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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
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
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
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).
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
n (eyes) Sphere (D) Cylinder (D) SphEq (D) BSCVA (logMAR) Kmax (D) CTmin (µm)
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
n (eyes) Sphere (D) Cylinder (D) SphEq (D) BSCVA (logMAR) Kmax (D) CTmin (µm)
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
n (eyes) Sphere (D) Cylinder (D) SphEq (D) BSCVA (logMAR) Kmax (D) CTmin (µm)
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.
Patients Mean ± SD; P value compared to baseline. Parameters Baseline 1 month 6 months
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.
Patients Mean ± SD; P value compared to baseline. Parameters Baseline 1 month 6 months
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).
LIGHT SCATTER CHANGE (AU)
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.
LIGHT SCATTER CHANGE (AU)
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®
Baseline 1 month 6 months
Baseline 1 month 6 months
Baseline 1 month 6 months
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.
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
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