Development and assessment of a novel ex vivo corneal culture technique involving an agarose-based dome scaffold for use as a model of in vivo corneal wound healing in dogs and rabbits

William M. Berkowski Jr. 1Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.

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Daniel J. Gibson 4Institute for Wound Research, Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, FL 32610.

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Serena L. Craft 3Department of Anatomic Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.

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Robert D. Whitley 1Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.
2Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.

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Gregory S. Schultz 4Institute for Wound Research, Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, FL 32610.

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Caryn E. Plummer 1Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.
2Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608.

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Abstract

OBJECTIVE

To develop and assess a novel ex vivo corneal culture technique involving an agarose-based dome scaffold (ABDS) for use as a model of in vivo corneal wound healing in dogs and rabbits.

SAMPLE

Corneas from clinically normal dogs (paired corneas from 8 dogs and 8 single corneas) and rabbits (21 single corneas).

PROCEDURES

8 single dog corneas (DCs), 1 DC from each pair, and 10 rabbit corneas (RCs) were wounded with an excimer laser; 1 DC from each pair and 11 RCs remained unwounded. Corneas were cultured for 21 days on ABDSs (8 pairs of DCs and all RCs) or on flat-topped scaffolds (8 single DCs). The surface area of corneal fluorescein retention was measured every 6 (DCs) or 12 (RCs) hours until full corneal epithelialization was detected. Changes in corneal clarity were evaluated at 0, 7, 14, and 21 days.

RESULTS

Median time to full epithelialization for wounded dog and rabbit corneas was 48 and 60 hours, respectively; among wounded DCs, time to full epithelization did not differ by scaffold type. After 21 days of culture on ABDSs, all DCs and RCs that epithelialized developed a circular, diffuse, cloud-like pattern of optical haze, whereas DCs cultured on flat-topped scaffolds developed a focal, crater-like region of optical haze. All corneas on the ABDSs maintained convex curvature throughout the study.

CONCLUSIONS AND CLINICAL RELEVANCE

Wounded ex vivo DCs and RCs cultured on ABDSs reliably epithelialized, formed optical haze (consistent with in vivo wound healing), and maintained convex curvature. This culture technique may be adaptable to other species.

Abstract

OBJECTIVE

To develop and assess a novel ex vivo corneal culture technique involving an agarose-based dome scaffold (ABDS) for use as a model of in vivo corneal wound healing in dogs and rabbits.

SAMPLE

Corneas from clinically normal dogs (paired corneas from 8 dogs and 8 single corneas) and rabbits (21 single corneas).

PROCEDURES

8 single dog corneas (DCs), 1 DC from each pair, and 10 rabbit corneas (RCs) were wounded with an excimer laser; 1 DC from each pair and 11 RCs remained unwounded. Corneas were cultured for 21 days on ABDSs (8 pairs of DCs and all RCs) or on flat-topped scaffolds (8 single DCs). The surface area of corneal fluorescein retention was measured every 6 (DCs) or 12 (RCs) hours until full corneal epithelialization was detected. Changes in corneal clarity were evaluated at 0, 7, 14, and 21 days.

RESULTS

Median time to full epithelialization for wounded dog and rabbit corneas was 48 and 60 hours, respectively; among wounded DCs, time to full epithelization did not differ by scaffold type. After 21 days of culture on ABDSs, all DCs and RCs that epithelialized developed a circular, diffuse, cloud-like pattern of optical haze, whereas DCs cultured on flat-topped scaffolds developed a focal, crater-like region of optical haze. All corneas on the ABDSs maintained convex curvature throughout the study.

CONCLUSIONS AND CLINICAL RELEVANCE

Wounded ex vivo DCs and RCs cultured on ABDSs reliably epithelialized, formed optical haze (consistent with in vivo wound healing), and maintained convex curvature. This culture technique may be adaptable to other species.

Ex vivo whole-organ culture (ie, corneal culture) has gained traction as a viable and desirable laboratory method for the study of the pathophysiology and treatment of ocular disease.1–6 Experimentally induced corneal wounding in laboratory animals for assessment of corneal wound-healing interventions typically provides excellent corneal clarity and is biologically realistic, but is limited by the high cost of maintaining a colony of live animals with a good quality of life, necessity for pain management, and potential influences on the wound-healing process that are difficult to control (eg, variability in tear film production, composition, and distribution; immune response; and degree of corneal vascularization and the potential for animal-animal interactions and corneal trauma). In addition, monitoring of corneal wound healing in live animals following experimentally induced wounding relies heavily on scoring systems for assessment of corneal clarity, which are subjective in nature and allow for observer error and bias.7,8 For this reason, ex vivo culture techniques can be useful for the study of wound healing and evaluation of experimental treatments and are consistent with the highest goals of animal research (ie, principles of replacement, refinement, and reduction).

At the same time, ex vivo culture of whole corneas presents many unique challenges that must be overcome to maintain cellular vitality and preserve normal tissue function.2,5 For instance, corneal clarity and healing behavior depend heavily on preservation of the precise structural organization of stromal collagen fibrils and the normal cellular function and metabolism of the epithelium and endothelium.9–12 Previously described2,5,13–17 corneal culture techniques have had variable degrees of success in maintaining viability and measuring epithelialization. The design of a corneal wound-healing model that approximates the in vivo formation of fibrosis within the 3-D space of the corneal stroma is dependent on maintenance of normal corneal architecture (especially curvature). Few previous approaches to corneal culture have successfully met all these challenges.2,17

Various methods are used for physically supporting corneal tissues during culture. In a previous study,17 an elaborate hydraulic support mechanism was devised, in which a rabbit cornea was attached to a polymer block with a medium-filled chamber beneath it; a constant flow of fresh culture medium in and out of the chamber (via inlet and outlet tubes) at 5 mL/h ensured that temperature and simulated intraocular pressure were adequate for maintenance of corneal turgidity and curvature. This technique17 provided unprecedented control over the endothelial environment with regard to fluid pressure characteristics and nutrient availability but was technically challenging and expensive. Other studies4,14 of rabbit corneas have revealed that use of the dome-shaped bottom of an inverted glass test tube for corneal support is simple, effective, cost efficient, and labor efficient. Investigators have also used an agarose-based conformer that is heated and poured onto the posterior aspect of the cornea, forming a transparent gel dome when cooled; this method maintains the unique curvature of each cornea, but initially exposes the corneal endothelium to high-temperature liquid agarose.13,15 The transparent agarose gel present immediately posterior to the cornea may also make imaging difficult because it contributes to light scatter associated with the polystyrene culture plates that are typically used. Another method5,6 involves the use of an air pocket between the posterior aspect of the cornea and an underlying flat-topped scaffold to provide a simple yet stable support base for the cornea during culture; however, eventual dissipation of the air pocket results in corneal flattening and potential aberrations in the biomechanical forces acting on tissue cells.

Historically, ex vivo corneal culture techniques have focused on the use of laboratory animals (eg, rabbits) and have each been limited to a single species.1,2,4 Although these techniques were each successfully applied to the targeted species, they could not be applied to other species without considerable modification. This lack of adaptability is especially important when considering the extreme diversity of species encountered in veterinary medicine. For instance, a rabbit cornea may have a thickness of 400 μm with a radius of 7 mm and a curvature of 44 diopters,18–20 whereas a dog cornea may have a thickness of 600 μm with a radius of 8.5 mm and a curvature of 39 diopters.21,22 An adult horse cornea may have a central thickness of 820 μm with a horizontal radius of 13 mm and a much flatter curvature of 17 diopters.23,24 Even within species, breed- and age-related characteristics can greatly influence the size and shape of corneal tissue. For example, the temporonasal diameter of an adult rabbit globe may be 3 times that of a neonatal rabbit globe.19 In horses, corneal curvature may differ by up to 5 diopters among breeds.24 To meet the needs of a diverse veterinary patient population, new techniques (or unique implementation of established techniques) should be considered. A corneal culture technique that could be readily adapted with minimal modification for use in a variety of species would be an invaluable tool for future research.

An aim of the study reported here was to develop a novel ex vivo corneal culture technique that would build on the success of previously described corneal culture techniques and potentially expand their applicability to other species. This novel corneal culture technique combined the positional stability of a polymer scaffold–microplate assembly with the individual tissue conformity of an agarose-based dome scaffold. In addition, to maintain a black background to facilitate the observation and imaging of optical haze (ie, decrease in corneal clarity), a new method was used for agarose preparation in which a sterile carbon suspension was added to the agarose mixture.

The primary objective of the study reported here was to demonstrate optical haze formation in experimentally wounded corneas cultured with this novel technique. Other objectives were to demonstrate that corneas cultured via this technique would successfully epithelialize and maintain convex corneal curvature (similar to in vivo conditions despite the absence of a rigid sclera and intraocular pressure) throughout a 21-day culture period and to successfully apply the same culture technique to dog and rabbit corneas with minimal modification. In addition, this novel corneal culture technique was compared with a previously described technique5,6 that used an air pocket between the posterior aspect of the cornea and its underlying flat-topped scaffold to explore the hypothesis that optical haze formation following experimentally induced corneal wounding is dependent on the biomechanical forces present in the wound bed.

Materials and Methods

Animals

All animals used in this study were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The study protocol was reviewed and approved by the University of Florida Institutional Animal Care and Use Committee.

Twenty-four globes were aseptically enucleated from 16 adult clinically normal mesaticephalic dogs that were euthanized for reasons unrelated to this study. Sixteen globes were paired left and right globes from 8 dogs; because of the limited and intermittent availability of dog globes, the remaining 8 dog globes were single (ie, not paired left and right) globes selected from 8 dogs by simple randomization during a concurrent study. All dog globes were determined to be free of gross anterior segment abnormalities on the basis of biomicroscopic examination.a

Twenty-one single rabbit globes with adnexa were acquired from a biological supply company.b The globes were harvested from clinically normal albino rabbits (age and body weight range of approx 8 to 12 weeks and 2.2 to 2.6 kg, respectively) that were euthanized for reasons unrelated to this study; globes were transported to the laboratory on ice, in a container filled with antimicrobial-fortified Dulbecco modified Eagle medium and Ham F-12 nutrient mixture.c All rabbit globes were determined to be free of gross anterior segment abnormalities on the basis of biomicroscopic examination, and their adnexal tissues were aseptically trimmed off and discarded.

Experimentally induced corneal wounding

Left and right corneas from globes of 8 dogs were unsystematically allocated to the wounded (n = 8 corneas) or unwounded (8 corneas) group. Corneas in the wounded group received an axial, stromal excimer laser–phototherapeutic keratectomy wound (6 mm in diameter and 250 μm in depth) with no transition zoned; corneas from both groups were cultured on the agarose-based dome scaffold. To compare the gross pattern of optical haze formation over time between culture techniques, the 8 single dog corneas were wounded according to the aforementioned procedure and cultured on a previously described5,6 flat-topped scaffold. Corneas of 10 rabbit globes received an axial, stromal excimer laser–phototherapeutic keratectomy wound (6 mm in diameter and 155 μm in depth) with no transition zone; wounded rabbit corneas and unwounded corneas from the remaining 11 rabbit globes were cultured on the agarose-based dome scaffold to assess interspecies compatibility of this culture technique (a flat-topped scaffold technique for culture of rabbit corneas was not available for this study). Following wounding, corneas were aseptically harvested from all globes by making a circumferential incision 4 mm posterior to the limbus and removing the uveal tract with gentle manual traction.

Corneal culture

Agarose-based dome scaffold technique

To provide a scaffold base for each cornea, solid cylinders were fabricated for dog (height, 8 mm; diameter, 12 mm) and rabbit (height, 8 mm; diameter; 11 mm) corneas from fused black acrylonitrile butadiene styrene filament material by use of a 3-D printer.e One black cylinder was centered in the floor of each well of a 6-well polystyrene (nontreated) microplatef and permanently affixed with methyl cyanoacrylate adhesive.g The finished cylinder scaffold-microplate assembly was cured for 24 hours, then sterilized with ethylene oxide gas.

Prior to the initiation of corneal culture, temporary supports were manufactured to provide a concave molding surface to maintain corneal curvature during filling of each of the corneoscleral rims with agarose gel. To accomplish this, each well of a 6-well polystyrene (nontreated) microplate was filled with sterile high-viscosity polyvinylsiloxane impression material.h A representative previously enucleated dog globe was pressed, with the cornea facing downward, into each well until 6 separate, near-identical concave impressions of the cornea with approximately 5 mm of the perilimbal sclera (1 impression/well; Figure 1) were obtained. The same process was repeated with a representative rabbit globe in a 12-well polystyrene (nontreated) microplatef to obtain 12 impressions of a rabbit cornea with perilimbal sclera. The dog and rabbit corneas used to obtain the impressions came from healthy animals that were euthanized for reasons unrelated to this study and were not subsequently cultured. Completed concave supports were sterilized and prepared for placement of corneas for culture.

Figure 1—
Figure 1—

Photographs to illustrate the step-by-step preparation of dog or rabbit corneoscleral rims for ex vivo corneal culture on an agarose-based dome scaffold. A–In a 6-well polystyrene (nontreated) microplate, each well is filled with sterile high-viscosity polyvinylsiloxane and a representative previously enucleated globe, with the cornea facing downward, is pressed into each well to create a concave support. The impressions in the 6 wells of the plate are almost identical. B–Each concave support is sterilized and prepared for placement of a corneoscleral rim. C–A cornea is placed (epithelial side facing down) into each concave support to maintain its curvature for agarose filling. D–Each cornea is filled with agarose mixture (2% agarose with carbon additive and maintenance medium) to the level of the limbus in preparation for placement onto a polymer cylindrical scaffold base. E–An example of a cylinder scaffold–microplate assembly for support of agarose-filled corneoscleral rims during culture. F–Each cornea (filled with black agarose gel) is positioned centrally on the cylindrical scaffold base, and the culture well is filled with starter medium to the level of the limbus.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

The dog and rabbit corneas harvested for culture were each gently placed (epithelial side facing down) into a concave support so that each cornea was maintained in an upside-down dome shape with no air bubbles between the corneal epithelium and the support (Figure 1). An agarose mixture consisting of fresh maintenance medium (Dulbecco modified Eagle medium and Ham F-12c nutrient mixture at a ratio of 1:1, supplemented with 10% fetal bovine serum,i HEPES buffer,j dextran 40,k and chondroitin sulfatek and fortified with 10% antimicrobial solution [streptomycin, penicillin, and amphotericin]l), 2% concentration of low-melting-point 2-hydroxyethyl agarose powder,m and 1.7% concentration of sterile carbon suspensionn was used to fill the corneas. The carbon additive was used to provide a dark background for imaging (ie, black agarose). Prior to filling the corneas, the agarose mixture was preheated to 65°C in a water bath, then transferred to and held inside a 5-mL serum pipette to air cool for approximately 30 seconds before application to the corneal endothelium. The posterior concavity of each cornea was filled to the level of the limbus with the agarose mixture. The agarose-filled corneas were then refrigerated (epithelial side facing down) at 1.6°C for 30 minutes to allow the filling to congeal.

Flat-topped scaffold technique

For comparison, 8 single dog corneas were cultured directly on a previously described flat-topped support scaffold.5,6 Each scaffold was prepared by molding a black polyurethane elastomero into a conical frustum (base radius, 20 mm; apical radius, 15 mm; height, 10 mm). One black scaffold was centered in the floor of each well of a 6-well polystyrene (nontreated) microplatef and permanently affixed with methyl cyanoacrylate adhesive.g The finished scaffold-microplate assembly was cured for 24 hours and then sterilized with ethylene oxide.

Culture conditions

The agarose-based dome and flat-topped scaffold techniques both used an air-liquid interface method in which the posterior surface of the cornea was submerged in culture medium and the anterior surface was exposed to air. Each cornea was placed (epithelial side facing up) in a separate presterilized culture well on top of its scaffold base. Culture wells were then filled with starter medium (identical to the maintenance medium, except fortified with 1% [instead of 10%] antimicrobial solution) until the fluid line reached the level of the limbus. Each microplate containing 6 cornea samples was covered with the manufacturer-provided top, placed on a nutating platep at 24 rotations/min, and incubated at 37°C, 95% humidity, and 5% CO2. For the first 24 hours, all corneas were cultured in the starter medium; thereafter, all corneas were cultured in maintenance medium.

Estimation of epithelialization rates

Fluorescein application

Fluorescein sodiumq was applied to each cornea at 0 hours (time corneas were placed on scaffold bases); corneas were then irrigated thoroughly with sterile water to flush away fluorescein prior to filling wells with starter medium. Every 6 hours (dog corneas) or 12 hours (rabbit corneas) thereafter, fluorescein was applied to each cornea after medium in the culture well was evacuated. Fluorescein application was repeated at these intervals until all corneas were negative for fluorescein retention (Figure 2).

Figure 2—
Figure 2—

Representative photographs of fluorescein retention in wounded rabbit (A) and dog (B) corneas cultured on agarose-based dome scaffolds and macrophotographed with cobalt blue illumination on day 2 of the culture period. On day 0, all corneas in the wounded group received an axial stromal excimer laser–phototherapeutic keratectomy wound (6 mm in diameter and 250 μm in depth for dog corneas and 6 mm in diameter and 155 μm in depth for rabbit corneas) with no transition zone and were placed on the scaffold base for culture. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

Macrophotography

After fluorescein application, each cornea was macrophotographed in a darkened room with cobalt blue illumination.a Images were obtained with a digital single-lens reflex camerar with a macro lens prefocused to a 1:1 reproduction ratio that was mounted on a tripod with a fixed lens working distance of 15.5 cm for each cornea. For image capture of all corneas, the camera was set to manual exposure with an International Organization for Standardization sensitivity of 400, an aperture setting of f/32, and an exposure time of 10 seconds. These settings were found to provide the most useful depth of focus given the many slight variations in individual corneal thicknesses and curvatures.

Image processing

For each cornea at each 6-hour or 12-hour time point, the total surface area of fluorescein retention (ie, wound surface area) was measured with image analysis software.s The time to full epithelialization for each wounded cornea was defined as the first time point at which there was no measurable fluorescein retention evident in the image for that cornea.

Statistical analysis of epithelialization rates

A Kaplan-Meier survival analysis was used to determine whether the time to full epithelialization (as determined by measurement of wound surface area) for the wounded dog corneas differed between the 2 culture techniques (ie, use of agarose-based dome vs flat-topped scaffold). Because the complete epithelialization of each wounded cornea was assumed to have occurred during the 6-hour interval between the time points when fluorescein retention was last observed and when no fluorescein retention was observed, time data were evaluated via interval censoring. The Kaplan-Meier distributions of time data obtained by use of the 2 culture techniques were compared with the Tarone-Ware test. Values of P < 0.05 were considered significant. Statistical analyses were performed with commercially available software.t,u

Subjective evaluation of corneal curvature

The curvature of each cornea was subjectively assessed at the time it was initially placed on top of its scaffold base and daily thereafter. This assessment consisted of visual inspection and slit-lamp biomicroscopy, the latter of which involved examination of each cornea with 10× or 16× magnification by use of a combination of diffuse light and cross-sectional illumination. Any gross distortions of corneal curvature were recorded. Although it would have been ideal to objectively measure the contour of the study corneas, keratometry and corneal topography were unavailable for this study.

Evaluation of optical haze

Macrophotography and hyperosmotic treatment

On day 0 (day that corneas were harvested and placed on scaffold base) and once every 7 days until day 21 of the culture period, all 24 corneas were macrophotographed with fluorescent ambient lighting by use of the aforementioned digital single-lens reflex camera apparatus with the addition of a lens-mounted macroflash. For this phase of the study, the camera was set to manual exposure with an International Organization for Standardization sensitivity of 400, an aperture setting of f/32, and an exposure time of 0.004 seconds. On day 21 (prior to macrophotography), each cornea underwent hyperosmotic treatment to remove any stromal edema (which naturally developed in all corneas throughout the culture period) that could contribute to optical haze; for hyperosmotic treatment, maintenance medium was removed from each well, and each cornea was immersed in 50% dextrosev for 10 minutes, then flushed with sterile eye irrigation solution after dextrose was removed (Figure 3).

Figure 3—
Figure 3—

Representative photographs of a wounded rabbit cornea (on day 21 of culture period) in pink maintenance medium (A) and after hyperosmotic treatment to remove stomal edema by immersing the cornea in 50% dextrose for 10 minutes, removing dextrose, then flushing the cornea with sterile eye irrigation solution (B). After hyperosmotic treatment, optical haze within the wound area is less obscured and more readily contrasted with the clear peripheral cornea. See Figures 1 and 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

Image processing

Day 0, 7, 14, and 21 images of dog and rabbit corneas were evaluated subjectively for the formation of optical haze. Optical haze formation was subjectively assessed by visual inspection and slit- lamp biomicroscopy (ie, it was not quantified). General comparisons were made between corneas cultured on the agarose-based dome scaffolds and flat-topped scaffolds (dog corneas only).

Histologic evaluation

Corneas were placed in Davidson fixative immediately after macrophotography on day 21. Thereafter, a subset of corneas (representing wounded and unwounded dog and rabbit corneas cultured on agarose-based dome scaffolds) was selected for histologic evaluation; the subset was used to conserve funding for the study. However, because the histologic evaluation was qualitative in nature, it was determined that evaluation of a subset of corneas would still provide valuable information about epithelial and stromal architecture. Selected corneas were transected through the axial wound area, and 5-μm sections were obtained; sections from each cornea were stained with H&E or Masson trichrome stain. A board-certified veterinary pathologist (SLC) assessed the corneal sections for epithelial morphology, cellularity of the corneal stroma, and uniformity of the stromal lamellae.

Results

Epithelialization and optical haze formation

Dog corneas

All 16 dog corneas that were cultured on agarose-based dome scaffolds remained in culture for the entire 21-day culture period. Seven of the 8 wounded corneas epithelialized fully and formed optical haze (Figure 4). One of the 8 wounded corneas completely failed to epithelialize or form optical haze during the 21-day culture period (despite this abnormal tissue behavior, the paraxial and perilimbal regions of this cornea maintained clarity that was similar to that of the other corneas). The phenol red–containing medium in the culture well of the cornea that failed to epithelialize continued to display a color change over time (a crude indicator of ongoing cellular metabolism), similar to the observed color change of the culture medium in wells that contained the other 7 wounded corneas, so it was kept in culture for the full 21 days. However, because this cornea failed to epithelialize, it was classified as nonsurviving and was not included in the analysis of epithelialization rates. All 8 wounded dog corneas that were cultured on flat-topped scaffolds epithelialized fully and remained in culture for the entire 21-day culture period. All unwounded dog corneas were negative for fluorescein retention and optical haze at all measured time points and maintained a grossly normal appearance until the end of the culture period.

Figure 4—
Figure 4—

Photographs of pairs of corneas from each of 4 dogs (A and B, C and D, E and F, and G and H) after 21 days of culture on agarose-based dome scaffolds. At the outset of the study, corneas of each dog were unsystematically allocated to the unwounded (A, C, E, and G) or wounded (B, D, F, and H) group. Notice the uniform optical clarity in the unwounded corneas and the presence of axial optical haze in the wounded corneas. See Figures 1 and 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

Rabbit corneas

Fourteen of the 21 (67%) rabbit corneas (9/11 unwounded and 5/10 wounded corneas) cultured on agarose-based dome scaffolds remained in culture for the entire 21-day culture period. One wounded cornea appeared healthy but was excluded from the study within the first 24 hours because it became dislodged from its cylindrical scaffold base during nutation and was found completely submerged in culture medium. Of the other 6 cor-neas for which the study could not be completed, all had a similar appearance to that of the other corneas until day 14 to 17 of the culture period; at that time, they began to develop a markedly edematous, contracted appearance that progressed rapidly until the corneas were no longer able to stay on their cylindrical scaffold bases, at which time they were removed from culture.

Of the 5 wounded corneas that survived the entire 21-day period, all developed gradual formation of focal, axial optical haze (Figure 5). Of the 9 unwounded corneas that survived for the 21-day period, all were negative for fluorescein retention and optical haze at all measured time points and maintained a grossly normal appearance until the end of the culture period.

Figure 5—
Figure 5—

Photographs of 2 unwounded (A and C) and 2 wounded (B and D) rabbit corneas after 21 days of culture on the agarose-based dome scaffold. Notice the uniform optical clarity in the unwounded corneas and the presence of axial optical haze in the wounded corneas. See Figures 1 and 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

Epithelialization rates

Dog corneas

Among the 8 wounded dog corneas cultured on the agarose-based dome scaffolds, the 7 that epithelialized fully all did so within 60 hours. The median time to full epithelialization of these 7 corneas was 48 hours (range, 36 to 60 hours; Figure 6). The median time to full epithelialization of the 8 wounded dog corneas cultured on flat-topped scaffolds was also 48 hours (range, 42 to 54 hours). The time to full epithelialization of the wounded corneas did not differ significantly (P = 0.96) between the 2 culture techniques (ie, use of agarose-based dome scaffold vs flat-topped scaffold).

Figure 6—
Figure 6—

Median surface area of fluorescein retention over time for wounded dog corneas that were cultured on agarose-based dome scaffolds (n = 7; squares [1 cornea that failed to epithelialize is not represented]) or flat-topped scaffolds (8; circles) for 21 days. Fluorescein was applied to each cornea at 0 hours (time corneas were placed on scaffold bases) and then every 6 hours until all corneas were negative for fluorescein retention. Corneas were macrophotographed in a darkened room with cobalt blue illumination at each time point, and the total surface area of fluorescein retention at each time point was measured with image analysis software. For each time point, the horizontal bars represent the SD for wound surface area measurements of corneas on the agarose-based dome (thin bars) and flat-topped (thick bars) scaffolds. See Figures 1 and 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

Rabbit corneas

Among the 10 wounded rabbit corneas cultured on agarose-based dome scaffolds, 9 were available for evaluation of epithelialization (1 cornea had become dislodged from its cylindrical scaffold base within the first 24 hours of the culture period). All 9 corneas had epithelialized fully within 84 hours, and 8 of the 9 corneas had epithelialized fully within 60 hours (median, 60 hours; range, 48 to 84 hours; Figure 7).

Figure 7—
Figure 7—

Surface area of fluorescein retention over time for 9 wounded rabbit corneas (each symbol type represents 1 rabbit cornea; 1 wounded cornea that became dislodged from its scaffold base within the first 24 hours of the culture period is not represented) that were cultured on agarose-based dome scaffolds. Fluorescein was applied to each cornea at 0 hours (time corneas were placed on scaffold bases) and then every 12 hours until all corneas were negative for fluorescein retention. No data were obtained at the 36-hour time point because no laboratory personnel were available to photograph the corneas. See Figures 1, 2, and 6 for remainder of key.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

Corneal curvature

All corneas cultured on agarose-based dome scaffolds maintained convex curvature for the duration of the 21-day culture period. However, corneas cultured on flat-topped scaffolds gradually lost convex curvature over the first 7 days of the culture period.

Characteristics of optical haze formation

For dog and rabbit corneas cultured on the agarose-based dome scaffolds, optical haze was subjectively characterized by a focal, circular, diffuse, cloud-like pattern, surrounded by a relatively clear periwound area (Figure 8). In contrast, the dog corneas that were cultured on flat-topped scaffolds developed a focal, crater-like region of optical haze with several linear radiations. Slit-lamp biomicroscopy of all corneas revealed that the optical haze was localized to the stroma, beginning immediately posterior to the epithelium and extending midway through the stroma.

Figure 8—
Figure 8—

Photographs of wounded dog corneas cultured on an agarose-based dome scaffold (A) or a flat-topped scaffold (B) on day 21 of the culture period. Notice that optical haze is more uniformly distributed throughout the axial wound area in the cornea cultured on the agarose-based dome scaffold because corneal curvature is preserved, whereas in the cornea cultured on the flat-topped scaffold, the loss of corneal curvature results in a crater-like optical haze pattern. See Figures 1 and 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

Histologic evaluation

Wounded corneas (collected after culture for 21 days on the agarose-based dome scaffolds) were characterized by a thin epithelium consisting predominantly of basal cells, with disruption of the epithelial basement membrane (Figure 9). Increased cellularity predominated throughout the anterior portion of the stroma of the wound area, and there was disruption and disorganization of the lamellar architecture of the stroma, compared with that of unwounded corneas.

Figure 9—
Figure 9—

Photomicrographs of sections of 2 representative wounded dog corneas collected after culture for 21 days on agarose-based dome scaffolds and stained with H&E stain (A) or Masson trichrome stain (B). Cellularity is increased in the anterior portion of the stroma (compared with that in unwounded corneas [not shown]), and there is disruption of stromal lamellar architecture within the wound area. In each panel, bar = 100 μm. See Figures 1 and 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 1; 10.2460/ajvr.81.1.47

Discussion

In the present study, the pattern of optical haze that developed in experimentally wounded dog and rabbit corneas during ex vivo culture was affected by the physical structure of the corneal stroma during the wound-healing process. Corneas cultured on the agarose-based dome scaffold maintained a natural convex curvature, which enabled corneas wounded by excimer laser to consistently form a focal, circular, diffuse, cloud-like pattern of optical haze that was compatible with the clinical appearance of corneal scar tissue formed in vivo. The observed optical haze pattern in corneas cultured on the agarose-based dome scaffold indicated that the migration of fibroblasts into the wound bed was affected by the arrangement of fibrils within the stromal lamellae, as determined by the shape of the underlying scaffold.25,26 In addition, with this novel culture technique, epithelialization and optical haze formation developed in both dog and rabbit corneas. To the authors’ knowledge, this corneal culture technique is the first to support these normal wound-healing behaviors in more than 1 species. By preserving the essential elements of this corneal culture technique (ie, the air-liquid interface, nutation, and basic constituents of the culture medium), it should be possible to adapt it for use with other species (eg, nonhuman primates, horses, and rodents) with only minor modifications to the scaffold and culture well dimensions.

It is important to note that the primary objective of the present study was to demonstrate formation of optical haze in wounded corneas cultured with this novel culture technique, given that light reflection in the cornea is of clinical importance to a patient with a corneal wound. The histologic presence of disorganized lamellae and high cellularity in the anterior portion of the stroma of the wounded corneas of the present study suggested that the observed optical haze represented corneal fibrosis (produced by fibroblast and myofibroblast activity). However, additional diagnostic tests (eg, immunohistochemical analysis for α– smooth muscle actin, histologic staining for collagen and elastin, or optical coherence tomography) would be needed for further characterization of the optical haze. These additional tests would be important for validation of this corneal culture technique and confirmation that the observed optical haze is caused by fibroblast and myofibroblast activity.

Experimental corneal wounding (by alkali burn) in dogs has been proposed as an in vivo model of corneal fibrosis.27 This wound healing model offers the benefit of the presence of intact innervation, tear film, and blood supply to encourage scar formation by stromal keratocytes and bone marrow–derived cells. However, the use of live dogs presents the challenge of providing them with routine care, optimal living conditions, and pain management following corneal wounding. Factors such as neovascularization, stromal edema, and the microbiota of the in vivo ocular surface can also introduce additional, potentially confounding variables. In addition, although the alkali burn method is widely used for experimental induction of corneal wounding, it may not produce as precise or repeatable wounding as that achieved with an excimer laser.28

Although agarose gel has been used with previously described corneal culture techniques,13,15 the technique used in the present study is the first to use a scaffold comprised of an agarose gel dome atop a polymer cylinder. The use of this scaffold prevented trauma to corneal tissue during nutation of the culture wells and kept corneas anchored in position for the duration of the 21-day culture period. Maintenance of each cornea in the same position within each culture well was also important for image analysis and for distribution of culture medium uniformly across the corneal epithelium during nutation within the air-liquid interface. In addition, the use of a 3-D extrusion printer offered a high degree of control over the exact dimensions of the polymer cylinders, which would be important when adapting this technique for other species or to address individual variations within a species.

To the authors’ knowledge, this ex vivo corneal culture technique is the first to use agarose with inert suspended carbon particles added to facilitate the documentation of optical haze. The carbon additive did not appear to affect the gross appearance or epithelialization of the corneas, likely because of its sterile and inert nature. Furthermore, the use of this additive resulted in excellent image contrast for detection of fluorescein retention and optical haze formation. Accordingly, future ex vivo cornea culture studies that use agarose gel might benefit from the use of a carbon additive as a means of facilitating photographic evaluation of opacities within otherwise clear corneas.

One challenge with the use of the agarose-based dome scaffold was the application of the heated agarose mixture directly onto the endothelial surface of the corneas. In the corneas of many species, the endothelium consists of a single cell layer that does not regenerate when individual cells become damaged or die.12,29 If a sufficiently high percentage of endothelial cells lose function, excessive stromal edema will develop, which is detrimental to image contrast and the evaluation of optical haze.11,30 In a study31 of rabbit corneas that underwent diode laser thermokeratoplasty, 50% of rabbit corneal endothelial cells died after being exposed to a temperature of 59°C for 60 seconds. This is concerning because most commercially available agarose powders have required melting temperatures of 65° to 95°C.32 An additional concern is the wide range (ie, 17° to 42°C) of gel points inherent in the different forms of agarose.32 These considerations are important given that corneal cultures are kept at a constant 37°C. To minimize temperature stress on the corneal endothelial cells and maintain the dome shape of the agarose supports, a low-melting-point agarose powder was used that had a melting temperature of ≤ 65°C, gel point of 26° to 30°C, and gel strength of ≥ 200 g/cm2 when used at a 2% concentration. As a precaution, the liquid agarose mixture was held inside a serum pipette for approximately 30 seconds before application to the corneal endothelium. Although the effect of this cooling technique on the temperature of the agarose mixture was not measured, the liquid was notably more viscous at the time of endothelial contact than when it was first removed from the water bath, suggesting that the temperature had cooled to < 65°C. As an additional precaution, corneas were refrigerated immediately after they were filled with the agarose mixture to minimize the time that endothelium was exposed to high temperatures.

Although there was no significant difference in the median epithelialization rates for wounded dog corneas cultured on the agarose-based dome scaffold and those cultured on the previously described5,6 flat-topped scaffold, 1 dog cornea failed to epithelialize on the dome scaffold. Interestingly, the region of fluorescein retention on this wounded cornea remained a constant size and shape throughout the 21-day culture period. If the corneal epithelium was adversely affected to the point of complete arrest of epithelialization, generalized sloughing or increasing exposure of the underlying stroma would have occurred, which would have resulted in a different pattern of fluorescein retention than what was observed. Likewise, contamination of the culture with bacteria that are resistant to the antimicrobial additives would likely have resulted in a distinct change in the character of the corneal tissue (eg, edema or visible colony formation) or the medium (eg, increasing turbidity or odor), neither of which was observed over the culture period. It is possible that damage occurred to the epithelial stem cells in the limbal crypts initially (caused by laser trauma or handling of corneal tissue at harvesting), yet enough endothelial function remained to delay the development of edema and maintain gross clarity in this cornea. Indeed, the dog cornea that failed to epithelialize maintained an appearance similar to that of the other 7 dog corneas that did epithelialize on the agarose-based dome scaffold. The other (unwounded) cornea from the same dog with the cornea that failed to epithelialize did not develop any gross abnormalities during the culture period.

Because images of rabbit corneas were acquired at 12-hour intervals (vs 6-hour intervals for dog corneas) and the first image without fluorescein retention was used to identify full epithelialization, it was not possible to compare epithelialization rates between rabbit and dog corneas. Furthermore, the longer interval between measurements for rabbit corneas may have artificially increased the median time to full epithelialization or the SD for these measurements (or both). However, this portion of the study satisfied the study objective of documenting epithelialization of wounded rabbit corneas cultured on the agarose-based dome scaffold.

Among the 21 rabbit corneas studied, 6 were lost between days 14 and 17. The narrow timeline of loss, acute change in tissue characteristics, and common abnormal appearance (eg, edema and a contracted appearance) of these nonsurviving corneas was suggestive of a pathological process. It was possible that overall tissue longevity was affected by the shipping and handling of rabbit globes, including the longer interval between enucleation and culture for rabbit globes (days), compared with that for dog globes (hours). The wounding process may have activated fibrogenesis in a much more fulminant manner in the nonsurviving (vs surviving) rabbit corneas, resulting in more contraction of the stromal wound bed and the grossly abnormal conformation observed between days 14 and 17 of the culture period. Histologic evaluation of nonsurviving and surviving rabbit corneas may offer important information about the etiopathogenesis of this process but has not yet been performed.

Overall, the ex vivo corneal culture technique involving an agarose-based dome scaffold evaluated in the present study represented a promising prototype for future clinical trials to assess topically administered ophthalmic medications. The ex vivo corneas cultured with this technique had wound-healing behavior that approximated what would be expected in vivo, and this fact supported the external validity of this technique. This novel culture technique provided an improved biomechanical support system that supported corneas of variable morphology (ie, dog and rabbit). The cultured corneas had a consistent pattern of optical haze formation, which indicated that this culture technique was a versatile platform that may serve as the basis for development of other experimental models of corneal wound healing.

Acknowledgments

This manuscript represents a portion of a thesis submitted by Dr. Berkowski to the University of Florida Department of Small Animal Clinical Sciences as partial fulfillment of the requirements for a Master of Science degree.

Supported in part by the University of Florida College of Veterinary Medicine Resident Intramural Competitive Research Grant and Fall Consolidated Faculty Research Development Award.

The authors declare that there were no conflicts of interest.

Presented in abstract form at the 48th Annual Conference of the American College of Veterinary Ophthalmologists, Baltimore, October 2017.

The authors thank Blanca Carbia and Patricia Lewis for technical assistance and Dr. William Berkowski Sr for advice and provision of project materials.

Footnotes

a.

Kowa SL-15, Kowa American Corp, Torrance, Calif.

b.

Pel Freez Biologicals, Rogers, Ark.

c.

Gibco DMEM/F12, Thermo Fisher Scientific Inc, Waltham, Mass.

d.

Nidek EC5000, Nidek Inc, Freemont, Calif.

e.

DaVinci 1.0, XYZ Printing Inc, New Kinpo Group, Taipei City, Taiwan.

f.

Falcon, Corning Inc, Corning, NY.

g.

Krazy Glue, Westerville, Ohio.

h.

Extrude-medium, Kerr, Orange, Calif.

i.

Neuromics, Edina, Minn.

j.

Thermo Fisher Scientific Inc, Waltham, Mass.

k.

Chem-Impex International Inc, Wood Dale, Ill.

l.

MP Biomedicals LLC, Irvine, Calif.

m.

A9414, Sigma-Aldrich Corp, St Louis, Mo.

n.

Endomark, PMT Corp, Chanhassen, Minn.

o.

Smooth-Cast ONYX, Smooth-On Inc, Macungie, Pa.

p.

UltraCruz, Santa Cruz Biotechnology, Dallas, Tex.

q.

BioGlo fluorescein ophthalmic strips, HUB Pharmaceuticals, Rancho Cucamonga, Calif.

r.

EOS Rebel XSi, Canon USA, Melville, NY.

s.

ImageJ, National Institutes of Health, Bethesda, Md.

t.

Excel for Microsoft Office 365 Pro Plus, 64 bit, Version 1910, Build 12130.20272; footnote u – November 2018.

u.

Real Statistics Resource Pack, release 6.2, Real Statistics Using Excel. Available at: www.real-statistics.com. Accessed Jun 11, 2018.

v.

VetOne, Boise, Idaho.

References

  • 1. Castro-Combs J, Noguera G, Cano M, et al. Corneal wound healing is modulated by topical application of amniotic fluid in an ex vivo organ culture model. Exp Eye Res 2008;87:5663.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Janin-Manificat H, Rovere MR, Galiacy SD, et al. Development of ex vivo organ culture models to mimic human corneal scarring. Mol Vis 2012;18:28962908.

    • Search Google Scholar
    • Export Citation
  • 3. Tsujita H, Brennan AB, Plummer CE, et al. An ex vivo model for suture-less amniotic membrane transplantation with a chemically defined bioadhesive. Curr Eye Res 2012;37:372380.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Sriram S, Gibson DJ, Robinson P, et al. Assessment of anti-scarring therapies in ex vivo organ cultured rabbit corneas. Exp Eye Res 2014;125:173182.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Proietto LR, Whitley RD, Brooks DE, et al. Development and assessment of a novel canine ex vivo corneal model. Curr Eye Res 2017;42:813821.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Berkowski WM, Gibson DJ, Seo S, et al. Assessment of topical therapies for improving the optical clarity following stromal wounding in a novel ex vivo canine cornea model. Invest Ophthalmol Vis Sci 2018;59:55095521.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Sanchez RF, Dawson C, Matas Riera M, et al. Preliminary results of a prospective study of inter- and intra-user variability of the Royal Veterinary College corneal clarity score (RVC-CCS) for use in veterinary practice. Vet Ophthalmol 2016;19:313318.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Sook Chun Y, Park IK. Reliability of 4 clinical grading systems for corneal staining. Am J Ophthalmol 2014;157:10971102.

  • 9. Goldman JN, Benedek GB, Dohlman CH, et al. Structural alterations affecting transparency in swollen human corneas. Invest Ophthalmol 1968;7:501519.

    • Search Google Scholar
    • Export Citation
  • 10. Meek KM, Fullwood NJ. Corneal and scleral collagens–a microscopist's perspective. Micron 2001;32:261272.

  • 11. Meek KM, Leonard DW, Connon CJ, et al. Transparency, swelling and scarring in the corneal stroma. Eye (Lond) 2003;17:927936.

  • 12. Ledbetter EC, Gilger BC. Diseases and surgery of the canine cornea and sclera. In: Gelatt KN, Gilger BC, Kern TJ, eds. Veterinary ophthalmology. 5th ed. Ames, Iowa: Wiley-Blackwell, 2013;9761049.

    • Search Google Scholar
    • Export Citation
  • 13. Carrington LM, Albon J, Anderson I, et al. Differential regulation of key stages in early corneal wound healing by TGF-β isoforms and their inhibitors. Invest Ophthalmol Vis Sci 2006;47:18861894.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Chuck RS, Behrens A, Wellik S, et al. Re-epithelialization in cornea organ culture after chemical burns and excimer laser treatment. Arch Ophthalmol 2001;119:16371642.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Harman RM, Bussche L, Ledbetter EC, et al. Establishment and characterization of an air-liquid canine corneal organ culture model to study acute herpes keratitis. J Virol 2014;88:1366913677.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Saghizadeh M, Epifantseva I, Hemmati DM, et al. Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing. Invest Ophthalmol Vis Sci 2013;54:81728180.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Tanelian DL, Bisla K. A new in vitro corneal preparation to study epithelial wound healing. Invest Ophthalmol Vis Sci 1992;33:30243028.

    • Search Google Scholar
    • Export Citation
  • 18. Bozkir G, Bozkir M, Dogan H, et al. Measurements of axial length and radius of corneal curvature in the rabbit eye. Acta Med Okayama 1997;51:911.

    • Search Google Scholar
    • Export Citation
  • 19. Hughes A. A schematic eye for the rabbit. Vision Res 1972;12:123138.

  • 20. Chan T, Payor S, Holden BA. Corneal thickness profiles in rabbits using an ultrasonic pachometer. Invest Ophthalmol Vis Sci 1983;24:14081410.

    • Search Google Scholar
    • Export Citation
  • 21. Murphy C, Samuelson D, Pollock R. The eye. In: Evans HE, de Lahunta A, eds. Miller's anatomy of the dog. 4th ed. St Louis: Elsevier, 2013;746785.

    • Search Google Scholar
    • Export Citation
  • 22. Montiani-Ferreira F, Petersen-Jones S, Cassotis N, et al. Early postnatal development of central corneal thickness in dogs. Vet Ophthalmol 2003; 6:1922.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Badial PR, Cisneros-Alvarez LE, Brandao CV, et al. Ocular dimensions, corneal thickness, and corneal curvature in quarter horses with hereditary equine regional dermal asthenia. Vet Ophthalmol 2015;18:385392.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Brooks DE, Matthews A, Clode AB. Diseases of the cornea. In: Gilger BC, ed. Equine ophthalmology. 3rd ed. Ames, Iowa: John Wiley & Sons, 2017;252368.

    • Search Google Scholar
    • Export Citation
  • 25. Dawson DG, Grossniklaus HE, McCarey BE, et al. Biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery: is there a difference between advanced surface ablation and sub-Bowman's keratomileusis? J Refract Surg 2008;24:S90S96.

    • Search Google Scholar
    • Export Citation
  • 26. Dupps WJ Jr, Wilson SE. Biomechanics and wound healing in the cornea. Exp Eye Res 2006;83:709720.

  • 27. Gronkiewicz KM, Giuliano EA, Kuroki K, et al. Development of a novel in vivo corneal fibrosis model in the dog. Exp Eye Res 2016;143:7588.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Mohan RR, Stapleton WM, Sinha S, et al. A novel method for generating corneal haze in anterior stroma of the mouse eye with the excimer laser. Exp Eye Res 2008;86:235240.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Andrew SE, Ramsey DT, Hauptman JG, et al. Density of corneal endothelial cells and corneal thickness in eyes of euthanatized horses. Am J Vet Res 2001;62:479482.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Meek KM, Dennis S, Khan S. Changes in the refractive index of the stroma and its extrafibrillar matrix when the cornea swells. Biophys J 2003;85:22052212.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Koop N, Wirbelauer C, Tüngler A, et al. Thermal damage to the corneal endothelium in diode laser thermokeratoplasty [in German]. Ophthalmologe 1999;96:392397.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Sigma-Aldrich. Agarose selection guide. Available at: www.sigmaaldrich.com/life-science/biochemicals/agarose/agarose-selection.html. Accessed Apr 1, 2017.

    • Search Google Scholar
    • Export Citation
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