Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Summary

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Abstract

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye's watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Introduction

Open globe (OG) injuries can result in permanent loss of vision when not treated or at least stabilized following injury¹. Delays, however, are prevalent in remote areas where access to ophthalmic intervention is not readily available, such as in rural areas or on the battlefield in military scenarios. When treatment is not readily available, the current standard of care is to protect the eye with a rigid shield until medical intervention is possible. In military medicine, this delay is currently up to 24 h, but it is anticipated to increase up to 72 h in future combat operations in urban environments where air evacuation is not possible

Protocol

Before performing this protocol, be aware that there are legal and ethical requirements in place for the use of animals in research and training. If live animals are used for the source of ocular tissue, seek approval by the local ethical or legal authority (IACUC or Ethics committee, etc.) before beginning. If there is any question in obtaining approval for the use of animals, do not proceed. We previously determined and reported that fresh porcine eyes obtained and used within 24 h post-mortem compared closest to in vivo physiology and fared well for these studies (Animal Technologies, Tyler, TX, USA)^10,1....

Results

Images captured via Optical Coherence Tomography (OCT) are shown for OG injured eyes to illustrate how a successful injury induction looks. Figure 3 shows images for control and OG injured AS tissue immediately after injury and 72 h later. Two views are shown: cross-sectional images through the injury site and top-down maximum intensity projection (MIPs) to visualize the surface area of the image. Control eyes show no noticeable disruption in the cornea, while clear injuries can be located t.......

Discussion

There are critical steps with the ASOC OG injury platform that should be highlighted to improve the likelihood of success when using the methodology. First, during the anterior segment dissection, preserving the trabecular meshwork is essential but challenging to do correctly. If the TM is disrupted, the eye will not maintain physiological pressure and will not meet eligibility criteria for experimental use. It is recommended to practice the dissection process under normal conditions first rather than introducing the add.......

Materials

Name	Company	Catalog Number	Comments
10-32 Polycarbonate straight plug, male threaded pipe connector	McMaster-Carr	51525K431
10-32 Socket cap screw, ½"	McMaster-Carr	92196A269
10 mL syringe	BD	302995
20 mL syringe	BD	302830
Anti-Anti	Gibco	15240-096
Ball-End L key	McMaster-Carr	5020A25
Betadine	Fisher Scientific	NC1696484
BD Intramedic PE 160 Tubing	Fisher Scientific	14-170-12E
Cotton swabs	Puritan	25-8061WC
DMEM media	ATCC	30-2002
FBS	ATCC	30-2020
Fine forceps	World Precision Instruments	15914
Gauze	Covidien	8044
Gentamicin	Gibco	15710-064
Glutamax	Gibco	35050-061
High temperature silicone O-ring, 2 mm wide, 4 mm ID	McMaster-Carr	5233T47
Large forceps	World Precision Instruments	500365
Large surgical scissors	World Precision Instruments	503261
Medium toothed forceps	World Precision Instruments	501217
Nail (puncture object)	McMaster-Carr	97808A503
Nylon syringe filters	Fisher	09-719C
PBS	Gibco	10010-023
Petri dish (100 mm)	Fisher	FB0875713
Polycarbonate, three-way, stopcock with male luer lock	Fisher	NC9593742
Razor blade	Fisher	12-640
Stainless steel 18 G 90 degree angle dispensing needle	McMaster-Carr	75165A81
Stainless steel 18 G straight ½'’ dispensing needle	McMaster-Carr	75165A675
Sterile 100 mL beakers with lids	VWR	15704-092
Vannas scissors	World Precision Instruments	WP5070