Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Tear plasmin activity with contact lens wear Antti Vannas, Deborah F. Sweeney, Brien A. Holden, Elina Sapyska, EevaMarjatta Salonen & Antti Vaheri To cite this article: Antti Vannas, Deborah F. Sweeney, Brien A. Holden, Elina Sapyska, EevaMarjatta Salonen & Antti Vaheri (1992) Tear plasmin activity with contact lens wear, Current Eye Research, 11:3, 243-251, DOI: 10.3109/02713689209001775 To link to this article: http://dx.doi.org/10.3109/02713689209001775
Published online: 02 Jul 2009.
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Current Eye Research
Volume I I iiumhcr 3 1992. 243-251
Tear plasmin activity with contact lens wear ~~~
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Antti Vanna\, Deborah F Sweeney, Brien A.Holden, Elina Sapyska, Eeva-Marjatta Salonen' and Antti Vaheri'
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Cornea and Contact Lens Research Unit, School of Optometry, University of New South Wales, Sydney, Australia and 'Department of Virology, University of Helsinki, Helsinki, Finland
ABSTRACT Plasmin, a proteolytic enzyme, has been detected in the tears of patients experiencing anterior ocular disease, and during contact lens wear. Using a radial caseinolysis procedure, we examined tear plasmin levels in 66 patients who were wearing soft and rigid lenses for daily and extended wear. Compared to non-contact lens wearers, patients wearing soft and rigid lenses for extended wear were significantly more likely to exhibit tear plasmin activity. Eight hours of open-eye thick HEMA lens wear did not induce tear plasmin activity in a group of 10 subjects. However, significant increases in tear plasmin activity were recorded after short-term (1 hour) eye closure with and without lens wear, and following overnight (8 hours) eye closure without lens wear. Overnight eye closure also resulted in significantly increased numbers of epithelial cells and leucocytes in the tear fluid. Our results suggest that increased tear plasmin activity during extended contact lens wear may be attributable to the effects of eye closure rather than hypoxia or the presence of the contact lens per se.
role of plasmin in the tear fluid involves extracellular proteolysis following trauma, infection and inflammation. It has been suggested that plasmin may play a pathogenic role in anterior ocular disease. Tervo er a1 have detected tear plasmin activity in 70% of eyes exhibiting corneal disease, or where there was conjunctival bacterial growth or mild subclinical viral infection (9). Tear plasmin levels are increased in patients with corneal ulcers (10,ll) and plasmin activity can be detected in the tears of allergic individuals within five minutes of topical exposure to allergens (12). Contact lens wear is also associated with increased tear plasmin activity (13,14). It has been suggested that the proteolytic activity of tear plasmin may contribute to the development of corneal epithelial pathology associated with contact lens wear ( 1 3 ~ 4 ) .Fibronectin, which plays a crucial
INTRODUCTION
Extended wear of hydrogel contact lenses can induce both
role in attaching migrating epithelial cells to the basement membrane during wound healing and repair, is particularly
acute and chronic anterior eye inflammation, and increases
susceptible to proteolysis by plasmin (10). The presence of
the risk of corneal and conjunctival bacterial infection (1-3).
plasmin in the tear fluid thus may delay wound healing
Although the pathogenesis of contact lens-related corneal
following contact lens-associated epithelial injury, increasing
infections is receiving considerable attention in the literature,
the risk of corneal infection. In addition, the chronic
the etiology of acute inflammatory responses such as the
presence of plasmin in the tear fluid during long-term
contact lens-induced 'acute red eye' (CLARE) reaction
extended lens wear may contribute to reduced epithelial
remains unclear. Several factors have been suggested as
adhesion, as described by Madigan et a l ( l 5 ) . Because
contributing to the onset of CLARE, including hypoxia ( 4 3 ,
plasmin is able to activate the complement system (16,17),
tight-fitting lenses ( 4 3 , and toxins released either from
generating factors which are chemotactic for polymorpho-
bacteria (6) or debris (7) trapped under lenses.
nuclear leucocytes (17), tear plasmin may also play a role in
Plasmin, a proteolytic enzyme, is formed from plasminogen, a proenzyme present in body tissues and fluids,
contact lens-induced inflammatory responses. This paper reports a preliminary series of studies which
including tears. Plasminogen activators, which mediate the
were conducted to compare the levels of tear plasmin activity
conversion of plasminogen to active plasmin, are present in
for soft and rigid contact lens wearers on daily and extended
the tear fluid and in corneal epithelial cells (8). The primary
wearing schedules, and for noncontact lens wearers. We also
Reccivcd o n Novenibcr 18. 1991. accepted on February 26. 1992
0 Oxford University Press
243
Current Eye Research evaluated the effects of eye dosure and contact lens-induced
of this assay has been reported to be O.lpg/ml (lo), our
hypoxia on tear plasmin levels.
plasmin standards did not always show activity at less than 1Ccg/d.
MATERIALS AND METHODS
Procedures
Tear samole collecth
Study A: Tear Dla Sm e v e l s for controls and contact Ienswear inp subiects. Ninety-four subjects participated in the
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Tears were collected from the inferior-temporal tear meniscus at the lower lid margin using a method developed
study after the procedures had been fully explained. No
by Sack et a1 (18). Five to six microlitres of tears were
subject had any sign of ocular disease. Subject details are
collected over a period of 5-10 minutes by trained technicians
listed in Table 1. Twenty-eight subjects acted as controls and
using disposable calibrated fire-polished lop1 glass capillary
were unadapted to contact lens wear. The remaining 66
tubes. Care was taken to ensure that no trauma was caused
subjects were participants in on-going clinical contact lens
and to avoid stimulation of reflex tearing. If any trauma was
trials at the Cornea and Contact Lens Research Unit
noted or reflex tearing suspected, the tear sample was
(CCLRU).
rejected. To guard further against sample contamination
Thirty-six of the contact lens-wearing subjects were
from reflex tearing, a subjective rating of the comfort of tear
wearing a mid-water content hydrogel lens (Etafilcon A, 58%
collection was also obtained from each subject at the time of
water content; Vistakon, Jacksonville, Florida) on either a
collection.
daily wear basis (n = 15), or for 6 to 13 nights of extended
After collection, capillary tubes were sealed with paraffin
wear (n = 21). Fourteen subjects were wearing a low Dk
and stored at 4'C. Analysis was conducted on the day of
RGP lens (Boston RxD, Dk/L = 22; Polymer Technology
collection, as repeated freezing and thawing of tear fluid
Corporation, Wilmington, Massachusetts) for daily wear,
samples has been shown to decrease enzyme activity (10).
while 16 were wearing moderate to high Dk RGP lenses
Assessment of oroteolvtic activify
(Fluoroperm, Dk/L = 38; Quantum 11, Dk/L = 39;
Proteolytic activity was measured by a radial caseinolysis
Equalens 11, Dk/L = 77; Polymer Technology Corporation)
procedure (10,19), which uses agarose gels loaded with bovine
on an extended wear basis (6 nights per week). The contact
milk casein as a substrate. Human plasmin (20 casein units
lens-wearing experience of these subjects, including their lens-
per mg; Kabi Diagnostica, Stockholm) was used as a
wearing history prior to participation in CCLRU clinical
standard. The areas of the lytic zones produced on the casein
trials, is summarised in Table 1.
agarose gels by tear fluid enzymatic activity were measured
Tear samples were collected between 11:OO and 17:OO at
after 24 and 48 hours of incubation at 37 * C, using the IBAS
routine aftercare visits, and analysed for plasmin activity, as
image analysis system (Kontron, Munich, West Germany).
described above.
The lytic zone areas of appropriately diluted standards,
Studv B: Effect of contact lens wear on tear 01asmin levels.
processed on the same day as the test samples, were
Ten subjects, 9 females and 1 male, aged between 19 and 27
averaged, and a plot of plasmin concentration versus lytic
years (mean age 22 years), participated in the study. All
zone area was produced. Tear fluid plasmin activity in the
subjects were noncontact lens wearers.
test samples was then derived with reference to this plot.
Subjects wore thick HEMA contact lenses (average
Results were expressed as micrograms of plasmin activity per
thickness 324pm, 38% water content, Dk/L = 2.5) in one
millilitre of tear fluid.
eye, chosen at random, for an eight hour period. The other
Previous investigations, using zymographic analysis
eye served as a non-lens-wearingcontrol. Tear samples were
(9,10,12-14) and monoclonal antibodies to plasmin (9,12-14).
collected from both eyes immediately prior to lens insertion
have demonstrated that proteolysis detected using this
(between 08:OO and 09:00), and following eight hours of open-
procedure is due to plasmin activity. Although the sensitivity
eye lens wear. Corneal thickness was also measured in three
244
Current Eye Research
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TABLE 1 Study A: Subject Details and Lens-Wearing History
subjects at these times using a Holden-Payor micro-
technique described by Fullard and Wilson (21). The
pachometer (20), to determine the level of corneal swelling
subject’s head was stabilised by resting the forehead on a
induced over the eight hour period of lens wear.
horizontally mounted headrest and the subject was asked to
Studv C: Effect of eve closure on tear levelS. Short-term (1 hour) studies: The same ten subjects from
fixate vertically downwards towards the irrigating chamber. The central irrigating tube was adjusted to lie 2-3mm below
Study B also participated in this study. Subjects were fitted
the corneal apex. The cornea was irrigated for ten seconds
in one eye, chosen at random, with a mid-water content
with approximately 2nd of sterile isotonic saline which had
hydrogel lens (Etafiicon A, 58% water content; Vistakon).
been fitered through a Millipore fiter (pore size 0.45~1).
Tear samples were collected from both eyes prior to lens
The washings from the corneal surface were collected in a
insertion and immediately following one hour of eye closure.
sterile autoclaved glass vial and the cell suspension was
The lens was then removed and further tear samples were
filtered through a polycarbonate filter paper with 5pm pore
collected 10 minutes and one hour after eye opening.
size. Specimens collected on the filter paper were stained
Diurnal variation in tear plasmin levels: Eight subjects,
with Diff-Quik (supplied by Bacto Laboratories pty. Ltd.,
5 females and 3 males, ranging in age from 20 to 26 years
Liverpool, NSW, Australia), and the numbers of leucocytes
(mean age 22 years), participated in the study after the
and epithelial cells were counted under a light microscope.
procedures had been fully explained. All subjects were non-
Delay between the collection and viewing of the slides was
contact lens wearers, and were not required to wear lenses
minimised in order to obviate cell damage and distortion.
during this study. Tear samples were collected from one eye,
Data analvu
chosen at random, at 14:OO and 22:30 on Day 1, and
To determine any differences in plasmin activity with daily
immediately on eye opening (07:00), and ten minutes (07: 10)
and extended contact lens wear (Study A), a proportions test
and one hour (08:OO) after eye opening on Day 2. Subjects
was performed. Wilcoxon’s matched pairs signed-rank test
slept overnight at the laboratory to ensure that tear samples
was used to examine the effect of hypoxia on tear plasmin
were collected immediately upon eye opening on Day 2.
levels (Study B). Friedman’s analysis of variance was used to
At the same times at which tears were collected (except at
investigate the effects of short periods of eye closure, and to
10 minutes after eye opening) the fellow eye was washed
test for diurnal variation in tear plasmin activity and numbers
using the nonantact corneal irrigation chamber and
of leucocytes and epithelial cells (Study C). When overall
245
Current Eye Research TABLE 2 Study A Incidence and Levels of Plasmin Activity in Tears of Controls and Contact Lens Wearers Controls
sort DW
I
RGP DW
soit EW
RGP EW
nlgb Plasmin +ve - patients - eyes pvalue*
0.009
Tear plasmin (Ig/ml) - mean - range
0 - 0.4
0-8
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* using proportions test, against control eyes
significance was detected, paired comparisons (using
(p = 0.175). The mean levels of tear plasmin were O.Olpg/ml
Wilcoxon’s test) were used to test data against ‘Day 1, 2pm’
and OSpg/ml, for RGP lenses worn on daily and extended
(baseline) results. For all tests, a critical significance level of
wear respectively.
0.05 was used to denote statistical significance.
Stuchr B: Effect of contact lens wear on tear daEight hours of open-eye wear of thick HEMA lenses in
RESULTS && A: Tear o h i n levels for controls and contact & -w subiects None of the control subjects showed evidence of plasmin
this study produced 7.1% corneal edema (range 2.8 to 9.3%)
in the three subjects whose corneal thicknesses were monitored. Table 3 presents the results for tear plasmin activity, for
Prior to lens insertion, eight of the ten subjects
activity in their tears at levels measurable with this method
all subjects.
(Table 2). Daily wear of soft contact lenses was associated
showed no measurable tear plasmin activity. Of the
with measurable plasmin activity in only one subject (7%),
remaining two subjects, one showed plasmin activity in both
with both eyes being positive. With extended wear of the
eyes, and the other in only one eye. The mean levels of tear
same type of soft contact lens, the tear fluid of four subjects
plasmin in the test and control eyes prior to lens insertion
(19%) and five eyes (12%) showed plasmin activity. This
were 1.7 and O.Spg/ml respectively (overall mean l.lpg/ml).
result with extended wear, compared to the control eyes, was
Following lens wear, two subjects showed plasmin activity
statistically significant (p = 0.008). However the difference in
in tears collected from the contact lens-wearing eye. One
plasmin activity between daily and extended lens wear did not
subject exhibited the same level of activity as prior to lens
reach statistical significance(p = 0.145). The mean levels of
insertion, while the second plasmin-positive subject had
tear fluid plasmin (Table 2) were 0.6pg/ml and l.lpg/nd, for
shown no plasmin activity prior to lens insertion. The
the daily and extended wear subjects respectively.
difference in plasmin activity prior to and following contact
One subject (7%) wearing RGP lenses on a daily wear
lens-induced hypoxia was not statistically significant
basis showed measurable tear plasmin activity, in one eye
(p = 0.655). The mean level of plasmin after lens wear was
only (4%; Table 2). Extended wear of RGP lenses was
1.8pg/ml. None of the control eyes exhibited plasmin activity
associated with plasmin activity in the tear fluid of three
at the end of the study.
subjects (19%), in one eye only (9%); this was significantly
Studv c: Effect of eve closure on tear d asmin leveh
different from the control eyes (p = 0.009), but was not
Short-term (1 hour) studies: Prior to lens insertion and eye
significantly different from the result for daily RGP lens wear
closure, no subjects exhibited measurable plasmin activity in
246
Current Eye Research TABLE 3 Study B Tear Plasmin Levels (pg/ml) Before and Atrer 8 Hours OF Thick HEMA Lens Wear
I
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CONTROLEYE
LENSWEARINGEYE-
L
0
0
R
0
0
L
R
0
0
L
R
0
0
L
Y
0
0
0
0
0
10.6
0
0
Plasmin t v e
pvalue.
- mean - median using Wilcoxon’s matched pairs signed-rank test
their tears (Table 4). Following one hour of eye closure, the
their tears (Table 5). Upon eye opening on Day 2, seven
mean level of tear plasmin activity in the control (non-lens-
subjects (88%) exhibited plasmin activity. This overnight
wearing) eye was 6.4pg/ml (median 4.3pg/ml), with five
increase in plasmin activity was statistically significant
subjects (50%) showing activity; this result was significantly
(p = 0.018). Ten minutes later only one subject exhibited any
different from the ‘before test’ result (p = 0.043). Ten
activity, and one hour after eye opening no subjects
minutes and one hour after eye opening, plasmin activity was
demonstrated any measurable tear plasmin activity. The
no longer detected in the tears of the control eyes.
mean levels of plasmin were 3.8pg/ml on eye opening
Following contact lens wear and eye closure, seven subjects (70%) showed plasmin activity (p = 0.018). This activity was
(median 3.9pg/ml), and 0.7pg/ml ten minutes later. Cell counts from eye washes of the fellow eye, performed
still present in three subjects ten minutes after eye opening
immediately following tear collection, followed a similar
but was no longer statistically significant (p = 0.108), and
pattern to the plasmin activity levels (Table 5). On Day 1,
only one subject showed tear plasmin activity one hour later.
low numbers of leucocytes and epithelial cells were present.
The mean levels of tear plasmin were 12.8pg/ml (median
Immediately following eye opening (07:OO) the cell counts
7.0pg/ml) following eye opening, 2.1pg/ml ten minutes later,
were high, but returned to daytime levels within one hour.
and 0.8pg/ml one hour after eye opening.
The increase in cell numbers on eye opening was statistically
Diurnal variation in tear plasmin levels: During the day on
significant (p
Day 1, no subjects showed measurable plasmin activity in
leucocytes).
-
0.012, for both epithelial cells and
247
Current Eye Research
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TABLE 4 Study C: Tear Plasmin Levels (pg/ml) Before and After One Hour of Eye Closure
using Wilcooron’s matched pairs signedmnk test, against ‘before test’ data
DISCUSSION
To investigate the effect of contact lens-induced hypoxia
This paper reports an investigation of tear plasmin levels
on tear plasmin levels, a group of unadapted subjects wore
with and without contact lens wear. In a group of contact
thick HEMA lenses for eight hours with the eyes open.
lens patients wearing hydrogel or RGP lenses on an extended
These lenses are known to produce significant hypoxia and
wear basis, 19% were found to be plasmin-positive. In
carbon dioxide accumulation in the cornea. However, the
comparison, only 7% of hydrogel and RGP daily lens wearers
presence of this lens per se, and the associated levels of
and none of the non-lens-wearing control subjects examined
corneal hypoxia and acidosis, did not induce tear plasmin
in this study showed tear plasmin activity at levels measurable by our assay procedure. Our results c o n f i i the earlier work
activity.
of Tervo el uf (13), although plasmin was encountered less
open-eye environment. In the closed eye, oxygen tension
frequently in the tear fluid of our subjects. Both studies used
decreases (22). carbon dioxide partial pressure increases (23),
a similar assay for plasmin activity. Subject or contact lens
temperature increases (22,24), there is a hyperosmotic shift in
characteristics could account for this difference in results.
the tears (Z),tear secretions diminish (26), and tear
Several reasons have been suggested for the increase in
The closed-eye environment is known to differ from the
composition changes (18). Results from the short-term
plasmin levels with contact lens wear. These include
(1 hour) closed-eye studies reported here demonstrate an
epithelial changes, allergic reactions, and bacterial
increase in tear plasmin activity with eye closure, with and
contamination.
without lens wear. Following eye opening the increased
-0
further factors which we chose to
evaluate were hypoxia and eye closure. 248
plasmin activity rapidly disappeared, with the majority of
Current Eye Research TABLE 5 Study C: Diurnal Variations in Tear Plasmin Levels and Cell Counts from Eye Washing
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0 0
0 0 0
s
Plasmin t v e 0
0
- pvaluet t
-
the l e u m e count contaii d a large n u of cells - the number of cells was estimated from the volume and cell density of the cell maw. using Wilcoxon’s matched airs signe4 rank test, against ‘Day 1, 2pm’ data
subjects showing no measurable activity ten minutes after eye opening. A significant increase in tear plasmin activity was also
Wilson er a1 also found an increase in the proportion of leucocytes, relative to epithelial cells, in the tear fluid following eye closure. Our results c o n f m this fmding and
found after eight hours of overnight sleep without lens wear.
raise the possibility that plasmin may act to mobilise
Again, the effect was found to disappear rapidly, with only
inflammatory cells during eye closure.
one subject out of eight showing measurable activity ten
In this study, caseinolytic activity was assumed to be due
minutes later. The corneal eye washes immediately after eye
to plasmin, on the basis of zymographic analyses performed
opening also showed a marked increase in the number of
by previous investigators (9,10,12-14). This proteolytic
epithelial cells and leucocytes, in accordance with an earlier
activity has also been found to be inhibited by a monoclonal
study (27). It is tempting to hypothesize that the increased
antibody to plasmin (9,12,14). However, recent work at our
number of epithelial cells on eye opening is a result of
laboratory (18) has demonstrated that the protein profile of
possible effects of increased tear plasmin levels on epithelial
the closed-eye tear fluid differs markedly from that in the
cell attachment and cell sloughing. However, as pointed out
open eye. It is possible, therefore, that the proteolytic activity
by Wilson et a1 (27), the absolute number of epithelial cells
of closed-eye tears may be partly due to other unidentified
recovered after eight hours sleep is surprisingly low,
proteins. Experiments are in progress to analyse proteins in
considering that removal of sloughed cells from the tear f h
closed-eye tears for proteolytic activity, and to quantify
is inhibited during eye closure. This suggests that epithelial
plasminogen activator and plasmin activity more accurately
cell sloughing may in fact be reduced during sleep. Our
under both closed- and open-eye conditions, using more
results do not bring any further understanding to this
sensitive techniques recently developed at our laboratory.
question.
In conclusion, on the basis of our fmdings increased tear 249
Current Eye Research plasmin activity with extended contact lens wear may be assumed to be related to the effects of eye closure rather than the effects of lens w e a r p s e . It is possible that some extended wear patients do not recover in some way from these effects, resulting in persistent tear plasmin activity during open-eye lens wear. ACKNOWLEDGEMENTS The authors wish to thank Niqui Sansey for valuable assistance during these studies, Reginald Wong for statistical guidance, and Peter Fagan, Helen Swarbrick, and Robert
Downloaded by [La Trobe University] at 02:21 15 March 2016
Sack for helpful comments and suggestions during preparation of the manuscript. This research was supported
by a grant from the Optometric Vision Research Foundation of Australia (grant no. 90.22).
8. Lantz, E. and Andersson, A. (1982) Release of fibrinolytic activators from the cornea and conjunctiva. 263-267. Graefes Arch. Clin. Exp. Ophthalmol. 9. Tervo, T., Salonen, E.M., Vaheri, A., Immonen, I., Van Setten, G.B., Himberg, J J . and Tarkkanen, A. (1988) Elevation of tear fluid plasmin in corneal disease. Acta Ophthalmol. 46 393-399. 10. Salonen, E.M., Tervo, T., Torma, E., Tarkkanen, A. and Vaheri, A. (1987) Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy. Acta Ophthalmol. 3-12. 11. Berman, M., k r y , R. and Gage, J. (1980) Evidence for a role of the platminogen activator-plasmin system in corneal ulceration. Invest. Ophthalmol. Vis. Sci. 1204-1221. 12. Salonen, E.M., Lauharanta, J., Sim,P.S.,Stephens, R. and Vaheri, A. (1988) Rapid appearance of plasmin in tear fluid after ocular allergen exposure. Clin. Exp. Immunol. ZS, 146-148. 13. Tervo, T., Van Setten, G.B., Andersson, R., Salonen, E.M., Vaheri, A., Immonen, I. and Tarkkanen, A. (1989) Contact lens wear is associated with the appearance of plasmin in the tear fluid: preliminary results. Graefes Arch. Clin. Exp. Ophthalmol. 42-44. 14. Van Setten, G.B., Tervo, T., Andersson, R., Perheentupa, J. and Tarkkanen, A. (1990) Plasmin and epidermal growth factor in the tear fluid of contact-lens wearers: effect of wearing different types of contact lenses and association with clinical findings. Ophthalmic Res.22, 233-240. 15. Madigan, M.C., Holden, B.A. and Kwok, L.S. (1987) Extended wear of contact lenses can compromise corneal epithelial adhesion. Curr. Eye Res. 4, 1257-1260. 16. Ratnoff, O.D. and Naff, G.B. (1967) The conversion of C'ls to C'1 esterase by plasmin and trypsin. J. Exp. Med. 125,337-358. 17. Ward, PA. (1967) A plasmin-split fragment of C'3 as a 189-206. new chemotactic factor. J. Exp. Med. 18. Sack, RA., Tan, KO. and Tan, A. (1992) Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid. Invest. Ophthalmol. Vis. Sci. (in press). 19. Saksela, 0. (1981) Radial caseinolysis in agarose: a simple method for detection of plasminogen activator in the presence of inhibitory substances and serum. Analyt. Biochem. u276-282. 20. Holden, B.A., Poke, K.A., Fonn, D. and Me-, G.W. (1982) Effects of cataract surgery on corneal function. Invest. Ophthalmol. Vis. Sci. 2 343-350. 21. Fullard, R J . and Wilson, G.S. (1986) Investigation of sloughed epithelial cells collected by non-invasive irrigation of the corneal surface. Curr. Eye Res. 5, 847-856. 22. Holden, B.A. and Sweeney, D.F. (1985) The oxygen tension and temperature of the superior palpebral conjunctiva. Acta Ophthalmol. @, 100-103. 23. Holden, B.A., Ross, R. and Jenkins, J. (1987) Hydrogel contact lenses impede carbon dioxide efflux from the human cornea. Cum. Eye Res.& 1283-1290. 24. Fatt, I. and Chaston, J. (1980) Temperature of a
a
CORRESPONDING AUTHOR Deborah F. Sweeney, Cornea and Contact Lens Research Unit, School of Optometry, University of New South Wales,
PO Box 1, Kensington, NSW 2033,AUSTRALIA REFERENCES 1. Zantos, S.G. and Holden, BA. (1978) Ocular changes associated with continuous wear of contact lenses. Aust. J. Optom. 418-426. 2. Mansmith, M.R., Korb, D.R., Greiner, J.V., Henriquez, A.S., Simon, MA. and Finnemore, V.M. (1977) Giant papillary conjunctivitis in contact lens wearers. Am. J. Ophthalmol. &, 697-708. 3. Poggio, E.C., Glynn, RJ., Schein, O.D., Seddon, J.M., Shannon, M.J., Scardino, VA. and Kenyon, K.R. (1989) The incidence of ulcerative keratitis among users of daily-wear and extended-wear soft contact lenses. New Eng. J. Med. U 779-783. 4. Kracher, G.P., Stark, WJ. and Hirst, L.W.(1981) Extended wear contact lenses for aphakia. Am. J. Optom. Physiol. Optics, 467-471. 5. Gordon, A. and Kracher, G.P. (1985) Corneal infitrates and extended-wear contact lenses. J. Am. Optom. Assoc. 198-201. 6. Phillips, A.J., Badenoch, P.R., Grutzmacher, R. and Roussel T.J. (1986) Microbial contamination of extended-wear contact lenses: an investigation of endotoxin as a cause of the acute ocular inflammation reaction. Int. Eyecare, 2 469-475. 7. Mertz, G.W. and Holden, BA. (1981) Clinical implications of extended wear research. Can. J. Optom. fi 203-205.
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contact lens on the eye. Int. Contact Lens Clin. 195198. 25. Terry, J.E. and Hill, R.M. (1978) Human tear osmotic pressure: diurnal variation and the closed lid. Arch. Ophthalmol. 120-122. 26. Baurn, J. (1990) A relatively dry eye during sleep. Cornea, % 1. 27. Wilson, G.S.,O’Leary, D.J. and Holden, B.A. (1989) Cell content of tears following overnight wear of contact lenses. Curr. Eye Res. S, 329-355.
251
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Tear plasmin activity with contact lens wear Antti Vannas, Deborah F. Sweeney, Brien A. Holden, Elina Sapyska, EevaMarjatta Salonen & Antti Vaheri To cite this article: Antti Vannas, Deborah F. Sweeney, Brien A. Holden, Elina Sapyska, EevaMarjatta Salonen & Antti Vaheri (1992) Tear plasmin activity with contact lens wear, Current Eye Research, 11:3, 243-251, DOI: 10.3109/02713689209001775 To link to this article: http://dx.doi.org/10.3109/02713689209001775
Published online: 02 Jul 2009.
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Date: 15 March 2016, At: 02:21
Current Eye Research
Volume I I iiumhcr 3 1992. 243-251
Tear plasmin activity with contact lens wear ~~~
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Antti Vanna\, Deborah F Sweeney, Brien A.Holden, Elina Sapyska, Eeva-Marjatta Salonen' and Antti Vaheri'
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Cornea and Contact Lens Research Unit, School of Optometry, University of New South Wales, Sydney, Australia and 'Department of Virology, University of Helsinki, Helsinki, Finland
ABSTRACT Plasmin, a proteolytic enzyme, has been detected in the tears of patients experiencing anterior ocular disease, and during contact lens wear. Using a radial caseinolysis procedure, we examined tear plasmin levels in 66 patients who were wearing soft and rigid lenses for daily and extended wear. Compared to non-contact lens wearers, patients wearing soft and rigid lenses for extended wear were significantly more likely to exhibit tear plasmin activity. Eight hours of open-eye thick HEMA lens wear did not induce tear plasmin activity in a group of 10 subjects. However, significant increases in tear plasmin activity were recorded after short-term (1 hour) eye closure with and without lens wear, and following overnight (8 hours) eye closure without lens wear. Overnight eye closure also resulted in significantly increased numbers of epithelial cells and leucocytes in the tear fluid. Our results suggest that increased tear plasmin activity during extended contact lens wear may be attributable to the effects of eye closure rather than hypoxia or the presence of the contact lens per se.
role of plasmin in the tear fluid involves extracellular proteolysis following trauma, infection and inflammation. It has been suggested that plasmin may play a pathogenic role in anterior ocular disease. Tervo er a1 have detected tear plasmin activity in 70% of eyes exhibiting corneal disease, or where there was conjunctival bacterial growth or mild subclinical viral infection (9). Tear plasmin levels are increased in patients with corneal ulcers (10,ll) and plasmin activity can be detected in the tears of allergic individuals within five minutes of topical exposure to allergens (12). Contact lens wear is also associated with increased tear plasmin activity (13,14). It has been suggested that the proteolytic activity of tear plasmin may contribute to the development of corneal epithelial pathology associated with contact lens wear ( 1 3 ~ 4 ) .Fibronectin, which plays a crucial
INTRODUCTION
Extended wear of hydrogel contact lenses can induce both
role in attaching migrating epithelial cells to the basement membrane during wound healing and repair, is particularly
acute and chronic anterior eye inflammation, and increases
susceptible to proteolysis by plasmin (10). The presence of
the risk of corneal and conjunctival bacterial infection (1-3).
plasmin in the tear fluid thus may delay wound healing
Although the pathogenesis of contact lens-related corneal
following contact lens-associated epithelial injury, increasing
infections is receiving considerable attention in the literature,
the risk of corneal infection. In addition, the chronic
the etiology of acute inflammatory responses such as the
presence of plasmin in the tear fluid during long-term
contact lens-induced 'acute red eye' (CLARE) reaction
extended lens wear may contribute to reduced epithelial
remains unclear. Several factors have been suggested as
adhesion, as described by Madigan et a l ( l 5 ) . Because
contributing to the onset of CLARE, including hypoxia ( 4 3 ,
plasmin is able to activate the complement system (16,17),
tight-fitting lenses ( 4 3 , and toxins released either from
generating factors which are chemotactic for polymorpho-
bacteria (6) or debris (7) trapped under lenses.
nuclear leucocytes (17), tear plasmin may also play a role in
Plasmin, a proteolytic enzyme, is formed from plasminogen, a proenzyme present in body tissues and fluids,
contact lens-induced inflammatory responses. This paper reports a preliminary series of studies which
including tears. Plasminogen activators, which mediate the
were conducted to compare the levels of tear plasmin activity
conversion of plasminogen to active plasmin, are present in
for soft and rigid contact lens wearers on daily and extended
the tear fluid and in corneal epithelial cells (8). The primary
wearing schedules, and for noncontact lens wearers. We also
Reccivcd o n Novenibcr 18. 1991. accepted on February 26. 1992
0 Oxford University Press
243
Current Eye Research evaluated the effects of eye dosure and contact lens-induced
of this assay has been reported to be O.lpg/ml (lo), our
hypoxia on tear plasmin levels.
plasmin standards did not always show activity at less than 1Ccg/d.
MATERIALS AND METHODS
Procedures
Tear samole collecth
Study A: Tear Dla Sm e v e l s for controls and contact Ienswear inp subiects. Ninety-four subjects participated in the
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Tears were collected from the inferior-temporal tear meniscus at the lower lid margin using a method developed
study after the procedures had been fully explained. No
by Sack et a1 (18). Five to six microlitres of tears were
subject had any sign of ocular disease. Subject details are
collected over a period of 5-10 minutes by trained technicians
listed in Table 1. Twenty-eight subjects acted as controls and
using disposable calibrated fire-polished lop1 glass capillary
were unadapted to contact lens wear. The remaining 66
tubes. Care was taken to ensure that no trauma was caused
subjects were participants in on-going clinical contact lens
and to avoid stimulation of reflex tearing. If any trauma was
trials at the Cornea and Contact Lens Research Unit
noted or reflex tearing suspected, the tear sample was
(CCLRU).
rejected. To guard further against sample contamination
Thirty-six of the contact lens-wearing subjects were
from reflex tearing, a subjective rating of the comfort of tear
wearing a mid-water content hydrogel lens (Etafilcon A, 58%
collection was also obtained from each subject at the time of
water content; Vistakon, Jacksonville, Florida) on either a
collection.
daily wear basis (n = 15), or for 6 to 13 nights of extended
After collection, capillary tubes were sealed with paraffin
wear (n = 21). Fourteen subjects were wearing a low Dk
and stored at 4'C. Analysis was conducted on the day of
RGP lens (Boston RxD, Dk/L = 22; Polymer Technology
collection, as repeated freezing and thawing of tear fluid
Corporation, Wilmington, Massachusetts) for daily wear,
samples has been shown to decrease enzyme activity (10).
while 16 were wearing moderate to high Dk RGP lenses
Assessment of oroteolvtic activify
(Fluoroperm, Dk/L = 38; Quantum 11, Dk/L = 39;
Proteolytic activity was measured by a radial caseinolysis
Equalens 11, Dk/L = 77; Polymer Technology Corporation)
procedure (10,19), which uses agarose gels loaded with bovine
on an extended wear basis (6 nights per week). The contact
milk casein as a substrate. Human plasmin (20 casein units
lens-wearing experience of these subjects, including their lens-
per mg; Kabi Diagnostica, Stockholm) was used as a
wearing history prior to participation in CCLRU clinical
standard. The areas of the lytic zones produced on the casein
trials, is summarised in Table 1.
agarose gels by tear fluid enzymatic activity were measured
Tear samples were collected between 11:OO and 17:OO at
after 24 and 48 hours of incubation at 37 * C, using the IBAS
routine aftercare visits, and analysed for plasmin activity, as
image analysis system (Kontron, Munich, West Germany).
described above.
The lytic zone areas of appropriately diluted standards,
Studv B: Effect of contact lens wear on tear 01asmin levels.
processed on the same day as the test samples, were
Ten subjects, 9 females and 1 male, aged between 19 and 27
averaged, and a plot of plasmin concentration versus lytic
years (mean age 22 years), participated in the study. All
zone area was produced. Tear fluid plasmin activity in the
subjects were noncontact lens wearers.
test samples was then derived with reference to this plot.
Subjects wore thick HEMA contact lenses (average
Results were expressed as micrograms of plasmin activity per
thickness 324pm, 38% water content, Dk/L = 2.5) in one
millilitre of tear fluid.
eye, chosen at random, for an eight hour period. The other
Previous investigations, using zymographic analysis
eye served as a non-lens-wearingcontrol. Tear samples were
(9,10,12-14) and monoclonal antibodies to plasmin (9,12-14).
collected from both eyes immediately prior to lens insertion
have demonstrated that proteolysis detected using this
(between 08:OO and 09:00), and following eight hours of open-
procedure is due to plasmin activity. Although the sensitivity
eye lens wear. Corneal thickness was also measured in three
244
Current Eye Research
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TABLE 1 Study A: Subject Details and Lens-Wearing History
subjects at these times using a Holden-Payor micro-
technique described by Fullard and Wilson (21). The
pachometer (20), to determine the level of corneal swelling
subject’s head was stabilised by resting the forehead on a
induced over the eight hour period of lens wear.
horizontally mounted headrest and the subject was asked to
Studv C: Effect of eve closure on tear levelS. Short-term (1 hour) studies: The same ten subjects from
fixate vertically downwards towards the irrigating chamber. The central irrigating tube was adjusted to lie 2-3mm below
Study B also participated in this study. Subjects were fitted
the corneal apex. The cornea was irrigated for ten seconds
in one eye, chosen at random, with a mid-water content
with approximately 2nd of sterile isotonic saline which had
hydrogel lens (Etafiicon A, 58% water content; Vistakon).
been fitered through a Millipore fiter (pore size 0.45~1).
Tear samples were collected from both eyes prior to lens
The washings from the corneal surface were collected in a
insertion and immediately following one hour of eye closure.
sterile autoclaved glass vial and the cell suspension was
The lens was then removed and further tear samples were
filtered through a polycarbonate filter paper with 5pm pore
collected 10 minutes and one hour after eye opening.
size. Specimens collected on the filter paper were stained
Diurnal variation in tear plasmin levels: Eight subjects,
with Diff-Quik (supplied by Bacto Laboratories pty. Ltd.,
5 females and 3 males, ranging in age from 20 to 26 years
Liverpool, NSW, Australia), and the numbers of leucocytes
(mean age 22 years), participated in the study after the
and epithelial cells were counted under a light microscope.
procedures had been fully explained. All subjects were non-
Delay between the collection and viewing of the slides was
contact lens wearers, and were not required to wear lenses
minimised in order to obviate cell damage and distortion.
during this study. Tear samples were collected from one eye,
Data analvu
chosen at random, at 14:OO and 22:30 on Day 1, and
To determine any differences in plasmin activity with daily
immediately on eye opening (07:00), and ten minutes (07: 10)
and extended contact lens wear (Study A), a proportions test
and one hour (08:OO) after eye opening on Day 2. Subjects
was performed. Wilcoxon’s matched pairs signed-rank test
slept overnight at the laboratory to ensure that tear samples
was used to examine the effect of hypoxia on tear plasmin
were collected immediately upon eye opening on Day 2.
levels (Study B). Friedman’s analysis of variance was used to
At the same times at which tears were collected (except at
investigate the effects of short periods of eye closure, and to
10 minutes after eye opening) the fellow eye was washed
test for diurnal variation in tear plasmin activity and numbers
using the nonantact corneal irrigation chamber and
of leucocytes and epithelial cells (Study C). When overall
245
Current Eye Research TABLE 2 Study A Incidence and Levels of Plasmin Activity in Tears of Controls and Contact Lens Wearers Controls
sort DW
I
RGP DW
soit EW
RGP EW
nlgb Plasmin +ve - patients - eyes pvalue*
0.009
Tear plasmin (Ig/ml) - mean - range
0 - 0.4
0-8
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* using proportions test, against control eyes
significance was detected, paired comparisons (using
(p = 0.175). The mean levels of tear plasmin were O.Olpg/ml
Wilcoxon’s test) were used to test data against ‘Day 1, 2pm’
and OSpg/ml, for RGP lenses worn on daily and extended
(baseline) results. For all tests, a critical significance level of
wear respectively.
0.05 was used to denote statistical significance.
Stuchr B: Effect of contact lens wear on tear daEight hours of open-eye wear of thick HEMA lenses in
RESULTS && A: Tear o h i n levels for controls and contact & -w subiects None of the control subjects showed evidence of plasmin
this study produced 7.1% corneal edema (range 2.8 to 9.3%)
in the three subjects whose corneal thicknesses were monitored. Table 3 presents the results for tear plasmin activity, for
Prior to lens insertion, eight of the ten subjects
activity in their tears at levels measurable with this method
all subjects.
(Table 2). Daily wear of soft contact lenses was associated
showed no measurable tear plasmin activity. Of the
with measurable plasmin activity in only one subject (7%),
remaining two subjects, one showed plasmin activity in both
with both eyes being positive. With extended wear of the
eyes, and the other in only one eye. The mean levels of tear
same type of soft contact lens, the tear fluid of four subjects
plasmin in the test and control eyes prior to lens insertion
(19%) and five eyes (12%) showed plasmin activity. This
were 1.7 and O.Spg/ml respectively (overall mean l.lpg/ml).
result with extended wear, compared to the control eyes, was
Following lens wear, two subjects showed plasmin activity
statistically significant (p = 0.008). However the difference in
in tears collected from the contact lens-wearing eye. One
plasmin activity between daily and extended lens wear did not
subject exhibited the same level of activity as prior to lens
reach statistical significance(p = 0.145). The mean levels of
insertion, while the second plasmin-positive subject had
tear fluid plasmin (Table 2) were 0.6pg/ml and l.lpg/nd, for
shown no plasmin activity prior to lens insertion. The
the daily and extended wear subjects respectively.
difference in plasmin activity prior to and following contact
One subject (7%) wearing RGP lenses on a daily wear
lens-induced hypoxia was not statistically significant
basis showed measurable tear plasmin activity, in one eye
(p = 0.655). The mean level of plasmin after lens wear was
only (4%; Table 2). Extended wear of RGP lenses was
1.8pg/ml. None of the control eyes exhibited plasmin activity
associated with plasmin activity in the tear fluid of three
at the end of the study.
subjects (19%), in one eye only (9%); this was significantly
Studv c: Effect of eve closure on tear d asmin leveh
different from the control eyes (p = 0.009), but was not
Short-term (1 hour) studies: Prior to lens insertion and eye
significantly different from the result for daily RGP lens wear
closure, no subjects exhibited measurable plasmin activity in
246
Current Eye Research TABLE 3 Study B Tear Plasmin Levels (pg/ml) Before and Atrer 8 Hours OF Thick HEMA Lens Wear
I
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CONTROLEYE
LENSWEARINGEYE-
L
0
0
R
0
0
L
R
0
0
L
R
0
0
L
Y
0
0
0
0
0
10.6
0
0
Plasmin t v e
pvalue.
- mean - median using Wilcoxon’s matched pairs signed-rank test
their tears (Table 4). Following one hour of eye closure, the
their tears (Table 5). Upon eye opening on Day 2, seven
mean level of tear plasmin activity in the control (non-lens-
subjects (88%) exhibited plasmin activity. This overnight
wearing) eye was 6.4pg/ml (median 4.3pg/ml), with five
increase in plasmin activity was statistically significant
subjects (50%) showing activity; this result was significantly
(p = 0.018). Ten minutes later only one subject exhibited any
different from the ‘before test’ result (p = 0.043). Ten
activity, and one hour after eye opening no subjects
minutes and one hour after eye opening, plasmin activity was
demonstrated any measurable tear plasmin activity. The
no longer detected in the tears of the control eyes.
mean levels of plasmin were 3.8pg/ml on eye opening
Following contact lens wear and eye closure, seven subjects (70%) showed plasmin activity (p = 0.018). This activity was
(median 3.9pg/ml), and 0.7pg/ml ten minutes later. Cell counts from eye washes of the fellow eye, performed
still present in three subjects ten minutes after eye opening
immediately following tear collection, followed a similar
but was no longer statistically significant (p = 0.108), and
pattern to the plasmin activity levels (Table 5). On Day 1,
only one subject showed tear plasmin activity one hour later.
low numbers of leucocytes and epithelial cells were present.
The mean levels of tear plasmin were 12.8pg/ml (median
Immediately following eye opening (07:OO) the cell counts
7.0pg/ml) following eye opening, 2.1pg/ml ten minutes later,
were high, but returned to daytime levels within one hour.
and 0.8pg/ml one hour after eye opening.
The increase in cell numbers on eye opening was statistically
Diurnal variation in tear plasmin levels: During the day on
significant (p
Day 1, no subjects showed measurable plasmin activity in
leucocytes).
-
0.012, for both epithelial cells and
247
Current Eye Research
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TABLE 4 Study C: Tear Plasmin Levels (pg/ml) Before and After One Hour of Eye Closure
using Wilcooron’s matched pairs signedmnk test, against ‘before test’ data
DISCUSSION
To investigate the effect of contact lens-induced hypoxia
This paper reports an investigation of tear plasmin levels
on tear plasmin levels, a group of unadapted subjects wore
with and without contact lens wear. In a group of contact
thick HEMA lenses for eight hours with the eyes open.
lens patients wearing hydrogel or RGP lenses on an extended
These lenses are known to produce significant hypoxia and
wear basis, 19% were found to be plasmin-positive. In
carbon dioxide accumulation in the cornea. However, the
comparison, only 7% of hydrogel and RGP daily lens wearers
presence of this lens per se, and the associated levels of
and none of the non-lens-wearing control subjects examined
corneal hypoxia and acidosis, did not induce tear plasmin
in this study showed tear plasmin activity at levels measurable by our assay procedure. Our results c o n f i i the earlier work
activity.
of Tervo el uf (13), although plasmin was encountered less
open-eye environment. In the closed eye, oxygen tension
frequently in the tear fluid of our subjects. Both studies used
decreases (22). carbon dioxide partial pressure increases (23),
a similar assay for plasmin activity. Subject or contact lens
temperature increases (22,24), there is a hyperosmotic shift in
characteristics could account for this difference in results.
the tears (Z),tear secretions diminish (26), and tear
Several reasons have been suggested for the increase in
The closed-eye environment is known to differ from the
composition changes (18). Results from the short-term
plasmin levels with contact lens wear. These include
(1 hour) closed-eye studies reported here demonstrate an
epithelial changes, allergic reactions, and bacterial
increase in tear plasmin activity with eye closure, with and
contamination.
without lens wear. Following eye opening the increased
-0
further factors which we chose to
evaluate were hypoxia and eye closure. 248
plasmin activity rapidly disappeared, with the majority of
Current Eye Research TABLE 5 Study C: Diurnal Variations in Tear Plasmin Levels and Cell Counts from Eye Washing
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0 0
0 0 0
s
Plasmin t v e 0
0
- pvaluet t
-
the l e u m e count contaii d a large n u of cells - the number of cells was estimated from the volume and cell density of the cell maw. using Wilcoxon’s matched airs signe4 rank test, against ‘Day 1, 2pm’ data
subjects showing no measurable activity ten minutes after eye opening. A significant increase in tear plasmin activity was also
Wilson er a1 also found an increase in the proportion of leucocytes, relative to epithelial cells, in the tear fluid following eye closure. Our results c o n f m this fmding and
found after eight hours of overnight sleep without lens wear.
raise the possibility that plasmin may act to mobilise
Again, the effect was found to disappear rapidly, with only
inflammatory cells during eye closure.
one subject out of eight showing measurable activity ten
In this study, caseinolytic activity was assumed to be due
minutes later. The corneal eye washes immediately after eye
to plasmin, on the basis of zymographic analyses performed
opening also showed a marked increase in the number of
by previous investigators (9,10,12-14). This proteolytic
epithelial cells and leucocytes, in accordance with an earlier
activity has also been found to be inhibited by a monoclonal
study (27). It is tempting to hypothesize that the increased
antibody to plasmin (9,12,14). However, recent work at our
number of epithelial cells on eye opening is a result of
laboratory (18) has demonstrated that the protein profile of
possible effects of increased tear plasmin levels on epithelial
the closed-eye tear fluid differs markedly from that in the
cell attachment and cell sloughing. However, as pointed out
open eye. It is possible, therefore, that the proteolytic activity
by Wilson et a1 (27), the absolute number of epithelial cells
of closed-eye tears may be partly due to other unidentified
recovered after eight hours sleep is surprisingly low,
proteins. Experiments are in progress to analyse proteins in
considering that removal of sloughed cells from the tear f h
closed-eye tears for proteolytic activity, and to quantify
is inhibited during eye closure. This suggests that epithelial
plasminogen activator and plasmin activity more accurately
cell sloughing may in fact be reduced during sleep. Our
under both closed- and open-eye conditions, using more
results do not bring any further understanding to this
sensitive techniques recently developed at our laboratory.
question.
In conclusion, on the basis of our fmdings increased tear 249
Current Eye Research plasmin activity with extended contact lens wear may be assumed to be related to the effects of eye closure rather than the effects of lens w e a r p s e . It is possible that some extended wear patients do not recover in some way from these effects, resulting in persistent tear plasmin activity during open-eye lens wear. ACKNOWLEDGEMENTS The authors wish to thank Niqui Sansey for valuable assistance during these studies, Reginald Wong for statistical guidance, and Peter Fagan, Helen Swarbrick, and Robert
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Sack for helpful comments and suggestions during preparation of the manuscript. This research was supported
by a grant from the Optometric Vision Research Foundation of Australia (grant no. 90.22).
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a
CORRESPONDING AUTHOR Deborah F. Sweeney, Cornea and Contact Lens Research Unit, School of Optometry, University of New South Wales,
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