye solution kit	AURAGEN, United States	P032IH
High and low precision electronic balance	Sdolis, Germany	https://www.solisinverters.com/global
Isopropanol	Shanghai Sinopharm Chemical Reagents Co., Ltd., China	https://en.reagent.com.cn/
Ki-67 antibody	Abcam, United Kingdom	ab16667
liquid nitrogen	Xiamen Yidong Gas Co., Ltd., China	https://www.china.tdk.com.cn/tdk_chn_en/tdk_xiamen/
Microhand holder	Suzhou XVI Vision Company, China	http://www.66vision.com/
Multiformaldehyde powder	Sigm, America	https://www.sigmaaldrich.cn
Normal temperature centrifuge 46	Eppendorf, Germany	https://www.thermofisher.cn
OCT	Shanghai Maokang Biotechnology Co., Ltd., China	4583
Ordinary biological micrographic system	Eclipse 50i, Nikon, Japan	https://www.microscope.healthcare.nikon.com/zh_CN/products/upright-microscopes/eclipse-ni-series
PBS	Shanghai Anjin Biotechnology Co., Ltd, China	SH30256.01
PCR, and the reactor apparatus	Biometra Thermocycler, Germany	https://www.analytik-jena.com/products/life-science/pcr-qpcr-thermal-cycler/thermal-cycler-pcr/biometra-tone-series/
Pipettes of various specifications	Eppendorf Company, Germany	https://www.eppendorf.com/cn-zh/
Primers for Rt-PCR	Shanghai Bio-Tech Co., Ltd.
Promeaine hydrochloride 0.5% eye drops	Alcon, Inc., USA	https://www.alcon.com/about-us
Rat PMN antibody	Fitzgerald, United States	20R-PR020
Real time fluorescence quantitative PCR	Applied Biosystems, United States	https://www.thermofisher.cn
Reverse transcription kit	TAKARA, Japan	https://www.takarabiomed.com.cn/
Slit lamp	TOPCON, Japan	https://topconchina.cn/
Smooth forceps	Suzhou XVI Vision Company, China	http://www.66vision.com/
Specimen-box	Lambolid (Fuzhou) Biotechnology Co., Ltd., China	http://chuangdianbio.com/
Superclean bench	New Plus Pi Art High Technology Company Limited, Singapore	https://www.hi-p.com/
Toothed forceps	Suzhou XVI Vision Company, China	http://www.66vision.com/
Toppicamide eye drops (Medol)	Alcon, Inc., USA	https://www.alcon.com/about-us
Trace nucleic acid protein concentration tester	Thermo Fisher Technologies Inc., USA	https://www.thermofisher.cn/cn/zh/home.html
Trizol	Invitrogen The United States	https://www.fishersci.com/us/en/brands/IIAM0WMR/invitrogen.html
Zeiss, with a surgical microscope	Carl ZEIS, Germany	https://www.zeiss.com/corporate/en/about-zeiss.html