pharmacological modification of the epithelial permeability by benzalkonium chloride in...

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INTRODUCTION

The combined topical application of riboflavin and UVA-light to the corneal stroma, called corneal colla-

gen cross-linking (CXL), is a newly accepted method for the treatment of progressing keratoconus1,2 and ectatic corneal diseases.3 The standard treatment protocol includes mechanical debridement of the cen-tral 9 mm of the corneal epithelium and subsequent dripping of riboflavin solution (0.1%) every 3 min for 30 min before the initiation of UVA-irradiation (370 nm; 3 mW/cm2), in combination with continued riboflavin dripping. Stiffening of the upper 200 µm of

Current Eye Research, 35(8), 715–721, 2010Copyright © 2010 Informa Healthcare USA, Inc.ISSN: 0271-3683 print/ 1460-2202 onlineDOI: 10.3109/02713683.2010.481068

ORIGINAL ARTICLE

Pharmacological Modification of the Epithelial Permeability by Benzalkonium

Chloride in UVA/Riboflavin Corneal Collagen Cross-Linking

Anja Kissner1, Eberhard Spoerl1, Roland Jung2, Kathrin Spekl2, Lutz E. Pillunat1, and Frederik Raiskup1

1Department of Ophthalmology, Carl Gustav Carus University Hospital, Dresden, Germany2Experimental Center of the Faculty of Medicine Carl Gustav Carus, Technical University of Dresden, Dresden,

Germany

ABSTRACT

Purpose: To examine the biomechanical effect and the UVA-absorption of a riboflavin/UVA cross-linking method, which suggests leaving the epithelium intact and applying benzalkonium chloride (BAC) on rabbits’ corneas.Methods: In total, 32 eyes from 16 rabbits were divided into 4 groups. Group 1 was treated with intact epithelium and without BAC. In groups 2 and 3, the epithelium was left intact and a hypoos-molar solution of riboflavin that contained BAC 0.02% or 0.04% was used. Group 4 was treated according to the standard protocol with mechanical debridement of the epithelium. After the treat-ment of both eyes, the rabbits were euthanized to prepare the corneas in order for the determination of the riboflavin absorption coefficient and biomechanical properties.Results: The absorption coefficients of groups 2, 3, and 4 were significantly increased compared to group 1. There were no significant differences between groups 2, 3, and 4. Stress-strain values and Young’s modulus for groups 2, 3, and 4 were significantly increased compared to group 1. The stiffening effects did not differ within groups 2, 3, and 4. The resistance to enzymatic digestion was significantly increased in groups 2, 3, and 4 as compared to group 1.Conclusions: Treatment with BAC 0.02% induces sufficient epithelial permeability for the passage of riboflavin, which enables its stromal diffusion and results in increased corneal stiffening after cross-linking as compared to the standard protocol. Further safety studies will be required before clinical use.

KEYWORDS: Benzalkonium chloride; Cross-linking; Intact epithelium; Keratoconus; Riboflavin and UVA

Received 05 November 2009; accepted 23 March 2010

Correspondence: Anja Kissner, M.D., Department of Ophthal-mology, Carl Gustav Carus University Hospital, Fetscherstraße 74, Dresden 01307, Germany. E-mail: [email protected]

05 November 2009

23 March 2010

© 2010 Informa Healthcare USA, Inc.

2010

Current Eye Research

0271-36831460-2202

10.3109/02713683.2010.481068

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mailto:[email protected]

716 A. Kissner et al.


the corneal stroma is affected through a photooxidative induction of collagen cross-links.4–10

Riboflavin, which is enriched in the corneal stroma by diffusion, serves as a photosensitizer and as a protector of the corneal endothelium11 and deeper intraocular structures, such as the lens and retina, due to its 370 nm absorption peak. Riboflavin-application has to start at least 30 min before the start of irradiation to enable sufficient intrastromal diffusion.12–15

The corneal epithelium with its tight junctions is considered to be the most important barrier to per-meability.16 Therefore, mechanical removal of the intact corneal epithelium is the standard protocol for CXL before the application of riboflavin. Unfortunately, the detached epithelium causes mild to severe postop-erative pain for the first three to four days and is con-nected with risk of corneal infections. Therefore, some surgeons tend to modify the standard protocol and to perform the treatment without epithelial removal.

Usage of benzalkonium chloride (BAC) as a pre-servative loosens the tight junctions of the corneal epithelium in order to enhance permeability to phar-maceutical agents. BAC is used in various concen-trations (0.0075 to 0.02%) in many ophthalmic drop solutions.

The advantages of this transepithelial cross-linking protocol include increased postoperative comfort and decreased risk of infections.

The aim of this study was to detect the biome-chanical effects of modifying the standard protocol as determined by treating the eyes of living rabbits. Thus, it was possible to determine the biomechanical proper-ties of the corneas of the enucleated bulbi immediately after CXL (only a few minutes after rabbits were eutha-nized) in order most closely reflect with the effect on human corneas in vivo.

METHODS

The study was approved by the animal experiments committee of the Technical University of Dresden (reference number 24D-9168.11-1/2008-44) and the procedure conformed to principles of animal treat-ment described in the Statement for Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology.

We selected the rabbit as the animal model so that the eyes were as similar as possible to the human cor-nea. Corneas from cattle or pig eyes are not suitable, because the CCT is too different from that of human corneas (pig, 1000 µm; cattle, 800 µm). Results from previous experimental CXL studies using pig eyes17 have only limited explanatory power.18,19 The corneas of sheep (CCT 500 µm) and rabbits (380 µm)20–22 seem

to have more suitable characteristics. Furthermore, the thickness of the epithelium should be comparable to that of the human for these investigations. Because the epithelium of pigs and cattle is too thick (100 µm), the rabbit epithelium (45 µm) seems to be a better model.21,22

According to the statistically estimated number of eyes, 32 eyes of 16 rabbits (New Zealand White Rabbits, female, 2.8 kg) were treated with intramuscular general anesthesia (ketamine i.m.; 35 mg/kg body weight and xylazine i.m.; 5 mg/kg body weight).

Both eyes of one rabbit were treated one after the other. During treatment of one eye, the fellow eye was closed with an eye patch. Before starting the treatment, we determined the central corneal thickness (CCT) of every eye (Pachy-Pen™XL, Accutome Inc., Malvern, Pennsylvania, USA).

The eyes were divided into four groups. Group 1 (11 eyes) was treated without removal of the epithelium; the riboflavin-solution was free of BAC. In groups 2 (8 eyes) and 3 (5 eyes), where the epithelium was left intact, a solution of riboflavin that contained BAC in two different concentrations (0.02% and 0.04%) was used. Group 4 (8 eyes) was treated according to the standard protocol with mechanical debridement of the epithelium in the central diameter (9 mm) and a hypoosmolar solution of riboflavin containing no BAC. There was no variation in any of the other aspects of the standard protocol (riboflavin had a concentration in solution of 0.1% and was dripped in the same inter-vals for the same period of time).

The particular treatment protocol used is shown in Tables 1 and 2. After treatment of the first eye was complete, treatment of the fellow eye was started immediately (Figure 1). Immediately after treatment of the fellow eye was complete, rabbits were eutha-nized before emerging from anesthesia by intravenous injection of T61® (embutramide, mebezoniumiodide, tetracaine; Intervet International; Unterschleißheim, Germany). Bulbi were enucleated to prepare the cor-neas for determination of the absorption coefficient, their biomechanical properties, and their resistance to enzymatic digestion.

After enucleation, the change in UVA-light absorp-tion by the prepared corneal discs was measured using a LASERMATE/Q (Roithner LaserTechnik, Vienna, Austria). The absorption coefficient was cal-culated.

After this procedure, vertical central corneal strips of 5 mm width and 8 mm length were prepared by using a special cutter with a double-bladed knife.

To measure stiffening, we used the biomaterial-tes-ter MINIMAT (Rheometric Scientific GmbH, Basewile, Germany), which is a device used to determine the stress-strain curves. Every single corneal strip was

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Corneal Cross-Linking with Benzalkonium Chloride 717


fixed between the two clamps of the device. The strain rate was 2 mm/min and the pre-stress force was 104 N/m2, as determined previously.6 Stress-strain values were fitted by the exponential function σ = A (exp (B · ε) -1). For statistical analysis Young’s modulus (elas-tic modulus, E) was calculated for 10% strain as the gradient of the mean stress-strain graphs with E = dσ/dε = A · B exp (B · ε). The values were compared using one-way analysis of variance (ANOVA) followed by the Sidak post-hoc test.

The lateral parts of some prepared corneas were exposed to collagenase solution and observed daily to evaluate the time to complete enzymatic digestion.

For statistical analysis SPSS 15.0 statistical software (SPSS GmbH, Munich, Germany) was used.

RESULTS

CCT was measured in all eyes preoperatively via ultrasonic pachymetry. These basic values were also used for stress-value-calculations (load per cross sectional area). There was no statistically sig-nificant difference among all groups with regard to basic CCT (ANOVA, P = 0.993). It was important to perform these measurements before starting the

treatment, because after epithelial debridement and dripping the hypoosmolar solution of riboflavin, the induced corneal swelling of about 30 µm would bias the calculation and lead to a lower stress value. In all groups CCT increased up to 30 µm after dripping hypoosmolar riboflavin solution, but this effect was not statistically significant.

TABLE 2 Treatment protocol for all groupsGroup Treatment protocol1 Application of riboflavin solution (without BAC)

at intervals of 3 min for a period of 30 min → UVA irradiation for 30 min with continuous riboflavin drip (without BAC)

2 Application of riboflavin solution (BAC-concentra-tion 0.02%) at intervals of 3 min for a period of 30 min → UVA irradiation for 30 min with continuous ribo-flavin drip (without BAC)

3 Application of riboflavin solution (BAC-concentra-tion 0.04%) at intervals of 3 min for a period of 30 min → UVA irradiation for 30 min with continuous ribo-flavin drip (without BAC)

4 Topical anesthesia → mechanical debridement of 3 min for a period of 30 min → UVA irradiation for 30 min with continuous riboflavin drip (without BAC)

FIGURE 1 Test arrangement: treatment of the rabbit eyes in general anesthesia.

01 2 3

Group

Abs

orpt

ion

coef

ficie

nt in

cm

−1

4

10

20

30

40

50

60

FIGURE 2 The absorption coefficients of group 2 (BAC 0.02%) as well as of groups 3 (BAC 0.04%) and 4 (epithelial debridement) significantly increased as compared to group 1 (intact epithelium). Statistical analysis found no difference among groups 2, 3, and 4 with regard to the absorption coefficient.

TABLE 1 Treatment parameters for all groups

Group Topical anesthesia Epithelium

Hypoosmolar solution of riboflavin

UVA irradiationConcentration of

riboflavinConcentration

of BAC1 0%2 None Intact

epithelium0.02% 370 nm

3 0.1% 0.04% 3 mW/cm2 30 min

4 Proparakain (0.005 % Con-centration of BAC)

Mechanical debridement

0%

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The absorption coefficients (Figure 2, Table 3) of group 2 (BAC 0.02%) as well as those of groups 3 (BAC 0.04%) and 4 (epithelial debridement) sig-nificantly increased as compared to those of group 1 (intact epithelium). Statistical analysis found no dif-ference in the absorption coefficient between groups 2, 3, and 4.

With regard to biomechanical properties, as reflected by stress-strain measurements (Figure 3, Table 3) and Young`s modulus (Figure 4, Table 3), stiffening effects were observed in groups 2, 3, and 4. Stiffening was visible when preparing the corneal discs; they did not lose their convex shape (Figure 5). These effects were statistically significant compared to group 1. The cor-neas of group 1 were flabby and foldable. There were no statistically significant differences between stress-strain values and Young’s modulus for groups 2, 3, and 4, which shows that the stiffening effect of the modified method including BAC treatment is comparable to that induced by the standard protocol involving epithelial removal. The mean stress-strain value of group 2 (BAC 0.02%) was 82% of the average of group 4 (standard protocol), but this difference was not statistically sig-nificant and a nearly similar cross-linking effect was detected.

Corneal pieces of group 1 (which were treated with intact epithelium and without BAC) had lower resis-tance to enzymatic digestion and dissolved after 6 days, whereas the pieces from groups 2, 3, and 4 had not dissolved completely after 14 days (Figure 6).

DISCUSSION

Riboflavin (C17H20N4O6) has a molar mass of 376.36 g/mol, which is identical to the absolute molec-ular mass (376.36 Da). Riboflavin also has hydrophilic character, which is why it is unable to penetrate the corneal epithelium.

It is presumed that properties, such as hydrophi-licity or lipophilicity, have more influence on perme-ability than does molecular mass.23 Underneath the epithelium, the corneal stroma is highly hydrophilic and allows the free passage of hydrophilic substances up to a molecular mass of 500 kDa (500,000 Da), but acts as a barrier for lipophilic molecules.24,25 BAC increases epithelial permeability by loosening the tight junctions.26–31 Such pharmacological modifica-tion of corneal epithelial permeability represents a newly suggested method to avoid epithelial debride-

TABLE 3 Absorption coefficient, stress values at 10% strain and Young`s modulus for all groupsGroup 1 (Intact epithelium) 2 (BAC 0.02%) 3 (BAC 0.04%) 4 (Standard protocol)Absorption coefficient (mean ± SD)

8.4 ± 2.4 30.2 ± 8.6 35.0 ± 7.9 42.4 ± 14.1

Stress-values for 10% strain in kPa (mean ± SD)

739.0 ± 136.8 1460.7 ± 578.9 1614.6 ± 545.8 1665.5 ± 478.3

Young`s modulus in MPa (mean ± SD)

11.1 ± 3.2 36.5 ± 4.5 48.9 ± 26.2 54.0 ± 21.8

01 2 3

Group

Stre

ss in

kP

a

4

500

1000

1500

2000

2500

FIGURE 3 Stress values for 10% strain in kPa. Significant stiffening effects were found in group 2 (BAC 0.02%), group 3 (BAC 0.04%), and group 4 (standard protocol) as compared to group 1 (intact epithelium). There were no statistically significant differences among groups 2, 3, and 4.

01 2 3

Group

You

ng’s

Mod

ulus

in M

Pa

4

2

4

6

8

FIGURE 4 Young`s modulus (in MPa) was signifi-cantly increased in group 2 (BAC 0.02%), group 3 (BAC 0.04%), and group 4 (standard protocol) as compared to group 1 (intact epithelium). The differ-ences among groups 2, 3, and 4 were not statistically significant.

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ment in CXL.32–34 However, the success of this treat-ment method was only evaluated with regard to the stability of visual acuity and corneal topography dur-ing follow-up of about two years.

So far, the only comparative experimental study was performed by Wollensak et al.35 The study used rabbit corneas with intact epithelium treated by CXL and BAC 0.005%, demonstrating biomechanical stiffen-ing effect of about one-fifth compared to the standard treatment protocol. However, the method used was not identical to the protocol suggested by Pinelli et al. Wol-lensak used Proparakain as a tensioactive substance (BAC-concentration 0.005%), while Pinelli applied a higher concentration of BAC (0.02%) in a hypoosmolar riboflavin solution. We sought to determine whether a higher concentration of BAC in combination with a hypoosmolar solution of riboflavin leads to a greater biomechanical effect.

Our study was the first investigation that adhered to the protocol suggested by Pinelli36 and used BAC at a concentration of 0.02% in a hypoosmolar riboflavin solution.

Wollensak et al.35 found no significant stiffening after CXL on intact epithelium (and without BAC) as compared to completely untreated corneas. For that reason, an untreated control group was not inves-tigated in our study; rather, the group treated with intact epithelium and without BAC (group 1) served as controls. We sought to compare the effect of CXL with BAC 0.02% and these controls on intact epithelium. The high absorption and stiffening values observed for group 4 confirm the efficacy of the standard protocol

with epithelial removal, which was reported in detail previously.8,15,37

Additionally, we also examined the effect of the tensioactive method with increased BAC concentra-tion (0.04%), which did not result in greater stiffening as compared to the standard protocol or treatment with BAC 0.02%. For this reason, and due to the toxic properties of BAC, we would not recommend further investigations of this modification to the protocol.

Our study was able to show clearly that BAC 0.02% clearly affects epithelial permeability to ribo-flavin, which increases the absorption coefficient and

group 4: standard protocol

group 1: intact epithelium

group 2: BAC 0.02 %

group 3: BAC 0.04 %

FIGURE 5 Stiffening effect: the corneas of group 1 (intact epithelium) were flabby and foldable, whereas the corneas of group 2 (BAC 0.02%), group 3 (BAC 0.04%), and group 4 (standard protocol) did not lose their convex shape during preparation for the examination.

group 2: BAC 0.02 %


group 3: BAC 0.04 %group 1: intact epithelium

group 2: BAC 0.02 %


group 3: BAC 0.04 %group 1: intact epithelium

FIGURE 6 Corneal pieces of group 1 (intact epithelium) had a lower resistance to enzymatic digestion compared to those of group 2 (BAC 0.02%), group 3 (BAC 0.04%), and 4 (standard protocol). The upper disc contains the corneal pieces exposed to collagenase solution on day 0. The lower disc shows the same pieces after six days.

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the stress-strain values as well as Young’s modu-lus. These factors represent corneal stiffening and cross-linking. Such a large effect was not measured in pig eyes with thicker epithelium (unpublished data). The increased resistance against enzymatic digestion observed represents further evidence of a cross-linking effect. The results indicate that CXL and BAC-mediated chemical modification of epithe-lial permeability seem to be effective. In absorption measurements, the groups treated with BAC were not different from the standard protocol group, which suggests that the permeability of the corneal epithe-lium was sufficient for the passage of riboflavin and its stromal accumulation.

The stress-strain measurement results support a comparable stiffening effect. The postoperatively increased resistance of the cornea to enzymatic digestion, previously described by Spoerl et al.,38 represents a simple but impressive biochemical tool and is seen as one of the most convincing effects of CXL. We observed this effect in groups 2, 3, and 4. The corneal pieces from corneas that underwent corneal debridement and treatment with BAC could be followed up to the point when they dissolved. Complete enzymatic degradation of the corneas in group 1 (intact epithelium without BAC) was as fast as that of the untreated corneas examined by Spoerl et al. Therefore, transepithelial cross-linking without BAC is ineffective.

In our investigations, we detected reduced resis-tance of the corneal epithelium to mechanical forces in the groups treated with BAC. Because the rabbits were killed immediately after the treatment we were not able to follow the epithelial healing process and we could not observe the incidence of postoperative corneal erosion.

The endothelial toxicity of BAC is also an impor-tant topic. The rabbits’ endothelial cells are able to regenerate,39,40 which is not the case in human endothe-lium. However, Hughes at al. described long-term recovery of human endothelial function after toxic injury, which took about one year.41

In the literature, there was no detailed information about the concentration and intracorneal accumulation of BAC after epithelial application. After loosening the epithelial tight junctions, the corneal stroma should not function as a barrier to BAC.

Our experiments did not investigate the potential endothelial damage caused by the intensive applica-tion of BAC contained in eye drops in short intervals for about 1 hr. The molecular mass of BAC cannot be determined exactly because of its mixture of various alkyl chain lengths. It ranges between about 270 Da and 420 Da, and seems to be small enough for free stromal passage.

Previous studies42 investigated only the effect of preservatives on endothelial cells after direct expo-sure. The challenge for future experiments would be to examine the effect of BAC in vivo during a pro-longed period of application and after transcorneal penetration on endothelial properties (polymorphism, polymegatism, etc.) in suitable animal species. As men-tioned before, rabbit eyes would not be suitable for this avenue of research due to the presence of regenerative endothelium.

CONCLUSION

The aim of our experiment was to prove the biome-chanical effect of CXL on intact epithelium and BAC. According to our results, the mixture with BAC 0.02% in a hypoosmolar riboflavin solution enables sufficient penetration of riboflavin through the corneal epithelium of rabbits with subsequent diffusion into the stroma, which is reflected in the high absorption coefficient and the effective biomechanical stiffening after CXL.

However, because of the uncertain endothelial toxic-ity of BAC after transcorneal penetration, we have to be aware of possible irreversible changes in corneal tis-sue induced by the use of this method. Further safety studies are necessary before the application of this technique in the clinic.

ACKNOWLEDGMENTS

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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pharmacological modification of the epithelial permeability by benzalkonium chloride in...

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