Ординатура / Офтальмология / Английские материалы / Mastering Corneal Collagen Cross-linking Techniques (C3-R CCL CxL)_Garg, Kanellopoulos, O'Brart, Lovisolo, Pinelli_2008

ABSTRACT

Purpose

Using the absorption coefficient of 53 cm-1, established in riboflavin soaked porcine corneas, it can be calculated that, using UVA 365 nm at 3.0 mW/cm2, the cytotoxic endothelial threshold is reached in corneas thinner than 400 µm. We have measured the absorption coefficient in postmortem human corneas after instilling riboflavin.

Setting

Department of Ophthalmology, University Hospital Antwerp,

Antwerp University

Methods

Corneal thickness was measured in 9 pairs of human donor eyes, using Pentacam Scheimpflug and ultrasound pachymetry. In one eye of each donor the epithelium was manually removed over an 8 mm zone; in the fellow eye the epithelium was left intact. In both eyes, riboflavin 0.1% in dextran 20% was instilled on the intact globe for 20 minutes. After this period the corneas were rinsed and a corneoscleral disc was trephined. The transmission of the central part of the cornea was measured in transillumination, using the UV light source and the UV detector of the cross-linking device.

Results

The average corneal thickness measured was 658.5 ± 51.5 µm after epithelial removal, and 758.3 ± 98.8 µm without epithelial removal. The average transmittance after instillation of riboflavin for 20 minutes was 12.89 ± 4.10% with epithelial abrasion and 28.52 ± 4.39% without (p<0.05). The resultant average absorption coefficient is 32 ± 5 cm-1 when the epithelium is removed, and 17 ± 2 cm-1 when it is left intact (p<0.05). These results show that the amount of riboflavin taken up by the cornea is much higher when the epithelium is removed. These findings may be relevant in the discussion on the necessity to remove the epithelium for corneal cross-linking.

Conclusion

The absorption coefficient (53 cm-1) established for porcine corneal strips soaked in riboflavin, is not reflecting the clinical situation. Using human donor globes, we established an absorption coefficient of 32 cm-1 implying a minimal corneal thickness of at least 490 µm to ensure endothelial safety.

INTRODUCTION

Corneal collagen cross-linking (C3R) by means of ultraviolet-A (UV-A) light and riboflavin (vitamin B2)

We also want to investigate whether riboflavin can penetrate into the cornea if the epithelium is left intact. In the standard treatment protocol it is obligatory to remove the corneal epithelium to allow for diffusion of the riboflavin into the stroma. Nevertheless, Pinelli and others report similar clinical results in C3-R treatment without removal of the epithelium.8 He suggests that the pretreatment use of topical anesthetics might allow for the penetration of riboflavin through the epithelium.

MATERIALS AND METHODS

Materials

Donor eyes were obtained from the Ocular Tissue Bank of the University Hospital Antwerp; they were discarded for use as donor tissue because of medical contraindications.

Riboflavin-5-phosphat 0.5% (G. Streuli and Co AG, Uznach, Switzerland) was diluted with a dextran T-500 20% solution in NaCl 0.9% to obtain a riboflavin 0,1% solution as used for clinical C3-R.

The corneal transmittance was measured by placing the excised cornea between the UV-source and the UV-detector of the cross-linking device, used for the clinical treatments (both provided by IROC AG, Zürich, Switzerland), as demonstrated in Figure 9.1.

Methods

1.Measurement of the absorption coefficient of the cornea, with and without epithelium after riboflavin solution instillation.

Eighteen eyes of 9 donors were used to calculate the absorption coefficient of the cornea for UV- A light with a wavelength of 365 nm and to investigate the influence of epithelial removal.

a.Measurement of postmortem corneal thickness. All 18 eyes of the 9 donors were used to calculate the average postmortem corneal thickness. The thickness of the donor corneas before and after epithelium removal was measured using Scheimpflug imaging (Pentacam, Oculus GmbH, Wetzlar) and/or ultrasound pachymetry.

b.Measurement of corneal transmittance.

In one eye of each pair (alternatively right or left) the epithelium was manually removed using a blunt spatula over about an 8 mm diameter zone under the microscope. The fellow eye was used as a control and its epithelium was left intact.

In both eyes, riboflavin 0.1% in dextran 20% was instilled on the intact globe for 20 minutes, 1 drop every 30 seconds to compensate for the fact that the drops run off the globe. At the end of 20 minutes the corneas were rinsed, gently wiped with a compress and a corneoscleral disc was trephined. The transmission of the central part of the cornea was measured in transillumination.

2.Influence of prolonged riboflavin 0.1% soaking or instillation on the absorption coefficient of the cornea, with and without epithelium.

In 2 pairs of the corneoscleral discs of the first experiment, additional measurements were made in order to establish whether it is possible to increase riboflavin uptake and increase UVabsorption by soaking the corneas or prolonging the instillation time.

a.Soaking experiments: Two corneas from one donor from the first experiment, one cornea with intact epithelium and one without epithelium, were soaked in riboflavin for an extra 20 minutes. A transmittance measurement was made every 5 minutes. These tests enabled us to determine a curve of riboflavin absorption by the cornea.

b.Prolonged instillation experiments. Two corneas from another donor, one with its epithelium intact and one with its epithelium removed, received extra drops on the anterior surface of the cornea. The transmittance was measured 40, 45, 55 and 65 minutes after the initial start of the instillation of drops.

c.Increase of riboflavin absorption by the cornea with respect to time: In hindsight we thought it would be interesting to have a complete analysis of the corneal riboflavin saturation through soaking to establish the steady-state value of the absorption coefficient: two extra corneoscleral discs, one with and one without epithelium, were soaked in riboflavin. Every 5 minutes, the corneas were rinsed, the surface gently wiped and the transmittance was measured.

d.Effect of riboflavin concentration on the absorption coefficient: In a final effort to obtain a higher value for the absorption coefficient, we removed the epithelium from 2 donor eyes and instilled undiluted riboflavin 0.5% (Streuli Gmbh) for 20 minutes. After instillation, the corneas were rinsed and gently wiped and the corneoscleral discs trephined. Based on the transmittance measurements of the central cornea the absorption coefficient can then be calculated.

3.Influence of proparacaine on riboflavin penetration of corneas with intact epithelium: Two eyes from the same donor were used to calculate the absorption coefficient of the cornea at 365 nm, in order to investigate the influence of proparacaine 0.5% eyedrops on riboflavin corneal penetration. For this experiment, we have followed the protocol as described in the communication of Pinelli. In both eyes, the epithelium was left intact. In one eye, proparacaine drops were instilled for 15 minutes, 2 drops every 5 minutes. In an oral communication at the 3rd Cross-linking Conference (December 2008, Zürich) Pinelli described a presoaking time with riboflavin of 5 minutes. We presoaked both eyeballs in riboflavin 0.1% solution even longer, for 10 minutes. At the end of the 10 minutes, the corneas were rinsed, gently wiped with a compress and a corneoscleral disc was trephined. The transmission of the central part of the cornea was measured in transillumination.

RESULTS

1-a. The average corneal thickness measured with the Scheimpflug technique was 658.5 ± 51.5 µm after

removal of the epithelium, and 758.3 ± 98.8 µm without epithelium removal.

1-b. The average transmittance after instillation of riboflavin for 20 minutes was 12.89 ± 4.10% with epithelial abrasion and 28.52 ± 4.39% without. A χ²-test shows that there is a significant difference between the transmittance of the cornea with or without epithelium abrasion (p<0.05).

The absorption coefficient can then be calculated taking into account the measured corneal thickness for the donor eyes using the LambertBeer formula:

T is transmittance, I is transmitted light intensity and I0 is the incident light intensity, x is the thickness of the cornea, µ is the absorption coefficient, e indicates the exponential function and ln is the neperian logarithm (the inverse function of an exponential).

The resulting average absorption coefficient is 32 ± 5 cm-1 when epithelium is removed, and 17

± 2 cm-1 when it is left intact. The average absorption coefficients are statistically different (p<0.05): the amount of absorbed riboflavin by the cornea is then much higher when the epithelium is removed.

Both corneas in Figure 9.2 have been instilled with riboflavin for 20 minutes. The right hand cornea shows the staining by the riboflavin in the zone where the epithelium has been removed; the distinction between the part saturated by riboflavin and the rest of the cornea can easily be seen. The cornea on the left is the one without

Figure 9.2: Photograph of the corneoscleral disks of both eyes of the same donor after instillation of riboflavin drops; 4 7 the epithelium is intact in the left cornea

epithelial removal; riboflavin seems not to have penetrated into the stroma through the intact epithelium.

2.We tried to establish whether it would be possible to increase UV absorption by corneas with and without intact epithelium by soaking the corneas or increasing the instillation time.

a.Soaking experiments

Measurement of transmittance was continued every 5 minutes from 20’ (moment of trephination) to 40 minutes after initiation of instillation. The corneas were soaked in riboflavin 0.1% from 20 minutes to 40 minutes. The absorption coefficients systematically increase throughout this experiment.

b.Prolonged instillation measurements

The corneal transmittance was measured from 20 (time point of trephination), and after 40, 45, 55 and 65 minutes after initiation of instillation. Instillation of riboflavin 0.1% was uninterruptedly continued. The absorption coefficient shows a slight increase which does not seem to be relevant.

the absorption coefficient in a relevant way, suggesting that prolonging the duration of instillation for the clinical treatment is not useful. Soaking on the other hand does increase the absorption coefficient, but this is not clinically applicable. It shows that the endothelial layer with its gap junction allows extra uptake of riboflavin into the cornea.

c.Increase of riboflavin absorption by the cornea with respect to time.

We were curious to know the absorption coefficient for a cornea maximally saturated with riboflavin, through soaking of fresh corneoscleral discs in riboflavin 0,1% for a total period of 45 minutes, one with the epithelium removed, both soaked in riboflavin solution from time point 0. The results are available in Figure 9.4. In these two eyes a steady-state is reached after 30 minutes with absorption coefficients leveling off between 30 and 35 cm-1 which indicates that the absorption coefficient of 32 cm-1, found in our first experiment after 20 minutes of instillation reflects a riboflavin saturated cornea.

d.Effect of the riboflavin concentration on the absorption coefficient.

The absorption coefficient of the cornea without epithelium and instilled with riboflavin 0.5% was 71 cm-1.

3.The transmittance of the corneas with and without proparacaine instillation was respectively 27.8% and 26.4%. The calculated absorption coefficients were then respectively: 18 cm-1 and 19 cm-1 respectively with and without proparacaine. These results are in the range of the absorption coefficient of corneas with intact epithelium.

DISCUSSION

In the first experiment we have determined that the absorption coefficient of human postmortem corneas, instilled with riboflavin 0,1% in dextran for 20’ – as applied in the standard C3R-treatment protocol – amounts to 32 ± 5 cm-1 with the epithelium removed, and to 17 ± 2 cm-1 when the epithelium is left intact. Prolonging the instillation time as in the second experiment, did not lead to an increased absorption coefficient; soaking corneas, allowing the riboflavin to enter through the endothelium, amounted to an absorption coefficient around 35 cm-1.

To limit the postmortem corneal edema and the resulting increase in corneal thickness which could be responsible for a falsely low value of the absorption coefficient, we have limited to less than 24 hours the interval between the donor’s decease and enucleation/ preparation time as much as possible. In animal eyes the experiments can be performed immediately after

death, but for logistical reasons postmortem times in animals often run up to several hours as well. We may have used a normal corneal thickness of 520 µm in the calculations because the bulk of the absorption occurs in the anterior part of the cornea; even then the absorption coefficients would be 40 cm-1 for epithelium off and 21 cm-1 for epithelium on, which is still well below the quoted values of 53 cm-1 for porcine corneas4 and 70 cm-1 for human corneas.7

If the absorption coefficient of the cornea for UVA 365 nm is 53 cm-1, the minimal corneal thickness for safe C3R-treatment must be 400 µm as recommended by Spoerl7 and Wollensak3,10, using the Lambert-Beer equation. This absorption coefficient was obtained using strips of porcine corneal tissue, soaked in riboflavin.4 If however the absorption coefficient is 32 ± 5 cm-1, as we have calculated in human corneas (with epithelium removed), the cornea should have a thickness of 660 µm to be safe for the endothelium (from 490 to 848 µm). The treatment of keratoconic eyes according to the clinical protocol using UVA 365 nm with 3 mW/cm2 irradiance, could therefore reach the critical threshold for damage of the endothelial cells. In the clinical experience and in confocal microscopy examinations11 however, until now there have been no reports on endothelial damage. Either the human endothelial cells are more resistant to UVA irradiation than their animal counterparts (the damage

thresholds have been determined in rabbit corneas3 4 9

and on porcine endothelial cell cultures10), or more extensive and prolonged studies of the human endothelium in clinical trials are necessary to show a possible detrimental effect in the longer term.

Based on our third experiment some conclusions can also be made on the necessity of epithelial abrasion to allow penetration of riboflavin in the cornea. The transmittance for UVA of the human cornea as quoted in other studies was 25%6 to 30%.9 We confirmed this number in the first group of eyes, instilled with riboflavin for 20 minutes, and in which the epithelium was left intact, thereby (indirectly) demonstrating that riboflavin does not enter the cornea if the epithelium is not removed. In our experiment with instillation of proparacaine before soaking the cornea with the riboflavin solution, the absorption coefficient remained unchanged, suggesting that the epithelial barrier for penetration of riboflavin remained intact. With regard to the safety of the endothelium, the treatment as proposed by Pinelli is not risky for the patient cornea.8 Wollensak established that the cytotoxic UV-A irradiance level of the corneal endothelium in absence of riboflavin is about 10 times lower than when the riboflavin (acting as a photosensitizer) is present. On porcine endothelial cells he found a UV-A damage threshold of 4 mW/cm² for 30 minutes.10

Further basic and clinical research is mandatory to fine-tune the safety threshold of corneal cross-linking with riboflavin for the human endothelial cells.

ACKNOWLEDGEMENTS

We would like to thank Nezahat Bostan, laboratory technician of the Antwerp University Hospital cornea

bank, for enabling this research with her supportive help and Rudi Leysen, our medical photographer, for the images of the corneas.

REFERENCES

1.Wollensak G, Spoerl E, Seiler T. Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking. J Cataract Refract Surg 2003;29:1780-85.

2.Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A- induced collagen cross-linking for the treatment of keratoconus. Am J Ophthalmol 2003;135:620-27.

3.Wollensak G, Spoerl E, Wilsch M, et al. Endothelial cell damage after riboflavin-ultraviolet-A treatment in the rabbit. J Cataract Refract Surg 2003;29:1786-90.

4.Spoerl E, Schreiber J, Hellmund K, et al. Untersuchungen zur Verfestigung der Hornhaut am Kaninchen. Ophthalmologe 2000;97:203-06.

5.Wollensak G, Spoerl E, Wilsch M, et al. Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment. Cornea 2004;23:43-49.

6.Kolozsvari L, Nogradi A, Hopp B, et al. UV absorbance of the human cornea in the 240to 400-nm range. Invest Ophthalmol Vis Sci 2002;43:2165-68.

7.Spoerl E, Mrochen M, Sliney D, et al. Safety of UVAriboflavin cross-linking of the cornea. Cornea 2007;26:385-89.

8.Pinelli R. Corneal collagen cross-linking with riboflavin: Results at six months follow-up in 20 eyes. Free paper, XXIV Congress of the ESCRS, 2006.

9.Boettner EA, Wolter JR. Transmission of the ocular media. Investigative Ophthalmology 1962;1:776-81.

10.Wollensak G, Spoerl E, Reber F, et al. Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro. Ophthalmic Res. 2003;35:324-28.

11.Mazzotta C, Traversi C, Baiocchi S, et al. Conservative treatment of keratoconus by riboflavin-UVA-induced cross-linking of corneal collagen: qualitative investigation. Eur J Ophthalmol 2006;16:530-35.

INTRODUCTION

Corneal collagen cross-linking with riboflavin is a new method to increase the biomechanical stability of the cornea by inducing additional cross-links between or within collagen fibers using UV-A light and riboflavin as photomediators. The procedure is variously known as CXL, CCL, and C3-R. It was developed from 1993 to 1997 by Prof. Theo Seiler and Prof. Eberhard Spoerl at the University of Dresden, Germany. The first patients were treated in 1998.

Clinical trials are continuing, but the treatment is seeing increasing adoption by the ophthalmological community, and has shown success in treating early cases of keratoconus, pellucid marginal degeneration, iatrogenic keratectasia after refractive lamellar surgery, and corneal melting that is unresponsive to

Figure 10.2: Cross-linked (top) and untreated (below) strips from the same cornea (Courtesy of Prof. Spoerl)

RIBOFLAVIN AND ULTRAVIOLET A LIGHT

Riboflavin, also known as Vitamin B2, is a naturally occurring photosensitizer. It is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), two coenzymes that are crucial for the metabolism of carbohydrates, fats and proteins into energy. Riboflavin is an essential constituent of all living cells. It is water soluble and only a trace amount is found in the human body.

The deleterious effects of ultraviolet light on ocular structures have been well documented. With the use of riboflavin as a photosensitizing agent, however, there is an ultraviolet-A transmission rate of only approximately 7% across the cornea, thus limiting

ultraviolet-A irradiance of the lens and retina. Nevertheless, concern remains about ultraviolet-A cytotoxicity to the corneal endothelium. In rabbits, the cytotoxic level for corneal endothelial damage induced by ultraviolet-A light was 0.36 mW/cm². Using the aforementioned surgical technique, this level could be reached in corneas with a central thickness of less than 400 µm, so it is imperative that surgeons measure corneal pachymetry preoperatively to ascertain that at least this threshold is met. The lens receives 0.65 J/cm² of UV-A irradiance, which is far less than the amount needed to produce cataract (70 J/cm²). As for retinal damage, research with rhesus monkeys has shown a threshold level of 81 mW/cm², which, again, is not achieved with a standard treatment.

PREPARATION OF 0.1% RIBOFLAVIN SOLUTION

•Dissolve Dextran T500 in physiological salt solution to achieve a 20% Dextran T500 solution.

•Dilute Riboflavin-5-phosphate 0.5% with Dextran solution to achieve a 0.1% Riboflavin solution (Ratio of mixture: 1 part of Riboflavin-5-phosphate 0.5% – 4 parts of Dextran T500 20%).

•Protect the solution from light, and use it within 24 hours.

STEP BY STEP TECHNIQUE

1.The procedure is typically performed under sterile conditions in the operating room. The patient is premedicated with two drops of topical anesthetics 2-5 minutes before surgery. A wire eyelid speculum is placed for exposure.

2.The corneal epithelium is removed (fully or

partially, in uniform pattern), by mechanical scraping over the central 8-9 μm of the cornea with a blunt spatula to allow for better diffusion of the riboflavin (riboflavin molecule is too large to penetrate intact epithelium).

3.The photosensitizer, 0.1% riboflavin solution (10 mg of riboflavin-5-phosphate in 10 mL of dextran-T-500 20% solution) is applied to the deepithelialized cornea every 3 minutes for approximately 20-30 minutes (Figure 10.3).

4.Using a slit-lamp inspection with blue light, the surgeon may ensure that riboflavin has reached the anterior chamber (Figure 10.4). UV-A light

Figure 10.3: After removal of the corneal epithelium riboflavin/dextran solution is instilled for 20-30 minutes (Courtesy of Prof. Theo Seiler)

Figure 10.4: Slit-lamp check of the riboflavin penetration. The anterior chamber has to be slightly yellow (Courtesy of Prof.Theo Seiler)

5.Measure corneal thickness at thinnest point after 30 min of riboflavin application. If under 400 µm, use hypotonic riboflavin solution without dextran (310.7 mOsm/l) to enforce swelling of the cornea. Apply 2 drops every 30 seconds until corneal thickness is at least 400 µm.

6.As an additional safety feature, the output energy intensity can be checked prior to each treatment using the UV light meter that is delivered with the system (Figure 10.5).

7.The irradiating source is placed 5 cm from the cornea’s center and applied for 30 minutes (Figure 10.6). The 370 nm wavelength allows approximately 93% of UV light to be absorbed into the cornea, thus, there is no risk for damage to the lens and retina. The UVA light interacts with the riboflavin, producing reactive oxygen molecules that cause the formation of chemical bonds between and within the corneal collagen fibrils, making them stiffer.

8.During irradiation treatment, a drop of riboflavin solution is applied every 5 minutes to sustain the necessary concentration of riboflavin and prevent desiccation of the cornea (Figure 10.7).

9.The surgeon keeps the cornea moist with a drop of balanced salt solution every 2 minutes.

10.At the conclusion of the procedure, the patient receives topical antibiotic and a bandage contact lens is applied for 72 hours or until corneal reepithelization is complete. This is followed by application of fluorometholone 0.1% eyedrops twice daily for 6 weeks. If necessary, artificial tears are prescribed.

SAFETY

Corneal cross-linking is considered to be a safe procedure, provided the recommended safeguards are observed.

1.A clinically used UV source should ensure a perfect homogenous irradiance. Hot spots may cause local damages of endothelium cells especially in thin corneas.

2.The irradiated area of the cornea must be limited to 8 μm. Only the cornea is irradiated. Sclera, goblet cells and limbus are not treated.

3.The following treatment parameters are chosen to reach a strong cross-linking effect and to avoid

Figure 10.5: Intensity check prior to each treatment

Figure 10.6: UV-A illumination of the cornea (Courtesy of Prof.Theo Seiler)

Figure 10.7: During UV-A irradiation, a drop of riboflavin solution is applied every 5 minutes (Courtesy of Prof.Theo Seiler)