Original Article Modulatory Effect of different Riboflavin Compositions on the Central Corneal Thickness of African Keratoconus Corneas during Collagen Crosslinking Timo Mark, Faustin Ngounou1, James Tamon1, Susanne Marx-Gross, Paul-Rolf Preussner
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ABSTRACT Purpose: A pilot investigation to transfer the established corneal collagen crosslinking (CXL) procedure in European eyes into clinically affected African eyes and to optimize the treatment by adapting the riboflavin composition. Materials and Methods: CXL was performed in 15 eyes (11 patients) with advanced stages of keratoconus in the Eye Clinic of Bafoussam in the West Region of Cameroon. The following six riboflavin compositions with different portions of active swelling additives were applied: Solution 1 (0.5% methylhydroxypropylcellulose [MHPC]), solution 2 (1.0% MHPC), solution 3 (1.7% MHPC), solution 4 (5% dextran), solution 5 (10% dextran) and solution 6 (no active swelling ingredient). The central corneal thickness (CCT) was measured by ultrasound pachymetry before and after de‑epithelialization and at least every 10 min during CXL. Results: The application of the riboflavin solutions resulted in the following mean final CCT values: 172 ± 15% using solution 1 (60 min/n = 5); 183 ± 8% using solution 2 (60 min/n = 5); 170% using solution 3 (60 min/n = 1); 80% using solution 4 (45 min/n = 1); 99% using solution 5 (45 min/n = 1) and 150 ± 13% using solution 6 (50 min/n = 2). Conclusions: The combination of riboflavin compositions with swelling and stabilizing effects on the corneal stroma seems necessary in African eyes with advanced keratoconus. Further studies are required to confirm these primary results.
Website: www.meajo.org DOI: 10.4103/0974-9233.124103 Quick Response Code:
Key words: Africa, Collagen Crosslinking, Corneal Thickness, Keratoconus, Riboflavin
INTRODUCTION
C
orneal collagen crosslinking (CXL), using ultraviolet‑A (UVA) radiation and the photosensitizer riboflavin, was recently introduced for the treatment of progressive keratoconus.1,2 In several studies, the method has shown a positive effect on the biomechanical1,3‑5 and biochemical6‑8 stability of the cornea. Thus, CXL has the potential to stop the progression of keratoconus, avoiding the need for penetrating keratoplasty.9 In regions of the world, where there is no access to corneal transplants, this simple and low priced procedure seems to offer
the only accessible treatment for keratoconus patients at this time. CXL requires a minimum central corneal thickness (CCT) of 400 µm to protect the endothelium from the cytotoxic irradiance.6 Often, late diagnosed keratoconus corneas reach values considerably below this threshold. However, during CXL the stromal thickness can be influenced by the chemical composition of the applied riboflavin solution. Exposed to a physiologic aqueous medium the deepithelialized cornea shows the tendency to increase its volume (swelling pressure), which is mainly due to the concentration of stromal polysaccharides.10 Recent in vitro studies in porcine corneas indicate that the osmolarity of the applied medium does not correlate with intraoperative corneal swelling.11
University Medical Center of the Johannes Gutenberg University Mainz, Department of Ophthalmology, Mainz, Germany, 1 Presbyterian Eye Services, Eye Clinic, Acha-Bafoussam, Bafoussam, Cameroon Corresponding Author: Mr. Timo Mark, Department of Ophthalmology, University Medical Center of the Johannes Gutenberg University, Mainz, Langenbeckstr 1, 55101 Mainz, Germany. E-mail: [email protected]
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Mark, et al.: Collagen Crosslinking in the West Region of Cameroon
Hence, the stroma can be hydrated or dehydrated during CXL, depending upon exposure to a riboflavin solution with a colloid osmotic pressure lower or higher than the physiological swelling pressure.
progression of keratoconus was confirmed from medical history. Central corneal scars were not regarded as an exclusion criterion, as there was no keratoplasty treatment available for these patients.
The colloid osmotic pressure of the commonly applied CXL drop composition is mainly given by the polysaccharide dextran (20%), which is added to increase the viscosity of the solution. Dextran shows abundant hydrophilic hydroxyl groups and its solution leads to a marked intraoperative reduction of CCT.12 Logically, these drops are not adequate in controlling the 400 µm threshold in patients with advanced stages of keratoconus.
Six different riboflavin solutions were used [Table 1]. Drops containing MHPC were prepared following a protocol of the University Hospital Carl Gustav Carus in Dresden. Drops with dextran and the drop composition without viscosity additive were produced according to protocols of the University Medical Center of the Johannes Gutenberg University in Mainz. The concentrations of the ingredients were adapted to our requirements. Solution 2 was produced in the Pharmacy of the University Medical Center of the Johannes Gutenberg University in Mainz. All other drops were prepared in the Pharmacy of the Eye Clinic Bafoussam.
However, a number of alternative treatment protocols for thin corneas have been proposed. 13,14 One possibility is to apply a solution containing 0.5% of the polysaccharide methylhydroxypropylcellulose (MHPC). In comparison with dextran, MHPC is missing the water binding hydroxyl groups and its solution is known to swell the stroma quite effectively.15 It should be noted in this context, that CXL is expected to be less effective in artificially swollen corneas, due to the lower relative concentration of collagen in the hydrated stroma.16,17 Therefore, the stroma should be swollen as much as necessary, but as little as possible. In this pilot study, we introduced CXL in the Eye Clinic of Bafoussam in the West Region of Cameroon. We applied variations of riboflavin solutions in order to deal with highly thinned out corneas. The major focus of this study was to determine the most effective treatment for each patient. In this every particular change of the riboflavin composition used is a result of our reaction to the given conditions.
MATERIALS AND METHODS CXL was performed in September 2011 and March 2012 in the Eye Clinic of Bafoussam in the West Region of Cameroon. When we started treatment with the well‑established European protocol, a “study” was not intended. We wanted to treat patients for whom no alternative treatment was available. However, during treatment, we found significant, unexpected differences between these African eyes compared with European eyes. Thus, we optimized our procedure step‑by‑step. Therefore, we did not apply for approval from the Ethics Committee and did not register the project with the Institutional Review Board. Nevertheless, all the procedures were in accordance with the ethical standards of the declaration of Helsinki. The diagnosis of keratoconus was based on corneal topography, ultrasound pachymetry and clinical signs, such as stromal thinning, Vogt striae and apical stromal scars. The pre‑operative
The treatment procedure was conducted under sterile conditions in the operating theater of the clinic. After instilling topical anesthesia (amethocaine 0.5%) and inserting a lid speculum, the central 9 mm of the corneal epithelium were abraded with a hockey stick spatula. The CCT was measured by ultrasound pachymetr y before and immediately after de‑epithelialization and at least every 10 min during the 60 min of eye drop application. In order to detect the thinnest point, at least five repeat measurements at different positions on the central cornea were performed. The riboflavin solution was applied every 3 min for 30 min prior to irradiation to ensure sufficient saturation of the stroma. After confirming that the thinnest point of the corneal stroma was thicker than 400 µm, UVA‑irradiation (3 mW/cm2, 365 nm) was started for a total of 30 min. The irradiation unit (UV‑365) is an in‑house production of the University Medical Center of the Johannes Gutenberg University in Mainz. The irradiance was measured by the UVA radiometer ILT 72 (International Light Technology, Peabody, USA). The application of the riboflavin solution was continued every 3 min during irradiation. Using solution 6, we reduced the dropping time before radiation from 30 to 20 min.
RESULTS A total of 15 eyes of 11 patients were treated. The average age of patients was 17.9 ± 4.7 years and ranged from 11 to 27 years. The treated eyes showed advanced stages of keratoconus with a mean maximum keratometry value of 61 ± 4.3 D ranging from 53.1 to 68.8 D. The mean minimal corneal pachymetry including epithelium was 379 ± 81 µm ranging from 266 to 515 µm [Table 2]. Figure 1 shows the mean CCT as the percentage of the CCT after de‑epithelialization. During 60 min of application, solution 1 caused an intraoperative swelling of the corneal stroma to an
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Mark, et al.: Collagen Crosslinking in the West Region of Cameroon Table 1: Initial weights for the production of 100 g riboflavin eye drops Ingredients Riboflavin MHPC Dextran Sodium chloride Aqua ad inj.
Solution 1
Solution 2
Solution 3
Solution 4
Solution 5
Solution 6
0.191 g 0.5 g ‑ 0.9 g 98.4 ml
0.127 g 1.0 g ‑ 1.1 g 97.8 ml
0.127 g 1.7 g ‑ 1.1 g 97.1 ml
0.127 g ‑ 5g 0.9 g 94.0 ml
0.127 g ‑ 10 g 0.9 g 89.0 ml
0.254 g ‑ ‑ 1.1 g 98.7 ml
MHPC: Methylhydroxypropylcellulose
Table 2: Pre‑operative and intra‑operative findings Patients
Age
K‑max (D)
Minimal CCT (µm)
1 2 3 4 5 1 6 7 4 8 9 8 3 10 11
11 15 16 18 27 11 16 17 18 22 13 22 16 18 24
68.8 60.0 61.7 59.6 63.7 65.7 54.5 59.4 63.9 66.2 59.7 53.1 56.3 59.9 60.6
337 344 272 355 352 333 480 432 282 431 266 429 410 339 515
Viscosity additive % MHPC 0.5
MHPC 1.0 MHPC 1.7 Dextran 5 Dextran 10 No additive
Drop application (min)
Pre‑swelling (µm)
Post‑swelling (µm)
Increase (µm)
60 60 60 60 60 60 60 60 60 60 60 45 45 50 50
320 350 358 399 316 315 422 370 398 408 380 517* 542* 413 470
609 619 569 618 567 583 746 675 781 715 647 510 436 658 659
289 269 211 219 251 268 324 305 383 307 273 −7 −106 245 189
Minimal CCT represents the thinnest point of the epithelialized cornea (single measurement). Pre‑and post‑swelling data represent the average of at least 5 repetitive measurements on different positions of the de‑epithelialized central cornea. The values marked with*characterize the CCT after an initial swelling period with solution 6. MHPC: Methylhydroxypropylcellulose, CCT: Central corneal thickness
Solution 4 and 5 were applied in the context of our modified treatment procedure, which required an initial swelling of the corneal stroma considerably over 400 µm and a subsequent stabilizing of the achieved CCT value with an adapted dextran composition. The swelling of the corneal stroma was achieved by applying solution 6 and stabilization was achieved by applying solution 4. Figure 2 compares this modified protocol with a treatment procedure using a MHPC solution.
DISCUSSION Figure 1: Mean central corneal thickness (CCT) in percentages of the CCT before treatment in 15 eyes after deepithelialization and application of various eye drop solutions. The graphs marked with * are measured after an initial swelling period with solution 6
average of 172 ± 15% (n = 5). Application of solution 2 for 60 min resulted in a final CCT value of 183 ± 8% (n = 5). In the same time span, solution 3 led to a swelling up to 170% (n = 1). Solution 4 seemed to be proper for stabilizing the stromal volume, reaching a mean final CCT of 99% after 45 min of application (n = 1). There was a marked thinning of final CCT (80%) using solution 5 for 45 min (n = 1). Application of solution 6 for 50 min resulted in an increase in corneal thickness to a mean value of 150 ± 13% after 50 min (n = 2). 68
Without exception, all known studies on CXL emphasize the challenge of keeping the stromal thickness of treated keratoconus corneas over the essential threshold of 400 µm.13,14 All the more surprising it seemed to us that we apparently had to deal with the contrary problem. Although we had to manage highly thinned out corneas, our main task consisted in keeping the intraoperative swelling within a limit. We treated five eyes with a riboflavin solution containing 0.5% MHPC (solution 1). This composition seemed to be particularly suitable for the treatment of thin African corneas, as a similar solution (riboflavin 0.2%/MHPC 0.5%/NaCl 0.7%) caused an increase of mean final CCT to 156% in Caucasian corneas
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with the drops based on MHPC, this solution showed a reduction of the intraoperative swelling, reaching a final CCT of 150%. Nevertheless, the achieved absolute values of 658 µm and 659 µm were still too high to ensure effective treatment [Table 2]. Considering these observations, it seemed that the intraoperative swelling could be modulated adequately by using a combination of swelling and stabilizing drop solutions. Swelling drops to bring the thin corneas over the necessary threshold of 400 µm and stabilizing eye drops to keep the corneal thickness in a range approximately between 400 and 500 µm which should ensure an effective treatment. Figure 2: Intra‑operative swelling of two treated eyes (patient 8). Graph (a) shows the swelling applying a methylhydroxypropylcellulose solution. Graph (b) shows the swelling and stabilizing period, using the modified protocol. The influence of the consecutive used solutions might overlap temporarily
(n = 7).15 Unexpectedly, this composition led to a considerably stronger intraoperative swelling in the treated African eyes, reaching a mean final CCT of 172%. Although the post‑abrasio values of the five treated eyes were below 400 µm, they all swelled to values around 600 µm during CXL [Table 2]. This intense swelling might be counter‑productive for the outcome of the procedure. CXL is expected to lose effectiveness in artificially swollen corneas, due to the lower relative concentration of collagen in the hydrated stroma.16,17 Furthermore, the currently used treatment parameters are assumed to treat the anterior 250 µm to 350 µm.18 This suggests that corneas that are swollen too much are probably just cross‑linked in the upper portion. Against this background our aim was to swell the corneal stroma as much as necessary and as little as possible. We increased the MHPC portion in the eye drops to 1.0% (soution 2) and 1.7% (solution 3). We tried to increase the colloid osmotic pressure of the solution, in order to counteract the swelling pressure of the cornea. With final CCTs up to 781 µm (solution 2) and 647 µm (solution 3) in individual corneas this step did not lead to the desired effect [Table 2]. Furthermore, the increased MHPC portion reduced the practicality of the drops due to the rising viscosity. However, in the next step we tried to counteract the swelling of the stroma by allowing some loss to evaporation. The film of the applied MHPC solutions covers the cornea for approximately 32 min before it breaks up. This makes evaporation of corneal fluid into the air improbable.19 To allow evaporation, we treated two eyes using eye drops without colloid viscosity additive (solution 6). The break up time of these drops was expected to be close to 90 s.19 Keeping a drop interval of 3 min, we assumed approximately 90 s after the break‑up of the corneal film where the stroma could lose volume through evaporation. Supportingly, the dropping time before radiation was shortened to 20 min. In comparison
Searching for a stabilizing riboflavin composition we referred to findings made in an in vitro study in porcine corneas. In this study, a negative correlation between the dextran concentration and the intraoperative swelling of the cornea was detected.11 Accordingly, a drop composition with less than the currently used 20% reduces the intraoperative deswelling of the stroma probably due to the lower amount of hydrophilic hydroxyl groups in the solution and the decreased colloid osmotic pressure. First of all we swelled the stroma using eye drops without colloid viscosity additive (solution 6) clearly over the threshold of 400 µm (542 µm). Afterward we tried to stabilize the achieved value using a drop composition with 10% dextran (solution 5). In vitro, this drop composition caused a very slight increase of corneal thickness.11 However, in vivo the 10% dextran drops showed still a clear decrease of CCT to 436 µm during 45 min of application [Table 2]. As these drops were obviously unsuitable for our intention we continued reducing the dextran concentration to 5% (solution 4). Once more we swelled the corneal stroma clearly over the 400 µm threshold (517 µm) using drops without viscosity additive (solution 6). In the following 45 min we applied the drops with 5% dextran. These drops seemed to be successful for stabilizing the stromal volume during treatment. They only decreased the value to 510 µm [Table 2 and Figure 2]. The question remains why the treated African corneas seemed to show a different swelling behavior in comparison with Caucasian eyes. We cannot determine the underlying etiological factors for this difference. It is unlikely that climate such as temperature or humidity has an influence, as Bafoussam (1500 m) is marked by a relatively mild climate. The assumption that the African origin itself might have an effect on the intraoperative swelling behavior is speculative. Apart from the awareness that African corneas are thinner than Caucasians,20,21 there are no other relevant differences described in the literature. However, we want to highlight one aspect, which does not necessarily have an influence on the swelling behavior, but
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which is remarkable nevertheless. Our treated patients in Bafoussam had a mean age of 17.9 ± 4.7 years, which on average, is 14 years younger than patients treated within the framework of a retrospective crosslinking study at the University Medical Center of the Johannes Gutenberg University in Mainz, Germany (n = 23).15 However, these young patients showed advanced stages of keratoconus. It is known that African American children have corneas that are on average 20 µm thinner than those of white and Hispanic children.22 However, this fact alone may not explain the early onset of the disease. Most likely this phenomenon is attributed to an increased eye rubbing, which was found in the medical history of all the treated patients. A multivariate study detected that the increased tendency of atopic patients to develop keratoconus can be attributed exclusively to eye rubbing.23 Eye rubbing leads to repeated microtrauma and therefore to increased release of interleukin‑1. This modulates the apoptosis of keratocytes. Due to the genetically determined higher expression of interleukin‑1‑receptors, in the keratocytes of patients with keratoconus, an increased release of interleukin‑1 could lead to a loss of keratocytes and stromal volume.24 There is no doubt that the use of individually adjusted swelling and stabilizing riboflavin compositions would bring the greatest benefit for each patient. However, this is not practical.
CONCLUSION
We gratefully acknowledge the research funding for tropical ophthalmology by the German Ophthalmological Society.
REFERENCES 1.
2. 3. 4.
5. 6.
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10.
To our findings, our recommended treatment protocol for thin African corneas provides an initial swelling of the corneal stroma to a value of 450 µm, using a drop solution without viscosity additive. This value should be subsequently stabilized applying a riboflavin solution with 5% dextran. Certainly, these drops may also cause a slight stromal swelling or deswelling depending upon the treated patient, but under pachymetric control this method provides adequate protection of the endothelium and should ensure the greatest benefit for patients. We believe the continuous monitoring of CCT with pachymetry is imperative for successful treatment. 10 repeat measurements on different regions of the central cornea should be performed every 5 min. The increased number of repetitive measurements should be performed in order not to overlook the thinnest point. None of the 10 values should be under 400 µm. This pilot study has some limitations. Our findings are not the result of an experimental set‑up, but reflect our reactions to the unexpected intraoperative swelling behavior of the treated African corneas. As keratoconus is a rare disease we have not treated enough patients yet to consolidate the results achieved so far. Further studies are necessary. In addition, the clinical effectiveness of CXL in African corneas has to be evaluated. 70
ACKNOWLEDGMENTS
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Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet‑a‑induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003;135:620‑7. Wollensak G. Crosslinking treatment of progressive keratoconus: New hope. Curr Opin Ophthalmol 2006;17:356‑60. Spoerl E, Huhle M, Seiler T. Induction of cross‑links in corneal tissue. Exp Eye Res 1998;66:97‑103. 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;29:1780‑5. Wollensak G, Spörl E, Seiler T. Treatment of keratoconus by collagen cross linking. Ophthalmologe 2003;100:44‑9. Wollensak G, Spoerl E, Wilsch M, Seiler T. Endothelial cell damage after riboflavin‑ultraviolet‑A treatment in the rabbit. J Cataract Refract Surg 2003;29:1786‑90. Iseli HP, Thiel MA, Hafezi F, Kampmeier J, Seiler T. Ultraviolet A/ riboflavin corneal cross‑linking for infectious keratitis associated with corneal melts. Cornea 2008;27:590‑4. Spoerl E, Wollensak G, Seiler T. Increased resistance of crosslinked cornea against enzymatic digestion. Curr Eye Res 2004;29:35‑40. Kanellopoulos AJ, Binder PS. Collagen cross‑linking (CCL) with sequential topography‑guided PRK: A temporizing alternative for keratoconus to penetrating keratoplasty. Cornea 2007;26:891‑5. Dohlman Ch, Hedbys BO, Mishima S. The swelling pressure of the corneal stroma. Invest Ophthalmol 1962;1:158‑62. Vetter JM, Brueckner S, Tubic‑Grozdanis M, Vossmerbäumer U, Pfeiffer N, Kurz S. Modulation of central corneal thickness by various riboflavin eyedrop compositions in porcine corneas. J Cataract Refract Surg 2012;38:525‑32. Kymionis GD, Kounis GA, Portaliou DM, Grentzelos MA, Karavitaki AE, Coskunseven E, et al. Intraoperative pachymetric measurements during corneal collagen cross‑linking with riboflavin and ultraviolet A irradiation. Ophthalmology 2009;116:2336‑9. Hafezi F, Mrochen M, Iseli HP, Seiler T. Collagen crosslinking with ultraviolet‑A and hypoosmolar riboflavin solution in thin corneas. J Cataract Refract Surg 2009;35:621‑4. Raiskup F, Spoerl E. Corneal cross‑linking with hypo‑osmolar riboflavin solution in thin keratoconic corneas. Am J Ophthalmol 2011;152:28‑321. Vetter JM, Tubic‑Grozdanis M, Faust M, Lorenz K, Gericke A, Stoffelns BM. Effect of various compositions of riboflavin eye drops on the intraoperative corneal thickness during UVA‑cross‑linking in keratoconus eyes. Klin Monbl Augenheilkd 2011;228:509‑14. Müller LJ, Pels E, Vrensen GF. The effects of organ‑culture on the density of keratocytes and collagen fibers in human corneas. Cornea 2001;20:86‑95. Ahearne M, Yang Y, Then KY, Liu KK. Non‑destructive mechanical characterisation of UVA/riboflavin crosslinked collagen hydrogels. Br J Ophthalmol 2008;92:268‑71. Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. Safety of UVA‑riboflavin cross‑linking of the cornea. Cornea 2007;26:385‑9.
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Mark, et al.: Collagen Crosslinking in the West Region of Cameroon 19. Wollensak G, Aurich H, Wirbelauer C, Sel S. Significance of the riboflavin film in corneal collagen crosslinking. J Cataract Refract Surg 2010;36:114‑20. 20. La Rosa FA, Gross RL, Orengo‑Nania S. Central corneal thickness of Caucasians and African Americans in glaucomatous and nonglaucomatous populations. Arch Ophthalmol 2001;119:23‑7. 21. Aghaian E, Choe JE, Lin S, Stamper RL. Central corneal thickness of Caucasians, Chinese, Hispanics, Filipinos, African Americans, and Japanese in a glaucoma clinic. Ophthalmology 2004;111:2211‑9. 22. Pediatric Eye Disease Investigator Group, Bradfield YS, Melia BM, Repka MX, Kaminski BM, Davitt BV, et al. Central
corneal thickness in children. Arch Ophthalmol 2011;129:1132‑8. 23. Bawazeer AM, Hodge WG, Lorimer B. Atopy and keratoconus: A multivariate analysis. Br J Ophthalmol 2000;84:834‑6. 24. Bron AJ, Rabinowitz YS. Corneal dystrophies and keratoconus. Curr Opin Ophthalmol 1996;7:71‑82.
Cite this article as: Mark T, Ngounou F, Tamon J, Marx-Gross S, Preussner P. Modulatory effect of different riboflavin compositions on the central corneal thickness of African keratoconus corneas during collagen crosslinking. Middle East Afr J Ophthalmol 2014;21:66-71. Source of Support: Research funding for tropical ophthalmology by the German Ophthalmological Society, Conflict of Interest: None declared.
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ABSTRACT Purpose: A pilot investigation to transfer the established corneal collagen crosslinking (CXL) procedure in European eyes into clinically affected African eyes and to optimize the treatment by adapting the riboflavin composition. Materials and Methods: CXL was performed in 15 eyes (11 patients) with advanced stages of keratoconus in the Eye Clinic of Bafoussam in the West Region of Cameroon. The following six riboflavin compositions with different portions of active swelling additives were applied: Solution 1 (0.5% methylhydroxypropylcellulose [MHPC]), solution 2 (1.0% MHPC), solution 3 (1.7% MHPC), solution 4 (5% dextran), solution 5 (10% dextran) and solution 6 (no active swelling ingredient). The central corneal thickness (CCT) was measured by ultrasound pachymetry before and after de‑epithelialization and at least every 10 min during CXL. Results: The application of the riboflavin solutions resulted in the following mean final CCT values: 172 ± 15% using solution 1 (60 min/n = 5); 183 ± 8% using solution 2 (60 min/n = 5); 170% using solution 3 (60 min/n = 1); 80% using solution 4 (45 min/n = 1); 99% using solution 5 (45 min/n = 1) and 150 ± 13% using solution 6 (50 min/n = 2). Conclusions: The combination of riboflavin compositions with swelling and stabilizing effects on the corneal stroma seems necessary in African eyes with advanced keratoconus. Further studies are required to confirm these primary results.
Website: www.meajo.org DOI: 10.4103/0974-9233.124103 Quick Response Code:
Key words: Africa, Collagen Crosslinking, Corneal Thickness, Keratoconus, Riboflavin
INTRODUCTION
C
orneal collagen crosslinking (CXL), using ultraviolet‑A (UVA) radiation and the photosensitizer riboflavin, was recently introduced for the treatment of progressive keratoconus.1,2 In several studies, the method has shown a positive effect on the biomechanical1,3‑5 and biochemical6‑8 stability of the cornea. Thus, CXL has the potential to stop the progression of keratoconus, avoiding the need for penetrating keratoplasty.9 In regions of the world, where there is no access to corneal transplants, this simple and low priced procedure seems to offer
the only accessible treatment for keratoconus patients at this time. CXL requires a minimum central corneal thickness (CCT) of 400 µm to protect the endothelium from the cytotoxic irradiance.6 Often, late diagnosed keratoconus corneas reach values considerably below this threshold. However, during CXL the stromal thickness can be influenced by the chemical composition of the applied riboflavin solution. Exposed to a physiologic aqueous medium the deepithelialized cornea shows the tendency to increase its volume (swelling pressure), which is mainly due to the concentration of stromal polysaccharides.10 Recent in vitro studies in porcine corneas indicate that the osmolarity of the applied medium does not correlate with intraoperative corneal swelling.11
University Medical Center of the Johannes Gutenberg University Mainz, Department of Ophthalmology, Mainz, Germany, 1 Presbyterian Eye Services, Eye Clinic, Acha-Bafoussam, Bafoussam, Cameroon Corresponding Author: Mr. Timo Mark, Department of Ophthalmology, University Medical Center of the Johannes Gutenberg University, Mainz, Langenbeckstr 1, 55101 Mainz, Germany. E-mail: [email protected]
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Hence, the stroma can be hydrated or dehydrated during CXL, depending upon exposure to a riboflavin solution with a colloid osmotic pressure lower or higher than the physiological swelling pressure.
progression of keratoconus was confirmed from medical history. Central corneal scars were not regarded as an exclusion criterion, as there was no keratoplasty treatment available for these patients.
The colloid osmotic pressure of the commonly applied CXL drop composition is mainly given by the polysaccharide dextran (20%), which is added to increase the viscosity of the solution. Dextran shows abundant hydrophilic hydroxyl groups and its solution leads to a marked intraoperative reduction of CCT.12 Logically, these drops are not adequate in controlling the 400 µm threshold in patients with advanced stages of keratoconus.
Six different riboflavin solutions were used [Table 1]. Drops containing MHPC were prepared following a protocol of the University Hospital Carl Gustav Carus in Dresden. Drops with dextran and the drop composition without viscosity additive were produced according to protocols of the University Medical Center of the Johannes Gutenberg University in Mainz. The concentrations of the ingredients were adapted to our requirements. Solution 2 was produced in the Pharmacy of the University Medical Center of the Johannes Gutenberg University in Mainz. All other drops were prepared in the Pharmacy of the Eye Clinic Bafoussam.
However, a number of alternative treatment protocols for thin corneas have been proposed. 13,14 One possibility is to apply a solution containing 0.5% of the polysaccharide methylhydroxypropylcellulose (MHPC). In comparison with dextran, MHPC is missing the water binding hydroxyl groups and its solution is known to swell the stroma quite effectively.15 It should be noted in this context, that CXL is expected to be less effective in artificially swollen corneas, due to the lower relative concentration of collagen in the hydrated stroma.16,17 Therefore, the stroma should be swollen as much as necessary, but as little as possible. In this pilot study, we introduced CXL in the Eye Clinic of Bafoussam in the West Region of Cameroon. We applied variations of riboflavin solutions in order to deal with highly thinned out corneas. The major focus of this study was to determine the most effective treatment for each patient. In this every particular change of the riboflavin composition used is a result of our reaction to the given conditions.
MATERIALS AND METHODS CXL was performed in September 2011 and March 2012 in the Eye Clinic of Bafoussam in the West Region of Cameroon. When we started treatment with the well‑established European protocol, a “study” was not intended. We wanted to treat patients for whom no alternative treatment was available. However, during treatment, we found significant, unexpected differences between these African eyes compared with European eyes. Thus, we optimized our procedure step‑by‑step. Therefore, we did not apply for approval from the Ethics Committee and did not register the project with the Institutional Review Board. Nevertheless, all the procedures were in accordance with the ethical standards of the declaration of Helsinki. The diagnosis of keratoconus was based on corneal topography, ultrasound pachymetry and clinical signs, such as stromal thinning, Vogt striae and apical stromal scars. The pre‑operative
The treatment procedure was conducted under sterile conditions in the operating theater of the clinic. After instilling topical anesthesia (amethocaine 0.5%) and inserting a lid speculum, the central 9 mm of the corneal epithelium were abraded with a hockey stick spatula. The CCT was measured by ultrasound pachymetr y before and immediately after de‑epithelialization and at least every 10 min during the 60 min of eye drop application. In order to detect the thinnest point, at least five repeat measurements at different positions on the central cornea were performed. The riboflavin solution was applied every 3 min for 30 min prior to irradiation to ensure sufficient saturation of the stroma. After confirming that the thinnest point of the corneal stroma was thicker than 400 µm, UVA‑irradiation (3 mW/cm2, 365 nm) was started for a total of 30 min. The irradiation unit (UV‑365) is an in‑house production of the University Medical Center of the Johannes Gutenberg University in Mainz. The irradiance was measured by the UVA radiometer ILT 72 (International Light Technology, Peabody, USA). The application of the riboflavin solution was continued every 3 min during irradiation. Using solution 6, we reduced the dropping time before radiation from 30 to 20 min.
RESULTS A total of 15 eyes of 11 patients were treated. The average age of patients was 17.9 ± 4.7 years and ranged from 11 to 27 years. The treated eyes showed advanced stages of keratoconus with a mean maximum keratometry value of 61 ± 4.3 D ranging from 53.1 to 68.8 D. The mean minimal corneal pachymetry including epithelium was 379 ± 81 µm ranging from 266 to 515 µm [Table 2]. Figure 1 shows the mean CCT as the percentage of the CCT after de‑epithelialization. During 60 min of application, solution 1 caused an intraoperative swelling of the corneal stroma to an
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Mark, et al.: Collagen Crosslinking in the West Region of Cameroon Table 1: Initial weights for the production of 100 g riboflavin eye drops Ingredients Riboflavin MHPC Dextran Sodium chloride Aqua ad inj.
Solution 1
Solution 2
Solution 3
Solution 4
Solution 5
Solution 6
0.191 g 0.5 g ‑ 0.9 g 98.4 ml
0.127 g 1.0 g ‑ 1.1 g 97.8 ml
0.127 g 1.7 g ‑ 1.1 g 97.1 ml
0.127 g ‑ 5g 0.9 g 94.0 ml
0.127 g ‑ 10 g 0.9 g 89.0 ml
0.254 g ‑ ‑ 1.1 g 98.7 ml
MHPC: Methylhydroxypropylcellulose
Table 2: Pre‑operative and intra‑operative findings Patients
Age
K‑max (D)
Minimal CCT (µm)
1 2 3 4 5 1 6 7 4 8 9 8 3 10 11
11 15 16 18 27 11 16 17 18 22 13 22 16 18 24
68.8 60.0 61.7 59.6 63.7 65.7 54.5 59.4 63.9 66.2 59.7 53.1 56.3 59.9 60.6
337 344 272 355 352 333 480 432 282 431 266 429 410 339 515
Viscosity additive % MHPC 0.5
MHPC 1.0 MHPC 1.7 Dextran 5 Dextran 10 No additive
Drop application (min)
Pre‑swelling (µm)
Post‑swelling (µm)
Increase (µm)
60 60 60 60 60 60 60 60 60 60 60 45 45 50 50
320 350 358 399 316 315 422 370 398 408 380 517* 542* 413 470
609 619 569 618 567 583 746 675 781 715 647 510 436 658 659
289 269 211 219 251 268 324 305 383 307 273 −7 −106 245 189
Minimal CCT represents the thinnest point of the epithelialized cornea (single measurement). Pre‑and post‑swelling data represent the average of at least 5 repetitive measurements on different positions of the de‑epithelialized central cornea. The values marked with*characterize the CCT after an initial swelling period with solution 6. MHPC: Methylhydroxypropylcellulose, CCT: Central corneal thickness
Solution 4 and 5 were applied in the context of our modified treatment procedure, which required an initial swelling of the corneal stroma considerably over 400 µm and a subsequent stabilizing of the achieved CCT value with an adapted dextran composition. The swelling of the corneal stroma was achieved by applying solution 6 and stabilization was achieved by applying solution 4. Figure 2 compares this modified protocol with a treatment procedure using a MHPC solution.
DISCUSSION Figure 1: Mean central corneal thickness (CCT) in percentages of the CCT before treatment in 15 eyes after deepithelialization and application of various eye drop solutions. The graphs marked with * are measured after an initial swelling period with solution 6
average of 172 ± 15% (n = 5). Application of solution 2 for 60 min resulted in a final CCT value of 183 ± 8% (n = 5). In the same time span, solution 3 led to a swelling up to 170% (n = 1). Solution 4 seemed to be proper for stabilizing the stromal volume, reaching a mean final CCT of 99% after 45 min of application (n = 1). There was a marked thinning of final CCT (80%) using solution 5 for 45 min (n = 1). Application of solution 6 for 50 min resulted in an increase in corneal thickness to a mean value of 150 ± 13% after 50 min (n = 2). 68
Without exception, all known studies on CXL emphasize the challenge of keeping the stromal thickness of treated keratoconus corneas over the essential threshold of 400 µm.13,14 All the more surprising it seemed to us that we apparently had to deal with the contrary problem. Although we had to manage highly thinned out corneas, our main task consisted in keeping the intraoperative swelling within a limit. We treated five eyes with a riboflavin solution containing 0.5% MHPC (solution 1). This composition seemed to be particularly suitable for the treatment of thin African corneas, as a similar solution (riboflavin 0.2%/MHPC 0.5%/NaCl 0.7%) caused an increase of mean final CCT to 156% in Caucasian corneas
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with the drops based on MHPC, this solution showed a reduction of the intraoperative swelling, reaching a final CCT of 150%. Nevertheless, the achieved absolute values of 658 µm and 659 µm were still too high to ensure effective treatment [Table 2]. Considering these observations, it seemed that the intraoperative swelling could be modulated adequately by using a combination of swelling and stabilizing drop solutions. Swelling drops to bring the thin corneas over the necessary threshold of 400 µm and stabilizing eye drops to keep the corneal thickness in a range approximately between 400 and 500 µm which should ensure an effective treatment. Figure 2: Intra‑operative swelling of two treated eyes (patient 8). Graph (a) shows the swelling applying a methylhydroxypropylcellulose solution. Graph (b) shows the swelling and stabilizing period, using the modified protocol. The influence of the consecutive used solutions might overlap temporarily
(n = 7).15 Unexpectedly, this composition led to a considerably stronger intraoperative swelling in the treated African eyes, reaching a mean final CCT of 172%. Although the post‑abrasio values of the five treated eyes were below 400 µm, they all swelled to values around 600 µm during CXL [Table 2]. This intense swelling might be counter‑productive for the outcome of the procedure. CXL is expected to lose effectiveness in artificially swollen corneas, due to the lower relative concentration of collagen in the hydrated stroma.16,17 Furthermore, the currently used treatment parameters are assumed to treat the anterior 250 µm to 350 µm.18 This suggests that corneas that are swollen too much are probably just cross‑linked in the upper portion. Against this background our aim was to swell the corneal stroma as much as necessary and as little as possible. We increased the MHPC portion in the eye drops to 1.0% (soution 2) and 1.7% (solution 3). We tried to increase the colloid osmotic pressure of the solution, in order to counteract the swelling pressure of the cornea. With final CCTs up to 781 µm (solution 2) and 647 µm (solution 3) in individual corneas this step did not lead to the desired effect [Table 2]. Furthermore, the increased MHPC portion reduced the practicality of the drops due to the rising viscosity. However, in the next step we tried to counteract the swelling of the stroma by allowing some loss to evaporation. The film of the applied MHPC solutions covers the cornea for approximately 32 min before it breaks up. This makes evaporation of corneal fluid into the air improbable.19 To allow evaporation, we treated two eyes using eye drops without colloid viscosity additive (solution 6). The break up time of these drops was expected to be close to 90 s.19 Keeping a drop interval of 3 min, we assumed approximately 90 s after the break‑up of the corneal film where the stroma could lose volume through evaporation. Supportingly, the dropping time before radiation was shortened to 20 min. In comparison
Searching for a stabilizing riboflavin composition we referred to findings made in an in vitro study in porcine corneas. In this study, a negative correlation between the dextran concentration and the intraoperative swelling of the cornea was detected.11 Accordingly, a drop composition with less than the currently used 20% reduces the intraoperative deswelling of the stroma probably due to the lower amount of hydrophilic hydroxyl groups in the solution and the decreased colloid osmotic pressure. First of all we swelled the stroma using eye drops without colloid viscosity additive (solution 6) clearly over the threshold of 400 µm (542 µm). Afterward we tried to stabilize the achieved value using a drop composition with 10% dextran (solution 5). In vitro, this drop composition caused a very slight increase of corneal thickness.11 However, in vivo the 10% dextran drops showed still a clear decrease of CCT to 436 µm during 45 min of application [Table 2]. As these drops were obviously unsuitable for our intention we continued reducing the dextran concentration to 5% (solution 4). Once more we swelled the corneal stroma clearly over the 400 µm threshold (517 µm) using drops without viscosity additive (solution 6). In the following 45 min we applied the drops with 5% dextran. These drops seemed to be successful for stabilizing the stromal volume during treatment. They only decreased the value to 510 µm [Table 2 and Figure 2]. The question remains why the treated African corneas seemed to show a different swelling behavior in comparison with Caucasian eyes. We cannot determine the underlying etiological factors for this difference. It is unlikely that climate such as temperature or humidity has an influence, as Bafoussam (1500 m) is marked by a relatively mild climate. The assumption that the African origin itself might have an effect on the intraoperative swelling behavior is speculative. Apart from the awareness that African corneas are thinner than Caucasians,20,21 there are no other relevant differences described in the literature. However, we want to highlight one aspect, which does not necessarily have an influence on the swelling behavior, but
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which is remarkable nevertheless. Our treated patients in Bafoussam had a mean age of 17.9 ± 4.7 years, which on average, is 14 years younger than patients treated within the framework of a retrospective crosslinking study at the University Medical Center of the Johannes Gutenberg University in Mainz, Germany (n = 23).15 However, these young patients showed advanced stages of keratoconus. It is known that African American children have corneas that are on average 20 µm thinner than those of white and Hispanic children.22 However, this fact alone may not explain the early onset of the disease. Most likely this phenomenon is attributed to an increased eye rubbing, which was found in the medical history of all the treated patients. A multivariate study detected that the increased tendency of atopic patients to develop keratoconus can be attributed exclusively to eye rubbing.23 Eye rubbing leads to repeated microtrauma and therefore to increased release of interleukin‑1. This modulates the apoptosis of keratocytes. Due to the genetically determined higher expression of interleukin‑1‑receptors, in the keratocytes of patients with keratoconus, an increased release of interleukin‑1 could lead to a loss of keratocytes and stromal volume.24 There is no doubt that the use of individually adjusted swelling and stabilizing riboflavin compositions would bring the greatest benefit for each patient. However, this is not practical.
CONCLUSION
We gratefully acknowledge the research funding for tropical ophthalmology by the German Ophthalmological Society.
REFERENCES 1.
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To our findings, our recommended treatment protocol for thin African corneas provides an initial swelling of the corneal stroma to a value of 450 µm, using a drop solution without viscosity additive. This value should be subsequently stabilized applying a riboflavin solution with 5% dextran. Certainly, these drops may also cause a slight stromal swelling or deswelling depending upon the treated patient, but under pachymetric control this method provides adequate protection of the endothelium and should ensure the greatest benefit for patients. We believe the continuous monitoring of CCT with pachymetry is imperative for successful treatment. 10 repeat measurements on different regions of the central cornea should be performed every 5 min. The increased number of repetitive measurements should be performed in order not to overlook the thinnest point. None of the 10 values should be under 400 µm. This pilot study has some limitations. Our findings are not the result of an experimental set‑up, but reflect our reactions to the unexpected intraoperative swelling behavior of the treated African corneas. As keratoconus is a rare disease we have not treated enough patients yet to consolidate the results achieved so far. Further studies are necessary. In addition, the clinical effectiveness of CXL in African corneas has to be evaluated. 70
ACKNOWLEDGMENTS
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Mark, et al.: Collagen Crosslinking in the West Region of Cameroon 19. Wollensak G, Aurich H, Wirbelauer C, Sel S. Significance of the riboflavin film in corneal collagen crosslinking. J Cataract Refract Surg 2010;36:114‑20. 20. La Rosa FA, Gross RL, Orengo‑Nania S. Central corneal thickness of Caucasians and African Americans in glaucomatous and nonglaucomatous populations. Arch Ophthalmol 2001;119:23‑7. 21. Aghaian E, Choe JE, Lin S, Stamper RL. Central corneal thickness of Caucasians, Chinese, Hispanics, Filipinos, African Americans, and Japanese in a glaucoma clinic. Ophthalmology 2004;111:2211‑9. 22. Pediatric Eye Disease Investigator Group, Bradfield YS, Melia BM, Repka MX, Kaminski BM, Davitt BV, et al. Central
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Cite this article as: Mark T, Ngounou F, Tamon J, Marx-Gross S, Preussner P. Modulatory effect of different riboflavin compositions on the central corneal thickness of African keratoconus corneas during collagen crosslinking. Middle East Afr J Ophthalmol 2014;21:66-71. Source of Support: Research funding for tropical ophthalmology by the German Ophthalmological Society, Conflict of Interest: None declared.
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