Evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in 10 cats

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Scientific publication about the use of accelerated cross-linking for the treatment of melting keratitis in cats.

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Evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in 10 cats

  1. 1. Veterinary Ophthalmology (2013) 1–10 DOI:10.1111/vop.12112 Evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in ten cats Frank Famose DVM, Cert. Veterinary Ophthalmology, Clinique Vétérinaire des Acacias, 42 avenue Lucien-Servanty, 31700 Blagnac, France Address communications to: F. Famose Tel.: +33 5 61 71 24 02 Fax: +33 5 61 71 65 52 e-mail: frankfamose@gmail.com Abstract Objectives Melting keratitis is a serious condition presenting a high risk of permanent blindness and is caused by infectious or noninfectious factors. In humans, the clinical efficacy of collagen cross-linking (CXL) has been described in the treatment of refractory infectious keratitis by arresting keratomalacia. The aim of this study was to evaluate the efficacy of accelerated CXL for the treatment of melting keratitis in cats. Animals studied Ten cats were treated for unilateral melting keratitis by accelerated CXL. Procedure Corneas were irradiated by UVA (370 nm) at 30 mW/cm² irradiance for 3 min after soaking with 0.1% riboflavin in 20% dextran for 30 min (D1). Follow-up was conducted 3, 7, 14, and 30 days after treatment. Results Pain improvement was noted for all cases at D4 examination. Epithelial healing was observed at D8 for 9 of 10 cases and at D15 for 1 of 10 cases. Resolution of cellular infiltration was observed for all cases at D8 examination. The corneal vascularization was reduced for 9 of 10 cats by D31. At D31, all cases presented a variable degree of corneal fibrosis, but all eyes had visual function. No recurrent infection was observed. Conclusion Accelerated CXL appears to be a valuable option for the treatment of melting keratitis in cats. All the cases have reached a satisfactory outcome despite the individual differences in the conditions prior to the CXL treatment and the variable presence of infectious agents. Key Words: accelerated cross-linking, cat, corneal melting, cross-linking, keratitis, optical coherence tomography INTRODUCTION In cats, melting keratopathies are serious conditions presenting a high risk of permanent blindness.1 In melting keratitis, stromal damage is initiated by various mechanisms including bacterial proliferation, toxin secretion, and microbial or corneal protease activation. An imbalance between the endogenous and exogenous matrix metalloproteinases (MMP) and the proteinases present in the cornea and the precorneal tear film leads to the destruction of corneal collagen.2–4 Microbial infection is usually suspected to be responsible for corneal melting, but cannot always be demonstrated.1 There are few corneal pathogens that are associated with primary corneal infections, and infectious corneal melting is typically due to secondary bacterial infections.1,5 Melting can also occur in the absence of infection and is thought to be secondary to an imbalance between proteolytic enzymes and protease inhibitors produced by resident and inflammatory cells.2–4 © 2013 American College of Veterinary Ophthalmologists Medical treatment is based on the administration of topical antibiotics and protease inhibitors either commercially available preparations or fortified compounded preparations.6,7 Despite treatment, vision loss can occur due to the progression of keratomalacia leading to corneal perforation. Perforations are managed by tectonic surgeries such as conjunctival grafts, biomaterial grafts, or amniotic membrane transplantation.8–11 Collagen cross-linking (CXL) is a technique that creates intrafibrillar covalent bonds in the collagen fibers of the corneal stroma via the photo-activation of riboflavin by ultraviolet-A (UVA) light and has been used since 1998 in humans for the treatment of progressive keratoconus, pellucid marginal degeneration, and ectatic complications of refractive surgeries.12–15 For these indications, safety and efficacy of this procedure have been widely established.12–21 The antimicrobial activity of CXL against numerous bacteria and fungi22 has been demonstrated under experimental conditions. In addition, an increased collagen resistance against
  2. 2. 2 famose enzymatic digestion23 has been demonstrated under experimental conditions in vitro. Recently, the clinical efficacy of CXL has been described in the treatment of presumed infectious keratitis and corneal melting in humans with promising results.22,24–31 Spiess et al. have recently described the application of CXL in three dogs and three cats in a pilot study,32 and Hellander-Edman et al. have described its use for the treatment of ulcerative keratitis in nine horses.33 Both studies used an adaptation of the treatment protocol used for human keratoconus described by Wollensack12 with the use of a compounded riboflavin solution. Accelerated cross-linking is a recent adaptation of this traditional technique based on the use of a higher irradiance UV light source and a shorter irradiation time. Efficacy and safety of this technique for the treatment of melting keratitis have been recently evaluated in eight dogs.34 The aim of the present study was to evaluate the efficacy of accelerated CXL for the treatment of melting keratitis in cats. MATERIALS AND METHODS Inclusion criteria This prospective, nonrandomized clinical study included cases referred for the evaluation after progression of clinical signs despite initial medical therapy. To be included in the study, cases had to have a clinical diagnosis of melting keratitis characterized by epithelial and anterior stromal loss, anterior stromal dissolution, cellular infiltration, and corneal vascularization. Cases with impending of confirmed corneal perforation were excluded from the study. Owners’ consent was obtained prior to the inclusion of the animals into the study. All procedures were performed in accordance with the French guidelines for animal care and followed the ARVO guidelines for animal use. Ophthalmologic examination Keratitis evaluation was made under the following criteria: • • • The specific clinical signs of keratitis were evaluated by slit-lamp examination (Hawkeye TM, Dioptrix, Toulouse, France). A clinical score modified from Tajima et al.35 (0–3; 0 = absent, 1 = mild, 2 = moderate, and 3 = severe) was used to grade the severity of mucopurulent discharge, corneal edema, corneal vascularization, conjunctivitis, blepharitis, and uveitis. The highest possible total score was 18. A pain score modified from the Melbourne University scoring36 (0–1; 0 = absent and 1 = present) was used to grade pain signs that included prostration, aggressive behavior, blepharospasm, enophthalmos, photophobia, ocular pruritus, and defense reaction to examination. The highest possible total score was seven. Cellular infiltration had generally a round or elliptic shape. The length of the major and minor axes (a and b) was measured with a manual caliper, and the area • was calculated as p*a/2*b/2 (in mm²) as described by Price et al.30 Ulceration size was measured with a manual caliper, and the length of the major and minor axes of the ulceration was recorded. The area was calculated as p*a/2*b/2 (in mm²). Fluorescein staining was not used for initial measurements of epithelial defect, because it can interfere with UVA absorption.37 Tear production was evaluated by Schirmer test I in all cats (Test de Schirmer, Virbac, Carros, France). A corneal sample was collected from each cat with a Kimura spatula (Moria-Surgical, Antony, France) and submitted for bacterial culture and antimicrobial sensitivity (Laboratoire Meynaud, Toulouse, France). A PCR test for FHV-1 was performed for all cats (Scanelis, Colomiers, France). Measurements of the corneal thickness The corneal thickness was evaluated by optical coherence tomography (OCT). The cats were anesthetized with IM 300 lg/m² medetomidine (DomitorTM, Pfizer, NY, USA) and 5 mg/kg ketamine (Imalgene 1000TM, Merial, Lyon, France). Cats were placed in dorsal recumbency, and the pachymetry was performed with an optical coherence tomography (OCT) device (iVue TM, Optovue, Fremont, CA, USA).38 Measurements were taken with the focus at the center of the corneal lesion. Minimal and maximal corneal thicknesses were evaluated with the OCT caliper tool (Fig. 1). For safety reasons, only the cats with a minimal corneal thickness >300 lm were included in the study. Treatment by accelerated cross-linking The eyelids were kept open with the use of a lid speculum. After the instillation of a topical anesthetic (Oxybuprocaine 0.4%, Cebesine 0. 4%TM, Bausch & Lomb-Chauvin, Montpellier, France), the ulcer margins were cleaned, and the debris was removed from the corneal surface with a microsurgery sponge as described by Rosetta et al.26 A solution of isotonic riboflavin (riboflavin 0.1%, dextran 20%, VibexTM, Avedro, Waltham, MA, USA) was instilled on the corneal surface for 30 min (one drop every 2 min). The corneal surface was rinsed with BSS at the end of riboflavin instillation. Penetration of riboflavin through the cornea was confirmed by visualizing the fluorescence of the riboflavin in the anterior chamber with slit-lamp biomicroscopy using the cobalt blue light. The corneas were irradiated with UVA (wave length = 370 nm) for a total dose of 5.4 J/cm², as described in human studies,22,24–29 delivered by the KXLTM system (Avedro) for 3 min with an irradiance of 30 mW/cm². The beam of 9 mm in diameter was centered and focused on the center of the corneal lesion. Care was taken not to irradiate the corneal limbus. One drop of riboflavin was instilled after 2 min of irradiation. All animals received a single CXL treatment at D1. © 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10
  3. 3. corneal melting in cats 3 (a) antibiotic treatment (Tobrex 0.3%TM, 1 drop q12 h) was continued until the next examination. Photographs were taken for each case at each follow-up appointment. RESULTS (b) Figure 1. Case 6. Central epithelial and stromal loss (green arrows), infiltration (red arrow), and vascularization (black arrow) are present (a). On OCT picture (b), the residual stromal thickness can be measured at the thinner part of the cornea (green arrows) at 447 lm. Cellular infiltration appears as a heterogeneous hyper-reflective zone (red arrow). Postoperative treatment Each eye was treated twice daily with one drop of a tobramycin solution (Tobrex 0.3%TM, Alcon, Rueil-Malmaison, France) after the CXL treatment until complete epithelial healing. All anticollagenase and other previous treatments were discontinued. Follow-up To evaluate corneal healing and the symptom reduction over a 30-day follow-up period, all eyes were examined 3, 7, 14, and 30 days (D4, D8, D15, and D31, respectively) after treatment using the same pretreatment protocol minus the Schirmer test. Epithelial integrity was evaluated after instillation of a drop of fluorescein solution. Fluorescein dye staining of the cornea was interpreted as a positive result. In cases of epithelial healing, topical treatment was discontinued. In cases of epithelial defect, topical Pre-operative features Ten cats were treated between April 2012 and February 2013 by the same clinician (Frank Famose). The pre-operative findings of each cat were recorded (Table 1). Five cats were Persians, four were Domestic Short-haired, and one was a Singapura. Keratitis duration before CXL treatment ranged from 7 days to 2 months. None of the cases had a bacteriologic analysis before initiation of the treatment by the referring veterinarian. Six of the 10 cats had received a surgical therapy prior to CXL (one epithelial debridement, three nictitans membrane flaps, one conjunctival pedicle graft, and one superficial keratectomy). All cases had received topical antibiotics, and 6 of 10 had received a topical anticollagenase prior to referral. Bacterial analysis was performed in all cases after referral, prior to CXL therapy. Four cultures were positive (three Pseudomonas aeruginosa and one Staphylococcus chromogenes). The bacteriological data and antimicrobial sensitivity are summarized in Table 2. Two cats were positive for FHV1 (case nos four and six). Maximal corneal thickness ranged from 650 to 1200 lm, and minimal corneal thickness ranged from 305 to 567 lm. The relative depth of the ulceration ranged from 16 to 75% of the corneal thickness. The size of epithelial defect ranged from 2 to 8 mm in maximal diameter. The surface of cellular infiltration ranged from 3.14 to 56.55 mm². Postoperative features The individual scores for clinical scores, pain scores, and the areas of epithelial defects and cellular infiltrates are summarized in Table 3. Epithelial healing was observed at D8 for 9 of 10 cases and at D15 for one case (Fig. 2). The mean clinical and pain scores showed a marked decrease at D4 and D8 (Fig. 2). Cellular infiltration of the cornea was present for all cases at the time of CXL and had resolved by D8 (Fig. 3). Corneal vascularization was present in all cases at D8, even in the cases in which it was not observed at D1. At 30 days post-treatment, it had resolved in 2 of 10 cases and was mild in 7 of 10 cases (Fig. 4) and moderate for 1 of 10 cases (Fig. 5). At 1 month, all cases had a variable degree of corneal fibrosis (Fig. 6), but all eyes were visual. No clinical sign of corneal infection (corneal ulceration, ocular discharge) was observed during the follow-up period. © 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10
  4. 4. 2 years OS 1 month Gentamicin (14 mg/ml)* NAC Framycetin Ciprofloxacin NAC Epithelial scraping 17 Sterile Negative 567 1020 54% 392 Conjunctival graft 15 Sterile Negative 493 1170 58% 695 292 696 Pseudomonas aeruginosa Positive 543 650 16% 12 15 Staphylococcus chromogenes Negative 380 660 42% 3rd eyelid flap Chloramphenicol Ciprofloxacin 2 months OS 1 month Singapura 4 None Ciprofloxacin Domestic Short-haired 8 years OD 1 month 3 *Gentamicin was used at fortified concentration of 14 mg/ml. † CT, corneal thickness. Previous surgical treatment Schirmer test (mm/min) Presurgical bacteriology PCR for FHV1 Min. CT† (lm) Max. CT (lm) Ulcer depth (% of corneal thickness) Ulcer size (mm) Age Affected eye Duration prior to CXL (from first symptoms) Previous topical medical treatment Persian Domestic Short-haired 1 year OD 3 weeks Breed 2 1 Case number Table 1. Individual cases data 897 Negative 305 1200 75% Sterile 17 None Gentamicin (14 mg/ml)* NAC 7 years OS 10 days Persian 5 898 Positive 447 1200 63% Sterile 18 3rd eyelid flap Chloramphenicol NAC 3 years OD 2 months Persian 6 896 Pseudomonas aeruginosa Negative 320 680 53% 14 3rd eyelid flap Framycetin NAC Domestic Short-haired 1 year OD 1 month 7 292 Negative 420 780 46% Sterile 13 None Framycetin NAC 8 years OS 15 days Persian 8 392 Negative 480 950 49% Sterile 16 Ciprofloxacin Gentamicin (14 mg/ml) * NAC None Domestic Short-haired 6 years OD 15 days 9 494 Pseudomonas aeruginosa Negative 420 720 42% Superficial keratectomy 15 Chloramphenicol Ciprofloxacin 4 years OD 7 days Persian 10 4 famose © 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10
  5. 5. corneal melting in cats 5 Table 2. Pre-operative bacterial culture results Sensitivity Case Bacterial species Aminoglycosides Quinolones Tetracyclines Chloramphenicol 3 4 7 10 Staphylococcus chromogenes Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa R S R R R R S S R S S S S NT NT NT S, sensitive; R, resistant; NT, not tested. DISCUSSION Table 3. Postoperative scores from D1 to D31 Case number Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Score D1 D4 D8 D15 D31 Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface Clinical score Pain score Ulcer surface Infiltration surface 13 5 23.56 32.99 13 6 4.71 9.42 13 4 3.14 7.07 10 5 28.27 28.27 12 5 43.98 56.55 10 7 50.27 50.27 10 4 37.7 49.48 11 4 3.14 3.14 12 6 4.71 9.42 10 5 12.57 15.71 11.4 9 2 4.71 9.42 8 3 1.57 4.71 10 2 0.79 3.14 6 3 3.14 3.14 8 2 11.78 18.85 7 4 9.42 15.71 7 4 11.78 18.85 4 1 0.79 0 7 4 0.79 3.14 4 3 3.14 7.07 7 3 0 0 0 5 0 0 0 5 0 0 0 3 0 0 0 6 0 0.79 0 4 0 0 0 5 1 0 0 4 0 0 0 2 0 0 0 2 0 0 0 3.9 2 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 2 0 0 0 2 0 0 0 3 0 0 0 2 0 0 0 1 0 0 0 2 0 0 0 1.7 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 2 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0.9 5.1 2.8 0.1 0 0 21.21 4.79 0.08 0 0 26.23 8.40 0 0 Average clinical score Average pain score Average (mm²) ulcer surface Average (mm²) infiltration surface 0 Accelerated collagen cross-linking was used for the treatment of feline melting keratitis in 10 cases. All treated animals showed reduced pain 3 days after treatment. This observation is similar to the results in humans in which ocular pain improvement was achieved over the same time duration.22,24,25 Complete epithelial healing was achieved in 7 days for 9 of 10 cats and at 14 days after treatment in one case. The healing rate in our study was shorter than that observed by Spiess et al.32 where epithelial healing was achieved in 7 days for one cat and 15 and 18 days for the two others. The difference of healing rate between these two studies could be explained by the difference in the initial size of the ulceration and the degree of the epithelial surface removal before riboflavin application. In the study by Spiess et al.,32 epithelium debridement was 5 to 11 mm in diameter, whereas in the present study, it was limited to the ulcer margins (from 2 to 8 mm) as described by Rosetta et al.26 Corneal melting resolved in all treated eyes within 7 days after CXL treatment. This was observed by biomicroscopic examination as a reduction in corneal thickness and cellular infiltration with recovery of corneal transparency. These results are similar to previous reports in the literature22,24–31,39 and can be attributable to direct effects of CXL. Clinical observations and experimental data have shown that CXL may have three distinct effects on cornea: a bactericidal effect, an increase in corneal resistance to mechanical forces and enzymatic digestion, and a reduction in corneal inflammation. A bactericidal effect has been established in experimental conditions40 with CXL. This effect is manifested by bacterial DNA and membrane alterations41 secondary to the liberation of free radicals by photo-activation of riboflavin. However, an immediate reduction in the bacterial load has not been demonstrated in the different clinical reports.22,24–31 Collagen cross-linking is reported to increase the corneal resistance to mechanical forces and enzymatic digestion secondary to mechanical and biochemical modifications of corneal structure42,43 and the creation of intralamellar covalent collagen bonds.44 This mechanism is thought to be limited to the first anterior 200 lm of the treated cornea45 and to contribute to increased resistance © 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10
  6. 6. 6 famose Figure 2. Progression of the average clinical score (red), average pain score (blue), average ulcer surface (gray), and average infiltration surface (green) during the observation period. (a) (a) (b) (b) Figure 3. Case 3. Pretreatment presentation (a) and 7 days after the treatment (b) Cellular infiltration has disappeared, and transparency has been recovered. Epithelial healing was complete. Figure 4. Case 2. Reduction in vascularization from pretreatment (a) to 1 month post-treatment (b) Residual vascularization was scored ‘mild’. © 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10
  7. 7. corneal melting in cats 7 (a) (a) (b) (b) Figure 5. Case 7. Pretreatment presentation (a) and 1 month after treatment (b) with persistent central superficial corneal vascularization scored as ‘moderate’. to proteases.23 Experimentally, an increase in mechanical rigidity and in resistance to proteolytic enzymes has been shown for human and swine corneas.23,46–48 Collagen cross-linking reduces the corneal inflammatory response by the induction of apoptosis of the keratocytes located in the anterior part of the stroma.49,50 This may contribute to modification of local immune response mediated by Langerhans and dendritic cells and may reduce corneal melting and vascularization.39 In the current study, a dramatic reduction in cellular infiltration and in corneal melting was observed. In addition, corneal vascularization increased in the first 7 days and regressed over the next 3 weeks, but was present at completion of the study in a reduced state in 8 of 10 eyes. Limited data have been published on the corneal effects of CXL in cats. A recent publication32 reports a modification of the treatment protocol used for human keratoconus described by Wollensack12 with the use of a compounded riboflavin solution. Accelerated cross-linking (with the KXLTM) is a recent adaptation of this technique Figure 6. Case 9. Pretreatment presentation (a) and 1 after CXL treatment (b) with two remaining areas of marked corneal fibrosis. and uses a higher irradiance UV light source and a shorter time of irradiation.51,52 The energy delivered by both protocols (30 mW/cm² for 3 min for the accelerated protocol or 3 mW/cm² for 30 min in the study by Wollensack et al.12) is the same (5.4 J/cm²) achieving the same biological effects.51,52 However, in experimental conditions, the biochemical stiffness of the cornea seems to decrease in higher irradiances due to rapid oxygen depletion, because CXL is an oxygen-dependent process.53 The influence on the treatment of the melting keratitis is unknown. Compared with the Wollensack et al.12 protocol, the use of accelerated cross-linking reduces operating time and thus the duration of anesthesia. In human patients, no additional adverse effects have been observed with irradiances >3 mW/cm².51,52 The results observed in the present study are similar to the previous evaluation of accelerated cross-linking for corneal melting in dogs.34 The isotonic riboflavin solution used in the present study (VibexTM) is commercially available in Europe, and its concentration is the same as the compounded solution used by Spiess et al.32 The absorption spectra of fluorescein and © 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10
  8. 8. 8 famose riboflavin for UV-A are very close. The presence of fluorescein in the anterior stroma limits UV-A absorption and could explain some differences observed in healing rates in human patients.37 Therefore, in the present study, fluorescein staining was used only in the follow-up period. Case inclusion was based on clinical diagnosis of melting keratitis by the observation of epithelial loss, corneal edema, cellular infiltration, and stromal dissolution. As stated earlier, microbial infection is usually suspected to drive the inflammatory state responsible for corneal melting by the contamination of a previous epithelial defect. However, it cannot always be demonstrated. Although bacterial contamination was suspected in all cases, only 4 of 10 cases had a positive culture in our study, which correlates with literature data.54 These results are similar to those presented by Spiess et al.32 in which none of the six cases (three dogs and three cats) had a positive bacteriologic culture. This can be either explained by the inhibition of bacteria by the previous medical treatments or related to a sterile keratitis and the activation of the proteases by other mechanisms.1,2 In our study, all the cases have achieved the same outcome regardless of the presence of bacteria or of the duration of the condition prior to CXL treatment. Similar observations have been reported in a series of 25 human cases.39 Makdoumi et al. concluded that CXL should be considered as the initial treatment for keratitis without the use of antibiotics, arguing that this could be a way of reducing the risk of antibiotic resistance.39 However, due to the limited data evaluating the efficacy of CXL in the treatment of melting keratitis in cats, topical antibiotic therapy was maintained until complete epithelial healing to prevent a secondary bacterial contamination. Tobramycin was used at twice daily for a preventive purpose although the frequency is unlikely to decrease bacterial growth. Although justified on a scientific basis to compare CXL with a traditional therapy for melting keratitis, no control group was included in our series. Animals were presented after a previous medical treatment (topical antibiotics and, in some cases, antiproteases) with no improvement in or worsening of the clinical signs at the time of referral. Treatment with antiproteases may have yielded similar results as described in this study. Because no control group was included, a comparison between the two treatment effects was not possible. However, all antiproteases treatments were stopped at the time of CXL; thus, the arrest of corneal melting could be attributed to the CXL procedure. In human patients, adverse effects of CXL have been described: postoperative infection, herpes virus exacerbation, and corneal endothelial lesions. With CXL treatment of human keratoconus, cases of postoperative infections have been described in the days or weeks following the procedure.55–57 In all cases, stromal infection was present 3–5 days after the procedure and was attributed to the surgical removal of corneal epithelium. In the present study, no postoperative infection was observed during the follow-up period, as described in the human studies.19–21,26 Herpetic keratitis has been described in a human patient 5 days after his treatment for keratoconus by CXL.58 In our series, two cats tested positive for FHV-1. However, there was no evidence of active disease in the immediate postoperative period. Therefore, no treatment against FHV-1 was prescribed. The risk of viral activation by CXL treatment in feline patients is unknown and should be investigated further because UV light has been used for reactivation of herpes simplex virus in animal models.59–63 However, the UV-B used in some experimental procedures61–63 had wave lengths (280–315 nm) significantly different from those used for collagen cross-linking (370 nm). Endothelial lesions may be directly attributable to the CXL treatment by the cytolytic effect of riboflavin photo-activation on endothelial cells.64,65 Experimentally, the maximal absorption depth for UVA in a riboflavin-saturated cornea is thought to be approximately 300 lm. UVA absorption does not stop at 300 lm, but absorption levels drop below the toxic threshold in the deeper parts of the cornea and eye as a whole. Stiffening effects of CXL seem to be limited to the more superficial 200 lm of the stroma,43,45 and apoptotic effects of the procedure appear to be rare in the deeper parts of the cornea (beyond 300 lm).45 This concept, often referred to as ‘riboflavin shielding’, correlates directly with the minimal corneal thickness necessary for safe CXL treatment. In this study, corneal thickness was >300 lm in all cases. No endothelial effects were observed in all treated cases. Before treatment, many eyes in this study presented with a thick cellular infiltration, which can interfere with UV penetration and produces a heterogeneous photo-activation of riboflavin. No difference in the results was noted between these different cases regardless of the corneal thickness or the severity of cellular infiltration. In human patients, infected corneas with a thickness less than 300 lm have been successfully treated by CXL after soaking with hypotonic riboflavin.26 Very thin corneas or corneas with impending perforation were not included in this study. However, the prospect of treating such corneas is very promising as perforation might be prevented by the stiffening effects of CXL. Extension of our study to a larger group should allow us to optimize treatment parameters according to the size and the depth of the corneal loss. In the study by Spiess et al.,32 a case of corneal sequestrum was observed within 15 days post-CXL. Keratocytes apoptosis has been hypothesized as a cause of sequestrum formation,66 and because CXL induces anterior apoptosis, corneal sequestrum formation could be a potential adverse effect. In the present study, no sign of sequestrum development was observed. In the study by Spiess et al.,32 it is not clear whether the keratitis or the CXL treatment or both were factors in the development of the © 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10
  9. 9. corneal melting in cats 9 sequestrum. In the present study, variable corneal fibrosis was observed in all cases. Corneal haze has been described in human patients with keratoconus after CXL treatment.14–18 In this study, it is not clear whether the corneal fibrosis can be attributed to the initial keratitis, the treatment procedure, or both. Follow-up for a longer period could be useful to evaluate the long-term effects of the treatment. All the cats treated in this study completely healed regardless of the presence of bacterial agents, the extension of the initial corneal lesions, the duration of the disease before treatment, and the previous treatments. The results achieved in this small series suggest that CXL could be a valuable therapeutic option for melting keratitis in cats. The CXL procedure requires a precise focusing of the UV beam and thus requires general anesthesia. Because the duration of the anesthesia is reduced in comparison with the traditional CXL protocol, accelerated cross-linking could present a practical advantage while providing the same biological effects. However, accelerated CXL is performed with a commercially available riboflavin (VibexTM) with a price significantly higher than that of compounded riboflavin. These disadvantages (anesthesia, price) have to be taken into account in the treatment decision. CXL could also be considered as primary treatment for keratitis with or without the use of antibiotics. REFERENCES 1. Gilger BC, Ollivier FJ, Bentley E. diseases and surgery of the canine cornea and sclera. In: Veterinary Ophthalmology. (ed.Gelatt KN) 4th edn. Ames, Blackwell Publishing, 2007; 690–752. 2. Ollivier FJ, Gilger BC, Barrie KP et al. Proteinases of the cornea and preocular tear film. Veterinary Ophthalmology 2007; 10: 199–206. 3. Wang L, Pan Q, Xue Q et al. Evaluation of matrix metalloproteinase concentration in precorneal tear film from dogs with Pseudomonas aeruginosa-associated keratitis. American Journal of Veterinary Research 2008; 69: 1341–1345. 4. Brejchova K, Liskova P, Cejkova J et al. Role of matrix metalloproteinase in recurrent corneal melting. Experimental Eye Research 2010; 90: 583–590. 5. Ollivier FJ. Bacterial corneal diseases in dogs and cats. Clinical Techniques in Small Animal Practice 2003; 18: 193–198. 6. Rgnier A. Clinical pharmacology and therapeutics part 2: e antimicrobial, antiinflammatory agents and antiglaucoma drugs. In: Veterinary Ophthalmology. (Gelatt KN) 4th edn. Ames, Blackwell Publishing, 2007, 288–331. 7. Brooks DE, Ollivier FJ. Matrix metalloproteinase inhibition in corneal ulceration. Veterinary Clinics of North America: Small Animal Practice 2004; 34: 611–622. 8. Hollingsworth SR. Corneal surgical techniques. Clinical Techniques in Small Animal Practice 2003; 16: 161–167. 9. 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