evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in 8 dogs

Evaluation of accelerated collagen cross-linking for the treatmentof melting keratitis in eight dogs

Frank FamoseService d’Ophtalmologie, Clinique V�et�erinaire 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]

AbstractObjective Melting keratitis is 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 dogs.

Animal studied and Procedure Eight dogs were treated for unilateral melting keratitis byaccelerated CXL. Corneas were irradiated by UVA (370 nm) at 30 mW/cm² irradi-ance for 3 min after soaking by 0.1% riboflavin in 20% dextran for 30 min. Follow-up was conducted 3, 7, 14, and 30 days after treatment.

Results Pain improvement was observed for all cases within 3 days after treatment.Epithelial healing was observed within 15 days for all cases.Disappearance of cellular infiltration was observed for all cases at day 7. The corneal

vascularization disappeared for 4 of 8 dogs and was reduced for 4 of 8 dogs within1 month. At 1 month, all cases presented a variable degree of corneal scarring, but all

eyes had visual function. No recurrent infection was observed.Conclusions The main observation of this study is that all the cases have presented with

the same clinical result regardless of the presence and the sensitivity of the infectiousagents and regardless of the duration of the condition prior to CXL treatment. Accel-

erated CXL appears to be a valuable option for the treatment of melting keratitis.

Key Words: accelerated CXL, cross-linking, dogs, melting keratitis, optical coherencetomography, riboflavin

INTRODUCTION

In dogs, melting keratitis is serious condition presentinga high risk of permanent blindness.1–3 In melting kerati-tis, stromal damage is initiated by various mechanismsincluding bacterial proliferation, toxin secretion, micro-bial or corneal proteases activation leading to keratomala-cia or corneal melting. An imbalance between theendogenous and exogenous matrix metalloproteinases(MMP) and the proteinases present in the cornea and theprecorneal tear film leads to the destruction of the colla-gen of the cornea.4–7 Microbial infection is usually sus-pected to drive the inflammatory state responsible forcorneal melting but cannot always be demonstrated.1

There are few corneal pathogens that are associated withprimary corneal infections,8,9 and infectious corneal melt-ing is typically due to secondary bacterial or fungal infec-tions.1–3 Risk factors for the development of secondaryinfectious keratitis include ocular trauma and corneal

exposition because of lagophthalmos, particularly inbrachycephalic breeds, with reduced corneal sensitivity,and ocular surface conditions such as keratoconjunctivitissicca or other quantitative or qualitative tear film defi-ciencies. Melting can also occur in the absence of infec-tion, and the imbalance between proteolytic enzymes andprotease inhibitors is thought to be due to the produc-tion of collagenolytic agents by resident and inflamma-tory cells.4–6

Medical treatment is based on the administration oftopical antibiotics either commercially available prepara-tions or reinforced compounded preparations10 and on thetopical use of protease inhibitors.7,11 Failure of the treat-ment can lead to loss of vision because of the evolution ofkeratomalacia and corneal perforation. These failures areusually managed by tectonic surgery. Traditional surgicalprocedures are conjunctival grafts, biomaterial grafts, oramniotic membrane transplantation.12–15 These techniqueslead to variable degrees of corneal opacity.

© 2013 American College of Veterinary Ophthalmologists

Veterinary Ophthalmology (2013) 1–10 DOI:10.1111/vop.12085

Collagen cross-linking (CXL) is a technique which cre-ates intrafibrillar covalent bonds in the collagen fibers ofthe corneal stroma via the photo-activation of riboflavinby ultraviolet-A (UVA) light. It has been used since 1998in human ophthalmology for the treatment of ectaticconditions of the cornea, such as progressive keratoconus,pellucid marginal degeneration, and ectatic complicationsof refractive surgeries.16–19 For these indications, safetyand efficacy of this procedure have been widely estab-lished.16–25 The antimicrobial activity of CXL againstnumerous bacteria and fungi26 has been demonstratedunder experimental conditions. Efficacy against fungalspecies is very variable: no effect on Candida and Fusari-um cultures;26,27 minimal/moderate effect on Fusarium invivo in rabbits;28 and an amphotericin B pretreatment–dependent effect on Candida albicans, Fusarium spp, andAspergillus fumigatus.29 An increased collagen resistanceagainst enzymatic digestion30 has been demonstratedunder experimental conditions in vitro. More recently,the clinical efficacy of CXL has been described in thetreatment of presumed infectious keratitis and cornealmelting.31–38

Because of the promising results obtained in humanophthalmology and the recently described application ofCXL in dogs and cats in a pilot study,39 the aim of thisstudy was to evaluate the efficacy of accelerated CXL forthe treatment of melting keratitis in dogs.

MATERIALS AND METHODS

Inclusion criteriaFor this prospective uncontrolled study, case recruitmentwas based on a clinical diagnosis of a present or impend-ing melting keratitis characterized by epithelial and ante-rior stromal loss, cellular infiltration, cornealvascularization, and loss of corneal integrity. The caseswere recruited after referral by the initial veterinarian afterworsening of ocular signs despite the initial medical treat-ment. Cases with impending or confirmed corneal perfo-ration were excluded from this study.

All animals were included after obtaining their owner’sconsent. All procedures were performed in accordancewith the French guidelines for animal care.

Ophthalmologic examinationKeratitis evaluation was made under the following criteria:

The specific signs of keratitis were evaluated by slit-lamp examination (HawkeyeTM, Dioptrix, Toulouse,France). A clinical score modified from Tajima et al.40

(0–3; 0 = absent, 1 = mild, 2 = moderate, 3 = severe) wasused to grade the severity of eye symptoms and signs,which included mucopurulent discharge, corneal edema,corneal vascularization, conjunctivitis, blepharitis, and uve-itis. The highest possible total score was 18. Cellular infil-tration was noted as ‘present’ or ‘absent’ and was acomponent of the diagnosis of the corneal melting, includ-

ing corneal edema and stromal dissolution.1,39 A painscore modified from the Hackett and McDonald scoring41

(0–1; 0 = absent, 1 = present) was used to grade pain signsthat included prostration, aggressive behavior, blepharo-spasm, enophthalmos, photophobia, ocular pruritus, anddefense reaction to examination. The highest possible totalscore was 7.

Tear production was evaluated by Schirmer test I withthe absorbent strip of paper (Test de Schirmer, Virbac,Carros, France) placed in the temporal third of the infe-rior conjunctival fornix and reading after 1 min (normalvalues 10–15 mm). A sample for bacteriological diagnosis(culture and antibiotic sensitivity) was performed on eachanimal by corneal scraping with a Kimura spatula (MoriaSurgical, Antony, France) and submitted for bacteriologicanalysis at a local laboratory dedicated to veterinary bacte-riology (Laboratoire Meynaud, Toulouse, France).

Measurements of the corneal thickness and the ulcerationsizeDogs were anesthetized with 300 lg/m² medetomidine(DomitorTM, Pfizer, New York, USA) and 5 mg/kg keta-mine (Imalgene 1000TM, Merial, Lyon, France) deliveredintravenously followed by 0.5–2% isoflurane (IsofluraneBelamontTM, Nicholas Piramal Ltd., London, UK) inoxygen after endotracheal intubation.

Dogs were maintained in dorsal recumbency with a vac-uum cushion. Pachymetry was performed with an OpticalCoherence Tomography (OCT) device (iVueTM, Optovue,Fremont, CA, USA) as described elsewhere42. Measure-ments were performed with the focus at the center of thecorneal lesion. Minimal and maximal corneal thicknesseswere evaluated with the caliper tool on the OCT picturesobtained (Fig. 1).

Ulceration size was measured with a manual caliper. Asthe corneal ulcers have sometimes an elliptic or irregular

Figure 1. OCT pachymetry. Corneal thickness is measured at its

maximum and its minimum. Case no 3.

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

2 f amo s e

shape, the greater axis length was noted as the ulcer size(in mm). Because it can interfere with UVA absorption,43

fluorescein staining was not used for initial measurementsof epithelial defects.

Treatment by accelerated cross-linkingThe eyelids were kept open with the use of a lid specu-lum. After the instillation of a topical anesthetic (oxybu-procaine 0.4%, Cebesine 0.4%TM, Bausch & Lomb-Chauvin, Montpellier, France), the ulcer margins werecleaned and the debris removed from the corneal surfacewith a microsurgery sponge. A solution of isotonic ribofla-vin (riboflavin 0,1%, dextran 20%, VibexTM, Avedro, Wal-tham MA, USA) was instilled on the cornea for 30 min(one drop every 2 minutes), and the corneal surface wasrinsed with balanced salt solution (BSS pour irrigation oc-ulaireTM, Alcon, Fort Worth, TX, USA) at the end ofriboflavin instillation. Penetration of riboflavin throughthe cornea was confirmed by visualizing the fluorescenceof the riboflavin in the anterior chamber with slit-lamp bi-omicroscopy using the cobalt blue light.

Corneas were irradiated with UVA (wave length =370 nm) for 3 min with an irradiance of 30 mW/cm²(5.4 J/cm² per dose) using the KXLTM System (Avedro,Waltham MA, USA). The beam of 9 mm in diameterwas centered and focused on the center of the corneallesion. Care was taken not to irradiate the corneal lim-bus. One drop of riboflavin was instilled after 2 min ofirradiation. All animals have received a single CXL treat-ment.

Postoperative treatmentWith patient’s safety in mind, each eye was treated twicedaily with one drop of a tobramycin solution (Tobrex0.3%TM, Alcon, Rueil-Malmaison, France) after the CXLtreatment until complete epithelial healing. All anticolla-genase treatments have been stopped.

Follow-upThe aim of the study was to evaluate corneal healing andsymptom reduction over a 30-day follow-up period. Eyeswere examined 3, 7, 14, and 30 days after treatment usingthe same pretreatment protocol minus the Schirmer test.Epithelial integrity was evaluated after instillation of adrop of fluorescein solution (fluorescein 0.5% collyre uni-dose TVM, Laboratoires TVM, Lempdes, France) andrinsing (Ocryl nettoyant oculaire, Laboratoires TVM,Lempdes, France) before cobalt-blue-light illumination(Hawkeye, Dioptrix, France). Fluorescein dye staining ofthe cornea was interpreted as a positive result. In cases ofepithelial healing, topical treatment was stopped. In casesof epithelial defect, topical antibiotic treatment (Tobrex0.3%TM two drops/day) was maintained until the nextexamination.

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

RESULTS

Preoperative featuresEight dogs were treated between April and December2012 at a single site (Clinique v�et�erinaire des Acacias,Toulouse-Blagnac, France) by the same clinician (FrankFamose). Table 1 presents individual preoperative fea-tures. Five of eight dogs were from brachycephalic breeds,two were Cavalier King Charles Spaniels with macro-palpebral fissures, and the last one was a Great Dane.

Keratitis duration before CXL treatment ranged from7 days to 1 month. None of the cases had a bacteriologicanalysis before initiation of the first treatment, and 5 of 8received a surgical treatment (one epithelial debridementand four nictitant membrane flaps).

Among eight samples submitted for bacteriologic analy-sis, three were positive. Pseudomonas aeruginosa was isolatedfrom case no. 2 and case no. °4 and was resistant to am-inosides (gentamicin and tobramycin) and sensitive to qui-nolones (marbofloxacin and enrofloxacin). In case no. 7, aPseudomonas aeruginosa was isolated and was sensitive toaminosides (gentamicin, tobramycin, kanamycin, framyce-tin) and to quinolones (marbofloxacin, enrofloxacin).

Maximal corneal thickness ranged from 543 to1370 lm, and minimal corneal thickness ranged from 292to 810 lm. The relative depth of the ulceration rangedfrom 29 to 56% of the corneal thickness. The size ofepithelial defect ranged from 2 to 8 mm in maximaldiameter.

Postoperative featuresIndividual scores are summarized on the Table 2.

Epithelial healing was observed at the 7th day post-treatment for 6 of 8 cases and at 15 days for the two lastcases (Fig. 2).

Mean clinical and pain score showed a marked decreasefrom the 3rd to the 7th day after treatment (Fig. 2). Cel-lular infiltration of the cornea was present for all cases atthe time of treatment and had disappeared 7 days afterCXL treatment (Fig. 3).

Corneal vascularization was present in all cases fromday 8, even in the cases in which it was not observed atD1. At 30 days post-treatment, it had disappeared in 4of 8 cases and was mild in 4 of 8 cases (Fig. 4). At1 month, all cases presented a variable degree of cornealfibrosis ranging from mild (Fig. 5) to marked (Figs 6 and7) and melanosis (Figs 5 and 7), but all eyes had visualfunction.

No recurrent infection was observed during the study.

DISCUSSION

We have used accelerated collagen cross-linking for thetreatment of melting keratitis in eight cases. All treatedanimals showed reduced pain 3 days after treatment. Thisobservation is similar to results in humans in which


t r e a tm ent o f m e l t i ng k e r a t i t i s 3

ocular pain improvement was achieved over the sametime duration.31–33

Complete epithelial healing was achieved in 7 days for6 of 8 dogs and within 14 days after treatment for 2 of 8.In the description of Spiess et al., epithelial healing wasachieved in 13 days for one dog and 30 and 40 days forthe two others. The difference in healing rates betweenthese two studies could be explained by the difference inthe epithelial removal before riboflavin application. Epi-thelium was removed on a surface of 9–11 mm in diame-ter in Spiess et al. study and was limited to the ulcermargins in the present study as described by Rosettaet al.34

Resolution of corneal melting was observed in all trea-ted eyes within 7 days after treatment. This effect wasobserved by biomicroscopic examination as a reduction incorneal thickness and cellular infiltration with recovery ofcorneal transparency. These results are similar to those inthe literature31–38,44 and could be attributable to directeffects of CXL. Clinical observations and experimentaldata have shown that CXL may have three distinct effectson cornea:

(1) A bactericidal effect because of free radical libera-tion by photo-activation of riboflavin. This effect ismanifested by bacterial DNA and membrane altera-tions.45 Although this effect has been well estab-lished in experimental conditions, immediatereduction in the bacterial load has not been demon-strated in the different clinical reports.31–39

(2) An increase in the corneal resistance because ofmechanical and biochemical modifications of cor-neal structure46,47 and the creation of intralamellarcovalent collagen bonds.48 This mechanism isthought to be limited to the first anterior 200 lmof the treated cornea49 and to contribute toincreased resistance to proteases.30 Experimentally,an increase in mechanical rigidity and in resistanceto proteolytic enzymes has been shown for humanand swine corneas.30,50–52 Similar data are not avail-able for dogs, but as long as architecture and colla-gen structure of canine and human corneas are verysimilar, it can be expected that the corneal effects ofCXL should be similar.

(3) A reduction in the corneal inflammatory response byinduction of apoptosis of the keratocytes located inthe anterior part of the stroma.53,54 This may con-tribute to modification of the local immune responsemediated by Langerhans and dendritic cells and mayreduce corneal melting and vascularization as suggestedby Makdoumi et al.44 In our cases, we observed a dra-matic reduction in cellular infiltration and in cornealmelting. Corneal vascularization increased in the first7 days and regressed over the next 3 weeks butpersisted in a reduced state in 4 of 8 eyes.

As far as we know, limited data have been published aboutthe corneal effects of CXL in dogs under clinical conditions.The first publication is very recent39 and presents an adap-

Table 1. Individual preoperative features

Case number Case no. 1 Case no. 2 Case no. 3 Case no. 4 Case no. 5 Case no. 6 Case no. 7 Case no. 8Breed Cavalier

King CharlesCarlin Shih Tzu Shih Tzu Pekingese Cavalier King

CharlesFrenchBulldog

Great Dane

Age 1 year 2 year 8 year 3 year 10 year 6 year 8 year 1 yearAffected eye OS OS OD OS OD OS OD ODDuration prior toCXL (from firstsymptoms)

3 weeks 1 week 1 month 2 weeks 2 weeks 2 weeks 2 weeks 1 week

Initial bacteriology NP* NP NP NP NP NP NP NPPrevious medicaltreatment (local)

FramycetinCiprofloxacinNAC†

Gentamicin14 mg/mLNAC

FramycetinNa-Hyaluronate

CiprofloxacinAtropine

Gentamicin14 mg/mLNAC

FramycetinTobramycinNAC

TobramycinNAC

Ciprofloxacin

Previous surgicaltreatment

Epithelialscraping

None 3rd eyelidflap

3rd eyelid flap None 3rd eyelid flap 3rd eyelidflap

None

Schirmer test(mm/min)

12 14 12 8 17 12 12 13

Presurgicalbacteriology

Sterile Pseudomonasaeruginosa

Sterile Pseudomonasaeruginosa

Sterile Sterile Pseudomonasaeruginosa

Sterile

Min. CT‡ (lm) 557 580 475 570 292 606 340 810Max. CT (lm) 1002 1300 703 800 660 980 543 1370Ulcer depth (%cornealThickness)

44% 55% 32% 29% 56% 38% 37% 41%

Ulcer size(mm)

3 8 8 3 2 4 8 2

*NP: Not performed.†NAC: N-acetylcysteine.‡CT: Corneal thickness.


4 f amo s e

tation of the treatment protocol used for human keratoc-onus described by Wollensack16 with the use of a com-pounded riboflavin solution. Accelerated cross-linking (withthe KXLTM) is a recent adaptation of this traditional tech-nique based on the use of a higher irradiance UV lightsource. The energy delivered by both protocols (30 mW/cm² for 3 min for the accelerated protocol or 3 mW/cm²for 30 min for the traditional one) is the same (5.4 J/cm²)achieving the same biological effects.55,56 However, inexperimental conditions, the biochemical stiffness of thecornea seems to decrease in higher influences due to rapidoxygen depletion, because CXL is an oxygen-dependantprocess.57 The influence on the treatment of melting kerati-tis is unknown. The use of accelerated cross-linking reducesoperating time and thus the duration of anesthesia. Inhuman patients, no additional adverse effects have beenobserved with irradiances >3 mW/cm².55,56 The isotonicriboflavin solution used in the present study (VibexTM) iscommercially available in Europe. Its concentration is thesame as the compounded solution used by Spiess et al. andis ready to use. The absorption spectra of fluorescein andriboflavin for UV-A are very close. The presence of fluores-cein in the anterior stroma limits UV-A absorption andcould explain some differences observed in healing rates inhuman patients43 by reducing the photochemical reactionof UV-A and riboflavin. In the present study, to avoid therisk of interference, fluorescein staining was used only inthe 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.3

However, a microbial infection cannot always be identi-fied. Although bacterial contamination was suspected in allcases, only 3 of 8 cases had a positive culture in our study,

Table 2. Summary of individual scores

Casenumber Score D1 D4 D8 D15 D31

Caseno. 1

Clinical score 10 6 4 1 0Pain score 5 2 0 0 0Epithelial defect + + � � �Cellular infiltration + + � � �

Caseno. 2


Caseno. 3

Clinical score 10 7 3 1 0Pain score 4 3 0 0 0Epithelial defect + + + � �Cellular infiltration + � � � �

Caseno. 4

Clinical score 7 5 2 1 0Pain score 4 3 1 0 0Epithelial defect + + � � �Cellular infiltration + � � � �

Caseno. 5


Caseno. 6


Caseno. 7

Clinical score 11 8 3 1 1Pain score 6 5 1 0 0Epithelial defect + + + � �Cellular infiltration + � � � �

Caseno. 8


Mean clinical score 10.375 6.5 2.625 1.125 0.5Mean pain score 5.125 3.25 0.375 0 0With a cellular infiltration(nb/8)

8 5 0 0 0

With an epithelial defect(nb/8)

8 8 2 0 0

Figure 2. Evolution of the mean clinical score

(blue), of the mean pain score (red), and of the

number of dogs with epithelial defect (green)

during the 30-day follow-up period.



which correlates with literature data.58 This fact can beexplained by the inhibition of bacteria by the medicaltreatments prescribed before the presentation and also bythe hypothesis of a sterile keratitis due to the activation ofthe proteases caused by mechanisms other than bacterialproliferation.1,3,4 This observation joins the results pre-sented by Spiess et al.39 in which none of the six cases(three dogs and three cats) had a positive bacteriologicculture. In the present study, the failure of the antibiotictreatments performed before referral could be explainedby the presence of resistant bacteria, but bacteriologicanalysis isolated them only in one case. Other causes, suchas the advanced stage of the disease when presented to theveterinarian, poor patient, and/or owner compliance couldinfluence the course of the disease.

The use of tobramycin in the postoperative phase maybe a bias for case no. 7 for which the germ identified wassensitive. In this case, the postoperative evolution wasexactly the same as the other cases, and the use of tobra-mycin did not show any added value in comparison.

The main observation of this study is that all the caseshave presented with the same outcome regardless of thepresence and the sensitivity of the isolated bacteria andregardless of the duration of the condition prior to CXLtreatment. Similar observations have been reported byMakdoumi in a series of 25 human cases.44 His conclusionis that CXL should be considered as the initial treatmentfor keratitis without the use of antibiotics, arguing that

this could be a way of reducing the risk of antibiotic resis-tance. In the present study, topical antibiotic therapy wasmaintained until complete epithelial healing, for the pre-vention of a secondary bacterial contamination, andaccording to the lack of data about efficacy of CXL treat-ment in dogs. The next step of the definitive evaluation ofthe CXL treatment would be the follow-up without anypostoperative treatment.

Although justified on a scientific basis to compare CXLwith a traditional treatment, no control group wasincluded in our series. Animals were presented after a pre-vious medical treatment (topical antibiotics and, in somecases, antiproteases) with no improvement in or worseningof the clinical signs at the time of referral. Treatment withantiproteases could have yielded similar results asdescribed in this study, and such therapy would have beena possible alternative. Because no control group wasincluded, a comparison of treatment effects was not possi-ble. However, all antiprotease treatments were stopped atthe time of CXL, and the arrest of corneal melting istherefore probably attributable to the CXL procedure.

In human patients, three kinds of adverse effects ofCXL have been described: postoperative infection, herpes-virus exacerbation, and corneal endothelial lesions. In thetreatment of human keratoconus by CXL, three cases ofpostoperative infections have been described in the daysor weeks following the procedure.59–61 In all cases, stromalinfection was present 3 to 5 days after the procedure andwas attributed to the surgical removal of corneal epithe-

(a)

(b)

Figure 4. Case no. 4 Evolution of vascularization from pretreatment

presentation (a) to 1 month post-treatment presentation (b). Residual

vascularization is scored ‘mild’.

(a)

(b)

Figure 3. Case no. 1 Initial presentation (a) and 7 days after the

treatment (b). Cellular infiltration and corneal edema are reduced,

and transparency of the corneal peripheral to the lesion has greatly

increased. Epithelial healing was complete.


6 f amo s e

lium. These observations are not transposable to thisstudy in which epithelial loss was an inclusion criterium,and previous bacterial infection was suspected. In the pres-ent study, no epithelial debridement was performed, butonly a careful cleaning of cellular debris before the instil-lation of riboflavin, as described by Rosetta et al.34 Nopostoperative infection was observed during the follow-upperiod, as described in the human studies.23–25,34 A case ofherpetic keratitis has been described in a human patient5 days after his treatment for keratoconus by CXL.62 Viralreactivation was described as a superficial keratitis andmaterialized by dendritic ulceration and as an anterioruveitis. In dogs, canine herpesvirus has a worldwide distri-bution and a high-exposure prevalence,63 and ocularsymptoms in adult dogs have been occasionallydescribed.64,65 The risk of viral activation by CXL treat-ment is unknown and should be investigated further.Endothelial lesions may be directly attributable to theCXL treatment by the cytolytic effect of riboflavin photo-activation on endothelial cells.66,67 Experimentally, themaximal absorption depth for UVA in a riboflavin-satu-rated cornea is thought to be approximately 300 lm forthis protocol. UVA absorption does not stop at 300 lm,but absorption levels drop below the toxic threshold inthe deeper parts of the cornea and eye as a whole. Stiffen-ing effects of CXL seem to be limited to the more superfi-cial 200 lm of the stroma,47,49 and apoptotic effects of theprocedure appear to be rare in the deeper parts of the cor-nea (deeper than 300 lm).49 This concept, often referred

to as ‘riboflavin shielding’, correlates directly with theminimal corneal thickness necessary for safe CXL treat-ment. In this study, corneal thickness was significantly>300 lm in 7 of 8 cases and was 292 lm in case no. 5.With the owner’s consent, we took the risk to treat such athin cornea. No endothelial effects were observed in anytreated cases. Before the treatment, many eyes presented athick cellular infiltration, which can interfere with UVpenetration and produce a heterogeneous photo-activationof riboflavin. No difference in the results was observedbetween these different cases regardless of the initial cor-neal thickness or infiltration. In cases of melting keratitis,corneal loss is sometimes much more pronounced thanthe cases we have treated, and the residual stroma can beless than 300 lm. In human patients, infected corneaswith a thickness <300 lm have been successfully treatedby CXL after soaking with hypotonic riboflavin34. Verythin corneas or corneas with impending perforation havenot been included in this study. An extension of our studyto a larger group should allow us to optimize treatmentparameters according to the size and the depth of the cor-neal loss.

In the study of Spiess et al.39 two brachycephalic dogswere treated, and one of these demonstrated the develop-ment of corneal melanosis during the second month follow-ing the CXL treatment. In the present study, at 30 daysafter the treatment, corneal melanosis was observed in 3 of5 brachycephalic dogs, but not in the two Cavalier KingCharles Spaniels that presented with macropalpebral fis-

(a)

(b)

Figure 5. Case no. 2. Pretreatment presentation (a) and 1 month

after treatment (b) with persistence of central superficial corneal

vascularization scored as ‘mild’ and central corneal melanosis.

(a)

(b)


after CXL, with central corneal fibrosis and ‘mild’ vascularization.

No corneal melanosis is visible (b).



sures nor in the Great Dane with euryblepharon. As statedby Spiess et al., it is not clear whether the keratitis or theCXL treatment or both were significant factors in the devel-opment of melanosis in these three patients. In this series, avariable degree of corneal fibrosis has been observed in allcases. Corneal haze has been described in human patientsafter CXL treatment for keratoconus.18–22 In this study, it isnot clear whether the corneal fibrosis can be attributable tothe initial keratitis, the treatment procedure, or both. Fol-low-up for a longer period could be useful to evaluate thelong-term effects of the treatment.

All the dogs treated in this study have presented a com-plete healing or at least limited sequelae of their keratitis,regardless of the presence of bacterial agents, the exten-sion of the initial corneal lesions, the duration of the dis-ease before treatment, and the previous treatments. Theexcellent results achieved in this small series with noobservable adverse effects in the short-term suggest thatCXL could be a valuable therapeutic option for meltingkeratitis. Previously published observations were con-firmed. The CXL procedure needs a precise focalizationof the UV beam and thus needs general anesthesia.Because the duration of the anesthesia is reduced in com-parison with the traditional CXL protocol, acceleratedcross-linking could present a practical advantage whileproviding the same biological effects. However, acceler-ated CXL is performed with a commercially availableriboflavin (VibexTM) whose price is significantly higher

than compounded riboflavin. These disadvantages (anes-thesia, price) have to be taken into account in the treat-ment decision. Based on the results of this study, CXLcould also be considered as primary treatment for keratitiswith or without the use of antibiotics. Further studies inthis direction will likely contribute to a better knowledgeof melting keratitis treatment and may also be useful incomparative ophthalmology.

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evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in 8 dogs

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