Forty-two patients (26 female, 16 male) who underwent PRK surgery
between the ages of 17 and 51 years (mean, 27 ± 9 [SD] years),
were selected for this study after obtaining informed consent in
adherence to the Declaration of Helsinki. For a given patient, the PRK
was performed one eye at a time with an interval of 1 to 2 weeks
between surgeries. Both PRK eyes were sampled except for seven patients
(three female, four male), who volunteered for sampling from only one
eye. Excluded from consideration were patients from whom a 15-μl tear
sample could not be obtained within 3 minutes. For 20 of the surgeries
for which additional informed consent could be obtained, the
contralateral eye was sampled as a control eye. This provided a sample
population of 77 eyes (41 right, 36 left) and 20 contralateral control
eyes. Data are means ± SD. Preoperative mean refractive error was−
3.0 ± 3.0 D (range, 5.0 to −10.0 D). Eighteen of the eyes had
preoperative astigmatism (mean, −1.5 ± 0.6 cylinder; range,−
1.0 to −2.75 cylinder). Seventeen of the patients had previous usage
of contact lenses (mean usage, 4 ± 2 years; range, 1–9 years).
PRK treatments with an excimer laser (Keratom II ArF; Schwind,
Kleinostheim, Germany; [193 nm]) were performed by the same
surgeon at Vital-Laser LLC, Department of Ophthalmology, University
Medical School of Debrecen, Hungary. De-epithelialization was performed
with a blunt keratome blade knife after epithelial marking with a 6.0-
to 6.5-mm Hoffer trephine for spherical correction and 7.5 to 8.0 mm
for astigmatic correction centered over the pupil. The epithelium was
scraped gently from periphery to center, with care taken to avoid
damaging the surface of Bowman‘s layer. Residual epithelial debris was
removed with a sterile microsponge. Epithelial anesthesia was induced
using 0.4% oxybuprocaine hydrochloride eyedrops. The diameter of the
ablation zone was 6.1 ± 0.2 mm (range, 6.0–6.5 mm) for patients
without astigmatism. For those with astigmatism, the diameter of the
astigmatic ablation mask was 7.5 ± 0.6 mm (range, 6.0–8.1 mm),
and the spherical ablation mask was 5.7 ± 0.1 mm (range, 5.3–6.0
mm). The mean ablation depth of the PRK surgery was 48 ± 22 μm
(range, 12–120 μm). The mean ablation depth (47 μm) of the
subgroup without astigmatism was not statistically significantly
different (P > 0.66) from the mean ablation depth (50μ
m) of the astigmatism subgroup.
The postoperative treatment included antibiotic eyedrops (Ciloxan;
Alcon, Fort Worth, TX), hourly on the first postoperative day and five
times daily during the next 5 days for each patient. The eyedrops were
withdrawn for at least 8 hours before tear sampling to avoid the
possibility of tear sample dilution. After the 5-day period, Flucon and
Tears Naturale (both from Alcon) were administered 5 times daily during
the first month, reduced to four times daily for the second month, and
to three times daily for the third month. No other treatment was used
during this period. All patients underwent follow-up examinations at 1,
3, and 6 months after the PRK procedure.
Tear samples for plasminogen activator analyses were obtained
immediately before and immediately after the PRK treatment and on the
third and fifth postoperative days from the PRK-treated eye and the
contralateral eye where it was used. Samples consisted of tears
collected with glass capillaries
16 19 under slit lamp
illumination from the lower tear meniscus (a horizontal thickening of
the precorneal tear film by the lower margin) at the lateral
canthus.
16 Care was taken not to touch the conjunctiva. We
used the same collection method throughout the study. The duration of
the sampling time was recorded, and the secretion rate was calculated
in microliters per minute, dividing the obtained tear volume by the
time of sample collection. Samples used in this investigation had
secretion rates of 5 to 15 μl/min for both the PRK eyes and the
contralateral controls. Samples were centrifuged (1800 rpm) for 8 to 10
minutes right after sample collection, and supernatants were
deep-frozen at −80°C and were thawed only once for measurements.
Plasminogen activator activity was measured in the sample tears by a
spectrophotometric method using human plasminogen and a
plasmin-specific chromogenic peptide substrate,
d-valyl-
l-leucyl-
l-lysine-
p-nitroanilide
(S-2251).
20 This assay is sensitive predominantly to
urokinase-like plasminogen activator.
16 Plasminogen and
the S-2251 were purchased from Chromogenix (Môlndal, Sweden).
Urokinase standard was purchased from Choay (Paris, France). This assay
is suitable to measure plasmin activity but can also be used for
determining plasminogen activator activity by adding plasminogen to the
reagents. Plasminogen activator activity was measured as described by
Shimada et al.
20 with the following modifications
according to Tözsér et al.
16 and
Tözsér and Berta
21 : 5 μl tear, or standard
urokinase, or plasmin was incubated in 100 μl of 0.05 Tris buffer (pH
7.4) at 37°C in the presence of 0.5 mM chromogenic substrate S-2251
and 1 μM human plasminogen in wells of microtiter plates. After 4
hours’ incubation, the reaction was terminated by the addition of 500μ
l of 8 M acetic acid. The absorption was measured at 405 nm with a
spectrophotometer (Multiscan MS; Labsystem, Helsinki, Finland).
Plasminogen independent amidolytic activity was measured similarly, but
plasminogen was omitted from the incubation mixture. The absorption
difference between the values obtained with and without plasminogen was
considered to be due to the plasminogen activator activity in tear,
whereas the absorbance value obtained without plasminogen was
considered to be plasminogen-independent amidolytic activity. Based on
the absorption values gained in the same system with urokinase standard
solutions with different concentrations we produced a calibration
curve. The plasminogen activator activities of the measured samples
were calculated with this calibration curve and were expressed in
international units per milliliter urokinase equivalent values.
Plasminogen-independent activity was found to be negligible in all the
tear samples.
Determination of haze was made without any knowledge of the plasminogen
activator levels for any of the patients. Therefore, there was no bias
in the determination of haze or in the correlation of plasminogen
activator activity with haze. The haze-grading system of Hanna was
adopted.
13
Standard statistical procedures were used to compare patient
characteristics between different groups (t-test for means
of correlated pairs). Plasminogen activator activities were compared
between different groups using t-tests for means with equal
variances. Comparisons with control eyes were performed using paired t-tests. Differences resulting in P < 0.05
were considered significant, and P < 0.001 was
considered highly significant.