Ocular surface epithelium and corneal vascularization in rabbits - IOVS

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Trephine. None. Linear. No. of eyes developing extensive vascularization. Source of new ..... syndrome, rosacea keratitis, and chemical burns, which are ...

Ocular surface epithelium and corneal vascularization in rabbits I. The role of wounding Richard A. Thoft, Judith Friend, and Hedwig S. Murphy A new model for rabbit corneal vascularization, created by making a penetrating wound, in corneas with epithelium of conjunctival origin, is described. Obligate resurfacing of the cornea from conjunctival epithelium usually leads to a small, but consistent peripheral superficial corneal vascularization. Subsequent penetrating wounds elicit, in 75% of cases, a marked vascular ingrowth. Normal eyes and eyes resurfaced by peripheral corneal epithelial cells do not vasadarize after such wounds. The vessels are located, in the anterior corneal stroma, and. the regenerated, epithelium has a conjunctival appearance. Although increased, hydration plays a role in this vascularization, the extent of vascularization was much greater in the presence of regenerated epithelium of conjunctival origin than in the presence of regenerated, epithelium of corneal origin. Key words: regenerating epithelium, wounds, corneal vascularization, corneal hydration, ocular surface

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"Orneal vascularization has been the subject of many studies which have recently been extensively reviewed by Fromer and Klintworth.1 Many methods have been used to induce vascularization, but unfortunately, many of the experimental models have proFrom the Department of Cornea Research, Eye Research Institute of Retina Foundation, the Cornea Service, Massachusetts Eye and Ear Infirmary, Boston, Mass., and the Kresge Eye Institute, and Department of Anatomy, Wayne State University School of Medicine, Detroit, Mich. Supported in part by Research Grant EY-01830, Institutional National Research Service Award EY-07018, Biomedical Research Support Grant PHS5S07RR05527, and Research Grant EY-01874 from the National Institutes of Health, United States Department Energy Contract EY-76-S-02-2401, and a Research to Prevent Blindness, Inc. grant; in part by the Massachusetts Lions Eye Research Fund, Inc. Submitted for publication Dec. 28, 1977. Reprint requests: Richard A. Thoft, M.D., Eye Research Institute of Retina Foundation, 20 Staniford St., Boston, Mass. 02114.

duced variable results in terms of regularity and degree of vessel ingrowth. In addition, previous methods have generally not duplicated the superficial vascularization which is a feature of a number of serious corneal diseases. Recent work in our laboratory has indicated that the ocular surface may play an important role in determining the corneal response to trauma. This paper presents the technique found to cause vascularization of the cornea when the epithelium has been removed. Blood vessel proliferation is produced by creating a penetrating wound in a cornea covered by regenerated ocular surface epithelium of conjunctival, as opposed to of corneal, origin. Methods Preparation of animals. Rabbits weighing between 2.5 and 3.5 kg were used for all experiments. Anesthesia was intravenous sodium pentobarbital with local proparacaine, maintained with

0146-0404/79/010085+08$00.80/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc.

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Table I. Ocular surface epithelium and corneal vascularization after iodine debridement and wounding* No. of eyes developing extensive vascularization Days after removal of epithelium 10

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83 170

Source of new epithelium Wound

Cornea

Conjunctiva

None Linear Trephine None Linear Trephine None Linear None Linear

0/11 0/6 0/6 0/12 0/5 0/7

1/13 (7.6) 5/5 (100.0) 6/8 (75.0) 2/16 (12.5) 5/5 (100.0) 6/11 (54.5) 0/3 1/3 (33.3) 0/2 1/2 (50.0)

0/5 0/3 —

*Values are given as the number of eyes developing extensive vascularization/total number of eyes in a category, with the percent following in parentheses. "Extensively vascularized" is defined in Methods as those with more than 4 mm of vessel ingrowth.

Table II. Ocular surface epithelium and corneal vascularization after heptanol washing and wounding* No. of eyes developing extensive vascularization Days after removal of epithelium 10

42

83

Source of new epithelium Wound

Cornea

Conjunctiva

None Linear Trephine None Linear Trephine None Linear

0/6 0/2 0/6 0/4 0/2 0/6 0/2 0/2

1/16 (6.25) 5/6 (83.3) 8/10 (80) 0/12 3/4 (75) 7/8 (87.5) 1/5 (20.0) 5/5 (100.0)

•"Values are given as the number of eyes developing extensive vascularization/total number of eyes in a category, with the percent following in parentheses. "Extensively vascularized" is defined in Methods as those with more than 4 mm of vessel ingrowth.

ether inhalation. When necessary, animals were killed with an overdose of sodium pentobarbital. Removal of epithelium. After anesthesia, either the whole corneal epithelium plus a 1 to 2 mm ring of limbal conjunctival epithelium or a 9 mm diameter area of central corneal epithelium was removed by iodine debridement followed by cocaine neutralization, by heptanol washing, or by scraping as described previously.2' 3 In the case of total epithelial removal, the regenerating epithelium originated in the conjunctiva. In the case of central epithelial removal, the epithelium regenerated from the remaining ring of corneal cells. Penetrating wounds. Ten days, 3 weeks, 6 weeks, and 3 months after the removal of the epithelium, when regenerating epithelium of corneal

or conjunctival origin had healed over the cornea, penetrating wounds were made. Two kinds of penetrating wounds were used, linear and trephine wounds. LINEAR WOUNDS. After anesthesia, an 8 mm

linear penetrating wound was made through the stroma across the visual axis, leaving 2 to 3 mm between the end of the wound and the limbus at either end. Two 10-0 nylon sutures were placed 5 mm apart to secure the wound. The anterior chamber reformed spontaneously with occasional eyes developing iris synechiae at \the wound edges. Initially, heparin was used to avoid aqueous clotting. It became apparent, however, that synechiae formed less frequently if the aqueous were allowed to clot. Antibiotic ointment was

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Table III. Ocular surface epithelium and corneal vascularization after scraping and wounding* No. of eyes developing extensive vascularization Source of new epithelium Days after removal of epithelium 10

42

83

Wound

Cornea

Conjunctiva

None Linear Trephine None Linear Trephine None Linear

0/7 0/2 0/5 0/5 0/3 0/2 0/3 0/3

0/20 6/6 (100.0) 9/14 (64.3) 0/10 3/5 (60.0) 3/5 (60.0) 0/5 3/5 (60.0)

*Values are given as the number of eyes developing extensive vascularization/total number of eyes in a category, with the percent following in parentheses. "Extensively vascularized" is defined in Methods as those eyes with more than 4 mm vessel ingrowth.

given immediately after the operation. The animals were examined three to five times weekly, and topical erythromycin was given routinely. Linear wounds were made in a total of 74 eyes. TREPHINE WOUNDS. After anesthesia, a 2 mm

diameter trephine was used to make a centrally placed 2 mm diameter penetrating wound as described previously.4 The anterior chamber reformed rapidly after the wound filled with a fibrin plug. Topical erythromycin was used immediately. The animals were examined three to five times weekly. Trephine wounds were made in a total of 88 eyes. Endothelial removal. After anesthesia, a 1 mm limbal incision was made in the cornea, and the endothelium was carefully scraped off with a specially designed instrument. Thirty-two eyes with regenerated epithelium of corneal or conjunctival origin which had been healed for 1 day and four eyes with normal epithelium had the endothelium scraped. Sixteen others with conjunctivally or corneally derived regenerated epithelium and four with normal corneal epithelium had the endothelium removed with benzalkonium chloride as described by Maurice and Perl man.0 Data from the two methods of endothelial destruction were pooled because the method used did not affect the results. Observations. Animals were examined three to five times weekly with a flashlight and at least once weekly by slit-lamp examination. The appearance of blood vessels and epithelial abnormalities were noted in detail. A 1 to 2 mm fringe of vessels at the limbus frequently occurs in eyes with regenerating epithelium of conjunctival origin prior to penetrating

wounds. 2 ' 3 For this study, therefore, vascularization was graded according to the following scale: 0 = corneas with no vessels 1+ = corneas with up to a 2 mm vessel ingrowth 2+ = corneas with up to a 4 mm vessel ingrowth 3+ = corneas with up to a 6 mm vessel ingrowth 4+ = corneas with vessels to the visual axis or a central wound In Tables I to III, only those corneas that fell into category 3+ or greater are considered "extensively vascularized." Corneal hydrations. Corneal hydrations were calculated as milligrams of water per milligram of dry weight of tissue after wet weights and postlyophilization dry weights of an 8 mm diameter central disc of cornea (usually with epithelium) were obtained by weighing the tissue on a Mettler analytical balance. For determining hydrations after various methods of epithelial removal (Table IV), only eyes with no epithelial defects, which were not extensively vascularized, were included. Histology. Since vascularization, in most cases, peaked approximately 2 weeks after a penetrating wound was made, the animals were killed at that time; the eyes were removed, fixed in 10% formalin, and prepared for routine histological examination with hematoxylin and eosin staining or periodic acid-Schiff reaction staining. A total of 41 eyes were studied histologically.

Results Observations. As has been reported previously, secondary defect formation, superficial

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Thoft, Friend, and Murphy

Fig. 1A. Vascularization after a linear wound. Two weeks after a linear wound had been made in an eye with regenerating epithelium of conjunctival origin that had been liealed over the surface for 1 day, vascularization extended to the edges of the wound.

\

i

A

Fig. IB. Vascularization after a trephine wound. Similarly, a trephine wound in a cornea with regenerating epithelium of conjunctival origin resulted in heavy vascularization 2 weeks later as shown here. This eye was debrided with iodine of all corneal and a ring ot conjunctival epithelium 34 days before and the trephine wound was made 2 weeks before this photo was taken. Vessels here reach % of the way to the wound.

punctate keratopathy, and a rough surface are characteristic of the eyes with epithelium of conjunctival origin, but not of the eyes with corneally derived epithelium. In addition, there are biochemical and functional differences between the two regenerated epi-

thelial types for at least 6 weeks after injury.2j 3 Removal of the total corneal epithelium, plus a ring of limbal conjunctival epithelium, usually resulted in the development of a 1 to 2 mm fringe of vessels at the limbus within 2 weeks, which remained stable for up to 3

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Table IV. Corneal hydration and ocular surface epithelium* Days after removal of epithelium

Method of epithelial removal

Source of regenerated epithelium

Corneal hydration (mg. Hjp/mg. dry wt.)

A. Normal corneal epithelium present on cornea: — None — B. Regenerated ocular surface epithelium present on cornea: 10 clays Iodine Cornea 10 days Iodine Conjunctiva Scraping 10 days Cornea Scraping 10 days Conjunctiva 10 days Cornea Heptanol 10 days Conjunctiva Heptanol 3 wk. Iodine Cornea 3wk. Iodine Conjunctiva 3 wk. Cornea Scraping 3 wk. Scraping Conjunctiva 3 wk. Heptanol Cornea 3 wk. Heptanol Conjunctiva

p value

3.4 ± 0.2 (10) 3.6 4.1 3.6 3.4 3.3 3.8 3.5 3.7 3.5 3.7 3.1 3.5

± ± ± ± ± ± ± ± ± ±

0.2 ( 4) 0.2 ( 6) 0.2 ( 4) 0.2 ( 6) 0.2 ( 4) 0.1 ( 6) ( 2) 0.3 ( 6) ( 2) 0.1( 8) 0.1 ( 4) 0.1 ( 5)

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