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

Evaluation of accelerated collagen cross-linking for the treatmentof melting keratitis in ten cats

Frank FamoseDVM, 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: [email protected]

AbstractObjectives Melting keratitis is a serious condition presenting a high risk of permanent

blindness and is caused by infectious or noninfectious factors. In humans, the clinicalefficacy of collagen cross-linking (CXL) has been described in the treatment of refrac-

tory infectious keratitis by arresting keratomalacia. The aim of this study was to evalu-ate 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-upwas conducted 3, 7, 14, and 30 days after treatment.

Results Pain improvement was noted for all cases at D4 examination. Epithelialhealing was observed at D8 for 9 of 10 cases and at D15 for 1 of 10 cases. Resolutionof 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 avariable 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 theindividual 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 pre-senting a high risk of permanent blindness.1 In meltingkeratitis, stromal damage is initiated by various mecha-nisms including bacterial proliferation, toxin secretion,and microbial or corneal protease activation. An imbalancebetween the endogenous and exogenous matrix metallo-proteinases (MMP) and the proteinases present in the cor-nea and the precorneal tear film leads to the destructionof corneal collagen.2–4 Microbial infection is usually sus-pected to be responsible for corneal melting, but cannotalways be demonstrated.1 There are few corneal pathogensthat are associated with primary corneal infections, andinfectious corneal melting is typically due to secondarybacterial infections.1,5 Melting can also occur in theabsence of infection and is thought to be secondary to animbalance between proteolytic enzymes and proteaseinhibitors produced by resident and inflammatory cells.2–4

Medical treatment is based on the administration of topi-cal antibiotics and protease inhibitors either commerciallyavailable preparations or fortified compounded prepara-tions.6,7 Despite treatment, vision loss can occur due tothe progression of keratomalacia leading to corneal perfo-ration. Perforations are managed by tectonic surgeriessuch as conjunctival grafts, biomaterial grafts, or amnioticmembrane transplantation.8–11 Collagen cross-linking(CXL) is a technique that creates intrafibrillar covalentbonds in the collagen fibers of the corneal stroma via thephoto-activation of riboflavin by ultraviolet-A (UVA) lightand has been used since 1998 in humans for the treatmentof progressive keratoconus, pellucid marginal degenera-tion, and ectatic complications of refractive surgeries.12–15

For these indications, safety and efficacy of this procedurehave been widely established.12–21 The antimicrobial activ-ity of CXL against numerous bacteria and fungi22 hasbeen demonstrated under experimental conditions. Inaddition, an increased collagen resistance against

© 2013 American College of Veterinary Ophthalmologists

Veterinary Ophthalmology (2013) 1–10 DOI:10.1111/vop.12112

enzymatic digestion23 has been demonstrated under exper-imental conditions in vitro. Recently, the clinical efficacyof CXL has been described in the treatment of presumedinfectious keratitis and corneal melting in humans withpromising results.22,24–31 Spiess et al. have recentlydescribed the application of CXL in three dogs and threecats in a pilot study,32 and Hellander-Edman et al. havedescribed its use for the treatment of ulcerative keratitis innine horses.33 Both studies used an adaptation of thetreatment protocol used for human keratoconus describedby Wollensack12 with the use of a compounded riboflavinsolution. Accelerated cross-linking is a recent adaptationof this traditional technique based on the use of a higherirradiance UV light source and a shorter irradiation time.Efficacy and safety of this technique for the treatment ofmelting keratitis have been recently evaluated in eightdogs.34 The aim of the present study was to evaluate theefficacy of accelerated CXL for the treatment of meltingkeratitis in cats.

MATERIALS AND METHODS

Inclusion criteriaThis prospective, nonrandomized clinical study includedcases referred for the evaluation after progression of clini-cal signs despite initial medical therapy. To be included inthe study, cases had to have a clinical diagnosis of meltingkeratitis characterized by epithelial and anterior stromalloss, anterior stromal dissolution, cellular infiltration, andcorneal vascularization. Cases with impending of con-firmed corneal perforation were excluded from the study.Owners’ consent was obtained prior to the inclusion ofthe animals into the study. All procedures were performedin accordance with the French guidelines for animal careand followed the ARVO guidelines for animal use.

Ophthalmologic examinationKeratitis evaluation was made under the following criteria:

• The specific clinical signs of keratitis were evaluated byslit-lamp examination (Hawkeye TM, Dioptrix, Tou-louse, France). A clinical score modified from Tajimaet al.35 (0–3; 0 = absent, 1 = mild, 2 = moderate, and3 = severe) was used to grade the severity of mucopuru-lent discharge, corneal edema, corneal vascularization,conjunctivitis, blepharitis, and uveitis. The highestpossible total score was 18.

• A pain score modified from the Melbourne Universityscoring36 (0–1; 0 = absent and 1 = present) was used tograde pain signs that included prostration, aggressivebehavior, blepharospasm, enophthalmos, photophobia,ocular pruritus, and defense reaction to examination.The highest possible total score was seven.

• Cellular infiltration had generally a round or ellipticshape. The length of the major and minor axes (a andb) was measured with a manual caliper, and the area

was calculated as p*a/2*b/2 (in mm²) as described byPrice et al.30

• Ulceration size was measured with a manual caliper,and the length of the major and minor axes of theulceration was recorded. The area was calculated asp*a/2*b/2 (in mm²). Fluorescein staining was not usedfor initial measurements of epithelial defect, because itcan interfere with UVA absorption.37

Tear production was evaluated by Schirmer test I in allcats (Test de Schirmer, Virbac, Carros, France). A cornealsample was collected from each cat with a Kimura spatula(Moria-Surgical, Antony, France) and submitted for bacte-rial culture and antimicrobial sensitivity (Laboratoire Mey-naud, Toulouse, France). A PCR test for FHV-1 wasperformed for all cats (Scanelis, Colomiers, France).

Measurements of the corneal thicknessThe corneal thickness was evaluated by optical coherencetomography (OCT). The cats were anesthetized with IM300 lg/m² medetomidine (DomitorTM, Pfizer, NY, USA)and 5 mg/kg ketamine (Imalgene 1000TM, Merial, Lyon,France). Cats were placed in dorsal recumbency, and thepachymetry was performed with an optical coherencetomography (OCT) device (iVue TM, Optovue, Fremont,CA, USA).38 Measurements were taken with the focus atthe center of the corneal lesion. Minimal and maximalcorneal thicknesses were evaluated with the OCT calipertool (Fig. 1). For safety reasons, only the cats with a mini-mal corneal thickness >300 lm were included in thestudy.

Treatment by accelerated cross-linkingThe eyelids were kept open with the use of a lidspeculum. After the instillation of a topical anesthetic(Oxybuprocaine 0.4%, Cebesine 0. 4%TM, Bausch &Lomb-Chauvin, Montpellier, France), the ulcer marginswere cleaned, and the debris was removed from the cor-neal surface with a microsurgery sponge as described byRosetta et al.26 A solution of isotonic riboflavin (riboflavin0.1%, dextran 20%, VibexTM, Avedro, Waltham, MA,USA) was instilled on the corneal surface for 30 min (onedrop every 2 min). The corneal surface was rinsed withBSS at the end of riboflavin instillation. Penetration ofriboflavin through the cornea was confirmed by visualizingthe fluorescence of the riboflavin in the anterior chamberwith slit-lamp biomicroscopy using the cobalt blue light.

The corneas were irradiated with UVA (wavelength = 370 nm) for a total dose of 5.4 J/cm², asdescribed in human studies,22,24–29 delivered by theKXLTM system (Avedro) for 3 min with an irradiance of30 mW/cm². The beam of 9 mm in diameter was centeredand focused on the center of the corneal lesion. Care wastaken not to irradiate the corneal limbus. One dropof riboflavin was instilled after 2 min of irradiation. Allanimals received a single CXL treatment at D1.

© 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10

2 f amo s e

Postoperative treatmentEach eye was treated twice daily with one drop of a tobra-mycin solution (Tobrex 0.3%TM, Alcon, Rueil-Malmaison,France) after the CXL treatment until complete epithelialhealing. All anticollagenase and other previous treatmentswere discontinued.

Follow-upTo evaluate corneal healing and the symptom reductionover 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 protocolminus the Schirmer test. Epithelial integrity was evaluatedafter instillation of a drop of fluorescein solution. Fluores-cein dye staining of the cornea was interpreted as a posi-tive result. In cases of epithelial healing, topical treatmentwas discontinued. In cases of epithelial defect, topical

antibiotic treatment (Tobrex 0.3%TM, 1 drop q12 h) wascontinued until the next examination.

Photographs were taken for each case at each follow-upappointment.

RESULTS

Pre-operative featuresTen cats were treated between April 2012 and February 2013by the same clinician (Frank Famose). The pre-operativefindings of each cat were recorded (Table 1). Five cats werePersians, four were Domestic Short-haired, and one was aSingapura.

Keratitis duration before CXL treatment ranged from7 days to 2 months. None of the cases had a bacteriologicanalysis before initiation of the treatment by the referringveterinarian. Six of the 10 cats had received a surgicaltherapy prior to CXL (one epithelial debridement, threenictitans membrane flaps, one conjunctival pedicle graft,and one superficial keratectomy). All cases had receivedtopical antibiotics, and 6 of 10 had received a topical anti-collagenase prior to referral.

Bacterial analysis was performed in all cases after refer-ral, prior to CXL therapy. Four cultures were positive(three Pseudomonas aeruginosa and one Staphylococcus chrom-ogenes). The bacteriological data and antimicrobial sensi-tivity are summarized in Table 2.

Two cats were positive for FHV1 (case nos four andsix).

Maximal corneal thickness ranged from 650 to1200 lm, and minimal corneal thickness ranged from 305to 567 lm. The relative depth of the ulceration rangedfrom 16 to 75% of the corneal thickness. The size of epi-thelial defect ranged from 2 to 8 mm in maximal diame-ter. The surface of cellular infiltration ranged from 3.14to 56.55 mm².

Postoperative featuresThe individual scores for clinical scores, pain scores, andthe areas of epithelial defects and cellular infiltrates aresummarized in Table 3.

Epithelial healing was observed at D8 for 9 of 10 casesand at D15 for one case (Fig. 2).

The mean clinical and pain scores showed a markeddecrease at D4 and D8 (Fig. 2). Cellular infiltration of thecornea was present for all cases at the time of CXL andhad 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. At30 days post-treatment, it had resolved in 2 of 10 casesand was mild in 7 of 10 cases (Fig. 4) and moderate for 1of 10 cases (Fig. 5). At 1 month, all cases had a variabledegree 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-upperiod.

(a)

(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).


co rn e a l m e l t i ng i n c a t s 3

Tab

le1.Individual

casesdata

Casenumber

12

34

56

78

910

Breed

Domestic

Short-haired

Persian

Domestic

Short-haired

Singapura

Persian

Persian

Domestic

Short-haired

Persian

Domestic

Short-haired

Persian

Age

1year

2years

8years

2months

7years

3years

1year

8years

6years

4years

Affectedeye

OD

OS

OD

OS

OS

OD

OD

OS

OD

OD

Durationpriorto

CXL

(from

first

symptoms)

3weeks

1month

1month

1month

10days

2months

1month

15days

15days

7days

Previoustopical

medical

treatm

ent

Framycetin

Ciprofloxacin

NAC

Gen

tamicin

(14mg/ml)*

NAC

Ciprofloxacin

Chloramphen

icol

Ciprofloxacin

Gen

tamicin

(14mg/ml)*

NAC

Chloramphen

icol

NAC

Framycetin

NAC

Framycetin

NAC

Ciprofloxacin

Gen

tamicin

(14mg/m

l)*

NAC

Chloramphen

icol

Ciprofloxacin

Previoussurgical

treatm

ent

Conjunctival

graft

Epithelial

scraping

None

3rdeyelid

flap

None

3rdeyelid

flap

3rdeyelid

flap

None

None

Superficial

keratectomy

Schirmer

test

(mm/m

in)

1517

1512

1718

1413

16

15

Presurgical

bacteriology

Sterile

Sterile

Staphylococcus

chromogenes

Pseudom

onas

aeruginosa

Sterile

Sterile

Pseudom

onas

aeruginosa

Sterile

Sterile

Pseudom

onas

aeruginosa

PCR

forFHV1

Negative

Negative

Negative

Positive

Negative

Positive

Negative

Negative

Negative

Negative

Min.CT

†(lm)

493

567

380

543

305

447

320

420

480

420

Max.CT

(lm)

1170

1020

660

650

1200

1200

680

780

950

720

Ulcer

depth

(%of

cornealthickn

ess)

58%

54%

42%

16%

75%

63%

53%

46%

49%

42%

Ulcer

size

(mm)

69

539

229

269

689

789

889

629

239

249

4

*Gen

tamicin

was

usedat

fortified

concentrationof14

mg/ml.

†CT,cornealthickn

ess.


4 f amo s e

DISCUSSION

Accelerated collagen cross-linking was used for the treat-ment of feline melting keratitis in 10 cases. All treated ani-mals showed reduced pain 3 days after treatment. Thisobservation is similar to the results in humans in whichocular pain improvement was achieved over the same timeduration.22,24,25

Complete epithelial healing was achieved in 7 days for9 of 10 cats and at 14 days after treatment in one case.The healing rate in our study was shorter than thatobserved by Spiess et al.32 where epithelial healing wasachieved in 7 days for one cat and 15 and 18 days for thetwo others. The difference of healing rate between thesetwo studies could be explained by the difference in the ini-tial size of the ulceration and the degree of the epithelialsurface removal before riboflavin application. In the studyby Spiess et al.,32 epithelium debridement was 5 to 11 mmin diameter, whereas in the present study, it was limitedto the ulcer margins (from 2 to 8 mm) as described byRosetta et al.26

Corneal melting resolved in all treated eyes within7 days after CXL treatment. This was observed by biomi-croscopic examination as a reduction in corneal thicknessand cellular infiltration with recovery of corneal transpar-ency. These results are similar to previous reports in theliterature22,24–31,39 and can be attributable to direct effectsof CXL. Clinical observations and experimental data haveshown that CXL may have three distinct effects on cornea:a bactericidal effect, an increase in corneal resistance tomechanical forces and enzymatic digestion, and a reduc-tion in corneal inflammation.

A bactericidal effect has been established in experimen-tal conditions40 with CXL. This effect is manifested bybacterial DNA and membrane alterations41 secondary tothe liberation of free radicals by photo-activation of ribo-flavin. However, an immediate reduction in the bacterialload has not been demonstrated in the different clinicalreports.22,24–31

Collagen cross-linking is reported to increase thecorneal resistance to mechanical forces and enzymaticdigestion secondary to mechanical and biochemical modi-fications of corneal structure42,43 and the creation of in-tralamellar covalent collagen bonds.44 This mechanism isthought to be limited to the first anterior 200 lm of thetreated cornea45 and to contribute to increased resistance

Table 2. Pre-operative bacterial culture results

Case Bacterial species

Sensitivity

Aminoglycosides Quinolones Tetracyclines Chloramphenicol

3 Staphylococcus chromogenes R R R S4 Pseudomonas aeruginosa S R S NT7 Pseudomonas aeruginosa R S S NT10 Pseudomonas aeruginosa R S S NT

S, sensitive; R, resistant; NT, not tested.

Table 3. Postoperative scores from D1 to D31

Casenumber Score D1 D4 D8 D15 D31

Case 1 Clinical score 13 9 3 2 1Pain score 5 2 0 0 0Ulcer surface 23.56 4.71 0 0 0Infiltration surface 32.99 9.42 0 0 0




Case 5 Clinical score 12 8 6 2 1Pain score 5 2 0 0 0Ulcer surface 43.98 11.78 0.79 0 0Infiltration surface 56.55 18.85 0 0 0



Case 8 Clinical score 11 4 4 2 1Pain score 4 1 0 0 0Ulcer surface 3.14 0.79 0 0 0Infiltration surface 3.14 0 0 0 0



Averageclinical score

11.4 7 3.9 1.7 0.9

Averagepain score

5.1 2.8 0.1 0 0

Averageulcer surface

(mm²) 21.21 4.79 0.08 0 0

Averageinfiltrationsurface

(mm²) 26.23 8.40 0 0 0



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)

(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.

(a)

(b)

Figure 4. Case 2. Reduction in vascularization from pretreatment (a)

to 1 month post-treatment (b) Residual vascularization was scored

‘mild’.


6 f amo s e

to proteases.23 Experimentally, an increase in mechanicalrigidity and in resistance to proteolytic enzymes has beenshown for human and swine corneas.23,46–48

Collagen cross-linking reduces the corneal inflammatoryresponse by the induction of apoptosis of the keratocyteslocated in the anterior part of the stroma.49,50 This maycontribute to modification of local immune response med-iated by Langerhans and dendritic cells and may reducecorneal melting and vascularization.39 In the currentstudy, a dramatic reduction in cellular infiltration and incorneal melting was observed. In addition, corneal vascu-larization increased in the first 7 days and regressed overthe next 3 weeks, but was present at completion of thestudy in a reduced state in 8 of 10 eyes.

Limited data have been published on the corneal effectsof CXL in cats. A recent publication32 reports a modifica-tion of the treatment protocol used for human keratoc-onus described by Wollensack12 with the use of acompounded riboflavin solution. Accelerated cross-linking(with the KXLTM) is a recent adaptation of this technique

and uses a higher irradiance UV light source and a shortertime of irradiation.51,52 The energy delivered by both pro-tocols (30 mW/cm² for 3 min for the accelerated protocolor 3 mW/cm² for 30 min in the study by Wollensacket al.12) is the same (5.4 J/cm²) achieving the same biologi-cal effects.51,52 However, in experimental conditions, thebiochemical stiffness of the cornea seems to decrease inhigher irradiances due to rapid oxygen depletion, becauseCXL is an oxygen-dependent process.53 The influence onthe treatment of the melting keratitis is unknown. Com-pared with the Wollensack et al.12 protocol, the use ofaccelerated cross-linking reduces operating time and thusthe duration of anesthesia. In human patients, no addi-tional adverse effects have been observed with irradiances>3 mW/cm².51,52 The results observed in the presentstudy are similar to the previous evaluation of acceleratedcross-linking for corneal melting in dogs.34 The isotonicriboflavin solution used in the present study (VibexTM) iscommercially available in Europe, and its concentration isthe same as the compounded solution used by Spiesset al.32 The absorption spectra of fluorescein and

(a)

(b)

Figure 5. Case 7. Pretreatment presentation (a) and 1 month

after treatment (b) with persistent central superficial corneal

vascularization scored as ‘moderate’.

(a)

(b)

Figure 6. Case 9. Pretreatment presentation (a) and 1 after CXL

treatment (b) with two remaining areas of marked corneal fibrosis.



riboflavin for UV-A are very close. The presence of fluo-rescein in the anterior stroma limits UV-A absorption andcould explain some differences observed in healing rates inhuman patients.37 Therefore, in the present study, fluores-cein staining was used only in the follow-up period.

Case inclusion was based on clinical diagnosis of melt-ing keratitis by the observation of epithelial loss, cornealedema, cellular infiltration, and stromal dissolution. Asstated earlier, microbial infection is usually suspected todrive the inflammatory state responsible for corneal melt-ing by the contamination of a previous epithelial defect.However, it cannot always be demonstrated. Althoughbacterial contamination was suspected in all cases, only 4of 10 cases had a positive culture in our study, which cor-relates with literature data.54 These results are similar tothose presented by Spiess et al.32 in which none of the sixcases (three dogs and three cats) had a positive bacterio-logic culture. This can be either explained by the inhibi-tion of bacteria by the previous medical treatments orrelated to a sterile keratitis and the activation of the prote-ases by other mechanisms.1,2

In our study, all the cases have achieved the same out-come regardless of the presence of bacteria or of the dura-tion of the condition prior to CXL treatment. Similarobservations have been reported in a series of 25 humancases.39 Makdoumi et al. concluded that CXL should beconsidered as the initial treatment for keratitis without theuse of antibiotics, arguing that this could be a way ofreducing the risk of antibiotic resistance.39 However, dueto the limited data evaluating the efficacy of CXL in thetreatment of melting keratitis in cats, topical antibiotictherapy was maintained until complete epithelial healingto prevent a secondary bacterial contamination. Tobramy-cin was used at twice daily for a preventive purposealthough the frequency is unlikely to decrease bacterialgrowth.

Although justified on a scientific basis to compare CXLwith a traditional therapy for melting keratitis, no controlgroup was included in our series. Animals were presentedafter a previous medical treatment (topical antibiotics and,in some cases, antiproteases) with no improvement in orworsening of the clinical signs at the time of referral.Treatment with antiproteases may have yielded similarresults as described in this study. Because no controlgroup was included, a comparison between the two treat-ment effects was not possible. However, all antiproteasestreatments were stopped at the time of CXL; thus, thearrest of corneal melting could be attributed to the CXLprocedure.

In human patients, adverse effects of CXL have beendescribed: postoperative infection, herpes virus exacerba-tion, and corneal endothelial lesions. With CXL treatmentof human keratoconus, cases of postoperative infectionshave been described in the days or weeks following theprocedure.55–57 In all cases, stromal infection was present3–5 days after the procedure and was attributed to the

surgical removal of corneal epithelium. In the presentstudy, no postoperative infection was observed duringthe follow-up period, as described in the human stud-ies.19–21,26 Herpetic keratitis has been described in ahuman patient 5 days after his treatment for keratoconusby CXL.58 In our series, two cats tested positive forFHV-1. However, there was no evidence of active diseasein the immediate postoperative period. Therefore, notreatment against FHV-1 was prescribed. The risk of viralactivation by CXL treatment in feline patients is unknownand should be investigated further because UV light hasbeen used for reactivation of herpes simplex virus in ani-mal models.59–63 However, the UV-B used in some exper-imental procedures61–63 had wave lengths (280–315 nm)significantly different from those used for collagencross-linking (370 nm). Endothelial lesions may bedirectly attributable to the CXL treatment by the cytolyticeffect of riboflavin photo-activation on endothelialcells.64,65 Experimentally, the maximal absorption depthfor UVA in a riboflavin-saturated cornea is thought to beapproximately 300 lm. UVA absorption does not stop at300 lm, but absorption levels drop below the toxicthreshold in the deeper parts of the cornea and eye as awhole. Stiffening effects of CXL seem to be limited to themore superficial 200 lm of the stroma,43,45 and apoptoticeffects of the procedure appear to be rare in the deeperparts of the cornea (beyond 300 lm).45 This concept,often referred to as ‘riboflavin shielding’, correlatesdirectly with the minimal corneal thickness necessary forsafe CXL treatment. In this study, corneal thickness was>300 lm in all cases. No endothelial effects were observedin all treated cases. Before treatment, many eyes in thisstudy presented with a thick cellular infiltration, whichcan interfere with UV penetration and produces a hetero-geneous photo-activation of riboflavin. No difference inthe results was noted between these different cases regard-less of the corneal thickness or the severity of cellularinfiltration. In human patients, infected corneas with athickness less than 300 lm have been successfully treatedby CXL after soaking with hypotonic riboflavin.26 Verythin corneas or corneas with impending perforation werenot included in this study. However, the prospect of treat-ing such corneas is very promising as perforation might beprevented by the stiffening effects of CXL. Extension ofour study to a larger group should allow us to optimizetreatment parameters according to the size and the depthof the corneal loss.

In the study by Spiess et al.,32 a case of corneal seques-trum was observed within 15 days post-CXL. Keratocytesapoptosis has been hypothesized as a cause of sequestrumformation,66 and because CXL induces anterior apoptosis,corneal sequestrum formation could be a potentialadverse effect. In the present study, no sign of seques-trum development was observed. In the study by Spiesset al.,32 it is not clear whether the keratitis or the CXLtreatment or both were factors in the development of the


8 f amo s e

sequestrum. In the present study, variable corneal fibrosiswas observed in all cases. Corneal haze has beendescribed in human patients with keratoconus after CXLtreatment.14–18 In this study, it is not clear whether thecorneal fibrosis can be attributed to the initial keratitis,the treatment procedure, or both. Follow-up for a longerperiod could be useful to evaluate the long-term effectsof the treatment.

All the cats treated in this study completely healedregardless of the presence of bacterial agents, the extensionof the initial corneal lesions, the duration of the diseasebefore treatment, and the previous treatments. The resultsachieved in this small series suggest that CXL could be avaluable therapeutic option for melting keratitis in cats.The CXL procedure requires a precise focusing of the UVbeam and thus requires general anesthesia. Because theduration of the anesthesia is reduced in comparison withthe traditional CXL protocol, accelerated cross-linkingcould present a practical advantage while providing thesame biological effects. However, accelerated CXL is per-formed with a commercially available riboflavin (VibexTM)with a price significantly higher than that of compoundedriboflavin. These disadvantages (anesthesia, price) have tobe taken into account in the treatment decision. CXLcould also be considered as primary treatment for keratitiswith 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. AmericanJournal 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. R�egnier A. Clinical pharmacology and therapeutics part 2:

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: SmallAnimal Practice 2004; 34: 611–622.

8. Hollingsworth SR. Corneal surgical techniques. ClinicalTechniques in Small Animal Practice 2003; 16: 161–167.

9. Bussi�eres M, Krohne SG, Stiles J et al. The use of porcine small

intestinal submucosa for the repair of full-thickness corneal defects

in dogs, cats and horses. Veterinary Ophthalmology 2004; 7: 352–359.10. Goulle F. Use of porcine small intestinal submucosa for corneal

reconstruction in dogs and cats: 106 cases. Journal of SmallAnimal Practice 2012; 53: 34–43.

11. Barachetti L, Giudice C, Mortellaro C. Amniotic membrane

transplantation for the treatment of feline corneal sequestrum: a

pilot study. Veterinary Ophthalmology 2010; 13: 326–330.12. Wollensack G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A-induced

collagen crosslinking for the treatment of keratoconus. AmericanJournal of Ophthalmology 2003; 135: 620–627.

13. Hafezi F, Kanellopoulos J, Wiltfrang J et al. Corneal collagen

cross-linking with riboflavin and ultraviolet A to treat induced

keratectasia after laser in situ keratomileusis ectasia and

keratoconus. Journal of Cataract and Refractive Surgery 2007; 33:

2035–2040.

14. Kymionis GD, Diakonis VF, Kalyvianaki M et al. One-year

follow-up of corneal confocal microscopy after corneal

cross-linking in patients with post laser in situ keratomileusis and

keratoconus.American Journal of Ophthalmology 2009; 147: 774–778.

15. Kymionis GD, Karavitaki AE, Kounis GA et al. Management of

pellucid marginal corneal degeneration with simultaneous

customized photorefractive keratectomy and collagen cross-linking.

Journal of Cataract and Refractive Surgery 2009; 35: 1298–1301.

16. Caporossi A, Baiocchi S, Mazzotta C et al. Parasurgical therapyfor keratoconus by riboflavin-ultraviolet type A rays induced

cross-linking of corneal collagen: preliminary refractive results in

an Italian study. Journal of Cataract and Refractive Surgery 2006;

32: 837–845.17. Raiskup-Wolf F, Hoyer A, Spoerl E et al. Collagen crosslinking

with riboflavin and ultraviolet-A light in keratoconus: long-term

results. Journal of Cataract and Refractive Surgery 2008; 34:

796–801.18. Spoerl E, Mrochen M, Sliney D et al. Safety of UVA-riboflavin

cross-linking of the cornea. Cornea 2007; 26: 385–389.19. Dhawan S, Rao K, Natrajan S. Complications of corneal collagen

cross-linking. Journal of Ophthalmology 2011. doi:10.1155/2011/

899015.

20. Koller T, Mrochen M, Seiler T. Complication and failure rates

after corneal crosslinking. Journal of Cataract and Refractive

Surgery 2009; 35: 1358–1362.21. Spoerl E, Hoyer A, Pillunat LE et al. Corneal cross-linking and

safety issues. The Open Ophthalmology Journal 2011; 5: 14–16.22. Panda A, Krishna SN, Kumar S. Photo-activated riboflavin

therapy of refractory corneal ulcers. Cornea 2012; 31: 1210–1213.23. Spoerl E, Wollensack G, Seiler T. Increased resistance of

cross-linked cornea against enzymatic digestion. Current EyeResearch 2004; 29: 35–40.

24. Makdoumi K, Mortensen J, Crafoord S. Infectious keratitis

treated with corneal crosslinking. Cornea 2010; 19: 1353–1358.25. Moren H, Malmsj€o M, Mortensen J et al. Riboflavin and

ultraviolet A collagen crosslinking for the treatment of keratitis.

Cornea 2010; 29: 102–104.

26. Rosetta P, Vinciguerra R, Romano MR et al. Corneal collagen

cross-linking window absorption. Cornea 2013; 32: 550–554.

27. Al-Sabai N, Koppen C, Tassignon MJ. UVA/riboflavin

crosslinking as treatment for corneal melting. Bulletin de la Soci�et�e

Belge d’Ophtalmologie 2010; 315: 13–17.28. Anwar HM, El-Danasoury AM, Hashem AN. Corneal collagen

crosslinking in the treatment of infectious keratitis. ClinicalOphthalmology 2011; 5: 1277–1280.

29. Iseli HP, Thiel MA, Hafezi F et al. Ultraviolet A/riboflavin

corneal cross-linking for infectious keratitis associated with

corneal melts. Cornea 2008; 27: 590–594.30. Price MO, Tenkman LR, Schrier M et al. Photoactivated

riboflavin treatment of infectious keratitis using collagen

cross-linking technology. Journal of Refractive Surgery 2012; 28:

706–713.



31. Alio JL, Abbouda A, Diaz Valle D et al. Corneal cross linking

and infectious keratitis: a systematic review with a meta-analysis

of reported cases. Journal of Ophthalmic Inflammation and Infection2013; 3: 47. doi:10.1186/1869-5760-3-47.

32. Spiess BM, Pot SA, Florin M et al. Corneal collagen

cross-linking (CXL) for the treatment of melting keratitis in cats

and dogs: a pilot study. Veterinary Ophthalmology 2013. Epub

Ahead of print. doi:10.1111/vop.12027.

33. Hellander-Edman A, Makdoumi K, Mortensen J et al. Corneal

cross-linking in 9 horses with ulcerative keratitis. BioMed Central

Veterinary Research 2013; 9: 128. doi:10.1186/1746-6148-9-128.

34. Famose F. Evaluation of accelerated cross-linking for the

treatment of melting keratitis in 8 dogs. Veterinary Ophthalmology

2013. Epub Ahead of Print. doi:10.1111/vop.12085.

35. Tajima K, Sinjyo A, Ito T et al. Methicillin-resistant

Staphylococcus aureus keratitis in a dog. Veterinary Ophthalmology2013; 16: 240–243.

36. Firth AM, Haldane SL. Development of a scale to evaluate

postoperative pain in dogs. Journal of American Veterinary Medical

Association 1999; 214: 651–659.37. Richoz O. UV-A absorption rate of fluorescein and its impact on

the efficacy of collagen cross-linking (CXL) for infectious corneal

melting. Abstract from the 8th International Congress of Corneal

Cross-Linking. December 7-8, 2012, Geneva, Switzerland.

38. Famose F. Assessment of the use of spectral domain optical

coherence tomography (SD-OCT) for evaluation of the healthy

and pathological cornea in dogs and cats. Veterinary

Ophthalmology 2013; Epub Ahead of print. doi:10.1111/vop.12028

39. Makdoumi K, Mortensen J, Sorkhabi O et al. UVA-Riboflavin

photochemical therapy of bacterial keratitis: a pilot study.

Graefe’s Archives of Clinical and Experimental Ophthalmology 2012;

250: 95–102.40. 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.

Investigative Ophthalmology & Visual Science 2008; 49: 3402–3408.41. Ruane PH, Edrich H, Gampp D et al. Photochemical inactivation

of selected viruses and bacteria in platelet concentrates using

riboflavin and light. Transfusion 2004; 44: 877–885.

42. Messmer EM, Meyer P, Herwig MC et al. Morphological and

immunohistochemical changes after corneal cross-linking. Cornea

2013; 32: 111–117.43. Wollensack G, Spoerl E, Seiler T. Stress-strain measurements of

human and porcine corneas after riboflavin-ultraviolet-A-induced

cross-linking. Journal of Cataract and Refractive Surgery 2003; 29:1780–1785.

44. McCall AS, Kraft S, Edelhauser HF et al. Mechanisms of corneal

tissue cross-linking in response to treatment with topical

riboflavin and long-wavelength ultraviolet radiation (UVA).

Investigative Ophthalmology & Visual Science 2010; 51: 129–138.

45. Kohlhaas M, Spoerl E, Schilde T et al. Biochemical evidence of

the distribution of cross-links in corneas treated with riboflavin

and ultraviolet A light. Journal of Cataract and Refractive Surgery2006; 32: 279–283.

46. Spoerl E, Huhle M, Kasper M et al. Artificial stiffening of the

cornea by induction of intrastromal cross-links. Ophthalmologe

1997; 94: 902–906.47. Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal

tissue. Experimental Eye Research 1998; 66: 97–103.48. Kontadakis GA, Ginis H, Karyotakis N et al. In vitro effect of

corneal collagen cross-linking on corneal hydration properties

and stiffness. Graefe’s Archives of Clinical and ExperimentalOphthalmology 2013; 251: 543–547.

49. Wollensack G, Iomdina E, Didert DD et al. Wound healing in

the rabbit cornea after corneal collagen cross-linking with

riboflavin and UVA. Cornea 2007; 26: 600–605.50. Wang F. UVA/riboflavin-induced apoptosis in mouse cornea.

Ophthalmologica 2008; 222: 2.51. Kanellopoulos AJ. Long term results of a prospective randomized

bilateral eye comparison trial of higher fluence, shorter

duration ultraviolet A radiation, and riboflavin collagen cross

linking for progressive keratoconus. Clinical Ophthalmology 2012;

6: 97–101.52. Celik HU, Alag€oz N, Yildirim Y et al. Accelerated corneal

crosslinking concurrent with laser in situ keratomileusis. Journalof Cataract and Refractive Surgery 2012; 38: 1424–1431.

53. Richoz O. Corneal Biomedical properties at different Corneal

Cross-Linking fluences. Abstract from the 8th International

Congress of Corneal Cross-Linking. December 7-8, 2012,

Geneva, Switzerland.

54. Massa KL, Murphy CJ, Hartmann FA et al. Usefulness of

aerobic microbial culture and cytologic evaluation of corneal

specimens in the diagnosis of infectious ulcerative keratitis in

animals. Journal of the American Veterinary Medical Association

1999; 215: 1671–1674.55. Sharma N, Maharana P, Singh G et al. Pseudomonas keratitis

after collagen crosslinking for keratoconus: case report and

review of literature. Journal of Cataract and Refractive Surgery

2010; 36: 517–520.56. Pollhammer M, Cursiefen C. Bacterial keratitis early after

corneal crosslinking with riboflavin and ultraviolet-A. Journal ofCataract and Refractive Surgery 2009; 35: 588–589.

57. P�erez-Santonja JJ, Artola A, Javaloy J et al. Microbial keratitis

after corneal collagen crosslinking. Journal of Cataract and

Refractive Surgery 2009; 35: 1138–1140.58. Kymionis GD, Portaliou DM, Bouzoukis DI et al. Herpetic

keratitis with iritis after corneal crosslinking with riboflavin and

ultraviolet A for keratoconus. Journal of Cataract and Refractive

Surgery 2007; 33: 1282–1284.59. Stanberry LR, Harrison CJ, Bravo FJ et al. Recurrent genital herpes

in the guinea pig augmented by ultraviolet irradiation: effects of

treatment with acyclovir.Antiviral Research 1990; 13: 227–235.

60. Stanberry LR. Animal model of ultraviolet-radiation-induced

recurrent herpes simplex virus infection. Journal of Medical

Virology 1989; 28: 125–128.

61. Rooney JF, Straus SE, Mannix ML et al. UV light-induced

reactivation of herpes simplex virus type 2 and prevention by

acyclovir. Journal of Infectious Diseases 1992; 166: 500–506.62. Goade DE, Nofchissey RA, Kusewitt DF et al. Ultraviolet light

induces reactivation in a murine model of cutaneous herpes simplex

virus-1 infection. Photochemistry and Photobiology 2001; 74: 108–114.

63. Morris J, Stuart PM, Rogge M et al. Recurrent herpetic stromal

keratitis in mice, a model for studying human HSK. Journal of

Visualized experiments: JoVE 2012; 70: e4276.64. Corneal GN. Corneal endothelial damage after collagen

cross-linking treatment. Cornea 2011; 30: 1495–1498.65. Bagga B, Pahuja S, Murthy S et al. Endothelial failure after

collagen cross-linking with riboflavin an UV-A: case report with

literature review. Cornea 2012; 31: 1197–1200.

66. Cullen CL, Wadowska DW, Singh A et al. Ultrastructural

findings in feline corneal sequestra. Veterinary Ophthalmology

2005; 8: 295–303.


10 f amo s e

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

Health & Medicine