new methods to evaluate the effect of conventional and modified

New Methods to Evaluate the Effect of Conventional and Modified Crosslinking Treatment for Keratoconus Jeannette Beckman Rehnman

Department of Clinical Sciences, Ophthalmology

Department of Radiation Sciences, Radiation Physics

and Biomedical Engineering

Umeå University

Umeå 2015

Responsible publisher under Swedish law: the Dean of the Medical Faculty

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

New Series No. 1746

ISBN: 978-91-7601-336-6

ISSN: 0346-6612

Electronic version available at http://umu.diva-portal.org/

Printed by: Print & Media

Umeå, Sweden, 2015

i

“Discovery consists of

seeing

what everybody has

seen

and

thinking

what nobody has

thought”

Albert Szent-Györgyi

Dedicated to my children – FILIP and EMMA

Tillägnad mina barn – FILIP och EMMA

ii

Table of contents

Abstract iv

Abbreviations and symbols v

Original papers vii

Sammanfattning på svenska viii

1. Introduction 1

1.1. The Cornea 1

1.2. Keratoconus 2

1.2.1. Epidemiology 2

1.2.2. Signs and symptoms 2

1.2.3. Diagnosis 2

1.2.4. Treatment options 3

1.3. Crosslinking 4

1.3.1. Background 4

1.3.2. Corneal crosslinking - three different mechanisms 4

1.3.3. The “Dresden protocol” 5

1.3.4. Biomechanical/physiochemical changes induced by CXL 5

1.4. Densitometric evaluation of corneal reflectivity 6

1.5. Corneal properties affecting IOP measurements 6

1.6. Biomechanical properties 7

1.6.1. Biomechanical properties of the normal cornea 7

1.6.2. Biomechanical properties in keratoconus 8

1.6.3. Determination of corneal biomechanical properties 8

1.7. Summary of the introduction 8

2. Aims 9

3. Materials and methods 10

3.1. Statistical power 10

3.2. Participants 10

3.2.1. Patients 11

3.2.2. Control subjects 11

3.3. Inclusion/exclusion criteria 11

3.3.1. Diagnosis and documentations of progressive keratoconus 12

3.4. Ophthalmologic examinations 12

3.5. Treatments 13

3.5.1. Conventional corneal crosslinking - CXL 13

3.5.2. Corneal reshaping and crosslinking - CRXL 13

3.6. Technical equipment 14

3.6.1. Pentacam® HR (Papers I+IV) 14

3.6.2. Goldmann Applanation Tonometry (Paper III) 15

3.6.3. Ocular Response Analyzer (Paper III) 15

3.6.4. Applanation Resonance Tonometry -technology (Paper III) 17

iii

3.7. Ethics 20

3.8. Statistical methods 20

4. Results 21

4.1. Overview over demographic data and parameters 21

4.2. Baseline Amsler-Krumeich stage and “Total Deviation” value 23

4.3. Occurrence of a demarcation line 23

4.4. Refractive results from the CRXL-study (Paper II) 23

4.5. Corneal light scattering after CXL (Paper I) 26

4.6. Corneal light scattering after CXL and CRXL (Paper IV) 29

4.6.1. Compared to baseline 30

4.6.2. Comparison between groups 32

4.6.3. Correlations 32

4.7. IOP and biomechanical outcomes after CXL (Paper III) 33

5. Discussion 36

5.1. The CRXL-study (Paper II) 36

5.2. Corneal light scattering after CXL and CRXL (Papers I+IV) 38

5.3. Corneal hysteresis after CXL (Paper III) 40

6. Conclusions 42

7. Future perspectives 43

8. Acknowledgements 44

9. References 46

iv

Abstract

Background: Today corneal crosslinking with ultraviolet-A photoactivation of riboflavin is an

established method to halt the progression of keratoconus. In some cases, when the refractive

errors are large and the visual acuity is low, conventional corneal crosslinking may not be

sufficient. In these cases it would be desirable with a treatment that both halts the progression

and also reduces the refractive errors and improves the quality of vision.

Aims: The aims of this thesis were to determine whether mechanical compression of the

cornea during corneal crosslinking for keratoconus using a sutured rigid contact lens could

improve the optical and visual outcomes of the treatment, and also to find methods to evaluate

the effect of different corneal crosslinking treatment regimens.

Methods: In a prospective, open, randomized case-control study, 60 eyes of 43 patients with

progressive keratoconus, aged 18-28 years, planned for routine corneal crosslinking, and a

corresponding age- and sex-matched control group was included. The patients were randomized

to conventional corneal crosslinking (CXL; n=30) or corneal crosslinking with mechanical

compression of the cornea during the treatment (CRXL; n=30).

Biomicroscopy, autorefractometry, best spectacle corrected visual acuity, axial length

measurement, Pentacam® HR Scheimpflug photography, pachymetry, intraocular pressure

measurements and corneal biomechanical assessments were performed before treatment

(baseline) and at 1 month and 6 months after the treatment.

One of the articles evaluated and compared the optical and visual outcomes between CXL and

CRXL, while the other three articles focused on methods to evaluate treatment effects. In Paper

I, the corneal light scattering was manually quantified from Scheimpflug images throughout the

corneal thickness at 8 measurements points, 0.0 to 3.0 mm from the corneal centre, in patients

treated with CXL. In Paper IV the corneal densitometry (light scattering) was measured with the

Pentacam® HR software, in 4 circular zones around the corneal apex and at 3 different depths of

the corneal stroma, in both CXL and CRXL treated corneas. Paper III quantified the

biomechanical effects of CXL in vivo.

Results: Corneal light scattering after CXL showed distinctive spatial and temporal profiles

and Applanation Resonance Tonometry (ART) -technology demonstrated an increased corneal

hysteresis 1 and 6 months after CXL. When comparing the refractive and structural results after

CXL and CRXL, CRXL failed to flatten the cornea, and the treatment did not show any benefits

to conventional CXL treatment, some variables even indicated an inferior effect. Accordingly,

the increase in corneal densitometry was also less pronounced after CRXL.

Conclusions: Analysis of corneal light scattering/densitometry shows tissue changes at the

expected treatment location, and may be a relevant variable in evaluating the crosslinking effect.

ART -technology is an in vivo method with the potential to assess the increased corneal

hysteresis after CXL treatment. By refining the method, ART may become a useful tool in the

future. Unfortunately, CRXL does not improve the optical and visual outcomes after corneal

crosslinking. Possibly, stronger crosslinking would be necessary to stabilize the cornea in a

flattened position.

v

Abbreviations and symbols

AL = Axial lengths

ART = Applanation Resonance Tonometer/ry

AU = Arbitrary units

BSCVA = Best spectacle corrected visual acuity

CCT = Central corneal thickness

CDVA = Corrected distance visual acuity

CH = Corneal hysteresis (measures corneal viscoelasticity)

CHART = CH measured with ART -technology

CHORA = CH measured with ORA®

CRF = Corneal resistance factor (measures corneal rigidity)

CRFORA = CRF measured with ORA®

CRXL = Corneal reshaping and crosslinking (advanced, modified

CXL)

CRXLctrl = Control group to patients treated with CRXL

CTmin = Corneal thickness at the thinnest point

CXL = Corneal crosslinking (conventional, routine)

CXLctrl = Control group to patients treated with CXL

D = Dioptre/s

DK value = Oxygen permeability of a contact lens

GAT = Goldmann Applanation Tonometer/ry

GSU = Gray scale units

IOP = Intraocular pressure

IOPART = IOP measured with ART -technology

IOPcc = Corneal compensated IOP

IOPg = Goldmann correlated IOP

IOPGAT = IOP measured with GAT

IOPORA = IOP measured with ORA®

K1 = Keratometry value for flat meridian

K2 = Keratometry value for steep meridian

KC = Keratoconus

Kmax = Maximum keratometry reading/value; maximum corneal

curvature

Kmean = Mean keratometry value, calculated as (K1+K2)/2

logMAR = Logarithm of the minimum angel of resolution (visual

acuity scale)

OCT = Optical coherence tomography

ORA® = Ocular Response Analyzer

PZT = Piezoelectric element

Rmin = Minimum corneal curvature; minimum corneal radius of

curvature

vi

SOD = Superoxide dismutase

SphEq = Spherical equivalent, calculated as sphere+cylinder/2

On a scan from Pentacam® HR, K1 corresponds to the flat corneal curvature

measured in a central 3 mm zone, K2 corresponds to the steep curvature and

Kmax corresponds to the steepest point of the anterior corneal surface (Gore

et al. 2013).

vii

Original papers

This thesis is based on the following publications (I-IV), which will be

referred to by their Roman numerals.

Reprints are reproduced with permission from the publisher.

I. Beckman Rehnman J, Janbaz CC, Behndig A, Lindén C.

Spatial distribution of corneal light scattering after corneal

collagen crosslinking. J Cataract Refract Surg 2011; 37:1939-

1944.

Copyright 2011 ASCRS and ESCRS. Published by Elsevier Inc. All rights

reserved.

II. Beckman Rehnman J, Behndig A, Hallberg P, Lindén C.

Initial results from mechanical compression of the cornea

during crosslinking for keratoconus. Acta Ophthalmol 2014;

92:644-649.

Copyright 2014 Acta Ophthalmologica Scandinavica Foundation.

Published by John Wiley & Sons Ltd.

III. Beckman Rehnman J, Behndig A, Hallberg P, Lindén C.

Increased corneal hysteresis after corneal collagen

crosslinking: A study based on applanation resonance

technology. JAMA Ophthalmol 2014; 132:1426-1432.

Copyright 2014 American Medical Association. All rights reserved.

IV. Beckman Rehnman J, Lindén C, Hallberg P, Behndig A.

Treatment effect and corneal light scattering with 2 corneal

cross-linking protocols: A randomized clinical trial. JAMA

Ophthalmol, published online August 27, 2015.

Copyright 2015 American Medical Association. All rights reserved.

viii

Sammanfattning på svenska

Keratokonus (KC) är en sjukdom i ögats hornhinna som drabbar ca 1/2000

personer. Vid KC sker en förtunning av vävnaden i hornhinnan, vilket gör att

hornhinnan börjar bukta utåt. Detta medför i sin tur brytningsfel och nedsatt

syn. Idag är korneal crosslinking (CXL) en etablerad metod för behandling

av lätt till måttlig KC. Behandlingen inleds med att ett fotosensiterande

ämne, riboflavin, ges i form av upprepade ögondroppar i det aktuella ögat

under 30 minuter. Ögat belyses sedan med en lampa, som avger UV-ljus,

under ytterligare 30 minuter. Kombinationen gör att kollagenet i

hornhinnan förstärks genom en kemisk process, s.k. korsbindning.

Hornhinnan blir därigenom mer mekaniskt stabil och sjukdomens förlopp

hejdas.

Då en del patienter redan har utvecklat betydande brytningsfel när de

kommer för behandling är CXL, som är en i huvudsak förebyggande

behandling, ofta otillräckligt. Vid dessa mer avancerade förändringar vore

det önskvärt med en behandling som både kan hejda sjukdomens förlopp

och minska brytningsfelen.

I denna avhandling utvärderas om en mekanisk kompression av hornhinnan

under CXL-behandling kan minska patienternas brytningsfel och därmed

också förbättra deras synskärpa, genom att ge en bestående utplaning av

hornhinnans utbuktning.

I avhandlingen utvärderas även olika kliniska mätmetoders förmåga att

kvantifiera behandlingseffekterna av olika varianter av CXL.

43 patienter (60 ögon), i åldern 18-28 år, med diagnosen KC inkluderades.

Patienterna randomiserades till behandling med korneal crosslinking utan

(CXL; n=30 ögon) eller med (CRXL; n=30 ögon) mekanisk kompression av

hornhinnan. Behandlingarna inleddes med att riboflavin gavs i form av

upprepade ögondroppar i det aktuella ögat under 30 minuter, efter det att

hornhinnans epitel avlägsnats i lokalbedövning. Vid CRXL syddes sedan en

flack, hård kontaktlins fast över hornhinnans yta med fyra stygn för att

pressa tillbaka utbuktningen. Med kontaktlinsen på plats bestrålades sedan

hornhinnan med UV-ljus för att åstadkomma en korsbindning av kollagenet,

med förhoppningen att hornhinnan därigenom skulle “låsas” i det nya, mer

utplanade läget. För att kompensera för kontaktlinsens absorption av UV-

ljus, förlängdes CRXL-gruppens bestrålningstid med 4 minuter, till totalt 34

minuter. En timme efter behandlingen avlägsnades kontaktlinsen. CXL-

gruppen fick samma behandling, frånsett kontaktlinsen och den extra

bestrålningstiden. Även en ålders- och könsmatchad kontrollgrupp

bestående av friska individer inkluderades.

ix

Patienterna undersöktes före samt 1 och 6 månader efter behandlingen. De

variabler som kontrollerades var bästa korrigerade synskärpa med glas,

hornhinnans brytkraft, hornhinnans tjocklek, ögats längd, ljusspridningen i

hornhinnan, ögontryck, hornhinnans elastiska egenskaper (CRF) och

hornhinnans viskoelastiska egenskaper (CH).

Avhandlingen består av fyra publicerade artiklar, varvid resultatet av CXL-

och CRXL-behandlingarna utvärderades och jämfördes i Artikel II. De övriga

artiklarna har fokuserat på utvärderingar av olika kliniska mätmetoders

förmåga att kvantifiera behandlingseffekterna av CXL och CRXL. I Artikel I

kvantifierades ljusspridningen i hornhinnan före och efter CXL-

behandlingen manuellt. Utifrån fotografier tagna med Scheimpflug kameran

analyserades totalt 8 tvärsnitt av hornhinnan, belägna 0, 1, 2 och 3 mm från

hornhinnans centrum (synaxeln). I Artikel IV analyserades ljusspridningen i

båda behandlingsgrupperna med hjälp av Pentacam® HR software, en

automatisk metod som kvantifierar ljudspridningens täthet. Denna metod

analyserar hornhinnan utifrån 4 cirkulära zoner, utgående från hornhinnans

apex (yttersta punkt), på 3 olika djup. I Artikel III kvantifierades

hornhinnans biomekaniska egenskaper (CRF och CH) efter CXL-

behandlingen.

Datamaterialet har analyserats utifrån kvantitativ metodik och en

signifikansnivå på P<0.05 har använts.

Ljusspridningen efter CXL-behandlingen visade tydliga mönster gällande

utbredning och tidsförlopp. Efter CXL-behandlingen kunde också en ökad

stabilitet i hornhinnan påvisas med hjälp av Applanation Resonans

Tonometri (ART) -teknologi (mätt som en ökning av CH-värdet). Vad gäller

CRXL-behandlingen klarade den inte av att “låsa” hornhinnan i det nya, mer

utplanade, läget och vid jämförelsen de båda behandlingarna emellan, var

CRXL inte lika effektiv som CXL. I överensstämmelse med ovan var också

ljusspridningen efter CRXL-behandlingen mindre framträdande.

Analyser av ljusspridningen gav värdefull information om

behandlingseffekten efter både CXL- och CRXL-behandlingen. Genom att

använda ART -teknologin har en ökad stabilitet i hornhinnan för första

gången varit möjlig att påvisa kliniskt efter en CXL-behandling. En

vidareutveckling av ART -metoden skulle kunna göra den till en potentiell

klinisk mätmetod vad gäller viskoelasticitet. Tyvärr lyckades inte CRXL-

behandlingen överträffa CXL-behandlingen. Möjligen skulle en starkare

korsbindningseffekt möjliggöra en stabilisering av hornhinnan i det

utplanade läget.

1

1. Introduction

1.1. The Cornea

The cornea is a transparent and a clear, dome-shaped structure that covers

the front part of the eye. This allows light to refract symmetrically to the

crystalline lens and then on towards the retina. An adult cornea is about 0.53

mm thick at the centre (Doughty & Zaman 2000) and becomes thicker with

increasing distance from the centre (Jonuscheit et al. 2015). The diameter is

about 11-12 mm horizontally and 10-11 mm vertically. With an average radius

of curvature on 7.8 mm, the cornea provides approximately 43 dioptres (D)

of the total 59 D of refractive power of the human eye.

Because of the corneas importance of transparency, it is a tissue without

blood vessels. The cornea receives most of its oxygen and nutrients via

diffusion from the tear film (through the outside surface) and by the aqueous

humour (through the inside surface).

The human cornea is a structure that consists of three layers with two

membranes in between. From the anterior to posterior part of the cornea,

these structures are: the epithelium, the Bowman´s membrane, the stroma,

the Descemet´s membrane and the endothelium.

The corneal epithelium is a thin layer of tightly packed cells covering the

corneal surface. It is normally five cell layers thick and it is easily

regenerated if the cornea is injured. If an injury goes deeper into the cornea,

it may causes opaque areas with decreased corneal transparency.

The Bowman´s membrane lies just beneath the epithelium and is a

membrane composed of collagen. This membrane is very tough and difficult

to penetrate and protects the cornea from deeper injuries.

The corneal stroma is the middle layer and it representing the largest part

of the corneal thickness. The stroma is consisting of highly regularly

arranged lamellae of collagen fibrils and extracellular matrix components

(for example proteoglycans) along with keratocytes (fibroblasts). The

keratocytes continually maintain the stroma by synthesizing collagen and

stromal molecules. The regular arrangement with a constant distance

between the lamellae is essential for the corneal transparency.

The Descemet´s membrane is a basement membrane of the corneal

endothelium. This membrane is composed mainly of collagen.

The endothelium consists of a one cell layer covering the inside of the

cornea. These cells are regulating the fluid and the solute transport between

the aqueous humour and the corneal stroma. If endothelium cells are

damaged, the cells do not regenerate. The overall cell density will then be

reduced which will impacts on the fluid regulation and if the endothelium no

longer can maintain a proper fluid balance, stromal swelling will occur. This

2

may cause corneal edema, opacification and loss of visual function.

(American Academy of Ophthalmology 2010-2011).

1.2. Keratoconus

Keratoconus (KC) is a progressive corneal degeneration with decreased

rigidity of the corneal structure. The definition of KC as a non inflammatory

process has been generally accepted (Krachmer et al. 1984; Rabinowitz

1998), but new research have found increased level of inflammatory

mediators such as cytokines (e.g. interleukin 6) in the tear fluid of KC

patients (Lema et al. 2009; Jun et al. 2011). These results indicate that KC

may involve inflammatory events (McMonnies 2015). Also a reduced level of

superoxide dismutase (SOD; Behndig et al. 2001) supports this theory. The

role of SOD is to remove reactive oxygen species, known to be associated

with inflammatory processes.

1.2.1. Epidemiology

The reported incidence of KC is approximately 1 in 2000 people (Rabinowitz

1998; American Academy of Ophthalmology 2010-2011) and affects both

men and women, but the ratio between men/women varies between reports

(Bawazeer et al. 2000; Lim & Vogt 2002; Saini et al. 2004; Ertan &

Muftuoglu 2008; Jonas et al. 2009). KC often debuts at puberty and

progress until the third or fourth decades of life, and then the progression

often halts (Krachmer et al. 1984; Rabinowitz 1998). It is most common as

an isolated condition, but can coexist with other disorders, for example with

Leber’s congenital amaurosis (Elder 1994), Down’s syndrome (Cullen &

Butler 1963) and mitral valve prolapse (Sharif et al. 1992). KC is also

associated with atopic dermatitis and eye rubbing (Bawazeer et al. 2000;

Jafri et al. 2004; Gunes et al. 2014), and about 6-10% of reported cases have

a positive family history (Rabinowitz 1998).

1.2.2. Signs and symptoms

The weakness of the corneal structures leads to corneal thinning and an

abnormal protrusion. The protrusion causes myopia and irregular

astigmatism, affecting the visual quality (Krachmer et al. 1984).

KC is a bilateral disease, although often asymmetrical (Rabinowitz 1998).

Initially, it is often unilateral (Krachmer et al. 1984; Kennedy et al. 1986),

but many patients develop bilateral KC before the progression halts (Li et al.

2004).

1.2.3. Diagnosis

Corneal topography based on the principles of Placido disc or Scheimpflug

imaging (Pentacam® HR; see 3.6.1.) are the most sensitive methods to

diagnose and quantify KC and have become gold standard methods to

3

diagnose and monitor KC (Maeda et al. 1994; Rabinowitz 1998; Gordon-

Shaag et al. 2015). By producing a topographic map over the corneal surface

and various indices, several quantitative methods have been developed,

based on these indices (Gordon-Shaag et al. 2015). By combining different

tomography parameters, Pentacam® HR can for example derive Amsler-

Krumeich stages (Krumeich et al. 1998; Table 1) and Belin-Ambrósio

enhanced ectasia assessment (Belin & Ambrósio 2013).

Table 1. Classification of keratoconus by Amsler-Krumeich stages. Stage is determined if one of the characteristics applies.

Stage Characteristics

I Eccentric corneal steepening

Myopia and/or astigmatism of 5.00 D

Central Kmean reading 48.00 D Vogt´s striae, no scars

II Myopia and/or astigmatism 5.00 to 8.00 D

Central Kmean reading 53.00 D Absence of scars

CTmin 400 μm

III Myopia and/or astigmatism 8.00 to 10.00 D

Central Kmean reading 53.00 D Absence of scars CTmin = 200 to 400 μm

IV Refraction not measurable

Central Kmean reading 55.00 D Central corneal scars, perforation

CTmin 200 μm

Kmean; mean keratometry value, calculated as (K1+K2)/2. CTmin; corneal thickness at the thinnest point.

1.2.4. Treatment options

In early stages of KC, the refractive errors can be corrected with glasses or

rigid contact lenses (Tan & Por 2007; Gordon-Shaag et al. 2015). None of

these interventions, however, have any effect on the progression of the

disease (Tuft et al. 1994; Gordon-Shaag et al. 2015). In late stages

keratoplasty may be the only treatment option (Tan & Por 2007; Gordon-

Shaag et al. 2015).

In recent years, a technique called corneal crosslinking (CXL) has been

introduced as a treatment for KC, particularly for the early stages of the

disease. This treatment halts the progression of KC (Gordon-Shaag et al.

2015). In attempts to reduce the refractive errors and improve the visual

acuity, combinations of CXL together with phototherapeutic keratectomy

(Kymionis et al. 2012), and phototherapeutic keratectomy and intrastromal

ring segments (Yeung et al. 2013) have been tried. The effect of these

treatments may be insufficient in more severe cases of KC (Park & Gritz

http://www.ncbi.nlm.nih.gov/pubmed/?term=Belin%20MW%5BAuthor%5D&cauthor=true&cauthor_uid=23925323

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ambr%C3%B3sio%20R%5BAuthor%5D&cauthor=true&cauthor_uid=23925323

4

2013). It is also considered controversial to remove corneal tissue in an

already thin cornea with decreased rigidity (Zhang & Zhang 2013).

1.3. Crosslinking

1.3.1. Background

Intermolecular crosslinking is a concept to enhance the rigidity of materials

and have been used in bioengineering as a technique to strengthen materials

for many years (Snibson 2010). Crosslinking is the creation of bonds,

covalent or ionic, that connects one polymer chain to another. A polymer is a

chain of monometric material, for example a synthetic polymer or a

biological molecule such as a protein (Sorkin & Varssano 2014). Crosslinking

changes the material’s physical properties. For example, when a rubber

molecule is crosslinked, its flexibility will be decreased and its rigidity and

melting temperature will be increased (Jenkins et al. 1996).

The idea behind today’s treatment of KC with corneal crosslinking using

ultraviolet (UV) irradiation as a way to stiffen the human cornea came after a

visit at a dentist who used UV irradiation to harden synthetic tooth filling

material (Snibson 2010).

1.3.2. Corneal crosslinking - three different mechanisms

In a normal cornea, aggregated forms of collagen monomers are

strengthened by intermolecular crosslinks. Collagen fibrils are naturally

enzymatically crosslinked (by enzyme lysyl oxidase) as a part of their

maturation process (Ashwin & McDonnell 2009).

Crosslinking also occurs during normal aging and in diabetes mellitus,

where the natural crosslinking increases during a non-enzymatic reaction

termed glycation (Maillard reaction; Malik et al. 1992; Sady et al. 1995;

Daxer et al. 1998).

When riboflavin molecules are exposed to UV-A light of 370 nm

wavelength, they absorb energy and reach an excited state. The process that

is oxygen depending (Richoz et al. 2013) can produce either singlet oxygen

molecules or superoxide radicals, in part depending on the availability of

oxygen (Wollensak 2006; Kamaev et al. 2012). These reactive oxygen species

are highly reactive and can induce covalent bonds (crosslinks) between many

different molecules including in the corneal stroma, e.g. collagen fibrils,

proteoglycans or nucleic acids (Pacifici & Davies 1990; Kolli & Aslanides

2010; Meek & Hayes 2013). In 1997 Spoerl et al. were first to achieve

crosslinking of corneal collagen by using riboflavin and UV irradiation in

enucleated porcine eyes (Spoerl et al. 1998). Later, this technique was

studied in vivo by Wollensak et al. They treated 22 progressive KC patients

with photosensitizing riboflavin drops and ultraviolet-A (UV-A) light after

epithelium removal. Afterwards, clinical follow-up showed that the

progression of KC was stopped in all eyes. In 70% of the eyes they also found

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20ZY%5BAuthor%5D&cauthor=true&cauthor_uid=23374574

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20XR%5BAuthor%5D&cauthor=true&cauthor_uid=23374574

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pacifici%20RE%5BAuthor%5D&cauthor=true&cauthor_uid=2233315

http://www.ncbi.nlm.nih.gov/pubmed/?term=Davies%20KJ%5BAuthor%5D&cauthor=true&cauthor_uid=2233315




5

a regression of the refractive error and the maximum keratometry readings

(Kmax; Wollensak et al. 2003a). Today CXL is a safe and efficient routine

method to halt the progression of KC (Goldich et al. 2010; Kolli & Aslanides

2010) and the effects of crosslinking treatment can be summarized in two

stages: first, a photochemical reaction takes place in the corneal stroma and

thereafter a wound healing process (Salomão et al. 2011; Touboul et al.

2012). Corneal haze and a corneal demarcation line after the crosslinking

treatment are signs of wound healing responses after the treatment

(Wollensak et al. 2003a; Seiler & Hafezi 2006; Mazzotta et al. 2012).

1.3.3. The “Dresden protocol”

The standard treatment protocol, named the “Dresden protocol” was first

described in 2003 by Wollensak et al. The protocol included the following

steps:

A topical anesthetic to anesthetize the eye.

Removal of the central 9 mm of the corneal epithelium.

Application of topical 0.1% riboflavin (vitamin B2) on the

deepithelized surface every 5 minutes during 30 minutes.

Irradiation with UV-A light (370 nm, 3 mW/cm2) during 30 minutes

with continued application of topical 0.1% riboflavin every 5

minutes.

Application of topical antibiotics and a soft contact lens.

(Wollensak et al. 2003a; Mazzotta et al. 2008).

1.3.4. Biomechanical/physiochemical changes induced by CXL

Today it is generally accepted that CXL acts through an increase in corneal

biomechanical strength (Beshtawi et al. 2013), but the exact mechanisms

behind CXL are not fully understood. There are several indirect evidences for

the presence of crosslinks in the cornea after CXL (Meek & Hayes 2013).

Most evidence are supplied in vitro by biomechanical (Wollensak et al.

2003b; He et al. 2010; Knox Cartwright et al. 2012; Dias et al. 2013),

thermomechanical (Spoerl et al. 2004a), biochemical (Spoerl et al. 2004b;

Brummer et al. 2011) and structural (Wollensak et al. 2004b) approaches.

Wollensak et al. (2003b) showed a 328.9% increase of the corneal rigidity

in human corneas (increase in Young´s modulus by factor of 4.5). This

stiffening effect is found to be more pronounced in the anterior layer of the

stroma (Kohlhaas et al. 2006) where a reduced swelling behavior also is

found (Wollensak et al. 2007). CXL also induces keratocyte apoptosis with

lacunar edema in the space where apoptotic keratocytes used to be. The area

next to the treated zone has a diffuse edema. The difference in the edema

pattern between the zones may explain the formation of the demarcation

line, visible at biomicroscopy after CXL (Wollensak & Herbst 2010b). This is

followed by disappearance of the corneal edema, keratocyte repopulation

http://www.ncbi.nlm.nih.gov/pubmed/?term=Meek%20KM%5BAuthor%5D&cauthor=true&cauthor_uid=23406488

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hayes%20S%5BAuthor%5D&cauthor=true&cauthor_uid=23406488

6

and an increased density of the extracellular matrix (Wollensak et al. 2004a;

Seiler & Hafezi 2006; Dhaliwal & Kaufman 2009; Wollensak & Herbst

2010b). These corneal reactions are visible as corneal haze and often cause a

slight decrease in visual acuity during the first months after a CXL treatment

(Wollensak et al. 2003a; Mazzotta et al. 2012).

By using gel electrophoresis, a formation of aggregated molecules

(polymers) of >1000 kDa was found in treated eyes (Wollensak & Redl

2008), which is a proof of the high-molecular-weight collagen polymers

forming crosslink bridges in the collagen (Ashwin & McDonnell 2010).

Another indirect evidence of crosslinked collagen in CXL treated corneas

is an increased shrinking temperature in the anterior stroma. Hydrothermal

shrinking was observed at 75C in the anterior stroma and at 70C the

posterior stroma (Spoerl et al. 2004a). Furthermore, increased resistance to

enzymatic digestion by pepsin, trypsin and collagenases (Spoerl et al. 2004b)

and increased collagen fiber diameters (Wollensak et al. 2004b) can be seen

after CXL.

1.4. Densitometric evaluation of corneal reflectivity

The corneal reactions (visible as corneal haze) after CXL can be estimated by

measuring light scattering. Optical coherence tomography (OCT) and

Scheimpflug images (Pentacam® HR) can both provide images of the cornea

with possibility to estimate the light scattering. OCT uses a longer

wavelength with higher tissue penetration compared to the blue light of the

Scheimpflug device and it therefore provides a more detailed image of the

corneal stroma (Doors et al. 2009). Still, Scheimpflug images are sufficiently

detailed for the densitometric measurements needed to quantify corneal

haze after CXL (Greenstein et al. 2010; Wittig-Silva et al. 2013).

1.5. Corneal properties affecting IOP measurements

Doughty & Zaman (2000) have according to a meta-analysis from 300 data

sets estimated the normal central corneal thickness (CCT) to be 534 µm.

Several studies have shown that the thickness of the cornea can affect the

measurement of the intraocular pressure (IOP). A thick (rigid) cornea

generally results in an overestimation of the IOP and vice versa for a thin

cornea (Ehlers et al. 1975; Whitacre & Stein 1993; Doughty & Zaman 2000).

Accordingly, KC patients often have lower IOP values due to thin corneas

(Rask & Behndig 2006).

The curvature of the cornea may also affect the IOP measurements, such

that true IOP will in steep cornea be overestimated and in flat cornea

underestimated (Whitacre & Stein 1993). A normal anterior corneal radius of

curvature is about 7.6-7.9 mm (Dubbelman et al. 2002; Eysteinsson et al.

2002).

7

Further investigations will be needed into how the increased corneal

mechanical stability after CXL treatment affects the IOP readings (Gkika et

al. 2012a).

1.6. Biomechanical properties

If a material deforms reversibly under stress, it is defined as elastic. If a

material flows when an external force is applied and then does not regain its

original shape when the force is removed, the material is defined as viscous.

If a material is viscoelastic, has it characteristics of both elasticity and

viscosity, resulting in energy dissipation when stress is applied. The energy

lost in the process is named “hysteresis” (Sorkin & Varssano 2014; Figure 1).

Figure 1. Schematic illustration of the concepts of elasticity and viscoelasticity.

Illustration by JB Rehnman

1.6.1. Biomechanical properties of the normal cornea

The cornea shows both elastic and viscoelastic properties (Dupps & Wilson

2006; Figure 1). The thickness of the corneal stroma is about 90% of the total

corneal thickness. The stroma is the main contributor to the corneal strength

and consists of both lamellae of collagen fibrils and proteoglycans (American

Academy of Ophthalmology 2010-2011; Sorkin & Varssano 2014). The

corneal stroma also consists of 80% water, which gives cornea its viscoelastic

properties. The water is thought to be the major contributor to the cornea’s

ability to absorb energy, corneal hysteresis (CH; Luce 2005; Sorkin &

Varssano 2014). The corneal resistance factor (CRF) is an indicator of the

cornea’s ability to resist external forces (Ortiz et al. 2007).

8

1.6.2. Biomechanical properties in keratoconus

Decreased mechanical stability is a cause of the protrusion of the cornea in

KC. Andreassen et al. (1980) have shown that KC corneas have about 40%

reduction in rigidity when compared with normal corneas. Accordingly,

Daxer & Fratzl (1997) have shown that the regularly arranged lamellae of

collagen fibrils in normal corneas appear to be altered in KC corneas. These

findings indicate altered crosslinks in KC corneas and possibly contribute to

the mechanical instability.

1.6.3. Determination of corneal biomechanical properties

Corneal biomechanical properties can be measured in vivo by using the

Ocular Response Analyzer (ORA®, CH and CRF; Luce 2005; Luce 2006).

With the Applanation Resonance Tonometry (ART) -technology (Eklund et

al. 2003; Hallberg et al. 2004; Hallberg et al. 2007; Jóhannesson et al.

2012) it is also possible to measure CH.

ORA® has been useful in detecting biomechanical changes related to

corneal refractive procedures (Chen et al. 2010; Ryan et al. 2011), aging

(Kamiya et al. 2009; Narayanaswamy et al. 2011) smoking (Hafezi 2009)

diabetes mellitus (Goldich et al. 2009; Hager et al. 2009) and Fuchs’

endothelial dystrophy (del Buey et al. 2009). Several studies have also shown

significant decrease in CH and/or CRF values in KC corneas when compared

with normal corneas (Shah et al. 2007; Fontes et al. 2011; Gkika et al.

2012b). Despite these findings, several studies have failed to measure

significant changes in CH and CRF in KC corneas after CXL (Sedaghat et al.

2010; Spoerl et al. 2011; Gkika et al. 2012b; Goldich et al. 2012).

1.7. Summary of the introduction

CXL has become an established routine method to halt progression of KC by

increasing the corneal rigidity (Goldich et al. 2010; Kolli & Aslanides 2010),

especially in early stages of the disease. In later stages, when the refractive

errors are large, there is yet no treatment able to stop the progression of KC,

modify the corneal shape and improve the visual quality.

Given the development of the method and the large scale use of CXL in

treatment of KC, there is need for tools to evaluate the treatments effects in

vivo (Seiler & Hafezi 2006) and for in vivo quantification of the

biomechanical status of the cornea.

9

2. Aims

The aims of this thesis were to determine whether mechanical compression

of the cornea during corneal crosslinking for keratoconus using a sutured

rigid contact lens could improve the optical and visual outcomes of the

treatment. In addition, we aimed to find methods to evaluate the effect of

different corneal crosslinking treatment regimens.

The aims were divided into these specific issues:

…to compare refractive changes after corneal crosslinking with and

without intra-treatment mechanical flattening of the cornea.

(Paper II)

…to quantify and measure the spatial distribution and time course of

the superficial light scattering using Scheimpflug photography or

Pentacam® HR software.

(Paper I, Paper IV)

…to quantify the biomechanical effects of CXL using ORA and

Applanation Resonance Tonometry (ART) -technology.

(Paper III)

…to assess the effect of CXL treatment on IOP measurement values

using three different tonometry techniques.

(Paper III)

10

3. Materials and methods

The clinical data used in the Papers (I-IV) were collected in a prospective,

open, randomized case-control study termed the CRXL-study (Figure 2).

Registered at ClinicalTrails.gov as Treatment of keratoconus with advanced

corneal crosslinking, NCT number 02425150 (ClinicalTrials.gov. 2015).

3.1. Statistical power

The group size of 60 eyes (30 eyes in each treatment group) was decided

using a power analysis. This study design will allow an 80% chance for

detection of differences, both between the treatments and between the time

points, according to the table below (α=0.05, power=0.80; Table 2).

Table 2. Power analyses.

Parameter Differences between Differences between treatments time points

BSCVA (logMAR) 0.2 0.1 SphEq (D) 1.9 0.8 K1 (D) 2.3 0.7 K2 (D) 2.9 0.8 Kmax (D) 3.8 0.6

Densitometry (GSU)/light scattering (AU) 3.1 0.6

IOPGAT (mmHg) 2.0 0.8

CHORA (mmHg) 1.0 0.4

CHART (mmHg) 1.1 1.1

CRFORA (mmHg) 1.1 0.4

BSCVA; best spectacle corrected visual acuity. SphEq; spherical equivalent, calculated as sphere+cylinder/2. K1; keratometry value for flat meridian. K2; keratometry value for steep meridian. Kmax; maximum keratometry value; maximum corneal curvature, obtained with the Pentacam® HR. IOPGAT; intraocular pressure measured with Goldmann Applanation tonometry. CHORA; corneal hysteresis measured with Ocular Response Analyzer. CRFORA; corneal resistance factor measured with Ocular Response Analyzer. CHART; corneal hysteresis measured with Applanation Resonance Tonometry -technology.

3.2. Participants

The study comprised 43 patients (60 eyes; 39 males, 4 females), age 18 to 28

years with age- and sex- matched control group (Figure 2).

11

Figure 2. Overview of the CRXL-study.

3.2.1. Patients

Patients with uni- or bilateral progressive KC, planned for routine corneal

crosslinking, were recruited to the study from the Department of Clinical

Sciences, Ophthalmology, Norrlands University Hospital, Umeå, Sweden,

between October 13, 2009 and December 15, 2011.

After inclusion the KC patients were randomly assigned to receiving either

conventional corneal crosslinking (CXL; n=30 eyes) using the “Dresden

protocol” (Wollensak et al. 2003a; Mazzotta et al. 2008) or a modified

treatment we termed corneal reshaping and crosslinking (CRXL; n=30 eyes).

A list of random numbers between 1 and 60 generated by a computer was

used for the randomization. The patients were included in running numbers

according to the computer-generated list, an even number was treated with

CXL and an uneven number was treated with CRXL.

Of the patients who had both eyes included in the study, 13 patients were

randomized to CXL in one eye and CRXL in the other eye. Three patients

were randomized to CRXL in both eyes, and one patient to CXL in both eyes.

3.2.2. Control subjects

To rule out natural time-dependent changes over time in the variables

assessed, we also included a control group of healthy subjects, recruited

between November 9, 2009 and May 3, 2012. Each treated eye of a KC

patient was matched with one eye of an age- and sex-matched control

subject. The control subjects followed the study protocol in every part, except

for the CXL- and CRXL-treatment.

3.3. Inclusion/exclusion criteria

The inclusion criteria were patients planned for routine corneal crosslinking

with progressive KC documented with repeated keratometry and subjective

refraction or Scheimpflug tomography using the Pentacam® HR (Oculus,

Inc. Lynnwood, WA, USA). A corneal thickness exceeding 400 µm at the

thinnest point after epithelial removal was also required for inclusion.

12

Ultrasonic pachymetry (ORA®; Ocular Response Analyzer, software version

1.02, Reichert, Inc. Depew, NY, USA) was performed before and after

epithelial removal and in borderline cases hypo-osmotic riboflavin was used

when deemed necessary (n=1). Inclusion required the KC patients to be

between 18 and 28 years, with no history of previous ocular surgery, no

corneal abnormalities except KC, and no cognitive insufficiency interfering

with the informed consent.

Conversely, exclusion criteria were age under 18 years or over 28 years,

previous ocular surgery, any corneal abnormalities except KC, and a

cognitive insufficiency interfering with the informed consent.

Patients wearing rigid contact lenses were instructed not to use their

lenses for a week before all examinations.

3.3.1. Diagnosis and documentations of progressive keratoconus

The KC diagnosis was based on the Amsler-Krumeich stages (Krumeich et al.

1998) and the “Total Deviation” keratoconus quantification value from the

Belin-Ambrósio enhanced ectasia assessment (Belin & Ambrósio 2013), both

obtained from the Pentacam® HR measurements.

An altered red fundus reflex and/or an irregular cornea seen as a

distortion of the keratometric mires were also required for diagnosis.

All KC patients had a history of progression, documented by repeated

keratometry and refraction (17 eyes) or by repeated Pentacam® HR

measurements (43 eyes). The 17 eyes documented by repeated keratometry

and refraction had a rapid, pronounced KC progression with increasing K

values (increasing corneal steepness) and astigmatism with decreasing best

spectacle corrected visual acuity (BSCVA) documented by the referring

clinic, which is why we chose not to await further progression before

inclusion and treatment in these cases.

3.4. Ophthalmologic examinations

The ophthalmologic examinations were preformed immediately before

treatment (baseline visit) and at 1 and 6 months after treatment. Each visit

included:

Slit-lamp biomicroscopic examination.

Autorefractometry (Oculus Park 1; Oculus, Inc.).

Best spectacle corrected visual acuity (BSCVA) using the ETDRS fast

protocol (Camparini et al. 2001).

Axial length measurement using the partial coherence

interferometry based IOL Master 500® (Carl Zeiss Meditec AG.,

Jena, Germany).

Corneal topography (e.g. keratometry) by Scheimpflug imaging

using Pentacam® HR.



13

Corneal tomography (e.g. light scattering) by Scheimpflug imaging

using Pentacam® HR.

Corneal thickness measurements with both Scheimpflug

tomography (Pentacam® HR) and ultrasonic pachymetry (Ultrasonic

pachymeter included in the ORA®).

Intraocular pressure by Goldmann Applanation Tonometry (GAT;

Haag-Streit, Inc. Bern, Germany), ORA® and ART -technology (see

3.6.4.).

Corneal biomechanical assessments employing ORA® and ART -

technology (see 3.6.4.).

3.5. Treatments

3.5.1. Conventional corneal crosslinking - CXL

The CXL treatment performed at the baseline visit was performed according

to the “Dresden protocol” (Wollensak et al. 2003a; Mazzotta et al. 2008) and

involved mechanical removal of the central 9 mm corneal epithelium after

topical anesthesia with tetracaine hydrochloride. After the epithelial removal

the corneal thickness was measured with ultrasonic pachymetry (ORA®) to

ensure that the corneal thickness did exceed 400 µm at the thinnest point. In

borderline cases, hypo-osmotic riboflavin was used when deemed necessary

(n=1, a patient randomized to CXL). Topical 0.1% riboflavin (Ricrolin®,

Sooft, Italy) was applied every 3 minutes during 30 minutes, and the cornea

was then irradiated with UV-A light for 30 minutes using a solid-state UV-A

illuminator (Caporossi/Baiocchi/Mazzotta, X-linker; Costruzione Strumenti

Oftalmici, Firenze, Italy). The shutter was set to deliver energy of 3 mW/cm2

and the diameter of the irradiation area was 8 mm. During the UV-A

irradiation, tetracaine hydrochloride and riboflavin were applied every 5

minutes.

3.5.2. Corneal reshaping and crosslinking - CRXL

In the CRXL group a 12.5 mm semi scleral rigid Boston XO contact lens

(Nordic Lenses, Inc. Kista, Sweden; DK value 100.0) was tightly sutured over

the cornea with four 6:0 Vicryl stitches (Ethicon Inc.) to the limbus, after the

riboflavin installation and before the UV-A irradiation. During the UV-A

irradiation, riboflavin was applied every 5 minutes under the contact lens

using a blunt cannula.

The back surface curvature of the contact lens was 11.0 mm,

corresponding to a corneal refractive power of +33.5 D. The contact lens was

used to induce a pronounced corneal flattening during the crosslinking

treatment, with the aim to “lock” cornea in this new position. The flattening

induced by the contact lens was based on the assumption that the anterior

surface of the cornea aligned to the back surface curvature of the lens. We

think this assumption is reasonable because of the four stitches were tightly

14

sutured to the limbal tissue, rendering concentric MD-folds indicating a

pronounced corneal flattening. The MD-folds were visible at the initiation of

the UV-A treatment and also one hour after the treatment, when the contact

lens was removed. UV-metre measurements revealed that the contact lens

absorbed 11% of the UV-A light, which was compensated for by increasing

the irradiation time to 34 minutes.

3.6. Technical equipment

3.6.1. Pentacam® HR (Papers I+IV)

Paper I, manual method: Each eye was photographed with the Scheimpflug

camera using the “25 pictures” program under standardized, mesopic light

conditions. The corneal light scattering, in this investigation expressed as

arbitrary units (AU) was analysed from two of the Scheimpflug images, with

a superior and a temporal camera position, using the “ImageJ” image-

analysis program. Using the “Line Tool” of “ImageJ”, the corneal light

scattering was measured at approximately 40 points along a line

perpendicular to the corneal surface at the visual axis and 1 mm, 2 mm and 3

mm inferior or temporal to the visual axis (Figure 14). Attempts to make

analyses at 4 mm were abandoned because of shadowing from eyelids or

light reflected from iris in too many cases. Each line measurement was

performed in duplicate and the mean value of the two measurements was

used for the analysis. Since there were no differences in the increase in the

corneal light scattering between the corresponding temporal and inferior

measurement points, a mean of the corresponding points was used in the

subsequent calculations. The corneal light scattering raw data thus obtained

was processed using the Microsoft Excel software (Microsoft Inc.).

To compare the corneal light scattering at different distances from the

visual axis, the values need to be corrected for the lager distance from the

illumination source of the Scheimpflug camera and for the angle of the

corneal surface at the given point. Both of these factors will decrease the

illumination and accordingly decrease the corneal light scattering obtained

at the point in question. By using Euclidian geometry, individual corrections

were calculated for each cornea, based on the working distance of 80 mm of

the Scheimpflug camera and the corneal curvature. Similar individual

corrections were also calculated for the UV-A irradiation (working distance

54 mm) during the treatment. The distance between the Scheimpflug camera

and the eye is within this system approximately 120 mm, which was not

corrected for in these calculations. The calculation was based on the facts

that the illumination is inversely proportional to the square of the distance

from the light source and inversely proportional to the angle of inclination.

The peak corneal light scattering value was assessed for each

measurement point. To obtain an overall measure of corneal light scattering

15

in the optical centre of each cornea, the mean of all peak values within 2 mm

from the visual axis was calculated for each cornea.

The individual photographs were also analysed manually for the

occurrence of a demarcation line, by a masked observer.

Paper IV, automated method: The corneal densitometry (light scattering),

in this investigation expressed as standardized gray scale units (GSU) was

measured with the Pentacam® HR software in 4 circular zones around the

corneal apex: a central 0-2 mm zone, a 2-6 mm zone, a 6-10 mm zone and a

10-12 mm zone. The corneal densitometry was also measured at 3 different

depths of the corneal stroma: in the anterior 120 µm layer, in the posterior

60 µm layer and in the layer between the two former layers (the central

layer).

3.6.2. Goldmann Applanation Tonometry (Paper III)

The GAT is considered the gold standard for estimating the intraocular

pressure. The method is based on Imbert Fick´s law which states that the

IOP is directly proportional to the ratio between the force (F) needed to

applanate a predefined contact area (A; Goldmann 1957; Figure 3).

Figure 3. Imbert Fick´s law. IOP = F / A

GAT has been shown to be dependent on CCT and corneal rigidity in that a

thick (rigid) cornea results in an overestimation of the IOP and vice versa for

a thin cornea (Whitacre & Stein 1993). Hence, a change in corneal rigidity

should be detectable as a change in the measured IOP value.

The GAT IOP measurements (IOPGAT) were performed in the central part

of the cornea with the tonometer tip prism set both horizontally (horizontally

split semi-circles) and vertically (vertically split semi-circles). Each

measurement was made in duplicate and the IOPGAT was presented as the

mean value of all four measurements.

3.6.3. Ocular Response Analyzer (Paper III)

The ORA® is a tonometer using an electro-optical system to measure the

corneal response to indentation by a rapid air-pulse when measuring the

IOP. It simultaneously measures the biomechanical properties of the cornea;

the corneal rigidity (CRF) and the corneal viscoelasticity (CH). The air-pulse

moves the cornea inwards past a defined point of applanation to a slight

concavity. Before the cornea then returns to its normal curvature, it’s again

passes the defined point of applanation. The corneal movements are

recorded and the abovementioned parameters CH and CRF are calculated.

16

The CH represents the difference between the pressure value at the inward

applanation (P1) and the corresponding value at the outward applanation

(P2). CRF is calculated from the formula (P1 - kP2). The constant k is defined

from an empirical analysis of the relationship between CCT and P1 and P2.

CRF has been shown to be more strongly associated with CCT than CH. The

ORA® also provides two different IOP parameters: Goldmann correlated IOP

(IOPg) and corneal compensated IOP (IOPcc). IOPg is the average of the two

pressure values at the applanation points. IOPcc is an IOP parameter

designed to be less dependent of corneal curvature and CCT (Luce 2006;

Figure 4 and 5).

Each eye was measured four times with the ORA® and the mean value of

the four measurements was used for IOPcc, CH and CRF (in this study

termed IOPORA, CHORA and CRFORA).

Figure 4. A plot of the air puff pressure function and the applanation signal.

Photography and illustration by JB Rehnman

17

Figure 5. Formulas for estimating the ORA® parameters.

IOPcc = P2 - kP1 IOPg = (P1 + P2)/2 CRF = P1 - kP2 CH = P1 - P2

3.6.4. Applanation Resonance Tonometry -technology (Paper III)

The ART is a recently introduced IOP tonometer (Hallberg et al. 2007;

Jóhannesson et al. 2012) based on resonance technology (Eklund et al.

2003). Two types of ART are available on the market, ART® Servo and ART®

Manual. Through the use of ART -technology it is possible to analyse the

changes in contact area and contact force during the applanation process,

which in turn makes it possible to get information about the biomechanical

properties of the cornea (Eklund et al. 2003; Hallberg et al. 2004).

Figure 6. The early research prototype of ART Servo used in the CRXL-study.

Photography by P Hallberg

In this study an early research prototype of ART Servo was used (Figure

6). A pipe shaped (30x5 mm, 1 mm wall thickness) piezoelectric element

(PZT) (Morgan Electroceramics, Southampton, UK) was moulded in an

aluminium container by silicon rubber (Wacker Elastosil RT622 Wacker

18

Chemie GmbH, München, Germany). To minimise friction forces, the

aluminium container was suspended between two flexible support washers,

0.1 mm thickness, in an outer container made of plastic.

At the rear of the sensor, a force transducer, PS-05KD (Kyowa, Tokyo,

Japan) was in contact with the aluminium case. A feedback circuit powered

the PZT-element in order to sustain the PZT in its resonance frequency

(approximately 60 kHz). The contact piece was made of PEEK-plastic

(Amersham Bioscience, Umeå, Sweden). The contact surface had a diameter

of 5 mm and a radius of curvature of 8 mm. A step motor (Haydon 21H4AC-

2.5-907, Couëron, France) was attached to the end of the sensor body

(Figure 7). Figure 7. Schematic picture of the ART sensor used in the CRXL-study.

Illustration by P Hallberg

The PZT formed a resonance sensor that measured the frequency shift (f,

Hz), which is proportional to the contact area. The contact force (F, mN) was

simultaneously measured by a force transducer. The data was sampled

continuously at 1000 Hz from the point when the sensor got in touch with

the cornea until the sensor was completely removed, i.e. during the

applanation and removal phases, respectively.

The step motor provided the sensor with a standardized servo-controlled

motion based on feedback from both force and area measurements. After

sufficient contact area was reached the sensor automatically reversed from

cornea. Applanation velocity was set to 4 mm/s and removal velocity was 7

mm/s.

The slope of the curve between force and frequency within certain

frequency ranges (i.e. contact area intervals) was interpreted as being

proportional to the IOP. A calibration constant transformed the slope of the

curve into an IOP value in millimetres of mercury (mmHg). The analysis

interval for the IOP during the applanation was calculated from data derived

19

from the analysis interval between 20 to 80 Hz in accordance with the

commercially available instrument (Jóhannesson et al. 2012). For choosing

the analysis interval during the removal phase an optimisation algorithm

was used (Eklund et al. 2003). It stepped through all combinations of start

points and interval lengths, increasing with steps of 10 Hz. The optimal

interval was determined as the frequency interval that produced the lowest

SD between the slope and the reference IOP measured with GAT. This

optimisation process showed that the interval between 120 to 60 Hz

produced the least SD. This interval was used in the study for measuring

removal IOP. (Figure 8a and 8b). The same calibration constant as for the

applanation phase was used during the removal phase.

The CH measured with ART -technology (CHART) was calculated as the

difference in IOP between the applanation and the removal phase (Hallberg

et al. 2004). For each patient four ART measurements were obtained and the

mean values for IOP and CH (in this study named IOPART and CHART) were

recorded.

Figure 8a, 8b shows IOP measurements of left (red; treated) and right (blue; untreated) eye before (8a) and after (8b) CXL treatment. The IOP is calculated from data originating from a

specific contact area interval (measured as a frequency shift, f), from applanation start to applanation end, and from removal start to removal end, marked in the figures. Corneal hysteresis (CH) was then calculated as the difference in IOP during applanation and IOP during removal. For the treated left eye the IOP before and after CXL were similar at applanation, whereas there was a difference in IOP (slope) during the removal. The treated left eye shows an increase in CH.

Figure 8a. Figure 8b.

??a.

Illustration by G Mannberg

contact area = f contact area = f

co

nta

ct f

orc

e (m

N)

co

nta

ct f

orc

e (m

N)

applanation end (80 Hz)

applanation start (20 Hz)

applanation end (80 Hz)

applanation start (20 Hz)

removal start (120 Hz) removal

end (60 Hz)

removal start (120 Hz)

removal end (60 Hz)

20

3.7. Ethics

The study was approved by the Regional Ethical Review Board in Umeå,

Sweden and was performed in accordance with the Declaration of Helsinki

(World Medical Association 2013).

All participants were provided both written and oral information before

giving written consent to participate in the study.

The participants in the control group received financial compensation for

their participation.

3.8. Statistical methods

Preoperative data were used as baseline values and were compared with data

obtained at 1 and 6 months after treatment (Paper I-IV). In Paper IV were

also the quotients between the central and the peripheral zones, at different

depths, determined for each individual cornea at 1 and 6 months after

treatment. In Paper II and III the above measurements were compared with

measurements in the control groups.

For statistical comparisons of normally distributed variables, Student´s

paired t test was used for analysis between different time points and between

the KC patients and their control group. For comparisons of normally

distributed data between the two treatment groups, Student’s unpaired t test

was used. Data were presented as mean standard deviation (SD) or mean

standard error of mean (SE or SEM).

For statistical comparison for non-normally distributed variables,

independent samples Kruskal-Wallis test was used for multiple groups (the

Amsler-Krumeich classification in Paper II, III). Non-normally distributed

data were presented as median interquartile range.

When comparing more than two groups, Bonferroni corrections were

applied (Paper I).

The confidence interval (CI) was calculated and presented in Paper III

(99% CI) and in Paper IV (95% CI).

To analyse linear relationships between different parameters, correlations

were assessed with Pearson’s bivariate correlation analysis for normally

distributed variables (Paper I, IV) and with Spearman’s rho for non-normally

distributed variables (Paper II).

SPSS, version 18 (SPSS Inc.) or Microsoft Excel software (Microsoft Inc.)

were used when processing raw data.

A P value less than 0.05 were considered statistically significant (Paper I-

IV).

http://www.ncbi.nlm.nih.gov/pubmed/?term=World%20Medical%20Association%5BCorporate%20Author%5D

21

4. Results

4.1. Overview over demographic data and parameters

Fifty-eight out of 60 eyes with KC were treated and followed up throughout

the 6-month period (Table 3; Figure 9). For parameters including in each

Paper, see Table 4.

Table 3. Overview over baseline demographic data.

Paper Time period Participants Treatment group n Sex Baseline - 6 months included eyes/P F/M

I 13.10.09 - 14.10.10 P CXL 13/13 0/13 II 13.10.09 - 05.11.12 P/C CXL/CRXL 60/43 4/39 III 13.10.09 - 05.11.12 P/C CXL 30/29 2/27 IV 13.10.09 - 31.05.12 P CXL/CRXL 60/43 4/39

P; patients. C; control subjects. CXL; conventional corneal crosslinking. CRXL; corneal reshaping and crosslinking. n; number of included eyes/number of included patients, in the treatment group/groups. Sex; number of included female/male patients, in the treatment group/groups. F; female. M; male.

Figure 9. CONSORT Flow Diagram. The number of eyes with KC enrolled and randomized to the CRXL-study, and KC eyes included in the 1 and 6 months follow-up.

60 eyes enrolled and

randomized

30 allocated to CXL

30 allocated to CRXL

30 received CXL

29 received CRXL

1 ♂ did not receive

intervention

30 analysed at baseline 29 followed up at 1 month

29 followed up at 6 months

30 analysed at baseline 28 followed up at 1 month

29 followed up at 6 months

1 ♀ lost to follow-up

at 1 and 6 months

1 ♂ lost to follow-up

at 1 month

22

One patient developed a keratitis after CXL but the infection was

subsequently successfully treated and the patient remained in the CRXL-

study. The patient’s data was excluded in Paper I, but was included in Paper

II, III and IV. In Paper I an additional patient was excluded due to technical

problems with the Scheimpflug camera at the 6-month visit. However, in

Paper IV this patient was included in all analyses except for the densitometry

analysis at the 6 months follow-up.

One control subject was included to match each KC patient. If the patient

had both eyes included in the CRXL-study, both eyes of the control subject

were also included.

In one of the control subjects a forme fruste KC was suspected and the

control subject was excluded and replaced with another control subject. One

control subject who was included to match both (CRXL) eyes of one patient

was lost to the 6 months follow-up.

Table 4. Overview over analysed parameters in Papers I-IV.

Parameter; Paper: I II III IV

BSCVA/CDVA x x x Sphere x Cylinder x SphEq x x Kmax x x Kmean x x Rmin x CTmin x x AL x Densitometry/light scattering x x IOPGAT x IOPORA x IOPART x CHORA x x CHART x x CRFORA x

BSCVA; best spectacle corrected visual acuity. CDVA; corrected distance visual acuity. SphEq; spherical equivalent, calculated as sphere+cylinder/2. Kmax; maximum keratometry value; maximum corneal curvature, obtained with the Pentacam® HR. Kmean; mean keratometry value, obtained with the Pentacam® HR, calculated as (K1+K2)/2. Rmin; minimum corneal radius of curvature, obtained with the Pentacam® HR. CTmin; corneal thickness at the thinnest point, obtained with the Pentacam® HR. AL; axial lengths. IOPGAT; intraocular pressure measured with Goldmann Applanation tonometry. IOPORA; intraocular pressure measured with Ocular Response Analyzer. IOPART; intraocular pressure measured with Applanation Resonance Tonometry -technology. CHORA; corneal hysteresis measured with Ocular Response Analyzer. CHART; corneal hysteresis measured with Applanation Resonance Tonometry -technology. CRFORA; corneal resistance factor measured with Ocular Response Analyzer.

23

Table 5. Age distribution of keratoconus patients and matched control subjects.

Patients Control subjects CXL CRXL CXLctrl CRXLctrl

n (eyes) 30 30 30 30

Mean SD (years) 23.7 3.1† 23.4 3.0# 23.7 3.1† 23.4 3.0# Range (years) 18.1 - 28.0 18.2 - 27.9 18.2 - 27.8 18.8 - 27.8 P=0.65; CXL vs. CRXL. † P=0.58; CXL vs. CXLctrl. # P=0.61; CRXL vs. CRXLctrl.

4.2. Baseline Amsler-Krumeich stage and “Total Deviation” value

The baseline Amsler-Krumeich stage was 2 (2-3) in both treatment groups

(median and interquartile range; P=0.94). The “Total Deviation”

keratoconus quantification value obtained from the Pentacam® HR

measurements was 8.5 6.7 standard deviations for the CXL group and 7.4

3.2 standard deviations for the CRXL group (P=0.43).

None of the included control subjects showed any signs of KC according to

these gradings.

4.3. Occurrence of a demarcation line

At the slit-lamp examination, a corneal demarcation line was seen in many of

the KC patients in both treatment groups at 1 month, but this was not

systematically registered in the study protocol.

On Scheimpflug images analysed by a masked observer, a demarcation

line was detected at 1 month in 21 eyes (72%) in the CXL group and in 20

patients (69%) in the CRXL group. Similarly, at 1 month, an increased

corneal light scattering was a consistent finding in all images after both

treatments.

4.4. Refractive results from the CRXL-study (Paper II)

This is a complete analysis including both the KC patients randomized to

CXL and CRXL and the healthy control subjects.

Before the treatments maximum keratometry readings (Kmax) and the

refractive astigmatism (cylinder) was higher (P<0.01; Table 6), the BSCVA

and the minimum corneal thickness (CTmin) was lower (P<0.01; Table 6;

Figure 10) in the KC patients than in the corresponding control subjects.

This difference between the KC patients and the control subjects persisted

throughout the 6-month period (P<0.01; Table 6; Figure 10).

In the KC patients at baseline, the refraction (sphere, cylinder and

spherical equivalent (SphEq)), BSCVA, axial lengths (AL), and Kmax and

CTmin (obtained with the Pentacam® HR) did not differ between the CXL and

CRXL groups (P>0.20).

24

Table 6. Visual and refractive outcomes, Kmax and CTmin at baseline and at 1 and 6 months follow-up after CXL and CRXL treatment, compared to control subjects.

Patients Mean ± SD; P value compared to baseline. Parameters Baseline 1 month 6 months

n (eyes) Sphere (D) Cylinder (D) SphEq (D) BSCVA (logMAR) Kmax (D) CTmin (µm)

CXL 29 -0.3 ± 2.7 -3.1 ± 2.1 -1.9 ± 2.8 0.19 ± 0.26 53.1 ± 4.9 481 ± 41

29 -0.2 ± 2.5; P=0.41 -3.3 ± 2.1; P=0.49 -1.8 ± 2.5; P=0.65 0.25 ± 0.23; P=0.09 53.9 ± 5.2; P<0.01 457 ± 44; P<0.01

29 0.1 ± 2.3; P=0.05 -3.1 ± 2.2; P=0.70 -1.4 ± 2.4; P=0.03 0.14 ± 0.18; P=0.03 52.6 ± 5.2; P=0.02 470 ± 42; P=0.01


CRXL 29 -0.5 ± 2.5 -3.0 ± 2.2 -1.9 ± 2.5 0.24 ± 0.29 54.1 ± 5.3 478 ± 30

28 -1.2 ± 3.1; P=0.08 -2.9 ± 1.9; P=0.67 -2.7 ± 3.0; P=0.06 0.28 ± 0.24; P=0.56 54.8 ± 5.3; P<0.01 472 ± 28; P=0.43

29 -1.0 ± 2.9; P=0.27 -2.9 ± 2.4; P=0.68 -2.4 ± 2.8; P=0.24 0.20 ± 0.21; P=0.20 53.5 ± 5.2; P=0.06 478 ± 29; P=0.97

Control subjects Mean ± SD; P value compared to patients at the same time point. Parameters Baseline 1 month 6 months


CXLctrl 29 -0.7 ± 1.9; P=0.51 -0.4 ± 0.4; P<0.01 -0.9 ± 1.9; P=0.09 -0.14 ± 0.07; P<0.01 44.5 ± 1.4; P<0.01 553 ± 33; P<0.01

29 -0.7 ± 1.9; P=0.36 -0.4 ± 0.4; P<0.01 -0.9 ± 1.9; P=0.10 -0.18 ± 0.06; P<0.01 44.5 ± 1.4; P<0.01 553 ± 32; P<0.01

29 -0.7 ± 1.9; P=0.15 -0.5 ± 0.4; P<0.01 -0.9 ± 1.8; P=0.30 -0.18 ± 0.06; P<0.01 44.5 ± 1.4; P<0.01 554 ± 32; P<0.01


CRXLctrl 29 -0.8 ± 1.6; P=0.59 -0.2 ± 0.4; P<0.01 -0.9 ± 1.8; P=0.09 -0.16 ± 0.06; P<0.01 44.1 ± 1.5; P<0.01 562 ± 24; P<0.01

29 -0.8 ± 1.7; P=0.58 -0.3 ± 0.3; P<0.01 -0.9 ± 1.8; P=0.02 -0.18 ± 0.06; P<0.01 44.0 ± 1.3; P<0.01 563 ± 27; P<0.01

27 -0.7 ± 1.7; P=0.57 -0.3 ± 0.3; P<0.01 -0.9 ± 1.7; P=0.02 -0.18 ± 0.06; P<0.01 44.1 ± 1.2; P<0.01 560 ± 23; P<0.01

SphEq; spherical equivalent, calculated as sphere+cylinder/2. BSCVA; best spectacle corrected visual acuity. Kmax; maximum keratometry value; maximum corneal curvature, obtained with the Pentacam® HR. CTmin; corneal thickness at the thinnest point, obtained with the Pentacam® HR.

At 1 month a small, insignificant decrease in BSCVA was seen for CXL

(P=0.09; P=0.56 for CRXL; Table 6; Figure 10). A significant increase in Kmax

(P<0.01) were seen after both treatments (Table 6). Also a significant

decrease in CTmin was observed 1 month after CXL treatment, but after CRXL

no corresponding corneal thinning was seen (P<0.01 for CXL; P=0.43 for

CRXL; Table 6).

At 6 months a significant improvement in both BSCVA and Kmax were seen

after CXL when comparing with the baseline value (P=0.03; P=0.02; Table

25

6; Figure 10), whereas the small improvement seen in the CRXL group was

insignificant (P=0.20; P=0.06; Table 6; Figure 10). The SphEq was also

significantly improved from baseline to 6 months after CXL treatment (+0.5

1.1 D), but after CRXL treatment the SphEq showed an insignificant

negative development (-0.5 2.1 D), which differed significantly from the

improvement seen after CXL (P=0.04). Comparing with the corresponding

control subjects, the CXL group did not change in SphEq during the 6-month

period (P=0.30; Table 6), whereas the impairment seen in the CRXL group

was significant (P=0.02; Table 6). The corneal thinning seen after CXL at 1

month was partially reversed at 6 months (P<0.01 for 1 versus 6 months). In

the CRXL group a similar but insignificant change was seen (P=0.06 for 1

versus 6 months).

Figure 10. Mean BSCVA at baseline and at 1 and 6 months follow-up after CXL and CRXL treatment, compared with control subjects.

In accordance with the corneal thinning, the AL decreased after CXL at 1

month (24.28 0.95 t0 24.25 0.98, P<0.01) and increased again at 6

month (24.26 0.96, P=0.03). In the CRXL group corresponding but less

pronounced changes were seen (P=0.01 and P=0.81, respectively).

CXL

CXLctrl

CRXL

CRXLctrl

logMAR BEST SPECTACLE CORRECTED VISUAL ACUITY

Baseline 1 month 6 months

26

No correlation was found between the degree of KC estimated from the

baseline “Total Deviation” keratoconus quantification value and the change

seen in BSCVA, Kmax and SphEq at 6 months after CXL and CRXL (P>0.20).

4.5. Corneal light scattering after CXL (Paper I)

This study involved 13 eyes of 13 KC patients (all men) randomized to CXL

and enrolled; 2 were excluded (see 4.1.). The result is therefore based on an

analysis of 11 eyes.

Table 7. Visual outcomes at baseline and at 1 and 6 months follow-up after CXL.


n (eyes) BSCVA/CDVA (logMAR)

CXL 11 0.13 ± 0.19

11 0.25 ± 0.22; P=0.048

11 0.09 ± 0.14; P=0.140

BSCVA; best spectacle corrected visual acuity. CDVA; corrected distance visual acuity.

Figure 11. The difference in BSCVA plotted against the difference in central corneal light scattering at 1 month after CXL treatment.

At 1 month a significant decrease in BSCVA (P=0.048; Table 7) were seen

after CXL treatment. The decrease in BSCVA was significantly correlated

with the overall (0+1+2 mm) increase in corneal light scattering in the

LIGHT SCATTER CHANGE (AU)

BSCVA CHANGE (logMAR)

27

optical centre of the cornea (R2=0.41, P=0.035; Figure 11). An improvement

in BSCVA was seen at 6 months (P=0.140 compared to baseline; Table 7;

P=0.003 compared to the 1 month values).

At the optical centre of the cornea a significant increase in the overall light

scattering was seen from baseline to 1 month (P<0.001; Table 8). The light

scattering was reduced at 6 months (P=0.002 compared to the 1 month

values), but was still higher than the baseline values (P<0.001 compared to

baseline; Table 8).

Table 8. Overall light scattering at baseline and at 1 and 6 months follow-up after CXL.


n (eyes) Light scattering (AU)

CXL 11 87.5 ± 10.0

11 124.5 ± 12.5; P<0.001

11 110.8 ± 15.0; P<0.001

AU; arbitrary units.

The increase in corneal light scattering at 1 month was more pronounced

at the visual axis than at 1 or 2 mm from the visual axis. Three millimetres

from the visual axis, only a slight increase in corneal light scattering was

seen. These differences also remained after the values were corrected for the

distance from the illumination source of the Scheimpflug camera and the

angle of the corneal surface at the measurement points (see 3.6.1.). After

applying factors correcting for these putative errors the means of the

maximum increases in light scattering at 0, 1, 2 and 3 mm from the visual

axis were shown in Figure 12 (P<0.002 when comparing 0 mm to 1 and 2

mm and when comparing 3 mm to all other measurement points). These

differences also remained when the values were corrected for the reduced

irradiation from the UV-A light according to the same principle (data not

shown).

At 6 months, there were no longer a significant difference in maximum

corneal light scattering between the visual axis and the 1 and 2 mm

measurement points (Figure 12), but there was still significantly less light

scattering at 3 mm (P=0.018 when comparing 3 mm to all other

measurement points; Figure 12).

28

Figure 12. The maximum increases in corneal light scattering at 1 and 6 months after CXL treatment at 0, 1, 2 and 3 mm from the visual axis (mean + SD).


1 month 6 months

In the superficial 70 µm of the cornea, corresponding to the corneal

epithelium, the corneal light scattering was unaltered from baseline values

both 1 and 6 months after treatment (Figure 13).

Figure 13. The mean increases in corneal light scattering at 1 and 6 months after CXL treatment at different corneal depths 0, 1, 2 and 3 mm from the visual axis.


1 month 6 months

Visual axis

1 mm

2 mm

3 mm

80

70

60

50

40

30

20

10

0

Visual axis

1 mm

2 mm

3 mm

Depth (µm)

29

At 1 month an increase in the corneal light scattering was seen from

approximately 80-240 µm depths at all measurement points. At 6 months

this peak was still remaining, albeit reduced, and a second peak of increased

corneal light scattering, seen as a demarcation line, occurred between 240-

340 µm (P=0.037 compared to 1 month values; Figure 13 and 14).

No increased corneal light scattering was seen deeper than 340 µm at

either time point (Figure 13).

Visual axis = 0 mm

4.6. Corneal light scattering after CXL and CRXL (Paper IV)

In this study the whole material of KC patients randomized to CXL or CRXL

were included. At baseline all 60 eyes were analysed. At 1 month, 29 eyes

were analysed in the CXL group and 28 in the CRXL group. At the 6-month

Figure 14. Example of Scheimpflug photographs showing the spatial distribution of the corneal light scattering before (baseline) and at 1 and 6 months after CXL treatment. The measurement points (0, 1, 2 and 3 mm) are indicated in the photographs. Note the increased corneal light scattering at 1 month and the demarcation line at 6 months.

30

visit 29 eyes in each group were analysed, except for the densitometry

analysis in the CXL group, where one more eye was excluded due to technical

problems with the Scheimpflug camera (see 4.1.).

At baseline there were no differences between the treatment groups

(P>0.05; Table 5, 9 and 10).

Table 9. Overview over baseline data for each treatment group.

Patients Mean ± SD; P value CXL vs. CRXL Parameters Baseline Baseline P value

n (eyes) Sphere (D) Cylinder (D) SphEq (D) BSCVA (logMAR) Kmax (D) Kmean (D) Rmin (µm) CTmin (µm)

CXL 30 -0.3 ± 2.7 -3.1 ± 2.1 -1.9 ± 2.7 0.19 ± 0.25 53.1 ± 4.8 45.9 ± 3.2 6.4 ± 0.6 480 ± 40

CRXL 30 -0.5 ± 2.5 -2.9 ± 2.2 -1.9 ± 2.5 0.23 ± 0.29 53.9 ± 5.4 46.7 ± 3.5 6.3 ± 0.6 479 ± 30

0.81 0.76 0.91 0.52 0.57 0.41 0.56 0.96

SphEq; spherical equivalent, calculated as (sphere+cylinder)/2. BSCVA; best spectacle-corrected visual acuity. Kmax; maximum keratometry value, maximum corneal curvature, obtained with the Pentacam® HR. Kmean; mean keratometry value, obtained with the Pentacam® HR, calculated as (K1+K2)/2. Rmin; minimum corneal radius of curvature, obtained with the Pentacam® HR. CTmin; corneal thickness at the thinnest point, obtained with the Pentacam® HR.

4.6.1. Compared to baseline

At 1 month a significant increase in corneal light scattering was seen in all

zones and layers, apart from the peripheral 10-12 mm zone in the CXL group

(P0.04; change from baseline in centre layer, zone 0-2 mm, 7.2 GSU; Table

10). A similar but smaller significant increase in corneal light scattering was

seen in zone 0-2 mm and 2-6 mm in all layers, and in zone 6-10 mm in the

anterior layer in the CRXL group (P=0.00; change from baseline in centre

layer, zone 0-2 mm, 2.2 GSU; Table 10). At 6 months a regression of the

corneal light scattering was seen in both groups, but the corneal light

scattering seen in the CXL group was still significant higher in all zones and

layers, apart from the peripheral 10-12 mm zone, compared to baseline

(P0.01; change from baseline in centre layer, zone 0-2 mm, 4.0 GSU; Table

10). In the CRXL group, only zone 0-2 mm and 2-6 mm in the anterior layer

still showed a significant increase in corneal light scattering compared to

baseline (P0.04; change from baseline in centre layer, zone 0-2 mm, 0.5

GSU; Table 10).

31

Table 10. Corneal densitometry (light scattering) and the differences in corneal densitometry, in all zones and layers, in both treatment groups (GSU). Baseline Layer/ Zone mm

CXL (mean SD)

n = 30

CRXL (mean SD)

n = 30

Diff. between CXL and CRXL

95% CI, diff. between CXL and CRXL

P value, CXL vs. CRXL

An

teri

or 0-2

2-6 6-10

10-12

21.1 4.0

18.3 3.1

15.3 2.7

24.7 8.2

23.2 4.8

19.9 3.9

16.5 3.3

27.3 8.1

-2.16 -1.65 -1.29 -1.87

-4.42 - 0.12 -3.48 - 0.18 -2.86 - 0.28 -6.80 - 1.64

0.06 0.08 0.10 0.23

Ce

ntr

e

0-2 2-6

6-10 10-12

13.3 2.5

11.6 2.1

10.8 1.9

16.3 3.8

14.4 2.6

12.5 2.3

11.4 2.4

18.5 5.0

-1.12 -0.98 -0.64 -2.18

-2.44 - 0.20 -2.11 - 0.15 -1.75 - 0.48 -4.49 - 0.13

0.10 0.09

0.18 0.06

Po

ster

ior

0-2 2-6

6-10 10-12

10.8 1.4

10.1 1.2

9.8 1.6

13.6 2.5

11.1 1.6

10.5 1.4

10.2 1.9

15.1 3.7

-0.29 -0.34 -0.45 -1.51

-1.07 - 0.49 -1.02 - 0.34 -1.37 - 0.46 -3.13 - 0.12

0.46 0.32 0.32 0.07

1 month Layer/ Zone mm

CXL (mean SD); P value, compared to baseline

n = 29

CXL CRXL (mean SD); P value, compared to baseline

n = 28

CRXL† Diff.

between CXL and CRXL

Diff. between

CXL and

CRXL†

95% CI, diff. between

CXL and

CRXL†

P value, diff. between

CXL and

CRXL†

An

teri

or

0-2 2-6

6-10 10-12

31.4 7.5; P=0.00

26.3 5.4; P=0.00

18.6 4.0; P=0.00

23.4 9.1; P=0.45

10.4 8.2 3.4 -1.4

30.3 6.7; P=0.00

24.8 5.2; P=0.00

17.9 3.7; P=0.00

25.7 8.5; P=0.28

7.3 5.0 1.5 -1.4

1.08 1.58 0.68 -2.25

3.11 3.19 1.96 0.08

0.36 - 5.86 1.36 - 5.02 0.68 - 3.23 -4.38 - 4.54

0.27 0.01 0.03 0.97

Ce

ntr

e

0-2 2-6

6-10 10-12

20.3 5.6; P=0.00

16.2 3.3; P=0.00

12.3 3.0; P=0.01

16.0 5.0; P=0.83

7.2 4.8 1.6 -0.2

16.6 3.2; P=0.00

14.2 2.8; P=0.00

11.6 2.4; P=0.11

17.4 5.2; P=0.21

2.2 1.7 0.3 -1.0

3.78 1.96 0.64 -1.44

5.02 3.05 1.30 -0.76

2.92 - 7.12 1.79 - 4.31 0.18 - 2.41 -1.85 - 3.37

0.00 0.00 0.02 0.56

Po

ster

ior

0-2 2-6

6-10 10-12

14.0 4.2; P=0.00

11.9 2.2; P=0.00

10.6 2.4; P=0.04

12.9 3.3; P=0.37

3.3 1.8 0.9 -0.6

11.8 1.9; P=0.00

11.1 1.6; P=0.00

10.3 1.9; P=0.27

14.2 3.6; P=0.14

0.7 0.7 0.1 -0.8

2.28 0.80 0.36 -1.29

2.61 1.16 0.78 -0.21

1.03 - 4.19 0.33 - 2.00 -0.08 - 1.64 -1.43 - 1.86

0.00 0.01 0.08 0.80

6 months Layer/ Zone mm

CXL (mean SD); P value, compared to baseline

n = 28

CXL CRXL (mean SD); P value, compared to baseline

n = 29

CRXL† Diff.

between CXL and CRXL

Diff. between

CXL and

CRXL†

95% CI, diff. between

CXL and

CRXL†

P value, diff. between

CXL and

CRXL†

An

teri

or

0-2 2-6

6-10 10-12

29.1 6.7; P=0.00

23.4 4.6; P=0.00

18.0 3.4; P=0.00

27.1 7.8; P=0.22

8.1 5.2 2.7 1.8

24.2 4.8; P=0.01

20.7 4.9; P=0.04

16.8 3.8; P=0.23

27.0 7.9; P=0.87

1.3 1.0 0.5 -0.2

4.93 2.73 1.15 0.11

6.75 4.18 2.22 2.03

4.23 - 9.27 2.27 - 6.09 0.70 - 3.74 -1.70 - 5.76

0.00 0.00 0.01 0.28

Ce

ntr

e

0-2 2-6

6-10 10-12

17.1 3.7; P=0.00

13.9 2.6; P=0.00

12.1 2.3; P=0.01

17.8 4.8; P=0.12

4.0 2.5 1.4 1.4

14.8 3.1; P=0.29

12.8 2.6; P=0.29

11.6 2.7; P=0.37

18.1 5.0; P=0.77

0.5 0.4 0.3 -0.2

2.33 1.08 0.51 -0.35

3.47 2.09 1.10 1.64

1.72 - 5.23 0.79 - 3.38 -0.01 - 2.21 -0.58 - 3.85

0.00 0.00 0.05 0.15

Po

ster

ior

0-2 2-6

6-10 10-12

12.1 2.0; P=0.00

11.1 1.4; P=0.00

10.8 1.7; P=0.01

14.5 3.7; P=0.18

1.3 1.0 1.0 1.0

11.2 1.9; P=0.42

10.7 1.7; P=0.21

10.4 2.2; P=0.21

14.9 4.2; P=0.91

0.2 0.3 0.3 -0.1

0.88 0.45 0.41 -0.39

1.12 0.77 0.71 1.04

0.24 - 2.00 0.04 - 1.49 -0.09 - 1.51 -0.79 - 2.87

0.01 0.04 0.08 0.26

CXL; densitometry values are the paired difference between values at 1 month and baseline or 6 months and baseline.

CRXL†; densitometry values are the paired difference between values at 1 month and baseline or 6 months and baseline.

32

In both treatment groups, the increase in corneal light scattering at 1

month was more pronounced in the central cornea and leveled off towards

the periphery. In the CRXL group this difference was less marked. At 6

months the difference in corneal light scattering between central and

peripheral cornea was still persisted in the anterior and central layers of the

CXL group, whereas it could no longer be detected in the CRXL corneas

(Table 10).

4.6.2. Comparison between groups

The increase in corneal light scattering in the CXL group was more persistent

in the central anterior part of the cornea compared with the CRXL group

(regression from 1 to 6 months -2.7 7.1 vs. -5.9 4.8 GSU).

The increase in corneal light scattering in the CXL group was also larger at

both 1 and 6 months in the deeper layers of the central zones compared with

the CRXL group (difference between the groups at 1 month in the posterior

layer, zone 0-2 mm, 2.61 GSU, 95% CI 1.03 to 4.19, P=0.00; difference

between the groups at 6 months in the posterior layer, zone 0-2 mm, 1.12

GSU, 95% CI 0.24 to 2.00, P=0.01; Table 10).

4.6.3. Correlations

At 6 months the increase in central corneal light scattering showed a

correlation with the reduction in Kmax at the same time point (R=-0.31 for the

whole material). If the two treatments were analysed separately, the

correlation was stronger (R=-0.45 for CXL; R=-0.56 for CRXL; a linear

model y=-2.19x + 6.49 (R2=0.21) was found for CXL and y=-0.99x + 0.66

(R2=0.31) was found for CRXL). Similar correlations were also found

between the increase in central corneal light scattering and minimum

corneal radius of curvature (Rmin; R=0.49 for the whole material).

No correlations were found between the increases in central corneal light

scattering at 6 months and the changes in: mean keratometry value (Kmean),

BSCVA, SphEq or CH (only for the CXL group). CH assessed as the change in

CH using ORA® or ART, measured at the same time point.

Comparison of the increase in corneal light scattering, measured with

corneal densitometry (Paper IV) with the increased in corneal light

scattering measured manually from the Scheimpflug photography (Paper I),

showed a strong correlation (R=0.90; Figure 15).

33

Figure 15. Correlation between the changes in densitometry measured in Paper IV and the changes in corneal light scattering measured manually in Paper I.

4.7. IOP and biomechanical outcomes after CXL (Paper III)

In this substudy were all KC patients randomized to CXL and their healthy

control subjects included. One patient (a female) discontinued the study

after the intervention. She was excluded from all analyses, which left 29 eyes

of 28 patients (1 female) to be evaluated (see 4.1.). Furthermore, some

measurements were missing due to technical problems with the tonometers

(GAT, ORA® and ART).

At baseline, both ORA® and ART showed a significant difference in CH

between KC patients and control subjects (-2.67 2.55 mmHg, 99% CI -4.05

to -1.32, P<0.01 for ORA®; -1.09 1.92 mmHg, 99% CI -2.26 to 0.07, P=0.01

for ART; Table 11; Figure 16a and 16b).

PAPER I: CORNEAL LIGHT SCATTERING (MANUAL)

PAPER IV: DESITOMETRY (AUTOMATED)

R=0.90

34

At 1 and 6 months after CXL treatment, ART showed a significant increase

in CH in KC patients (1.2 2.4 mmHg, 99% CI -0.1 to 2.5, P=0.02 difference

between 1 month and baseline; 1.1 2.7 mmHg, 99% CI -0.3 to 2.6, P=0.04

difference between 6 months and baseline; Table 11; Figure 16a), whereas

ORA® did not (Table 11; Figure 16b).

The increase in CH after CXL treatment measured with ART leveled off

the difference in CH between KC patients and control subjects at 1 and 6

months (P=0.16 for 1 month; P=0.20 for 6 months; Table 11; Figure 16a).

The difference measured with ORA® persisted (P<0.01 for 1 month; P<0.01

for 6 months; Table 11; Figure 16b) and the CHORA values were unaltered by

CXL treatment. Also, the CRFORA values were unaltered through the study

(Table 11).

CH-ART

3

4

5

Pre 1-month 6-month

ART CXL

ART Ctrl

CH-ORA

7

8

9

10

11

12

Pre 1-month 6-month

ORA CXL

ORA Ctrl

Corneal hysteresis with ORA and ART after CXL

7

8

9

10

11

12

Pre 1-month 6-month

3

4

4

5

5

6

ORA CXL

ART CXL

Figure 16a and 16b shows CH measured with ART and ORA®

(mean SEM) on KC patients and their control subjects. Before CXL treatment patients had a significant lower CH compared to the control subjects with both ART and ORA®. After CXL treatment a significant increase in CH was measured with ART, but not with ORA®.

Figure 16c shows CH measured with ART and ORA® in the same figure. After CXL treatment ART measured a significant increased CH.

CHART

CHORA

CH measured with ART and ORA®




Patients Controls

Controls

Patients

ART

ORA®

16a.

16b.

16c.

35

Table 11. IOP and corneal biomechanical outcomes, CTmin and Kmean at baseline and at 1 and 6 months follow-up after CXL treatment, compared with control subjects.

Patients

Mean ± SD; P value compared to baseline (paired t test), the corresponding numbers of eyes are in parentheses, indicating the numbers of eyes available for comparison for that variable. ORA® ART GAT CTmin Kmean IOPORA CHORA CRFORA IOPART CHART IOPGAT

n (eyes ) Baseline

28 14.0 ± 2.7

28 8.5 ± 1.4

28 7.4 ± 1.6

28 10.6 ± 3.1

28 3.8 ± 1.6

29 11.0 ± 2.8

29 481 ± 41

29 45.9 ± 3.3

n (eyes) 1 month P value (n)

29 14.4 ± 2.5 0.30 (28)

29 7.9 ± 1.5 0.08 (28)

29 6.8 ± 1.5 0.10 (28)

28 11.1 ± 3.1 0.53 (27)

27 4.9 ± 2.1 0.02 (26)

28 11.8 ± 2.6 <0.01 (28)

29 457 ± 44 <0.01 (29)

29 45.8 ± 3.9 0.85 (29)

n (eyes) 6 months P value (n)

29 13.9 ± 2.6 0.67 (28)

29 8.5 ± 1.3 0.86 (28)

29 7.3 ± 1.6 0.65 (28)

29 10.2 ± 2.7 0.13 (28)

29 4.8 ± 2.0 0.04 (28)

29 11.1 ± 2.9 0.78 (29)

29 470 ± 42 0.01 (29)

29 45.3 ± 3.5 <0.01 (29)

Control subjects

Mean ± SD; P value compared to patients at the same time point (paired t test), the corresponding numbers of eyes are in parentheses, indicating the numbers of eyes available for comparison for that variable. ORA® ART GAT CTmin Kmean IOPORA CHORA CRFORA IOPART CHART IOPGAT

n (eyes) Baseline P value (n)

28 14.1 ± 2.8 0.86 (27)

28 11.1 ± 1.5 <0.01 (27)

28 10.6 ± 1.8 <0.01 (27)

27 10.8 ± 2.6 0.56 (26)

23 4.3 ± 1.7 0.01 (22)

29 12.0 ± 2.2 0.13 (29)

29 553 ± 33 <0.01 (29)

29 43.6 ± 1.4 <0.01 (29)

n (eyes) 1 month P value (n)

29 13.4 ± 2.8 0.08 (29)

29 11.1 ± 1.4 <0.01 (29)

29 10.4 ± 1.8 <0.01 (29)

28 10.0 ± 2.9 0.25 (27)

26 4.1 ± 2.0 0.16 (24)

29 11.7 ± 2.3 0.95 (28)

29 553 ± 32 <0.01 (29)

29 43.6 ± 1.4 0.01 (29)

n (eyes) 6 months P value (n)

29 13.6 ± 3.1 0.71 (29)

29 10.9 ± 1.5 <0.01 (29)

29 10.2 ± 1.8 <0.01 (29)

29 10.6 ± 2.9 0.59 (29)

29 4.3 ± 1.9 0.20 (29)

29 11.6 ± 2.6 0.54 (29)

29 554 ± 32 <0.01 (29)

29 43.6 ± 1.4 0.04 (29)

IOPORA; intraocular pressure measured with Ocular Response Analyzer. CHORA; corneal hysteresis measured with Ocular Response Analyzer. CRFORA; corneal resistance factor measured with Ocular Response Analyzer. IOPART; intraocular pressure measured with Applanation Resonance Tonometry -technology. CHART; corneal hysteresis measured with Applanation Resonance Tonometry -technology. IOPGAT; intraocular pressure measured with Goldmann Applanation tonometry. CTmin; corneal thickness at the thinnest point, obtained with the Pentacam® HR. Kmean; mean keratometry value, obtained with the Pentacam® HR, calculated as (K1+K2)/2.

In patients, the IOP did not change significantly with any method through

the study, except for an increase of 1.0 ± 1.7 mmHg at 1 month with GAT

(P<0.01; Table 11). There was a significantly decrease in CTmin by -24 ± 26

µm (P<0.01) at 1 month after treatment and by -11 ± 21 µm (P=0.01) 6

months after treatment, when compared to baseline (Table 11). A significant

change of -0.6 ± 0.7 D was seen in corneal curvature (measured as Kmean) at 6

months after treatment (P<0.01; Table 11).

36

5. Discussion

Corneal crosslinking with UV-A photoactivation of riboflavin (Wollensak et

al. 2003a) is an established routine method to halt the progression of ectasia

in KC patients by increasing the corneal rigidity (Goldich et al. 2010; Kolli &

Aslanides 2010). Several studies report convincing results (Wollensak et al.

2003a; Wollensak 2006; Raiskup-Wolf et al. 2008; Vinciguerra et al. 2009;

Goldich et al. 2012). A few standardized treatment protocols are available,

but the optimal treatment protocol has not yet been determined (Touboul et

al. 2012; Kymionis et al. 2012; Sorkin & Varssano 2014; Tomita et al. 2014),

maybe due to the lack of available tools to evaluate the treatments effects in

vivo.

There are a lot of in vitro studies confirming the stiffening effect of the

crosslinking treatment in corneal tissue (Spoerl et al. 1998; Wollensak et al.

2003b; Wollensak et al. 2004b; Spoerl et al. 2004a; Kohlhaas et al. 2006;

Wollensak & Redl 2008; Knox Cartwright et al. 2012; Dias et al. 2013). It is

generally accepted that the effect of the crosslinking treatment is caused by

an increase in corneal biomechanical strength, which remains stable over

time (Beshtawi et al. 2013). Still, repeated attempts to show an increase in

CH in vivo with ORA® after crosslinking treatment have failed (Sedaghat et

al. 2010; Spoerl et al. 2011; Gkika et al. 2012b; Goldich et al. 2012).

5.1. The CRXL-study (Paper II)

The idea behind the CRXL-study was to find a treatment able to stop the

progression of KC and to modify the corneal shape with improvement of the

visual quality in those patients with large refractive errors. In an attempt to

create a corneal flattening during the crosslinking treatment we used a 12.5

mm semi scleral rigid contact lens with a back surface curvature of 11.0 mm,

corresponding to a corneal refractive power of +33.5 D. The contact lens was

used to induce a pronounced corneal flattening during the crosslinking

treatment, with the aim to “lock” the cornea in this new position. The

selection of lens was based on the assumption that some degree of regression

was to be expected.

Eyes with more severe KC have been reported to respond better to corneal

crosslinking (Greenstein & Hersh 2013; Yam & Cheng 2013), but in this

study no differences in baseline values were found between the treatment

groups. Apart from the rigid contact lens in the CRXL group, we tried to keep

all other treatment variables equal between the CXL and the CRXL groups,

by installing riboflavin under the contact lens and by increasing the

irradiation time in the CRXL group to compensate for the blocking effect of

the contact lens.

37

Six months after the treatments we found no correlation between the

degree of KC and the treatment effect for either of the treatment groups.

After CXL treatment we found significant improvements of the SphEq and

BSCVA, and a decrease in Kmax at 6 months. This is in accordance with the

findings of Goldich et al. (2012). Over a period of 2 years, several studies

have shown a tendency for the corneal curvature to continuously decrease

(Raiskup-Wolf et al. 2008; Vinciguerra et al. 2009; Goldich et al. 2012), but

the mechanism underlying the continuous corneal flattening after corneal

crosslinking remains unclear (Goldich et al. 2012). In the present study we

saw no significant improvements of the variables examined up to 6 months

after CRXL treatment, which also makes subsequent improvements unlikely.

The study continues, however, and future analyses will answer this question.

Despite our attempt to install the riboflavin under the contact lens, the

riboflavin film may have differed between the treatment groups, because the

contact lens in the CRXL group was tightly aligned to corneal surface. This

may have affected the riboflavin film and accordingly the outcome. The

influence of the riboflavin film on the outcomes of the crosslinking effect has

been demonstrated in 2010 (a) by Wollensak et al. An alternate explanation

to the inferior treatment effect with CRXL could be that oxygen was blocked

by the rigid contact lens and did not reach the stroma, a known feature of

rigid contact lenses (Compañ et al. 2014). Also after accelerated corneal

crosslinking some studies have reported an inferior effect compared to

conventional corneal crosslinking, which may also be attributed to oxygen

depletion (Touboul et al. 2012; Kymionis et al. 2014). Whatever the reason

for the reduced corneal light scattering in the CRXL group is (lower UV-A

effect, reduced riboflavin concentration or oxygen depletion), they all will

result in a reduced oxidative stress and thereby a reduced corneal

crosslinking effect.

Other attempts to combine crosslinking treatment with other procedures

to change the corneal shape have shown various degrees of success

(Kanellopoulos & Binder 2007; Coskunseven et al. 2009; Kymionis et al.

2010; Cheema et al. 2012; Kremer et al. 2012; Vega-Estrada et al. 2012),

although the contribution of crosslinking to the summarized effect may not

have been fully elucidated (Cakir et al. 2013). Similar to us, Vega-Estrada et

al. (2012) were unsuccessful in maintaining the flattened corneal shape

induced by microwave treatment in KC corneas with crosslinking. Perhaps

the molecular nature of the crosslinking effect on the corneal stroma can

explain why the crosslinking treatment fails to maintain the changes in

corneal shape induced by mechanical compression and microwave

treatment, or maybe the regression is dose dependent.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kanellopoulos%20AJ%5BAuthor%5D&cauthor=true&cauthor_uid=17667633

http://www.ncbi.nlm.nih.gov/pubmed/?term=Binder%20PS%5BAuthor%5D&cauthor=true&cauthor_uid=17667633

38

5.2. Corneal light scattering after CXL and CRXL (Papers I+IV)

In Paper I we demonstrated that the alterations in corneal light scattering

after CXL treatment are readily assessable with non-invasive Scheimpflug

photography.

The increased corneal light scattering seen after the CXL treatment was

not uniformly distributed through the entire thickness of the cornea. In the

superficial 70 µm, corresponding to the epithelium, no increase in corneal

light scattering could be demonstrated, which aligns well to a previous in

vivo confocal microscopy investigation showing complete epithelial

restitution two weeks after treatment (Knappe et al. 2011).

In the stroma, the treatment effect was reduced with increasing corneal

depth, in accordance with the Lambert-Beer law (Wollensak et al. 2010a). At

1 month after treatment, our findings with a more pronounced increase in

corneal light scattering in the superficial stroma, gradually diminishing

down to zero at 240 µm depth is in accordance with this principle and

indicates that the corneal light scattering is proportional to the level of

irradiation at this time point. Our results are also in accordance with

confocal microscopy findings demonstrated by Knappe et al. (2011). They

showed that in the early healing phase, the space where the apoptotic

keratocytes used to be (keratocytes apoptosis is caused by the crosslinking

treatment; see 1.3.4.), are replaced by “large, strongly hyperreflective

structures” in the anterior and middle corneal stroma. At about 4 months, in

connection with the return of the keratocyte population, these structures

gradually lose their hyper reflectivity. These findings correlate well with our

findings of increased corneal light scattering.

At 6 months after treatment a significant increase in corneal light

scattering occurred at the depth of 240-340 µm. This finding corresponds to

the “demarcation line” described by Seiler & Hafezi. The demarcation line

does not appear to be directly correlated to the irradiation effect or to the

degree of keratocyte apoptosis, such as the earlier, more superficial light

scattering. Seiler & Hafezi have assumed that this demarcation line

represents “differences in the refractive index and/or reflection properties of

untreated versus X-linked corneal stroma” (Seiler & Hafezi 2006). Our

impression is that our finding is indeed a demarcation line. Behind this line

no effect on the corneal stroma can be detected.

The UV-A light source used in the CRXL-study, expect to have a

homogeneous irradiation in the whole 8 mm diameter of the treated area

and before each treatment, the effect of the UV-A light beam was verified

with a UV-metre. Despite this, there were significant differences in the light

scattering at the visual axis, compared to 1-2 mm from the visual axis at 1

month, with more pronounced light scattering at the visual axis. These

differences cannot be explained by artifacts induced by the UV-A light or the

illumination source of the Scheimpflug camera, nor can it be attributed to

39

differences in irradiation due to the corneal curvature, since we have made

individual corrections for each cornea, based on the distances of the UV-A

light and the Scheimpflug camera.

At the corneal apex the collagen structure is different in KC corneas

compared to normal corneas (Radner et al. 1998). KC corneas also have a

defective defence against reactive oxygen species, which is even less efficient

in the central part of the cornea (Kenney et al. 2000; Behndig et al. 2001;

Chwa et al. 2008). Irradiation of riboflavin molecules with UV-A light is

known to generate superoxide radicals (Segura-Aguilar 1993; Viggiano et al.

2003), and extracellular SOD, which is a major corneal scavenger of

superoxide radicals exists in lower amounts in the central cornea (Behndig et

al. 2001). It is possible that structural and biochemical differences between

the corneal centre and the periphery in KC may contribute to the light

scattering patterns seen in Paper I.

In Paper IV we showed that 6 months after treatment the increase in

corneal light scattering are related to the degree of corneal flattening. We

also showed that the increase in corneal light scattering, measured with

corneal densitometry (Paper IV) shows a strong correlation to the increased

corneal light scattering measured manually from the Scheimpflug

photography (Paper I). In Paper IV our impression is that the demarcation

line often seen after crosslinking treatment (Seiler & Hafezi 2006) is a part of

what we measured as an increase in corneal densitometry, since it occurs at

approximately the same depth and has a similar time course as the increase

of corneal light scattering in Paper I. Furthermore, the increased corneal

densitometry coincides with the zone where the biomechanical stability is

believed to increase after crosslinking treatment (Wollensak et al. 2003b;

Kohlhaas et al. 2006).

In accordance with the results in Paper II, where we showed that the

refractive effects of CRXL were inferior to those of CXL in some respects,

Paper IV showed that the increase in corneal densitometry was also less

pronounced in the CRXL group. Even if the increase in corneal densitometry

is lower after CRXL, the peripheral cornea shows more densitometry

increase relative to the corneal centre. Perhaps the corneal flattening

induced by the rigid contact lens caused a more uniform irradiation pattern

during the CRXL treatment, compared to the CXL treatment, where the

cornea was more steep and protruding.

In the CXL group, the increased densitometry goes deeper in the cornea

and has a more prolonged time course, compared to the CRXL group. This

finding is in accordance with the finding of a deeper demarcation line after

conventional corneal crosslinking compared to accelerated corneal

crosslinking reported by Kymionis et al. (2014). It may be that the oxygen

tension in the corneal stroma is higher during conventional crosslinking

treatment.

40

5.3. Corneal hysteresis after CXL (Paper III)

In Paper III we demonstrated an increase in CH in vivo with ART after CXL

treatment for KC. At baseline we found a significant difference in CH

between KC patients and control subjects with both ART and ORA®, but

after CXL treatment we only found a significant increase in CH with ART.

The CHORA value was unaltered after CXL treatment, in accordance with

previous studies (Sedaghat et al. 2010; Spoerl et al. 2011; Gkika et al. 2012b;

Goldich et al. 2012).

ART utilizes resonance technology and it is based on the assumption of

constant acoustic impedance in the corneal tissue. Theoretically, it would be

possible that the corneal acoustic impedance could be changed by the CXL

treatment, but then also the measured IOPART would have changed

accordingly, which did not occur. In addition, CHART was calculated as the

difference in IOP between the applanation and the removal phase (Hallberg

et al. 2004) and a change in the corneal acoustic impedance would not

change this difference, consequently it would not affect CHART.

In viscoelastic materials, the counterforce decreases at lower speed, i.e.

the difference in IOP between the applanation and the removal phase

decreases and CH at low speed will be smaller. ART measures CH at a lower

speed than ORA® and therefore have the CHART lower values.

By using a sensor tip with a radius of curvature of only 10 µm, Murayama

et al. (2004) have shown that resonance technology is useful to detect elastic

properties of small cell membranes. The sensor tip of the ART has a radius of

curvature of 8 mm. For measuring crosslinking treatment induced corneal

changes, ART would probably benefit from a sensor tip with a steeper

curvature (smaller radius of curvature). This would probably increase the

sensitivity of the CHART measurements, since the only tissue of interest is the

tissue in the corneal centre. The ORA® utilizes a standardized air puff that

causes a rather large area of corneal flattening. It is possible that the corneal

bending may occur partially outside the treated area, which could lead to

difficulties in detecting the stiffening effect from the crosslinking. On the

other hand, ORA® shows a significant difference in CH when comparing KC

patients with healthy control subjects. In KC patients the thickness of the

peripheral cornea is preserved while the central cornea is affected (Fontes et

al. 2011). Possibly, the inability of ORA® to detect increased CH after CXL

may be caused by the fact that the interlamellar cohesion of the corneal

stroma is unaffected by crosslinking, which have been demonstrated by

Wollensak et al. (2011). This, however, does not explain the increase in CH

seen with ART after CXL.

The decrease in CTmin and Kmean after CXL treatment cannot have

contributed to the increase in CHART, because a thinner and flatter cornea

would rather have decreased the CH (Narayanaswamy et al. 2011; Hwang et

al. 2013). It is generally accepted that a thick (rigid) cornea and a steep

41

cornea results in an overestimation of the applanation IOP and vice versa for

a thin cornea and a flat cornea (Whitacre & Stein 1993). Accordingly, KC

patients often have lower IOP due to thin corneas (Rask & Behndig 2006).

ART and ORA® are two tonometers developed to be less sensitive to different

corneal biomechanical properties, such as thickness and curvature (Luce

2005; Hallberg et al. 2004). We found that after CXL treatment, IOPART and

IOPORA were unaltered. At 1 month after CXL treatment IOPGAT was

significantly increased compared to baseline, despite the corneal thinning

seen at the same time point. We interpret the increased IOPGAT value as a

sign of increased corneal rigidity induced by the CXL treatment.

42

6. Conclusions

To summarize our results, eyes treated with CXL stabilized or improved, and

we can confirm the positive results with conventional crosslinking reported

by others (Raiskup-Wolf et al. 2008; Vinciguerra et al. 2009; Goldich et al.

2012). After CRXL, however, the results were less convincing. Corneal haze

(increased light scattering) was a consistent finding in both treatment

groups, which indicates that a crosslinking effect took place in all our

subjects. In accordance with the inferior refractive effects of CRXL, we

showed that the increase in corneal densitometry was also less pronounced

after this treatment.

We also found that the corneal densitometry increase differs in different

parts of the cornea and appears to be related to the crosslinking effect. This

suggests that the increase in densitometry is a potential indicator of the

treatment effect.

The increase in corneal light scattering measured manually showed a

strong correlation with the corneal light scattering measured with automated

corneal densitometry. Since the automated densitometry method is

considerably faster, the automated method has a greater potential to become

a clinically useful tool.

Finally, using ART technology, we have been able to assess an increase in

CH in vivo after CXL, which has not been demonstrated previously.

43

7. Future perspectives

Given the widespread use of CXL in today’s KC treatment arsenal, tools

capable of quantifying the treatment effect in vivo after CXL treatment has a

great potential value. Scheimpflug photography and corneal densitometry

appear to be useful tools to map the increased corneal light scattering after

corneal crosslinking treatment. The light scattering may give valuable

information about the corneal response to the crosslinking treatment. It

provides an impression of the stromal changes that occur after the treatment

and also the depth of the treatment effect. Likewise, a device based on ART -

technology can also become a useful tool in future assessment of corneal

biomechanical properties after corneal crosslinking treatment.

Further evaluation of the CRXL method and modification of the treatment

parameters will be needed. Possibly, the crosslinking effect could be

augmented, and the corneal riboflavin concentration and/or the oxygen

tension under the rigid contact lens could be increased. It could even be

considered to leave the contact lens in the eye for a longer time after the

treatment to stabilize the cornea in a flattened position.

44

8. Acknowledgements

I wish to express my sincere gratitude to the following persons who have

made the completion of this thesis possible:

First of all I want to thank…

Professor Christina Lindén, my main supervisor, for your always positive

attitudes, your energy and your enthusiasm. For teaching me a lot of

ophthalmology and for organizing all practical aspects regarding my PhD

studies.

Professor Anders Behndig, my co-supervisor, for your major knowledge

and interest in the project. For always being there, with me and the patients.

For your encouraging comments and support, whenever I needed it.

Associate professor Per Hallberg, my co-supervisor, for your technical

skills and your positive attitudes. For teaching me a lot about technique and

formulas.

I also want to thank…

My examiner and Head of Unit, Ophthalmology, Umeå University, Professor

Fatima Pedrosa Domellöf, for being a part in my progress during these

years.

Engineers Göran Mannberg, Kenji Claesson and Marcus Karlsson,

for technical support.

Research nurse Michael Hansson, for your help and support in the

beginning of the data collection.

Nurse Gertrud Skogman, for all help with scheduling the patients.

The administrators Birgit Johansson, Johanna Lidgren, Marika Falk,

Tomas Jacobsson and Birgitta Bäcklund, for all assistance with the

administration.

The staff at the Eye Clinic, Norrlands University Hospital, for helping me

with lot of things, necessary for the patients when they getting their

treatment.

45

The former and the present PhD students at the Department of

Clinical Sciences, Ophthalmology, for company and support.

Professor Lene Martin, for introducing me to the world of ophthalmology

research and for being my role model.

The former Head of the Eye Clinic, Västerbotten County Council, Elisabeth

Zackrisson and the former Department manager of the Eye Clinic,

Skellefteå, Eva Hedström Boman, for believing in me and for helping me

getting my first contact with the Department of Clinical Sciences,

Ophthalmology.

My former colleagues and staff at the Eye Clinic at Skellefteå hospital, for

making it possible for me, being away from the clinic, while I was working

with the CRXL-study.

My present colleagues and staff at the Rheumatology Clinic, Västerbotten

County Council, for making it possible for me, being away from the clinic,

while I was working on this thesis.

All the patients and healthy volunteers who participated in the CRXL-

study.

Last, but not least, my family…

My parents, Monica and Steneric Beckman, for always being there to

help me and my family when needed.

My husband Hans, for being a part of my life.

My children, Filip and Emma, for just being you, the very best in my life.

The CRXL-study and/or the work with this thesis were supported by grants

from the Västerbotten County Council (ALF and FoU), the KMA Fund

(Kronprinsessan Margaretas Arbetsnämnd för synskadade),

Ögonfonden, Stiftelsen JC Kempes Minnes Stipendiefond, the

European Regional Development Fund, Stiftelsen för medicinsk

forskning i Skellefteå and Riksföreningen för ögonsjukvård.

46

9. References

American Academy of Ophthalmology, section 8. External disease and

cornea 2010-2011; pp. 6-9 and 296-300.

Andreassen TT, Simonsen AH, Oxlund H. Biomechanical properties of

keratoconus and normal corneas. Exp Eye Res 1980; 31:435-441.

Ashwin PT, McDonnell PJ. Collagen cross-linkage: a comprehensive review

and directions for future research. Br J Ophthalmol 2010; 94:965-70.

Bawazeer AM, Hodge WG, Lorimer B. Atopy and keratoconus: a multivariate

analysis. Br J Ophthalmol 2000; 84:834-836.

Behndig A, Karlsson K, Johansson BO, Brännström T, Marklund SL.

Superoxide dismutase isoenzymes in the normal and diseased human

cornea. Invest Ophthalmol Vis Sci 2001; 42:2293-2296.

Belin MW, Ambrósio R. Scheimpflug imaging for keratoconus and ectatic

disease. Indian J Ophthalmol 2013; 61:401-406.

Beshtawi IM, O'Donnell C, Radhakrishnan H. Biomechanical properties of

corneal tissue after ultraviolet-A-riboflavin crosslinking. J Cataract Refract

Surg 2013; 39:451-462.

Brummer G, Littlechild S, McCall S, Zhang Y, Conrad GW. The role of

nonenzymatic glycation and carbonyls in collagen cross-linking for the

treatment of keratoconus. Invest Ophthalmol Vis Sci 2011; 52:6363-6369.

Cakir H, Pekel G, Perente I, Genç S. Comparison of intrastromal corneal ring

segment implantation only and in combination with collagen crosslinking for

keratoconus. Eur J Ophthalmol 2013; 23:629-634.

Camparini M, Cassinari P, Ferrigno L, Macaluso C. ETDRS-fast:

implementing psychophysical adaptive methods to standardized visual

acuity measurement with ETDRS charts. Invest Ophthalmol Vis Sci 2001;

42:1226-1231.

Cheema AS, Mozayan A, Channa P. Corneal collagen crosslinking in

refractive surgery. Curr Opin Ophthalmol 2012; 23:251-256.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ashwin%20PT%5BAuthor%5D&cauthor=true&cauthor_uid=19666925

http://www.ncbi.nlm.nih.gov/pubmed/?term=McDonnell%20PJ%5BAuthor%5D&cauthor=true&cauthor_uid=19666925

http://www.ncbi.nlm.nih.gov/pubmed/19666925






http://www.ncbi.nlm.nih.gov/pubmed/?term=Brummer%20G%5BAuthor%5D&cauthor=true&cauthor_uid=21724915

http://www.ncbi.nlm.nih.gov/pubmed/?term=Littlechild%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21724915

http://www.ncbi.nlm.nih.gov/pubmed/?term=McCall%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21724915

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=21724915

http://www.ncbi.nlm.nih.gov/pubmed/?term=Conrad%20GW%5BAuthor%5D&cauthor=true&cauthor_uid=21724915

http://www.ncbi.nlm.nih.gov/pubmed/?term=the+role+of+nonenzymatic+glycation+and+carbonyls

http://www.ncbi.nlm.nih.gov/pubmed/?term=Cakir%20H%5BAuthor%5D&cauthor=true&cauthor_uid=23483496

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pekel%20G%5BAuthor%5D&cauthor=true&cauthor_uid=23483496

http://www.ncbi.nlm.nih.gov/pubmed/?term=Perente%20I%5BAuthor%5D&cauthor=true&cauthor_uid=23483496

http://www.ncbi.nlm.nih.gov/pubmed/?term=Gen%C3%A7%20S%5BAuthor%5D&cauthor=true&cauthor_uid=23483496


http://www.ncbi.nlm.nih.gov/pubmed/?term=Cheema%20AS%5BAuthor%5D&cauthor=true&cauthor_uid=22569468

http://www.ncbi.nlm.nih.gov/pubmed/?term=Mozayan%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22569468

http://www.ncbi.nlm.nih.gov/pubmed/?term=Channa%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22569468

http://www.ncbi.nlm.nih.gov/pubmed/?term=Corneal+collagen+crosslinking+in+refractive+surgery+cheema

47

Chen S, Chen D, Wang J, Lu F, Wang Q, Qu J. Changes in ocular response

analyzer parameters after LASIK. J Refract Surg 2010; 26:279-288.

Chwa M, Atilano SR, Hertzog D, Zheng H, Langberg J, Kim DW, Kenney MC.

Hypersensitive response to oxidative stress in keratoconus corneal

fibroblasts. Invest Ophthalmol Vis Sci 2008; 49:4361-4369.

Cullen JF, Butler HG. Mongolism (Down’s Syndrome) and keratoconus. Br J

Ophthalmol 1963; 47:321-330.

ClinicalTrials.gov. Treatment of keratoconus with advanced corneal

crosslinking. NCT02425150. Accessed September 14, 2015.

https://clinicaltrials.gov/ct2/show/NCT02425150?term=NCT02425150&ra

nk=1.

Compañ V, Oliveira C, Aguilella-Arzo M, Mollá S, Peixoto-de-Matos SC,

González-Méijome JM. Oxygen diffusion and edema with modern scleral

rigid gas permeable contact lenses. Invest Ophthalmol Vis Sci 2014; 55:6421-

6429.

Coskunseven E, Jankov MR 2nd, Hafezi F, Atun S, Arslan E, Kymionis GD.

Effect of treatment sequence in combined intrastromal corneal rings and

corneal collagen crosslinking for keratoconus. J Cataract Refract Surg 2009;

35:2084-2091.

Daxer A, Fratzl P. Collagen fibril orientation in the human corneal stroma

and its implication in keratoconus. Invest Ophthalmol Vis Sci 1997; 38:121-

129.

Daxer A, Misof K, Grabner B, Ettl A, Fratzl P. Collagen fibrils in the human

corneal stroma: structure and aging. Invest Ophthalmol Vis Sci 1998;

39:644-648.

del Buey MA, Cristobal JA, Ascaso FJ, Lavilla L, Lanchares E. Biomechanical

properties of the cornea in Fuchs' corneal dystrophy. Invest Ophthalmol Vis

Sci 2009; 50:3199-3202.

Dhaliwal JS, Kaufman SC. Corneal collagen cross-linking: a confocal,

electron, and light microscopy study of eye bank corneas. Cornea 2009;

28:62-67.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Coskunseven%20E%5BAuthor%5D&cauthor=true&cauthor_uid=19969212

http://www.ncbi.nlm.nih.gov/pubmed/?term=Jankov%20MR%202nd%5BAuthor%5D&cauthor=true&cauthor_uid=19969212

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hafezi%20F%5BAuthor%5D&cauthor=true&cauthor_uid=19969212

http://www.ncbi.nlm.nih.gov/pubmed/?term=Atun%20S%5BAuthor%5D&cauthor=true&cauthor_uid=19969212

http://www.ncbi.nlm.nih.gov/pubmed/?term=Arslan%20E%5BAuthor%5D&cauthor=true&cauthor_uid=19969212

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kymionis%20GD%5BAuthor%5D&cauthor=true&cauthor_uid=19969212

http://www.ncbi.nlm.nih.gov/pubmed/?term=Effect+of+treatment+sequence+in+combined+intrastromal+corneal+rings+and+corneal

http://www.ncbi.nlm.nih.gov/pubmed/?term=Daxer%20A%5BAuthor%5D&cauthor=true&cauthor_uid=9501878

http://www.ncbi.nlm.nih.gov/pubmed/?term=Misof%20K%5BAuthor%5D&cauthor=true&cauthor_uid=9501878

http://www.ncbi.nlm.nih.gov/pubmed/?term=Grabner%20B%5BAuthor%5D&cauthor=true&cauthor_uid=9501878

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ettl%20A%5BAuthor%5D&cauthor=true&cauthor_uid=9501878

http://www.ncbi.nlm.nih.gov/pubmed/?term=Fratzl%20P%5BAuthor%5D&cauthor=true&cauthor_uid=9501878

http://www.ncbi.nlm.nih.gov/pubmed/?term=daxer+misof

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lavilla%20L%5BAuthor%5D&cauthor=true&cauthor_uid=19255149

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lanchares%20E%5BAuthor%5D&cauthor=true&cauthor_uid=19255149

48

Dias J, Diakonis VF, Kankariya VP, Yoo SH, Ziebarth NM. Anterior and

posterior corneal stroma elasticity after corneal collagen crosslinking

treatment. Exp Eye Res 2013; 116:58-62.

Doors M, Tahzib NG, Eggink FA, Berendschot TTJM, Webers CAB, Nuijts

RMMA. Use of anterior segment optical coherence tomography to study

corneal changes after collagen cross-linking. Am J Ophthalmol 2009;

148:844-851.

Doughty MJ, Zaman ML. Human corneal thickness and its impact on

intraocular pressure measures: A review and meta-analysis approach. Surv

Ophthalmol 2000; 44:367-408.

Dubbelman M, Weeber HA, van der Heijde RG, Völker-Dieben HJ. Radius

and asphericity of the posterior corneal surface determined by corrected

Scheimpflug photography. Acta Ophthalmol Scand 2002; 80:379-383.

Dupps WJ Jr, Wilson SE. Biomechanics and wound healing in the cornea.

Exp Eye Res 2006; 83:709-720.

Ehlers N, Bramsen T, Sperling S. Applanation tonometry and central corneal

thickness. Acta Ophthalmol (Copenh) 1975; 53:34-43.

Elder MJ. Leber congenital amaurosis and its association with keratoconus

and keratoglobus. J Pediatr Ophthalmol Strabismus 1994; 31:38-40.

Eklund A, Hallberg P, Lindén C, Lindahl OA. An applanation resonator

sensor for measuring intraocular pressure using combined continuous force

and area measurement. Invest Ophthalmol Vis Sci 2003; 44:3017-3024.

Ertan A, Muftuoglu O. Keratoconus clinical findings according to different

age and gender groups. Cornea 2008; 27:1109-1113.

Eysteinsson T, Jonasson F, Sasaki H, Arnarsson A, Sverrisson T, Sasaki K,

Stefánsson E; Reykjavik Eye Study Group. Central corneal thickness, radius

of the corneal curvature and intraocular pressure in normal subjects using

non-contact techniques: Reykjavik Eye Study. Acta Ophthalmol Scand 2002;

80:11-15.

Fontes BM, Ambrosio R, Jr., Velarde GC, Nose W. Ocular response analyzer

measurements in keratoconus with normal central corneal thickness

compared with matched normal control eyes. J Refract Surg 2011; 27:209-

215.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Dubbelman%20M%5BAuthor%5D&cauthor=true&cauthor_uid=12190779

http://www.ncbi.nlm.nih.gov/pubmed/?term=Weeber%20HA%5BAuthor%5D&cauthor=true&cauthor_uid=12190779

http://www.ncbi.nlm.nih.gov/pubmed/?term=van%20der%20Heijde%20RG%5BAuthor%5D&cauthor=true&cauthor_uid=12190779

http://www.ncbi.nlm.nih.gov/pubmed/?term=V%C3%B6lker-Dieben%20HJ%5BAuthor%5D&cauthor=true&cauthor_uid=12190779


http://www.ncbi.nlm.nih.gov/pubmed/?term=Eysteinsson%20T%5BAuthor%5D&cauthor=true&cauthor_uid=11906297

http://www.ncbi.nlm.nih.gov/pubmed/?term=Jonasson%20F%5BAuthor%5D&cauthor=true&cauthor_uid=11906297

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sasaki%20H%5BAuthor%5D&cauthor=true&cauthor_uid=11906297

http://www.ncbi.nlm.nih.gov/pubmed/?term=Arnarsson%20A%5BAuthor%5D&cauthor=true&cauthor_uid=11906297

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sverrisson%20T%5BAuthor%5D&cauthor=true&cauthor_uid=11906297

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sasaki%20K%5BAuthor%5D&cauthor=true&cauthor_uid=11906297

http://www.ncbi.nlm.nih.gov/pubmed/?term=Stef%C3%A1nsson%20E%5BAuthor%5D&cauthor=true&cauthor_uid=11906297

http://www.ncbi.nlm.nih.gov/pubmed/?term=Reykjavik%20Eye%20Study%20Group%5BCorporate%20Author%5D


49

Gkika MG, Labiris G, Kozobolis VP. Tonometry in keratoconic eyes before

and after riboflavin/UVA corneal collagen crosslinking using three different

tonometers. Eur J Ophthalmol 2012a; 22:142-152.

Gkika M, Labiris G, Giarmoukakis A, Koutsogianni A, Kozobolis V.

Evaluation of corneal hysteresis and corneal resistance factor after corneal

cross-linking for keratoconus. Graefes Arch Clin Exp Ophthalmol 2012b;

250:565-573.

Goldich Y, Barkana Y, Gerber Y, Rasko A, Morad Y, Harstein M, Avni I,

Zadok D. Effect of diabetes mellitus on biomechanical parameters of the

cornea. J Cataract Refract Surg 2009; 35:715-719.

Goldich Y, Marcovich AL, Barka Y, Avni I, Zadok D. Safety of corneal

collagen cross-linking with UV-A and riboflavin in progressive keratoconus.

Cornea 2010; 29:409-411.

Goldich Y, Marcovich AL, Barkana Y, Mandel Y, Hirsh A, Morad Y, Avni I,

Zadok D. Clinical and corneal biomechanical changes after collagen cross-

linking with riboflavin and UV irradiation in patients with progressive

keratoconus: results after 2 years of follow-up. Cornea 2012; 31:609-614.

Goldmann H. Applanation tonometry, In: Newell FW, ed. Glaucoma,

Transactions of the second conference. New York. Josiah Macy Jr.

Foundation 1957; pp. 167-220.

Gordon-Shaag A, Millodot M, Shneor E, Liu Y. The Genetic and

Environmental Factors for Keratoconus. Biomed Res Int 2015; Article ID

795738. Published online 2015 May 17.

Gore DM, Shortt AJ, Allan BD. New clinical pathways for keratoconus. Eye

(Lond) 2013; 27:329-339.

Greenstein SA, Fry KL, Bhatt J, Hersh PS. Natural history of corneal haze

after collagen crosslinking for keratoconus and corneal ectasia: Scheimpflug

and biomicroscopic analysis. J Cataract Refract Surg 2010; 36:2105-2114.

Greenstein SA, Hersh PS. Characteristics influencing outcomes of corneal

collagen crosslinking for keratoconus and ectasia: implications for patient

selection. J Cataract Refract Surg 2013; 39:1133-1140.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Rasko%20A%5BAuthor%5D&cauthor=true&cauthor_uid=19304094

http://www.ncbi.nlm.nih.gov/pubmed/?term=Morad%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=19304094

http://www.ncbi.nlm.nih.gov/pubmed/?term=Harstein%20M%5BAuthor%5D&cauthor=true&cauthor_uid=19304094

http://www.ncbi.nlm.nih.gov/pubmed/?term=Avni%20I%5BAuthor%5D&cauthor=true&cauthor_uid=19304094

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zadok%20D%5BAuthor%5D&cauthor=true&cauthor_uid=19304094

http://www.ncbi.nlm.nih.gov/pubmed/?term=Mandel%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22378112

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hirsh%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22378112

http://www.ncbi.nlm.nih.gov/pubmed/?term=Morad%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22378112

http://www.ncbi.nlm.nih.gov/pubmed/?term=Avni%20I%5BAuthor%5D&cauthor=true&cauthor_uid=22378112

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zadok%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22378112

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449900/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449900/

http://www.ncbi.nlm.nih.gov/pubmed/?term=Gore%20DM%5BAuthor%5D&cauthor=true&cauthor_uid=23258309

http://www.ncbi.nlm.nih.gov/pubmed/?term=Shortt%20AJ%5BAuthor%5D&cauthor=true&cauthor_uid=23258309

http://www.ncbi.nlm.nih.gov/pubmed/?term=Allan%20BD%5BAuthor%5D&cauthor=true&cauthor_uid=23258309



http://www.ncbi.nlm.nih.gov/pubmed/?term=Greenstein%20SA%5BAuthor%5D&cauthor=true&cauthor_uid=23889865

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hersh%20PS%5BAuthor%5D&cauthor=true&cauthor_uid=23889865

http://www.ncbi.nlm.nih.gov/pubmed/?term=characteristics+influencing+outcomes+of+corneal+collagen

50

Gunes A, Tok L, Tok O, Seyrek L. The Youngest Patient with Bilateral

Keratoconus Secondary to Chronic Persistent Eye Rubbing. Semin

Ophthalmol 2015; 21:1-3.

Hafezi F. Smoking and corneal biomechanics. Ophthalmology 2009;

116:2259.

Hager A, Wegscheider K, Wiegand W. Changes of extracellular matrix of the

cornea in diabetes mellitus. Graefes Arch Clin Exp Ophthalmol 2009;

247:1369-1374.

Hallberg P, Lindén C, Lindahl OA, Bäcklund T, Eklund A. Applanation

resonance tonometry for intraocular pressure in humans. Physiol Meas

2004; 25:1053-1065.

Hallberg P, Eklund A, Backlund T, Lindén C. Clinical evaluation of

applanation resonance tonometry: a comparison with Goldmann

applanation tonometry. J Glaucoma 2007; 16:88-93.

He X, Spoerl E, Tang J, Liu J. Measurement of corneal changes after collagen

crosslinking using a noninvasive ultrasound system. J Cataract Refract Surg

2010; 36:1207-1212.

Hwang HS, Park SK, Kim MS. The biomechanical properties of the cornea

and anterior segment parameters. BMC Ophthalmol 2013; 13:49.

Jafri B, Lichter H, Stulting RD. Asymmetric keratoconus attributed to eye

rubbing. Cornea 2004; 23:560-564.

Jenkins A D, Kratochvíl P, Stepto RFT, Suter UW. Glossary of basic terms in

polymer science. Pure & Appl. Chem 1996; 68:2287-2311.

Jóhannesson G, Hallberg P, Eklund A, Lindén C. Introduction and clinical

evaluation of servo-controlled applanation resonance tonometry. Acta

Ophthalmol 2012; 90:677-682.

Jonas JB, Nangia V, Matin A, Kulkarni M, Bhojwani K. Prevalence and

associations of keratoconus in rural Maharashtra in central India: the

Central India Eye and Medical Study. Am J Ophthalmol 2009; 148:760-765.



51

Jonuscheit S, Doughty MJ, Martin R, Rió-Cristóbal A, Cruikshank V, Lang S.

Peripheral nasal-temporal corneal asymmetry in relation to corneal

thickness: a Scheimpflug imaging study. Ophthalmic Physiol Opt 2015;

35:45-51.

Jun AS, Cope L, Speck C, Feng X, Lee S, Meng H, Hamad A, Chakravarti S.

Subnormal cytokine profile in the tear fluid of keratoconus patients. PLoS

One 2011; 6:e16437.

Kamaev P, Friedman MD, Sherr E, Muller D. Photochemical kinetics of

corneal cross-linking with riboflavin. Invest Ophthalmol Vis Sci 2012;

53:2360-2367.

Kamiya K, Shimizu K, Ohmoto F. Effect of aging on corneal biomechanical

parameters using the Ocular Response Analyzer. J Refract Surg 2009;

25:888-893.

Kanellopoulos AJ, Binder PS. Collagen cross-linking (CCL) with sequential

topography-guided PRK: a temporizing alternative for keratoconus to

penetrating keratoplasty. Cornea 2007; 26:891-895.

Kennedy RH, Bourne WM, Dyer JA. A 48-year clinical and epidemiologic

study of keratoconus. Am J Ophthalmol 1986; 101:267-273.

Kenney MC, Brown DJ, Rajeev B. Everett Kinsey Lecture. The elusive causes

of keratoconus: a working hypothesis. CLAO J 2000; 26:10-13.

Knappe S, Stachs O, Zhivov A, Hovakimyan M, Guthoff R. Results of

confocal microscopy examinations after collagen cross-linking with

riboflavin and UVA light in patients with progressive keratoconus.

Ophthalmologica 2011; 225:95-104.

Knox Cartwright NE, Tyrer JR, Marshall J. In vitro quantification of the

stiffening effect of corneal cross-linking in the human cornea using radial

shearing speckle pattern interferometry. J Refract Surg 2012; 28:503-508.

Kohlhaas M, Spoerl E, Schilde T, Unger G, Wittig C, Pillunat LE.

Biomechanical evidence of the distribution of cross-links in corneas treated

with riboflavin and ultraviolet A light. J Cataract Refract Surg 2006; 32:279-

283.

Kolli S, Aslanides IM. Safety and efficacy of collagen crosslinking for the

treatment of keratoconus. Expert Opin Drug Saf 2010; 9:949-957.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kamaev%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22427580

http://www.ncbi.nlm.nih.gov/pubmed/?term=Friedman%20MD%5BAuthor%5D&cauthor=true&cauthor_uid=22427580

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sherr%20E%5BAuthor%5D&cauthor=true&cauthor_uid=22427580

http://www.ncbi.nlm.nih.gov/pubmed/?term=Muller%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22427580


http://www.ncbi.nlm.nih.gov/pubmed/?term=Kanellopoulos%20AJ%5BAuthor%5D&cauthor=true&cauthor_uid=17667633

http://www.ncbi.nlm.nih.gov/pubmed/?term=Binder%20PS%5BAuthor%5D&cauthor=true&cauthor_uid=17667633

http://www.ncbi.nlm.nih.gov/pubmed/?term=Collagen+cross-linking+(CCL)+with+sequential

52

Krachmer JH, Feder RS, Belin MW. Keratoconus and related

noninflammatory corneal thinning disorders. Survey of ophthalmology 1984;

28:293-322.

Kremer I, Aizenman I, Lichter H, Shayer S, Levinger S. Simultaneous

wavefront-guided photorefractive keratectomy and corneal collagen

crosslinking after intrastromal corneal ring segment implantation for

keratoconus. J Cataract Refract Surg 2012; 38:1802-1807.

Krumeich JH, Daniel J, Knulle A. Live-epikeratophakia for keratoconus. J

Cataract Refract Surg 1998; 24:456-463.

Kymionis GD, Kontadakis GA, Naoumidi TL, Kazakos DC, Giapitzakis I,

Pallikaris IG. Conductive keratoplasty followed by collagen cross-linking

with riboflavin-UV-A in patients with keratoconus. Cornea 2010; 29:239-

243.

Kymionis GD, Grentzelos MA, Kounis GA, Diakonis VF, Limnopoulou AN,

Panagopoulou SI. Combined transepithelial phototherapeutic keratectomy

and corneal collagen cross-linking for progressive keratoconus.

Ophthalmology 2012; 119:1777-1784.

Kymionis GD, Tsoulnaras KI, Grentzelos MA, Plaka AD, Mikropoulos DG,

Liakopoulos DA, Tsakalis NG, Pallikaris IG. Corneal stroma demarcation line

after standard and high-intensity collagen crosslinking determined with

anterior segment optical coherence tomography. J Cataract Refract Surg

2014; 40:736-740.

Lema I, Sobrino T, Dur´an JA, Brea D, D´ıez-Feijoo E. Subclinical

keratoconus and inflammatory molecules from tears. Br J Ophthalmol 2009;

93:820-824.

Li X, Rabinowitz YS, Rasheed K, Yang H. Longitudinal study of the normal

eyes in unilateral keratoconus patients. Ophthalmology 2004; 111:440-446.

Lim N, Vogt U. Characteristics and functional outcomes of 130 patients with

keratoconus attending a specialist contact lens clinic. Eye 2002; 16:54-59.

Luce DA. Determining in vivo biomechanical properties of the cornea with

an ocular response analyzer. J Cataract Refract Surg 2005; 31:156-162.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kremer%20I%5BAuthor%5D&cauthor=true&cauthor_uid=22858060

http://www.ncbi.nlm.nih.gov/pubmed/?term=Aizenman%20I%5BAuthor%5D&cauthor=true&cauthor_uid=22858060

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lichter%20H%5BAuthor%5D&cauthor=true&cauthor_uid=22858060

http://www.ncbi.nlm.nih.gov/pubmed/?term=Shayer%20S%5BAuthor%5D&cauthor=true&cauthor_uid=22858060

http://www.ncbi.nlm.nih.gov/pubmed/?term=Levinger%20S%5BAuthor%5D&cauthor=true&cauthor_uid=22858060



http://www.ncbi.nlm.nih.gov/pubmed/?term=Kontadakis%20GA%5BAuthor%5D&cauthor=true&cauthor_uid=20023583

http://www.ncbi.nlm.nih.gov/pubmed/?term=Naoumidi%20TL%5BAuthor%5D&cauthor=true&cauthor_uid=20023583

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kazakos%20DC%5BAuthor%5D&cauthor=true&cauthor_uid=20023583

http://www.ncbi.nlm.nih.gov/pubmed/?term=Giapitzakis%20I%5BAuthor%5D&cauthor=true&cauthor_uid=20023583

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pallikaris%20IG%5BAuthor%5D&cauthor=true&cauthor_uid=20023583



http://www.ncbi.nlm.nih.gov/pubmed/?term=Grentzelos%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=22683058

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kounis%20GA%5BAuthor%5D&cauthor=true&cauthor_uid=22683058

http://www.ncbi.nlm.nih.gov/pubmed/?term=Diakonis%20VF%5BAuthor%5D&cauthor=true&cauthor_uid=22683058

http://www.ncbi.nlm.nih.gov/pubmed/?term=Limnopoulou%20AN%5BAuthor%5D&cauthor=true&cauthor_uid=22683058

http://www.ncbi.nlm.nih.gov/pubmed/?term=Panagopoulou%20SI%5BAuthor%5D&cauthor=true&cauthor_uid=22683058



http://www.ncbi.nlm.nih.gov/pubmed/?term=Tsoulnaras%20KI%5BAuthor%5D&cauthor=true&cauthor_uid=24630796

http://www.ncbi.nlm.nih.gov/pubmed/?term=Grentzelos%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=24630796

http://www.ncbi.nlm.nih.gov/pubmed/?term=Plaka%20AD%5BAuthor%5D&cauthor=true&cauthor_uid=24630796

http://www.ncbi.nlm.nih.gov/pubmed/?term=Mikropoulos%20DG%5BAuthor%5D&cauthor=true&cauthor_uid=24630796

http://www.ncbi.nlm.nih.gov/pubmed/?term=Liakopoulos%20DA%5BAuthor%5D&cauthor=true&cauthor_uid=24630796

http://www.ncbi.nlm.nih.gov/pubmed/?term=Tsakalis%20NG%5BAuthor%5D&cauthor=true&cauthor_uid=24630796

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pallikaris%20IG%5BAuthor%5D&cauthor=true&cauthor_uid=24630796

53

Luce D. Methodology for corneal compensated IOP and corneal resistance

factor for the Reichert Ocular Response Analyzer. American Research in

Vision and Ophthalmology (ARVO). Fort Lauderdale, FL, USA: Invest

Ophthalmol Vis Sci 2006; 47: E-Abstract 2266.

Maeda N, Klyce SD, Smolek MK, Thompson HW. Automated keratoconus

screening with corneal topography analysis. Invest Ophthalmol Vis Sci 1994;

35:2749-2757.

Malik NS, Moss SJ, Ahmed N, Furth AJ, Wall RS, Meek KM. Ageing of the

human corneal stroma: structural and biochemical changes. Biochim

Biophys Acta 1992; 1138:222-228.

Mazzotta C, Traversi C, Baiocchi S, Caporossi O, Bovone C, Sparano MC,

Balestrazzi A, Caporossi A. Corneal healing after riboflavin ultraviolet-A

collagen cross-linking determined by confocal laser scanning microscopy in

vivo: early and late modifications. Am J Ophthalmol 2008; 146:527-533.

Mazzotta C, Caporossi T, Denaro R, Bovone C, Sparano C, Paradiso A,

Baiocchi S, Caporossi A. Morphological and functional correlations in

riboflavin UV A corneal collagen cross-linking for keratoconus. Acta

Ophthalmol 2012; 90:259-265.

McMonnies CW. Inflammation and keratoconus. OptomVis Sci 2015;

92:e35-41.

Meek KM, Hayes S. Corneal cross-linking--a review. Ophthalmic Physiol Opt

2013; 33:78-93.

Murayama Y, Constantinou CE, Omata S. Micro-mechanical sensing

platform for the characterization of the elastic properties of the ovum via

uniaxial measurement. J Biomech 2004; 37:67-72.

Narayanaswamy A, Chung RS, Wu RY, Park J, WongWL, Saw SM,. Wong TY,

Aung T. Determinants of corneal biomechanical properties in an adult

Chinese population. Ophthalmology 2011; 118:1253-1259.

Ortiz D, Piñero D, Shabayek MH, Arnalich-Montiel F, Alió JL. Corneal

biomechanical properties in normal, post-laser in situ keratomileusis, and

keratoconic eyes. J Cataract Refract Surg 2007; 33:1371-1375.

Pacifici RE, Davies KJ. Protein degradation as an index of oxidative stress.

Methods Enzymol 1990; 186:485-502.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Malik%20NS%5BAuthor%5D&cauthor=true&cauthor_uid=1547284

http://www.ncbi.nlm.nih.gov/pubmed/?term=Moss%20SJ%5BAuthor%5D&cauthor=true&cauthor_uid=1547284

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ahmed%20N%5BAuthor%5D&cauthor=true&cauthor_uid=1547284

http://www.ncbi.nlm.nih.gov/pubmed/?term=Furth%20AJ%5BAuthor%5D&cauthor=true&cauthor_uid=1547284

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wall%20RS%5BAuthor%5D&cauthor=true&cauthor_uid=1547284




http://www.ncbi.nlm.nih.gov/pubmed/?term=Mazzotta%20C%5BAuthor%5D&cauthor=true&cauthor_uid=20456255

http://www.ncbi.nlm.nih.gov/pubmed/?term=Caporossi%20T%5BAuthor%5D&cauthor=true&cauthor_uid=20456255

http://www.ncbi.nlm.nih.gov/pubmed/?term=Denaro%20R%5BAuthor%5D&cauthor=true&cauthor_uid=20456255

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bovone%20C%5BAuthor%5D&cauthor=true&cauthor_uid=20456255

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sparano%20C%5BAuthor%5D&cauthor=true&cauthor_uid=20456255

http://www.ncbi.nlm.nih.gov/pubmed/?term=Paradiso%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20456255

http://www.ncbi.nlm.nih.gov/pubmed/?term=Baiocchi%20S%5BAuthor%5D&cauthor=true&cauthor_uid=20456255

http://www.ncbi.nlm.nih.gov/pubmed/?term=Caporossi%20A%5BAuthor%5D&cauthor=true&cauthor_uid=20456255

http://www.ncbi.nlm.nih.gov/pubmed/?term=morphological+and+functional+correlations+of+riboflavin.

http://www.ncbi.nlm.nih.gov/pubmed/?term=morphological+and+functional+correlations+of+riboflavin.


http://www.ncbi.nlm.nih.gov/pubmed/?term=Hayes%20S%5BAuthor%5D&cauthor=true&cauthor_uid=23406488

http://www.ncbi.nlm.nih.gov/pubmed/?term=meek+corneal+cross+linking+a+review

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pacifici%20RE%5BAuthor%5D&cauthor=true&cauthor_uid=2233315



54

Park J, Gritz DC. Evolution in the use of intrastromal corneal ring segments

for corneal ectasia. Curr Opin Ophthalmol 2013; 24:296-301.

Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998; 42:297-319.

Radner W, Zehetmayer M, Skorpik C, Mallinger R. Altered organization of

collagen in the apex of keratoconus corneas. Ophthalmic Res 1998; 30:327-

332.

Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen crosslinking with

riboflavin and ultraviolet-A light in keratoconus: long-term results. J


Rask G, Behndig A. Effects of corneal thickness, curvature, astigmatism and

direction of gaze on Goldmann applanation tonometry readings. Ophthalmic

Res 2006; 38:49-55.

Richoz O, Hammer A, Tabibian D, Gatzioufas Z, Hafezi F. The biomechanical

effect of corneal collagen cross-linking (CXL) with riboflavin and UV-A is

oxygen dependent. Transl Vis Sci Technol 2013; 2:6.

Ryan DS, Coe CD, Howard RS, Edwards JD, Bower KS. Corneal

biomechanics following epi-LASIK. J Refract Surg 2011; 27:458-464.

Sady C, Khosrof S, Nagaraj R. Advanced Maillard reaction and crosslinking

of corneal collagen in diabetes. Biochem Biophys Res Commun 1995;

214:793-797.

Saini JS, Saroha V, Singh P, Sukhija JS, Jain AK. Keratoconus in Asian eyes

at a tertiary eye care facility. Clin Exp Optom 2004; 87:97-101.

Salomão MQ, Chaurasia SS, Sinha-Roy A, Ambrósio R Jr, Esposito A,

Sepulveda R, Agrawal V, Wilson SE. Corneal wound healing after ultraviolet-

A/riboflavin collagen cross-linking: a rabbit study. J Refract Surg 2011;

27:401-407.

Sedaghat M, Naderi M, Zarei-Ghanavati M. Biomechanical parameters of the

cornea after collagen crosslinking measured by waveform analysis. J


Segura-Aguilar J. A new direct method for determining superoxide

dismutase activity by measuring hydrogen peroxide formation. Chem Biol

Interact 1993; 86:69-78.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Park%20J%5BAuthor%5D&cauthor=true&cauthor_uid=23665526

http://www.ncbi.nlm.nih.gov/pubmed/?term=Gritz%20DC%5BAuthor%5D&cauthor=true&cauthor_uid=23665526


http://www.ncbi.nlm.nih.gov/pubmed/?term=Sady%20C%5BAuthor%5D&cauthor=true&cauthor_uid=7575546

http://www.ncbi.nlm.nih.gov/pubmed/?term=Khosrof%20S%5BAuthor%5D&cauthor=true&cauthor_uid=7575546

http://www.ncbi.nlm.nih.gov/pubmed/?term=Nagaraj%20R%5BAuthor%5D&cauthor=true&cauthor_uid=7575546

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sady+khosrof

http://www.ncbi.nlm.nih.gov/pubmed/?term=Salom%C3%A3o%20MQ%5BAuthor%5D&cauthor=true&cauthor_uid=21162471

http://www.ncbi.nlm.nih.gov/pubmed/?term=Chaurasia%20SS%5BAuthor%5D&cauthor=true&cauthor_uid=21162471

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sinha-Roy%20A%5BAuthor%5D&cauthor=true&cauthor_uid=21162471

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ambr%C3%B3sio%20R%20Jr%5BAuthor%5D&cauthor=true&cauthor_uid=21162471

http://www.ncbi.nlm.nih.gov/pubmed/?term=Esposito%20A%5BAuthor%5D&cauthor=true&cauthor_uid=21162471

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sepulveda%20R%5BAuthor%5D&cauthor=true&cauthor_uid=21162471

http://www.ncbi.nlm.nih.gov/pubmed/?term=Agrawal%20V%5BAuthor%5D&cauthor=true&cauthor_uid=21162471

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wilson%20SE%5BAuthor%5D&cauthor=true&cauthor_uid=21162471

http://www.ncbi.nlm.nih.gov/pubmed/?term=wound+healing+after+ultraviolet-A%2Friboflavin

55

Seiler T, Hafezi F. Corneal cross-linking-induced stromal demarcation line.

Cornea 2006; 25:1057-1059.

Shah S, Laiquzzaman M, Bhojwani R, Mantry S, Cunliffe I. Assessment of the

biomechanical properties of the cornea with the Ocular Response Analyzer in

normal and keratoconic eyes. Invest Ophthalmol Vis Sci 2007; 48:3026-

3031.

Sharif KW, Casey TA, Colart J. Prevalence of mitral valve prolapse in

keratoconus patients. J R Soc Med 1992; 85:446-448.

Snibson GR. Collagen cross-linking: a new treatment paradigm in corneal

disease - a review. Clin Experiment Ophthalmol 2010; 38:141-53.

Sorkin N, Varssano D. Corneal collagen crosslinking: A systematic Review.

Ophthalmologica 2014; 232:10-27.

Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal tissue. Exp

Eye Res 1998; 66:97-103.

Spoerl E, Wollensak G, Dittert DD, Seiler T. Thermomechanical behavior of

collagen-cross-linked porcine cornea. Ophthalmologica 2004a; 218:136-140.

Spoerl E, Wollensak G, Seiler T. Increased resistance of crosslinked cornea

against enzymatic digestion. Curr Eye Res 2004b; 29:35-40.

Spoerl E, Terai N, Scholz F, Raiskup F, Pillunat LE. Detection of

biomechanical changes after corneal cross-linking using Ocular Response

Analyzer software. J Refract Surg 2011; 27:452-457.

Tan DT, Por YM. Current treatment options for corneal ectasia. Curr opin

ophthalmol 2007; 18:284-289.

Tomita M, Mita M, Huseynova T. Accelerated versus conventional corneal

collagen crosslinking. J Cataract Refract Surg 2014; 40:1013-1020.

Touboul D, Efron N, Smadja D, Praud D, Malet F, Colin J. Corneal confocal

microscopy following conventional, transepithelial, and accelerated corneal

collagen cross-linking procedures for keratoconus. J Refract Surg 2012;

28:769-776.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Snibson%20GR%5BAuthor%5D&cauthor=true&cauthor_uid=20398104


http://www.ncbi.nlm.nih.gov/pubmed/?term=Spoerl%20E%5BAuthor%5D&cauthor=true&cauthor_uid=9533835

http://www.ncbi.nlm.nih.gov/pubmed/?term=Huhle%20M%5BAuthor%5D&cauthor=true&cauthor_uid=9533835

http://www.ncbi.nlm.nih.gov/pubmed/?term=Seiler%20T%5BAuthor%5D&cauthor=true&cauthor_uid=9533835



56

Tuft SJ, Moodaley LC, Gregory WM, Davison CR, Buckley RJ. Prognostic

factors for the progression of keratoconus. Ophthalmology 1994; 101:439-

447.

Vega-Estrada A, Alió JL, Plaza Puche AB, Marshall J. Outcomes of a new

microwave procedure followed by accelerated cross-linking for the treatment

of keratoconus: a pilot study. J Refract Surg 2012; 28:787-793.

Viggiano A, Viggiano D, Viggiano A, De Luca B. Quantitative histochemical

assay for superoxide dismutase in rat brain. J Histochem Cytochem 2003;

51:865-871.

Vinciguerra P, Albe E, Trazza S, Seiler T, Epstein D. Intraoperative and

postoperative effects of corneal collagen cross-linking on progressive

keratoconus. Arch Ophthalmol 2009; 127:1258-1265.

Whitacre MM, Stein R. Sources of error with use of Goldmann-type

tonometers. Surv Ophthalmol 1993; 38:1-30.

Wittig-Silva C, Chan E, Pollock G, Snibson GR. Localized changes in stromal

reflectivity after corneal collagen cross-linking observed with different

imaging techniques. J Refract Surg 2013; 29:410-416.

Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A-induced collagen

crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003a;

135:620-627.

Wollensak G, Spoerl E, Seiler T. Stress-strain measurements of human and

porcine corneas after riboflavin–ultraviolet-A-induced cross-linking. J

Cataract Refract Surg 2003b; 29:1780-1785.

Wollensak G, Spoerl E, Reber F, Seiler T. Keratocyte cytotoxicity of

riboflavin/UVA-treatment in vitro. Eye 2004a; 18:718-722.

Wollensak G, Wilsch M, Spoerl E, Seiler T. Collagen fiber diameter in the

rabbit cornea after collagen crosslinking by riboflavin/UVA. Cornea 2004b;

23:503-507.

Wollensak G. Crosslinking treatment of progressive keratoconus: new hope.

Curr Opin Ophthalmol 2006; 17:356-360.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Vega-Estrada%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23347373

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ali%C3%B3%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=23347373

http://www.ncbi.nlm.nih.gov/pubmed/?term=Plaza%20Puche%20AB%5BAuthor%5D&cauthor=true&cauthor_uid=23347373

http://www.ncbi.nlm.nih.gov/pubmed/?term=Marshall%20J%5BAuthor%5D&cauthor=true&cauthor_uid=23347373

http://www.ncbi.nlm.nih.gov/pubmed/?term=Outcomes+of+a+new+microwave+procedure+followed+by+acceler

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wollensak%20G%5BAuthor%5D&cauthor=true&cauthor_uid=16900027


57

Wollensak G, Aurich H, Pham DT, Wirbelauer C. Hydration behavior of

porcine cornea crosslinked with riboflavin and ultraviolet A. J Cataract

Refract Surg 2007; 33:516-521.

Wollensak G, Redl B. Gel electrophoretic analysis of corneal collagen after

photodynamic cross-linking treatment. Cornea 2008; 27:353-356.

Wollensak G, Aurich H, Wirbelauer C, Sel S. Significance of the riboflavin

film in corneal collagen crosslinking. J Cataract Refract Surg 2010a; 36:114-

120.

Wollensak G, Herbst H. Significance of the lacunar hydration pattern after

corneal cross linking. Cornea 2010b; 29:899-903.

Wollensak G, Spörl E, Mazzotta C, Kalinski T, Sel S. Interlamellar cohesion

after corneal crosslinking using riboflavin and ultraviolet A light. Br J

Ophthalmol 2011; 95:876-880.

World Medical Association. World Medical Association Declaration of

Helsinki: ethical principles for medical research involving human subjects.

JAMA 2013; 310:2191-2194.

Yam JC, Cheng AC. Prognostic factors for visual outcomes after crosslinking

for keratoconus and post-LASIK ectasia. Eur J Ophthalmol 2013; 23:799-

806.

Yeung SN, Low SA, Ku JY, Lichtinger A, Kim P, Teichman J, Iovieno A,

Rootman DS. Transepithelial phototherapeutic keratectomy combined with

implantation of a single inferior intrastromal corneal ring segment and

collagen crosslinking in keratoconus. J Cataract Refract Surg 2013; 39:1152-

1156.

Zhang ZY, Zhang XR. Refractive surgery for keratoconus. Ophthalmology

2013; 120:434.


http://www.ncbi.nlm.nih.gov/pubmed/?term=Herbst%20H%5BAuthor%5D&cauthor=true&cauthor_uid=20508511

http://www.ncbi.nlm.nih.gov/pubmed/?term=significance+of+the+lacunar+hydration+pattern+after


http://www.ncbi.nlm.nih.gov/pubmed/?term=Sp%C3%B6rl%20E%5BAuthor%5D&cauthor=true&cauthor_uid=21357598

http://www.ncbi.nlm.nih.gov/pubmed/?term=Mazzotta%20C%5BAuthor%5D&cauthor=true&cauthor_uid=21357598

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kalinski%20T%5BAuthor%5D&cauthor=true&cauthor_uid=21357598

http://www.ncbi.nlm.nih.gov/pubmed/?term=Sel%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21357598

http://www.ncbi.nlm.nih.gov/pubmed/?term=Interlamellar+cohesion+after

http://www.ncbi.nlm.nih.gov/pubmed/?term=Interlamellar+cohesion+after

http://www.ncbi.nlm.nih.gov/pubmed/?term=World%20Medical%20Association%5BCorporate%20Author%5D


http://www.ncbi.nlm.nih.gov/pubmed/?term=Yam%20JC%5BAuthor%5D&cauthor=true&cauthor_uid=23787452

http://www.ncbi.nlm.nih.gov/pubmed/?term=Cheng%20AC%5BAuthor%5D&cauthor=true&cauthor_uid=23787452

http://www.ncbi.nlm.nih.gov/pubmed/?term=yam+cheng+prognostic+factor

http://www.ncbi.nlm.nih.gov/pubmed/?term=Yeung%20SN%5BAuthor%5D&cauthor=true&cauthor_uid=23706927

http://www.ncbi.nlm.nih.gov/pubmed/?term=Low%20SA%5BAuthor%5D&cauthor=true&cauthor_uid=23706927

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ku%20JY%5BAuthor%5D&cauthor=true&cauthor_uid=23706927

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lichtinger%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23706927

http://www.ncbi.nlm.nih.gov/pubmed/?term=Kim%20P%5BAuthor%5D&cauthor=true&cauthor_uid=23706927

http://www.ncbi.nlm.nih.gov/pubmed/?term=Teichman%20J%5BAuthor%5D&cauthor=true&cauthor_uid=23706927

http://www.ncbi.nlm.nih.gov/pubmed/?term=Iovieno%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23706927

http://www.ncbi.nlm.nih.gov/pubmed/?term=Rootman%20DS%5BAuthor%5D&cauthor=true&cauthor_uid=23706927

http://www.ncbi.nlm.nih.gov/pubmed/?term=yeung+transepithelial+phototherapeutic+keratectomy++intrastromal+ring

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20ZY%5BAuthor%5D&cauthor=true&cauthor_uid=23374574

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20XR%5BAuthor%5D&cauthor=true&cauthor_uid=23374574


new methods to evaluate the effect of conventional and modified

Documents