Exp. Eye Res. (2001) 72, 631±641doi:10.1006/exer.2001.0999, available online at http://www.idealibrary.com on

Human Tear Fluid PDGF-BB, TNF-aaaaa and TGF-bbbbb1 vs Corneal Hazeand Regeneration of Corneal Epithelium and Subbasal Nerve

Plexus after PRK

ILPO S. J. TUOMINENa*, TIMO M.T. TERVOa, ANNA-MAIJA TEPPOb, TUULI U. VALLEa,CAROLA GROÈ NHAGEN-RISKAb AND MINNA H. VESALUOMAa

aThe Department of Ophthalmology, University of Helsinki, Helsinki, Finland and bThe Department ofMedicine, Division of Nephrology, University of Helsinki, Helsinki, Finland

(Received Washington 7 June 2000, accepted in revised form 23 February 2001 and published

myo®brobtaxis and

0014-483

* Addressof OphthalmFIN-00029

electronically 20 April 2001)

The aim was to determine the association of tear ¯uid cytokine levels and post-PRK corneal hazeevaluated by in vivo confocal microscopy. In addition, the possible association between subbasal neuralregeneration and haze formation, or epithelial regeneration were investigated. Twenty eyes of 20patients (16 women and four men, age 30.7+7.5 years, range 21±48 years) underwent a myopicPRK. The spherical equivalent (SE) of the intended correction was ÿ4.7+1.5 D (range ÿ2.75 toÿ9.00 D). ELISA-methods were used to assess tear ¯uid concentrations of TGF-b1, PDGF-BB and TNF-apre-operatively, and post-operatively on day 2 and at 3 months. Tear ¯uid ¯ow in the collection capillarywas recorded, and rates of cytokine release (� tear ¯uid ¯ow-corrected concentrations) were calculated.In vivo confocal microscopy was performed at 3 months to evaluate the corneal morphology and todetermine numerical haze estimate. There was wide interindividual variation between pre-operative andpost-operative concentrations and rates of release of TGF-b1, PDGF-BB and TNF-a. Subepithelial hazewas observed in all corneas and the mean haze estimate was 506+401 U (100±1410 U). However, noassociation was found between tear ¯uid cytokine levels and post-PRK haze. Regenerating subbasalnerve plexus was found in 18 out of 20 corneas; in two corneas it was absent or could not be visualizeddue to subepithelial haze. The density of the subbasal nerve ®ber bundles had a positive correlation withthe epithelial thickness (Pearson correlation, r � 0.56, P � 0.011), but not with the haze estimate orthe thickness of the haze area. At 3 months post-PRK, haze could be observed in all patients. The resultssuggest that tear ¯uid cytokine analysis, as measured, may not be suitable for screening the potentialcandidates for haze formation. We did not ®nd any correlation between haze and regeneration ofsubbasal nerve plexus, but we demonstrated that the regeneration of subbasal nerve plexus might havesigni®cant in¯uence on regulation of epithelial healing. # 2001 Academic Press

Key words: In vivo confocal microscopy; CMTF; TNF-a; TGF-b1; PDGF-BB; PRK; photorefractiveelium

keratectomy; cornea; subbasal nerve plexus; epith

1. Introduction

Excimer laser photorefractive keratectomy (PRK) is awidely accepted surgical method for correction of mildto moderate refractive errors. Corneal haze andregression are the main post-operative complications,but the underlying mechanisms have not yet beenclearly elucidated. However, information on cornealwound healing has increased signi®cantly during therecent years. Corneal cells are known to expressdifferent cytokines or their receptors potentiallymodulating wound healing (Wilson, He and Lloyd,1992; Wilson et al., 1994a,b, 1996b; Li and Tseng,1995), and cytokine effects have been investigated inanimal models or in vitro. Transforming growthfactor-b1 (TGF-b1), e.g. is a multifunctional cytokineinvolved in the regulation of keratocyte activation,

last transformation, proliferation, chemo-wound healing after refractive surgery

5/01/060631�11 $35.00/0

correspondence to: I. S. J. Tuominen, The Departmentology, University of Helsinki, Eye Bank, P.O. Box 220,

HUS, Finland. E-mail: ilpo.tuominen@helsinki.®

; haze.

(Jester et al., 1996; Andresen, Ledet and Ehlers,1997; Myers et al., 1997; Andresen and Ehlers, 1998,Mùller-Pedersen et al., 1998a,b; Jester et al., 1999).Platelet-derived growth factor-BB (PDGF-BB) has alsobeen shown to have mitogenic, chemotactic andmigratory effects on corneal keratocytes in vitro(Hoppenreijs et al., 1993; Andresen et al., 1997;Andresen and Ehlers, 1998, Kim et al., 1999). Tumornecrosis factor-a (TNF-a), on the other hand, is aninductor of apoptosis, which is supposed to play animportant role in the ®rst steps of corneal woundhealing after PRK (Wilson et al., 1996a,b; Helenaet al., 1998).

PRK induces increased release of TGF-b1, TNF-aand PDGF-BB in human tear ¯uid during the earlydays of wound healing (Vesaluoma et al., 1997a,b,c),but the clinical signi®cance of this ®nding has notbeen examined earlier. Lohmann, Hoffmann andReischl (1998) have, however, been able to show

that patients with high tear epidermal growth factor(EGF) levels at 1 week after PRK had a refraction at

# 2001 Academic Press

and oral diazepam 5±10 mg (Diapam; Orion, Hel-

6 months, which was +1.0 D outside the intendedcorrection. Consequently, it would be of great interestand importance to ®nd a tear ¯uid mediator, thatcould be used as a screening instrument for evalua-tion of the potential risks for post-operative complica-tions in individual patients.

A novel investigation methodÐin vivo confocalmicroscopyÐoffers a unique possibility to evaluatethe association between TGF-b1, TNF-a and PDGF-BBtear levels and the severity of post-PRK haze, asconfocal microscopy through focusing (CMTF)enables numeric estimation of the density of cornealsubepithelial haze (Mùller-Pedersen et al., 1997).Furthermore, our aim was to evaluate the potential

632

correlation between the grade of subbasal neural

concentrations and rates of release in pre-operative vs

regeneration and haze formation after PRK.

2. Materials and Methods

Patients

The present study was approved by the EthicalReview Committee of Helsinki University Eye Hospitaland performed according to The Declaration ofHelsinki. An informed consent was obtained from allpatients. Twenty eyes of 20 patients (16 females andfour males, age 30.7+5.9 years, range 21±48 years)who had undergone myopic PRK were examined onceat 3 months after surgery by in vivo confocal micro-scopy. The spherical equivalent (SE) of the intendedcorrection was ÿ4.7+1.5 D (range ÿ2.75 toÿ9.00 D). The astigmatic correction was performedto 12 patients out of 20. The intended cylindercorrection was ÿ0.73+0.27 D (range ÿ0.50 toÿ1.50 D). Before operation the patients were carefullyexamined, and showed no signs of ocular in¯am-mation, allergy or other ocular diseases. One eye,however, had a history of recurrent corneal erosionsyndrome. The patients were advised not to wear theircontact lenses 2 weeks prior to operation. The clinicalcorneal haze was estimated on slit lamp according to

Fantes scale (Fantes et al., 1990) post-operatively at 3 months.

Excimer Laser Photorefractive Keratectomy

PRK was performed after surgical abrasion of theepithelium (6.5 mm dia. ) using a Beaver Eye Blade(Becton Dickinson, Franklin Lakes, NJ, U.S.A.). Six mmwide PRKs of varying ablation depths were performedusing a VisX 20/20 excimer laser (VisX Co, SunnyvaleCA, U.S.A.) or NIDEK EC 5000 excimer laser (Nidek,

Gamagoni, Aichi, Japan). The mean ablation depth was 60.7+14.1 mm (range 36±93 mm).

Eye-patching and Post-operative Medication

Each eye was pressure-patched for 3 days followingPRK. In the morning of the ®rst and/or second

post-operative day the patch was removed and the lidswere gently cleaned with a paper wipe. After waitingfor about 30 sec the tear ¯uid sample was collected.Then the chloramphenicol ointment (Oftan Chlora;Santen, Tampere, Finland) was applied and the eyewas repatched. In addition to the ointment twice aday for 4 days, the post-operative medication included¯uorometholone drops (Liqui®lm-FML; Allergan,Irvine, CA, U.S.A.) starting on the fourth post-operative day three times a day for 1±3 months,oral diclofenac sodium 25 mg (Voltaren; Ciba-Geigy,Basel, Switzerland) 30 min before the operation andtwo to three times a day for the ®rst days after PRK

I . S . J . TUOMINEN ET AL.

sinki, Finland) for the ®rst two post-operative nights.

Tear Fluid Collection

All tear ¯uid samples were collected with a scaled 5or 25 ml ®re-polished microcapillary tube as previouslydescribed (van Setten et al., 1989). Special attentionwas paid not to irritate the cornea or conjunctiva.Tears were collected pre-operatively (day 0), on thesecond (day 2) post-operative day and at 3 monthspost-operatively. The volume of tear ¯uid needed forour three assays was 5 ml. The samples wereimmediately transferred to Eppendorf tubes and storedat ÿ708C until assessed. The tear ¯uid ¯ow in thecollection capillary (ml minÿ1) was calculated bydividing the volume of the tear ¯uid sample by thetear ¯uid collection time. The tear ¯ow-correctedconcentration, i.e. rate of release was calculated bymultiplying the concentration in the sample (ng lÿ1)by the tear ¯uid ¯ow in the collection capillary(ml minÿ1) (Vesaluoma et al., 1997a,b,c). As theinterindividual variations in the tear ¯uid cytokineconcentrations were high, changes ( %) in the cytokine

post-operative samples were also calculated.

Cytokine Immunoassays

The concentrations of TGF-b1 in tear ¯uid weremeasured by an enzyme immunoassay as follows(Vesaluoma et al., 1997a). Microtiter plates (Max-isorp, Nunc Intermed, Denmark) were coated withmonoclonal mouse anti-TGF-b1, ÿb2, ÿb3 (Gen-zyme Diagnostics, Cambridge, MA, U.S.A.) 0.1 mg perwell in 0.05 M Na2CO3 buffer, pH 9.2 overnight at48C. After washing the wells with 0.05 M phosphate-buffered saline, pH 7.3, containing 0.05 % Tween 20,100 ml of acid-activated and neutralized (0.1 N HCl48C 1 hr) standard dilutions (natural human TGF-b1,Code BDP 1, R & D systems, London, U.K.) andsamples (®nal dilutions 18-fold) were added to thewells and incubated overnight at 48C. The unboundmaterial was removed with above washing buffer, and

100 ml of 1000-fold diluted antibodies to humanTGF-b1 (Code 27.283.29, Jansen Biochimica, Beerse,

lymphotoxins. The detection limit of the assay wasÿ1

produced per each eye. Mean values of the measure-

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Belgium) conjugated with alkaline phosphatase wereadded and incubated at 378C for 2 hr. After washingthe amount of alkaline phosphatase ®xed to the tubeswas determined in diethanolamine (1.0 mol lÿ1)-magnesium chloride (0.5 mol lÿ1) buffer, pH 10.0,using p-nitro-phenylphosphate as substrate for 1 hr atroom temperature. The absorbance of the p-nitro-phenolate liberated was measured at 405 nm with a340 ATC microtitration plate reader (SLT, Lab-instruments, Vienna, Austria). The detection limitfor the assay was 5 ng lÿ1.

PDGF-BB concentrations were measured by asandwich enzyme immunoassay (Vesaluoma et al.,1997b). Microtiter plates (Maxisorp2, NuncInstrumed, Denmark) were coated with goat IgG-type antibody to highly puri®ed Escherichia coli-derivedrecombinant human PDGF-BB (Code AB-220-Na, R &D Systems Europe Ltd, Abingdon, U.K.) (0.25 mg perwell) in 0.05M Na2CO3 buffer, pH 9.2) overnight at48C. After washing the wells with 350 ml of water,100 ml of recombinant human PDGF-BB homodimer(Code 2038-01, Genzyme Diagnostics, Cambridge,MA, U.S.A.) as standard (serial dilutions from 1000to 5 ng lÿ1), or of ®ve to 20-fold diluted tears wereadded to the wells, and incubated at 248C for 60 minon a horizontal rotating table at 80 rpm. The unboundmaterial was removed and wells washed twice withsaline, containing 0.05 % Tween 20. One hundred mlof 100-fold diluted rabbit antibody to human PDGF-BB(Code ZP-215, Genzyme Diagnostics, Cambridge, MA,U.S.A.) was added and incubated for 1 hr at roomtemperature as above. After washing 100 ml of 500-fold diluted alkaline phosphatase conjugated swineantibody to rabbit IgG (Code 67850, Orion Diagnos-tica, Espoo, Finland) was added and incubated asabove. After that, the plates were washed three timeswith 350 ml of washing solution. The amount ofalkaline phosphatase ®xed to the tubes was deter-mined in 1.0 M diethanolamine-0.5M MgCl2 buffer,pH 10.0 (Code 170057, Reagena Ltd, Kuopio,Finland) at room temperature for 3 hr in the dark,using p-nitrophenolphosphate (Code 104-105, SigmaChemical Co., St. Louis, MO, U.S.A.) as substrate. Theabsorbance of p-nitrophenolate liberated wasmeasured at 405 nm with 340 ATC microtitrationplate reader (SLT-Labinstruments, Vienna, Austria).The absorbance of the 0 ng lÿ1 standard was sub-tracted from all the other absorbances, and the deltaabsorbances were used for calculations. The detectionlimit of the assay was 20 ng lÿ1.

TNF-a concentrations were determined by a doubleantibody radioimmunoassay developed for measuringof serum TNF (Teppo and Maury, 1987) as previouslydescribed (Vesaluoma et al., 1997b). A volume of10 ml of tear ¯uid samples was ®rst diluted by addingassay buffer to reach a volume of 100 ml. TNF-a fromtears competed with a ®xed amount of 125 I labeled

TEAR FLUID CYTOKINES, NERVES AND HAZE AFT

TNF-a (10 000 counts per min for 50 ml) for thebinding sites of 30 000-fold diluted speci®c rabbit

antibodies. The bound TNF-a was precipitated withSepharose bound antirabbit IgG and then centrifugedand the radioactivity of the pellets was counted. E. coliderived recombinant human TNF-a (Code TNF-H,Genzyme Diagnostics, Cambridge, U.S.A.) was used asstandard. It had a molecular weight of 36 kDa, and aspeci®c activity 51 � 107 U mgÿ1 of protein, asmeasured by bioassay with mouse L 929 cells. Rabbitantiserum to human TNF-a (Code P-300A, Endogen,MA, U.S.A.) showed 51 % cross reaction with

RK 633

10 ng l .

In vivo Confocal Microscopy

A tandem scanning confocal microscope (TSCM,Model 165A, Tandem Scanning Corporation, Reston,VA, U.S.A.) was used for examining the central corneaof the patients at the Helsinki University Eye Hospital,Finland. The setup and operation of the confocalmicroscope has been described previously (Petroll,Jester and Cavanagh, 1996; Mùller-Pedersen et al.,1997). Brie¯y, a 24 � 0.6 NA variable workingdistance objective lens was used. The ®eld-of-viewwith this lens was 450 � 360 mm, and the z-axisresolution 9 mm. Images were detected using a DageVE1000 low-light level camera and recorded on SVHStape. In addition, confocal microscopy through-focusscans (CMTF) were obtained as previously described(Li et al., 1997; Mùller-Pedersen et al., 1997). Videoimages of interest were digitized using a PC-basedimaging system with custom software (University ofTexas Southwestern Medical Center at Dallas, TX,U.S.A.), and printed using an Epson Stylus Color 800printer (Seiko Epson Corporation, Nagano, Japan)without image enhancement. Using the custom soft-ware, the CMTF data was digitized onto the PC,intensity pro®le curves were calculated, and aquantitative estimate of the increased back-scatteringfrom the subepithelial haze (CMTF-haze estimate) wasachieved. The thickness of the haze area was alsocalculated. One to four acceptable CMTF scans were

ments were used for all statistical calculations.

Grading of Subbasal Innervation

One of our interests was to evaluate the regener-ation of the subbasal nerve plexus. Special attentionwas thus paid to viewing of the subbasal nerve plexus.Grading was based on the confocal microscopicimage, with the most subbasal nerve ®ber bundles,which were then counted. It is important to note thatthe resolution of the confocal microscopy is, however,not high enough to distinguish single nerve ®bers, butonly nerve ®ber bundles. Regeneration of stromal

innervation could not be estimated, as the ®eld of viewwas too small for that purpose.

TABLE I

Concentrations and releases of PDGF-BB, TGF-b1 and TNF-a

Concentrationday 0 (ng lÿ1)

Concentrationday 2 (ng lÿ1)

Concentration3 month(ng lÿ1)

Release day 0(pg minÿ1)

Release day 2(pg minÿ1)

Release 3month

(pg minÿ1)

PDGF-BB:below detection limit/n (13/19) (7/19) (10/20) (13/19) (7/19) (10/19)median 20 1230 660 0.02 51 2mean 1700 7280 2920 13 301 7S.D. 4330 13 500 5900 41 553 14min 20 20 20 0.02 0.02 0.02max 17 780 56 950 21 200 180 2373 59P 0.075 0.248 0.008* 0.721

TGF-b1below detection limit/n (12/19) (13/19) (7/20) (12/19) (13/19) (7/19)median 5 5 255 0.005 0.005 1mean 820 210 840 1 5 3S.D. 2850 440 1500 2 10 4min 5 5 5 0.005 0.005 0.005max 12 560 1720 6730 10 36 17P 0.445 0.198 0.203 0.116

TNF-abelow detection limit/n (0/19) (0/19) (0/20) (0/19) (0/19) (0/19)meadian 1760 920 770 7 27 4mean 1920 1020 990 10 37 5S.D. 1560 480 970 8 27 5min 120 240 90 2 11 0.8max 6910 1930 4150 30 120 18P 0.039* 0.002* 0.000** 0.085

Concentrations and releases of PDGF-BB, TGF-b1 and TNF-a in human tear ¯uid before PRK, and 2 days and 3 months post-operatively.Mean, median, standard deviations, ranges and number of values below the detection limit are shown. P-values shown indicate the differencescompared to the pre-operative levels. Signi®cance level of P 5 0.05 is marked with * and signi®cance level P 5 0.001 is marked with **(Wilcoxon Signed Ranks Test).

634 I . S . J . TUOMINEN ET AL.

Statistical Analyses

Statistical analyses were performed using SPSS forWindows (version 8.0). Normality was tested usingShapiro Wilk test, and Wilcoxon's signed-rank test(the two group-paired test) were performed forcomparison of the groups. Bonferroni correction wasused when adequate. Pearson or Spearman's corre-lation coef®cients (r/rho) were used to evaluate thecorrelations between the variables. Data are given asmean+ standard deviation (S.D.) and the differenceswere considered statistically signi®cant when prob-ability (P) values were less than 0.05. All concen-trations below the detection limits were expressed asthe values of the detection limits and were included inthe statistical analyses. The rates of release of those

samples were considered as ®xed values, i.e. the same value as the concentration, in order to minimize error.

3. Results

Tear Fluid Samples

The tear ¯uid ¯ow in the collection capillary

was (mean+ S.D.) 9.6+11.1 ml minÿ1 (0.7±42.9 ml minÿ1) pre-operatively, 43.0+30.7 ml

minÿ1 (7.1±125.0 ml; P 5 0.001) on day 2, and8.8+8.2 ml minÿ1 (0.7±33.3 ml minÿ1; P � 0.003)at 3 months post-operatively. The volumes of the tear¯uid samples were 14.4+8.6 ml (range 5±30) pre-operatively, 34.0+27.8 ml (range 10±110) on day 2and 16.0+5.3 ml (range 5±24) 3 months post-operatively. A weak positive correlation (r � 0.498,P � 0.03) was observed between the ablation depthand the tear ¯uid ¯ow on day 2. However, at 3 monthspost-operatively, there was no correlation between theablation depth and the tear ¯uid ¯ow (r � ÿ0.285,P � 0.237). The data of cytokine concentrations andrates of release are given in Table I. No signi®cantcorrelations were found between the levels of variouscytokines at certain time points, except for a weakpositive correlation between the PDGF-BB and TNF-aconcentrations at 3 months post-operatively(r � 0.534, P � 0.015). The cytokine levels of thepatient with basement membrane dystrophy did notdiffer from those of other patients, except for clearlyhigher than average PDGF-BB concentration(11 400 ng lÿ1) on day 2. The pre-operative sample,however, was not available.

The changes in the tear ¯uid cytokine concentra-tions and rates of release from pre-operative values to

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day 2 values showed extremely wide variations:for TGF-b1, the changes in the concentrationranged from ÿ99 to �16 700 % and in the rate ofrelease from ÿ99 to �470 000 %; for PDGF-BB, thechange in the concentration ranged from ÿ57 % to�119 000 % and in the rate of release from ÿ32 to�2 480 000 %, and for TNF-a, the change in the

TEAR FLUID CYTOKINES, NERVES AND HAZE AFT

concentration ranged from ÿ88 to 1000 % and in the

cytokines did not show correlations with the CMTF-

rate of release from ÿ19 % to 3450 %.

Confocal Microscopy at 3 Months Post-operatively

The surface epithelial cells, posterior stromalkeratocytes and endothelial cells of all corneaspresented with normal shape and re¯ectivity (imagesnot shown). The basal epithelial cell area of twocorneas showed pathological ®ndings. The basalepithelial cells of a cornea with a history of recurrenterosions presented with presumably intracellulardeposits (Rosenberg et al., 2000). Similar depositscould be discerned among subepithelial haze resultingfrom highly re¯ective keratocyte nuclei and visiblekeratocyte processes [Fig. 1(A)]. Another corneashowed highly re¯ective particles, round to oval inshape, in the subepithelial area [Fig. 1(B)]. The basalepithelial cells of all other patients appeared normal,and the Bowman's layer was absent in all corneas.The epithelial thickness varied from 29 to 47 mm(39.4+5.5 mm), and the epithelium appeared thusthinner than in normal corneas (Li et al., 1997). Thedata on patient characteristics at 3 months after PRKare shown in Table II.

The subbasal nerve plexus was absent in twocorneas, and 18 corneas showed 1±5 (mean 2.1+1.4) regenerating nerve ®ber bundles [Fig. 1(C)].Subepithelial haze was observed in all corneas, andin two cases it was very highly re¯ecting and appearedvacuolized [Fig. 1(D)] (Mùller-Pedersen et al., 1997).The ®rst anterior keratocytes exhibited brightlyre¯ecting nuclei and thickened keratocyte processes,as signs of ongoing keratocyte activation as describedby Mùller-Pedersen et al. (1997) [Fig. 1(D)]. In one ofthese two corneas, subbasal nerve plexus was absent[Fig. 1(D)]. In the other 18 corneas, haze was moresubtle, and subbasal nerve ®ber bundles were visible[Fig. 1(C)], except for one case. For comparison,normal subbasal nerve plexus and the ®rst anteriorkeratocyte layer in a healthy unoperated cornea areshown in Fig. 1(E) and (F). The CMTF-haze estimatesand the thickness of the haze area are given inTable II. On slit-lamp, haze was mild (from 0 to 1) inall eyes. In general, the CMTF-haze estimates were alsolow, which was in accordance with the clinicalscoring. CMTF-pro®les of a cornea with subtle hazeor vacuolized dense haze are shown in Fig. 2(A) and(B). For comparison, a pro®le obtained from a normalunoperated cornea is presented in Fig. 2(C).

The number of subbasal nerve ®ber bundles had apositive correlation with the epithelial thickness

(r � 0.56, P � 0.011; Fig. 3). The number of thesubbasal nerve ®ber bundles had no correlation withthe CMTF-haze estimate (r � ÿ0.281, P � 0.230) orthe thickness of the haze area (r � ÿ0.398,P � 0.082). The ablation depth showed no corre-lation with the nerve count (r � 0.240, P � 0.308),CMTF-haze estimate (r � 0.041, P � 0.865) or thethickness of the haze area (r � ÿ0.006, P � 0.979).The CMTF-haze estimate, as was expected, showed agood correlation with the thickness of the haze area(r � 0.783, P � 0.000).

No correlations were found between the cytokineconcentrations or rates of release at any time pointsand the CMTF-haze estimate at 3 months. Themagnitude of the changes from day 0 to day 2 inthe concentrations or rates of release of the three

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haze estimate at 3 months (data not shown).

4. Discussion

The present study was designed to evaluate thepossible clinical association of tear ¯uid cytokinelevels with the degree of corneal subepithelial hazeafter PRK. The cytokines TGF-b1, TNF-a and PDGF-BB were chosen for their potential in¯uence oncorneal keratocytes and stromal wound healing,including keratocyte repopulation of the ablatedarea after initial keratocyte apoptosis, keratocyteactivation and subsequent production of extracellularmatrix (Ohji et al., 1994; Jester et al., 1996, 1999;Wilson et al., 1996a, Andresen et al., 1997; Myerset al., 1997; Andresen and Ehlers, 1998). We haveearlier developed enzyme immunoassays for themeasurement of these cytokines in low-volume tear¯uid samples (Vesaluoma et al., 1997a,b,c). Thedisadvantages and potential sources of error usingthe glass microcapillary method for tear collectionhave thus been discussed elsewhere (Vesaluoma et al.,1997b). In the present study, only TNF-a, of themeasured cytokines, was readily measurable in allsamples at all time points. TGF-b1 and PDGF-BB weredetected in 8/20 and 7/20 pre-operative samples, in6/19 and 13/19 samples on day 2 and in 7/20 and10/20 samples at 3 months, respectively. As inter-individual variation in the cytokine concentrationsappeared wide, the magnitude of the changes incytokine concentrations during the early days afterPRK were also calculated. For TGF-b1 and PDGF-BB,highly increased concentrations from day 0 to day 2were observed in several eyes (up to �16 700 and�119 000 %, respectively), but the increases of TNF-a concentration were less remarkable (up to�1000 %). There are several potential sources fortear ¯uid cytokines, including the main and accessorylacrimal glands, corneal epithelial and stromal cells,conjunctival or in¯ammatory cells, extracellular

matrix or leakage from the conjunctival vessels.Recent studies suggest that corneal wounding results

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636 I . S . J . TUOMINEN ET AL.

FIG. 1. (A) Deposits among subepithelial haze in a cornea with a history of recurrent erosions. The patient was a 31 year oldmale, and the ablation depth was 50 mm. (B) Highly re¯ective particles, presumably of in¯ammatory origin, were observed inthe subepithelial area of one cornea. (C) Regenerating nerve ®ber bundles and subtle haze in cornea of a 26 year old femalewith the ablation depth of 81 mm. The CMTF-pro®le of this patient is shown in Fig. 2(A). (D) In two corneas a very highlyre¯ecting vacuolized haze was observed. Brightly re¯ecting nuclei and thickened keratocyte processes were suggestive ofongoing keratocyte activation. The subbasal nerve plexus was absent, or could not be detected due to dense haze. The patientwas a 29 year old male. The CMTF-pro®le of this patient is shown in Fig. 2(B). (E) Subbasal nerve plexus with normalbranching in a unoperated cornea. The patient was a 31 year old male without any history of ocular disease. The CMTF-pro®le

tocy

TEAR FLUID CYTOKINES, NERVES AND HAZE AFTER PRK 637

in increased lacrimal gland mRNA levels for TNF-a,HGF, KGF and EGF (Thompson and Beuerman, 1992;Thompson et al., 1994; Wilson, Liang and Kim,

1999). Whether PRK wound induces cytokine pro-duction in the human lacrimal gland is not known.The current study showed that there were no

correlations between the pre- or post-operative tear¯uid levels of TGF-b1, TNF-a or PDGF-BB andsubepithelial post-PRK haze estimated at 3 monthsby in vivo confocal microscopy. The eyes with high

of this patient is shown in Fig. 2(C). (F) The ®rst anterior kerain (E). The size of each image is 320 � 250 mm.

increases in cytokine concentrations did not presentwith higher CMTF-haze estimates, either. On the

other hand, the clinical healing of all eyes proceededwithout problems, and the maximal clinical hazeobserved at 3 months was scored 1. One of the

limitations of the study was the small size of thepatient group, while no severe complications, such asdense haze (score 3 or 4) occurred in this group. The

CMTF-haze estimates were also low, correspondingly,which might have contributed to the result. Never-theless, we observed high cytokine concentrations,e.g. of TGF-b1, in individual eyes without haze

te layer in a healthy unoperated cornea; the same patient as

formation, which may suggest that tear ¯uid analysisis not suitable for screening the potential candidates

FIG. 2. CMTF-pro®les of a cornea with subtle haze (A) orvacuolized dense haze (B) are shown. For comparison, apro®le obtained from a normal unoperated cornea ispresented in (C). a, surface epithelial peak; b, basal epithelialpeak; c, the subbasal nerve ®ber bundles; d, the ®rst anteriorkeratocytes; e, stromal keratocytes; f, endothelial cell peak;0 0

638

for haze formation even if the patient cohort was morepresentative in terms of different degrees of sub-epithelial haze.

The confocal microscopic features of the operatedcorneas were much the same as previously publishedby others (Corbett et al., 1996; Linna and Tervo,1997; Mùller-Pedersen et al., 1997; Frueh, Cadez andBoÈhnke, 1998; BoÈhnke, Thaer and Schipper, 1998).The most interesting area to be viewed more closelywas the subepithelial area with regenerating nervesand subepithelial haze. All corneas presented withdetectable subepithelial layer of high re¯ectivity (haze)by confocal microscopy. Two eyes showed vacuolizedhaze that appeared active with highly re¯ectingkeratocyte nuclei and visible keratocyte processes,and the other eyes revealed more subtle hazesuggestive of gradual disappearance. BoÈhnke et al.(1998) showed a very discrete layer of subepithelialscar tissue in PRK-corneas even 8±43 months post-operatively, and according to our preliminary resultsdelicately increased subepithelial layer of abnormalextracellular matrix can be detected even 6 years after

c , regenerating subbasal nerve plexus; d , altered kerato-cytes and corneal haze.

PRK (Moilanen et al., unpublished data). Further-more, BoÈhnke et al. (1998) described rod- and

needle-shaped re¯ective bodies in the stroma monthsafter PRK, but we did not observe such structures inour patients.

In¯ammatory cells, such as macrophages appar-ently play an essential role in wound healing afterPRK (O'Brien et al., 1998). They also demonstratedthe presence of T- and B-lymphocytes and Langerhanscells in the operated corneas. Just like epithelial cellsand keratocytes (Li and Tseng, 1995), in¯ammatorycells e.g. macrophages, granulocytes and lymphocytesare also potential local sources of cytokines involvedin wound healing (Lohmann-Matthes, Steinmullerand Franke-Ullmann, 1994; Liles and Van Voorhis,1995). PRK also transiently increases the number ofin¯ammatory cells in tears (Ramirez-Florez andMaurice, 1996). By confocal microscopy we wereable to ®nd cells, possibly of in¯ammatory origin, injust one cornea. These round to oval, highly re¯ectivecells were located in the subepithelial area whereactivated keratocytes were also observed. It is, ofcourse, possible that subepithelial haze masks thein¯ammatory cells in the area. Mùller-Pedersen et al.(1998b) have previously imaged a layer of in¯am-matory cells in a rabbit cornea after transepithelialPRK. Similar ®ndings on humans are, however, notavailable.

The cornea is one of the most densely innervatedtissues in the human body. MuÈ ller, Pels and Vrensen(1996) have shown that even individual epithelialcells and keratocytes are innervated. It is, therefore, tobe expected that proper innervation is important forthe physiological status of epithelial cells and thequiescent keratocytes. During PRK, the anteriorstromal nerve trunks and subbasal nerve plexus areablated. In addition, it has long been known thatproper innervation is needed for normal cornealepithelial regeneration (Beuerman and Schimmelp-fennig, 1980). It is a fascinating hypothesis thatinnervation might have a direct effect on cornealwound healing. To our knowledge, we are the ®rst todemonstrate that the degree of subbasal nerve plexusregeneration after PRK in human in vivo cornea hasimpact on restoration of corneal epithelium. This is anadditional piece of indirect evidence for the trophicand regulatory functions of corneal innervation. Ourobservation is in line with earlier in vitro orexperimental ®nding showing that innervation hasin¯uence on epithelial proliferation (Araki et al.,1994; Nakamura et al., 1997). Whether nerves alsoexert an effect on the healing of the stromal tissue isnot known. In the current study we did not ®ndstatistically signi®cant correlation between the num-ber of subbasal nerve ®ber bundles and the CMTF-haze estimate, or the thickness of the haze area.

After the initial anterior keratocyte loss after PRK,the area is repopulated with new migratory kerato-cytes, which will shortly transform to activated

I . S . J . TUOMINEN ET AL.

keratocytes that produce extracellular matrix (Mùller-Pedersen et al., 1997). The return of the keratocytes

FIG. 3. A positive correlation between the nerve count and the epithelial thickness at 3 months after PRK suggesting that theregeneration state of subbasal nerve plexus has impact on epithelial regeneration and regulation. Epithelial thickness is

(CMles, w

TEAR FLUID CYTOKINES, NERVES AND HAZE AFTER PRK 639

into quiescence takes months (Linna and Tervo,1997; BoÈhnke et al., 1988; Frueh et al., 1998), andthe timing coincidences with the gradual disappear-ance of the subepithelial haze (as observed on slitlamp) and regeneration of the innervation. Mùller-Pedersen et al. (1997) showed delicate subbasal nerve®bers in nine out of 17 patients at 1 month after PRK,whereas according to Frueh et al. (1998) only 1/18corneas presented with subbasal nerves at that time.In their study, seven out of 18 corneas showed nerveregeneration by 4 months, and 13 out of 18 corneasby 12 months. BoÈhnke et al. (1998) examined 15 eyes8±43 months post-operatively, and they foundregenerated nerve ®bers in all eyes. In our study,only two corneas showed total absence of subbasalnerve plexus at 3 months, whereas in other 18corneas at least single nerve ®ber bundles wereobserved in the central area, although the branchingpattern was not normal by 3 months. There arecertain limitations in grading of neural regeneration,e.g. dense haze impedes the observation of thin nerve®ber bundles. The resolution of confocal microscopyequipment also has a limited threshold.

Regrowth of the nerve ®bers into the ablated area isalso essential for the return of corneal sensitivity afterrefractive surgery. After PRK, the central sensitivity,measured using Cochet-Bonnet esthesiometer, returnsback to the pre-operative level by 3 months (PeÂrez-

measured by in vivo confocal microscopy trough focusingmicroscopy image, with the most subbasal nerve ®ber bund

Santonja et al., 1999). Until then the corneo-lacrimalre¯ex arc, which regulates tear ¯uid release from the

lacrimal glands, is supposed to be damaged, andseveral patients complain about dryness of the ocularsurface for the ®rst few months after PRK. Damagedlid re¯ex may also result in inadequate protection ofthe cornea. In our study we did not measure cornealsensitivity, but based on the results by PeÂrez-Santonjaet al. (1999), we conclude that the general degree ofregeneration of subbasal innervation at 3 months wellcorresponds to the clinically normal ®nding oncorneal sensitivity by that time.

In conclusion, our present study brings out thefollowing ®ndings. (1) Pre- or early post-operativelevels of the cytokines TGF-b1, TNF-a or PDGF-BB didnot correlate with the development of corneal haze asestimated by in vivo confocal microscopy at 3 monthsafter PRK; it is, however, noteworthy that none of ourpatients showed grade 3 or 4 haze. (2) Subbasalinnervation of the central cornea had regenerated in18 out of 20 eyes. (3) Even corneas with a clinical hazescore of zero presented with increased re¯ectivity inthe subepithelial layer. (4) In general, the CMTF-hazeestimates were low. (5) A positive correlation wasobserved between the regeneration of subbasal inner-vation (as based on the number of the nerve ®berbundles in the confocal microscopic image with mostnerves) and the thickness of epithelium, however therewas no correlation with the CMTF-haze estimate andregeneration of subbasal nerve plexus. The clinical

TF) analysis. The nerve count is based on the confocalhich were counted.

outcome of the patients was, however, preferable, nosevere cases of haze were observed.

Acknowledgements

The authors are grateful to the Finnish Medical Council,Finland, Finnish Eye Foundation, Finland, Finnish Eye andTissue Bank Foundation, Finland, Ella and Georg EhnroothFoundation, Finland, The Friends of the Blind, Finland,

640

Instrumentarium Scienti®c Foundation, Finland andHelsinki University Central Hospital, Finland.

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