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Abstract

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Biology

Modeling Cataract Surgery in Mice

Published: December 1st, 2023

DOI:

10.3791/66050

1Department of Biological Sciences, University of Delaware

This method models cataract surgery in vivo by removing lens fiber cells from adult mice and leaving behind the capsular bag with attached lens epithelial cells (LECs). The injury response is then assessed at various times post-surgery using molecular and morphological criteria.

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms. Recently, our laboratory and others have successfully used genetically modified mice to study the molecular mechanisms that drive the induction of proinflammatory signaling and LEC epithelial to mesenchymal transition, leading to new insight into PCO pathogenesis. Here, we report the established protocol for modeling cataract surgery in mice, which allows for robust transcriptional profiling of the response of LECs to lens fiber cell removal via RNAseq, the evaluation of protein expression by semi-quantitative immunofluorescence, and the use of modern mouse genetics tools to test the function of genes that are hypothesized to participate in the pathogenesis of acute sequelae like inflammation as well as the later conversion of LECs to myofibroblasts and/or aberrant lens fiber cells.

The lens is a highly organized, transparent tissue that refracts light to produce a clearly focused image onto the retina1,2,3. This specialized organ is surrounded by an uninterrupted basement membrane (the capsule), which isolates the lens from other parts of the eye. The inner anterior surface of the capsule anchors a monolayer of lens epithelial cells (LECs), which then differentiate at the lens equator into lens fiber cells, which comprise the vast majority of the lens3. A cataract occurs when the lens loses its transparency due to factors like ag....

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The following protocol was approved by the University of Delaware Institutional Animal Care and Use Committee under protocol #1039-2021-1. In compliance with the Association for Research in Vision and Ophthalmology (ARVO) for the Use of Animals in Ophthalmic and Vision Research24, all ocular survival surgeries can only be performed in one eye. See Table of Materials for details about reagents and instruments used in this protocol. Alternative approaches for extracting the lens fib.......

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Based on the results from this surgical method, the Duncan Lab has used this mouse model of cataract surgery to construct an injury response time course of lens epithelial cells post-surgery (Figure 5). By 6 h after surgery, 5% of the lens epithelial transcriptome is differentially expressed (Figure 6A), including the upregulation of numerous immediate early response genes and proinflammatory cytokines23 (Figure 6B

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This surgical technique requires advanced mouse handling and micro-surgical skills that take practice to develop. The most difficult to master is the placement of fine sutures in the cornea to close the wound. Suppose the experimenter is a total novice to suturing. In that case, it is recommended to first practice using string and cloth, then move to practice using large-diameter sutures on small fruits to master the needed hand movements to make surgical square knots. Then, the experimenter must practice making incision.......

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This work was supported by the National Eye Institute (EY015279 and EY028597) and Delaware INBRE (P20 GM103446).

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NameCompanyCatalog NumberComments
0.5% Erythryomycin opthalmic ointment USPBaush Lomb24208-910-55
1% Atropine sulfate ophthalmic solutionAmneal60219-1748-2
1% Tropicamide opthalmic solution USPAkorn17478-102-12
10-0 Nylon sutureEthicon7707G
2.5% Phenylephrine hydrochloride ophtahlmic solution USPAkorn17478-200-12
26 G 1/2 Needles - straightBD PrecisionGlide5111
27 G 45° bent dispensing tip 1"Harfington
Balanced saline solutionPhoenix57319-555-06
Bupranorphine (0.1 mg/kg)APP Pharmaceticals401730D
Chlorhexidine solution
Needle holder 2-110Duckworth & Kent2-110-3E
Noyes scissors, straightFine Science Tools12060-02
SMZ800 Nikon model microscopeNikon
Sterile disposable scalpel No.11Feather2975#11
Tweezers #5 DumontElectron Microscopy Sciences72700-D
Xylazine/Ketamine/Acepromazine (35 mg/kg, 80 mg/kg, 0.5 mg/kg) solutionAPP Pharmaceticals401730D

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