LASEK vs PRK Chun Chen Chen, Dimitri T. Azar

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13.1

Introduction

Laser subepithelial keratomileusis (LASEK) is a newly developed, modified PRK technique that is based on the detachment of an epithelial flap after the application of dilute alcohol solution, and subsequent repositioning of the flap following laser ablation. The repositioned flap is thought to act as a natural mechanical barrier that diminished post-operative pain and de- creases haze formation [27]. LASEK theoretical- ly offers the advantages of avoiding the flap complications of LASIK and, also, addresses the drawbacks of discomfort and delayed recovery associated with conventional PRK.

Laser in situ keratomileusis (LASIK) contin- ues to be the dominant procedure in refractive surgery [15]. It offers more comfort in the early post-operative period, faster visual rehabilitation and minimal haze by maintaining the central corneal epithelium. However, there are increasing reports of LASIK complications, particularly related to flap creation [24, 40, 43].

These include free caps, incomplete pass of the microkeratome, flap wrinkles, epithelial in- growth, flap melting, diffuse lamellar keratitis, keratectasia and an increase in high-order aberrations [22–24, 33, 40, 43]. Furthermore, LASIK is difficult to perform safely in certain situa- tions, such as very steep or flat corneas, deep-set eyes, anterior scleral buckles and previous glau- coma filtering surgery [4].

Photorefractive keratectomy (PRK) remains an excellent option for mild to moderate correc- tions, particularly for cases associated with thin corneas, recurrent corneal erosions, or a predis- position to trauma [3]. PRK does not structural-

LASEK vs PRK

Chun Chen Chen, Dimitri T. Azar

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∑Photorefractive keratectomy (PRK) has been used widely because of its predictability and safety in treating low to moderate myopia

∑Laser subepithelial keratomileusis (LASEK), a modification of PRK, involves the creation of an epithelial flap that is repositioned after laser ablation of Bowman’s membrane and the anterior stroma. The epithelial sheet can be generated mechanically or using alcohol

∑The viability and integrity of the epithelial flap during the LASEK procedure is crucial for achieving adhesion after flap repositioning and minimising the wound healing process

∑Meticulous titration of the dose and exposure time of dilute alcohol solution, and reproducible LASEK technique will be helpful to preserve the viability of the epithelial flap

∑Although potential theoretical advantages of LASEK over PRK are decreased post- operative discomfort, faster visual rehabilitation and less haze, several studies have failed to confirm these potential advantages

∑LASEK may be a viable alternative for patients with low myopia, thin corneas and life styles that predispose them to flap trauma

Core Messages

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ly weaken the cornea. Significant post-operative pain, slower visual recovery and haze might happen and be deterrents to the patients.

LASEK has become a viable alternative to PRK and LASIK in selected patients with thin corneas and patients with lifestyles or profes- sions that predispose them to flap trauma, in- cluding athletes or military personnel, for ex- ample [7]. Early studies suggest that refractive and visual results of LASEK are comparable to those of PRK and LASIK. Lower level of haze formation, relatively fast visual recovery, unifor- mity of corneal topography and better contrast sensitivity were reported in patients after LASEK surgery [12, 27, 32, 35–37].

13.2

Surgical Technique of LASEK and PRK

By salvaging the central epithelium, LASEK is in essence a hybrid of PRK and LASIK [13]. In the LASEK procedure, the epithelial is partially removed from Bowman’s layer after the application of dilute alcohol, connected at the hinge area. Laser ablation is applied directly to Bow- man’s layer as with traditional PRK. The epithelial flap is repositioned in its original position over the laser ablated stromal surface.

The procedure of LASEK was first performed at the Massachusetts Eye and Ear Infirmary by one of the authors (DTA). The original surgical technique involves preplaced epithelial marked for accurate realignment, a Carones alcohol dispenser to weaken the epithelial sheet by exposure to 18 % alcohol for 25 s, a jeweller’s forceps to locate the dissection, a Merocel sponge to peel the epithelial sheet and a 30-gauge Rycroft irrigation cannula to reposition the flap [7].

Camellin and Cimberle described a similar technique that uses a Janach trephine (Janach, Italy) to perform a pre-incision of corneal epithelium, an alcohol solution cone to reserve 20 % alcohol for 30 s, and the short side of an epithelial micro-hoe to detach and fold the epithelium [9]. Epithelial sheet viability and adhesion are the basis for achieving the potential advantages of LASEK [7]. Hence, various techniques are developed in an attempt to preserve epithelial flap viability.

13.2.1

Personal Experience (Azar’s Technique)

After anaesthesia and application of a lid specu- lum, the cornea is marked with overlapping 3.0-mm circles around the corneal periphery (Fig. 13.1). An alcohol dispenser consisting of a customised 7- or 9-mm semi-sharp marker, at- tached to a hollow metal handle, with a reser- voir for the 18 % alcohol (Fig. 13.2), allows irrigation/aspiration of the alcohol after applying firm pressure on the central cornea (ASICO, Westmont, IL) (Fig. 13.3) [7, 11]. After 25–30 s, the solution is absorbed using the suction port (Fig. 13.4) and a dry cellulose sponge (Fig. 13.5).

One arm of the Azar LASEK Scissors (ASICO;

right and left, Fig. 13.6) is inserted under the epithelium and traced around the delineated margin of the epithelium, leaving 2–3 clock hours of intact margin. The loosened epithelium is peeled as a single sheet using a Merocel sponge, leaving a flap with the hinge still at- tached (Fig. 13.7). After laser ablation, an anteri- Fig. 13.1 a, b. Marking the paracentral corneal portion (a) with an overlapping floral pattern (b)

a

b

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or-chamber cannula was used to hydrate the stroma and epithelial flap with balanced salt solution. The epithelial flap was replaced on the stroma under intermittent irrigation. Care was taken to realign the epithelial flap using the previous marks and to avoid epithelial defects. The flap then was allowed to dry for 5 min.

13.2 Surgical Technique of LASEK and PRK 205

Fig. 13.2. Azar-Carones LASEK I/A trephine (ASICO AE-2918)

Fig. 13.3. Alcohol circulation Fig. 13.4. Alcohol absorption

Fig. 13.5. Epithelial flap edge revelation

Fig. 13.6 a, b. Flap elevation by the jeweller’s forceps (a) or the Azar LASEK Scissor (b) (ASICO AE-5489, AE-5499)

a

b

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13.2.2

Camellin’s Technique

The pre-cut incision is performed with a special micro-trephine (Janach, Italy). The depth of mi- cro-trephine is designed to be 70 mm, 80 mm in 8-mm trephines and 90 mm in 9-mm trephine.A blunt portion of the blade at the 12-o’clock position protects the area of the hinge. An alcohol solution cone (Janach, Italy), which is about 0.5mm or 1mm larger than the trephine, will be placed on the eye after trephination. Two to three drops of alcoholic solution will be in- stilled inside the well and left for 25 to 30 s. The pre-cut incision is then dried and thoroughly washed with water. The pre-cut margin is lifted with a hockey spatula to detach the epithelium, and the epithelial flap is gently detached, gath- ered and folded up at the 12 o’clock position [9, 10, 27].

As the pre-incision is not always perfect, an epithelial micro-hoe is used to complete it. The hoe is pressed firmly downward and pulled about 1 mm toward the pupil centre. Alterna- tively, the epithelium is detached with the short side of a hockey spatula, making tiny move- ments almost perpendicular to the margin. The flap is generated and completed along the entire area of trephination up to the hinge.

13.2.3

Vinciguerra’s Techinique (Butterfly Technique)

To maintain the viability of epithelial cells, Dr.

Vinciguerra proposed a modification of the LASEK technique that preserves the connection between the corneal flap and limbus [42].

The butterfly technique requires the use of the Vinciguerra PRK/LASEK spatula (ASICO, Westmont, IL) to impart a thin abrasion to the paracentral corneal epithelium, from 8 to 11 o’clock in order to spare the optic zone. After positioning the LASEK OZ chamber that is connected to the LASEK pump, apply 20 % alcohol for several seconds. The length of time depends on the firmness of the epithelial adhesion noted during the initial abrasion, with a firmer adhesion requiring a slightly longer time. With the Vinciguerra-Carones LASEK spatula, cautiously dissect the epithelium from Bowman’s membrane up to the limbus. It is mandatory to keep the cornea well hydrated in order to preserve the obtained loosening effect otherwise the sec- ond half of the flap will be dehydrated following completion of dissection of the first half of the flap.

13.2.4

Gel Assisted LASEK

Dr. McDonald used viscous gel (hydroxypropyl cellulose 0.3 %) to aid in the separation of the epithelial sheet. The syringe filled with the gel was connected to the cannula. After epithelial trephination, a 2.25 round knife scored down to Bowman’s layer for a distance of 1–2 mm. Ten drops of sodium chloride 5 % were adminis- tered in order to slightly stiffen the cells and were then removed. By sawing back and forth, the epithelial sheet could be lifted. Gel was in- jected under the epithelium before the cut was made in the middle by scissors. The flap was pushed away after application of gel.

Fig. 13.7. Generation of corneal epithelial flap

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Summary for the Clinician

∑ LASEK using dilute alcohol is a simple, inexpensive and reproducible technique

∑ The integrity of the epithelial flap and hinge should be preserved during the LASEK procedure

13.3

Clinical Results of LASEK vs PRK

Azar et al. reported the clinical results of treating 101 myopic patients (131 eyes) with the LASEK procedure [18]. The patients were en- rolled between 1996 and 2002. The epithelial defect was complete in 98.8 % of eyes by 1 week.

Subjective mild pain was reported in 65 % of patients. All but one eye had uncorrected visual acuity (UCVA) of 20/40 or better at 6 months.At 1 year, UCVA was 20/40 or better in 94 % of eyes.

The overall rate of haze formation was 33.1 %.

No patients had his corneal haze recorded as greater than “mild”. The epithelial flap can be consistently created, peeled and returned using the technique previously described (see Sect.

13.2.1).

After treating 249 patients, Camellin reported that intraoperative flap management was easy in 60 % of cases, average in 28 %, and difficult in 12 % [10]. No pain was experienced by 44 % of cases in the first 24 h after surgery, and 80 % of the post-operative best spectacle corrected visual acuity (BSCVA) was achieved by 90 % of patients 10 days post-operatively.

The series of patients treated with LASEK show promising results [5, 7, 12, 18, 26, 27, 31, 32, 34–37]. Scerrati compared the results in two groups of 15 patients treated with LASIK or LASEK. In post-operative corneal topography, BSCVA, and contrast sensitivity, the results of LASEK were superior to those of LASIK [35].

Lohmann et al. and Rouweyha et al. reported the results of treating eyes with high myopia (21 eyes and 32 eyes, respectively) for up to 6 months follow-up [31, 34]. Lohmann et al. pre- sented results whereby all patients were within

±1.0 D of emmetropia. On slit lamp biomi- croscopy, all corneas were transparent and no haze was noted at the post-operative points.

Rouweyha et al. also reported UCVA of 20/40 or better in 33 of eyes at day 1; 71 % at 2 weeks; and 100 % at 3 months.

Lee et al. and Litwak et al. conducted studies comparing LASEK performed in one eye and PRK in the other eye [27, 30]. Lee et al. found that the epithelial defect was healed by the fourth day in eyes that underwent PRK and by the fifth day in eyes that underwent LASEK [27]. The mean epithelial healing time was 3.18±0.50 days and 3.64±0.63 days, respectively, while the difference was not statistically significant (p=0.10). Similarly, Litwak et al. reported that the epithelial defect was completely healed by the fourth day in the PRK and LASEK eyes [30].

The mean epithelial healing time was 3.3±0.5 days and 3.6±0.5 days, respectively (p=0.07).

The subjective pain scores recorded by Lee et al. at 7 days was significantly higher in the PRK eye than the LASEK eye (2.36±0.67 versus 1.63±0.81, p=0.047) [27]. At 1 week, UCVA was 20/25 or better in ten eyes that underwent PRK (37 %) and 16 eyes that underwent LASEK (59 %). At 3 months, it was 20/25 or better in 15 eyes that underwent PRK (56 %) and 17 eyes that underwent LASEK (63 %). A total of 17 patients (63 %; p>0.05) preferred the LASEK procedure because of faster visual rehabilitation (three eyes), painless recovery (ten eyes), and better visual acuity (four eyes). At 1 month, the mean haze score was 0.86±0.45 in eyes that underwent PRK and 0.46±0.24 eyes that underwent LASEK; this was statistically significant (p=0.02). At 3 months, the difference was not statistically significant (p=0.22).

In the series by Litwak et al., 18 patients (72 %) reported more ocular discomfort in the LASEK eye compared to six patients (24 %) who complained more about the PRK eye at 1 day [30]. At 3 days, the difference was higher: 80 % complained about the LASEK eye and 4 % complained about the PRK eye. At 1 week, the UCVA was 20/25 or better in 12 PRK eyes and 12 LASEK eyes (48 %). At 1 month the UCVA was 20/25 or better in 19 PRK eyes (76 %) and 20 LASEK eyes (80 %). No eye had lost one or more lines of best spectacle corrected visual acuity (BSCVA) at the 1-month follow-up examination. At 1 day, patients reported better vision in four LASEK eyes (16 %) and 20 PRK eyes (80 %). At day 3 patients

13.3 Clinical Results of LASEK vs PRK 207

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reported better vision in one LASEK eye (4 %) and 24 PRK eyes (96 %). There was no develop- ment of post-operative corneal haze at 1 month in PRK and LASEK groups.

The comparative studies of LASEK versus PRK showed discrepancies regarding immedi- ate ocular discomfort, subjective UCVA in these two studies. The difference may be inherent in the study population (race and age of the en- rolled patient), concentration and duration of dilute alcohol solutions, techniques of epithelial flap elevation and reposition.

∑ LASEK is as safe and effective as PRK and LASIK

∑ It is not clear whether LASEK is associated with substantially less pain and haze or faster visual rehabilitation than PRK

13.4

Electron Microscopy

13.4.1

Preparation of Specimens for Electron Microscopy

The epithelial sheet specimens were obtained from patients undergoing PRK. The epithelial sheets were fixed in half-strength Karnovsky fixative (2 % paraformaldehyde and 2.5 % glu- taraldehyde) in 0.2 M sodium cacodylate buffer (pH 7.4) overnight and post-fixed in 1 % osmi- um tetroxide in 0.2 M sodium cacodylate for 1.5 h. After dehydration in graded alcohol, the eyes were embedded in epoxy resin (Epon- Araldite). Thick sections (1 mm) were stained with toluidine blue, and a suitable area containing basal layers was chosen. The blocks were trimmed accordingly, thin sectioned (80–90 Å), stained with 2 % uranyl acetate Reynold’s lead nitrate, and examined with a transmission electron microscope (model 410; Philips, Eind- hoven, The Netherlands).

13.4.2

Electron Microscopic Analysis of Epithelial Sheets Removed Using 20 % Alcohol

Normal corneal epithelia are non-keratinizing, stratified, squamous epithelia five to seven layers thick. Desmosomes are present along all cell membranes abutting other cell membranes.

The cells of the basal layer are columnar, and hemidesmosomes are present along their basal plasma membrane adjacent to the basement membrane. Beneath the epithelium is a unilamellar basement membrane that overlies a thick collagen stroma through which anchoring fibres extend from the lamina densa [6, 38].

Azar et al. and Chen et al. studied the electron micrograph of freed epithelial sheets, which were obtained from 20 % alcohol exposure for 20 s [7, 11]. The freed epithelial sheet dis- played normal stratification. The basal epithelial surface of isolated epithelial sheets showed blebbing of the basal cell membrane and au- tophagic vacuoles within the cytoplasm of the epithelial basal cells of the freed sheet in two of the four specimens. They also observed variable basement membrane complex configurations beneath the epithelial basal cells: unilamellar basement membrane with focal disruptions (Fig. 13.8 a), irregular and discontinuous basement membrane with intact hemidesmo- some (Fig. 13.8 b), disruptions of basal cell membranes with absent basement membrane (Fig. 13.8 c) and duplicated basement membrane containing dense bundles of anchoring fibrils (Fig. 13.8 d). The basement membrane layer showed discontinuous and irregular extracellular matrix fragments. The adherence of the basement membrane to the basal layer of the epithelium is vital because it is believed that the basement membrane provides the stability and support that keeps the epithelium intact even with manipulation, thereby preserving the integrity and viability of the entire epithelium.

The presence of desmosomes provides anchoring mechanisms for the epithelium to adhere to the ablated stroma. In addition, Gabler et al. also demonstrated that the plane of separation after ethanol exposure in human cadaver eyes was

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between the lamina densa and the Bowman’s layer [20]. By immunofluorescence studies, the cleavage plane of alcohol induced corneal flap was located between the lamina lucida and lamina densa of the basement membrane [16].

∑ The electron microscopic and histopatho- logical evaluation indicates that the point of separation during the LASEK procedure was likely to be within the basement mem- brane or between the basement membrane and the Bowman’s layer

∑ The variation of epithelial-basement membrane configuration after dilute ethanol exposure may be due to variability

between individuals in relation to the adherence of the epithelium to the base- ment membrane or to the variability of the effect of alcohol on adhesion of epithelial cells

13.5

Corneal Wound Healing After the Refractive Process

While laser refractive surgery offers the prom- ise to correct visual refractive error, biologic variability in the wound healing response is thought to be the major factor limiting the predictability of the outcome of refractive surgery.

13.5 Corneal Wound Healing After the Refractive Process 209

Fig. 13.8 a–d. Transmission electron micrographs of freed epithelial sheets after 20 % alcohol application for 25 s (Specimen I,a; II,b; III,c; and IV,d).Vari- able separation of the basement membrane zone was seen.aSpecimen I showing a localised area of irregu- lar basement membrane zone (arrow) and basal cell membrane disruption (arrowheads) (original magni- fication ¥17,750).bDiscontinuous basement mem- brane zone beneath the basal epithelial cells (arrows), evident at higher magnification, was associated with a decreased number of electron-dense hemidesmo- somes (arrowheads) (original magnification ¥30,000).

cThe basal cell membranes and the basement mem- brane (arrows) were disrupted in Specimen III. Auto- graphic vacuole formation (arrowheads) was exten- sive in the cytoplasm (original magnification ¥1650).

dSpecimen IV: The freed epithelial sheet retained a duplicated basement membrane zone. Pockets of cross-banded anchoring fibrils were arranged in a network between the layers of basal lamina (arrows).

Electron-dense hemidesmosomes (arrowheads) were present along the basal cell membrane (original magnification ¥17,750). Bar=1 mm. (Reproduced from [11])

a b

c d

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The corneal wound healing cascade is complex and involves epithelial mitosis and migration, keratocyte necrosis and apoptosis, myofibroblast transformation, extracellular matrix depo- sition and remodelling and inflammatory cell infiltration [17, 21 ,25, 29, 44].

13.5.1

Epithelial Wound Healing

During PRK, the central epithelium is completely removed, while in LASEK, the injury to the epithelium is limited to an incision or abrasion through the mid-peripheral epithelium. The epithelial cells immediately adjacent to the damaged areas flatten, shed their microvilli and develop pseudopodial extensions. The epithelial cell starts sliding and migration along the tissue until the epithelial defect is covered [19]. During the process, the epithelium also plays an active role in corneal stromal wound healing. The epithelium can produce both stimulatory and in- hibitory cytokines related to plasminogen acti- vation that can affect the release of collagenase and other proteases, as well as inhibitors of collagenase [28]. The epithelial–mesenchymal interaction will maintain a balance between a synthesis of new collagen and proteoglycans with normal assembly and the degradation of the extracellular matrix to allow the restoration of normal structure.

13.5.2

Stromal Wound Healing

As soon as the epithelial barrier is broken by the incision during PRK and LASEK, the stroma be- gins to imbibe fluid and becomes oedematous adjacent to the wound. After the injury of the corneal epithelium, an underlying keratocyte loss occurs within 1 h. This phenomenon was first recognised by Dohlman et al. [14]. Kerato- cyte apoptosis, subsequent replenishment with activated keratocytes, is an initiator of the wound healing process that occurs following PRK and LASEK. Animal studies demonstrated that superficial keratocytes undergo pro- grammed cell death mediated by cytokines

released from the injured epithelium, such as interleukin (IL)-1a, Fas/Fas ligand, bone morphogenic protein (BMP) 2, BMP 4 and tu- mour necrosis factor (TNF)-a [8, 45, 46]. Fur- thermore, tear fluid also contains a wide range of peptide growth factors and is secreted in increased amount after laser ablation of the stroma [47]. Following PRK, an increased amount of transforming growth factor (TGF)-1 in tear fluid was observed, thus leading to keratocyte pro- liferation, migration, myofibroblast transformation and synthesis of stromal extracellular matrix components such as fibronectin and collagen [28, 41].

13.6

Epithelial Cell Viability

Corneal epithelial integrity is essential to maintaining balanced epithelial–mesenchymal interactions, which play an active role in the chemo- kinetics of corneal wound healing, keratocyte apoptosis, myofibroblast transformation and corneal neovascularization [11]. It is hypothe- sised that the viability of epithelial flap decreas- es changes in stromal keratocytes and reduces the production of extracellular matrix and collagen. This may result in less post-operative haze formation with LASEK than PRK [4].

The viability of the ethanol-treated epithelial sheet was further studied in tissue culture for cell migration and attachment [11]. One of the three specimens showed outgrowth and attachment of epithelial cells from the epithelial sheet at days 1–15 (Fig. 13.9). These findings were rein- forced by the electron microscopic evaluations of the epithelial tissue specimen in vivo.

Concentrations of ethanol ranging from 10 % to 30 % are widely used to remove the corneal epithelium before PRK [2]. Stein et al. reported that using dilute alcohol (25 %) in 91 cases of PRK was a safe, effective and predictive method of removing the epithelium [39]. Abad et al.

found that chemical de-epithelialization with dilute ethanol (18 %) appears to be safe and effective and might promote faster rehabilitation [1]. Gabler et al. used 0.1 % trypan blue to test the viability of the epithelial flap of human cadaver eyes after alcohol treatment. They ob-

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13.6 Epithelial Cell Viability 211

Fig. 13.9 a, b. Inverted phase contrast photographs of the tissue culture from one of the three freed epithelial sheets generated after 20 % ethanol treatment for 25 s.aEpithelial outgrowth was observed at day 1

extending from the original sheet border (arrow- heads) to the 1-day outer border (arrows).bThe cell attachment and epithelial outgrowth were persistent until day 15. Bar=50 mm. (Reproduced from [11])

a b

Fig. 13.10 a–g. Fluorescein viability stain with calcein-AM/ethidium homodimer of the cells after 10 % (a), 20 % (b), 24 % (c), 25 % (d), 26 % (e), and 40 % (f) EtOH-H₂O treatment for 20 s. Metabolically active cells convert non-fluorescent calcein-AM into green fluorescent polyanionic calcein and exclude ethidium homodimer (a). Damaged cell membranes allow per- meation of ethidium homodimer and its binding to

nucleic acids resulting in red fluorescence (f).

Bar=50 mm.gCellular survival after different concentrations of alcohol treatment for 20 s. The percentage of viable cells (with exclusive green fluorescence) was calculated by counting cells per ten fields at ¥400 magnification. (Reproduced from [11].) Figure 13.10 g see next page

a b c

d e f

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served that the epithelial cells were vital for up to 45 s of 20 % ethanol exposure [20].

Chen et al. detected a dose- and time-dependent effect of dilute alcohol on cultured corneal epithelial cells [11]. The 25 % concentration of dilute alcohol was the inflection point of epithelial survival (Fig. 13.10). A significant increase in cellular death occurred after 35 s of 20 % alcohol exposure (Fig. 13.11). Also, 40 s of exposure further increased apoptosis after 8 h of incubation (Fig. 13.12). These findings are consistent with the clinical observations of var- ied epithelial attachment to the stromal bed after LASEK surgery.

∑ The effect of dilute alcohol on corneal epithelial cell viability is dose- and time- dependent

∑ Application of the optimal dose and duration of dilute ethanol will facilitate epithelial flap generation, achieve maximal epithelial survival and subsequent adhesion of the repositioned epithelial flap to the stromal bed

Fig. 13.10 g. Cellular survival after different concentrations of alcohol treatment for 20 s. The percentage of viable cells (with exclusive green fluorescence)

was calculated by counting cells per ten fields at ¥400 magnification. (Reproduced from [11])

g

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13.6 Epithelial Cell Viability 213

Fig. 13.11 a–g. Fluorescein viability stain with calcein-AM/ethidium homodimer of cells exposed to 20 % EtOH-H₂O for 20 s (a), 25 s (b), 30 s (c), 35 s (d), 40 s (e), or 45 s (f). Calcein-positive green fluorescence indicates metabolically active cells, and ethidium homodimer-positive red fluorescence indicates damage to the cell membrane and binding to nucleic

acids. Bar=50 mm.gCellular survival with different exposure times. The percentage of viable cells was calculated from the number of green, red, and bicol- ored cells counter per ten fields at ¥400 magnifica- tion. The control group was treated with 100 % KSFM (0 % ethanol). (Reproduced from [11])

a b c

d e f

g

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Fig. 13.12 a–j. TUNEL labelling of cultured corneal epithelial cells exposed to 20 % EtOH-H₂O for 20 s (a–c) and 40 s (d–f) and to EtOH-KSFM for 40 s (g–i).

The TUNEL positivity was evaluated after 8 h (a,d,g), 12 h (b, e, h) and 24 h (c, f, i) of incubation. Maximal TUNEL positivity after 20 s of EtOH-H₂O exposure was detected at 24 h of incubation (c, 58.05±33.10) and after 40 s of EtOH-H₂O exposure at 8 h of incubation

(d, 94.12±1.21 %). Substantially lower TUNEL positivity was seen after 8, 12, and 24 h of incubation with EtOH-KSFM for 40 s (g, 0.65± 0.02 %;h, 7.11±1.49 %;i, 4.52±1.05 %).jTUNEL positivity after 8, 12, and 24 h of incubation of 20 % EtOH-H₂O for 20 and 40 s and 2 % EtOH-KSFM for 20 and 40 s compared to controls.

Control groups were treated with 100 % KSFM for 20 s. (Reproduced from [11])

a b c

d e f

j

g h i

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