Session 273 Corneal Extracellular matrix and biomechanics

Transcription

ARVO 2016 Annual Meeting Abstracts
273 Corneal Extracellular matrix and biomechanics
Monday, May 02, 2016 3:45 PM–5:30 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 2355–2400/A0084–A0129
Organizing Section: Cornea
Program Number: 2355 Poster Board Number: A0084
Presentation Time: 3:45 PM–5:30 PM
Identifying the role of PDGFRα in the fibrotic pathway of
TGF -β3
Sriniwas Sriram1, 2, Jennifer A. Tran1, Xiaoqing Q. Guo1, 2,
Hetian Lei1, 2, Andrius Kazlauskas1, 2, James D. Zieske1, 2. 1Schepens
Eye Research Institute/MEE, Boston, MA; 2Ophthalmology, Harvard
Medical School, Boston, MA.
Purpose: The difference in function between two highly similar
transforming growth factor beta (TGF-β) isoforms, TGF-β1 and -β3
(T1 and T3, respectively), still has not been clearly elucidated in the
literature. The purpose of this study was to develop a 3-dimensional
(3D) cell culture model that aimed to identify this difference in
function in vitro. Our hypothesis was that T1 and T3 show varied
effects on fibrosis by differentially activating the platelet derived
growth factor receptor alpha (PDGFRα).
Methods: Human corneal fibroblasts (HCF) were infected with
lentivirus containing optimized short-hairpin RNA (shRNA)
sequences for PDGFRα, which permanently knocked down the
PDGFRα in these cells (HCF-P). 3D constructs of HCF or HCF-P
were cultured on polycarbonate inserts for 4 weeks ± 0.1ng/ml T1
or T3. At the end of 4 weeks, the constructs were processed for
immunofluorescence (IF) and RT-qPCR analysis. RNA from these
constructs also was analyzed with a custom mRNA array that looked
at 86 key fibrosis genes.
Results: IF and RT-qPCR results showed that when compared to
HCF no growth factor control, SMA was not stimulated as much
by T3 as T1; however, in HCF-P, SMA expression remarkably
increased with the presence of T3. The mRNA array results showed
that in HCF, T3 stimulated a majority of the fibrotic markers that
T1 stimulated but to a lesser degree—ITGB6, CTGF, PDGF and
SERPINA1. There also were some signaling factors that were
downregulated by T3 when compared to T1—Integrin’s (ITGAV,
ITGA2), MMPs (MMP3, MMP14), STATs (STAT1, 6) and SMADs
(SMAD2-7). When we looked at the difference between T3 treatment
in HCF and HCF-P to identify the source of T3’s fibrotic response,
STAT3, SRC and ITGAV were some of the signaling growth factors
that were highly upregulated in HCF-P.
Conclusions: The presence or absence of PDGFRα elicited
contrasting fibrotic responses to the same T3 treatment. Identifying
the signaling factors that make T3 cause fibrosis may help in
understanding how it differs from T1, and may potentially help in
developing a treatment that stimulates wounding with reduced levels
of scarring.
Commercial Relationships: Sriniwas Sriram, None;
Jennifer A. Tran, None; Xiaoqing Q. Guo, None; Hetian Lei,
None; Andrius Kazlauskas, None; James D. Zieske, None
Support: NIH Grant R01EY005665 (JDZ), NIH Grant P30EY03790
(core), and Massachusetts Lions Research Fund 2014-15
Utility of TGFBI screening in the diagnosis of suspected
paraproteinemic keratopathy
Marina Zakharevich1, Tova Mannis2, Gabriel Mannis2,
Jennifer Rose -Nussbaumer2, Anthony J. Aldave1. 1Stein Eye Institute,
Los Angeles, CA; 2University of California San Francisco, San
Francisco, CA.
Purpose: To describe the clinical utility of molecular genetic analysis
in the diagnosis or exclusion of suspected corneal dystrophies.
Methods: Slit lamp examination and optical coherence tomography
(OCT) were performed on a 52 year old female referred for
evaluation of bilateral corneal stromal opacities. After informed
consent was obtained, genomic DNA was collected, and PCR
amplification and cycle sequencing of all 17 exons and exon-intron
boundaries of TGFBI were performed.
Results: Slit lamp examination demonstrated bilateral discrete
anterior stromal opacities, which were also demonstrated by OCT
to be confined to a single plane in the anterior corneal stroma.
While the appearance of the opacities was consistent with granular
corneal dystrophy, they were located in only the temporal cornea
in a crescentic pattern, sparing the central cornea. This pattern, the
late onset and the absence of a family history were atypical of GCD.
Screening of TGFBI did not reveal a pathogenic mutation, excluding
GCD1 and other TGFBI-related corneal dystrophies as the cause
of the observed corneal phenotype. The patient was subsequently
referred for evaluation to exclude paraproteinemic keratopathy, which
identified chronic lymphocytic leukemia (CLL) as the potential
systemic cause of the corneal findings.
Conclusions: Paraproteinemic keratopathies may present with
phenotypic similarities to a variety of corneal dystrophies. Genetic
testing, which is available for most corneal dystrophies, provides the
clinician with a definitive means of differentiating between inherited
and acquired disorders.
Commercial Relationships: Marina Zakharevich; Tova Mannis,
None; Gabriel Mannis, None; Jennifer Rose-Nussbaumer, None;
Anthony J. Aldave, None
Support: NEI Grant 1R01 EY022082 (A.J.A.), NEI Grant P30
EY000331 (core grant), the Walton Li Chair, the Stottr fund and an
unrestricted grant from Research to Prevent Blindness
p38/TGFβ-signaling pathway involved in induction of
thrombospondin-1 in human corneal fibroblasts
Audrey E. Hutcheon1, 2, Xiaoqing Q. Guo1, 2, Sriniwas Sriram1, 2,
Jennifer A. Tran1, James D. Zieske1, 2, Audrey E. Hutcheon1, 2.
1
Schepens Eye Research Institute/MEE, Boston, MA;
2
Ophthalmology, Harvard Medical School, Boston, MA.
Purpose: Previous studies have shown that competitive inhibition of
the SMAD/TGFβ-signaling pathway does not reduce the expression of
Thrombospondin-1 (TSP1), a known downstream TGFβ-target protein,
in human corneal fibroblasts (HCF). In addition, when HCF were
stimulated with TGF-β1 (T1), they were induced to produce more TSP1,
as well as, fibrotic markers, cellular fibronectin (cFN) and collagen type
III (Col III). Therefore, in this study, we hypothesized that an alternate
TGFβ-signaling pathway—the p38 pathway—induces TSP1 in HCF,
and may also affect cFN and Col III.
Methods: Human corneal fibroblasts (HCF) were grown in EMEM
+ 10% FBS until they were 60-70% confluent, at which time the
cells were cultured overnight in EMEM only, to serum starve the
cells. The next day, HCF were cultured as follows for 24 hours: 1)
Control: EMEM only; 2) T1: EMEM + 2ng/ml T1; 3) p38inh: EMEM
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+ 10μM of p38 inhibitor (SB202190); and 4) Both: EMEM + 2ng/ml
T1 + 10μM p38 inhibitor. Cells then were harvested and processed
for western blot or quantitative real-time polymerase chain reaction
(qRT-PCR) for TSP1, cFN, and Col III. Experiments were repeated at
least three times.
Results: TSP1 protein and mRNA is present in serum starved HCF
(control), and as expected, upon treating these cells with T1, TSP1
protein increased; however, the mRNA remained relatively similar
to control. In the p38inh samples, both TSP1 protein and mRNA
dramatically decreased when compared with both the control and
T1 samples. When both T1 and p38 inhibitor were introduced, TSP1
mRNA levels dramatically decreased, similar to the p38inh samples;
however, TSP1 protein levels only decreased compared to T1
samples. Col III and cFN responded in a similar manner as TSP1.
Conclusions: Interestingly, the p38 inhibitor did decrease the protein
and/or mRNA levels of TSP1, Col III, and cFN in HCF, thus showing
that in HCF these proteins are downstream from the p38/TGFβsignaling pathway. Also, the discrepancy between mRNA and protein
levels for TSP1 suggests that there are other factors involved in either
the stabilization or degradation of TSP1 in HCF.
Commercial Relationships: Audrey E. Hutcheon, None;
Xiaoqing Q. Guo, MEE 2015-575 - Zieske; ML 36770-549P01US
(P); Sriniwas Sriram, None; Jennifer A. Tran, None;
James D. Zieske, MEE 2015-575 - Zieske; ML 36770-549P01US
(P); Audrey E. Hutcheon, None
Support: NIH Grant R01EY005665 (JDZ), NIH Grant P30EY03790
(core)
Comparison of human corneal fibroblasts cultured in human or
fetal bovine serum in an in vitro 3D model
Wenjing Wu1, 2, Shrestha Priyadarsini3, James D. Zieske1, 2,
Dimitrios Karamichos3, 4. 1Schepens Eye Research Institute/MEE,
Boston, MA; 2Ophthalmology, Harvard Medical School, Boston,
MA; 3Ophthalmology/Dean McGee Eye Institute, University of
Oklahoma Health Sciences Center, Oklahoma City, OK; 4Cell
Biology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK.
Purpose: TGF-β isoforms are known for their effects on corneal
fibrosis with TGF-β1 and -β2 being pro-fibrotic and TGF-β3
being anti-fibrotic. The purpose of this study was to investigate the
differences between human serum (HS) and fetal bovine serum (FBS)
on human corneal fibroblasts (HCF) in an in vitro 3D culture model,
as well as, examine HS’s influence on corneal stromal fibrosis.
Methods: HCF were cultured for 2 weeks in EMEM + 0.5mM
Vitamin C (2-O-α-D-glucopyranosyl-L-ascorbic acid) with either
HS or FBS ± TGF-β1, -β2, or -β3. After 2 weeks, samples were
collected, processed for qRT-PCR and/or western blot, and examined
for alterations in fibrotic markers—α-smooth muscle actin (α-SMA)
and collagen types III (Col III) and V (Col V)—as well as, focal
adhesion kinase (FAK) and matrix metalloproteinases 1 (MMP1) and
3 (MMP3).
Results: In HCF controls, Col III was significantly downregulated
(p<0.05) and MMPs 1 and 3 (p<0.0001 for both) were upregulated
in HS compared to FBS; however, there was no difference in
α-SMA or Col V expression. For all TGFβ isoforms, α-SMA
showed no significant difference between serums; however, Col III
was significantly decreased (p<0.004) and Col V was significantly
increased (p<0.0005) in HS as compared with FBS samples. When
compared with their respective serum controls, both MMP1 and
3 in HS were significantly downregulated by the TGFβ isoforms,
especially TGF-β3 (p<0.05); however, in the FBS samples, MMPs
1 and 3 were both upregulated with TGFβ stimulation. Interestingly,
TGF-β1 stimulated FAK (p<0.05), while TGF-β3 appeared to have
no effect.
Conclusions: Our results show that HS could potentially replace
FBS in our in vitro culture model, and offer a more translational in
vitro corneal stroma equivalent. The use of HS may also provide
insights into the development of a fibrotic phenotype. For example,
HCF cultured with HS and TGF-β3 appeared to significantly inhibit
degradation enzymes (ie. MMPs 1 and 3) and promote a non-fibrotic
phenotype.
Commercial Relationships: Wenjing Wu, None;
Shrestha Priyadarsini, None; James D. Zieske, None;
Dimitrios Karamichos, None
Support: NIH Grant R01EY020886 (JDZ and DK), NIH Grant
R01EY005665 (JDZ), NIH Grant P30EY03790 (core)
RAFT tissue equivalent model of keratoconus: A novel 3D
system useful for interrogating disease phenotype and functional
differences of corneal stromal cells
Alvena Kureshi1, Christine Putri1, James Phillips2,
James L. Funderburgh3, Julie T. Daniels1. 1Institute of
Ophthalmology, University College London, London, United
Kingdom; 2UCL Eastman Dental Institute, London, United Kingdom;
3
University of Pittsburgh, Pittsburgh, PA.
Purpose: Keratoconus (KC) is a corneal disorder characterized by
progressive structural thinning, resulting in a conical-shaped cornea
causing loss of vision. Cell-extracellular matrix interactions are
thought to control normal tissue organization and transparency and it
is expected that these interactions may be perturbed in KC. The aim
of this study was to develop a 3D RAFT tissue equivalent (TE) model
of KC to compare the phenotypic and functional behavior of stromal
cells.
Methods: Stromal cells were characterised by phenotypic assessment
using a variety of stromal cell markers and a contraction assay to
compare their contractile capacity. RAFT TE were seeded with
normal and KC stromal cells (n=3) and cultured for 3 weeks.
Functional characterization of RAFT TE included investigations of
material properties using an Electroforce Bose tensile testing device;
MMP activity in stromal cells using gelatin zymography and ELISA;
collagen production using a Sircol collagen assay and assessment of
collagen fibril organization using scanning electron microscopy.
Results: A 3D in vitro model of KC was achieved using RAFT TE.
Normal and KC stromal cells both exhibited stromal cell markers in
culture. KC stromal cells exhibited a significantly greater contractile
capacity at 72 hours of culture with 75 ± 3% compared to normal
cells at 29 ± 7% and acellular gels at 3 ± 1% (n=3) (p<0.0001).
Statistically significant differences in RAFT TE material properties
were observed. Mean break strength of KC and normal RAFT TE
were 0.17 ± 0.02 MPa and 0.25 ± 0.03 MPa respectively (n=3)
(p<0.05). A reduced stiffness of 0.01 ± 0.002 MPa in KC RAFT TE
compared to 0.02 ± 0.002 MPa in normal RAFT TE was observed.
Increased activity of MMP-2 in normal RAFT TE was observed
but no differences in pro-MMP-1 or collagen production. Collagen
fibril ultrastructure in KC RAFT TE appeared markedly thinner and
loosely packed compared to normal RAFT TE.
Conclusions: To our knowledge, this is the first 3D tissue equivalent
model of KC. RAFT TE serves as an ideal biomimetic model to
investigate normal and abnormal tissue organization in the corneal
stroma. Significant differences in contractile capacity of KC cells
and strength and stiffness of KC RAFT TE suggests the remodeling
capacity of stromal cells is perturbed in KC, leading to ultrastructural
changes of the corneal stroma.
Commercial Relationships: Alvena Kureshi, None;
Christine Putri, None; James Phillips, None;
James L. Funderburgh, None; Julie T. Daniels, None
Support: Special Trustees of Moorfields Eye Hospital
The distribution of elastic fibres in keratoconic corneas using en
bloc tannic acid and orcein staining
Tomas L. White2, Philip N. Lewis2, Koji Kitazawa1, Tsutomu Inatomi1,
Shigeru Kinoshita1, Keith M. Meek2. 1Opthalmology, Kyoto
Prefectural University of Medicine, Kyoto, Japan; 2Optometry and
Vision Science, Cardiff Univeristy, CARDIFF, United Kingdom.
Purpose: We have recently demonstrated the presence and
distribution of elastic fibres in normal human cornea using serial
block face scanning electron microscopy (SBF-SEM). These fibres
formed sheets at the limbus before becoming long fibres that run
transversely and longitudinally, along the plane of the cornea,
concentrated in the ‘pre-Descemet’s layer’ (PDL). The aim of
the current study was to use SBF-SEM and transmission electron
microscopy (TEM) to determine if these findings are consistent, or
differ, in human keratoconic corneas.
Methods: Keratoconic corneal buttons were fixed in 4%
paraformaldehyde (PFA) following penetrating keratoplasty. Buttons
were then dissected, fixed in modified Karnovsky’s fixative and en
bloc stained using 2 different protocols. The first involved using
multiple staining solutions including osmium tetroxide, tannic acid,
uranyl acetate and lead acetate, prior to dehydration and embedding
in CY212 resin. The second protocol substituted tannic acid with
orcein, with normal cornea and sclera samples being stained as
controls. Polymerised blocks were attached to Gatan specimen pins
and transferred to a Zeiss Sigma FEG equipped with a Gatan 3View
system. Images of the block face surface were acquired every 50 nm
by automated serial sectioning. 100nm gold sections were obtained
from the same specimen pins and visualised using a Joel1010 TEM.
Results: No fibres could be clearly identified using SEM due
to a lack of contrast. However, fibres were clearly visible above
Descemet’s membrane in the orcein stained control samples. TEM
confirmed the presence of elastic fibres in the control samples and
also revealed that there were very few fibres in the PDL region of
both central and peripheral keratoconic samples. Many dark stained
fibres were seen below the epithelium, in the anterior stroma of the
thinned cone region in keratoconus tissue. Furthermore, the central
scarred region of cornea contained a distinct mass of staining where
numerous amounts of fibres were evident.
Conclusions: Elastic fibre specific en bloc staining using tannic acid
and orcein protocol has revealed a lack of fibres in the PDL region
in keratoconic corneal buttons and an increased presence of fibres in
the anterior stroma, below the epithelium. These findings are directly
opposite to those that we have reported in normal cornea and may
influence keratoconus morphology and/or pathogenesis.
Commercial Relationships: Tomas L. White, None;
Philip N. Lewis, None; Koji Kitazawa, None; Tsutomu Inatomi,
None; Shigeru Kinoshita, None; Keith M. Meek, None
Support: MRC
Cellularization of 3D printed Recombinant Human Collagen type
III scaffolds using corneal mesenchymal stem cells
Steffi Matthyssen1, Kurt Coppens2, Eleonora Ferraris2,
Jennifer Patterson3, Marie-José Tassignon1, 4, Nadia Zakaria1, 4.
1
Ophthalmology, University of Antwerp, Antwerp, Belgium;
2
Advanced Manufacturing laboratory, Faculty of Engineering
Technology, KU Leuven, Leuven, Belgium; 3Materials for Living
Systems, Department of Materials Engineering, KU Leuven, Leuven,
Belgium; 4Ophthalmology, Antwerp University Hospital, Edegem,
Belgium.
Purpose: To investigate cellularization of 3D printed human
recombinant collagen type III (RHC III) scaffolds in vitro, using
corneal mesenchymal stem cells (MSCs).
Methods: Corneal MSC cultures were established using either a
collagenase digestion (1.5hrs vs 4hrs) of the stroma or by plating
stromal explants. Cells were seeded in either DMEM + 10% FBS or
Epilife +5% FBS. At passage three, cells were characterized using
Flow Cytometry and trilineage differentiation was performed using
specific differentiation media. MSCs were seeded onto 3D printed
(n=6) and non-printed (n=6) samples at a density of 20,000 cells/
sample. For controls, cells were seeded on glass (n=6). At day 10,
scaffolds were processed for immunocytochemistry (n=15) and
Scanning Electron Microscopy (SEM) (n=3).
Results: Flow cytometry showed that corneal MSCs were positive
for CD73, CD90, CD105, CD13, CD29, CD44, CD166 and
negative for CD11b, CD14, CD19, CD34, CD45, CD79a and
HLA-DR. Corneal MSCs were able to differentiate into osteocytes,
chondrocytes and adipocytes. There was no significant difference
between the MSC phenotypes using the different isolation or culture
protocols. Light microscopy showed that corneal MSCs proliferated
on both printed and non-printed RHC III scaffolds and SEM showed
the circular print pattern of the RHC III. On immunocytochemistry
we observed collagen type III fibril alignment in the printed samples
but not in the non-printed samples. The MSCs actin filaments of
the cytoskeleton stained positive for phalloidin and we observed
alignment orthogonally to the direction of the collagen.
Conclusions: Our results demonstrate that 3D printed RHC III is a
suitable substrate for cultivating corneal MSCs.
Commercial Relationships: Steffi Matthyssen, None;
Kurt Coppens, None; Eleonora Ferraris; Jennifer Patterson,
None; Marie-José Tassignon, None; Nadia Zakaria, None
Collagen-Proteoglycan Interactions in the Corneal Stroma: A
Computer Simulation
Carlo Knupp, Rob D. Young, Andrew J. Quantock, Keith M. Meek.
Cardiff University, Cardiff, United Kingdom.
Purpose: Recent three-dimensional electron microscopy studies
carried out by our group (e.g. Lewis et al, Structure,18, 1-7, 2010)
confirmed that the spatial distribution of collagen fibrils in the corneal
stroma is regulated by the interaction of proteoglycans with the
collagen fibrils. From these studies, we suggested that the relatively
flexible proteoglycan chains make temporally transient links
between adjacent collagen fibrils in a non-systematic, asymmetric
way and that they give rise to forces that are ultimately responsible
for the spatial distribution of the collagen fibrils in the cornea. Our
purpose is to simulate these interactions computationally and verify
whether they can explain the most salient structural and functional
characteristics of the cornea.
Methods: Ad-hoc software was written to simulate the behaviour
of the collagen fibrils under the influence of external forces brought
about by the action of the proteoglycans.
Results: Our computer simulation covers a 1 second-long timedependent evolution of a portion of the corneal stroma (with up
to 1000 fibrils) in which the collagen fibrils are subject to thermal
motion and proteoglycan induced forces. The appearance of
the simulated corneas is remarkably similar to what is seen in
electron micrographs, as confirmed by the calculation of Fourier
transforms and radial projection functions for the simulations and the
micrographs. The calculation of transparency and X-ray diffraction
patterns from the simulated corneas are also in good agreement with
the data collected from real corneas. In addition, we have probed
the possibility of other proteoglycan unrelated forces acting on the
collagen fibrils, such as the pressure exercised by adjacent corneal
lamellae, which shows that they may contribute substantially to the
overall collagen fibril distribution in the cornea.
Conclusions: The fibril distributions obtained in our simulations
compare favourably with electron microscopical, X-ray diffraction
and corneal transparency data, indicating that the local interactions
between collagen and proteoglycans can explain some of the main
structural and functional aspects of the cornea.
Commercial Relationships: Carlo Knupp, None; Rob D. Young,
None; Andrew J. Quantock, None; Keith M. Meek, None
Support: MRC Grant MR/K000837/1
Cytosporone B inhibits the TGF-β–induced expression of
a–smooth muscle actin and contractility in human corneal
fibroblasts
Naoyuki Morishige, Yukiko Morita, Shizuka Murata. Ophthalmology,
Yamaguchi Univ Grad Sch of Med, Ube, Japan.
Purpose: Cytosporone B (CsnB) is an antagonist for the nuclear
receptor transcription factor NR4A1, known as Nur77, which
contributes to the regulation of apoptosis and glucose homeostasis.
Recently, it is reported that NR4A1 is related to tissue scarring by
regulating transforming growth factor–b (TGF-β) signaling. To
investigate the role of NR4A1 in human corneal fibroblasts (HCFs),
we examined the effects of CsnB on the expression of a–smooth
muscle actin (α-SMA) and contractility in these cells induced by
stimulation with TGF-β.
Methods: Cultured HCFs were stimulated with TGF-β (10 ng/ml)
in the presence of various concentrations (0 to 10 μM) of CsnB for 3
days, after which the cells were lysed and subjected to immunoblot
analysis with antibodies to α-SMA. HCFs were also cultured in
collagen gels and exposed to CsnB (1 μM), TGF-β (10 ng/ml),
or neither or both agents for 3 days, after which the change in gel
diameter was determined as a measure of cell contractility.
Results: The TGF-β–induced expression of α-SMA in HCFs
was inhibited by CsnB in a concentration-dependent manner. The
diameter of HCF-containing collagen gels after culture for 3 days
was 7.8 ± 0.8, 8.3 ± 0.3, 7.0 ± 0.3, or 8.0 ± 0.9 mm in the absence
of additions or in the presence of CsnB, TGF-β, or both agents,
respectively.
Conclusions: CsnB inhibited TGF-β–induced contraction of HCFs,
likely as a result of its attenuation of the up-regulation of α-SMA
expression. Our results suggest that NR4A1 mediates these effects
of TGF-β in HCFs, and that CsnB warrants further investigation as
a potential therapeutic modulator of corneal stromal contraction and
scarring.
Commercial Relationships: Naoyuki Morishige, None;
Yukiko Morita; Shizuka Murata, None
Support: JSPS KAKENHI grant number 15K20264
Chitosan membrane-cultivated keratocyte spheres suppress
stromal fibrosis via inhibition of epithelial mesenchymal
transition (EMT)
Chi-Chin Sun1, 2, Kuo-Ying Lee1, Jong-Hwei S. Pang3. 1Department of
Ophthalmology, Chang Gung Memorial Hospital, Keelung, Keelung,
Taiwan; 2Department of Chinese Medicine, School of Medicine,
Chang Gung University, Taoyuan, Taiwan; 3Institute of Clinical
Medical Sciences, Chang Gung University, Taoyuan, Taiwan.
Purpose: Previously, we demonstrated the anti-fibrotic effects of
keratocyte spheres cultivated on chitosan membrane. The purpose of
this study is to investigate the underlying mechanisms.
Methods: Human corneas stripped of both endothelium and
epithelium were placed in a solution of collagenase overnight
at 37°C. Confluent cell layers were treated with trypsin-EDTA
and seeded onto the chitosan coatings or plastic dishes. We used
Affymetrix oligonucleotide microarrays to analyze common
transcriptomes and thereby learn about the core gene expression
profile in human keratocytes cultured on chitosan membrane
on plastic dishes. The advantage of microarray analysis of gene
expression is that it can be used to disclose associating genes
that were previously thought to be unrelated to a physiological
or pathological event. However, interpreting complex biological
processes from gene expression profiles often requires extensive
knowledge mining in biomedical literature. Therefore, we adopted a
commercially available biological database and software program,
MetaCore (GeneGo Inc.), for functional network analysisto reveal the
common functions of the interacting genes. Real-time PCR, western
blotting analysis and immunofluorescence staining were performed to
validate the expressions of these genes at RNA and protein levels.
Results: Analysis of gene expression by microarray and MetaCore
pathway mapping revealed that regulation of epithelial-tomesenchymal transition (EMT) was the most important pathway
involved in keratocyte spheres cultured on chitosan membrane as
compared with on plastic dishes (4.193E-12). Transforming growth
factor beta receptor type 1 was significantly down-regulated in
chitosan-cultivated keratocyte spheres. Cell motility-associated
genes including caldesmon, tropomyosin-1, ACTA2 and ACTB were
all down-regulated in keratocyte spheres. Real-time PCR, western
blotting analysis and immunofluorescence staining confirmed the
findings observed at transcriptional level.
Conclusions: Our studies demonstrated that chitosan membranecultivated keratocyte spheres repaired corneal scarring through
inhibition of transforming growth factor beta-induced epithelial
mesenchymal transition (EMT).
Commercial Relationships: Chi-Chin Sun, None; Kuo-Ying Lee,
None; Jong-Hwei S. Pang, None
Support: NSC99-2314-B-182A-025-MY3
Confocal microscopy findings after 3 different techniques: epi-off,
trans-epithelial and iontophoresis-assisted cross-linking
Manuel Ramirez, Emmanuel Cabrera, Everardo Hernandez-Quintela.
Cornea and Refractive Surgery, APEC, Mexico City, Mexico.
Purpose: To evaluate the stromal corneal findings in 3 different
cross-linking techniques (epi-off, trans-epithelial and iontophoresisassisted).
Methods: 18 eyes of 18 keratoconus patients (group A) were treated
by epi-off corss-linking technique, 15 eyes of 15 keratoconus
patients (group B) were treated by trans-epithelial crosslinking
technique and 22 eyes of 26 keratoconus patients (group C) were
treated by iontophoresis-assisted cross-linking technique. All eyes
were examined with the confocal microscope (Confoscan 4, Fortune
Technologies, Italy), a central scan of the total corneal thickness
was taken before, 1 week and 1 month after cross-linking treatment.
Corneal stroma was evaluated by using the Navis v. 3.5.0. Software
(NIDEK, Multi-Instrument Diagnostic System, Japan).
Results: In group A we found, after cross-linking treatment, activated
keratocites from superficial stroma to 275.1 ± 85.9 µm stromal depth
at 1 week and from superficial stroma to 324.9 ± 66.0 µm stromal
depth at 1 month after treatment. In group B and group C we did
not found activated keratocites in all stromal depth at 1 week and 1
month after cross-linking treatment.
Conclusions: Activated keratocites were found at the corneal stroma
in patients treated after epi-off cross-linking technique, in contrast
patients treated with trans-epithelial and iontophoresis-assisted
crosslinking techniques did not show activated keratocites at the
corneal stroma after treatment.
Commercial Relationships: Manuel Ramirez, None;
Emmanuel Cabrera, None; Everardo Hernandez-Quintela, None
Human Stromal Lamellar Morphology and Its Relationship to
Central to Peripheral Thickness Change
Jan P. Bergmanson, Carmen L Mosqueda, Alan R Burns. College of
Optometry, University of Houston, Houston, TX.
Purpose: The human cornea is thicker peripherally than centrally, but
is this due to a greater number of lamellae in the peripheral cornea or
an increase in lamellar thickness, or both? The purpose of this study
was to determine the total number of lamellae present and the average
lamellar thickness in the anterior, middle and posterior stroma, both
in the central and extreme peripheral normal cornea.
Methods: Three eye bank corneas were processed and examined
with a Tecnai G2 12 Twin transmission electron microscope (FEI
Company Hillsboro, OR) operating at 100 kV and magnified
at 4200X. Montages of overlapping images from epithelium to
endothelium were assembled for each cornea for extreme periphery
and central regions. Lamellae on each montage were marked and
counted using an established protocol. The stroma was divided into
anterior, mid and posterior segments. Data were expressed as means
± SD and differences assessed using Analysis of Variance (ANOVA)
and Tukey post-hoc tests with significance set at P ≤ 0.05.
Results: The central/peripheral lamellar counts for each cornea
(260/260; 231/235; 247/257) were remarkably similar. As the
lamellae approach the central region they were frequently observed
to branch. However, peripheral posterior lamellae were on average
twice as thick as those in the central cornea (3.2 ± 0.4 and 1.6 ± 0.3
µm, respectively), with some lamellae reaching 10 µm in thickness.
Anterior lamellae were thinner than mid-stromal lamellae, but only at
the periphery.
Conclusions: The human cornea is thicker in the periphery not
because it has more lamellae but because it has thicker posterior
lamellae. While lamellar branching is common, the total number of
lamellae (~250) does not increase centrally. Therefore, some lamellae
must stop short of crossing the cornea. Given that keratoconic
cornea has considerably more lamellae (~350) at the cone apex, it
will be important to establish whether this increase is also present
peripherally.
Commercial Relationships: Jan P. Bergmanson, None; Carmen
L Mosqueda, None; Alan R Burns
Support: NIH/NEI Grants EY018239 and P30EY007551
Differentiation of Normal Human Corneal Induced Pluripotent
Stem Cells to Retinal Progenitor Cells
Roy Joseph2, Om P. Srivastava2, Roswell R. Pfister1. 1Ophthalmology,
Univ of Alabama at Birmingham, Birmingham, AL; 2Vision Sciences,
University Of Alabama at Birmingham, Birmingham, AL.
Purpose: To differentiate human corneal induced pluripotent stem
cells (iPSC) to retinal progenitor cells.
Methods: Normal human corneal fibroblasts from three donors were
reprogramed directly to pluripotent stem cells (PSC) by delivering
reprogramming factors in a single virus using 2A “self-cleaving”
peptides, using a single polycistronic lentiviral vector co-expressing
four transcription factors (Oct 4, Sox2, Klf4 and Myc) to yield
induced pluripotent stem cells (iPSC). Phase-contrast images were
obtained using our Olympus 1X71 inverted microscope. These iPS
cells were immunofluorescentally characterized for stem cell markers
(SSEA4, Oct4 and Sox2). The iPS cells were grown in embryoid
medium (EM) [(DMEM F-12 medium containing 10% knockout
serum replacement, 2% B27 supplement, 1% N2 supplement, 1%
L-glutamine, 1% 100X NEAA, 1% penicillin/streptomycin, 1 ng/
ml noggin, 1 ng/ml DKK-1, 1 ng/ml IGF-1 and 0.5 ng/ml bFGF],
and plated at a density of 50 cell clumps/cm2 in ultralow attachment
plates. Cell clumps were cultured for 5 days, and then embryoid
bodies were removed and differentiated in EM +10 ng/ml noggin, 10
ng/ml DKK-1, 10 ng/ml IGF-1 and 1 ng/ml bFGF (Differentiation
medium 1) in 6-well culture plates coated with poly-D-lysine,
collagen, laminin and fibronectin. Media were changed every other
day for 10 days with Differentiation media 1, then every other day
for an additional 18 days with Differentiation media 1 +10 nm DAPT
(N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl
ester, a γ- secretase inhibitor).
Results: Immunofluorescence analysis of the colonies showed that
they were positive for all the three stem cell markers (SSEA4, Oct4
and Sox2). The iPS cells were grown to differentiate for 33 days, and
then immunocytochemical analysis of the cells showed expression of
retinal photoreceptor markers, recoverin and rhodopsin.
Conclusions: We successfully reprogramed normal human corneal
fibroblasts using single polycistronic virus co-expressing four
transcription factors. We were also able to differentiate the iPSC
to retinal precursor cells which expressed photoreceptor markers,
rhodopsin and recoverin. These retinal precursor cells are a valuable
source which could be used for transplantation in retinal degenerative
diseases.
Commercial Relationships: Roy Joseph; Om P. Srivastava, None;
Roswell R. Pfister, None
Cellular Factor XIII is Present in the Corneal Stroma
Zsuzsanna Z. Orosz1, 2, Amir H. Shemirani2, Helga Bárdos3,
Bence Nagy4, Andrea Facskó1, András Berta5, Róza Ádány3,
László Muszbek2, 6. 1Department of Ophthalmology, University
of Szeged, Szeged, Hungary; 2Division of Clinical Laboratory
Science, Department of Laboratory Medicine, University of
Debrecen, Debrecen, Hungary; 3Department of Preventive Medicine,
University of Debrecen, Debrecen, Hungary; 4Department of
Pathology, University of Szeged, Szeged, Hungary; 5Department
of Ophthalmology, University of Debrecen, Debrecen, Hungary;
6
Thrombosis, Hemostasis and Vascular Biology Research Group
of the Hungarian Academy of Sciences, University of Debrecen,
Debrecen, Hungary.
Purpose: Transglutaminases (TGs) are a family of enzymes that
cross-link proteins by e(g-glutamyl)lysyl bonds and most of them
have been implicated in the modulation of extracellular matrix. Here
we investigated the presence of three TGs, keratinocyte TG (TG1), tissue transglutaminase (TG-2) and the cellular form of blood
coagulation factor XIII (cFXIII) in the corneal tissue.
Methods: Frozen sections of normal human cornea obtained
from enucleated bulbus were stained for cFXIII, TG-1 and TG-2
using poly-, or monoclonal antibodies. Detection of cFXIII was
also combined with labeling for CD11b, CD34, CD45, CD68 and
CD163 using double immunofluorescent staining. FITC-labeled or
biotinylated secondary antibodies with Texas red-labeled streptavidin
were used for the visualization of immunoreactions. Western blot
analysis was also performed on corneal stroma, and the expression of
cFXIII mRNA was analyzed by real-time qPCR.
Results: A significant part of keratocytes showed intensive staining
for cFXIII, but not for TG-1 and TG-2. Neither epithelial nor
endothelial cells were labeled by anti-cFXIII antibody. cFXIII
positive keratocytes were unevenly distributed in the corneal stroma;
they were abundant in the subepithelial tertile of stroma (120±10/
visual field), while they were sparse (38±6/visual field) in the
subendothelial tertile. cFXIII+ cells showed co-staining for CD34,
however, a significant number of CD34+ cells were negative for
cFXIII. CD34+ cells were evenly distributed throughout the stroma.
Only a few cells were stained for CD11b and CD45, they were also
labeled by anti-cFXIII antibody. No cell showed positivity for CD68
and CD163. cFXIII expression was confirmed by Western blotting,
and real-time qPCR demonstrated the presence of cFXIII mRNA in
the corneal stroma.
Conclusions: This is the first report demonstrating the presence
of cFXIII in the cornea. A significant part of CD34+ keratocytes
contained cFXIII, their transglutaminase activity might play a role
in wound healing of the cornea. Further investigations are needed to
explore the role of FXIII in corneal wound healing.
Commercial Relationships: Zsuzsanna Z. Orosz; Amir
H. Shemirani, None; Helga Bárdos, None; Bence Nagy, None;
Andrea Facskó, None; András Berta, None; Róza Ádány, None;
László Muszbek, None
Support: TÁMOP 4.2.2/A-11/1/KONV-2012-0045; TÁMOP
4.2.2/B-10/1-2010-0024
Investigation of the Corneal Biomechanics in Healthy Chinese
Subjects Using a Corneal Visualization Scheimpflug Technology
Miao He1, Hui Ding2, Xingwu Zhong2, 1. 1Zhongshan Ophthalmic
Center, Sun Yat-sen University, Guangzhou, China; 2Hainan Eye
Hospital, Zhongshan Ophthalmic Center, Sun Yat-senUniversity,
Haikou, China.
Purpose: In vivo quantification of corneal biomechanics became
local focus since the introduction of Corneal Visualization
Scheimpflug Technology (CorVis ST) in recent years. The aim of this
study was to evaluate corneal biomechanical properties in a Chinese
healthy population using this device.
Methods: Healthy subjects with 20 years and older were enrolled.
All eyes underwent complete ocular examinations, including slitlamp examination, auto refractometer, intraocular pressure (IOP), and
CorVis ST. Corneal biomechanical parameters included maximum
deformation amplitude (MDA), first applanation time (A1T), first
applanation length (A1L), first applanation velocity (A1V), second
applanation time (A2T), second applanation length (A2L), second
applanation velocity (A2V), highest concavity time (HCT), peak
distance (PD), and central radius of curvature (CCR). The Pearson
correlation analysis was used to evaluate the correlations of corneal
biomechanical parameters with age, central corneal thickness (CCT),
spherical equivalent of refractive error (SE), gender, and IOP.
Results: 150 subjects were included with a mean age was 56.4±6.5
years. Mean SE was -1.65±1.07 diopters, the mean IOP was
14.5±2. 4 mmHg. Mean CCT was 549.72±29.96mm. There were no
significant differences in SE, IOP, or CCT between male and female.
Mean deformation amplitude was 1.07 ±0.04 mm. Highest concavity
time was 17.38 ± 0.10 mm. A1T, A2T were 7.16 ± 0.46 ms and 22.10
±0.37 ms, respectively. Most (9/10) CorVis ST parameters were
independently associated with IOP, but only 4 parameters (MDA,
A1L, A2L, A2V) were significantly correlated with CCT or age.
Gender and SE did not influence CorVis ST parameters.
Conclusions: The CorVis ST provided informative measurements of
corneal biomechanical properties. No significant difference of corneal
biomechanics existed between male and female. IOP, age, and CCT
were correlated to corneal biomechanics.
Commercial Relationships: Miao He, None; Hui Ding, None;
Xingwu Zhong, None
Support: Science and Technology Planning Project of Guangdong
and Hainan Province (ZDXM2014077,2013B090200057)
Comparative Study of the Effect of Pilocarpine and Tropicamide
Eye Drops on Corneal Topography
Abbas Azimikhorasani, Shahrzad Mirzahoseini, Javad Heravian,
Azamalsadat Parizad. Mashhad University of Medical Sciences,
Mashhad, Iran (the Islamic Republic of).
Purpose: This study was conducted to evaluate the effect of
contraction and relaxation of ciliary muscle caused by pilocarpine
and tropicamide eye drops on corneal radius, central corneal power
and corneal astigmatism.
Methods: This prospective study was performed on sixty normal and
healthy eyes of sixty volunteers with a mean age of 38.19 year (range
18 to 49 years) and without any ocular pathology. Volunteers divided
into 2 groups of thirty, in the first group corneal topography of both
eye were measured before and 30 minutes after instillation of topical
tropicamide 0.5% in only one eye, and the other eye was the control
eye and no drop was given. In the second group, the same routine
was performed, except that subject received on drop of pilocarpine
1% in one eye. Statistical comparison between groups for the
central corneal power, corneal radius and corneal astigmatism were
performed using paired t test.
Results: In group 1, no significant changes were found in corneal
radius, power and astigmatism, however, in group 2 subjects who
have received pilocarpine eye drop, mean corneal radius value
decreased significantly by 0.05 mm and mean corneal power
increased by a plus power of +0.32 D. There was no significant
change in corneal astigmatism in both groups.
Conclusions: It seems that pilocarpine induced ciliary muscle
contraction which may cause pressure on corneal limbus of cornea
and scleral spur that resulted in changes in corneal curvature.
But tropicamide eye drop did not affect corneal radius and other
corneal parameters and corneal topography can be carried out after
instillation of tropicamide eye drop.
Commercial Relationships: Abbas Azimikhorasani, None;
Shahrzad Mirzahoseini, None; Javad Heravian, None;
Azamalsadat Parizad, None
Depth-dependent extrafibrillar matrix stiffness of the human
cornea predicts spherical aberration induced by laser refractive
surgery
Mengchen Xu1, 2, Mark Buckley3, Amy L. Lerner1, 3,
Geunyoung Yoon2, 3. 1Mechanical Engineering, University of
Rochester, Rochester, NY; 2Flaum Eye Institute, University of
Rochester, Rochester, NY; 3Biomedical Engineering, University of
Rochester, Rochester, NY.
Purpose: To investigate the influence of depth-dependent corneal
extrafibrillar matrix stiffness on wavefront aberrations induced by
laser refractive surgery.
Methods: Three-dimensional anisotropic hyperelastic corneal models
with a regional fibril distribution (Pandolfi, 2008) were implemented
in finite element software (ABAQUS 6.14). Two material cases were
compared: (1) averaged uniform extrafibrillar matrix stiffness along
depth, (2) five material layers with depth-dependent extrafibrillar
matrix stiffness based on previous experimental data (Sloan, 2014).
Three preoperative (preOP) model eyes with different axial lengths to
induce 5, 7 and 9 diopters (D) myopic refractive error were generated
for each material case. An iterative algorithm was used to identify
the stress-free configurations of preOP eyes. Myopic corrections
were simulated by removing tissue with ablation thickness calculated
by the Munnerlyn equation for a 6 mm optical zone. Anterior and
posterior corneal surface profiles were exported from each preOP and
postoperative (postOP) model eye and used to calculate wavefront
aberrations for a 6 mm pupil using a custom-developed surface fitting
and ray-tracing program.
Results: For the uniform matrix stiffness model, the trend observed
for induced spherical aberration (SA) was opposite to the clinical
observation. In contrast, the depth-dependent extrafibrillar matrix
stiffness model showed a significant positive linear correlation
between the induced positive SA and the amount of attempted diopter
correction, in accordance with clinical data. Induced SA predicted
after 5, 7 and 9D correction was 0.35, 0.48 and 0.63μm, respectively.
PostOP refractive power results predicted that the biomechanical
response to the tissue ablation would cause an under-correction in
myopia of 15 – 20%. An increasing trend was also found for other
aberrations such as astigmatism and quadrafoil with increasing
amount of myopic correction.
Conclusions: The uniform and depth-dependent extrafibrillar matrix
stiffness of the human cornea lead to different trends of wavefront
aberrations induced by laser refractive surgery when fibril distribution
remains unchanged. Characterization of the depth-dependent matrix
stiffness improves reliability in predicting clinical trends and may be
key to understanding the role of biomechanics in optical outcomes
after laser refractive surgery.
Commercial Relationships: Mengchen Xu, None; Mark Buckley,
None; Amy L. Lerner, None; Geunyoung Yoon, None
Support: NIH EY014999
Anisotropic Diffusion in the Corneal Stroma Measured by ThreeDimensional Fluorescence Recovery After Photobleaching
R Glenn Hepfer1, Peng Chen1, George O Waring2, 1, Hai Yao1.
1
Bioengineering, Clemson University, Charleston, SC;
2
Ophthalmology, Medical University of South Carolina, Charleston,
SC.
Purpose: This study investigates transport properties within the
corneal stroma. The corneal stroma is a highly organized tissue,
with collagen fibers that run primarily in the lateral direction. Such
organization may give rise to anisotropic diffusion, i.e. diffusion that
is dependent on direction. We hypothesize that anisotropic diffusion
can be detected in the corneal tissue of whole ex-vivo eyes through
three-dimensional fluorescence recovery after photobleaching (3D
FRAP).
Methods: Fresh, intact porcine eyes were obtained from a local
slaughterhouse. The epithelia of the eyes were removed, and a
solution containing a fluorescent probe was applied to the cornea. 3D
FRAP was performed on the corneas and a custom Matlab program
was used to calculate the diffusion coefficient in three directions:
nasal-temporal (X), superior-inferior (Y) and anterior-posterior (Z).
3D FRAP was also performed on glutaraldehyde-treated corneas.
Results: The diffusion coefficient in the Z direction was significantly
decreased when compared to the diffusion coefficients in the X
(P=0.004, ANOVA) and Y (P<0.0001, ANOVA) directions. There
was no significant difference between the diffusion coefficients in
the X and Y directions. The corneas treated with glutaraldehyde had
significantly reduced diffusion coefficients in all directions when
compared to normal corneas (P<0.0001, ANOVA). Furthermore,
there was no significant difference between diffusion coefficients in
different directions in the glutaraldehyde-treated corneas.
Conclusions: Native porcine corneas exhibit anisotropic diffusion;
the diffusion in directions parallel to collagen fibers is faster
than the diffusion in directions orthogonal to collagen fibers. The
glutaraldehyde treatment decreases the diffusion in the cornea and
eliminates the anisotropy. The technique presented will be useful
in examining the effects of certain corneal treatments (e.g. corneal
cross-linking) as well as in developing tissue-engineered corneas that
mimic the material properties of the native tissue.
Commercial Relationships: R Glenn Hepfer, None; Peng Chen,
None; George O Waring, None; Hai Yao, None
Corneal Thickness is Positively Correlated with Tissue Elasticity
by Elastography Imaging
Michael D. Twa1, Srilatha Vantipalli2, Manmohan Singh3, Jiasong Li3,
Kirill Larin3. 1Vision Science, University of Alabama at Birmingham,
Birmingham, AL; 2Optometry, University of Houston, Houston, TX;
3
Biomedical Engineering, University of Houston, Houston, TX.
Purpose: Corneal thickness and hydration varies considerably in
normal and diseased eyes. There are conflicting reports from previous
studies regarding the influence of tissue hydration on corneal
biomechanics. Here we evaluated corneal elasticity as a function of
corneal thickness using optical coherence elastography (OCE).
Methods: Fresh whole rabbit eyes (n=10) were deepithelialized, then
treated with 0.9% saline every 5 minutes for 60 minutes, followed
by 20% Dextran every 5 minutes for 60 minutes. A focused air
pulse system was used to induce low amplitude (≤ 10 μm) tissue
deformation at the corneal apex that was imaged using a phasestabilized swept source optical coherence tomography system. Tissue
elasticity was measured along with total corneal thickness every
20 minutes. Tissue elasticity was quantified by the dynamic natural
frequency, which previous work has shown is linearly related to the
square root of Young’s modulus.
Results: Although corneal thickness changed little over the saline
treatment period, measured elasticity changed by 4% (P > .05).
Dextran treatment reduced corneal thickness by 250 μm and
reduced measured corneal elasticity by 30% (P < .0001). These two
parameters were highly correlated (R2 = .980; P = .0001).
Conclusions: Corneal thickness changes due to variations in tissue
hydration are positively correlated with measured tissue elasticity
by OCE. Further studies are needed to help separate the effects due
to corneal thickness alone and those due to corneal hydration, which
may affect both elastic and viscous components of the observed
viscoelastic response.
tissue section. Video tracking and Matlab were used to analyze bar
displacement and calculate the force required to separate EDM from
stroma over the course of the peel.
Results: No significant difference was found in the average peel
force/width (g/mm) necessary to separate EDM form stroma in
non-diabetic (0.082 ± 0.011) and non-severe diabetic (0.079 ± 0.004)
donor tissue (p = 0.907). In contrast, severe diabetic donor tissue
required significantly greater force/width to peel EDM (0.103 ±
0.028) compared to both non-diabetic (p = 0.038) and non-severe
diabetic (p = 0.029) tissue samples.
Conclusions: We have for the first time quantified the adhesion
strength between Descemet membrane and the underlying stroma
in human corneas. Differences in adhesion strength amongst nondiabetic, non-severe diabetic, and severe diabetic tissues suggest
that severe diabetes mellitus may be a risk factor influencing donor
preparation failures.
Commercial Relationships: Chaid Schwarz, None;
Benjamin T. Aldrich, None; Kimberlee Burckart, None;
Gregory Schmidt, None; Cynthia Reed, None; Mark A. Greiner,
None; Edward A. Sander, None
Average central corneal thickness and elasticity of whole fresh rabbit
eyes (n=10) over time. Deepithelialized corneas were treated with
0.9% saline every 5 minutes for 60 minutes, then with 20% dextran.
Central corneal thickness was measured using OCT and elasticity was
quantified as the dynamic natural frequency from optical cohernce
elastograhy imaging.
Commercial Relationships: Michael D. Twa; Srilatha Vantipalli,
None; Manmohan Singh, None; Jiasong Li, None; Kirill Larin,
None
Support: NIH/NEI R01-EY022362
Decreased corneal biomechanical properties in children with
osteogenesis imperfecta
Brenda L. Bohnsack, Lisa Lagrou, Sayoko E. Moroi. University of
Michigan, Ann Arbor, MI.
Purpose: Osteogenesis imperfecta (OI) is a genetic disease due to
type I collagen defects. In adults, OI is associated with degenerative
ocular pathologies, however, eye findings in children are not welldescribed.
Methods: A prospective, comparative, cross-sectional study of
pediatric patients (2-19 years) diagnosed with OI. Thirteen patients
with OI and five control patients underwent complete ophthalmic
examinations. Additional tests included ocular response analyzer,
pachymetry, axial length measurement, automated perimetry, and,
retinal nerve fiber layer (RNFL) thickness. Statistically significant
differences between OI and control groups were determined using
independent-samples t test.
Results: We found significantly (p<0.05) decreased corneal hysteresis
Results: We found significantly (p<0.05) decreased corneal
hysteresis (8.2±1.4mmHg vs. 10.5±1.3), and central corneal
thickness (CCT; 451.9 ± 29.1mm vs. 550.5±30.7 mm) in OI patients
compared to controls. There was no significant difference in corneal
resistance factor (9.9±1.3 mmHg vs. 10.5 ±0.8 mmHg). None of
these differences correlated with age, race, or gender. IOP corrected
for corneal thickness and hysteresis was significantly higher in OI
patients than controls (19.1 ± 3.0 mmHg vs. 13.7 ± 0.7 mmHg).
There were no differences in keratometry, axial length and RNFL
thickness in OI patients compared to controls.
Conclusions: Strength, flexibility and organization of type-I-collagen
are critical for corneal structure and function. In our study, we found
that children with OI had decreased corneal hysteresis and CCT,
which resulted in artificially low IOP readings. In adults, OI has
been associated with primary open angle glaucoma and keratoconus.
Our findings suggest that in OI, alterations in corneal biomechanical
properties are present at a young age and affected individuals should
be routinely screened for glaucoma and corneal pathologies.
Commercial Relationships: Brenda L. Bohnsack, None;
Lisa Lagrou, None; Sayoko E. Moroi, None
Adhesion Strength of Descemet Membrane in Healthy and
Diabetic Donor Corneas
Chaid Schwarz1, Benjamin T. Aldrich2, 3, Kimberlee Burckart3,
Gregory Schmidt3, Cynthia Reed3, Mark A. Greiner2, 3,
Edward A. Sander1. 1Biomedical Engineering, The University of
Iowa, Iowa City, IA; 2Department of Ophthalmology and Visual
Sciences, The University of Iowa, Iowa City, IA; 3Iowa Lions Eye
Bank, Coralville, IA.
Purpose: Descemet membrane endothelial keratoplasty (DMEK)
is an increasingly popular surgical procedure for treating diseases
involving the corneal endothelium. Anecdotal evidence suggests that
differences in the force required to peel the Endothelium-Descemet
membrane complex (EDM) during DMEK surgical preparation may
contribute to the greater incidence of preparation failures observed
in diabetic compared to non-diabetic donor tissues (Greiner et al.,
2014). Here, we report on the development of a mechanical testing
method for quantifying the peel strength of human donor corneas,
and our progress on quantitatively assessing the putative differences
between non-diabetic and diabetic tissues.
Methods: Corneoscleral tissues stored less than 14 days from
procurement from control non-diabetic, non-severe diabetic, and
severe diabetic donors 50-75 years old were used in this study.
To reflect loading conditions used in surgical preparation, we
applied cantilever bending to rectangular sections of corneal EDM
during a continuous peel test. Using a thin Nitinol bar anchored to
peripheral tissue with a calibrated force-displacement relationship,
a displacement of 0.25 mm/s was applied to the EDM in order
to separate EDM from the stromal layer over the length of the
Support: NIH Grant K08EY022912-01; Research to Prevent
Blindness Career Development Award; Edward Mallinckrodt Jr
Foundation Grant; Paul Lichter, M.D. Research Discovery Fund;
Alcon Research Institute Young Investigators Grant
Patient-Specific Computational Analysis of Laser in situ
Keratomileusis: A clinical validation study
Ibrahim Seven, Ali Vahdati, William J. Dupps. Ophthalmology,
Cleveland Clinic Cole Eye Institute, Mayfield Hts, OH.
Purpose: To develop a microstructural based, 3D, patient specific,
finite element model (FEM) of LASIK and compare the outcomes of
the simulations to actual post-LASIK follow-up tomographies.
Methods: Patient-specific tomographic data from 20 eyes of 12
patients undergoing sphero-cylindrical LASIK treatment were
meshed including epithelium, flap, wound, and residual stromal bed
using a custom meshing software. Case-specific treatment settings
were simulated. Simulated keratometry values (SimK) including the
average curvature of the steep (K1), flat (K2) meridians, and central
3mm circular region (Kmean) were obtained from the anterior surfaces
of both the actual tomographies and their generated FEMs. Mean
difference (MD) and mean absolute difference (MAD) between these
values were calculated to assess prediction error. Pearson correlation
values (R) between individual errors in predicted Kmean and age,
corneal hysteresis (CH), and corneal resistance factor (CRF) were
calculated.
Results: MD and MAD between simulated and actual post-LASIK
cases were -0.13 ± 0.36 D and 0.28 ± 0.25 D respectively. The
differences between simulated and actual SimK values were not
statistically significant (p values for Kmean=0.1, K1=0.4, K2=0.5). R
values between the individual differences in Kmean and CH, CRF, and
age were 0.63, 0.53, and -0.5, respectively (p = 0.004, = 0.01, and =
0.02, respectively).
Conclusions: The investigated computational modeling approach
demonstrated low prediction errors and may be useful for clinical
guidance in planning LASIK. Clinical biomechanical metrics and
surrogates explain some of the variance in prediction error and may
be useful to tailor generic material properties into patient specific
material properties to increase prediction accuracy.
Commercial Relationships: Ibrahim Seven, None; Ali Vahdati,
None; William J. Dupps, Cleveland Clinic/Optoquest (P)
Support: R01 EY023381, Ohio Third Frontier Innovation Platform
Award TECH 13-059, Unrestricted Grant from Research to Prevent
Blindness to the Dept. of Ophthalmology of the Cleveland Clinic
Lerner College of Medicine of Case Western Reserve University.
WST11-D and Near Infra Red (NIR) light induces ex-vivo and
in-vivo corneal stiffening using low energy and short irradiation
time
Jurriaan Brekelmans1, 2, Alexandra Goz2, 3, Alexander Brandis4,
Mor Dickman1, Rudy M. Nuijts1, Avigdor Scherz2, Arie Marcovich2, 3.
1
Ophthalmology, University Eye Clinic Maastricht, Maastricht,
Netherlands; 2Plant and Environmental Sciences, Weizmann Institute
of Science, Rehovot, Israel; 3Ophthalmology, Kaplan Medical Center,
Rehovot, Israel; 4Biological Services, Weizmann Institute of Science,
Rehovot, Israel.
Purpose: Controversy exists regarding the efficacy of shortened
corneal cross-linking (CXL) protocols. We previously described
the safety and efficacy of WST11-D/NIR in corneal stiffening using
parameters similar to the Dresden protocol for Riboflavin/UVA CXL.
This ex-vivo and in-vivo laboratory study sets out to determine the
stiffening effect of shortened WST11-D/NIR protocols with 1, 5 and
30 minutes of NIR irradiation in a rabbit model.
Methods: Corneas of 3 months old, male, New Zealand White
rabbits were treated in-vivo (n=24) and ex-vivo (n=54) using three
irradiation times and biomechanically tested immediately (ex-vivo)
or one month after treatment (in-vivo). One cornea of each pair was
mechanically de-epithelialized and topically impregnated with 2.5
mg/ml WST11 (Steba-Biotech, France) combined with dextran-500
(WST11-D) for 20 minutes. Next, irradiation with NIR light at
10mW/cm2 was applied for either 30 minutes (nin=3, nex=11), 5
minutes (nin=6, nex=5) or 1 minute (nin=3, nex=11) using a diode laser
at 755nm (CeramOptics, Israel). Untreated fellow eyes served as
controls. Corneal strips, 4±0.2mm in width, underwent biomechanical
stress-strain measurements using a biomaterial tester (Minimat,
Rheometric Scientific GmbH, Germany) to obtain the tangent
modulus. Results were compared by paired Student-T tests and linear
mixed modelling using SPSS (IBM Corp., Armonk, NY).
Results: Ex-vivo treatment resulted in a similar significant
increase (>100%) in Young’s modulus (p<0.001) for all three
irradiation protocols, figure 1. One month after in-vivo treatment
Young’s modulus increased significantly after 30 and 5 minutes of
irradiation (125% and 35% respectively, p<0.05) but did not increase
significantly after 1 minute of irradiation, figure 2.
Conclusions: Shortening NIR irradiation time to 5 minutes without
increasing total delivered energy results in significant corneal
stiffening both ex-vivo and in-vivo. Differences between ex-vivo
and in-vivo stiffening may result from living tissue behavior, making
1 minute irradiation in-vivo not sufficient. This novel ultra-fast
treatment with reduced irradiation time and overall irradiation energy
may provide a safe alternative to Riboflavin/UVA CXL for the
treatment of corneal ectasia.
Fig. 1 Young’s Modulus, ex-vivo experiments.
The stromal rise thinned the overlying epithelium by -18 ± 5.1
microns, equivalent to 35% of the preoperative epithelial thickness.
The volume of the epithelium thinned was redistributed peripheral
to the inlay diameter (2 mm), extending the change at the anterior
corneal surface to about twice the inlay diameter. The central anterior
corneal surface rose by 10 ± 3.2 microns, creating a progressive Add
power profile with a central peak of -5 D.
Conclusions: When a meniscus shaped corneal inlay is placed
beneath a corneal flap, epithelial remodeling redistributed most of the
inlay’s volume, with a much smaller biomechanical remodeling of the
stroma anterior to the inlay.
*CAUTION: Investigational device. Limited by Federal (United
States) law to investigational use.
Commercial Relationships: Alan J. Lang, ReVision Optics, Inc.;
Arturo Chayet, ReVision Optics, Inc. (C); Enrique BarragánGarza, ReVision Optics, Inc. (C)
Fig. 2 Young’s Modulus, in-vivo experiments.
Commercial Relationships: Jurriaan Brekelmans, None;
Alexandra Goz, None; Alexander Brandis, Int. publication
Number: WO 2013/027222 Al (P); Mor Dickman, None;
Rudy M. Nuijts, None; Avigdor Scherz, Int. publication Number:
WO 2013/027222 Al (P); Arie Marcovich, Int. publication Number:
WO 2013/027222 Al (P)
Remodeling of the Cornea Induced by a Shape-Changing
Hydrogel Inlay
Alan J. Lang1, Arturo Chayet2, Enrique Barragán-Garza3. 1R&D,
ReVision Optics, Lake Forest, CA; 2Codet Vision Institute, Tijuana,
Mexico; 3Laser Ocular Hidalgo, Monterrey, Mexico.
Purpose: Assess epithelial and stromal remodeling anterior to a
hydrogel inlay placed within the cornea.
Methods: The analysis was performed on subjects (N = 30) enrolled
in a prospective clinical investigation of the Raindrop® Near Vision
Inlay, implanted in corneal of the non-dominate eye of emmetropic
presbyopic subjects. The inlay had a meniscus shape with a 2 mm
diameter and a 34 micron central thickness. The inlay was placed
beneath a targeted 150 microns corneal flap (femtosecond laser), over
the center of the light constricted pupil. The change to the anterior
corneal surface profile (ΔC) was measured using postoperative minus
preoperative wavefront measurements. The change to the epithelial
thickness profile (ΔE) was measured using Fourier domain optical
coherence tomography (OCT). Changes to the anterior stroma (flap)
were reflected in alteration of Bowman’s layer shape (ΔBow) and
visible OCT images, but this changes was more accurately calculated
from the above two measurements; ΔBow = ΔC - ΔE.
Results: When placed on the flap bed, the inlay’s volume displaced
stroma anterior to the inlay, with 91% of the inlay volume reflected
in the change to Bowman’s layer directly above the inlay’s diameter.
The 8% loss is approximately accounted for by a slight increase in
stromal volume just outside the inlay diameter. Bowman’s layer rose
centrally by 28 ± 8.5 microns, 85% of the inlay’s central thickness.
Retroprosthetic Membrane Formation in Boston
Keratoprothesis: A Case Control Matched Comparison of
Titanium versus PMMA Back Plate
Joelle Hallak1, Rushi K. Talati2, Jose De la Cruz1, Maria S. Cortina1.
1
Ophthalmology, University of Illinois at Chicago, Chicago, IL;
2
Ophthalmology, Northwestern University, Chicago, IL.
Purpose: Retroprosthetic membrane (RPM) formation is the most
common complication following Boston keratoprosthesis (Kpro)
implantation. While design changes have been made to improve
tissue compatibility, little evidence exists comparing the frequency of
RPM formation in various Kpro models. This retrospective, casecontrol matched study compared the frequency of RPM formation in
patients implanted with a titanium back plate Kpro versus patients
implanted with a polymethyl methacrylate (PMMA) back plate Kpro.
Methods: Twenty-one eyes from 21 consecutive patients implanted
with a titanium back plate Kpro were case-matched with 21 eyes
from 21 patients implanted with a PMMA backplate Kpro by a
single surgeon at a large academic center. Case-matching was
based on preoperative diagnosis, type of surgery (e.g. aphakic vs
pseudophakic, Kpro only vs Kpro combined with vitrectomy and/
or glaucoma shunt) and age. Formation of visually significant RPM
requiring YAG laser treatment or surgical membranectomy was the
primary outcome measure. Initial visual acuity (VA), best corrected
visual acuity (BCVA), and VA at follow-up were secondary outcome
measures. Paired and unpaired t-tests were performed for statistical
significance.
Results: Ten patients implanted with a titanium Kpro developed
visually significant RPM compared to 7 patients implanted with a
PMMA Kpro (p=0.38). Time from surgery to YAG treatment was
not significantly different between the titanium and PMMA groups
(227.10 ± 44.58 days vs 275.86 ± 75.50 days). Initial VA prior to
surgical intervention was better for titanium Kpro patients (14.3%
vs 0% had VA of 20/200 or better; p=0.03 for mean VA difference).
Following surgery, patients implanted with a titanium Kpro achieved
BCVA earlier (63.19 ± 16.44 days vs 173.90 ± 34.16 days; p=0.01),
however the BCVA was not found to be statistically different
(p=0.19). VA at most recent follow-up was not significantly different
between the two groups (p=0.92).
Conclusions: Titanium Kpro back plates do not seem to significantly
reduce RPM formation compared to PMMA Kpro back plates when
patients are controlled for preoperative diagnosis. However, a larger
prospective study would be necessary to confirm these findings.
While differences in visual outcomes exist between the two Kpro
models in the short-term, they are temporal and insignificant in the
long-term.
Commercial Relationships: Joelle Hallak; Rushi K. Talati, None;
Jose De la Cruz, Alcon (C); Maria S. Cortina, None
Support: Research to Prevent Blindness Departmental Support
The Resistance to Collagenase Dissolution of Cross-Linked
Cryopreserved Donor Corneas
Darrell R. Lewis, Mark Seamone, Christopher Seamone.
Ophthalmology, Dalhousie University, Halifax, NS, Canada.
Purpose: To determine if UV-A riboflavin cross-linking (CXL) of
donor corneas after cryopreservation confers resistance to keratolysis
as has been described for fresh donor corneas.
Methods: Three methods of donor cornea cryopreservation were
compared to donor corneas stored in Optisol-GS at 4°C for less than
2 weeks (control). The cryopreservation arms consisted of corneas
stored in Optisol-GS at -15°C, Optisol-GS at -70°C, and ≥99%
anhydrous glycerol at -80°C. Within each cryopreservation method,
corneas were randomized to either receive CXL or not. The primary
outcome measure was the time to complete corneal dissolution in a
collagenase-A solution.
Donor corneas cryopreserved in Optisol-GS at -15°C (n=4), OptisolGS at -70°C (n=4), and ≥99% glycerol at -80°C (n=4) were thawed
for 30 minutes in a room temperature balanced salt solution. Corneas
were mounted on artificial anterior chambers and de-epithelialized.
Pre-treatment with 0.1% riboflavin in dextran solution every 2
minutes for 15 minutes was undertaken. High fluence, accelerated
CXL (9 mW/cm2, 10 min) was then performed. As controls, 4 donor
corneas matching each of the cryopreservation methods were thawed
but did not undergo CXL. Additionally, 4 fresh donor corneas did not
undergo CXL. Corneas were punched with an 8mm Baron trephine.
These 8 mm corneal buttons were agitated in a 0.3% collagenase-A
solution at 37°C. Corneas were examined every 30 minutes until
complete dissolution occurred.
Data was analysed via Mann-Whitney U-test and Kruskal-Wallis
analysis where appropriate.
Results: Donor corneas cryopreserved in ≥99% glycerol at -80°C
required a statistically significantly longer time for complete corneal
dissolution than fresh donor corneas (p=0.03). Dissolution times for
donor corneas cryopreserved in Optisol-GS at either -15°C or -70°C
did not statistically significantly differ from fresh donor corneas,
p=0.13 and p=0.26 respectively.
Conclusions: Cross-linking donor corneas cryopreserved in ≥99%
glycerol at -80°C increases their resistance to keratolysis. Donor
corneas cryopreserved in Optisol-GS have a similar resistance to
keratolysis as fresh donor corneas.
The ability to enhance cryopreserved donor corneas via CXL may
prove advantageous to reduce graft failure rates. Cryopreserved tissue
may be appropriate for deep anterior lamellar keratoplasty, glaucoma
tube shunt coverage, Boston keratoprosthesis carriers, and tectonic
grafting for melts.
Commercial Relationships: Darrell R. Lewis, None;
Mark Seamone, None; Christopher Seamone, None
Biomechanical properties of the cornea measurement using
Scheimpflug noncontact tonometry in aniridia patients
Bogumil Wowra, Dariusz Dobrowolski, Dorota Tarnawska,
Edward Wylegala. Railway Hospital Katowice. II School of Medicine
with the Division of Dentistry in Zabrze Medical University of
Silesia, Katowice, Poland.
Purpose: To evaluate biomechanical properties of the cornea in
patients with congenital aniridia.
Methods: 26 patients (16 women, 10 men) with congenital aniridia
aged from 6 to 55 underwent corneal examination using Scheimpflug
noncontact tonometry. CCT, deformation amplitude, first applanation
time, second applanation time, highest concavity and IOP were
evaluated.
Results: CCT ranged from 496 μm to 718 μm (mean 623 μm).
Deformation amplitude range from 0.36 to 1.01 mm (mean 0.62 mm)
corelated with IOP result range from 15.6 mmHg to 42.8 mmHg
(mean 22.82 mmHg).
Conclusions: Evaluation of corneal biomechanical properties is
useful in estimation real IOP in aniridia patients.
Commercial Relationships: Bogumil Wowra;
Dariusz Dobrowolski, None; Dorota Tarnawska, None;
Edward Wylegala, None
Cross-linking biomechanical effect in human corneas by
same energy, different UV-A fluence: an enzymatic digestion
comparative evaluation
George Asimellis1, 2, A. J. Kanellopoulos2, 3. 1Kentucky College of
Optometry, PikeVille, KY; 2LaserVision.gr Clinical & Research Eye
Institute, Athens, Greece; 3Ophthalmology, NY University, NY, NY.
Purpose: Collagenase has been known to contribute to breakdown of collagen in the corneal stroma. This breakdown is a
vigorous biochemical process used as an indirect metric of corneal
biomechanical properties. The stabilizing biochemical effect of
corneal cross-linking (CXL) may be thus reflected by an increased
resistance to collagenase digestion. Clinical results suggesting
comparable effectiveness of Higher-fluence (irradiance) CXL
variations to the conventional protocol (3 mW/cm2) in stabilizing
keratoconus progression have been reported. The enzymatic
degradation resistance modulations achieved via CXL application at
different UV-A irradiances has not been studied in human corneas.
The purpose of this study is to comparatively evaluate ex-vivo these
enzymatic degradation resistance differences.
Methods: The study involved twenty-five human donor corneas,
randomly allocated to 5 groups (n=5 each). CXL was applied with
UV-A irradiance of 3-mW/cm2, 9-mW/cm2, 18-mW/cm2, 30-mW/
cm2, and 45-mW/cm2, maintaining equal cumulative energy dose
of 5.4 J/cm2. UV-A was delivered on half of the cornea. The nonirradiated halves served as controls. Specimens were subjected to
collagenase-A enzymatic digestion. The time to complete dissolution
in each specimen was recorded.
Results: Time to dissolution in group-A (3-mW/cm2 for 30’) was
321±13.4 minutes (range: 300 to 330) compared to 171±8.2 (range:
165 to 180) for their control. In group-B (9-mW/cm2 for 10’)
282±19.6 minutes (range: 270 to 315) compared to 177±6.7 (165
to 180) for their control. In group-C (18 mW/cm2 for 5’) 267±19.6
minutes (range: 240 to 285) compared to 177±7.7 (range 165 to 180)
for their control. In group-D (30-mW/cm2 for 3‘) 252±12.5 minutes
(range: 240 to 270) compared to 180±10.6 minutes (range: 165 to
195) for their control. In group-E (45-mW/cm2 for 2‘) 204±17.1
minutes (range: 180 to 225) compared to 186±8.2 minutes (range:
180 to 195) for their control.
Conclusions: The data in this ex-vivo human corneal study indicate
that the biomechanical effect of CXL studied by resistance to
enzymatic digestion in human corneas is comparable between
irradiance of 9, 18 and 30-mW/cm2, and appears to be reduced at
fluence of 45mW/cm2.
Design and execution of the patterned half-area illumination of the
treated corneas.
Commercial Relationships: George Asimellis, None;
A. J. Kanellopoulos, i-Optics (C), ISP Surgical (C), Keramed (C),
Avedro (C), Allergan (C), Alcon (C)
Penetrating keratoplasty versus deep anterior lamellar
keratoplasty: which technique establishes healthy corneal
biomechanics?
HANS R. VELLARA, Noor Q. Ali, Akilesh Gokul, Jason Turuwhenua,
Charles N. McGhee, Dipika Patel. Ophthalmology, University of
Auckland, Auckland, New Zealand.
Purpose: The cornea is biomechanically compromised in
keratoconus. Ideal management options would restore the corneal
biomechanics to that of a healthy cornea. This study analysed the
corneal biomechanical properties of keratoconic corneas which had
undergone either a penetrating keratoplasty (PKP) or a deep anterior
lamellar keratoplasty (DALK) and compared them to those of healthy
control corneas
Methods: All eyes were examined by slit-lamp biomicroscopy,
Pentacam tomography, and the CorVis ST (CST). The CST is a
non-contact tonometer coupled with an ultra-high speed Scheimpflug
camera which provides descriptors of ocular biomechanics.
Furthermore, specialised software was designed in house to remove
the collateral globe displacement to the air-pulse to isolate the
maximum corneal deformation (MCD). A multiple regression
analysis was used to compare CST outputs and additionally derived
parameters between groups after controlling for patient age, intraocular pressure (IOP), central corneal thickness (CCT), maximum
simulated keratometry (KMAX), suture number, and time from surgery.
Results: This study included 42 PKP, 27 DALK, and a 152 healthy
control eyes. There were no significant differences in patient age,
IOP, time from surgery, KMAX, donor size, and donor age, between
treated groups (p>0.05). CCT was significantly higher in DALK-
treated corneas (590 ± 52µm, p<0.001) (compared to PKP-treated
(542 ± 50µm), and healthy control corneas (544 ± 34µm)).
Furthermore, there were no significant differences in patient age, IOP,
and KMAX between treated and control groups (p>0.05). However,
multiple regression analysis revealed a significantly higher MCD
in PKP-treated corneas (0.86 ± 0.02mm) than in DALK (0.80 ±
0.02 mm, p=0.03) and healthy controls (0.79 ± 0.01mm, p=0.02).
Additionally, the PKP-treated corneas had a greater number of
parameters significantly different to control corneas than DALKtreated corneas.
Conclusions: DALK-treated corneas were biomechanically more
similar to healthy controls than PKP-treated corneas. This might be
due to the scar tissue interface in DALK-treated corneas redirecting
the force applied to the peripheral cornea and the scar interface in
PKP-treated corneas resulting in an incomplete transfer of energy to
the peripheral cornea.
Commercial Relationships: HANS R. VELLARA, None;
Noor Q. Ali, None; Akilesh Gokul, None; Jason Turuwhenua,
None; Charles N. McGhee, None; Dipika Patel, None
The Mechanics of Corneal Deformation and Rupture for
Penetrating Injury in the Human Eye
Andrew Rau3, Scott Lovald3, Steven Nissman1, John McNulty2,
Jorge Ochoa3, Michael Baldwinson4. 1University of Pennsylvania
Perelman School of Medicine, Scheie Eye Institute, Philadelphia, PA;
2
Materials and Corrosion Engineering, Exponent, Inc., Menlo Park,
CA; 3Biomedical Engineering Practice, Exponent, Inc., Philadelphia,
PA; 4Google[X], Mountain View, CA.
Purpose: A penetrating eye injury is a surgical emergency with a
guarded visual prognosis. The purpose of this study was to determine
the force required to rupture the cornea with a penetrating object, and
to study how this force is affected by the object geometry.
Methods: Thirty-six human cadaveric eye specimens were used
for the study. Spherical indenters of three different diameters
(1.0, 1.5, and 2.0 mm) were pressed into the apex of the cornea
at two displacement rates (1.0 mm/s and 5.0 mm/s) until rupture
of the specimen occurred. Low strain stiffness (between 3-5 mm
indenter displacement), high strain stiffness (between 80%-90%
of the maximum failure force), indenter displacement at failure,
and the force at failure were determined from the test data and
used to characterize the mechanical tissue response. Multi-variable
regression analysis was performed on the output parameters in order
to determine associations of the input variables (indenter size, test
speed, tissue postmortem time, and specimen geometry) on the
mechanics of the tissue response.
Results: Twenty-nine of the 36 specimens failed at the location
where the indenter contacted the cornea, four specimens failed at the
limbus, and three specimens failed in the sclera near sites of muscle
attachment. The average force at failure caused by the 1.0 mm, 1.5
mm, and 2.0 mm diameter indenters increased from 30.5±5.5 N to
40.5±8.3 N to 58.2±14.5 N, respectively (p<0.002) (Table 1, Figure
1). The force at failure was also determined to be associated with
the donor age (p<0.001), and globe diameter (p<0.041), but was not
associated with intraocular pressure, tissue postmortem time, axial
length, or indenter speed.
Conclusions: This study has quantified the force-displacement
response of a large series of human cadaveric eyes subjected to
penetrating indentation loads on the cornea. The results provide
useful data for characterizing the relationship between corneal
rupture and the geometry of a penetrating object.
The outcome measures for all test groups. Note: results presented as
Avg. ± St. Dev.
Force (N) vs. displacement (mm) curves for the 1.0 mm indenter
tests (specimens 25-36). Specimens that failed at the contact location
in the cornea are indicated with a circle at the failure point, while
specimens that failed in other regions of the eye are indicated with an
X at the failure point.
Commercial Relationships: Andrew Rau, Google[X]
(F); Scott Lovald, Google[X] (F); Steven Nissman, None;
John McNulty, Google[X] (F); Jorge Ochoa, Google[X] (F);
Michael Baldwinson, Google[X]
Support: Partial support for this study was provided by Google[X]
Comparison between the diagnostic ability of corneal
Tomography and novel biomechanical parameters in keratoconus
Vishal Jhanji1, Tommy Chan1, Marco Yu2. 1Ophthalmology,
Chinese Univ of Hong Kong, Kowloon, Hong Kong; 2Hang Seng
Management College, Kowloon, Hong Kong.
Purpose: Early and accurate diagnosis of keratoconus is of
paramount importance for effective management of these cases.
Corneal tomography as well as biomechanical evaluation are being
increasingly employed for this purpose. This study compared
the diagnostic ability of tomographic and novel biomechanical
parameters in cases with keratoconus
Methods: Forty-two eyes of 42 participants were included (21
normal, 21 keratoconus). Corneal tomographic parameters were
measured using Scheimpflug imaging (Pentacam, Oculus, Wetzlar,
Germany). Biomechanical parameters were obtained from a
Scheimpflug camera (Corvis, Oculus, Wetzlar, Germany). The
area under receiver operating curve (AUC) and partial AUC for
specificity ≥ 80% for each parameter was calculated to assess the
discrimination ability and compared between devices. Correlation
analysis was performed between parameters obtained from both
devices. A p-value < 0.05 was considered statistically significant. In
addition, deformation amplitude (DA) ratio 1 and 2, a ratio between
deformation amplitude at apex and at 1mm and 2 mm from the centre
of the corneal apex respectively, was proposed and validated on
Corvis.
Results: Corneal parameters were selected for comparison based
on their diagnostic performance. Belin/Ambrosio Enhanced Ectasia
Display (BAD) showed the highest AUC and partial AUC followed
by Ambrosio’s relational thickness maximum (ARTmax) with AUC
≥ 0.95 and partial AUC ≥ 0.18. DA ratio 1 and 2 also demonstrated
the highest AUC and partial AUC amongst other Corvis parameters.
There was no significant difference for AUC and partial AUC
of BAD and ARTmax compared to those of DA Ratio 1 and 2
(p>0.186). Significant correlation was found between BAD and DA
Ratio 1 and 2 (p<0.003) as well as ARTmax and DA Ratio 1 and 2
(p<0.002) for eyes with keratoconus.
Conclusions: Novel biomechanical parameters were able to
provide discriminative ability between normal and keratoconic eyes
comparable to tomographic analysis using Scheimpflug imaging.
Corneal biomechanics may have the potential to effectively diagnose
keratoconus.
Commercial Relationships: Vishal Jhanji, None; Tommy Chan,
None; Marco Yu, None
Physical properties of electron-beam irradiated corneas stored in
recombinant human serum albumin
Khoa D. Tran1, Mark A. Terry2, Christopher Stoeger1. 1Lions
VisionGift, Portland, OR; 2Devers Eye Institute, Portland, OR.
Purpose: While electron-beam (e-beam) irradiated cornea tissue have
been successfully used in ophthalmic procedures, structural changes
within the tissue as a result of e-beam irradiation and the resulting
functional outcomes have not been described in detail. Here, we
report preliminary studies comparing the physical properties of fresh
versus e-beam treated corneas.
Methods: The clarity, structural properties, and surgical handling
of e-beam irradiated corneas were examined and compared to
fresh donor corneas. Dark field microscopy and computer-aided
analysis using Matlab were performed to determine corneal clarity.
Differential scanning calorimetry and tissue histology were used
to examine the corneal collagen matrix organization. The rigidity
(elastic modulus) of the cornea were determined using Brillouin
optical microscopy. Tissue and suture handling evaluations were
performed by two different fellowship-trained cornea surgeons who
were masked to tissue treatment conditions prior to handling.
Results: The clarity of fresh and e-beam irradiated corneas were 92.4
± 3.5% and 89.7 ± 2.7%, respectively (N=18, P=0.009). Differential
scanning calorimetry revealed that the denaturing temperature of
e-beam irradiated tissue is approximately 2°C lower than fresh
corneas (N=5), although histological sections comparing fresh and
e-beam treated tissue revealed minimal changes in the collagen
matrix (N=8). Brillouin optical microscopy and analysis suggests that
irradiated corneas are slightly more rigid than fresh corneas, although
this change did not affect the tissue’s ability to handle external forces
as no statistically significant differences were detected in the shear
moduli of the two groups (7.3 kPa vs. 6.2 kPa, N=6). Likewise,
cornea surgeons evaluating e-beam treated tissue did not note major
differences in the tissue’s elasticity or ability to handle sutures.
Conclusions: Our preliminary results suggest that e-beam treated
corneas possess similar properties to fresh donor corneas. Although
there is a 2.7% decrease in clarity and slight changes to the
structure of the corneal tissue after irradiation, these changes do
not appear to have adverse effects on their clinical and functional
properties. Additional studies are required to strengthen our current
understanding of e-beam irradiation on corneal tissue, including
examination of e-beam treated tissues after prolonged storage (>1
year).
Commercial Relationships: Khoa D. Tran, None; Mark A. Terry,
None; Christopher Stoeger, None
Hierarchical changes in corneal collagen structure under load
James S. Bell1, Sally Hayes1, Charles Whitford2, Ahmed Elsheikh2,
Keith M. Meek1. 1Cardiff University, Cardiff, United Kingdom;
2
University of Liverpool, Liverpool, United Kingdom.
Purpose: Current understanding of corneal biomechanics is based
upon hierarchical maps of collagen distribution, but these maps
are obtained from corneas at rest. To quantify the mechanical and
microstructural response to load, we carried out mechanical tests of
corneal samples in an X-ray beam.
Methods: 14 human corneas with scleral rims were obtained from
Bristol Eye Bank and transported in organ culture medium to
Diamond Light Source, where they were deswelled in 15% dextran
solution for one day. Full thickness super-inferior tensile strips 3mm
in width were sectioned from the corneas and attached to a custombuilt extensometer that could be mounted on the stages of the I02 and
I22 beamlines.
X-ray scatter images were obtained in the wide angle (WAXS – I02)
and small angle (SAXS – I22) configurations, allowing changes
in the collagen structure and fibrillar architecture to be measured
at length scales ranging from angstroms (e.g. the helical period of
tropocollagen molecules) up to tens of nanometres (e.g. the diameter
of collagen fibrils). Variations across the cornea were assessed by
scanning the X-ray beam across the sample.
Results: X-ray scattering images were taken following static strains
of 1.4%, 2.8%, 5% and 8%. The direction of preferential molecular
and fibrillar orientation shifted from naso-temporal to superiorinferior following the 1.4% uniaxial strain application and the degree
of anisotropy increased with strain. The largest change in molecular
architecture took place between 2.8% and 5% uniaxial strain
increments, after which the total collagen aligned in the superiorinferior direction was 24±10% greater than in other directions and
the ratio of collagen aligned in the superior-inferior direction to
that in the naso-temporal direction changed from 0.86±0.04 at rest
to 1.90±0.40. Beyond 5% uniaxial strain the rate of reorientation
diminished.
Conclusions: Quantifying changes in the structure and fibrillar
architecture of the cornea under load provides a means to relate
findings from mechanical tests with changes in microstructure.
We find here that in healthy human corneas there is a reorientation
“cut-off” at around 5% tensile strain, after which resistance to load
is mediated by other means, such as fibril slipping. The ability of the
corneal collagen network to reorientate informs us about binding and
interconnectivity, and the ability of the cornea to adapt in what is a
variable mechanical environment.
Commercial Relationships: James S. Bell, None; Sally Hayes,
None; Charles Whitford, None; Ahmed Elsheikh, None;
Keith M. Meek, None
Support: MRC Programme Grant 503626
3D Inflation Strains in Porcine Corneas
Keyton Clayson1, 2, Elias Pavlatos1, Jun Liu1. 1Biomedical
Engineering, The Ohio State University, Columbus, OH; 2Biophysics,
The Ohio State University, Columbus, OH.
Purpose: To measure the 3D deformation of the cornea using highresolution ultrasound speckle tracking, and to evaluate the presence
of shear during inflations of the cornea.
Methods: Seven porcine globes were tested within 72 hours postmortem. Whole globes were immersed in a 10% dextran solution
for 1 hour to reduce swelling, then secured to a custom-built holder
and immersed in 0.9% saline. The anterior chamber was infused
with Optisol GS (Bausch and Lomb) via a column system to control
intraocular pressure (IOP). The globes were preconditioned with 25
pressure cycles from 10 to 12 mmHg, then equilibrated at 10 mmHg
for 1 hour. Testing involved raising the pressure to 11 mmHg and
12mmHg consecutively, with a 15 minute equilibration time at each
pressure. For each step, a 55 MHz ultrasound probe with an imaging
window of 5.5 mm was aligned along the nasal-temporal direction
of the cornea, and consecutive 2D frames of radiofrequency data
were acquired at 14 µm steps scanned over 2 mm in the superiorinferior direction to form a volume centered on the corneal apex.
The 3D principal strains (ε1, ε2, ε3), max shear, and volume ratio
were calculated using an ultrasound speckle tracking technique (Cruz
Perez et al, ABME, 2015). Volumetric strain maps and vector plots
were visualized in Paraview (Kitware Inc).
Results: The average principal strains, max shear, and volume ratio
in porcine corneas are summarized in Table 1. Representative maps
of the principal strains and principal vector directions are shown
in Figure 1. During corneal inflation, the principal strain of largest
magnitude was through-thickness compression (ε3), while the other
two principals (ε1, ε2) were tensile and largely in-plane. Significant
shear with a magnitude larger than all principals was observed. The
volume ratios close to one confirmed minimum swelling and nearincompressibility during inflation.
Conclusions: 3D ultrasound speckle tracking showed that the cornea
experiences compressive, tensile, and shear strains during IOP
increase. The predominance of shear deformation during pressure
increase suggests that weakness in resisting shear may be involved in
corneal ectasia and warrants future investigations.
Table 1: Mean ± SD of principal strains and maximum shear during
inflation (n=7).
Figure 1: Maps of principal vector directions (top) and principal
strains (bottom) in a representative cornea (A-P: anterior-posterior,
S-I: superior-inferior, N-T: nasal-temporal).
Commercial Relationships: Keyton Clayson, None;
Elias Pavlatos; Jun Liu, None
Support: NIH RO1EY020929
A Multicenter Study of Ocular Response Analyzer Custom
Variables in Disqualified and Candidate Refractive Surgery
Screening Patients
Vinicius S. De Stefano1, 2, Ibrahim Seven1, James B. Randleman3,
William J. Dupps1. 1Cole Eye Institute, Cleveland Clinic, Cleveland,
OH; 2Ophthalmology, Federal University of Sao Paulo, Sao Paulo,
Brazil; 3Emory Eye Center, Emory University, Atlanta, GA.
Purpose: To assess the ability of air-puff derived measures of
biomechanical behavior to predict clinical decisions on refractive
surgery candidacy.
Methods: Data were retrospectively collected from consecutive
refractive surgery screening exams by two surgeons from different
academic centers (Cleveland Clinic Cole Eye Institute (CC) and
Emory Eye Center (EEC)). Only right eyes were analyzed. The
disqualified subject groups consisted of 19 (CC) and 28 (EEC) eyes
and included subjects who were disqualified as candidates based
on clinical impression after reviewing available clinical, topo/
tomographic (Placido and Scheimpflug), and biomechanical data
(Ocular Response Analyzer (ORA)). Control groups consisted of eyes
deemed clinically to be safe candidates for LASIK (n=26 (CC) and
23 (EEC)). Keratometric and pachymetric data from the Pentacam
were compared for each group. While standard ORA variables were
available to surgeons at the time of the decision, the 3 investigatorderived variables (Table 1, previously described by Hallahan et al,
Ophthalmol 2014) were calculated retrospectively and surgeons were
effectively masked to them. Comparisons were performed using
t-tests for independent samples.
Results: Anterior curvature and corneal thickness data were
significantly different between groups (Table 1). Hysteresis loop area
(HLA) was the only biomechanical variable that was significantly
different between disqualified and control subjects across both
centers (Table 2).
Conclusions: Across two clinical sites, a custom biomechanical
variable, HLA, was predictive of which refractive surgery screening
patients were selected for LASIK and which were disqualified.
Certain measures of the corneal dynamic response to an air puff may
serve as objective correlates to clinically perceived risk of ectasia.
Table 1. Disqualified patients group and control group baseline and
pre-operative corneal features.
Table 2. Pre-operative variables derived from the ORA for
disqualified and control (candidate) patient groups.
Commercial Relationships: Vinicius S. De Stefano,
None; Ibrahim Seven, None; James B. Randleman, None;
William J. Dupps
Support: CAPES Grant PDSE - 99999.007333/2015-03; R01
EY023381; Ohio Third Frontier Innovation Platform Award TECH
13-059; Unrestricted Grant from RPB to the Dept. of Ophthalmology
of the Cleveland Clinic Lerner College of Medicine of Case Western
Reserve University; Unrestricted Grant from RPB to the Dept. of
Ophthalmology of the Emory University
A New Stiffness Parameter in Air Puff Induced Corneal
Deformation Analysis
Cynthia J. Roberts1, Ashraf M. Mahmoud1, Jeffrey Bons4,
Arif Hossain4, Ahmed Elsheikh5, Riccardo Vinciguerra6,
Paolo Vinciguerra7, 8, Renato Ambrosio2, 3. 1Ophthalmology &
VIsual Science; and Biomedical Engineering, The Ohio State
University, Columbus, OH; 2Rio de Janeiro Corneal Tomography and
Biomechanics Study Group, Rio de Janeiro, Brazil; 3Ophthalmology,
Federal University of São Paulo, Rio de Janeiro, Brazil; 4Mechanical
& Aerospace Engineering, The Ohio State University, Columbus,
OH; 5School of Engineering, University of Liverpool, Liverpool,
United Kingdom; 6Division of Ophthalmology, University of
Insubria, Varese, Italy; 7Eye Center, Humanitas Clinical and Research
Center, Rozzano, Italy; 8Vincieye Clinic, Milano, Italy.
Purpose: To investigate a new stiffness parameter in corneal
deformation analysis and compare responses in normal (NL) and
keratoconic (KC) subjects, matched for intraocular pressure (IOP).
Methods: A new stiffness parameter (SP) is defined as the resultant
pressure at inward applanation, divided by corneal deflection
amplitude at highest concavity (HCDeflectAmp). The spatial
and temporal profiles of the Corvis ST air puff (Oculus, Wetzlar,
Germany) were characterized using hot wire anemometry from 0
to 16mm from the nozzle. Measured velocity was correlated with
the pressure profile exported by the Corvis ST, measured within the
nozzle. The z position of the cornea at the time of inward applanation
was used to calculate an adjusted air pressure value (adjAP1) at the
time and position of first applanation. An algorithm to correct IOP
estimation based on finite element modeling, termed IOPfem, was
used for the equation: SP = (adjAP1 - IOPfem)/ HCDeflectAmp.
Linear regression analyses between corneal deformation parameters
(CDP’s) and SP were performed on a retrospective dataset of 258
KC eyes and 460 NL eyes. CDP’s from a subset of 119 eyes of 119
subjects in each group were matched for IOPfem and compared using
t-tests. Significance threshold was p < 0.05.
Results: All CDP’s evaluated showed a significant difference
between NL and KC, except peak distance. (Table 1) The KC
group had lower SP values, thinner pachymetry, shorter applanation
lengths, greater absolute values of applanation velocities, earlier first
applanation times and later second applanation times, greater HC
deformation and HC deflection amplitudes, and lower HC radius of
concave curvature (greater concave curvature). All CDP’s evaluated
showed a significant relationship with SP in both groups. Stiffer eyes
were associated with greater pachymetry, longer applanation lengths,
lower absolute value of applanation velocities, later first applanation
times, earlier second applanation times, lower HC deformation and
HC deflection amplitudes, shorter peak distances, greater HC radius
of concave curvatures (flatter), and higher values of IOPfem.
Conclusions: Keratoconic eyes demonstrated less resistance
to deformation than normal eyes with similar IOP. All of the
deformation parameters investigated showed a significant relationship
with the new stiffness parameter. This may be useful in future
biomechanical studies comparing populations without having to
match IOP.
corneal apex during deformation-recovery process together with the
reference pressure value enabled generation of corneal hysteresis
representing viscoelastic properties of the cornea (Figs.1A-B). The
corneal response to the air pulse was determined for ascending (5
to 35 mmHg) and descending (from 35 to 5 mmHg) IOP levels.
Later on, the corneal response to the air pulse before and after CXL
procedure under constant IOP condition was measured. In all cases,
the parameters of recorded hysteresis loops (applied pressure vs.
deformation amplitude) were analyzed.
Results: Air-puff SS-OCT measurements enabled to calculate
corneal hysteresis loop parameters such as the hysteresis curve area
or maximum deformation amplitude. The corneal hysteresis area
became lower with ascending IOP value (Fig. 1B). Additionally,
viscoelastic behavior of ex vivo porcine corneas was manifested
as a difference in the hysteresis area measured for ascending and
descending IOP values (Fig. 1C). There was a statistically significant
correlation between hysteresis area and maximum deformation
amplitude (R=0.99, p=0.001) (Fig. 1D). The central corneal thickness
drops immediately after CXL procedure (p=0.05) (Fig. 2A). CXL
made the corneas more stiff resulting in lower hysteresis area values
(Figs. 2B-C).
Conclusions: OCT combined with the air-puff gives additional
information about IOP and biomechanical properties of pig eye ex
vivo. Further development of this method has potential of being
used as a tool for minimally invasive assessment of IOP and central
biomechanical properties in healthy and diseased eyes in humans.
Commercial Relationships: Cynthia J. Roberts, Oculus
(C), Oculus (F); Ashraf M. Mahmoud, None; Jeffrey Bons,
None; Arif Hossain, None; Ahmed Elsheikh, Oculus
(F); Riccardo Vinciguerra, None; paolo vinciguerra;
Renato Ambrosio, Oculus (C)
Support: OCULUS Optikgeräte GmbH
Assessment of biomechanical properties of porcine corneas with
air-puff swept source OCT
Ewa C. Maczynska1, Karol M. Karnowski1, Bartlomiej Kaluzny2,
Ireneusz Grulkowski1, Maciej D. Wojtkowski1. 1Faculty of Physics,
Astronomy and Informatics, Nicolaus Copernicus University, Torun,
Poland; 2Collegium Medicum, Nicolaus Copernicus University,
Bydgoszcz, Poland.
Purpose: A novel approach to assess Intraocular Pressure (IOP) and/
or specific biomechanical properties of the cornea were proposed.
Biomechanical properties of the porcine corneas ex vivo at wellcontrolled levels of the intraocular pressure, and the impact of crosslinking (CXL) on corneal biomechanics were studied.
Methods: Thirty fresh, enucleated porcine eyes were measured
with high-speed swept source Optical Coherence Tomography
(SS-OCT) instrument combined with the air-puff chamber for
non-contact generation of corneal deformation. Data acquired from
minutes of dextran and, therefore, preferable in clinical use. Lower
viscosity of imbibition solution as well as higher hydration of corneal
stroma could be identified as factors for accelerating the diffusion of
riboflavin. With correct settings two-photon microscopy is a precise
way to determine the concentration distribution of riboflavin in
corneal stroma.
Commercial Relationships: Ewa C. Maczynska, None;
Karol M. Karnowski, None; Bartlomiej Kaluzny, None;
Ireneusz Grulkowski, None; Maciej D. Wojtkowski
Support: Foundation for Polish Science (TEAM; #TEAM/20118/8), Polish Ministry of Science and Higher Education (IUVENTUS
PLUS; #IP2014 014073)
Two-photon fluorescence microscopy (TFM) and UVA
transmission of corneas with HPMC and dextran solutions prior
to corneal crosslinking (CXL)
Theo G Seiler1, 2, Tobias Ehmke3, Isaak Fischinger1, 2,
Alexander Heisterkamp4, Beatrice E. Frueh1. 1Universitätsklinik
für Augenheilkunde, Inselspital Bern, Bern, Switzerland; 2IROC
AG, Zuerich, Switzerland; 3Laserzentrum Hannover e.V., Hanover,
Germany; 4Institut für Quantenoptik, Universität Hannover, Hanover,
Germany.
Purpose: To evaluate the corneal stroma riboflavin gradient and
its implications to safety prior to CXL in commercially available
dextran/HPMC solutions at different time points.
Methods: Four different groups of porcine corneas, 5 each, were
compared regarding the riboflavin concentration in the anterior
stroma. Before all experiments, stable corneal hydration conditions
were established for the corresponding solution. Two groups (A and
B) were treated with 0.1% riboflavin in 20% dextran and 2 groups
with 0.1% riboflavin in 1.1% HPMC in 1.1% NaCl (C and D).
After imbibition, two-photon microscopy was used to determine
fluorescence intensity after 10 minutes (group A and C) and 30
minutes (group B and D). For signal attenuation and concentration
determination corneas were saturated and measured a second time by
TFM. Additionally, the distribution was simulated mathematically
and compared to the experimental results. UVA transmission was
determined in all 4 groups after the imbibition.
Results: Riboflavin concentration is decreasing with depth for all
solutions. Analysis of the fit functions leads to diffusion coefficients
of D = 7.67x10-7 for the HPMC solution for 10 minutes and D=
1.31x10-7cm2/s for the dextran solution for 30 minutes. The riboflavin
distribution after 10 minutes of imbibition is similar to the one after
30 minutes of 20% dextran (Fig.1). Transmission values of the
HPMC solutions were 0.16±0.01% after 10 and 0.05±0.01 after 30
minutes, respectively 0.26±0.02% and 0.19±0.01% in the dextran
group. Physiologically hydrated corneas imbibed with 20% dextran
after 30 minutes lead to a transmission of 0.16±0.01%.
Conclusions: The riboflavin distribution after 10 minutes of
imbibition of HPMC is comparable to the distribution after 30
Commercial Relationships: Theo G Seiler; Tobias Ehmke,
None; Isaak Fischinger, None; Alexander Heisterkamp, None;
Beatrice E. Frueh, None
Multivariate analyses of in vivo anisotropic, hyperelastic
biomechanical properties of the human cornea with age, IOP and
corneal thickness
Abhijit Sinha Roy, Mathew Francis, Sushma Tejwani, Jyoti Matalia,
Himanshu Matalia, Rohit Shetty. Narayana Nethralaya, Bangalore,
India.
Purpose: We performed multivariate analyses of anisotropic,
hyperelastic biomechanical properties of the human cornea using in
vivo air-puff applanation and inverse finite element technique.
Methods: 50 eyes of 25 normal subjects, ranging in age from 1150 years, were imaged with Corvis-ST and Pentacam (OCULUS
Optikgerate Gmbh, Germany). Inclusion criteria were spherical
equivalent less than -3D, no prior ocular surgery, no inflammation
and no corneal degenerations. Cornea deformation from Corvis-ST
and 3-D geometry from Pentacam was used to construct patientspecific inverse finite element models based on a recent study
(Sinha Roy et al., J Mech Behav Biomed Mat 2015; 48:173-82).
The recent study quantified the properties using a fiber and depth
dependent hyperelastic model with 4 material constants (C10, C20,
k1 and k2). C10 and C20 quantified the elastic response of the
cellular matrix. k1 and k2 quantified the non-linear stiffening effect
of the collagen fibers, arranged in a lamellar network with depthdependent crosslinks. Multivariate analyses of covariance of material
constants was performed with age, intraocular pressure (IOP) and
central corneal thickness (CCT) as covariates. Association between
the eyes of a subject was also studied in the multivariate model.
Mean±standard error of the mean is reported here.
Results: Mean age, IOP and CCT was 22.4±1.26 years, 534.6±3.8
μm and 15.5±0.4 mmHg, respectively. Mean C10, C20, k1 and k2
was 87.41±6.3 kPa, 5.1±0.25 MPa, 35±0.6 kPa and 154.4±23.5,
respectively. C10 (p=0.62), C20 (p=0.42) and k1 (p=0.20) were
similar for the left and right eye of the same subject. However, k2
of the left eye was significantly higher than the same of the right
eye (p=0.01). k2 was also positively correlated with age (p=0.02)
indicating a signficant age related stiffening effect in the human
cornea. C10 was positively correlated with CCT (p=0.01). The other
constants did not correlate with either age, IOP or CCT (p>0.05).
Conclusions: We studied the in vivo anisotropic properties of the
human cornea as a function of IOP, CCT and age for the first time.
Increase in k2 with age indicated some age related stiffening effect
in the human cornea. CCT may also influence the biomechanical
property of the cellular matrix (C10). Further study with a larger
sample size and older subjects is needed to confirm the trends.
Commercial Relationships: Abhijit Sinha Roy, Carl Zeiss (F),
Carl Zeiss (R), Bioptigen (F), Avedro (F), Topcon (F), Cleveland
Clinic Innovations (P), Narayana Nethralaya Foundation (P);
Mathew Francis; Sushma Tejwani, None; Jyoti Matalia, None;
himanshu Matalia, None; rohit shetty, Carl Zeiss (F), Narayana
Nethralaya (P), Allergan (F)
Effect of cataract surgery on the biomechanical property of the
cornea
Yoshiaki Kiuchi1, Yoshitaka Nakao1, Noriko Ihara1,
Shunsuke Nakakura2, Ryo Asaoka3, Makoto Aihara3. 1Ophthalmology,
Hiroshima University, Minami-Ku, Japan; 2２) Department of
Ophthalmology, Tsukazaki Hospital, Himeji, Japan; 3３)
Department of Ophthalmology, The University of Tokyo, Tokyo,
Japan.
Purpose: Cataract surgery may change the ocular biomechanical
property. Corneal visualization scheimpflug technology (Corvis ST
Tonometry: CST) provides several parameters that represent the
corneal biomechanical property. The purpose of our study was to
investigate the effect of phacoemulsification and intraocular lens
implantation on the CST parameters.
Methods: Data from 59 normal eyes of 59 subjects (mean age=74.0
years old) were prospectively acquired at Tsukazaki Hospital
and Hiroshima University Hospital between February 2014 and
December 2014. The intraocular pressure (IOP) with Goldmann
applanation tonometry (IOP-G), central corneal thickness, axial
length, corneal curvature, and CST parameters were measured
before and one week, one month and 3 months after cataract surgery.
Measurements with CST and IOP-G were carried out three times
per patient and the averages of all three measurements were used in
statistical analyses (linear mixed effect model).
Results: IOP-G significantly decreased from 15.2±4.3 mmHg to
11.8±3.1 mmHg at 3 months after cataract surgery (p<0.05). The
maximum deformation amplitude and peak distance increased
significantly. The changes after cataract surgery of A1 time, A2 time,
A1 length and A2 length were not significant. Although in normal
subjects A2 velocity decreases with IOP reduction (Asaoka, PLOS
ONE 2015), A2 velocity in our subjects increased significantly after
cataract surgery (p<0.05).
Conclusions: The corneal biomechanical property might be changed
by cataract surgery.
Commercial Relationships: Yoshiaki Kiuchi, None;
Yoshitaka Nakao, None; Noriko Ihara; Shunsuke Nakakura,
None; Ryo Asaoka, None; Makoto Aihara, None
Support: Research Grant 26462688 (to YK) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
Small incision lenticule extraction (SMILE) vs LASIK: an ex vivo
biomechanical evaluation of low and high myopic corrections
Briana C. Gapsis1, Adam Ahlquist1, Henry Perry1, 4,
George Asimellis2, A. J. Kanellopoulos2, 3. 1Department of
Ophthalmology, Nassau University Medical Center, East Meadow,
NY; 2Laservision.gr Clinical & Research Eye Institute, Athens,
Greece; 3Department of Ophthalmology, New York University
Medical School, New York, NY; 4Ophthalmic Consultants of Long
Island, East Meadow, NY.
Purpose: The small-incision lenticule extraction (SMILE) procedure
and laser in situ keratomileusis (LASIK) are both safe, effective, and
predictable surgical techniques for the correction of myopia. SMILE
presents an alternative to LASIK, which currently may be considered
the most established form of laser vision correction. The purpose of
this study is to evaluate corneal biomechanical changes associated
with low and high myopic correction performed with small incision
lenticule extraction (SMILE, employing the Visumax laser, Carl Zeiss
Meditec, Jena Germany) compared to Femtosecond-laser assisted
LASIK (FS200 & EX500 lasers, Alcon Surgical, Ft. Worth, TX)
Methods: Thirty human donor corneas were subjected to myopic
SMILE or LASIK. These corneas were randomly allocated to
one of four investigative groups (n=5 each), subjected to the
following treatments: group-A -3.00 diopters (D) SMILE; group-B
-8.00D SMILE; group-C -3.00D LASIK; group-D -8.00D LASIK.
Additionally, two control groups (n=5 each) were formed, one
for each procedure: group-E SMILE and group-F LASIK. The
corneas in these control groups were subjected to the corresponding
femtosecond-laser lamellar cuts but not to tissue removal.
Biomechanical evaluation of tensile strength was conducted by
biaxial force application. Primary outcome measures were stress at
10% and 15% strain, and Young’s modulus at 10% and 15% strain.
Results: In SMILE, the average relative difference (Δ) of the four
metrics evaluated was -35.6% between the -3.00D correction and
control and -50.2% between the -8.00D correction and control. In
LASIK, average Δ was -21.2% between the -3.00D correction and
control, and -50.4% between the -8.00D correction and control. When
evaluating the same degree of myopic correction, SMILE, compared
to LASIK, appears to result in greater biomechanical reduction
for the -3.00D correction (-25.9%) while the difference at -8.00D
correction is not statistically significant.
Conclusions: Biomechanical tensile strength is reduced with
increasing amounts of myopia corrected in both procedures. LASIK
results in less strength reduction in smaller myopes while, in higher
myopes, SMILE and LASIK appear to result in similar corneal tensile
strength reduction.
Commercial Relationships: Briana C. Gapsis, None;
Adam Ahlquist, None; Henry Perry, Blephex (C), Omidria (C),
Ophthalmic Consultants of Long Island, Allergan (C), Alcon (C);
George Asimellis, None; A. J. Kanellopoulos, Alcon/WaveLight
(C), i-Optics (C), Avedo (C), ISP Surgical (C), Keramed (C), Allergan
(C)
Blood thyroid hormone levels and corneal tomographical,
topographical and biomechanical properties
Zsuzsa Flaskó1, 2, Elena Zemova2, Timo Eppig3, László Módis4,
Achim Langenbucher3, Zisis Gatzioufas5, Nóra Szentmáry2, 6,
Berthold Seitz2. 1Department of Ophthalmology, Kenézy Hospital,
Debrecen, Hungary; 2Department of Ophthalmology, Saarland
University Medical Center, Homburg/Saar, Germany; 3Experimental
Ophthalmology, Saarland University, Homburg/Saar, Germany;
4
Department of Ophthalmology, Debrecen Medical University Center,
Debrecen, Hungary; 5Moorfields Eye Hospital, London, United
Kingdom; 6Department of Ophthalmology, Semmelweis University,
Budapest, Hungary.
Purpose: Hypothyroidism could be associated with keratoconus
and may be one of its causing factors. Our aim was to determine
the potential impact of blood thyroid hormone levels on corneal
topography, tomography and biomechanics.
Methods: 757 eyes (376 left eyes, 500 males) were included in our
study. Patients were classified as euthyroid (n=563) or hypothyroid
(n=194) based on blood thyroid hormone status (TSH, FT3, FT4).
We further subdivided hypothyroid patients in those with (n=122)
and without (n=72) medication. Patients were examined using
TMS-5 topographer (Tomey, Tennenlohe, Germany)(keratoconus
index: KCI; keratoconus severity index: KSI; keratoconus prediction
index: KPI), Pentacam HR tomographer (Oculus Optikgeräte GmbH,
Wetzlar, Germany)(keratoconus index: KI; central keratoconus index:
CKI) and Ocular Response Analyzer (ORA, Reichert Ophthalmic
Instruments, Buffalo, NY) (corneal resistance factor: CFR; corneal
hysteresis: CH; keratoconus match index: KMI).
Results: Using Mann-Whitney U test, KCI, KSI, KPI of TMS-5 and
KI, CKI of Pentacam were significantly lower in hypothyroid patients
with medication compared to euthyoids or hypothyroid patients
without medication (p<0.001 for all). In addition, KMI, CH and CRF
of ORA were significantly greater in hypothyreosis patients with
medication compared to euthyroids or hypothyroid patients without
medication (p<0.001 for all). Using a generalised linear model, KMI
depended on blood FT3 level (p=0.029), however, other parameters
of TMS-5, Pentacam or ORA were not affected significantly by blood
TSH, FT3 or FT4 levels (p>0.136).
Conclusions: Blood TSH, FT3 and FT4 levels may affect
keratoconus specific indices of corneal topography and tomography,
and corneal biomechanical properties. Unlike in keratoconus, an
abnormally increased KMI index of ORA may be indicative for
reduced FT3 blood level and hypothyreosis.
Commercial Relationships: Zsuzsa Flaskó, None;
Elena Zemova, None; Timo Eppig, None; László Módis,
None; Achim Langenbucher, None; Zisis Gatzioufas, None;
Nóra Szentmáry, None; Berthold Seitz, None
Support: HARVO (Hungarian Association for Research in Vision
and Ophthalmology) Travel Grant
New dynamic corneal biomechanical parameters derived from
curvature versus pressure analysis of Ocular Response Analyzer
(ORA) data
David Luce. Ophthalmology, Luce Technical Consulting, Clarence
Center, NY.
Purpose: To present calculation and visualization methods for new
and existing corneal biomechanical properties using curvature versus
pressure analysis of Ocular Response deformation data.
Methods: Corneal deformation, induced by the ORA air jet, is
detected optically, This signal undergoes sign changes, inversion and
calibration to provide corneal curvature that is then plotted versus the
applied eye pressure.. Energy conversion (to heat) and the dynamic
viscoelastic moduli during the 30 msec deformation are explicitly
visible in the “hysteresis” loop that is created. Numerical results are
calculated from the loop’s geometric properties, especially areas of
the sub-loops of positive and negative curvature. A Corneal Bending
Modulus (CBM) is determined from the slope of the initial curvature
following first applanation (zero curvature).
Retrospective ORA data from 34 eyes of 17 subjects from a regular
clinical practice and with varied ocular conditions were analyzed.
Results: We report here on statistics for a single parameter, the
Corneal Bending Modulus (CBM), the average slope of the pressure/
curvature function in a region starting at first applanation and ending
before peak pressure and any iris interference. The KC and 4 norm.
diverse population’s mean/std dev / age is 47 ± 9 yrs. and includes
normal, keratoconus, LASIK, INTACS, PRK, PKP subjects. The
CV for the CBM between right and left eyes is 26% and 9.`% for
unaltered eyes, normal and KC, N=7.
Conclusions: In all cases in the 34 diverse eyes normal eyes
have lower compliance than the KC eyes (N=7). In all cases the
populations examined here are small. Despite ourconclusions are
that there are indications that the technique at using curvature shows
significant promise. Muc that justifies continued work. It should be
pointed out that the mathematical treatment taken here differs fom the
current ORA in that the inversion of the raw ORA data emphasizes
the low signal levels regions, the opposite of the current ORA data,
The new biomechanical method method extracts informartion during
the initinal negative curvature, a unique balance point. In addition
any significant interference/with the iris\lens is cleaarly visible and
detected.
Diverse CBM images
parameters in patients (i.e. keratoconus and cross-linking treatment).
Also, it allows absolute estimates of IOP, independent of mechanical
parameters, of high relevance to monitoring of glaucoma.
Commercial Relationships: Nandor Bekesi; Carlos Dorronsoro;
Susana Marcos, None
Support: Comunidad de Madrid & EU Marie Curie (FP7/2007-2013/
REA 291820), ERC Advanced Grant 294099, Spanish Government
Grant FIS2014-56643-R
Figure 2 - Summary of CBM trial resultsCommercial Relationships:
David Luce
Biomechanical properties and IOP reconstruction from air-puff
corneal deformation imaging: validations in model and porcine
eyes
Nandor Bekesi, Carlos Dorronsoro, Susana Marcos. Institute of
Optics, Consejo Superior de Investig Cientif CSIC, Madrid, Spain.
Purpose: Keratoconus is a disease that alters the mechanical
behavior of the cornea. Air-puff corneal dynamic deformation
depends on the corneal biomechanical properties, corneal thickness
and Intraocular Pressure (IOP). The aim of the study is to estimate
both mechanical properties and IOP by means of inverse Finite
Element modeling.
Methods: Corneal deformation of artificial hydrogel corneal models
and enucleated porcine eyes (n = 8) to an air puff were measured by
high speed Scheimpflug imaging (Corvis ST) at different IOPs (15
to 45 mmHg). A computational optimization incorporating inverse
modeling was utilized in order to obtain mechanical properties from
Corvis deformation images. Corneal Finite Element Models were
programmed in ANSYS software, using Scheimpflug-based corneal
geometry measurements, and literature values for the sclera. Airpuff pressure dynamics was measured in a previous study (Kling
et al PLOS One 2014). The parameters of the hyperelastic material
model and the IOP were changed in an iterative simulation to fit the
simulated deformed shape and the deformation history of the corneas
with the measured ones.
Results: The maximum deformation amplitudes (DA) of the artificial
corneal model were 0.69, 0.52 and 0.47 mm at IOP of 15, 30 and
45 mmHg, respectively. Simulations resulted in Young’s moduli of
0.475, 0.465 and 0.478 MPa for the three IOP levels respectively.
DA of porcine corneas were 1.25, 0.869 and 0.624 mm at IOP of
15, 30 and 45 mmHg, respectively. Resulting Young’s moduli of the
cornea were 0.462, 0.436 and 0.454 MPa. The simulated IOP values
matched the nominal IOP values within the step size (1 mmHg) of the
optimization.
Conclusions: High speed imaging together with numerical
optimization allows simultaneous accurate reconstruction of both
corneal mechanical properties and IOP, as validated with models
ex vivo. This study extends previous work where the reconstructed
mechanical parameters were obtained ex vivo with fixed, known IOP.
The proposed method can be useful to monitor corneal mechanical
Finite Element Analysis of Penetrating Injury to the Human Eye
Scott Lovald1, Andrew Rau1, Steven Nissman2, Nicoli Ames3,
John McNulty4, Jorge Ochoa1, Michael Baldwinson5. 1Biomedical
Engineering, Exponent, Menlo Park, CA; 2Scheie Eye Institute,
University of Pennsylvania Perelman School of Medicine,
Philadelphia, PA; 3Mechanical Engineering, Exponent, Boulder, CO;
4
Materials and Corrosion Engineering, Exponent, Menlo Park, CA;
5
Google[x], Mountain View, CA.
Purpose: Penetrating injuries to the eye are among the most frequent
causes of permanent visual impairment. The purpose of this study
was to determine the strain at which rupture occurs in the cornea due
to a penetrating object.
Methods: For the experiment, probes of varying diameters (1.0, 1.5,
and 2.0 mm) were pressed into the apex of the cornea in 36 human
cadaveric globes until perforation of the specimen. To match the
experiment, an axisymmetric finite element model of the idealized
human globe was created in Abaqus 6.14 (Figure 1). The cornea and
sclera were modeled as isotropic nonlinear hyperelastic materials. To
evaluate model sensitivity, three separate models for the sclera were
constructed (minimum, medium, and maximum stiffness) within the
range of experimentally-observed material behavior. In addition, two
separate internal pressure conditions were implemented: 1) a sealed
fluid cavity with an initial pressure, and 2) a constant pressure applied
directly to the cavity surfaces. The model was used to map the
force-displacement response of the experiments and quantitatively
determine a peak strain at which the eye ruptures.
Results: For the experiments, the average force at failure increased
from 30.5±5.5 N (1.0 mm probe) to 40.5±8.3 N (1.5 mm probe) to
58.2±14.5 N (2.0 mm probe) as the probe size increased (p<0.002).
The force-displacement responses of the finite element models of
all three probe sizes bounded and tracked the experimental data.
The peak strain at failure in the cornea was located on its posterior
surface. This strain was in the range of 29% to 33% for all models
analyzed. Figure 2 shows strain contours for the 1 mm probe.
Conclusions: The current study has developed a validated finite
element model of the human eye for analysis of penetrating injury
to the cornea. The results have determined an objective failure strain
of corneal tissue, which is consistent between sensitivity studies of
varying material models, pressure conditions, and penetrating objects
sizes. These results provide critical, quantitative information for
understanding the risk of penetrating eye injuries.
Finite element model set-up for globe indentation. Material sections
are indicated in the middle image. The figure on the right shows the
indenter probe displaced into the cornea.
Purpose: To investigate changes in corneal tangent elastic
modulus and other corneal biomechanics following short-term
orthokeratology.
Methods: Seventeen Chinese subjects (age, 18-25 years) were fitted
with orthokeratology (ortho-k) lenses for 1 month in both eyes.
Their baseline spherical equivalent was between -2.13 and -4.38D
with a corneal toricity within 2D. Subjective refraction and ocular
measurements were monitored in 4 visits, namely at the baseline
and after the first overnight wear, the first week, and first month
of ortho-k treatment. Corneal topography and pachymetry were
performed. The corneal tangent elastic modulus (E) was measured
using a novel corneal indentation device. Corneal hysteresis (CH)
and the corneal resistance factor (CRF) were measured using the
Ocular Response Analyzer. All measurements were conducted in
the morning. One eye from each subject with a better uncorrected
visual acuity (UCVA) after 1 month of ortho-k treatment was used for
analysis.
Results: The mean spherical equivalent decreased from -3.15 +/0.72D at the baseline to -0.11 +/- 0.22D at 1 month, final UCVA
-0.07 +/- 0.07. Significant corneal flattening (both the steepest
and flattest meridians) was observed as quickly as after 1 night of
ortho-k treatment (Friedman test, p<0.001). Similar rapid central
corneal thinning was demonstrated (Repeated measures analysis
of variance, RMANOVA, p<0.001). Compared with the baseline,
the CRF decreased significantly throughout the ortho-k treatment
(RMANOVA, p=0.001) (from 10.4 to 9.8mmHg, a reduction of
5.8%). No significant change was observed in CH (RMANOVA,
p=0.066). A significant increase in E was observed (RMANOVA,
p=0.001) (from 0.456 to 0.509MPa, an increase of 11.8%). No
correlation was found between initial corneal tangent elastic modulus
and the speed of myopia reduction.
Conclusions: Similar to previous studies, short-term ortho-k
treatment altered corneal biomechanics in terms of the CRF with
no change in CH. The increase in corneal tangent elastic modulus
revealed a stiffer cornea from ortho-k treatment. Should corneal
tangent elastic modulus be a more suitable parameter for evaluating
the corneal response to ortho-k treatment requires more studies.
The maximum principal strain in the cornea is shown for each
1.0 mm probe analysis at the step corresponding to the average
experimental failure force.
Commercial Relationships: Scott Lovald; Andrew Rau,
Google[x] (F); Steven Nissman, None; Nicoli Ames, Google[x]
(F); John McNulty, Google[x] (F); Jorge Ochoa, Google[x] (F);
Michael Baldwinson, Google[x]
Support: Partial financial support for this study was provided by
Google[X]
Short-term orthokeratology on corneal tangent elastic modulus a pilot study
Andrew K. Lam1, Ying Hon1, Angel Wong1,
Jimmy Tse1, Guo-Zhen Chen2, Shu-Hao Lu2, David C. C. Lam2.
1
School of Optometry, The Hong Kong Polytechnic University,
Hong Kong, China; 2Department of Mechanical and Aerospace
Engineering, The Hong Kong University of Science and Technology,
Hong Kong, China.
Corneal hysteresis and corneal resistance factor in ortho-k treatment.
Each error bar indicates 1 standard deviation.
Corneal tangent elastic modulus in ortho-k treatment. Each error bar
indicates 1 standard deviation.
Commercial Relationships: Andrew K. Lam, None; Ying Hon,
None; Angel Wong, None; Jimmy Tse, None; Guo-Zhen Chen,
None; Shu-Hao Lu, None; David C. C. Lam

Session 273 Corneal Extracellular matrix and biomechanics

Transcription

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