REVIEW URRENT C OPINION
Current and future applications of corneal cross-linking Sumitra S. Khandelwal a and J. Bradley Randleman b,c
Purpose of review To review current concepts and future directions of corneal cross-linking (CXL) as a treatment for keratoconus, ectasia after refractive surgery and infectious keratitis. Recent findings Several important laboratory and clinical studies have established the safety and success of corneal crosslinking for the treatment of keratoconus and other corneal ectasias. Recently, additional studies have analyzed new directions and controversies in corneal cross-linking, exploring new indications, comparing new techniques and analyzing results of new protocols. Summary The results of bench and clinical research are providing the foundation to allow for protocol modifications of the standard cross-linking protocols and expansion of cross-linking concepts for techniques such as accelerated cross-linking, epithelium-sparing protocols and measurement of progression and success. Keywords accelerated corneal cross-linking, corneal cross-linking, cross-linking, ectasia, infectious keratitis, keratoconus
INTRODUCTION Corneal cross-linking (CXL) represents the epitome of translational research in the cornea. From its initial inspiration on a visit to the dentist by Professor Theo Seiler, to the remarkable increase in corneal rigidity demonstrated in laboratory models, to impressive results demonstrated in prospective clinical trials, CXL has revolutionized the treatment of corneal ectasias. Since its advent, CXL has evolved to include alternative indications, parameters and protocols. This article serves to review the fundamental aspects of this historic treatment, to discuss its current and future indications and to evaluate the data regarding new techniques and protocols in the treatment of corneal diseases.
FUNDAMENTAL CONCEPTS IN CORNEAL CROSS-LINKING Observations that the rate of keratoconus was lower in patients with diabetes and increased age sparked the idea that natural cross-linking of the corneal collagen fibrils may result in strengthening and stiffening of tissue [1]. This led to the development of CXL, a process in which a combination of a photoinducer, ultraviolet (UV) light and a photochemical reaction induces free radicals, leading to a www.co-ophthalmology.com
chemical bond between collagen fibrils. Initial CXL animal studies resulted in up to a 70% increase in corneal rigidity [2], whereas the first clinical study by Wollensak et al. [3] showed halting of corneal progression and topographic flattening in patients with keratoconus. Since then, several prospective studies have echoed these results in patients with keratoconus [4,5,6 ,7–9,10 ] and ectasia after corneal refractive surgery (hereafter termed ‘ectasia’) [11,12]. &&
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Photoinducers Riboflavin functions as an optimal photoinducer in the biochemical reaction in CXL. It possesses properties such as a wide range of absorption, although it is still well tolerated for systemic absorption [13]. Riboflavin is, however, a relatively large molecule; thus, its primary limitation is sufficient absorption a
Baylor College of Medicine, Cullen Eye Institute, Houston, Texas, USA, Department of Ophthalmology, Emory University and cEmory Vision, Emory Eye Center, Atlanta, Georgia, USA b
Correspondence to J. Bradley Randleman, MD, Emory Eye Center, 5671 Peachtree Dunwoody Rd NE, Suite 400 Atlanta, GA 30342, USA. E-mail: [email protected] Curr Opin Ophthalmol 2015, 26:206–213 DOI:10.1097/ICU.0000000000000146 Volume 26 Number 3 May 2015
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KEY POINTS CXL has shown to be well tolerated and efficacious in the treatment of keratoconus and ectasia after refractive surgery. Standard protocols have shown stable long-term improvement in vision and corneal topography. New variations including epithelium-sparing techniques, accelerated protocols and alternate imaging of the cornea are in early stages, but may provide alternate ways to treat corneal disease.
into the corneal stroma through an intact epithelium [14]. Various techniques, termed ‘epithelium on’ CXL, have been studied to navigate around this issue, although the success of these protocols remains controversial.
Ultraviolet light UV light serves as the second important component of CXL. Key parameters include wavelength, irradiance and time of irradiation; these parameters are specific to the success and safety of treatment. The range of absorption peak of riboflavin for CXL ranges from 360 to 370 nm as determined through initial studies [15]. Variations to intensity and duration in preclinical studies established that maximum stiffening involved the use of 3 mW/cm2 of energy for 30 min, which corresponded to a total energy dose (fluence) of 5.4 J/cm2. This guided the development of the standard Dresden protocol, which allowed for greatest efficiency of tissue stiffening. Variations to this protocol have been termed accelerated CXL. The actual photochemical reaction that leads to stiffening involves riboflavin absorbing the ultraviolet light (UVA) energy and exciting the molecule to create reactive oxygen species. This induces covalent bonds between corneal collagen molecules and between collagen and proteoglycans [16] leading to the biomechanical stiffening. Oxygen plays a fundamental role in this reaction, although further studies are needed to fully understand this relationship.
CORNEAL CROSS-LINKING SAFETY PARAMETERS Safety parameters center on the protection of ocular structures such as the corneal endothelium, lens and retina. A pretreatment-anticipated corneal stromal thickness of 400 mm serves to limit penetration to the corneal endothelium [17]. However once the epithelium is removed, there is a risk of
dehydration, causing significant corneal thinning in some cases [18]. Many keratoconus and ectasia patients already have marked corneal thinning prior to treatment. Modified protocols utilize hypoosmotic riboflavin in an effort to cause stromal swelling up to 25% in order to obtain the minimal corneal thickness [19]. More recent studies have utilized isoosmolar riboflavin without Dextran to allow corneal thickening [20]. For all iatrogenic thickened corneas, concern exists for safety and efficacy. The response of CXL may be less because of the decreased concentration of collagen fibrils in a hydrated cornea [21]. Other safety concerns include complications as a result of the treatment itself. Epithelium removal creates a variety of risks such as infiltrates, delayed reepithelialization and infectious keratitis [22]. Corneal haze following CXL has been studied, although its impact on vision is not established [23]. Keratocyte damage is also a concern even with epithelium-on approaches [24]; however, studies have suggested that repopulation occurs within weeks or months of treatment [25]. Other studies suggest keratocyte apoptosis may serve as an indicator for CXL success [26].
CLINICAL USES Cross-linking has shown promise in treating a myriad of conditions, including corneal ectatic disorders in isolation, in combination with refractive surgery modalities and for infectious keratitis.
KERATOCONUS The original application for CXL was for progressive keratoconus, which presents as progressive focal corneal thinning and steepening that starts as early as childhood and continues through the first several decades. Although weakening of the corneal stroma occurs in these patients, the pathophysiology behind keratoconus is still unknown and likely multifactorial. The first clinical study included patients with progressive keratoconus who underwent standard Dresden protocol, with riboflavin presoaking followed by 3 mol/l/cm2 of UV energy for 30 min, resulting in halting of disease progression and topographic corneal flattening [3]. The first randomized controlled trial showed significant corneal flattening in their initial 1-year follow-up [9] that continued up to 4 years after treatment [6 ]. Several studies followed with similar results, including improvement in visual acuity, transient corneal thinning that resolved within 6 months and improvement in topographic parameters leading to corneal flattening
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[7,8]. Raiskup et al. [10 ] published the longest clinical follow-up of 10 years in patients with keratoconus, confirming improvement in vision and topographic parameters in the long term (Figs 1 and 2). Although keratoconus typically presents clinically in young adulthood, early topographic changes likely occur during childhood [27]. Considering the young age of onset and progressive nature of keratoconus, pediatric patients may benefit the most from CXL. Early studies confirmed that pediatric patients show a significant improvement in topography and visual acuity following CXL [28]. However, a 3-year follow-up suggests that despite the initial improvement, some pediatric patients may still progress in the long term [29]. Additional studies are required and should focus on the appropriate protocol and techniques in order to provide long-term results, maintaining safety in pediatric patients. Proper counseling is vital for parents to understand the progressive nature of this disease. However, the general recommendation is progression over 3–6 months indicates a consideration for CXL in pediatric patients to avoid corneal transplantation [30 ]. &&
IATROGENIC CORNEAL ECTASIA Ectasia after corneal refractive surgery (ectasia) occurs from biomechanical instability between collagen fibrils resulting in corneal thinning and weakening that is similar to keratoconus [31]. Risk factors for ectasia after refractive surgery include abnormal preoperative topography, thin corneas and large amounts of tissue ablation [32]. Recent studies by Santhiago et al. [33 ] described the percentage tissue altered at the time of laser-assisted in-situ keratomileusis (LASIK) as a risk factor in postLASIK ectasia. Similarities to the biomechanical properties between ectasia and keratoconus led to the use of CXL in this disease process. Initial studies showed CXL to halt progression of ectasia [11] with &&
subsequent prospective studies confirming topography corneal flattening and improvement in visual acuity at 1 year following treatment [12,34]. Specific risks to CXL in the treatment of ectasia after LASIK include risk of haze, flap complications and epithelial ingrowth.
NEW INDICATIONS FOR CORNEAL CROSS-LINKING In addition to initial indications for stabilizing the ectatic disease process, recent advanced indications have been evaluated. These include combining CXL with refractive surgeries to enhance visual outcomes in patients with ectasias, termed CXL plus [35 ] and using the principles of CXL to treat corneal infections, termed photoactivated chromophore for keratitis (PACK-CXL) [36]. &&
CORNEAL CROSS-LINKING PLUS Although CXL studies show halting of corneal steepening and improvement in topography and vision, the effect on visual function is limited. The term CXL plus pertains to treatment with CXL combined with additional refractive treatment. Topographyguided photorefractive keratectomy (PRK) allows shaping of the irregular cornea without addressing the progressive nature of the disease. Studies have shown improvement in visual acuity and stability with topography-guided PRK with CXL [35 ]. Parameters such as combining CXL with refractive ablations in simultaneous versus sequential treatments, depth of maximal treatment and use of mitomycin C are still debated. Topography-guided PRK with CXL requires longer follow-up, but holds promise for visual rehabilitation as well as halting progression of disease. Other refractive options with CXL include intrastromal ring segments, intraocular lenses and combinations of all of the above-mentioned with preliminary success [35 ]. Once again, long-term follow-up and additional randomized controlled studies are needed for these treatment options. &&
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PHOTOACTIVATED CHROMOPHORE FOR KERATITIS–CORNEAL CROSS-LINKING: INFECTIOUS KERATITIS
FIGURE 1. Corneal demarcation line following CXL with the standard protocol. Note the prominent demarcation line at approximately 300 mm depth. CXL ¼ corneal cross-linking. 208
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In addition to the treatment of ectasia, the basic principles of CXL, a photoinducer combined with ultraviolet light, serves as a treatment option for infectious keratitis termed ‘photoactivated chromophore for keratitis’ or PACK-CXL [36]. Theories include the ability to directly damage microbes, increase resistance to enzymatic damage, prevent Volume 26 Number 3 May 2015
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FIGURE 2. Corresponding difference map for the eye in figure 3 (right image). Topography obtained before (left image) and after (central image) CXL with the standard protocol. CXL ¼ corneal cross-linking.
microbial replication, release free radicals and alter the ocular surface to create a more hostile environment for microorganisms. In-vivo and animal studies show efficacy in treating challenging keratitis pathogens [37], whereas clinical data show improvement in keratitis with the concurrent use of antimicrobial [38,39 ]. The only comparative prospective trial by Said et al. [39 ] determined when comparing medication alone to medication and PACK-CXL, time to healing were the same with a tendency for the PACK-CXL group to heal quicker. Systemic review and metaanalysis reviewed 12 case series that suggested better time to healing in bacterial cases with fungal, Acanthamoeba and culture-negative cases with the worse prognosis and high risk of requiring transplantation [40 ]. Accelerated protocols may also be able to treat and kill pathogens, allowing for shorter treatment with equivalent killing of pathogen [41]. Case reports suggest PACK-CXL alone may assist in the treatment of infectious keratitis [42], but larger studies are required. Although a randomized prospective trial comparing medical to PACK-CXL alone would be ideal, there are ethical considerations. In any case, PACK-CXL offers another treatment option for difficult cases of microbial keratitis. &&
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CORNEAL CROSS-LINKING CHALLENGES AND CONTROVERSIES Cross-linking concepts are still early in their development, and among the most controversial areas today include determining optimal treatment protocols, solving the epithelial barrier to riboflavin and reproducibly measuring treatment success.
ACCELERATED PROTOCOLS In order to accelerate the treatment process, which takes more than 1 h with the standard protocol, variations to this protocol have been studied with the goal to shorten time of treatment, still creating an optimal photochemical reaction by maintaining the same fluence. According to the Bunsen–Roscoe law of reciprocity, the effect of a photochemical reaction is directly proportional to the total irradiation dose, regardless of how the combination of time and intensity are distributed [43]. When one reduces the duration of UV light exposure, one must then increase the intensity of treatment in order to maintain the appropriate fluence. This process, however, may not apply to all reactions occurring in biological systems. Stress strain models on porcine corneas found treatment with standard CXL and accelerated CXL (10 mW/cm2, 9 min) were equivocal with increasing intensity to 40–45 mW/cm2 with corresponding illumination times of approximately 2 min resulting in reduced biomechanical stiffening [44]. Hammer et al. [45] reported significant decrease in stress–strain models in ex-vivo corneas with increased irradiance and decreased treatment times. The authors theorized that oxygen plays an important role and that accelerated treatments may not allow enough recovery of oxygen at the treatment surface. Despite the controversial laboratory data, clinical studies have explored accelerated CXL as well. Retrospective cohorts have shown patients with mild to moderate keratoconus undergoing 10 mW/cm2 treatment for 9 min with improvement in topographic parameters and no complications at 6 month [46] and 1 year [47]. A randomized controlled trial comparing accelerated and conventional CXL showed no difference at 1 year in visual acuity, maximum keratometry, anterior stromal
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keratocyte density and subbasal nerve density or endothelial cell count [48 ] (Figs 3 and 4). Despite limited laboratory and clinical data to support accelerated CXL, there may be expanding roles for its use. A study focusing on pediatric patients revealed improvement in visual acuity, topography and corneal aberrations using an accelerated protocol of 30 mW/cm [2] for 4 min, corresponding to a total dose of 7.2 J [49]. Accelerated CXL may be applicable in children or uncooperative adults who cannot tolerate long treatment or require anesthesia. &
EPITHELIUM: A BARRIER TO RIBOFLAVIN The tight junctions of the corneal epithelium act as a barrier to absorption of adequate riboflavin for CXL success [14]. Standard protocols call for removal of the epithelium, with various techniques available including manual debridement, brush, or laser removal with phototherapeutic keratectomy [50]. Removal of the epithelium increases the risk of pain, infections and other complications. Epithelium-on techniques, if efficacious, would reduce these complications and be particularly well-suited for pediatric patients. Multiple laboratory and clinical studies have focused on modifications to overcome the epithelium as a barrier to riboflavin. In animal models, several studies have shown riboflavin with permeability enhancers such as BAC and EDTA induced equivalent permeability and increased stiffness compared with standard CXL in the short term [51]. However, other animal studies have shown standard epithelium-off protocol to cause less cell death and be equivalent in regard to biomechanical changes [24]. In the clinical setting, various transepithelial riboflavin formulations using permeability enhancers show some penetration [52] but not equal to
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standard protocol in comparative studies [53–55]. Confocal imaging reveals epithelium on protocols to have almost one-third less apoptotic effect than the standard protocols [56]. Newer techniques may allow for better riboflavin penetration. Iontophoresis techniques utilize a low electric gradient to enhance molecular transport allowing for ocular drug delivery and may allow for riboflavin to cross the tight junctions of the epithelium. Mastropasqua et al. [57 ] showed that in human cadaver eyes iontophoresis resulted in greater and deeper saturation of riboflavin compared with conventional epithelium-on protocols but not to the level of standard epithelium-off protocols. Clinical results from case series have shown topographic flattening and improved visual acuity [58,59 ]. In comparative clinical studies, iontophoresis was associated with less damage of corneal subbasal nerves and anterior keratocytes compared with conventional procedures, but the demarcation line was present in less than 50% of the cases [60]. The demarcation line appears less pronounced than traditional epi-on CXL but still better than standard transepithelial procedures [61]. Additional studies are required to determine whether the benefits of sparing the epithelium with these procedures outweigh the risk of failure of treatment of CXL. &
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MEASURING TREATMENT SUCCESS Evidence of effectiveness in laboratory studies shows objective data that confirm that CXL is in fact effective with increase in collagen fiber diameter on electron microscopy [62], resistance to enzymatic digestion, increase in biomechanical stiffness indicated by increase in Young modulus [2] and other imagining modalities such as atomic force microscopy [63] and second harmonic generation microscopy. Second harmonic generation microscopy offers an opportunity to image collagen fibrils
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FIGURE 3. Corneal demarcation line following CXL with an accelerated protocol. The demarcation line is present, but it is more faint and less deep, at approximately 220 mm. CXL ¼ corneal cross-linking. 210
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FIGURE 4. Corresponding difference map for the eye in figure 3 (right image). Topography obtained before (left image) and after (central image) CXL with the accelerated protocol. CXL ¼ corneal cross-linking.
in a high-contrast manner, generates a three-dimensional reconstruction and detects differences in patterns of lamellae between treated and untreated corneas [64]. Measurement in the clinical setting becomes more difficult. Visual acuity serves as a primary goal for patients with several studies confirming improvement in best-corrected visual acuity following treatment. Although suggested that it may correlate with improved topographic or higher order aberrations, other studies have not confirmed this finding. Most clinical studies have utilized maximum topographic keratometry to determine treatment success; however, this number does not correlate well with improvement in visual acuity following treatment [12]. Clinically, a demarcation line can be seen with anterior segment OCT and confocal microscopy, which tend to correlate with each other and may indicate depth of treatment [65]. This is also seen in nonkeratoconic eyes treated with CXL prior to PRK [66]. The demarcation line likely represents the transition between cross-linked and untreated stroma but does not confirm a biomechanical reaction. Demarcation lines do not appear to correlate with change in visual acuity or maximum keratometry [60,67]. The cornea has dynamic and elastic properties, so topography likely only provides a fraction of the information needed to determine progression, regression and stability of disease. The study of corneal biomechanics provides additional information that may lead to a better determination of what success means after CXL. The first in-vivo technology, the Ocular Response Analyzer (Reichert Ophthalmic Instruments, Rochester, NY), provides data of corneal hysteresis, showing a transient increase in parameters following CXL [68,69].
Advanced waveform variables may be better suited to evaluate corneal changes after CXL [70]. The Corvis ST (Oculus Optikera te Gmbh, Wetzlar, Germany) utilizes Scheimpflug technology in the visualization of corneal dynamic deformation. It has shown high repeatability in normal and keratoconus eyes [71], and specifically has been shown to accurately measure keratoconus progression [72]. It may provide a better measure for keratoconus progression and success following treatment, although additional data is required [73].
CONCLUSION With over 400 articles in the peer-reviewed literature and new ideas and directions discussed weekly, CXL remains a hot topic for the treatment of ectasia and beyond. Its development emphasizes the importance of translational science, whereas new directions remain exciting but still in early stages. As new protocols and indications become available, the importance of study design and results is necessary to continue to provide this well tolerated and efficacious treatment for patients.
METHODS OF LITERATURE SEARCH Medline and PubMed databases were searched using the keywords cornea cross linking, collagen cross linking, keratoconus, ectasia, infectious keratitis. Acknowledgements None. Financial support and sponsorship This work is supported in part by separate unrestricted departmental grants to Emory University Department of
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Ophthalmology and Baylor College of Medicine, Cullen Eye Institute from the Research to Prevent Blindness, Inc. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Daxer A, Misof K, Grabner B, et al. Collagen fibrils in the human corneal stroma: structure and aging. Invest Ophthalmol Vis Sci 1998; 39:644–648. 2. 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; 29:1780–1785. 3. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003; 135:620–627. 4. Caporossi A, Mazzotta C, Baiocchi S, Caporossi T. Long-term results of riboflavin ultraviolet a corneal collagen cross-linking for keratoconus in Italy: the Siena eye cross study. Am J Ophthalmol 2010; 149:585–593. 5. Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: long-term results. J Cataract Refract Surg 2008; 34:796–801. 6. Wittig-Silva C, Chan E, Islam FM, et al. A randomized, controlled trial of && corneal collagen cross-linking in progressive keratoconus: three-year results. Ophthalmology 2014; 121:812–821. Long-term follow-up over 3 years in a randomized controlled trial of CXL for keratoconus showing improvement in visual acuity and topography flattening in the treatment groups. The treatment arms had topographic flattening that continued at 1, 2, and 3 years. Meanwhile, the control group continued to have progressive topographic steepening and corneal thinning. Two patients had minor complications that did not affect visual acuity. 7. Asri D, Touboul D, Fournie P, et al. Corneal collagen crosslinking in progressive keratoconus: multicenter results from the French National Reference Center for Keratoconus. J Cataract Refract Surg 2011; 37:2137–2143. 8. Vinciguerra P, Albe E, Trazza S, et al. Refractive, topographic, tomographic, and aberrometric analysis of keratoconic eyes undergoing corneal crosslinking. Ophthalmology 2009; 116:369–378. 9. Wittig-Silva C, Whiting M, Lamoureux E, et al. A randomized controlled trial of corneal collagen cross-linking in progressive keratoconus: preliminary results. J Refract Surg 2008; 24:S720–S725. 10. Raiskup F, Theuring A, Pillunat LE, Spoerl E. Corneal collagen crosslinking & with riboflavin and ultraviolet-A light in progressive keratoconus: ten-year results. J Cataract Refract Surg 2015; 41:41–46. Longest follow-up to date of CXL for keratoconus showing improvement in visual acuity and topographic flattening over a 10-year period while maintaining stable endothelial cell count and safety profile. 11. Hafezi F, Kanellopoulos J, Wiltfang R, Seiler T. Corneal collagen crosslinking with riboflavin and ultraviolet A to treat induced keratectasia after laser in situ keratomileusis. J Cataract Refract Surg 2007; 33:2035–2040. 12. Hersh PS, Greenstein SA, Fry KL. Corneal collagen crosslinking for keratoconus and corneal ectasia: one-year results. J Cataract Refract Surg 2011; 37:149–160. 13. Edwards AM. Structure and general properties of flavins. Methods Mol Biol 2014; 1146:3–13. 14. Baiocchi S, Mazzotta C, Cerretani D, et al. Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium. J Cataract Refract Surg 2009; 35:893–899. 15. Spoerl E, Mrochen M, Sliney D, et al. Safety of UVA-riboflavin cross-linking of the cornea. Cornea 2007; 26:385–389. 16. Zhang Y, Conrad AH, Conrad GW. Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking. J Biol Chem 2011; 286:13011–13022. 17. Kymionis GD, Portaliou DM, Diakonis VF, et al. Corneal collagen cross-linking with riboflavin and ultraviolet-A irradiation in patients with thin corneas. Am J Ophthalmol 2012; 153:24–28. 18. Holopainen JM, Krootila K. Transient corneal thinning in eyes undergoing corneal cross-linking. Am J Ophthalmol 2011; 152:533–536. 19. 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; 35:621–624. 20. Oltulu R, Satirtav G, Donbaloglu M, et al. Intraoperative corneal thickness monitoring during corneal collagen cross-linking with isotonic riboflavin solution with and without dextran. Cornea 2014; 33:1164–1167.
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21. Hafezi F. Limitation of collagen cross-linking with hypoosmolar riboflavin solution: failure in an extremely thin cornea. Cornea 2011; 30:917–919. 22. Abbouda A, Abicca I, Alio JL. Infectious keratitis following corneal crosslinking: a systematic review of reported cases: management, visual outcome, and treatment proposed. Semin Ophthalmol 2014; 13:1–7. 23. Greenstein SA, Fry KL, Bhatt J, Hersh PS. Natural history of corneal haze after collagen crosslinking for keratoconus and corneal ectasia: Scheimpflug and biomicroscopic analysis. J Cataract Refract Surg 2010; 36:2105– 2114. 24. Armstrong BK, Lin MP, Ford MR, et al. Biological and biomechanical responses to traditional epithelium-off and transepithelial riboflavin-UVA CXL techniques in rabbits. J Refract Surg 2013; 29:332–341. 25. Mencucci R, Paladini I, Sarchielli E, et al. Transepithelial riboflavin/ultraviolet. A corneal cross-linking in keratoconus: morphologic studies on human corneas. Am J Ophthalmol 2013; 156:874–884. 26. Mencucci R, Marini M, Paladini I, et al. Effects of riboflavin/UVA corneal crosslinking on keratocytes and collagen fibres in human cornea. Clin Experiment Ophthalmol 2010; 38:49–56. 27. Leoni-Mesplie S, Mortemousque B, Touboul D, et al. Scalability and severity of keratoconus in children. Am J Ophthalmol 2012; 154:56–62. 28. Vinciguerra P, Albe E, Frueh BE, et al. Two-year corneal cross-linking results in patients younger than 18 years with documented progressive keratoconus. Am J Ophthalmol 2012; 154:520–526. 29. Chatzis N, Hafezi F. Progression of keratoconus and efficacy of pediatric [corrected] corneal collagen cross-linking in children and adolescents. J Refract Surg 2012; 28:753–758. 30. Hafezi F, Randleman JB. Corneal collagen cross linking. Thorofare, NJ: && SLACK; 2013. pp. xv, 167. Comprehensive book with summary of the cross-linking process, including the basic science foundations, treatment protocols and outcomes, recognition and management of potential complications, and modifications of the standard protocol for special circumstances. 31. Dawson DG, Randleman JB, Grossniklaus HE, et al. Corneal ectasia after excimer laser keratorefractive surgery: histopathology, ultrastructure, and pathophysiology. Ophthalmology 2008; 115:2181–2191. 32. Randleman JB, Woodward M, Lynn MJ, Stulting RD. Risk assessment for ectasia after corneal refractive surgery. Ophthalmology 2008; 115:37–50. 33. Santhiago MR, Smadja D, Gomes BF, et al. Association between the && percentage tissue altered and postlaser in situ keratomileusis ectasia in eyes with normal preoperative topography. Am J Ophthalmol 2014; 158:87–95. Determination that percentage tissue ablated at the time of LASIK was significantly correlated with development of post-LASIK ectasia, above all other parameters. 34. Richoz O, Mavrakanas N, Pajic B, Hafezi F. Corneal collagen cross-linking for ectasia after LASIK and photorefractive keratectomy: long-term results. Ophthalmology 2013; 120:1354–1359. 35. Kymionis GD, Grentzelos MA, Portaliou DM, et al. Corneal collagen cross&& linking (CXL) combined with refractive procedures for the treatment of corneal ectatic disorders: CXL plus. J Refract Surg 2014; 30:566–576. Establishment of CXL plus: review of combined CXL with refractive surgical techniques to improve visual acuity in additional to corneal stability with CXL alone. 36. Hafezi F, Randleman JB. PACK-CXL: defining CXL for infectious keratitis. J Refract Surg 2014; 30:438–439. 37. Martins SA, Combs JC, Noguera G, et al. Antimicrobial efficacy of riboflavin/ UVA combination (365 nm) in vitro for bacterial and fungal isolates: a potential new treatment for infectious keratitis. Invest Ophthalmol Vis Sci 2008; 49:3402–3408. 38. Price MO, Tenkman LR, Schrier A, et al. Photoactivated riboflavin treatment of infectious keratitis using collagen cross-linking technology. J Refract Surg 2012; 28:706–713. 39. Said DG, Elalfy MS, Gatzioufas Z, et al. Collagen cross-linking with && photoactivated riboflavin (PACK-CXL) for the treatment of advanced infectious keratitis with corneal melting. Ophthalmology 2014; 121:1377– 1382. Prospective clinical study comparing medical treatment of infectious keratitis with CXL and medical treatment. The combined treatment did not shorten the time of healing; however, the medical-only treatment group had a 21% rate of perforation or recurrence compared with no patients in the CXL plus medical treatment. 40. Alio JL, Abbouda A, Valle DD, et al. Corneal cross linking and infectious & keratitis: a systematic review with a meta-analysis of reported cases. J Ophthalmic Inflamm Infect 2013; 3:47. Meta-analysis of reported case series of infectious keratitis treatment with PACKCXL suggesting that this treatment works better in bacterial cases and less well in fungal or culture-negative cases. 41. Richoz O, Kling S, Hoogewoud F, et al. Antibacterial efficacy of accelerated photoactivated chromophore for keratitis-corneal collagen cross-linking (PACK-CXL). J Refract Surg 2014; 30:850–854. 42. Tabibian D, Richoz O, Riat A, et al. Accelerated photoactivated chromophore for keratitis-corneal collagen cross-linking as a first-line and sole treatment in early fungal keratitis. J Refract Surg 2014; 30:855–857. 43. Bunsen RW, Roscoe RE. Photochemical researches, part V: on the measurement of the chemical action of direct and diffuse sunlight. Proc R Soc Lond 1862; 12:306–312.
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Corneal cross-linking Khandelwal and Randleman 44. Wernli J, Schumacher S, Spoerl E, Mrochen M. The efficacy of corneal crosslinking shows a sudden decrease with very high intensity UV light and short treatment time. Invest Ophthalmol Vis Sci 2013; 54:1176–1180. 45. Hammer A, Richoz O, Arba Mosquera S, et al. Corneal biomechanical properties at different corneal cross-linking (CXL) irradiances. Invest Ophthalmol Vis Sci 2014; 55:2881–2884. 46. Mita M, Waring GO 4th, Tomita M. High-irradiance accelerated collagen crosslinking for the treatment of keratoconus: six-month results. J Cataract Refract Surg 2014; 40:1032–1040. 47. Elbaz U, Shen C, Lichtinger A, et al. Accelerated (9-mW/cm2) corneal collagen crosslinking for keratoconus-A 1-year follow-up. Cornea 2014; 33:769–773. 48. Hashemian H, Jabbarvand M, Khodaparast M, Ameli K. Evaluation of corneal & changes after conventional versus accelerated corneal cross-linking: a randomized controlled trial. J Refract Surg 2014; 30:837–842. Randomized control trial comparing convention and accelerate CXL showing equivalent improvement in visual acuity and topographic parameters in both groups. 49. Ozgurhan EB, Kara N, Cankaya KI, et al. Accelerated corneal cross-linking in pediatric patients with keratoconus: 24-month outcomes. J Refract Surg 2014; 30:843–849. 50. Kymionis GD, Grentzelos MA, Kounis GA, et al. Combined transepithelial phototherapeutic keratectomy and corneal collagen cross-linking for progressive keratoconus. Ophthalmology 2012; 119:1777–1784. 51. Torricelli AA, Ford MR, Singh V, et al. BAC-EDTA transepithelial riboflavinUVA crosslinking has greater biomechanical stiffening effect than standard epithelium-off in rabbit corneas. Experiment Eye Res 2014; 125:114– 117. 52. Filippello M, Stagni E, O’Brart D. Transepithelial corneal collagen crosslinking: bilateral study. J Cataract Refract Surg 2012; 38:283–291. 53. Taneri S, Oehler S, Lytle G, Dick HB. Evaluation of epithelial integrity with various transepithelial corneal cross-linking protocols for treatment of keratoconus. J Ophthalmol 2014; 2014:614380. 54. Alhamad TA, O’Brart DP, O’Brart NA, Meek KM. Evaluation of transepithelial stromal riboflavin absorption with enhanced riboflavin solution using spectrophotometry. J Cataract Refract Surg 2012; 38:884–889. 55. Koppen C, Wouters K, Mathysen D, et al. Refractive and topographic results of benzalkonium chloride-assisted transepithelial crosslinking. J Cataract Refract Surg 2012; 38:1000–1005. 56. Caporossi A, Mazzotta C, Baiocchi S, et al. Transepithelial corneal collagen crosslinking for keratoconus: qualitative investigation by in vivo HRT II confocal analysis. Eur J Ophthalmol 2012; 22 (Suppl 7):S81–S88. 57. Mastropasqua L, Nubile M, Calienno R, et al. Corneal cross-linking: intras& tromal riboflavin concentration in iontophoresis-assisted imbibition versus traditional and transepithelial techniques. Am J Ophthalmol 2014; 157:623–630; e1. Laboratory study in human cadaver eyes showing iontophoresis-assisted CXL yielded improved riboflavin compared with epi-on protocols but not to the level of epi-off protocols. 58. Bikbova G, Bikbov M. Transepithelial corneal collagen cross-linking by iontophoresis of riboflavin. Acta Ophthalmol 2014; 92:e30–e34.
59. Vinciguerra P, Randleman JB, Romano V, et al. Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: initial clinical outcomes. J Refract Surg 2014; 30:746–753. Prospective nonrandomized study showing that transepithelial iontophoresis CXL is a well tolerated and efficacious procedure for the treatment of keratoconus with improvement in visual acuity. 60. Bouheraoua N, Jouve L, El Sanharawi M, et al. Optical coherence tomography and confocal microscopy following three different protocols of corneal collagen-crosslinking in keratoconus. Invest Ophthalmol Vis Sci 2014; 55:7601–7609. 61. Bonnel S, De Riboyre BM, Bedubourg B, et al. Demarcation line evaluation of iontophoresis-assisted transepithelial corneal collagen cross-linking for keratoconus. J Refract Surg 2015; 31:36–40. 62. Wollensak G, Wilsch M, Spoerl E, Seiler T. Collagen fiber diameter in the rabbit cornea after collagen crosslinking by riboflavin/UVA. Cornea 2004; 23:503–507. 63. Seifert J, Hammer CM, Rheinlaender J, et al. Distribution of Young’s modulus in porcine corneas after riboflavin/UVA-induced collagen cross-linking as measured by atomic force microscopy. PLoS One 2014; 9:e88186. 64. Tan HY, Chang YL, Lo W, et al. Characterizing the morphologic changes in collagen crosslinked-treated corneas by Fourier transform-second harmonic generation imaging. J Cataract Refract Surg 2013; 39:779–788. 65. Kymionis GD, Grentzelos MA, Plaka AD, et al. Correlation of the corneal collagen cross-linking demarcation line using confocal microscopy and anterior segment optical coherence tomography in keratoconic patients. Am J Ophthalmol 2014; 157:110–115. 66. Malta JB, Renesto AC, Moscovici BK, et al. Stromal demarcation line induced by corneal cross-linking in eyes with keratoconus and nonkeratoconic asymmetric topography. Cornea 2015; 34:199–203. 67. Yam JC, Chan CW, Cheng AC. Corneal collagen cross-linking demarcation line depth assessed by Visante OCT After CXL for keratoconus and corneal ectasia. J Refract Surg 2012; 28:475–481. 68. Goldich Y, Marcovich AL, Barkana Y, et al. Clinical and corneal biomechanical changes after collagen cross-linking with riboflavin and UV irradiation in patients with progressive keratoconus: results after 2 years of follow-up. Cornea 2012; 31:609–614. 69. Vinciguerra P, Albe E, Mahmoud AM, et al. Intra- and postoperative variation in ocular response analyzer parameters in keratoconic eyes after corneal crosslinking. J Refract Surg 2010; 26:669–676. 70. Hallahan KM, Rocha K, Roy AS, et al. Effects of corneal cross-linking on ocular response analyzer waveform-derived variables in keratoconus and postrefractive surgery ectasia. Eye Contact Lens 2014; 40:339–344. 71. Tian L, Ko MW, Wang LK, et al. Assessment of ocular biomechanics using dynamic ultra high-speed Scheimpflug imaging in keratoconic and normal eyes. J Refract Surg 2014; 30:1–7. 72. Ali NQ, Patel DV, McGhee CN. Biomechanical responses of healthy and keratoconic corneas measured using a noncontact Scheimpflug-based tonometer. Invest Ophthalmol Vis Sci 2014; 55:3651–3659. 73. Bak-Nielsen S, Pedersen IB, Ivarsen A, Hjortdal J. Dynamic Scheimpflugbased assessment of keratoconus and the effects of corneal cross-linking. J Refract Surg 2014; 30:408–414.
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Current and future applications of corneal cross-linking Sumitra S. Khandelwal a and J. Bradley Randleman b,c
Purpose of review To review current concepts and future directions of corneal cross-linking (CXL) as a treatment for keratoconus, ectasia after refractive surgery and infectious keratitis. Recent findings Several important laboratory and clinical studies have established the safety and success of corneal crosslinking for the treatment of keratoconus and other corneal ectasias. Recently, additional studies have analyzed new directions and controversies in corneal cross-linking, exploring new indications, comparing new techniques and analyzing results of new protocols. Summary The results of bench and clinical research are providing the foundation to allow for protocol modifications of the standard cross-linking protocols and expansion of cross-linking concepts for techniques such as accelerated cross-linking, epithelium-sparing protocols and measurement of progression and success. Keywords accelerated corneal cross-linking, corneal cross-linking, cross-linking, ectasia, infectious keratitis, keratoconus
INTRODUCTION Corneal cross-linking (CXL) represents the epitome of translational research in the cornea. From its initial inspiration on a visit to the dentist by Professor Theo Seiler, to the remarkable increase in corneal rigidity demonstrated in laboratory models, to impressive results demonstrated in prospective clinical trials, CXL has revolutionized the treatment of corneal ectasias. Since its advent, CXL has evolved to include alternative indications, parameters and protocols. This article serves to review the fundamental aspects of this historic treatment, to discuss its current and future indications and to evaluate the data regarding new techniques and protocols in the treatment of corneal diseases.
FUNDAMENTAL CONCEPTS IN CORNEAL CROSS-LINKING Observations that the rate of keratoconus was lower in patients with diabetes and increased age sparked the idea that natural cross-linking of the corneal collagen fibrils may result in strengthening and stiffening of tissue [1]. This led to the development of CXL, a process in which a combination of a photoinducer, ultraviolet (UV) light and a photochemical reaction induces free radicals, leading to a www.co-ophthalmology.com
chemical bond between collagen fibrils. Initial CXL animal studies resulted in up to a 70% increase in corneal rigidity [2], whereas the first clinical study by Wollensak et al. [3] showed halting of corneal progression and topographic flattening in patients with keratoconus. Since then, several prospective studies have echoed these results in patients with keratoconus [4,5,6 ,7–9,10 ] and ectasia after corneal refractive surgery (hereafter termed ‘ectasia’) [11,12]. &&
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Photoinducers Riboflavin functions as an optimal photoinducer in the biochemical reaction in CXL. It possesses properties such as a wide range of absorption, although it is still well tolerated for systemic absorption [13]. Riboflavin is, however, a relatively large molecule; thus, its primary limitation is sufficient absorption a
Baylor College of Medicine, Cullen Eye Institute, Houston, Texas, USA, Department of Ophthalmology, Emory University and cEmory Vision, Emory Eye Center, Atlanta, Georgia, USA b
Correspondence to J. Bradley Randleman, MD, Emory Eye Center, 5671 Peachtree Dunwoody Rd NE, Suite 400 Atlanta, GA 30342, USA. E-mail: [email protected] Curr Opin Ophthalmol 2015, 26:206–213 DOI:10.1097/ICU.0000000000000146 Volume 26 Number 3 May 2015
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KEY POINTS CXL has shown to be well tolerated and efficacious in the treatment of keratoconus and ectasia after refractive surgery. Standard protocols have shown stable long-term improvement in vision and corneal topography. New variations including epithelium-sparing techniques, accelerated protocols and alternate imaging of the cornea are in early stages, but may provide alternate ways to treat corneal disease.
into the corneal stroma through an intact epithelium [14]. Various techniques, termed ‘epithelium on’ CXL, have been studied to navigate around this issue, although the success of these protocols remains controversial.
Ultraviolet light UV light serves as the second important component of CXL. Key parameters include wavelength, irradiance and time of irradiation; these parameters are specific to the success and safety of treatment. The range of absorption peak of riboflavin for CXL ranges from 360 to 370 nm as determined through initial studies [15]. Variations to intensity and duration in preclinical studies established that maximum stiffening involved the use of 3 mW/cm2 of energy for 30 min, which corresponded to a total energy dose (fluence) of 5.4 J/cm2. This guided the development of the standard Dresden protocol, which allowed for greatest efficiency of tissue stiffening. Variations to this protocol have been termed accelerated CXL. The actual photochemical reaction that leads to stiffening involves riboflavin absorbing the ultraviolet light (UVA) energy and exciting the molecule to create reactive oxygen species. This induces covalent bonds between corneal collagen molecules and between collagen and proteoglycans [16] leading to the biomechanical stiffening. Oxygen plays a fundamental role in this reaction, although further studies are needed to fully understand this relationship.
CORNEAL CROSS-LINKING SAFETY PARAMETERS Safety parameters center on the protection of ocular structures such as the corneal endothelium, lens and retina. A pretreatment-anticipated corneal stromal thickness of 400 mm serves to limit penetration to the corneal endothelium [17]. However once the epithelium is removed, there is a risk of
dehydration, causing significant corneal thinning in some cases [18]. Many keratoconus and ectasia patients already have marked corneal thinning prior to treatment. Modified protocols utilize hypoosmotic riboflavin in an effort to cause stromal swelling up to 25% in order to obtain the minimal corneal thickness [19]. More recent studies have utilized isoosmolar riboflavin without Dextran to allow corneal thickening [20]. For all iatrogenic thickened corneas, concern exists for safety and efficacy. The response of CXL may be less because of the decreased concentration of collagen fibrils in a hydrated cornea [21]. Other safety concerns include complications as a result of the treatment itself. Epithelium removal creates a variety of risks such as infiltrates, delayed reepithelialization and infectious keratitis [22]. Corneal haze following CXL has been studied, although its impact on vision is not established [23]. Keratocyte damage is also a concern even with epithelium-on approaches [24]; however, studies have suggested that repopulation occurs within weeks or months of treatment [25]. Other studies suggest keratocyte apoptosis may serve as an indicator for CXL success [26].
CLINICAL USES Cross-linking has shown promise in treating a myriad of conditions, including corneal ectatic disorders in isolation, in combination with refractive surgery modalities and for infectious keratitis.
KERATOCONUS The original application for CXL was for progressive keratoconus, which presents as progressive focal corneal thinning and steepening that starts as early as childhood and continues through the first several decades. Although weakening of the corneal stroma occurs in these patients, the pathophysiology behind keratoconus is still unknown and likely multifactorial. The first clinical study included patients with progressive keratoconus who underwent standard Dresden protocol, with riboflavin presoaking followed by 3 mol/l/cm2 of UV energy for 30 min, resulting in halting of disease progression and topographic corneal flattening [3]. The first randomized controlled trial showed significant corneal flattening in their initial 1-year follow-up [9] that continued up to 4 years after treatment [6 ]. Several studies followed with similar results, including improvement in visual acuity, transient corneal thinning that resolved within 6 months and improvement in topographic parameters leading to corneal flattening
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[7,8]. Raiskup et al. [10 ] published the longest clinical follow-up of 10 years in patients with keratoconus, confirming improvement in vision and topographic parameters in the long term (Figs 1 and 2). Although keratoconus typically presents clinically in young adulthood, early topographic changes likely occur during childhood [27]. Considering the young age of onset and progressive nature of keratoconus, pediatric patients may benefit the most from CXL. Early studies confirmed that pediatric patients show a significant improvement in topography and visual acuity following CXL [28]. However, a 3-year follow-up suggests that despite the initial improvement, some pediatric patients may still progress in the long term [29]. Additional studies are required and should focus on the appropriate protocol and techniques in order to provide long-term results, maintaining safety in pediatric patients. Proper counseling is vital for parents to understand the progressive nature of this disease. However, the general recommendation is progression over 3–6 months indicates a consideration for CXL in pediatric patients to avoid corneal transplantation [30 ]. &&
IATROGENIC CORNEAL ECTASIA Ectasia after corneal refractive surgery (ectasia) occurs from biomechanical instability between collagen fibrils resulting in corneal thinning and weakening that is similar to keratoconus [31]. Risk factors for ectasia after refractive surgery include abnormal preoperative topography, thin corneas and large amounts of tissue ablation [32]. Recent studies by Santhiago et al. [33 ] described the percentage tissue altered at the time of laser-assisted in-situ keratomileusis (LASIK) as a risk factor in postLASIK ectasia. Similarities to the biomechanical properties between ectasia and keratoconus led to the use of CXL in this disease process. Initial studies showed CXL to halt progression of ectasia [11] with &&
subsequent prospective studies confirming topography corneal flattening and improvement in visual acuity at 1 year following treatment [12,34]. Specific risks to CXL in the treatment of ectasia after LASIK include risk of haze, flap complications and epithelial ingrowth.
NEW INDICATIONS FOR CORNEAL CROSS-LINKING In addition to initial indications for stabilizing the ectatic disease process, recent advanced indications have been evaluated. These include combining CXL with refractive surgeries to enhance visual outcomes in patients with ectasias, termed CXL plus [35 ] and using the principles of CXL to treat corneal infections, termed photoactivated chromophore for keratitis (PACK-CXL) [36]. &&
CORNEAL CROSS-LINKING PLUS Although CXL studies show halting of corneal steepening and improvement in topography and vision, the effect on visual function is limited. The term CXL plus pertains to treatment with CXL combined with additional refractive treatment. Topographyguided photorefractive keratectomy (PRK) allows shaping of the irregular cornea without addressing the progressive nature of the disease. Studies have shown improvement in visual acuity and stability with topography-guided PRK with CXL [35 ]. Parameters such as combining CXL with refractive ablations in simultaneous versus sequential treatments, depth of maximal treatment and use of mitomycin C are still debated. Topography-guided PRK with CXL requires longer follow-up, but holds promise for visual rehabilitation as well as halting progression of disease. Other refractive options with CXL include intrastromal ring segments, intraocular lenses and combinations of all of the above-mentioned with preliminary success [35 ]. Once again, long-term follow-up and additional randomized controlled studies are needed for these treatment options. &&
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PHOTOACTIVATED CHROMOPHORE FOR KERATITIS–CORNEAL CROSS-LINKING: INFECTIOUS KERATITIS
FIGURE 1. Corneal demarcation line following CXL with the standard protocol. Note the prominent demarcation line at approximately 300 mm depth. CXL ¼ corneal cross-linking. 208
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In addition to the treatment of ectasia, the basic principles of CXL, a photoinducer combined with ultraviolet light, serves as a treatment option for infectious keratitis termed ‘photoactivated chromophore for keratitis’ or PACK-CXL [36]. Theories include the ability to directly damage microbes, increase resistance to enzymatic damage, prevent Volume 26 Number 3 May 2015
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FIGURE 2. Corresponding difference map for the eye in figure 3 (right image). Topography obtained before (left image) and after (central image) CXL with the standard protocol. CXL ¼ corneal cross-linking.
microbial replication, release free radicals and alter the ocular surface to create a more hostile environment for microorganisms. In-vivo and animal studies show efficacy in treating challenging keratitis pathogens [37], whereas clinical data show improvement in keratitis with the concurrent use of antimicrobial [38,39 ]. The only comparative prospective trial by Said et al. [39 ] determined when comparing medication alone to medication and PACK-CXL, time to healing were the same with a tendency for the PACK-CXL group to heal quicker. Systemic review and metaanalysis reviewed 12 case series that suggested better time to healing in bacterial cases with fungal, Acanthamoeba and culture-negative cases with the worse prognosis and high risk of requiring transplantation [40 ]. Accelerated protocols may also be able to treat and kill pathogens, allowing for shorter treatment with equivalent killing of pathogen [41]. Case reports suggest PACK-CXL alone may assist in the treatment of infectious keratitis [42], but larger studies are required. Although a randomized prospective trial comparing medical to PACK-CXL alone would be ideal, there are ethical considerations. In any case, PACK-CXL offers another treatment option for difficult cases of microbial keratitis. &&
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CORNEAL CROSS-LINKING CHALLENGES AND CONTROVERSIES Cross-linking concepts are still early in their development, and among the most controversial areas today include determining optimal treatment protocols, solving the epithelial barrier to riboflavin and reproducibly measuring treatment success.
ACCELERATED PROTOCOLS In order to accelerate the treatment process, which takes more than 1 h with the standard protocol, variations to this protocol have been studied with the goal to shorten time of treatment, still creating an optimal photochemical reaction by maintaining the same fluence. According to the Bunsen–Roscoe law of reciprocity, the effect of a photochemical reaction is directly proportional to the total irradiation dose, regardless of how the combination of time and intensity are distributed [43]. When one reduces the duration of UV light exposure, one must then increase the intensity of treatment in order to maintain the appropriate fluence. This process, however, may not apply to all reactions occurring in biological systems. Stress strain models on porcine corneas found treatment with standard CXL and accelerated CXL (10 mW/cm2, 9 min) were equivocal with increasing intensity to 40–45 mW/cm2 with corresponding illumination times of approximately 2 min resulting in reduced biomechanical stiffening [44]. Hammer et al. [45] reported significant decrease in stress–strain models in ex-vivo corneas with increased irradiance and decreased treatment times. The authors theorized that oxygen plays an important role and that accelerated treatments may not allow enough recovery of oxygen at the treatment surface. Despite the controversial laboratory data, clinical studies have explored accelerated CXL as well. Retrospective cohorts have shown patients with mild to moderate keratoconus undergoing 10 mW/cm2 treatment for 9 min with improvement in topographic parameters and no complications at 6 month [46] and 1 year [47]. A randomized controlled trial comparing accelerated and conventional CXL showed no difference at 1 year in visual acuity, maximum keratometry, anterior stromal
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keratocyte density and subbasal nerve density or endothelial cell count [48 ] (Figs 3 and 4). Despite limited laboratory and clinical data to support accelerated CXL, there may be expanding roles for its use. A study focusing on pediatric patients revealed improvement in visual acuity, topography and corneal aberrations using an accelerated protocol of 30 mW/cm [2] for 4 min, corresponding to a total dose of 7.2 J [49]. Accelerated CXL may be applicable in children or uncooperative adults who cannot tolerate long treatment or require anesthesia. &
EPITHELIUM: A BARRIER TO RIBOFLAVIN The tight junctions of the corneal epithelium act as a barrier to absorption of adequate riboflavin for CXL success [14]. Standard protocols call for removal of the epithelium, with various techniques available including manual debridement, brush, or laser removal with phototherapeutic keratectomy [50]. Removal of the epithelium increases the risk of pain, infections and other complications. Epithelium-on techniques, if efficacious, would reduce these complications and be particularly well-suited for pediatric patients. Multiple laboratory and clinical studies have focused on modifications to overcome the epithelium as a barrier to riboflavin. In animal models, several studies have shown riboflavin with permeability enhancers such as BAC and EDTA induced equivalent permeability and increased stiffness compared with standard CXL in the short term [51]. However, other animal studies have shown standard epithelium-off protocol to cause less cell death and be equivalent in regard to biomechanical changes [24]. In the clinical setting, various transepithelial riboflavin formulations using permeability enhancers show some penetration [52] but not equal to
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standard protocol in comparative studies [53–55]. Confocal imaging reveals epithelium on protocols to have almost one-third less apoptotic effect than the standard protocols [56]. Newer techniques may allow for better riboflavin penetration. Iontophoresis techniques utilize a low electric gradient to enhance molecular transport allowing for ocular drug delivery and may allow for riboflavin to cross the tight junctions of the epithelium. Mastropasqua et al. [57 ] showed that in human cadaver eyes iontophoresis resulted in greater and deeper saturation of riboflavin compared with conventional epithelium-on protocols but not to the level of standard epithelium-off protocols. Clinical results from case series have shown topographic flattening and improved visual acuity [58,59 ]. In comparative clinical studies, iontophoresis was associated with less damage of corneal subbasal nerves and anterior keratocytes compared with conventional procedures, but the demarcation line was present in less than 50% of the cases [60]. The demarcation line appears less pronounced than traditional epi-on CXL but still better than standard transepithelial procedures [61]. Additional studies are required to determine whether the benefits of sparing the epithelium with these procedures outweigh the risk of failure of treatment of CXL. &
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MEASURING TREATMENT SUCCESS Evidence of effectiveness in laboratory studies shows objective data that confirm that CXL is in fact effective with increase in collagen fiber diameter on electron microscopy [62], resistance to enzymatic digestion, increase in biomechanical stiffness indicated by increase in Young modulus [2] and other imagining modalities such as atomic force microscopy [63] and second harmonic generation microscopy. Second harmonic generation microscopy offers an opportunity to image collagen fibrils
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FIGURE 3. Corneal demarcation line following CXL with an accelerated protocol. The demarcation line is present, but it is more faint and less deep, at approximately 220 mm. CXL ¼ corneal cross-linking. 210
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FIGURE 4. Corresponding difference map for the eye in figure 3 (right image). Topography obtained before (left image) and after (central image) CXL with the accelerated protocol. CXL ¼ corneal cross-linking.
in a high-contrast manner, generates a three-dimensional reconstruction and detects differences in patterns of lamellae between treated and untreated corneas [64]. Measurement in the clinical setting becomes more difficult. Visual acuity serves as a primary goal for patients with several studies confirming improvement in best-corrected visual acuity following treatment. Although suggested that it may correlate with improved topographic or higher order aberrations, other studies have not confirmed this finding. Most clinical studies have utilized maximum topographic keratometry to determine treatment success; however, this number does not correlate well with improvement in visual acuity following treatment [12]. Clinically, a demarcation line can be seen with anterior segment OCT and confocal microscopy, which tend to correlate with each other and may indicate depth of treatment [65]. This is also seen in nonkeratoconic eyes treated with CXL prior to PRK [66]. The demarcation line likely represents the transition between cross-linked and untreated stroma but does not confirm a biomechanical reaction. Demarcation lines do not appear to correlate with change in visual acuity or maximum keratometry [60,67]. The cornea has dynamic and elastic properties, so topography likely only provides a fraction of the information needed to determine progression, regression and stability of disease. The study of corneal biomechanics provides additional information that may lead to a better determination of what success means after CXL. The first in-vivo technology, the Ocular Response Analyzer (Reichert Ophthalmic Instruments, Rochester, NY), provides data of corneal hysteresis, showing a transient increase in parameters following CXL [68,69].
Advanced waveform variables may be better suited to evaluate corneal changes after CXL [70]. The Corvis ST (Oculus Optikera te Gmbh, Wetzlar, Germany) utilizes Scheimpflug technology in the visualization of corneal dynamic deformation. It has shown high repeatability in normal and keratoconus eyes [71], and specifically has been shown to accurately measure keratoconus progression [72]. It may provide a better measure for keratoconus progression and success following treatment, although additional data is required [73].
CONCLUSION With over 400 articles in the peer-reviewed literature and new ideas and directions discussed weekly, CXL remains a hot topic for the treatment of ectasia and beyond. Its development emphasizes the importance of translational science, whereas new directions remain exciting but still in early stages. As new protocols and indications become available, the importance of study design and results is necessary to continue to provide this well tolerated and efficacious treatment for patients.
METHODS OF LITERATURE SEARCH Medline and PubMed databases were searched using the keywords cornea cross linking, collagen cross linking, keratoconus, ectasia, infectious keratitis. Acknowledgements None. Financial support and sponsorship This work is supported in part by separate unrestricted departmental grants to Emory University Department of
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Ophthalmology and Baylor College of Medicine, Cullen Eye Institute from the Research to Prevent Blindness, Inc. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Daxer A, Misof K, Grabner B, et al. Collagen fibrils in the human corneal stroma: structure and aging. Invest Ophthalmol Vis Sci 1998; 39:644–648. 2. 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; 29:1780–1785. 3. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003; 135:620–627. 4. Caporossi A, Mazzotta C, Baiocchi S, Caporossi T. Long-term results of riboflavin ultraviolet a corneal collagen cross-linking for keratoconus in Italy: the Siena eye cross study. Am J Ophthalmol 2010; 149:585–593. 5. Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: long-term results. J Cataract Refract Surg 2008; 34:796–801. 6. Wittig-Silva C, Chan E, Islam FM, et al. A randomized, controlled trial of && corneal collagen cross-linking in progressive keratoconus: three-year results. Ophthalmology 2014; 121:812–821. Long-term follow-up over 3 years in a randomized controlled trial of CXL for keratoconus showing improvement in visual acuity and topography flattening in the treatment groups. The treatment arms had topographic flattening that continued at 1, 2, and 3 years. Meanwhile, the control group continued to have progressive topographic steepening and corneal thinning. Two patients had minor complications that did not affect visual acuity. 7. Asri D, Touboul D, Fournie P, et al. Corneal collagen crosslinking in progressive keratoconus: multicenter results from the French National Reference Center for Keratoconus. J Cataract Refract Surg 2011; 37:2137–2143. 8. Vinciguerra P, Albe E, Trazza S, et al. Refractive, topographic, tomographic, and aberrometric analysis of keratoconic eyes undergoing corneal crosslinking. Ophthalmology 2009; 116:369–378. 9. Wittig-Silva C, Whiting M, Lamoureux E, et al. A randomized controlled trial of corneal collagen cross-linking in progressive keratoconus: preliminary results. J Refract Surg 2008; 24:S720–S725. 10. Raiskup F, Theuring A, Pillunat LE, Spoerl E. Corneal collagen crosslinking & with riboflavin and ultraviolet-A light in progressive keratoconus: ten-year results. J Cataract Refract Surg 2015; 41:41–46. Longest follow-up to date of CXL for keratoconus showing improvement in visual acuity and topographic flattening over a 10-year period while maintaining stable endothelial cell count and safety profile. 11. Hafezi F, Kanellopoulos J, Wiltfang R, Seiler T. Corneal collagen crosslinking with riboflavin and ultraviolet A to treat induced keratectasia after laser in situ keratomileusis. J Cataract Refract Surg 2007; 33:2035–2040. 12. Hersh PS, Greenstein SA, Fry KL. Corneal collagen crosslinking for keratoconus and corneal ectasia: one-year results. J Cataract Refract Surg 2011; 37:149–160. 13. Edwards AM. Structure and general properties of flavins. Methods Mol Biol 2014; 1146:3–13. 14. Baiocchi S, Mazzotta C, Cerretani D, et al. Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium. J Cataract Refract Surg 2009; 35:893–899. 15. Spoerl E, Mrochen M, Sliney D, et al. Safety of UVA-riboflavin cross-linking of the cornea. Cornea 2007; 26:385–389. 16. Zhang Y, Conrad AH, Conrad GW. Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking. J Biol Chem 2011; 286:13011–13022. 17. Kymionis GD, Portaliou DM, Diakonis VF, et al. Corneal collagen cross-linking with riboflavin and ultraviolet-A irradiation in patients with thin corneas. Am J Ophthalmol 2012; 153:24–28. 18. Holopainen JM, Krootila K. Transient corneal thinning in eyes undergoing corneal cross-linking. Am J Ophthalmol 2011; 152:533–536. 19. 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; 35:621–624. 20. Oltulu R, Satirtav G, Donbaloglu M, et al. Intraoperative corneal thickness monitoring during corneal collagen cross-linking with isotonic riboflavin solution with and without dextran. Cornea 2014; 33:1164–1167.
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21. Hafezi F. Limitation of collagen cross-linking with hypoosmolar riboflavin solution: failure in an extremely thin cornea. Cornea 2011; 30:917–919. 22. Abbouda A, Abicca I, Alio JL. Infectious keratitis following corneal crosslinking: a systematic review of reported cases: management, visual outcome, and treatment proposed. Semin Ophthalmol 2014; 13:1–7. 23. Greenstein SA, Fry KL, Bhatt J, Hersh PS. Natural history of corneal haze after collagen crosslinking for keratoconus and corneal ectasia: Scheimpflug and biomicroscopic analysis. J Cataract Refract Surg 2010; 36:2105– 2114. 24. Armstrong BK, Lin MP, Ford MR, et al. Biological and biomechanical responses to traditional epithelium-off and transepithelial riboflavin-UVA CXL techniques in rabbits. J Refract Surg 2013; 29:332–341. 25. Mencucci R, Paladini I, Sarchielli E, et al. Transepithelial riboflavin/ultraviolet. A corneal cross-linking in keratoconus: morphologic studies on human corneas. Am J Ophthalmol 2013; 156:874–884. 26. Mencucci R, Marini M, Paladini I, et al. Effects of riboflavin/UVA corneal crosslinking on keratocytes and collagen fibres in human cornea. Clin Experiment Ophthalmol 2010; 38:49–56. 27. Leoni-Mesplie S, Mortemousque B, Touboul D, et al. Scalability and severity of keratoconus in children. Am J Ophthalmol 2012; 154:56–62. 28. Vinciguerra P, Albe E, Frueh BE, et al. Two-year corneal cross-linking results in patients younger than 18 years with documented progressive keratoconus. Am J Ophthalmol 2012; 154:520–526. 29. Chatzis N, Hafezi F. Progression of keratoconus and efficacy of pediatric [corrected] corneal collagen cross-linking in children and adolescents. J Refract Surg 2012; 28:753–758. 30. Hafezi F, Randleman JB. Corneal collagen cross linking. Thorofare, NJ: && SLACK; 2013. pp. xv, 167. Comprehensive book with summary of the cross-linking process, including the basic science foundations, treatment protocols and outcomes, recognition and management of potential complications, and modifications of the standard protocol for special circumstances. 31. Dawson DG, Randleman JB, Grossniklaus HE, et al. Corneal ectasia after excimer laser keratorefractive surgery: histopathology, ultrastructure, and pathophysiology. Ophthalmology 2008; 115:2181–2191. 32. Randleman JB, Woodward M, Lynn MJ, Stulting RD. Risk assessment for ectasia after corneal refractive surgery. Ophthalmology 2008; 115:37–50. 33. Santhiago MR, Smadja D, Gomes BF, et al. Association between the && percentage tissue altered and postlaser in situ keratomileusis ectasia in eyes with normal preoperative topography. Am J Ophthalmol 2014; 158:87–95. Determination that percentage tissue ablated at the time of LASIK was significantly correlated with development of post-LASIK ectasia, above all other parameters. 34. Richoz O, Mavrakanas N, Pajic B, Hafezi F. Corneal collagen cross-linking for ectasia after LASIK and photorefractive keratectomy: long-term results. Ophthalmology 2013; 120:1354–1359. 35. Kymionis GD, Grentzelos MA, Portaliou DM, et al. Corneal collagen cross&& linking (CXL) combined with refractive procedures for the treatment of corneal ectatic disorders: CXL plus. J Refract Surg 2014; 30:566–576. Establishment of CXL plus: review of combined CXL with refractive surgical techniques to improve visual acuity in additional to corneal stability with CXL alone. 36. Hafezi F, Randleman JB. PACK-CXL: defining CXL for infectious keratitis. J Refract Surg 2014; 30:438–439. 37. Martins SA, Combs JC, Noguera G, et al. Antimicrobial efficacy of riboflavin/ UVA combination (365 nm) in vitro for bacterial and fungal isolates: a potential new treatment for infectious keratitis. Invest Ophthalmol Vis Sci 2008; 49:3402–3408. 38. Price MO, Tenkman LR, Schrier A, et al. Photoactivated riboflavin treatment of infectious keratitis using collagen cross-linking technology. J Refract Surg 2012; 28:706–713. 39. Said DG, Elalfy MS, Gatzioufas Z, et al. Collagen cross-linking with && photoactivated riboflavin (PACK-CXL) for the treatment of advanced infectious keratitis with corneal melting. Ophthalmology 2014; 121:1377– 1382. Prospective clinical study comparing medical treatment of infectious keratitis with CXL and medical treatment. The combined treatment did not shorten the time of healing; however, the medical-only treatment group had a 21% rate of perforation or recurrence compared with no patients in the CXL plus medical treatment. 40. Alio JL, Abbouda A, Valle DD, et al. Corneal cross linking and infectious & keratitis: a systematic review with a meta-analysis of reported cases. J Ophthalmic Inflamm Infect 2013; 3:47. Meta-analysis of reported case series of infectious keratitis treatment with PACKCXL suggesting that this treatment works better in bacterial cases and less well in fungal or culture-negative cases. 41. Richoz O, Kling S, Hoogewoud F, et al. Antibacterial efficacy of accelerated photoactivated chromophore for keratitis-corneal collagen cross-linking (PACK-CXL). J Refract Surg 2014; 30:850–854. 42. Tabibian D, Richoz O, Riat A, et al. Accelerated photoactivated chromophore for keratitis-corneal collagen cross-linking as a first-line and sole treatment in early fungal keratitis. J Refract Surg 2014; 30:855–857. 43. Bunsen RW, Roscoe RE. Photochemical researches, part V: on the measurement of the chemical action of direct and diffuse sunlight. Proc R Soc Lond 1862; 12:306–312.
Volume 26 Number 3 May 2015
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Corneal cross-linking Khandelwal and Randleman 44. Wernli J, Schumacher S, Spoerl E, Mrochen M. The efficacy of corneal crosslinking shows a sudden decrease with very high intensity UV light and short treatment time. Invest Ophthalmol Vis Sci 2013; 54:1176–1180. 45. Hammer A, Richoz O, Arba Mosquera S, et al. Corneal biomechanical properties at different corneal cross-linking (CXL) irradiances. Invest Ophthalmol Vis Sci 2014; 55:2881–2884. 46. Mita M, Waring GO 4th, Tomita M. High-irradiance accelerated collagen crosslinking for the treatment of keratoconus: six-month results. J Cataract Refract Surg 2014; 40:1032–1040. 47. Elbaz U, Shen C, Lichtinger A, et al. Accelerated (9-mW/cm2) corneal collagen crosslinking for keratoconus-A 1-year follow-up. Cornea 2014; 33:769–773. 48. Hashemian H, Jabbarvand M, Khodaparast M, Ameli K. Evaluation of corneal & changes after conventional versus accelerated corneal cross-linking: a randomized controlled trial. J Refract Surg 2014; 30:837–842. Randomized control trial comparing convention and accelerate CXL showing equivalent improvement in visual acuity and topographic parameters in both groups. 49. Ozgurhan EB, Kara N, Cankaya KI, et al. Accelerated corneal cross-linking in pediatric patients with keratoconus: 24-month outcomes. J Refract Surg 2014; 30:843–849. 50. Kymionis GD, Grentzelos MA, Kounis GA, et al. Combined transepithelial phototherapeutic keratectomy and corneal collagen cross-linking for progressive keratoconus. Ophthalmology 2012; 119:1777–1784. 51. Torricelli AA, Ford MR, Singh V, et al. BAC-EDTA transepithelial riboflavinUVA crosslinking has greater biomechanical stiffening effect than standard epithelium-off in rabbit corneas. Experiment Eye Res 2014; 125:114– 117. 52. Filippello M, Stagni E, O’Brart D. Transepithelial corneal collagen crosslinking: bilateral study. J Cataract Refract Surg 2012; 38:283–291. 53. Taneri S, Oehler S, Lytle G, Dick HB. Evaluation of epithelial integrity with various transepithelial corneal cross-linking protocols for treatment of keratoconus. J Ophthalmol 2014; 2014:614380. 54. Alhamad TA, O’Brart DP, O’Brart NA, Meek KM. Evaluation of transepithelial stromal riboflavin absorption with enhanced riboflavin solution using spectrophotometry. J Cataract Refract Surg 2012; 38:884–889. 55. Koppen C, Wouters K, Mathysen D, et al. Refractive and topographic results of benzalkonium chloride-assisted transepithelial crosslinking. J Cataract Refract Surg 2012; 38:1000–1005. 56. Caporossi A, Mazzotta C, Baiocchi S, et al. Transepithelial corneal collagen crosslinking for keratoconus: qualitative investigation by in vivo HRT II confocal analysis. Eur J Ophthalmol 2012; 22 (Suppl 7):S81–S88. 57. Mastropasqua L, Nubile M, Calienno R, et al. Corneal cross-linking: intras& tromal riboflavin concentration in iontophoresis-assisted imbibition versus traditional and transepithelial techniques. Am J Ophthalmol 2014; 157:623–630; e1. Laboratory study in human cadaver eyes showing iontophoresis-assisted CXL yielded improved riboflavin compared with epi-on protocols but not to the level of epi-off protocols. 58. Bikbova G, Bikbov M. Transepithelial corneal collagen cross-linking by iontophoresis of riboflavin. Acta Ophthalmol 2014; 92:e30–e34.
59. Vinciguerra P, Randleman JB, Romano V, et al. Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: initial clinical outcomes. J Refract Surg 2014; 30:746–753. Prospective nonrandomized study showing that transepithelial iontophoresis CXL is a well tolerated and efficacious procedure for the treatment of keratoconus with improvement in visual acuity. 60. Bouheraoua N, Jouve L, El Sanharawi M, et al. Optical coherence tomography and confocal microscopy following three different protocols of corneal collagen-crosslinking in keratoconus. Invest Ophthalmol Vis Sci 2014; 55:7601–7609. 61. Bonnel S, De Riboyre BM, Bedubourg B, et al. Demarcation line evaluation of iontophoresis-assisted transepithelial corneal collagen cross-linking for keratoconus. J Refract Surg 2015; 31:36–40. 62. Wollensak G, Wilsch M, Spoerl E, Seiler T. Collagen fiber diameter in the rabbit cornea after collagen crosslinking by riboflavin/UVA. Cornea 2004; 23:503–507. 63. Seifert J, Hammer CM, Rheinlaender J, et al. Distribution of Young’s modulus in porcine corneas after riboflavin/UVA-induced collagen cross-linking as measured by atomic force microscopy. PLoS One 2014; 9:e88186. 64. Tan HY, Chang YL, Lo W, et al. Characterizing the morphologic changes in collagen crosslinked-treated corneas by Fourier transform-second harmonic generation imaging. J Cataract Refract Surg 2013; 39:779–788. 65. Kymionis GD, Grentzelos MA, Plaka AD, et al. Correlation of the corneal collagen cross-linking demarcation line using confocal microscopy and anterior segment optical coherence tomography in keratoconic patients. Am J Ophthalmol 2014; 157:110–115. 66. Malta JB, Renesto AC, Moscovici BK, et al. Stromal demarcation line induced by corneal cross-linking in eyes with keratoconus and nonkeratoconic asymmetric topography. Cornea 2015; 34:199–203. 67. Yam JC, Chan CW, Cheng AC. Corneal collagen cross-linking demarcation line depth assessed by Visante OCT After CXL for keratoconus and corneal ectasia. J Refract Surg 2012; 28:475–481. 68. Goldich Y, Marcovich AL, Barkana Y, et al. Clinical and corneal biomechanical changes after collagen cross-linking with riboflavin and UV irradiation in patients with progressive keratoconus: results after 2 years of follow-up. Cornea 2012; 31:609–614. 69. Vinciguerra P, Albe E, Mahmoud AM, et al. Intra- and postoperative variation in ocular response analyzer parameters in keratoconic eyes after corneal crosslinking. J Refract Surg 2010; 26:669–676. 70. Hallahan KM, Rocha K, Roy AS, et al. Effects of corneal cross-linking on ocular response analyzer waveform-derived variables in keratoconus and postrefractive surgery ectasia. Eye Contact Lens 2014; 40:339–344. 71. Tian L, Ko MW, Wang LK, et al. Assessment of ocular biomechanics using dynamic ultra high-speed Scheimpflug imaging in keratoconic and normal eyes. J Refract Surg 2014; 30:1–7. 72. Ali NQ, Patel DV, McGhee CN. Biomechanical responses of healthy and keratoconic corneas measured using a noncontact Scheimpflug-based tonometer. Invest Ophthalmol Vis Sci 2014; 55:3651–3659. 73. Bak-Nielsen S, Pedersen IB, Ivarsen A, Hjortdal J. Dynamic Scheimpflugbased assessment of keratoconus and the effects of corneal cross-linking. J Refract Surg 2014; 30:408–414.
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