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Histologic and Ultrastructural Findings in Human Corneas After Successful Laser In Situ Keratomileusis
Nicole J. Anderson, MD;
Henry F. Edelhauser, PhD;
Nariman Sharara, MD;
Keith P. Thompson, MD;
Roy S. Rubinfeld, MD;
Dawn M. Devaney, OD;
Nancy L'Hernault, MA;
Hans E. Grossniklaus, MD
Arch Ophthalmol. 2002;120:288-293.
ABSTRACT
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Objective To examine the histologic and ultrastructural features of human corneas
after successful laser in situ keratomileusis (LASIK) in 2 patients post mortem.
Methods Portions of 4 corneas were processed for histology, transmission electron
microscopy, and scanning electron microscopy.
Results Case 1 had undergone LASIK 3 months prior to death and case 2 had undergone
LASIK 20 months prior to death. A Hansatome (Bausch & Lomb Surgical Inc,
Clarement, Calif) microkeratome with a 180-µm plate had been used for
case 1 and an Automated Corneal Shaper (Chiron Corporation, Munich, Germany)
with a 160-µm plate had been used for case 2. Histologically, the LASIK
flap measured 160 µm and 150 µm in thickness in case 1 and case
2, respectively. Corneas from both cases exhibited minor epithelial ingrowth
into the wound, reactive keratocytes at the wound margin, irregular collagen
fibrils in the wound bed, and severed collagen bundles at the flap hinge.
These findings were more pronounced in case 1 than in case 2, and the wound
interface was virtually imperceptible in case 2. Additionally, the corneas
from case 1 contained periodic acid-Schiff–positive electron dense material
and wide-spaced collagen at the wound interface, and there was an absence
of corneal nerves.
Conclusions These findings show that changes caused by wound repair that are present
at 3 months are minor 20 months after LASIK.
INTRODUCTION
WOUND HEALING following laser in situ keratomileusis (LASIK) has been
studied in animal models1-2 and
clinically in human corneas.3 The changes caused
by the wound healing process after LASIK have been compared with those that
occur after photorefractive keratectomy (PRK).3-6
The Bowman layer is disrupted by PRK and the resulting corneal haze ensues
secondary to the wound repair response.7-11
The Bowman layer is preserved, however, after LASIK in humans. The role of
the Bowman layer in corneal wound healing is under investigation.12-13 Histologic and ultrastructural studies
in rabbits have shown parallel collagen lamellae in the flap bed and disarranged
collagen alignment with associated reactive keratocytes at the keratectomy
wound margin in rabbit corneas after LASIK.2, 4-5
The few reports of ultrastructural changes in human corneas following LASIK
are in corneal button specimens after penetrating keratoplasty14-16
or in blind phthisical eyes in which LASIK was performed just prior to enucleation.17
In this study, we examined the histologic and ultrastructural findings
after uncomplicated, successful LASIK. Our findings show active wound healing
changes 3 months after LASIK. These changes include reactive keratocytes,
collagen disarray, and eosinophilic periodic acid-Schiff (PAS)–positive
electron dense material at the flap interface. There are minimal changes at
the flap interface 20 months after LASIK.
CASE REPORTS
CASE 1
A 49-year-old man had uncomplicated LASIK performed in July 2000. A
preoperative consensus refraction (2 manifest refractions and 1 cycloplegic
refraction) was –3.00 + 3.00 x 178 OD and –3.25 + 3.24 x
172 OS. His best-corrected visual acuity was 20/20 OU. Corneal thickness measurements
were obtained using ultrasonic pachymetry and measured 533 µm OD and
530 µm OS. Keratometry values determined by computed corneal topography
were 43.75 x 173/41.75 x 83 OD and 43.75 x 174/42.75 x
84 OS. The patient was corrected for full-distance acuity in both eyes. A
9.5-mm Hansatome (Bausch & Lomb Surgical Inc, Clarement, Calif) microkeratome
with a plate thickness of 180 µm was used to construct the flap in both
eyes. Laser ablation was performed with the Summit Autonomous (Summit, Waltham,
Mass), achieving an ablation depth of 35.8 µm OD and 38.6 µm OS.
On postoperative day 1, the patient's visual acuity was 20/20 OU without correction
and he had an uncomplicated course. At his last postoperative visit (2 weeks
after LASIK), his manifest refraction was –0.25 + 0.75 x 001 OD
and –0.25 + 0.50 x 038 OS. The patient died from an assault just
prior to his 3-month postoperative visit and the corneas were obtained post
mortem. The death-to-preservation time in Optisol-GS (Chiron Ophthalmics,
Irvine, Calif) was 4 hours 35 minutes.
CASE 2
A 55-year-old man had uncomplicated LASIK performed in March 1999. A
manifest refraction to determine the laser inputs was –2.75 OD and –3.00
+ 0.75 x 76 OS. His best-corrected visual acuity was 20/20 OU. Corneal
thickness measurements using ultrasonic pachymetry measured 542 µm OD
and 534 µm OS. Keratometry values were determined by computed corneal
topography and measured 44.34 OD and 44.64 x 83/43.78 x 173 OS.
The patient was corrected for full distance in both eyes. An Automated Corneal
Shaper (Chiron Corporation, Munich, Germany) with a plate thickness of 160
µm was used to make a flap in both eyes. Laser ablation was performed
with the VISX Star Excimer Laser (VISX Inc, Santa Clara, Calif), achieving
an ablation depth of 30.0 µm OD and 25.0 µm OS. The patient's
visual acuity was 20/20 uncorrected on the first postoperative day and he
had an uncomplicated course. At the time of his last examination (1 year postoperatively),
his manifest refraction was plano OD and plano + 0.50 x 30 OS. The patient
died from congestive heart failure 20 months after LASIK and the corneas were
obtained post mortem. The death-to-preservation time in Optisol-GS medium
was 4 hours 10 minutes.
RESULTS
The corneas from both cases were trisected. One third was placed in
10% neutral buffered formalin and each remaining third was placed in 2.5%
glutaraldehyde. The formalin-fixed portions were routinely processed through
increasing concentrations of alcohol, cleared in xylene, and embedded in paraffin.
Sections including the LASIK flap were stained with hematoxylin-eosin, PAS,
and Bodian. One third of each cornea in the 2.5% glutaraldehyde solution was
postfixed with 0.1 M cacodylate buffer and 1% osmium tetroxide, and was embedded
in epoxy resin. Semithin sections were stained with uranyl acetate–
lead citrate and examined with a JEOL 100 CX II (JEOL, Tokyo, Japan) transmission
electron microscope. The LASIK flaps from the remaining corneal thirds were
separated with jeweler's forceps from the corneal bed and the beds and undersurface
of the flaps were processed and scanned with a JEOL 35CF scanning electron
microscope. The flap thickness measured 160 µm and 150 µm in the
corneas from case 1 and case 2, respectively. There was minor epithelial ingrowth,
with microscopic epithelial plugging in the superficial microkeratome wound
in the corneas from both cases (Figure 1).
There was PAS-positive electron dense material at the wound interface in the
corneas from case 1 (Figure 2) but
not in the corneas from case 2. The interface between the flap and bed in
case 2 was barely perceptible, with occasional keratocytes associated with
separations between the flap and bed (Figure
3). There was a focal area of thickened basement membrane of the
basilar epithelium in one cornea from case 2 (Figure 4). This change was overlying a focal, microscopic area of
epithelial debris in the flap-bed interface. Reactive keratocytes were present
at the wound interface in the corneas from case 1 and in the area of the flap
hinge in both cases (Figure 5).
These keratocytes exhibited nuclear chromatin margination and abundant cytoplasm
containing distended rough endoplasmic reticulum. The collagen lamellae in
the corneal bed and undersurface of the flap exhibited a whorled pattern in
both corneas from case 1, which was not present in both corneas from case
2 (Figure 6). A Bodian stain showed
no corneal nerves in case 1 and rare, scattered, short corneal nerves in case
2. The deep stroma, the Descemet membrane, and the endothelium were normal
in both cases. The endothelial cell counts performed on the hematoxylin-eosin–stained
slides averaged 10 and 12 for cases 1 and 2, respectively.
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Figure 1. Case 1, left eye. A, There is
a break in the Bowman layer (between arrowheads) and minor epithelial ingrowth
present (arrow) (hematoxylin-eosin, original magnification x100). B,
The ultrastructure of the area shown in A shows epithelium (epi) extending
into the wound around the edge of the disrupted Bowman layer (arrowhead) (hematoxylin-eosin,
original magnification x4750).
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Figure 2. Case 1, left eye. A, A line of
eosinophilic, periodic acid-Schiff–positive material (arrowhead) with
associated reactive keratocytes (arrow) is present at the interface between
the flap and bed (hematoxylin-eosin, original magnification x100). B,
The corresponding ultrastructure of the area shown in A shows a reactive keratocyte
(arrow) associated with electron dense material (arrowhead) (hematoxylin-eosin,
original magnification, x4750).
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Figure 3. Case 2, right eye. A, The interface
between the flap and bed is not perceptible. Occasional keratocytes (arrows)
are associated with stromal clefts in this area (hematoxylin eosin, original
magnification x100). B, Rare keratocytes (arrow) are associated with
a cleft between the flap and bed (asterisks) (hematoxylin-eosin, original
magnification, x4750).
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Figure 4. Case 2, left eye. There is no
perceptible interface between the flap and bed in this specimen. There is
focal thickening of the basement membrane of the epithelium (arrowheads) (hematoxylin-eosin,
original magnification x100).
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Figure 5. A, Case 1, right eye. B, Case
2, right eye. There are reactive keratocytes (arrows) surrounded by wavy collagen
lamellae in the right cornea from case 1 (A) and the right cornea from case
2 (B) in the area of the flap hinge (original magnification x4750).
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Figure 6. Scanning electron micrographs.
A, Case 1, left eye. The stromal bed exhibits irregular whorls of collagen
lamellae (original magnification x100). B, Case 2, right eye. The stromal
bed is smooth, without a whorling pattern of collagen lamellae (original magnification
x100).
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COMMENT
Studies in rabbits have shown a minimal corneal wound healing response
following LASIK.2, 4-6
In rabbits, well-arranged regular collagen lamellae are present at the interface,
with collagen lamellar irregularities and reactive keratocytes appearing only
at the edge of the flap.4-5 In
one of these studies, prominent epithelial plugs and keratocytes appeared
early in the postoperative period and were no longer present at the wound
margin at 2 and 5 months after surgery.2
The activated keratocytes were in close proximity with the epithelium and
were no longer observed at 5 months when the epithelial plugs were much smaller.
Fibronectin and tenascin were shown only at the incision site of the microkeratome
in rabbit corneas,2, 6 suggesting
an active wound healing process at the flap edge. Periodic acid-Schiff–positive
material has been identified at the wound interface in rabbits as late as
9 months after LASIK.1, 3
In vivo confocal microscopy has been used to study stromal changes in
human corneas after LASIK.18-19
Keratocyte activity at the interface seems to peak in the early postoperative
course (1-2 weeks), and by 6 months there appears to be a loss of keratocytes
in the anterior portion of the flap.19 Up until
now, histologic confirmation of these findings in humans has been limited
to diseased corneas requiring penetrating keratoplasty14-16
and blind eyes that underwent LASIK just prior to enucleation (Table 1). 17 Wright and coworkers14 described one patient with epithelial ingrowth leading
to penetrating keratoplasty 6 weeks after undergoing LASIK. In the areas free
of epithelial ingrowth, the interface was nearly invisible, with only slight
irregularities in stromal lamellar thickness at the base of the LASIK treatment
area and a few apoptotic keratocytes seen by transmission electron microscopy.
Jabbur and coworkers15 recently described a
cornea removed after penetrating keratoplasty for ectasia, which occurred
following LASIK. The cornea showed interruption of the Bowman layer and mild
anterior cellularity.15 Geggel and Talley16 described a patient with iatrogenic keratoectasia
examined 22 months after undergoing LASIK. That case exhibited central stromal
thinning and the flap interface exhibited a thin line of PAS-positive material
22 months postoperatively.16 Latvala and coworkers17 studied the effects of LASIK on blind, phthisical
eyes at 8 days, 54 days, and 4 months prior to enucleation. All eyes contained
epithelial plugs at the interface and exhibited fibronectin and tenascin in
the wound. The fibronectin and tenascin were identified only at the flap margin
and were associated with the epithelial plugs at 4 months after LASIK.17
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Clinicopathologic Studies of Human Corneas After LASIK*
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Our findings describe the histologic and ultrastructural findings 3
and 20 months after successful LASIK. There seems to be active wound healing
at 3 months that virtually disappears at 20 months after uncomplicated LASIK.
The wound repair response includes altered collagen alignment, reactive keratocytes,
and PAS-positive electron dense material in the wound interface. Although
it is possible that the pattern of collagen lamellae at the interface in case
1 is an artifact, we feel that is unlikely since both corneas looked the same
and the same technique was used to prepare all 4 corneas. At 20 months postoperatively,
there were occasional areas of separation between the flap and interface,
and only a few reactive keratocytes at the interface, most notably at the
flap hinge. Microscopic epithelial plugs were present at 3 and 20 months following
LASIK. A Bodian stain was negative for corneal nerves at 3 months although
it was positive for rare, short superficial corneal nerves at the edge of
the flap at 20 months. These findings are consistent with the previous reports
that transection of the anterior stromal/epithelial nerve plexus after LASIK
results in reduced corneal sensation 3 to 6 months after LASIK.19-20
Studies in rabbits19 and humans17
have shown an absence of superficial stromal/epithelial nerves shortly after
flap creation and laser ablation. The nerves appear to sprout and regenerate
to almost normal density as early as 2 months postoperatively. In this study,
we found a lack of corneal nerves at 3 months and a few small superficial
nerves at 20 months after LASIK. These findings may account for clinical variability
in the return of corneal sensation after LASIK.21-22
Previous studies have also shown variability in flap thickness after
LASIK. Microkeratomes have plates to cut flaps of various thickness. This
is important when considering the amount of tissue to be ablated; at least
250 µm of corneal thickness should be retained to minimize the possibility
of keratectasia.23-26
Our study is consistent with a previous study that showed overestimation of
flap thickness.14 In case 1, a 180-µm
plate resulted in a 160-µm-thick flap, and in case 2, a 160-µm
plate resulted in a 150-µm-thick flap. Latvala and coworkers16 showed a flap thickness of 60 to 100 µm for
an intended 140-µm-thick flap. It is unknown if this is due to undercutting
by the microkeratome or due to tissue processing. The endothelial cell counts
in cases 1 and 2 were 10 and 12, respectively, which are comparable to age-matched
controls.27 Histologic and ultrastructural
changes following LASIK show a wound healing response occurring at the flap-bed
interface. This wound healing response decreases with time, resulting in minimal
changes at 20 months after LASIK in our cases. Additional pathologic studies
of corneas after successful LASIK are needed since variabilities in surgical
technique, different surgeons, different lasers, and different patients cannot
be discounted in our limited study.
AUTHOR INFORMATION
Submitted for publication April 20, 2001; final revision received September
25, 2001; accepted October 26, 2001.
This study was supported in part by grants RO1 EY00933, P30 EY06360,
and T32 EY07092 from the National Eye Institute, Bethedsa, Md, and an unrestricted
departmental grant from Research to Prevent Blindness Inc, New York, NY.
Corresponding author: Hans E. Grossniklaus, MD, L. F. Montgomery
Ophthalmic Pathology Laboratory, BT428 Emory Eye Center, 1365 Clifton Rd,
Atlanta, GA 30322 (e-mail: ophtheg{at}emory.edu).
From the Department of Ophthalmology, Emory University School of Medicine,
Atlanta, Ga (Drs Anderson, Edelhauser, Sharara, Thompson, and Grossniklaus,
and Ms L'Hernault); and Washington Eye Physicians and Surgeons, Chevy Chase,
Md (Drs Rubinfeld and Devaney). The authors have no propriety interest in
any of the products mentioned.
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ABSTRACT
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