Journal of Refractive Surgery

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Experimental Model of Corneal Haze in Chickens

Jesús Merayo-Lloves, MD; Bety Yáñez, MD; Agustín Mayo, BS; Raúl Martín, OD; José Carlos Pastor, MD

Abstract

ABSTRACT

PURPOSE: To develop an experimental animal model of corneal haze following photorefractive keratectomy (PRK).

METHODS: Fifteen Iber Braun hens underwent unilateral PRK for -9.00 D of myopia. The animals were sacrificed at 1, 3, and 6 months postoperatively, and light microscopy was performed.

RESULTS: Slit-lamp microscopy showed haze in the PRK-treated eyes. Histopathologic study disclosed epithelial hyperplasia, basement membrane abnormalities, and extensive anterior stromal disorganization.

CONCLUSIONS: An easy and inexpensive model of haze after PRK was developed in an animal with Bowman's layer. This new model could be useful to understand the pathophysiology and pharmacologic modulation of corneal haze. [J Refract Surg 2001;17:696-699]

Abstract

ABSTRACT

PURPOSE: To develop an experimental animal model of corneal haze following photorefractive keratectomy (PRK).

METHODS: Fifteen Iber Braun hens underwent unilateral PRK for -9.00 D of myopia. The animals were sacrificed at 1, 3, and 6 months postoperatively, and light microscopy was performed.

RESULTS: Slit-lamp microscopy showed haze in the PRK-treated eyes. Histopathologic study disclosed epithelial hyperplasia, basement membrane abnormalities, and extensive anterior stromal disorganization.

CONCLUSIONS: An easy and inexpensive model of haze after PRK was developed in an animal with Bowman's layer. This new model could be useful to understand the pathophysiology and pharmacologic modulation of corneal haze. [J Refract Surg 2001;17:696-699]

A transient loss of corneal transparency or "haze" is one of the major complications of photorefractive keratectomy (PRK) with the argon fluoride (ArF) excimer laser at 193 nm, especially when correction of high amounts of myopia is attempted.14

The limited information about human eye pathology has been gained almost exclusively from eyes removed within a few months of ablation. Most pathologic studies of wound healing subsequent to excimer laser radiation were conducted in animals, in particular rabbits and monkeys.1,5-10 Rabbits are a poor model of the human cornea because they do not have Bowman's layer and they develop an aggressive inflammatory response.1,5*6 This layer is present in primates an? a few other species, most notably in the class Aves.11 In addition, animal models in which monkeys are used are very expensive.

In this study, we report an experimental animal model of PRK that reproduced corneal haze and we describe the histopathologic characteristics of corneal wound healing after photoablation in hens.

MATERIALS AND METHODS

Animals

Fifteen Iber Braun adult hens (weight, 2 kg) were housed in the animal facilities of the Institute of Ophthalmobiology (IOBA), University of Valladolid. The animals were cared for following the guidelines of the Association for Research in Vision and Ophthalmology for use of animals in ophthalmic and vision research.12

Photorefractive Keratectomy

Photorefractive keratectomy was performed using the Summit OmniMed (Summit, Waltham, MA) 193-nm excimer laser with a fluence of 180 mJ/cm2 per pulse at 10 Hz. The laser was controlled each operative day by analyzing the patterns etched on black photopaper. Five animals were randomly assigned to three groups according to the amount of follow-up: Group 1 had 1 month, Group 2 had 3 months, and Group 3 had 6 months of followup after PRK.

The 15 animals were anesthetized with intravenous Ketolar (ketamine, 40 mg/kg) and locally by application of tetracaine 0.5%. After excision of the nictitating membrane, a lid speculum was placed. One eye of each animal underwent PRK of -9.00 diopters (D) (87 ?ta) after a 5.0-mm circular deepithelialization with scraping. Fellow eyes served as controls. At the end of the surgery, Tobrex (tobramycin) ointment was applied.

Table

TableHistologic Parameters of PRK-treated Chicken EyesFigure 1. Slit-lamp microscopy of a hen cornea 1 month after myopic ablation shows diffuse haze, grade 2+ (arrows).

Table

Histologic Parameters of PRK-treated Chicken Eyes

Figure 1. Slit-lamp microscopy of a hen cornea 1 month after myopic ablation shows diffuse haze, grade 2+ (arrows).

Two animals died from the effect of anesthetics and one control group eye had a self-inflicted injury that produced an epithelial defect and loss of corneal transparence.

Evaluation of Haze

Haze was graded on a scale from 0 to 4+ (Fantes and colleagues8) by a masked observer at 1, 3, and 6 months.

At these time-points, five animals per group were sacrificed and the corneas were removed immediately with a 7.75-mm trephine. The corneas then were fixed in 4% formaldehyde and stained with hematoxylin eosin (HE), periodic acid-Schiff (PAS), and Masson trichrome (MT) for light microscopy. A masked observer evaluated all specimens based on the parameters in the Table. Basement membrane parameters (excess and irregularity) were graded as follows: present (1) and absent (0), and anterior stromal scarring as mild (1), moderate (2), and severe (3).

Statistical Analysis

The differences between groups were analyzed using Student's i-test for independent samples with SAS (SAS-Institute Inc., Cary, NC). A P-value of ss .05 was considered statistically significant.

RESULTS

All ablated corneas were re-epithelialized by 3 to 5 days after surgery. No recurrent erosions or delayed epithelial losses were observed.

Following PRK, all eyes had haze as observed by slit-lamp microscopy. In the control (untreated) corneas, haze ranged from 0.5+ to 2+ (mean 0.6 ? 0.67). PRK-treated corneas had significantly (P 5S .05) more stromal haze than controls (mean 1.5 ± 0.7) (Fig 1). Progressive clearing of the corneal opacity occurred over time.

Light Microscopy

The principal features of corneal healing response in this model are summarized in the Table. These findings included a marked epithelial hyperplasia, excess basement membrane and irregularity, an increased number of keratocytes and inflammatory cells, and anterior stromal infiltration. The most important changes were seen at 1 month after PRK and were still observed to a lesser degree 6 months postoperatively.

Figure 2. Light micrograph of a section from a hen cornea 30 days after PRK shows marked epithelial hyperplasia, undulating pattern of epithelial basement membrane (short arrows), and an increased number of keratocytes (long arrows). Note the presence of vacuoles (asterisk) and irregularly arranged collagen (arrowheads) (MT X 400).

Figure 2. Light micrograph of a section from a hen cornea 30 days after PRK shows marked epithelial hyperplasia, undulating pattern of epithelial basement membrane (short arrows), and an increased number of keratocytes (long arrows). Note the presence of vacuoles (asterisk) and irregularly arranged collagen (arrowheads) (MT X 400).

Epithelial hyperplasia was significantly different compared with the control eyes. The number of epithelial layers was significantly inferior at 6 months after PRK compared with 1 month after PRK.

In the epithelial basement membrane, zones of excess and irregularities were observed. The undulating appearance of this membrane and anterior stroma were significantly reduced at 6 months postoperatively.

Morphologic anterior stromal changes, such as the presence of vacuoles, amorphous extracellular material, and irregular collagen fibers, were detected in PRK-treated eyes 4 weeks after surgery (Fig 2). An increased number of keratocytes with cellular polymorphism in the photoablated area was observed in PRK-treated corneas, which was significantly different from the control corneas (P *s .05). Only PRK-treated corneas had inflammatory cells (polymorphonucleocytes and neutrophils).

DISCUSSION

This study describes a new experimental model of corneal haze in hens, and shows a similar histopathologic response to previously reported models. This model may be useful in understanding the pathophysiology and pharmacologic modulation of corneal haze.

Rabbits are used most frequently in models of PRK, but because rabbit eyes do not have Bowman's layer, the results are not directly applicable to humans.1-6 Studies carried out in monkeys require expensive and sophisticated devices.7-10

The animal used in the present study was advantageous compared with other experimental models; Iber Braun hens are readily obtained, can be handled easily by inexperienced personal, and most importantly, their maintenance is inexpensive. Some important characteristics of the hen cornea: diameter is about 7 to 8 mm, epithelium has three to seven layers and is 35 to 40 µm thick, Bowman's layer is 5 to 8 µm thick, and the corneal stroma is approximately 270 to 350 µm thick and forms about 90% of the cornea.13

The results of prefiminary studies demonstrate that it is necessary to ablate through Bowman's layer for scarring to develop.9*10 We produced corneal opacity in all animals after photoablation to a depth of 87 µm. Bowman's layer in hens is approximately 55 µm in depth.

In primate and avian species, the collagen fibrils in the anterior stromal layer adjacent to the epithelial basal lamina are densely interwoven to form a feltlike layer that is referred to as Bowman's layer,u'u In humans, this layer is approximately 8 to 12 µm thick.14 Diverse experimental and clinical studies9?10?15 suggest that the ablation of this layer has an important role in the initial woundhealing response after excimer laser surgery because of a direct interaction between epithelial and stromal cells.

In our study, mild reticular haze between the epithelium and stroma was present 4 weeks after photoablation and tended to resolve over time? similar to that observed in rabbits and humans.1"6 An important characteristic of this model was that we observed a precorneal thin film of lipid secretion in some eyes that made evaluation of mild opacities difficult.

Anterior stromal scarring consisted of an irregular pattern of collagen lamellae, vacuolization, and increased density of keratocytes, as has been reported in rabbits and monkeys.1?5"10 The undulating stromal appearance was most marked, as reported in primate models710, and could be associated with a more severe response in hens.

The laser system used in this study is not in clinical use today. With present-day technology, this type of stromal response might not be found. However, it was necessary for us to obtain substantive corneal haze in order for future applications of this model to evaluate the pharmacological modulation of this opacity.

REFERENCES

1. Marshall J, Trokel SL, Rothery S, Krueger RR. Photoablative reprofiling of the cornea using an excimer laser: photorefractive keratectomy. Lasers Ophthalmol 1986;1:21-48.

2. Seiler T, Holschbach A, Derse M, Jean B, Genth U. Complications of myopic photorefractive keratectomy with the excimer laser. Ophthalmology 1994;101:153-160.

3. Sher N, Chen V, Bowers RA, Frantz JM, Brown DC, Eiferman R, Lane SS, Parker P, Ostrov C, Doughman D. The use of the 193 nm excimer laser for myopic photorefractive keratectomy in sighted eyes: a multicenter study. Arch Ophthalmol 1991;109:1525-1530.

4. Gartry DS, Kerr Muir MG, Marshall J. Excimer laser photorefractive keratectomy: 18-month follow-up. Ophthalmology 1992;99:1209-1219.

5. Hanna KD, Pouliquen YM, Waring GO 3rd, Savoldelli M, Cotter J, Morton K, Menasche M. Corneal stromal healing in rabbits after 193 nm excimer laser surface ablation. Arch Ophthalmol 1989;107:895-901

6. Tuft SJ, Gartry DS, Rawe IM, Meek KM. Photorefractive keratectomy: implications of corneal wound healing. Br J Ophthalmol 1993;77:243-247.

7. Marshall J, Trokel SL, Rothery S, Krueger RR. Long-term healing of the central cornea after photorefractive keratectomy using an excimer laser. Ophthalmology 1988:95: 1411-1421.

8. Fantes FE, Hanna KD, Waring GO III, Pouliquen Y, Thompson KP, Savoldelli M. Wound healing after excimer1 laser keratomileusis (photorefractive keratectomy) in monkeys. Arch Ophthalmol 1990;108:665-675.

9. Del Pero RA, Gigstad JE, Roberts AD, Klintworth GK, Martin CA, L'Esp?rance FA Jr, Taylor DM. A refractive and histopathologic study of excimer laser keratectomy in primates. Am J Ophthalmol 1990;109:419-429.

10. Hanna KD, Pouliquen YM, Salvodelli M, Fantes F, Thompson KP, Waring GO 3rd, Samson J. Corneal wound healing in monkeys 18 months after excimer laser photorefractive keratectomy. Refract Corneal Surg 1990;6:340-345.

11. Cameron JD. Biomedical Foundations of Ophthalmology, Vol 3, Chap 6. In: Tasman W, Jaeger E, eds. Duane's Foundations of Clinical Ophthalmology. Philadelphia, PA: Lippincott; 1994:9-11.

12. The Association for Research in Vision and Ophthalmology (ARVO). Statement for the Use of Animals in Ophthalmic and Vision Research. In: Handbook for the Use of Animals in Biomedical Research. Animals in Research Committee (2nd edition). Bethesda, MD: The Association for Research in Vision and Opthalmology; 1993:15-16.

13. Lucio A, Smith RL. Architecture of the corneal stroma of the hen. Acta Anat (Basel) 1984;120:196-201.

14. Gipeon Hi, Sugrue SP. Cell biology of the corneal epithelium. In: Albert DM, Jakobiec FA, eds. Principles and Practice of Ophthalmology: Basic Sciences. New York, NY: WB Saunders; 1994:3-16.

15. Wilson SE, Liu JJ, Mohan RR. Stromal-epithelial interactions in the cornea. Prog Retin Eye Res 1999;18:293-309.

Table

Histologic Parameters of PRK-treated Chicken Eyes

Figure 1. Slit-lamp microscopy of a hen cornea 1 month after myopic ablation shows diffuse haze, grade 2+ (arrows).

10.3928/1081-597X-20011101-11

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