Wound Healing After Excimer Laser Keratomileusis (Photorefractive Keratectomy) in Monkeys Francisco E. Fantes, MD; Khalil D. Hanna, MD; Keith P. Thompson, MD; Michelle Savoldelli
George O. Waring III, MD; Yves Pouliquen, MD;
\s=b\ Laser myopic keratomileusis (photorefractive keratectomy) performed on 29 rhesus monkey corneas with an argon fluoride (193-nm) excimer laser and a computer-controlled, moving slit delivery system. The 4-mm-diameter central ablation zone ranged in depth from 11 \g=m\m (-2 diopters effect) to 46 \g=m\m (-8 diopters effect). Corneas were studied for the 9 months postoperwas
atively by clinical slit-lamp microscopy, and periodically with light and transmission electron microscopy. By 6 weeks, mild to moderate subepithelial haze was apparent in 93% of the corneas, with considerable variability in density. Progressive clearing occurred so that by 6 to 9 months 12 of 13 surviving corneas (92%) were either completely clear (4 corneas) or trace hazy (8 corneas). The epithelium was thickened at 21 days after ablation and returned to normal thickness by 3 months. At 3 weeks, subepithelial fibroblasts were three times the density of normal keratocytes and returned to nearly normal numbers by 9 months. We concluded that the anterior monkey cornea demonstrated a mild, typical wound healing response after excimer laser keratomileusis. {Arch Ophthalmol. 1990;108:665-675)
Table 1.—Argon Fluoride Excimer Laser Keratomileusis and Follow-up of Monkey Corneas Planned
Monkey No.
OD OS OD OS OD OS OS OD OS OD OS OD OS OD OS OD OS
far UV, 193-nm radiation emitted by the argon fluoride excimer laser has demonstrated a unique ability to ablate corneal tissue with submicrometer accuracy, leaving minimal residual tissue damage.1·2 Marshall et al3 suggested that ablation of the central anterior corneal stroma (anterior laser keratomileu¬
The
sis, large area ablation, photorefractive keratectomy) could be performed by the excimer laser to achieve a new radius of curvature without causing corneal scarring. Indeed, it is only necessary to remove 5 to 12
Accepted publication March 7, 1990. From the Department of Ophthalmology, Emory University School of Medicine and the Yerkes Regional Primate Center, Atlanta, Ga (Drs Fantes, Hanna, Waring, and Thompson), and INSERM Unit 86, Paris, France (Dr Pouliquen and Ms Savoldelli). Reprint requests to Department of Ophthalmology, Emory University School of Medicine, 1327 Clifton Rd NE, Atlanta, GA 30322 for
(Dr Waring).
Eye
12
Optical Effect, Diopters
Amount of Total
Subepithelial
Follow-up Time, mo
Haze at Last
Final
Examination*
Statust
-2
3
2.5
Alive
-4
3
2.5
Alive
-2
2.5
2.0
-8
2.5
2.0
-4
9
0.5
-2
9
0.5
Killed
2.0
Alive Alive
-2
1.5
2.0
Alive
-2
2.5
1.5
Killed
-4
2.5
-2
1.5
1.5
Alive
-4
3
0.5
Killed
-8
5.5
2.0
Killed
-4
9
0.5
Alive
OD
-8
9
0.5
Killed
OD OS OD OS
-4
9
0
Alive
-8
9
1
Alive
OD
-8
6
0.5
Killed
OD
-4
5 min
NA
Killed
0
Killed
7 d
OD
-4
OS
Not treated
OD OS
Unablated Controls
3 wk
Killed ...
...
*The scale of 0 to 4 is defined in the "Clinical Examination" section. NA indi¬ cates not applicable. fFinal status was at the time of the last examination.
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2 Weeks
6 Weeks
000 ooo
0) 0) CD
a
fDDDODDD
ti
ooo
A
ooooo
Clear
-4
-2
Attempted
-I
Correction, D
-2
-4
Attempted Correction, D 6 Months
3 Months ±r
"'
Q
Clear -2
-4
Attempted
-8
Correction, D
-2
-4
Attempted Correction,
D
9 Months
Scattergrams of the density of haze of individual according to each group of attempted correction (—2, —4, and —8 diopters [D]) and at different time inter¬ vals after ablation. The opacities were graded 0 to 4+ as Fig
>
1.
corneas
—
Q
Clear
defined in the "Clinical Examination" section. -2
-4
Attempted Correction,
D
of Bowman's layer and stroma per diopter of in¬ tended refractive change for ablation zones from 4 to 6 mm in diameter.4 Corneal wound healing after excimer laser kera¬ tomileusis poses two problems. The first is the optical clarity of the ablated cornea. Published information concerning subepithelial corneal clarity following ex¬ cimer ablation is contradictory. We have previously reported clinical and histological subepithelial opacification and clear corneas after laser keratomileusis in rabbits using the scanning slit delivery system.5 Marshall and colleagues6 used a constricting dia¬ phragm that moved only a few steps in monkeys and reported mild stromal haze that cleared over 1 year. Similarly, McDonald and colleagues7 used a constrict¬ ing diaphragm with more steps and reported opaque corneas in an initial series of monkeys; following µ
technical improvements, the same researchers re¬ ported a milder transient haze in monkeys7 and blind human eyes,8 which cleared in a few months. Seiler (oral communication, September 7, 1989, German Ophthalmological Society, Bonn, West Germany) and McDonald (oral communication, August 27-31, 1989, European Intraocular Implant Council, Zurich, Swit¬ zerland) have reported excimer laser keratomileusis in sighted human eyes with a transient subepithelial
haze. The second corneal wound healing problem con¬ cerns the predictability and stability of the refractive result. Either epithelial hyperplasia or stromal re¬ generation could fill in the ablated zone and alter the calculated refractive correction. An initial achieved correction might change with time if the thickness of the epithelium or the regenerated stroma changes.
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histopathologic study of these wounds over time is important. We report here the clinical (29 eyes) and histopathologic and ultrastructural (16 eyes) results of argon fluoride (193-nm) excimer laser keratomileusis in rhesus monkey eyes.
Thus,
a
MATERIALS AND METHODS Monkey Model and Experimental Design
This study conformed to the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. A total of 29 rhesus monkey eyes were ablated. Monkeys were anesthesized prior to surgery and examined with an intramuscular injection of ketamine hydrochloride (10 mg/kg). One eye was selected for study of short-term injury, one eye for 7 days, and one eye for 21 days. Twentysix additional eyes were treated for longer follow-up in three categories: 8 eyes to correct —2 diopters (D), 8 eyes for
—4 D, and 10 eyes for —8 D (Table 1). Two normal corneas and the peripheral unablated portion of the treated cornea served as control tissue. Laser Variables and Study Groups
An excimer laser (Lambda Physik EMG103, Coherent Radiation Corp, Alton, Mass) was used for all experiments. The resonator cavity was filled with helium, argon, and flu¬ orine gases to specifications by the manufacturer and was configured with stable optics. Pulse energy was 160 mJ ( ± 10% ) for a radiant exposure of 250 mJ/cm2 at the cornea, as measured with a joulemeter (Gentec, Sainte-Foy, Cana¬ da). The pulse frequency was 5 Hz. We calculated the ablation depth per pulse by measuring the thickness of de-epithelialized corneas in freshly enucle¬ ated rabbit eyes with an ultrasonic pachometer (speed of sound in cornea, 1640 m/s), counting the number of pulses needed to perforate the corneas. The value of 0.45 µ per pulse was similar to those reported by others.910 In the three groups of eyes, the calculated maximal central ablation depths were 11 µ (166 pulses, —2 D effect), 23 µ (326 pulses, —4 D effect), and 46 µ (646 pulses, -8 D effect). These calculated values were derived from proprietary mathematical algorithms developed by Hanna et al" and the International Business Machines (IBM) Scientific Cen¬ ter, Paris, France, and correlated well with the ones calcu¬ lated by other investigators.9
Delivery and Coupling Systems
Fig 2.—Graphic presentation of the average density corneal haze in each ablation group (—2, —4, and —8 diopters [D]) at different time in¬ tervals.
The delivery of the 193-nm laser beam was carried out by the Hanna-IBM computer-controlled delivery system de¬ scribed in detail elsewhere." Briefly, a slit mask 2.8 mm long with a mathematically defined configuration for the correc¬ tion of myopia shaped the beam. The slit image was moved by a prism and the lens was controlled by computer-regu¬ lated servomotors. There was no optical device in the deliv¬ ery system to homogenize the beam. The delivery system contained room air. A total of four translation movements across the 90°, 180°, 45°, and 135° meridians were per¬ formed during each operation. For each translation move¬ ment, 42, 82, and 162 pulses were used for the —2, —4, and —8 D theoretical corrections, respectively. The ablated op¬ tical zone was 4 mm in diameter. As the beam emerged from the delivery system, it passed through a conical fixation de¬ vice, similar to that used in the Hanna suction trephine,12 which was attached to both the delivery system and the eye. In addition, there was low flow of humidified, 99.9% purified
Slit-lamp photographs of the rhesus monkey cornea before and after ablation. Left, Indirect oblique illumination of a clear cornea shows only the normal reticulated pattern (arrowheads). Center, 0.5-grade opacity (arrowheads) could be seen and photographed only by indirect oblique illumination. Right, Grade 1+ opacity (arrowheads) could be easily seen by direct diffuse illumination. Fig 3.
—
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Fig 4.—Control
cornea. Left, Light micrograph demonstrates epithelium (E) consisting of six to eight layers of stratified squamous cells (40 to 50 pm thick), with one layer of tall uniform basal cells, three layers of polygonal cells, and approxi¬ mately three layers of surface squamous cells. Bowman's layer (BL) is present. The anterior stroma(s) showed interdigitating collagen lamellae with a few scattered keratocytes. The lamellar organization became better approximately 40 to 50 pm pos¬ terior to Bowman's layer (toluidine blue, original magnification X20). Right, Transmission electron micrograph shows basal epithelial cells with cytoplasmic filaments connecting to hemidesmosomal plaques at the basal plasma membrane. A thin ep¬ ithelial basement membrane is continuous. Bowman's layer is approximately 8 µ thick and consists of fine filaments. The anterior stroma shows multiple short interdigitating collagen bundles studded with focal amorphous electron dense deposits and with normal keratocytes (arrowhead) (original magnification X9500).
Fig 5.—Cornea immediately after ablation, a, Light micrograph shows a smooth stromal surface without steps and the ab¬ sence of Bowman's layer. The interdigitating anterior stromal lamellae are undisturbed (toluidine blue, original magnification X20). There was a marked decrease in the number of keratocytes in the anterior approximately 40 pm of the stroma (be¬ tween arrowheads), b, Transmission electron micrograph demonstrates small undulations of the surface with a discontinuous, electron-dense condensate and some debris, c and d, Keratocytes in the anterior stroma show disintegration and fragmen¬ tation (arrowheads) (original magnification X25 000).
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Fig 6.—Cornea 7 days after ablation. Top, Light micrograph shows thickened epithelium with approximately 10 layers, demonstrating loss of orderly maturation. Epithelium is approximately 60 µ thick. Dark cells, presumably active, occupy approximately 10% of the basal area (arrowheads). The epithelial-stromal junction shows small areas of vacuolization. Fibrocytes are scattered throughout the stroma (toluidine blue, original magnification X40). Center, Transmission electron micro¬ graph demonstrates two rare inflammatory cells within the epithelium (arrowheads). No inflammatory cells were seen in the stroma (asterisk). Increased fibrocytes are present in the anterior stroma, some showing vacuolization. The normal anterior lamellar structure appears unaltered (original magnification X3500). Bottom, Transmission electron micro¬ graph shows focal formation of basement membrane with hemidesmosomes and lamina densa (between arrowheads). Intraepithelial (E) and subepithelial vacuoles (V) are present (original magnification X55 400). S indicates anterior stroma.
nitrogen delivered across the surface of the cornea through the fixation device.
Uniformity of Laser Beam Before surgery on each monkey, the optical elements of the laser and the delivery system were aligned and opti¬ mized to improve the uniformity of the beam The homoge¬ neity of the laser beam was studied quantitatively by pro¬ jecting it onto a fluorescent screen and using a video frame grabber to capture an image that was digitized and dis¬ played as a three-dimensional profile of beam energy. Qualitative assessment was done by evaluating the patterns etched upon blackened photographic paper. The two meth¬ ods compared favorably when examining the beam quality.
Surgical Technique After topical proparacaine hydrochloride was applied to the eye, a 6-mm-diameter disc of filter paper was moistened with 4% cocaine hydrochloride and applied to the surface of the central cornea for a few seconds. Using an operating microscope, the surgeon removed the epithelium with a blunt spatula, leaving 1 to 2 mm of peripheral epithelium intact. The eye was coupled to the delivery system with the fixation cone. Immediately after the surgery, 1 drop of gentamicin sulfate was applied. No further topical medication was given; specifically, no corticosteroids were used. In ad¬ dition, in monkey No. 13, the right eye had a complete tarsorrhaphy for 2 days, and the left eye had a 72-hour bovine collagen contact lens (Bausch and Lomb, Rochester, NY). Clinical Examination
Slit-lamp microscopic examinations and photographs (Zeiss) were done every 2 days until epithelialization was complete, then every week for the first month, and monthly thereafter. One observer (F.E.F.) did all examinations and recorded the findings on prospective data sheets without reference to previous results, with special attention to the location and density of any central haze. Criteria for the density of the opacity follow: grade 0, to¬ tally clear, such that no opacity could be seen by any method of slit-lamp microscopic examination; grade 0.5, a trace or a faint corneal haze seen only by indirect broad tangential illumination; grade 1, haze of minimal density seen with difficulty with direct and diffuse illumination; grade 2, a mild haze easily visible with direct focal slit illumination; grade 3, a moderately dense opacity that partially obscured the iris details; and grade 4, a severely dense opacity that obscured completely the details of intraocular structures.
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Fig 7.—Cornea 3 weeks after ablation. Left, Light micrograph shows epithelium with a gen¬ erally normal pattern of maturation but contin¬ ued thickening (approximately 50 Mm). There is mild vacuolization of the epithelial-stromal junction. The anterior 50 to 60 µ of the stroma (between arrowheads) shows a striking in¬ crease in active fibrocytes (toluidine blue, original magnification X40). Right, Transmis¬ sion electron micrograph shows mild vacu¬ olization beneath the epithelium (E) and the stroma (S). The anterior stroma is almost com¬ pletely occupied by active fibroblasts (aster¬ isks) with poorly organized, presumably newly secreted, extracellular matrix between them. a fairly sharp zone of demarcation
There is
(arrowhead) between this streaming layer of fi¬ brocytes and more normal lamellar collagen structure (original magnification X3500).
Three other observers (K.D.H, G.O.W., K.P.T.) also ex¬ amined the monkeys periodically. When there was a differ¬ ence of opinion on the density grading, the more opaque value was used, but never with a difference of more than 0.5 grade among observers. Refractive results were not reported because the variable results obtained on repeated measurements indicated that the retinoscopic technique was inaccurate in these nonfixating monkeys with changing corneal opacities. Tissue Preparation
Eyes were enucleated according to a prospective schedule (Table 1). The anesthesized monkey was killed immediately after enucleation with an overdose of sodium pentobarbital. After a few drops of 2.5% glutaraldehyde were applied to the corneal surface, the central 7.5 mm of the cornea was trephined, and the corneal button was bisected with a razor blade, epithelial side up. One half was fixed for light and electron microscopy in refrigerated 2.5% glutaraldehyde buffered in 0.1 mmol/L of sodium cacodylate. The tissue was postfixed in 2% osmium tetroxide buffer in 0.1 mmol/L of sodium cacodylate, dehy¬ drated in a graded series of alcohols, and embedded in Ar-
aldite. One-micrometer-thick sections were stained with toluidine blue for orientation and light microscopy. Thin sections were mounted on copper grids and stained with uranyl acetate and lead citrate before being examined by a transmission electron microscope. The other half of each specimen was processed for immunohistochemistry as described elsewhere.13 RESULTS Clinical Observations
Surgery was performed uneventfully in all eyes. Immediately after ablation, the treated areas had a uniform, ground-glass appearance. The profile of the ablated zone was graduated and the edges blended smoothly with the surrounding nonablated area. All corneas were epithelialized by 7 days. The stroma of the normal rhesus monkey cornea had a re¬ ticulated appearance that extended to the periphery,
most apparent anteriorly, and did not disappear after ablation. During the first 2 weeks, 63% of the corneas were clear (Figs 1 to 3). By 6 weeks, a faint subepithelial haze was apparent in all but two corneas (Fig 1), the amount of haze varying among individual corneas. In general, the amount of opacity was worse in the shallower ablations. Some progressive clearing occurred in all corneas so that in 11 of the 13 corneas followed up for 9 months, 3 were clear, 7 had only a trace haze, and 1 had 1+ haze (Fig 1). The thickness of the subepithelial opacity varied in different areas in different corneas but was no more than approxi¬ mately 10% of the stromal thickness. The opacities were 3 to 4 mm in diameter, had a ground-glass ap¬ pearance that was not completely uniform, showed feathered edges, and were distinct from the normal reticulated pattern of the rhesus monkey stroma. was
Light and Transmission Electron Microscopic Observations
The histopathologic structure and ultrastructure of the two control corneas and the areas outside the ab¬ lated zones were normal in all specimens examined (Fig 4). Epithelial thickness ranged from 38 to 52 µ . The number of keratocytes in the anterior 50 µ of stroma was 30 to 50 cells per 0.0132 mm2 (one high-
field). Changes Immediately After Ablation.—The most strik¬ ing acute findings were the relatively smooth surface and the marked reduction in numbers of keratocytes and the presence of damaged keratocytes in the ante¬ rior 30 to 50 µ of the stroma (Fig 5). Seven Days After Ablation.—The epithelium had power
covered the ablated
zone and was thickened to 45 to 70 µ (normal, 40 to 50 µ ). Focal areas of basement membrane and hemidesmosome formation were present. Quiescent keratocytes were few, and fibroblasts appeared in the anterior stroma, approxi-
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10 to 12 weeks after ablation. Top left, top right, and center left, Cornea with clinical opacity of 1.5 density. Top left, Light mi¬ crograph shows epithelium of normal thickness with persistent increase in number of dark basal cells and moderately severe vacuolization (v) at the epithelial-stromal interface. The anterior stroma (between arrow¬ heads) shows densely packed fibroblasts and a generally lamellar pat¬ tern of organization (toluidine blue, original magnification X40). Top right, Transmission electron micrograph shows focally attached basal epithelial cells (E) surrounded by vacuolar spaces (V). Increased num¬ ber of fibroblasts (F) show active rough endoplasmic reticulum (inset, arrowhead). Electron dense deposits consisting of amorphous material and apparent collagenous fibrils occupy stroma (main figure, original magnification X10 200; inset, original magnification X54 000). Center left, Subepithelial stroma contains numerous clumps of microfibrils (ar¬ rowheads), presumably secreted by the fibroblasts (F). Subepithelial vacuoles (V) are present, and the epithelium is focally detached (E)
Fig 8.—Cornea
(original magnification X3500). Center right and bottom, Histologie structure and ultrastructure of cornea with a haze graded 0.5 density. Center right, Light micrograph shows epithelium with normal maturation and thickness with a few dark basal cells associated and mild subep¬ ithelial vacuolization (arrowhead). Anterior stroma contains few active fibroblasts and has well-preserved lamellar configuration (toludine blue, original magnification X40). Bottom, Transmission electron microscopy shows basal epithelial cells with multiple cytoplasmic tonofilaments, a discontinuous basement membrane, a few small subepithelial vacuoles (arrowheads), and fibroblast (F) in the anterior stroma (original magni¬
fication X9500).
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Fig 9.—Cornea 6 months after ablation. Transmission electron micrographs show a difference between hazy and clear corneas. Left, Cornea graded with a clinical density of 1.5 shows extensive thickening of the base¬ ment membrane (between arrowheads), marked vacuolization of the epithelial-stromal junction (V), and focal accumulations of elec¬ tron dense deposits throughout the anterior stroma (arrows) (original magnification X28 000). Right, In a cornea graded with a clinical density of 0.5, basal epithelial cells ap¬ proximate stroma, and basement membrane is present with focal disruptions (arrowheads). The underlying stroma appears normal (origi¬ nal magnification X33 000).
Fig 10.—Cornea 9 months after ablation. Cornea with clinical haze graded 0.5 density. Left, Epithelium shows normal mat¬ uration, dark basal cells, and normal junctional zone. The anterior stroma shows a slightly increased number of quiescent keratocytes (toluidine blue, original magnification X40). Right, Transmission electron micrograph shows normal basal epithe¬ lial cell (E) with normal basement membrane (arrowheads) consisting of a lamina lucida and a lamina densa, with an occa¬ sional focal
disruption.
Anterior stroma has electron dense
mately 35 to 50 cells per high-power field, many showing dilated rough endoplasmic reticulum and cytoplasmic vacuolization (Fig 6). Three Weeks After Surgery.—As the epithelium was restored to a more normal pattern of maturation, a
marked increase in the number of fibroblasts in the anterior 40 to 60 µ of the stroma occurred, with a density of approximately 200 cells per high-power field. The normal anterior stromal lamellar structure appeared somewhat disorganized and more electron dense due to the increased cellularity. The active fi¬ brocytes showed marked increase in rough endoplas¬ mic reticulum, suggesting active synthetic activity, as demonstrated by the appearance of clumps of appar¬ ently newly secreted extracellular matrix between the fibrocytes (Fig 7). The preexisting stromal colla-
deposits.
gen fibrils of normal diameter were still present. Ten to Twelve Weeks After Surgery.—At approxi¬
mately 3 months, the histopathologic distinctions be¬ corneas with denser clinical haze (grade 1.0 to 2.0) and trace haze (grade 0.5) were more marked (Fig 8). In the corneas with the greater amount of haze, there were more dark basal epithelial cells, greater amounts of vacuolization at the epithelial-stromal junction, more fragmentation of the newly secreted basement membrane, larger numbers of activated fi¬ brocytes in the anterior stroma, and larger amounts of extracellular matrix deposits, both amorphous and tween
fibrillar. The general architecture of the anterior stroma was preserved in corneas with both trace and denser haze; we did not observe large-diameter collagen
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present in Descemet's membrane. The endothelium
Table 2.—Summary of Wound Healing Events After Argon Fluoride Excimer Laser Keratomileusis in Monkey Corneas*
Severity Grade by 2-3
7d
21 d
Haze
was
mo
Clear
6 Haze
mo
Clear
9
mo
thickening Dark basal cells
detachment
Subepithelial disruption
Activated anterior stromal fibrocytes Clumps of extracellular stromal microfibrils
0-0.5
2-3
vacuolization Basement membrane 1-2
1
1-2
1-2
2-3
2-3
0-0.5
1-2
0-0.5
0-0.5
0-0.5
0-0.5
severity grades follow: 0 indicates normal; 1, nearly normal; 2, moderately abnormal; and 3, highly abnormal. The clinical grades follow: haze was defined as corneas with grade 1 or more opacity; clear, corneas with grade 0.5 or less opac¬ ity. The
disorganized cicatricial tissue suggestive of layer of subepithelial fibrosis. Six Months After Surgery.—The epithelium showed normal maturation with six to eight cell layers and a
fibrils
or
a new
central thickness of 35 to 40 µ . A few scattered dark basal cells persisted with minimal vacuolization at the epithelial-stromal junction. There was no major distinction in the appearance of the epithelium be¬ tween the clearer and more hazy corneas. The base¬ ment membrane appeared more normal in the clearer corneas, with focal hemidesmosomes and anchoring fibrils and only occasional interruptions, while in the hazier corneas the basement membrane was more ir¬ regular, focally thickened, and more frequently in¬ terrupted. The anterior stroma of the clearer corneas had a normal lamellar pattern with only a slightly increased number of keratocytes (90 cells per highpower field), whereas in the hazier corneas, there was persistence of active fibroblasts with adjacent clumps of amorphous material and microfibrils, presumably newly secreted extracellular matrix (Fig 9). Nine Months After Surgery.—The corneal structure has returned to almost normal at this time. The ep¬ ithelium demonstrated normal maturation and dif¬ ferentiation with a continuous basement membrane studded by hemidesmosomal-attachment complexes. Rarely were these focal discontinuities in the base¬ ment membrane. The keratocytes were generally quiescent, although a few showed increased rough endoplasmic reticulum, cytoplasmic vacuoles, and adjacent clumps of microfibrils and amorphous ma¬ terial. The overall architecture of the anterior stroma was normal, without focal cicatrization (Fig 10, Table
2).
COMMENT
Time After Keratomileusis
Epithelial epithelial Epithelial
normal throughout.
Posterior Stroma, Descemet's Membrane, and Endothelium.—No abnormalities were seen in posterior stromal keratocytes. No newly produced material was
Of the approximately 15 different refractive surgi¬ cal procedures that have evolved over the past century,14 excimer laser keratomileusis holds the greatest promise to achieve accurate and predictable refractive results because the laser theoretically can create any anterior radius of curvature in the central cornea by removing small amounts of tissue with minimal disruption of the underlying stroma, without significantly changing the biomechanics of the cornea.15 The use of an excimer laser for any refractive cor¬ neal surgery is termed photorefractive keratecto¬ my— photo referring to the laser light, refractive re¬ ferring to the purpose of the operation, and keratectomy referring to the removal of tissue by the ablative process. There are numerous types of exci¬ mer laser photorefractive keratectomy: anterior keratomileusis to correct myopia, hyperopia, or astig¬ matism by removing tissue from the central anterior cornea (corneal sculpting, corneal reprofiling, large area ablation)2,3; linear radial keratectomy to correct astigmatism by making deep narrow troughs in the cornea analogous to those made with a diamond knife16; radial keratectomy17; and stromal kerato¬ mileusis in which the laser replaces the cryolathe and the refractive bench to remove tissue from the stro¬ mal side of a resected lenticule, or replaces the microkeratome to remove tissue from the stromal bed in the in situ technique. There are two major areas that must be developed and controlled for excimer laser keratomileusis to be successful: technical aspects of the laser and delivery system, and biological responses of the cornea. Technical improvements in argon fluoride lasers and delivery systems have occurred steadily since this technique was first described in 1983.2 Among the nu¬ merous technical factors that must be defined and controlled are the homogeneity of the laser beam, the amount of radiant exposure delivered to the cornea, the repetition rate of the ablation, the total number of pulses and the total amount of energy delivered, and the shape of the laser beam as it impacts the cor¬ nea. Investigators reporting the results of argon flu¬ oride excimer laser keratomileusis have used differ¬ ent types of lasers and delivery systems with different ablation parameters, so it is difficult to generalize about the results of the surgery except to acknowledge that improvements continue to occur. We have de¬ scribed in detail here and elsewhere1118"20 the laser, delivery system, and technical parameters that we have used. The second area that must be controlled is the structural and biological response of the cornea. This includes the contour and uniformity of the ablated surface as well as the variable wound healing re¬ sponses of individual corneas. Previous reports have described a spectrum of results, from frank opacification to crystal clear corneas.68·21·22 In this study, we
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have characterized the wound healing response in the rhesus monkey cornea, demonstrating that the epi¬ thelium heals well without undergoing significant hyperplasia, and that there is a transient anterior stroma fibroplasia with the production of new extra¬ cellular matrix in eyes that were clinically graded clear to moderately hazy (density of 0 to 2.5 on a scale of 0 to 4). Characteristics of the Ablated Surface
A common underlying assumption of excimer laser corneal surgery is that a smoother keratectomy bed will have less scarring. Although this has never been proved experimentally, practical experience in the field of lamellar corneal surgery, as well as experience in the field of plastic surgery, supports the idea that a clean, smooth incision margin leaves a less promi¬ nent scar. We achieved a smooth surface with very regular transitions in the change in curvature by us¬ ing the scanning slit delivery system. Epithelial Healing We found no clinical problems with epithelial heal¬ ing during the 9 months of observation: no delay in
re-epithelialization, no recurrent epithelial erosion, and no persistent epithelial hyperplasia. We observed a transient thickening of the epithelium during the first few weeks of epithelial healing, but persistent hyperplasia was not present, presumably because of the smooth transitions in curvature that were achieved, such that the epithelium received no stim¬ ulus to thicken and form a large facet. However, it is possible that deeper excisions might stimulate epi¬ thelial hyperplasia, particularly if the contours were not smooth. Epithelial-Stromal Junction Areas of basement membrane discontinuity per¬ sisted for 3 months, associated with dark cells and focal areas of basal cell detachment and vacuolization at the interface. Discontinuity of the basement mem¬ brane is typically seen with epithelial healing over bare stroma.23·24 These irregularities at the epithelialstromal junction may have contributed to the tran¬ sient corneal haze. Anterior Stroma
We think the changes in the anterior stroma were the major cause for the corneal haze. Clinically, the majority of corneas remained clear when they epithelialized by 2 weeks after surgery, presumably be¬ cause there was minimal disruption of the anterior stroma after the acute injury; the normal lamellar architecture persisted, and the keratocytes degener¬ ated and disappeared. This degeneration of kerato¬ cytes was not a specific response to the excimer laser ablation, because it can occur after simple epithelial scrape injury. For example, Crossen25 observed that scrape injuries in rabbits produced visible signs of keratocyte degeneration 30 minutes after scraping and complete absence of cells in the anterior 40% of the stroma at 15 hours. By 24 hours, with the epithe-
Hal wound two thirds closed, the keratocytes had repopulated the subepithelial stroma. Similarly, Binder and collaborators26 described keratocyte degenera¬ tion as far as 300 µ from radial keratotomy inci¬
sions. The subepithelial stromal haze appeared at 2 to 3 weeks after ablation and peaked by 6 to 8 weeks. Dur¬ ing this time, activated fibroblasts migrated into the existing normal structure in numbers many times that of the normal keratocytes and secreted new ex¬ tracellular matrix containing type III collagen and protokeratan, as demonstrated by immunohistochemical staining.13 Ultrastructurally, this material appeared as clumps of microfibrils and dense depos¬ its. In general, we were able to correlate the number of active fibroblasts and the amount of new extracelluar matrix with the amount of clinical haze on a
qualitative—but not quantitative—basis. After approximately 3 months of intense activity,
the number of fibroblasts decreased, and less new ex¬ tracellular matrix was apparent, so that the stromal structure became more normal and the amount of corneal haze diminished. We found no distinct new layer of subepithelial fibrosis on the ablated surface. However, because new extracellular matrix was pro¬ duced, it is possible that there were changes in the volume of the subepithelial stroma that might pro¬ duce alterations in the anterior curvature of the cor¬ nea and might confound the attempt to create an ac¬ curate and stable change in refraction. Among Individual Eyes Both our clinical and histopathologic examinations demonstrated individual differences in the amount of opacity and in the wound healing response. There are two possible explanations for this variability: (1) the injury was different in each individual, and (2) there was a different biological response for each individ¬ ual. Because we standardized the laser ablation and because reliable computer programs controlled both the laser and the delivery system, we do not think that variability in the laser ablation explains the range of responses observed. We think that individual vari¬ ability in wound healing response was the major rea¬ son that some corneas were clear and other corneas exhibited a moderate haze. Improvements in the technical quality of the laser beam and its delivery may decrease the magnitude of the wound healing response, but there is no obvious reason why variations in wound healing after excimer laser keratomileusis should be categorically different from variations in wound healing after radial kera¬ Variation
totomy
or
penetrating keratoplasty—a variability
with which most corneal surgeons are unpleasantly familiar. This lack of uniform wound healing re¬ sponse may significantly affect the predictability of excimer laser keratomileusis. We observed that shallow ablations had a greater amount of haze than the deeper ablations, the oppo¬ site of what we predicted. This might have occurred because the anterior stromal lamellae interdigitated more than the midstromal lamellae, and thus the an-
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terior lamellae might be more disrupted with result¬ ant light scattering and haze. Another possible ex¬ planation relates to the method of delivery of the pulses with the scanning slit delivery system: shal¬ lower ablations required fewer pulses per translation, possibly creating a less uniform surface that could be associated with an increased amount of haze. We did not identify differences in histologie response be¬ tween deeper and shallower ablations. Clinical Significance of Experimental Findings We conclude that argon fluoride excimer laser keratomileusis stimulates a normal wound healing response in both the corneal epithelium and anterior stroma. This is likely to occur after ablation with any laser and delivery system combination. Irregularities in the basal epithelial cells, increased numbers of fi¬ broblasts, and production of extracellular matrix cause light scattering and corneal haze. The intensity, duration, and clinical significance of the haze will vary from one system to another and one individual to another. As the epithelium, the basement mem¬ brane zone, and the anterior stroma heal and remodel, the amount of haze gradually diminishes. In our monkey model, the haze reached subclinical levels
(clear to 0.5 intensity) in the majority of eyes. If this process could be better controlled and the haze reduced to only a transient event, it might be accept¬
able in humans. However, if the haze persists as a distinct opacity, the procedure would not be clinically acceptable. Because we were unable to accurately re¬ fract these animals due to the amount of opacity present, we could not correlate our intended refrac¬ tive change with the achieved refractive change, a task that must be left to human experimentation. Modulation of the corneal wound healing response to excimer laser ablation by corticosteroids or other drugs remains to be investigated. Support for this study was provided by grant EY007388-02 from the National Institutes of Health, Bethesda, Md; a grant-in-aid from Coherent Radiation Corp; a grant-in-aid from INSERM, Paris, France; and the Division of Research Resources at the Yerkes Regional Primate Center, Atlanta, Ga. The Yerkes Regional Primate Center is fully accredited by and a recipient of grant 07BR05364 from the American Association for Accreditation of Lab¬ oratory Animal Care and Biomedicai Research Support. Dr Hanna has a proprietary interest in the laser delivery system used in this study. None of the other authors has a proprietary in¬ terest in the companies or materials involved in the study. Dr Thompson is a receipient of departmental health training grant award T32-EY07092 from the National Institutes of Health.
References 1. Waring GO. Development of a system for excimer laser corneal surgery. Trans Am Ophthalmol Soc. 1989;87:854-983. 2. Trokel SL, Srinivasan R, Braren B. Excimer laser surgery of the cornea. Am J Ophthalmol. 1983;96:710-715. 3. Marshall J, Trokel S, Rothery S, Krueger RR. Photoablative reprofiling of the cornea using an excimer laser: photorefractive keratectomy. Lasers Ophthalmol. 1986;1:21-48. 4. Munnerlyn CR, Koons SJ, Marshall JM. Photorefractive keratectomy: a technique for laser refractive surgery. J Cataract
Refract Surg. 1988;14:46-52. 5. Hanna KD, Pouliquen Y, Waring Go, Savoldelli M. Corneal wound healing after excimer laser ablation in rabbits. Invest Ophthalmol Vis Sci. 1988;29(suppl):390. 6. Marshall J, Trokel S,Rothery S, Krueger R. Long-term healing of the central cornea after photorefractive keratectomy using an excimer laser. Ophthalmology. 1988;95:1411-1421. 7. McDonald M, Frantz J, Santana E, et al. Excimer laser surface
shaping of the primate cornea for the correction of myopia. Invest Ophthalmol Vis Sci. 1988;29(suppl):310. 8. McDonald M, Shofner S, Klyce S, et al. Clinical results of central photorefractive keratectomy (PRK) with the 193nm excimer laser for the treatment of myopia: the blind eye study. Invest Ophthalmol Vis Sci. 1989;30(suppl):216. 9. Krueger RR, Trokel SL. Quantitation of corneal ablation by ultraviolet laser light. Arch Ophthalmol. 1985;103:1741-1742. 10. Puliafito CA, Wong H, Steinert RF. Quantitative and ultra-
structural studies of excimer laser ablation of the cornea at 193 and 248 nanometers. Lasers Surg Med. 1987;7:155-159. 11. Hanna KD, Chastang JC, Asfar L, Samson J, Pouliquen Y, Waring GO. Scanning slit delivery system. J Cataract Refract Surg.
1989;15:390-396. 12. Waring GO, Hanna KD. The Hanna suction punch block and trephine system for penetrating keratoplasty. Arch Ophthalmol. 1989;107:1536-1539. 13. Geiss MJ, Fantes FE, SunderRaj N, et al. Healing of excimer
laser ablated monkey corneas: an immunohistochemical evaluation. Invest Ophthalmol Vis Sci. 1989;30(suppl):189. 14. Waring GO. Making sense of keratospeak: a classification of
refractive corneal surgery. Arch Ophthalmol. 1986;103:1472-1477. 15. Hanna K, Jouve FE, Waring G. Computer simulation of lamellar keratectomy and laser myopic keratomileusis. J Refract
Surg. 1988;4:222-231. 16. Seiler T, Bende T, Wollensak J, Trokel S. Excimer laser keratectomy for correction of astigmatism. Am J Ophthalmol.
1988;105:117-124. 17. Cotliar AM, Schubert HD, Mandel ER, Trokel SL. Excimer laser radial keratotomy. Ophthalmology. 1985;92:206-208. 18. Hanna KD, Chastang JC, Pouliquen Y, Renard G, Asfar L, Waring GO. Excimer laser keratectomy for myopia with a rotating-slit delivery system. Arch Ophthalmol. 1988;106:245-250. 19. L'Esperance FA, Warner JW, Telfair WB, Yoder PR, Martin
CA. Excimer laser instrumentation and technique for human corneal surgery. Arch Ophthalmol. 1989;107:131-139. 20. Hanna KD, Pouliquen Y, Waring GO, et al. Corneal stromal wound healing in rabbits after 193-nm excimer laser surface ablation. Arch Ophthalmol. 1989;107:895-901. 21. Fantes FF, Waring GO. Effect of excimer laser radiant exposure on uniformity of ablated corneal surface. Lasers Surg Med. 1989;9:533-542. 22. Gaster R, Binder B, McCord R, Berns MW, Burstein N. Excimer laser ablation and wound healing of superficial cornea in rabbits and primates. Invest Ophthalmol Vis Sci. 1988;29
(suppl):309. 23. Taylor DM, L'Esperance FA, Del Pero RA, et al. Human excimer laser lamellar keratectomy. Ophthalmology. 1989;96:654-663. 24. Gipson IK, Spurr-Michaud S, Tisdale A, Keough M. Reassembly of the anchoring structures of the corneal epithelium during wound healing repair in the rabbit. Invest Ophthalmol Vis Sci. 1989;30:425-434. 25. Crossen CE. Cellular changes following epithelial abrasion. In: Beuerman RW, Crossen CE, Kaufman AG, eds. Healing Process in the Cornea. Houston, Tex: Gulf Publishing Co; 1989:314. 26. Binder PS, Stainer GA, Zavala EY, Deg J, Akers P. Acute morphologic features of radial keratotomy. Arch Ophthalmol. 1983;101:1113-1116.
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George O. Waring III, MD; Yves Pouliquen, MD;
\s=b\ Laser myopic keratomileusis (photorefractive keratectomy) performed on 29 rhesus monkey corneas with an argon fluoride (193-nm) excimer laser and a computer-controlled, moving slit delivery system. The 4-mm-diameter central ablation zone ranged in depth from 11 \g=m\m (-2 diopters effect) to 46 \g=m\m (-8 diopters effect). Corneas were studied for the 9 months postoperwas
atively by clinical slit-lamp microscopy, and periodically with light and transmission electron microscopy. By 6 weeks, mild to moderate subepithelial haze was apparent in 93% of the corneas, with considerable variability in density. Progressive clearing occurred so that by 6 to 9 months 12 of 13 surviving corneas (92%) were either completely clear (4 corneas) or trace hazy (8 corneas). The epithelium was thickened at 21 days after ablation and returned to normal thickness by 3 months. At 3 weeks, subepithelial fibroblasts were three times the density of normal keratocytes and returned to nearly normal numbers by 9 months. We concluded that the anterior monkey cornea demonstrated a mild, typical wound healing response after excimer laser keratomileusis. {Arch Ophthalmol. 1990;108:665-675)
Table 1.—Argon Fluoride Excimer Laser Keratomileusis and Follow-up of Monkey Corneas Planned
Monkey No.
OD OS OD OS OD OS OS OD OS OD OS OD OS OD OS OD OS
far UV, 193-nm radiation emitted by the argon fluoride excimer laser has demonstrated a unique ability to ablate corneal tissue with submicrometer accuracy, leaving minimal residual tissue damage.1·2 Marshall et al3 suggested that ablation of the central anterior corneal stroma (anterior laser keratomileu¬
The
sis, large area ablation, photorefractive keratectomy) could be performed by the excimer laser to achieve a new radius of curvature without causing corneal scarring. Indeed, it is only necessary to remove 5 to 12
Accepted publication March 7, 1990. From the Department of Ophthalmology, Emory University School of Medicine and the Yerkes Regional Primate Center, Atlanta, Ga (Drs Fantes, Hanna, Waring, and Thompson), and INSERM Unit 86, Paris, France (Dr Pouliquen and Ms Savoldelli). Reprint requests to Department of Ophthalmology, Emory University School of Medicine, 1327 Clifton Rd NE, Atlanta, GA 30322 for
(Dr Waring).
Eye
12
Optical Effect, Diopters
Amount of Total
Subepithelial
Follow-up Time, mo
Haze at Last
Final
Examination*
Statust
-2
3
2.5
Alive
-4
3
2.5
Alive
-2
2.5
2.0
-8
2.5
2.0
-4
9
0.5
-2
9
0.5
Killed
2.0
Alive Alive
-2
1.5
2.0
Alive
-2
2.5
1.5
Killed
-4
2.5
-2
1.5
1.5
Alive
-4
3
0.5
Killed
-8
5.5
2.0
Killed
-4
9
0.5
Alive
OD
-8
9
0.5
Killed
OD OS OD OS
-4
9
0
Alive
-8
9
1
Alive
OD
-8
6
0.5
Killed
OD
-4
5 min
NA
Killed
0
Killed
7 d
OD
-4
OS
Not treated
OD OS
Unablated Controls
3 wk
Killed ...
...
*The scale of 0 to 4 is defined in the "Clinical Examination" section. NA indi¬ cates not applicable. fFinal status was at the time of the last examination.
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2 Weeks
6 Weeks
000 ooo
0) 0) CD
a
fDDDODDD
ti
ooo
A
ooooo
Clear
-4
-2
Attempted
-I
Correction, D
-2
-4
Attempted Correction, D 6 Months
3 Months ±r
"'
Q
Clear -2
-4
Attempted
-8
Correction, D
-2
-4
Attempted Correction,
D
9 Months
Scattergrams of the density of haze of individual according to each group of attempted correction (—2, —4, and —8 diopters [D]) and at different time inter¬ vals after ablation. The opacities were graded 0 to 4+ as Fig
>
1.
corneas
—
Q
Clear
defined in the "Clinical Examination" section. -2
-4
Attempted Correction,
D
of Bowman's layer and stroma per diopter of in¬ tended refractive change for ablation zones from 4 to 6 mm in diameter.4 Corneal wound healing after excimer laser kera¬ tomileusis poses two problems. The first is the optical clarity of the ablated cornea. Published information concerning subepithelial corneal clarity following ex¬ cimer ablation is contradictory. We have previously reported clinical and histological subepithelial opacification and clear corneas after laser keratomileusis in rabbits using the scanning slit delivery system.5 Marshall and colleagues6 used a constricting dia¬ phragm that moved only a few steps in monkeys and reported mild stromal haze that cleared over 1 year. Similarly, McDonald and colleagues7 used a constrict¬ ing diaphragm with more steps and reported opaque corneas in an initial series of monkeys; following µ
technical improvements, the same researchers re¬ ported a milder transient haze in monkeys7 and blind human eyes,8 which cleared in a few months. Seiler (oral communication, September 7, 1989, German Ophthalmological Society, Bonn, West Germany) and McDonald (oral communication, August 27-31, 1989, European Intraocular Implant Council, Zurich, Swit¬ zerland) have reported excimer laser keratomileusis in sighted human eyes with a transient subepithelial
haze. The second corneal wound healing problem con¬ cerns the predictability and stability of the refractive result. Either epithelial hyperplasia or stromal re¬ generation could fill in the ablated zone and alter the calculated refractive correction. An initial achieved correction might change with time if the thickness of the epithelium or the regenerated stroma changes.
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histopathologic study of these wounds over time is important. We report here the clinical (29 eyes) and histopathologic and ultrastructural (16 eyes) results of argon fluoride (193-nm) excimer laser keratomileusis in rhesus monkey eyes.
Thus,
a
MATERIALS AND METHODS Monkey Model and Experimental Design
This study conformed to the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. A total of 29 rhesus monkey eyes were ablated. Monkeys were anesthesized prior to surgery and examined with an intramuscular injection of ketamine hydrochloride (10 mg/kg). One eye was selected for study of short-term injury, one eye for 7 days, and one eye for 21 days. Twentysix additional eyes were treated for longer follow-up in three categories: 8 eyes to correct —2 diopters (D), 8 eyes for
—4 D, and 10 eyes for —8 D (Table 1). Two normal corneas and the peripheral unablated portion of the treated cornea served as control tissue. Laser Variables and Study Groups
An excimer laser (Lambda Physik EMG103, Coherent Radiation Corp, Alton, Mass) was used for all experiments. The resonator cavity was filled with helium, argon, and flu¬ orine gases to specifications by the manufacturer and was configured with stable optics. Pulse energy was 160 mJ ( ± 10% ) for a radiant exposure of 250 mJ/cm2 at the cornea, as measured with a joulemeter (Gentec, Sainte-Foy, Cana¬ da). The pulse frequency was 5 Hz. We calculated the ablation depth per pulse by measuring the thickness of de-epithelialized corneas in freshly enucle¬ ated rabbit eyes with an ultrasonic pachometer (speed of sound in cornea, 1640 m/s), counting the number of pulses needed to perforate the corneas. The value of 0.45 µ per pulse was similar to those reported by others.910 In the three groups of eyes, the calculated maximal central ablation depths were 11 µ (166 pulses, —2 D effect), 23 µ (326 pulses, —4 D effect), and 46 µ (646 pulses, -8 D effect). These calculated values were derived from proprietary mathematical algorithms developed by Hanna et al" and the International Business Machines (IBM) Scientific Cen¬ ter, Paris, France, and correlated well with the ones calcu¬ lated by other investigators.9
Delivery and Coupling Systems
Fig 2.—Graphic presentation of the average density corneal haze in each ablation group (—2, —4, and —8 diopters [D]) at different time in¬ tervals.
The delivery of the 193-nm laser beam was carried out by the Hanna-IBM computer-controlled delivery system de¬ scribed in detail elsewhere." Briefly, a slit mask 2.8 mm long with a mathematically defined configuration for the correc¬ tion of myopia shaped the beam. The slit image was moved by a prism and the lens was controlled by computer-regu¬ lated servomotors. There was no optical device in the deliv¬ ery system to homogenize the beam. The delivery system contained room air. A total of four translation movements across the 90°, 180°, 45°, and 135° meridians were per¬ formed during each operation. For each translation move¬ ment, 42, 82, and 162 pulses were used for the —2, —4, and —8 D theoretical corrections, respectively. The ablated op¬ tical zone was 4 mm in diameter. As the beam emerged from the delivery system, it passed through a conical fixation de¬ vice, similar to that used in the Hanna suction trephine,12 which was attached to both the delivery system and the eye. In addition, there was low flow of humidified, 99.9% purified
Slit-lamp photographs of the rhesus monkey cornea before and after ablation. Left, Indirect oblique illumination of a clear cornea shows only the normal reticulated pattern (arrowheads). Center, 0.5-grade opacity (arrowheads) could be seen and photographed only by indirect oblique illumination. Right, Grade 1+ opacity (arrowheads) could be easily seen by direct diffuse illumination. Fig 3.
—
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Fig 4.—Control
cornea. Left, Light micrograph demonstrates epithelium (E) consisting of six to eight layers of stratified squamous cells (40 to 50 pm thick), with one layer of tall uniform basal cells, three layers of polygonal cells, and approxi¬ mately three layers of surface squamous cells. Bowman's layer (BL) is present. The anterior stroma(s) showed interdigitating collagen lamellae with a few scattered keratocytes. The lamellar organization became better approximately 40 to 50 pm pos¬ terior to Bowman's layer (toluidine blue, original magnification X20). Right, Transmission electron micrograph shows basal epithelial cells with cytoplasmic filaments connecting to hemidesmosomal plaques at the basal plasma membrane. A thin ep¬ ithelial basement membrane is continuous. Bowman's layer is approximately 8 µ thick and consists of fine filaments. The anterior stroma shows multiple short interdigitating collagen bundles studded with focal amorphous electron dense deposits and with normal keratocytes (arrowhead) (original magnification X9500).
Fig 5.—Cornea immediately after ablation, a, Light micrograph shows a smooth stromal surface without steps and the ab¬ sence of Bowman's layer. The interdigitating anterior stromal lamellae are undisturbed (toluidine blue, original magnification X20). There was a marked decrease in the number of keratocytes in the anterior approximately 40 pm of the stroma (be¬ tween arrowheads), b, Transmission electron micrograph demonstrates small undulations of the surface with a discontinuous, electron-dense condensate and some debris, c and d, Keratocytes in the anterior stroma show disintegration and fragmen¬ tation (arrowheads) (original magnification X25 000).
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Fig 6.—Cornea 7 days after ablation. Top, Light micrograph shows thickened epithelium with approximately 10 layers, demonstrating loss of orderly maturation. Epithelium is approximately 60 µ thick. Dark cells, presumably active, occupy approximately 10% of the basal area (arrowheads). The epithelial-stromal junction shows small areas of vacuolization. Fibrocytes are scattered throughout the stroma (toluidine blue, original magnification X40). Center, Transmission electron micro¬ graph demonstrates two rare inflammatory cells within the epithelium (arrowheads). No inflammatory cells were seen in the stroma (asterisk). Increased fibrocytes are present in the anterior stroma, some showing vacuolization. The normal anterior lamellar structure appears unaltered (original magnification X3500). Bottom, Transmission electron micro¬ graph shows focal formation of basement membrane with hemidesmosomes and lamina densa (between arrowheads). Intraepithelial (E) and subepithelial vacuoles (V) are present (original magnification X55 400). S indicates anterior stroma.
nitrogen delivered across the surface of the cornea through the fixation device.
Uniformity of Laser Beam Before surgery on each monkey, the optical elements of the laser and the delivery system were aligned and opti¬ mized to improve the uniformity of the beam The homoge¬ neity of the laser beam was studied quantitatively by pro¬ jecting it onto a fluorescent screen and using a video frame grabber to capture an image that was digitized and dis¬ played as a three-dimensional profile of beam energy. Qualitative assessment was done by evaluating the patterns etched upon blackened photographic paper. The two meth¬ ods compared favorably when examining the beam quality.
Surgical Technique After topical proparacaine hydrochloride was applied to the eye, a 6-mm-diameter disc of filter paper was moistened with 4% cocaine hydrochloride and applied to the surface of the central cornea for a few seconds. Using an operating microscope, the surgeon removed the epithelium with a blunt spatula, leaving 1 to 2 mm of peripheral epithelium intact. The eye was coupled to the delivery system with the fixation cone. Immediately after the surgery, 1 drop of gentamicin sulfate was applied. No further topical medication was given; specifically, no corticosteroids were used. In ad¬ dition, in monkey No. 13, the right eye had a complete tarsorrhaphy for 2 days, and the left eye had a 72-hour bovine collagen contact lens (Bausch and Lomb, Rochester, NY). Clinical Examination
Slit-lamp microscopic examinations and photographs (Zeiss) were done every 2 days until epithelialization was complete, then every week for the first month, and monthly thereafter. One observer (F.E.F.) did all examinations and recorded the findings on prospective data sheets without reference to previous results, with special attention to the location and density of any central haze. Criteria for the density of the opacity follow: grade 0, to¬ tally clear, such that no opacity could be seen by any method of slit-lamp microscopic examination; grade 0.5, a trace or a faint corneal haze seen only by indirect broad tangential illumination; grade 1, haze of minimal density seen with difficulty with direct and diffuse illumination; grade 2, a mild haze easily visible with direct focal slit illumination; grade 3, a moderately dense opacity that partially obscured the iris details; and grade 4, a severely dense opacity that obscured completely the details of intraocular structures.
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Fig 7.—Cornea 3 weeks after ablation. Left, Light micrograph shows epithelium with a gen¬ erally normal pattern of maturation but contin¬ ued thickening (approximately 50 Mm). There is mild vacuolization of the epithelial-stromal junction. The anterior 50 to 60 µ of the stroma (between arrowheads) shows a striking in¬ crease in active fibrocytes (toluidine blue, original magnification X40). Right, Transmis¬ sion electron micrograph shows mild vacu¬ olization beneath the epithelium (E) and the stroma (S). The anterior stroma is almost com¬ pletely occupied by active fibroblasts (aster¬ isks) with poorly organized, presumably newly secreted, extracellular matrix between them. a fairly sharp zone of demarcation
There is
(arrowhead) between this streaming layer of fi¬ brocytes and more normal lamellar collagen structure (original magnification X3500).
Three other observers (K.D.H, G.O.W., K.P.T.) also ex¬ amined the monkeys periodically. When there was a differ¬ ence of opinion on the density grading, the more opaque value was used, but never with a difference of more than 0.5 grade among observers. Refractive results were not reported because the variable results obtained on repeated measurements indicated that the retinoscopic technique was inaccurate in these nonfixating monkeys with changing corneal opacities. Tissue Preparation
Eyes were enucleated according to a prospective schedule (Table 1). The anesthesized monkey was killed immediately after enucleation with an overdose of sodium pentobarbital. After a few drops of 2.5% glutaraldehyde were applied to the corneal surface, the central 7.5 mm of the cornea was trephined, and the corneal button was bisected with a razor blade, epithelial side up. One half was fixed for light and electron microscopy in refrigerated 2.5% glutaraldehyde buffered in 0.1 mmol/L of sodium cacodylate. The tissue was postfixed in 2% osmium tetroxide buffer in 0.1 mmol/L of sodium cacodylate, dehy¬ drated in a graded series of alcohols, and embedded in Ar-
aldite. One-micrometer-thick sections were stained with toluidine blue for orientation and light microscopy. Thin sections were mounted on copper grids and stained with uranyl acetate and lead citrate before being examined by a transmission electron microscope. The other half of each specimen was processed for immunohistochemistry as described elsewhere.13 RESULTS Clinical Observations
Surgery was performed uneventfully in all eyes. Immediately after ablation, the treated areas had a uniform, ground-glass appearance. The profile of the ablated zone was graduated and the edges blended smoothly with the surrounding nonablated area. All corneas were epithelialized by 7 days. The stroma of the normal rhesus monkey cornea had a re¬ ticulated appearance that extended to the periphery,
most apparent anteriorly, and did not disappear after ablation. During the first 2 weeks, 63% of the corneas were clear (Figs 1 to 3). By 6 weeks, a faint subepithelial haze was apparent in all but two corneas (Fig 1), the amount of haze varying among individual corneas. In general, the amount of opacity was worse in the shallower ablations. Some progressive clearing occurred in all corneas so that in 11 of the 13 corneas followed up for 9 months, 3 were clear, 7 had only a trace haze, and 1 had 1+ haze (Fig 1). The thickness of the subepithelial opacity varied in different areas in different corneas but was no more than approxi¬ mately 10% of the stromal thickness. The opacities were 3 to 4 mm in diameter, had a ground-glass ap¬ pearance that was not completely uniform, showed feathered edges, and were distinct from the normal reticulated pattern of the rhesus monkey stroma. was
Light and Transmission Electron Microscopic Observations
The histopathologic structure and ultrastructure of the two control corneas and the areas outside the ab¬ lated zones were normal in all specimens examined (Fig 4). Epithelial thickness ranged from 38 to 52 µ . The number of keratocytes in the anterior 50 µ of stroma was 30 to 50 cells per 0.0132 mm2 (one high-
field). Changes Immediately After Ablation.—The most strik¬ ing acute findings were the relatively smooth surface and the marked reduction in numbers of keratocytes and the presence of damaged keratocytes in the ante¬ rior 30 to 50 µ of the stroma (Fig 5). Seven Days After Ablation.—The epithelium had power
covered the ablated
zone and was thickened to 45 to 70 µ (normal, 40 to 50 µ ). Focal areas of basement membrane and hemidesmosome formation were present. Quiescent keratocytes were few, and fibroblasts appeared in the anterior stroma, approxi-
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10 to 12 weeks after ablation. Top left, top right, and center left, Cornea with clinical opacity of 1.5 density. Top left, Light mi¬ crograph shows epithelium of normal thickness with persistent increase in number of dark basal cells and moderately severe vacuolization (v) at the epithelial-stromal interface. The anterior stroma (between arrow¬ heads) shows densely packed fibroblasts and a generally lamellar pat¬ tern of organization (toluidine blue, original magnification X40). Top right, Transmission electron micrograph shows focally attached basal epithelial cells (E) surrounded by vacuolar spaces (V). Increased num¬ ber of fibroblasts (F) show active rough endoplasmic reticulum (inset, arrowhead). Electron dense deposits consisting of amorphous material and apparent collagenous fibrils occupy stroma (main figure, original magnification X10 200; inset, original magnification X54 000). Center left, Subepithelial stroma contains numerous clumps of microfibrils (ar¬ rowheads), presumably secreted by the fibroblasts (F). Subepithelial vacuoles (V) are present, and the epithelium is focally detached (E)
Fig 8.—Cornea
(original magnification X3500). Center right and bottom, Histologie structure and ultrastructure of cornea with a haze graded 0.5 density. Center right, Light micrograph shows epithelium with normal maturation and thickness with a few dark basal cells associated and mild subep¬ ithelial vacuolization (arrowhead). Anterior stroma contains few active fibroblasts and has well-preserved lamellar configuration (toludine blue, original magnification X40). Bottom, Transmission electron microscopy shows basal epithelial cells with multiple cytoplasmic tonofilaments, a discontinuous basement membrane, a few small subepithelial vacuoles (arrowheads), and fibroblast (F) in the anterior stroma (original magni¬
fication X9500).
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Fig 9.—Cornea 6 months after ablation. Transmission electron micrographs show a difference between hazy and clear corneas. Left, Cornea graded with a clinical density of 1.5 shows extensive thickening of the base¬ ment membrane (between arrowheads), marked vacuolization of the epithelial-stromal junction (V), and focal accumulations of elec¬ tron dense deposits throughout the anterior stroma (arrows) (original magnification X28 000). Right, In a cornea graded with a clinical density of 0.5, basal epithelial cells ap¬ proximate stroma, and basement membrane is present with focal disruptions (arrowheads). The underlying stroma appears normal (origi¬ nal magnification X33 000).
Fig 10.—Cornea 9 months after ablation. Cornea with clinical haze graded 0.5 density. Left, Epithelium shows normal mat¬ uration, dark basal cells, and normal junctional zone. The anterior stroma shows a slightly increased number of quiescent keratocytes (toluidine blue, original magnification X40). Right, Transmission electron micrograph shows normal basal epithe¬ lial cell (E) with normal basement membrane (arrowheads) consisting of a lamina lucida and a lamina densa, with an occa¬ sional focal
disruption.
Anterior stroma has electron dense
mately 35 to 50 cells per high-power field, many showing dilated rough endoplasmic reticulum and cytoplasmic vacuolization (Fig 6). Three Weeks After Surgery.—As the epithelium was restored to a more normal pattern of maturation, a
marked increase in the number of fibroblasts in the anterior 40 to 60 µ of the stroma occurred, with a density of approximately 200 cells per high-power field. The normal anterior stromal lamellar structure appeared somewhat disorganized and more electron dense due to the increased cellularity. The active fi¬ brocytes showed marked increase in rough endoplas¬ mic reticulum, suggesting active synthetic activity, as demonstrated by the appearance of clumps of appar¬ ently newly secreted extracellular matrix between the fibrocytes (Fig 7). The preexisting stromal colla-
deposits.
gen fibrils of normal diameter were still present. Ten to Twelve Weeks After Surgery.—At approxi¬
mately 3 months, the histopathologic distinctions be¬ corneas with denser clinical haze (grade 1.0 to 2.0) and trace haze (grade 0.5) were more marked (Fig 8). In the corneas with the greater amount of haze, there were more dark basal epithelial cells, greater amounts of vacuolization at the epithelial-stromal junction, more fragmentation of the newly secreted basement membrane, larger numbers of activated fi¬ brocytes in the anterior stroma, and larger amounts of extracellular matrix deposits, both amorphous and tween
fibrillar. The general architecture of the anterior stroma was preserved in corneas with both trace and denser haze; we did not observe large-diameter collagen
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present in Descemet's membrane. The endothelium
Table 2.—Summary of Wound Healing Events After Argon Fluoride Excimer Laser Keratomileusis in Monkey Corneas*
Severity Grade by 2-3
7d
21 d
Haze
was
mo
Clear
6 Haze
mo
Clear
9
mo
thickening Dark basal cells
detachment
Subepithelial disruption
Activated anterior stromal fibrocytes Clumps of extracellular stromal microfibrils
0-0.5
2-3
vacuolization Basement membrane 1-2
1
1-2
1-2
2-3
2-3
0-0.5
1-2
0-0.5
0-0.5
0-0.5
0-0.5
severity grades follow: 0 indicates normal; 1, nearly normal; 2, moderately abnormal; and 3, highly abnormal. The clinical grades follow: haze was defined as corneas with grade 1 or more opacity; clear, corneas with grade 0.5 or less opac¬ ity. The
disorganized cicatricial tissue suggestive of layer of subepithelial fibrosis. Six Months After Surgery.—The epithelium showed normal maturation with six to eight cell layers and a
fibrils
or
a new
central thickness of 35 to 40 µ . A few scattered dark basal cells persisted with minimal vacuolization at the epithelial-stromal junction. There was no major distinction in the appearance of the epithelium be¬ tween the clearer and more hazy corneas. The base¬ ment membrane appeared more normal in the clearer corneas, with focal hemidesmosomes and anchoring fibrils and only occasional interruptions, while in the hazier corneas the basement membrane was more ir¬ regular, focally thickened, and more frequently in¬ terrupted. The anterior stroma of the clearer corneas had a normal lamellar pattern with only a slightly increased number of keratocytes (90 cells per highpower field), whereas in the hazier corneas, there was persistence of active fibroblasts with adjacent clumps of amorphous material and microfibrils, presumably newly secreted extracellular matrix (Fig 9). Nine Months After Surgery.—The corneal structure has returned to almost normal at this time. The ep¬ ithelium demonstrated normal maturation and dif¬ ferentiation with a continuous basement membrane studded by hemidesmosomal-attachment complexes. Rarely were these focal discontinuities in the base¬ ment membrane. The keratocytes were generally quiescent, although a few showed increased rough endoplasmic reticulum, cytoplasmic vacuoles, and adjacent clumps of microfibrils and amorphous ma¬ terial. The overall architecture of the anterior stroma was normal, without focal cicatrization (Fig 10, Table
2).
COMMENT
Time After Keratomileusis
Epithelial epithelial Epithelial
normal throughout.
Posterior Stroma, Descemet's Membrane, and Endothelium.—No abnormalities were seen in posterior stromal keratocytes. No newly produced material was
Of the approximately 15 different refractive surgi¬ cal procedures that have evolved over the past century,14 excimer laser keratomileusis holds the greatest promise to achieve accurate and predictable refractive results because the laser theoretically can create any anterior radius of curvature in the central cornea by removing small amounts of tissue with minimal disruption of the underlying stroma, without significantly changing the biomechanics of the cornea.15 The use of an excimer laser for any refractive cor¬ neal surgery is termed photorefractive keratecto¬ my— photo referring to the laser light, refractive re¬ ferring to the purpose of the operation, and keratectomy referring to the removal of tissue by the ablative process. There are numerous types of exci¬ mer laser photorefractive keratectomy: anterior keratomileusis to correct myopia, hyperopia, or astig¬ matism by removing tissue from the central anterior cornea (corneal sculpting, corneal reprofiling, large area ablation)2,3; linear radial keratectomy to correct astigmatism by making deep narrow troughs in the cornea analogous to those made with a diamond knife16; radial keratectomy17; and stromal kerato¬ mileusis in which the laser replaces the cryolathe and the refractive bench to remove tissue from the stro¬ mal side of a resected lenticule, or replaces the microkeratome to remove tissue from the stromal bed in the in situ technique. There are two major areas that must be developed and controlled for excimer laser keratomileusis to be successful: technical aspects of the laser and delivery system, and biological responses of the cornea. Technical improvements in argon fluoride lasers and delivery systems have occurred steadily since this technique was first described in 1983.2 Among the nu¬ merous technical factors that must be defined and controlled are the homogeneity of the laser beam, the amount of radiant exposure delivered to the cornea, the repetition rate of the ablation, the total number of pulses and the total amount of energy delivered, and the shape of the laser beam as it impacts the cor¬ nea. Investigators reporting the results of argon flu¬ oride excimer laser keratomileusis have used differ¬ ent types of lasers and delivery systems with different ablation parameters, so it is difficult to generalize about the results of the surgery except to acknowledge that improvements continue to occur. We have de¬ scribed in detail here and elsewhere1118"20 the laser, delivery system, and technical parameters that we have used. The second area that must be controlled is the structural and biological response of the cornea. This includes the contour and uniformity of the ablated surface as well as the variable wound healing re¬ sponses of individual corneas. Previous reports have described a spectrum of results, from frank opacification to crystal clear corneas.68·21·22 In this study, we
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have characterized the wound healing response in the rhesus monkey cornea, demonstrating that the epi¬ thelium heals well without undergoing significant hyperplasia, and that there is a transient anterior stroma fibroplasia with the production of new extra¬ cellular matrix in eyes that were clinically graded clear to moderately hazy (density of 0 to 2.5 on a scale of 0 to 4). Characteristics of the Ablated Surface
A common underlying assumption of excimer laser corneal surgery is that a smoother keratectomy bed will have less scarring. Although this has never been proved experimentally, practical experience in the field of lamellar corneal surgery, as well as experience in the field of plastic surgery, supports the idea that a clean, smooth incision margin leaves a less promi¬ nent scar. We achieved a smooth surface with very regular transitions in the change in curvature by us¬ ing the scanning slit delivery system. Epithelial Healing We found no clinical problems with epithelial heal¬ ing during the 9 months of observation: no delay in
re-epithelialization, no recurrent epithelial erosion, and no persistent epithelial hyperplasia. We observed a transient thickening of the epithelium during the first few weeks of epithelial healing, but persistent hyperplasia was not present, presumably because of the smooth transitions in curvature that were achieved, such that the epithelium received no stim¬ ulus to thicken and form a large facet. However, it is possible that deeper excisions might stimulate epi¬ thelial hyperplasia, particularly if the contours were not smooth. Epithelial-Stromal Junction Areas of basement membrane discontinuity per¬ sisted for 3 months, associated with dark cells and focal areas of basal cell detachment and vacuolization at the interface. Discontinuity of the basement mem¬ brane is typically seen with epithelial healing over bare stroma.23·24 These irregularities at the epithelialstromal junction may have contributed to the tran¬ sient corneal haze. Anterior Stroma
We think the changes in the anterior stroma were the major cause for the corneal haze. Clinically, the majority of corneas remained clear when they epithelialized by 2 weeks after surgery, presumably be¬ cause there was minimal disruption of the anterior stroma after the acute injury; the normal lamellar architecture persisted, and the keratocytes degener¬ ated and disappeared. This degeneration of kerato¬ cytes was not a specific response to the excimer laser ablation, because it can occur after simple epithelial scrape injury. For example, Crossen25 observed that scrape injuries in rabbits produced visible signs of keratocyte degeneration 30 minutes after scraping and complete absence of cells in the anterior 40% of the stroma at 15 hours. By 24 hours, with the epithe-
Hal wound two thirds closed, the keratocytes had repopulated the subepithelial stroma. Similarly, Binder and collaborators26 described keratocyte degenera¬ tion as far as 300 µ from radial keratotomy inci¬
sions. The subepithelial stromal haze appeared at 2 to 3 weeks after ablation and peaked by 6 to 8 weeks. Dur¬ ing this time, activated fibroblasts migrated into the existing normal structure in numbers many times that of the normal keratocytes and secreted new ex¬ tracellular matrix containing type III collagen and protokeratan, as demonstrated by immunohistochemical staining.13 Ultrastructurally, this material appeared as clumps of microfibrils and dense depos¬ its. In general, we were able to correlate the number of active fibroblasts and the amount of new extracelluar matrix with the amount of clinical haze on a
qualitative—but not quantitative—basis. After approximately 3 months of intense activity,
the number of fibroblasts decreased, and less new ex¬ tracellular matrix was apparent, so that the stromal structure became more normal and the amount of corneal haze diminished. We found no distinct new layer of subepithelial fibrosis on the ablated surface. However, because new extracellular matrix was pro¬ duced, it is possible that there were changes in the volume of the subepithelial stroma that might pro¬ duce alterations in the anterior curvature of the cor¬ nea and might confound the attempt to create an ac¬ curate and stable change in refraction. Among Individual Eyes Both our clinical and histopathologic examinations demonstrated individual differences in the amount of opacity and in the wound healing response. There are two possible explanations for this variability: (1) the injury was different in each individual, and (2) there was a different biological response for each individ¬ ual. Because we standardized the laser ablation and because reliable computer programs controlled both the laser and the delivery system, we do not think that variability in the laser ablation explains the range of responses observed. We think that individual vari¬ ability in wound healing response was the major rea¬ son that some corneas were clear and other corneas exhibited a moderate haze. Improvements in the technical quality of the laser beam and its delivery may decrease the magnitude of the wound healing response, but there is no obvious reason why variations in wound healing after excimer laser keratomileusis should be categorically different from variations in wound healing after radial kera¬ Variation
totomy
or
penetrating keratoplasty—a variability
with which most corneal surgeons are unpleasantly familiar. This lack of uniform wound healing re¬ sponse may significantly affect the predictability of excimer laser keratomileusis. We observed that shallow ablations had a greater amount of haze than the deeper ablations, the oppo¬ site of what we predicted. This might have occurred because the anterior stromal lamellae interdigitated more than the midstromal lamellae, and thus the an-
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terior lamellae might be more disrupted with result¬ ant light scattering and haze. Another possible ex¬ planation relates to the method of delivery of the pulses with the scanning slit delivery system: shal¬ lower ablations required fewer pulses per translation, possibly creating a less uniform surface that could be associated with an increased amount of haze. We did not identify differences in histologie response be¬ tween deeper and shallower ablations. Clinical Significance of Experimental Findings We conclude that argon fluoride excimer laser keratomileusis stimulates a normal wound healing response in both the corneal epithelium and anterior stroma. This is likely to occur after ablation with any laser and delivery system combination. Irregularities in the basal epithelial cells, increased numbers of fi¬ broblasts, and production of extracellular matrix cause light scattering and corneal haze. The intensity, duration, and clinical significance of the haze will vary from one system to another and one individual to another. As the epithelium, the basement mem¬ brane zone, and the anterior stroma heal and remodel, the amount of haze gradually diminishes. In our monkey model, the haze reached subclinical levels
(clear to 0.5 intensity) in the majority of eyes. If this process could be better controlled and the haze reduced to only a transient event, it might be accept¬
able in humans. However, if the haze persists as a distinct opacity, the procedure would not be clinically acceptable. Because we were unable to accurately re¬ fract these animals due to the amount of opacity present, we could not correlate our intended refrac¬ tive change with the achieved refractive change, a task that must be left to human experimentation. Modulation of the corneal wound healing response to excimer laser ablation by corticosteroids or other drugs remains to be investigated. Support for this study was provided by grant EY007388-02 from the National Institutes of Health, Bethesda, Md; a grant-in-aid from Coherent Radiation Corp; a grant-in-aid from INSERM, Paris, France; and the Division of Research Resources at the Yerkes Regional Primate Center, Atlanta, Ga. The Yerkes Regional Primate Center is fully accredited by and a recipient of grant 07BR05364 from the American Association for Accreditation of Lab¬ oratory Animal Care and Biomedicai Research Support. Dr Hanna has a proprietary interest in the laser delivery system used in this study. None of the other authors has a proprietary in¬ terest in the companies or materials involved in the study. Dr Thompson is a receipient of departmental health training grant award T32-EY07092 from the National Institutes of Health.
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