ARVO 2016 Annual Meeting Abstracts
339 Glaucoma Biomechanics
Tuesday, May 03, 2016 11:00 AM–12:45 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 3540–3573/A0238–A0271
Organizing Section: Glaucoma
Program Number: 3540 Poster Board Number: A0238
Presentation Time: 11:00 AM–12:45 PM
Aqueous Humor Flow in the Posterior Chamber of the Eye in the
Case of Iridotomy
Jennifer H. Tweedy1, Mariia Dvoriashyna2, Rodolfo Repetto2.
1
Department of Bioengineering, Imperial College London, London,
United Kingdom; 2Department of Civil, Chemical and Environmental
Engineering, University of Genoa, Genoa, Italy.
Purpose: The choice of optimal size and location of an iridotomy is
still poorly understood. We study aqueous flow driven by production
in the ciliary body in the posterior chamber of the eye, and also
consider the effect of miosis on the flow. We use a theoretical
approach that allows the problem to be solved semi-analytically. We
use the results find both the pressure difference between anterior and
posterior chambers, which gives insight into the risk of angle closure
glaucoma, and also the stress generated on the surrounding tissues.
We use it to predict the range of iridotomy sizes that maintain the
pressure within safe limits and avoid large stresses.
Methods: We derive the shape of the posterior chamber from
ultrasound images. Since its geometry is long and thin, we use
lubrication theory to simplify the problem and we justify a quasisteady approach to model miosis. In our model we treat the
iridotomy as a point sink in the iris and we assume the flux through
it is proportional to the pressure drop across it. We can achieve a
numerically tractible model by mathematically regularising the
pressure, and we implement it using a finite difference method.
Results: The geometry of the posterior chamber significantly
influences the pressure and flow, and in particular the height and
length of the iris-lens channel and the diameter of the iridotomy.
Conversely, the location of the iridotomy on the iris and the velocity
of the iris during miosis do not have a significant effect. We find the
iridotomy diameter that maximizes the velocity of the jet of aqueous
passing through the iridotomy, and show it is much higher during
miosis than otherwise. In the case of pupillary block an iridotomy
with a diameter of at least 20 um is needed to avoid dangerously high
pressures.
Conclusions: The ideal size and location of an iridotomy is
influenced by various geometrical and fluid mechanical factors. We
find the most significant ones are the size of the hole, the width and
height of the narrow iris-lens channel and the possible presence of
pupillary block. For certain iridotomy diameters, we cannot rule
out the possibility that the jet velocity through the iridotomy during
miosis might become large enough so as to cause corneal damage.
Pressure distribution and depth-averaged velocity vectors of the
flow in the posterior chamber in the presence of an iridotomy with
diameter 100 um.
Commercial Relationships: Jennifer H. Tweedy, Ophtec (C);
Mariia Dvoriashyna, None; Rodolfo Repetto, Ophtec (C)
Program Number: 3541 Poster Board Number: A0239
Presentation Time: 11:00 AM–12:45 PM
Regionally Discrete Aqueous Humor Outflow Quantification
using Fluorescein Canalograms
Eric N. Brown1, Ralitsa Loewen2, 3, Pritha Sengupta2, 3,
Joel S. Schuman2, 3, Ian A. Sigal2, 3, Nils A. Loewen2, 3. 1Department of
Ophthalmology, Vanderbilt University, Nashville, TN; 2Department
of Ophthalmology, University of Pittsburgh, Pittsburgh, PA; 3The
Louis J Fox Center for Vision Restoration, University of Pittsburgh,
Pittsburgh, PA.
Purpose: To quantify conventional outflow directly in its anatomic
location in a common anterior segment culture model.
Methods: We obtained fluorescein canalograms in six porcine whole
eyes (WhEs) and six porcine anterior segment cultures (ACs). Eyes
were perfused with a constant pressure of 15 mmHg using media
containing 0.017 mg/ml fluorescein. Flow patterns were visualized
using a stereo dissecting microscope equipped for fluorescent
imaging as previously described. Canalograms were analyzed for
filling time per quadrant using a custom designed R software package
and a generalized additive model. Changes in outflow was quantified
and compared over time in anterior segment cultures. The code and
examples of its use will be publically available at GitHub.com.
Results: In WhEs, quantifiably faster flow was seen nasally
compared to other regions. ACs had reduced numbers of functional
collectors on day 1 without exhibiting a preferential flow pattern.
Statistical analysis revealed correlations and increase in nasal filling
in ACs on day 3. Automated imaging analysis algorithm classified
areas of perilimbal outflow based on the total amount of flow and the
rate of flow. Filling rates uncovered convergent perilimbal flow.
Conclusions: An algorithm was developed that analyzes regional
and circumferential outflow patterns. Altered flow was detected in a
common anterior segment culture model.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Methods: In order to examine the effects of AMPK activation on
IOP, a previously published system of perfused constant flow ex vivo
human anterior segments was employed. All donor pairs of eyes
(aged 71, 82, and 84 years) were obtained within 24 hours after death
from National Disease Research Interchange. No donors were known
to have a history of glaucoma or other ocular disorder. After a process
that included removal of vitreous, lens, and iris, anterior segments
were rinsed thoroughly with culture media and mounted into custom
plexiglass culture chambers. Anterior segments were perfused at
a constant flow rate of 2.5 µL/min. Perfused tissue was allowed
to equilibrate at 37oC and 5% CO2 until a stable baseline IOP was
achieved. Then one eye was perfused with 2.5 µl of 1x PBS per 1
mL of ex vivo media as a control while the opposite eye received
2.5 µL of 200 mM 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4carboxamide (AICAR)—an adenosine analog and AMPK activator—
per 1 mL of ex vivo media.
Results: Treatment with 2.5µL of 200mM AICAR in 1mL of ex vivo
media results in a mean decrease in IOP of 18.54±1.78% by day 7
(p<0.05 for paired t-tests; n=3), compared with paired opposite eye
controls.
Conclusions: Our findings in perfused ex vivo human anterior
segments suggests that AMPK is a regulatory element for IOP and
possible novel therapeutic target in POAG. Assessing the effects
of topical administration of AMPK-activating agents in an in vivo
animal model will yield a more complete understanding of the role
of AMPK signaling in regulating aqueous humor outflow and may
further strengthen the possibility of future therapeutic applications in
humans.
Quantification of conventional aqueous humor outflow.
Commercial Relationships: Eric N. Brown, None;
Ralitsa Loewen, None; Pritha Sengupta, None; Joel S. Schuman,
None; Ian A. Sigal, None; Nils A. Loewen, None
Support: National Eye Institute KO8-EY022737; Research to
Prevent Blindness; National Eye Institute 5R01EY013178-16;
National Eye Institute 2T32EY017271-06A1 (IVS Training
Program); National Eye Institute 5R01EY023966-02
Program Number: 3542 Poster Board Number: A0240
Presentation Time: 11:00 AM–12:45 PM
Treatment with AICAR decreases intraocular pressure in
perfused ex vivo human anterior segments
Ayan Chatterjee1, Guadalupe Villarreal3, Dong-Jin Oh2,
Douglas J. Rhee2. 1Wills Eye Hospital, Philadelphia, PA; 2Department
of Ophthalmology and Visual Sciences, Case Western Reserve
University, Cleveland, OH; 3Wilmer Eye Institute, Baltimore, MD.
Purpose: Primary open-angle glaucoma (POAG) is a leading cause
of irreversible blindness worldwide and elevated intraocular pressure
(IOP) is the only rigorously tested modifiable risk factor. IOP is a
function of aqueous humor production in the ciliary body and outflow
facility through the eye’s primary drain, the trabecular meshwork
(TM). AMP-activated protein kinase (AMPK) has been shown
to regulate extracellular matrix (ECM) turnover and cytoskeletal
organization in primary cultured human TM cells. Mice who lack the
gene encoding the catalytic alpha-2 subunit of AMPK have increased
intraocular pressure as well as decreased aqueous humor outflow
facility.
Commercial Relationships: Ayan Chatterjee, WIPO Patent
Application WO/2015/138391 (P); Guadalupe Villarreal, WIPO
Patent Application WO/2015/138391 (P); Dong-Jin Oh, WIPO
Patent Application WO/2015/138391 (P); Douglas J. Rhee, WIPO
Patent Application WO/2015/138391 (P)
Support: HHMI Med Fellows Research Grant
Program Number: 3543 Poster Board Number: A0241
Presentation Time: 11:00 AM–12:45 PM
Estimating human trabecular meshwork stiffness by numerical
modeling and advanced OCT imaging
Ke Wang1, Murray A. Johnstone2, Chen Xin3, Steven Padilla2,
Ruikang K. Wang3, C R. Ethier1. 1Biomedical Engineering, Georgia
Institute of Technology, Atlanta, GA; 2Ophthalmology, University of
Washington, Seattle, WA; 3Bioengineering, University of Washington,
Seattle, WA.
Purpose: The cause of increased outflow resistance leading to
ocular hypertension in glaucoma remains unknown. Human
trabecular meshwork (hTM) stiffness, measured by AFM, was
markedly elevated in glaucomatous vs. normal eyes (Last et al.,
2011). However, in that study, the TM was dissected free from its
physiological environment and mechanically loaded differently
than in vivo. Our goal was to estimate hTM stiffness using an
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
alternate approach, based on advanced OCT imaging and numerical
biomechanical modeling.
Methods: Anterior regions of normal post mortem human eyes
(n=2; ages: 74, 79 years; post mortem time: 24, 9 hours; Sightlife
Eyebank) were dissected into corneoscleral wedges. A cannula was
inserted into Schlemm’s canal (SC) to allow SC luminal pressure to
be controlled by a variable height reservoir. Tissue displacements
were captured by OCT in a series of cross-sections through the TM/
SC at each reservoir pressure. Based on the observed anatomy in one
cross-section at low SC pressure, a quasi-3D specimen-specific finite
element model (FEM) was created (Fig. 1). Loading and boundary
conditions were applied to the model as delivered experimentally.
Tissues were treated as isotropic and hyperelastic. FEM simulations
were carried out using a range of stiffness values for TM while other
tissues were assigned a stiffness based on literature or best estimates.
TM stiffness was varied until the L2-norm difference between
OCT-observed and computed SC displacement was minimized.
A sensitivity analysis was performed to investigate the influence
of surrounding tissue stiffness and ciliary body (CB) boundary
delineation on TM stiffness estimation.
Results: Estimated hTM stiffnesses were 114 and 159 kPa, with an
unambiguous “best match” value for each of the 2 eyes examined to
date. Sensitivity analysis suggested that predicted TM stiffness was
insensitive to CB boundary delineation and stiffness of CB, scleral
and cornea.
Conclusions: Estimated hTM stiffness in normal eyes is c. 20x
greater than reported by Last et al. Combining FEM and OCT has the
potential to provide an alternative approach to assess hTM stiffness
in a physiologically relevant manner. Future work will improve FEM
geometry to better depict local structures, thus obtaining a more
accurate estimate of outflow tissue stiffnesses.
Figure 1: Representative finite element mesh of a cross-section from
one corneoscleral wedge.
Commercial Relationships: Ke Wang, None;
Murray A. Johnstone, None; Chen Xin, None; Steven Padilla,
None; Ruikang K. Wang, None; C R. Ethier, None
Program Number: 3544 Poster Board Number: A0242
Presentation Time: 11:00 AM–12:45 PM
Bidirectional interactions between trabecular meshwork cells and
their extracellular matrix in the presence of dexamethasone
Vijaykrishna Raghunathan2, Christopher J. Murphy1, 2, Paul Russell2.
1
Ophthalmology & Vision Science, University of California Davis,
Davis, CA; 2Surgical & Radiological Sciences, University of
California Davis, Davis, CA.
Purpose: Treatment with corticosteroids can result in ocular
hypertension, and steroid induced glaucoma. The extent to which
biomechanical changes in trabecular meshwork (TM) cells and
extracellular matrix (ECM) contribute towards this dysfunction is
poorly understood. Further, the impact that dysregulated ECM has
on cellular function in exacerbating disease is unknown. We wanted
to determine how (i) dexamethasone (dex) modulated cell & ECM
biomechanics, and ECM composition & morphology, and (ii) ECM
derived from cells treated with dex might further influence cell
biomechanics and cytoskeletal dynamics.
Methods: Primary and hTERT immortalized human trabecular
meshwork (HTM & htHTM respectively) cells (25000/cm2) were
each plated on aminated glass and were cultured in 10-7M dex
or vehicle control (ethanol) for 4 weeks. htHTM cells were then
dissociated and the mechanics of the ECM that was deposited by the
treated or the control cells was measured by AFM. Decellularization
was verified by immunostaining. ECM derived from cells was
solubilized for further analyses. To determine the impact that these
ECM had on cellular behavior, htHTM cells were plated on matrices
derived, after chronic control or dex treatment, for 3days. Cell
mechanics, immunocytochemistry, qPCR and Western blotting were
performed to ascertain cytoskeletal dynamics.
Results: Dex treatment resulted in 2 fold increases in HTM cell
stiffness. The matrix deposited after chronic dex treatment was 4 fold
stiffer than that deposited by vehicle control cells. Changes in ECM
mechanics were associated with significant differences in fibronectin
morphology with no changes in their expression level. htHTM cells
cultured on ECM derived after dex treatment were ~2 fold stiffer,
than those cultured on ECM derived from cells without steroid
treatment, accompanied by elevated mRNA expression of myocilin.
Conclusions: We have demonstrated in vitro ECM derived from dex
treated cells are stiffer than ECM deposited by control HTM cells
associated with compositional changes and fibronectin morphology
the most significantly altered. Further, ECM derived from cells with
steroid treatment significantly altered cytoskeletal dynamics and
gene expression. These results demonstrate glucocorticoid treatment
can initiate substantial changes in the biophysical and biochemical
environment of cells which can further alter cellular phenotype.
Commercial Relationships: Vijaykrishna Raghunathan, None;
Christopher J. Murphy, None; Paul Russell, None
Support: Funded by Bright Focus Foundation grant (G2015078),
NIH Grant P30EY12576 and an unrestricted grant from Research to
Prevent Blindness.
Program Number: 3545 Poster Board Number: A0243
Presentation Time: 11:00 AM–12:45 PM
Decreased Intraocular Pressure after Tarsal Strip for Ectropion
Repair
Chad Kaplan, Timothy Nees, Larissa Ghadiali, Dana Blumberg,
Lama Al-Aswad, George A. Cioffi, C Gustavo De Moraes,
Bryan Winn, Jeffrey M. Liebmann. Ophthalmology, Columbia
University, New York, NY.
Purpose:
Blinking and lid anatomy have been reported to affect intraocular
anatomy and intraocular pressure (IOP).(1,2,3) We hypothesized
that a lax lower lid decreases lid-related IOP fluctuation and aqueous
movement during blinking which may impact trabecular function.
This study seeks to determine the effect of ectropion repair by tarsal
strip on IOP.
Methods: In this retrospective study, we enrolled patients who
underwent a single tarsal strip procedure from 2010-2015 by a single
surgeon with at least 2 pre- and 2 post-operative IOP measurements.
Exclusion criteria included a history of glaucoma treatment, posterior
segment surgery, complex lid surgery, and repeat lid surgery
within 1 year. Pseudophakic patients were included, although IOP
measurements within three months of cataract surgery were excluded.
If the cataract surgery was after the tarsal strip procedure, all IOP
data after the date of the cataract surgery were excluded. Pre-and
postoperative IOP measurements were compared.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Results: 49 eyes from 28 patients were enrolled (12 men, 16 women;
mean age 78.7 ± 9.2 years). Mean IOP before and after tarsal strip
surgery was 15.1 ± 2.2 vs. 13.8 ± 2.1 mm Hg, respectively (P< 0.001,
95% CI: -1.89 to -0.81; Mixed-effects linear model). Average IOP
reduction was 8.6% (range -36.2% to +15.3%).
Conclusions: IOP was reduced following tarsal strip repair for lower
lid ectropion, suggesting that lid laxity may have an adverse effect
on aqueous outflow. Further investigation is warranted to assess the
relationships between lid laxity, aqueous movement, blinking, and
glaucoma risk. Maintenance of normal lid position and function may
be important for IOP regulation.
1. IOP Reduction in Tarsal Strip Procedure
Kaplan C, Grover S, Chalam KV
Investigative Ophthalmology & Visual Science April 2014, Vol.55,
151. ARVO Poster 2014
2. Direct-Recorded Intraocular Pressure Variations in a Human
Subject
Coleman DJ, Trokel S
Arch Ophthalmol. 1969;82(5):637-640.
3. Tight orbit syndrome: a previously unrecognized cause of openangle glaucoma.
Lee GA, Ritch R, Liang SY, Liebmann JM, Dubois P, Bastian-Jordan
M, et al.
Acta Ophthalmol. 2010;88:120–4
Commercial Relationships: Chad Kaplan, None; Timothy Nees;
Larissa Ghadiali, None; Dana Blumberg, None; Lama Al-Aswad,
None; George A. Cioffi, None; C Gustavo De Moraes, None;
Bryan Winn, None; Jeffrey M. Liebmann, None
Program Number: 3546 Poster Board Number: A0244
Presentation Time: 11:00 AM–12:45 PM
Change in biomechanical parameters analyzed with the Corvis
ST following trabeculectomy
Karin R. Pillunat, Eberhard Spoerl, Lutz E. Pillunat. Ophthalmology,
Univ Clinic Carl Gustav Carus Dresden, Dresden, Germany.
Purpose: To evaluate biomechanical changes after trabeculectomy
using the Corvis Scheimpflug technology.
Methods: 29 consecutive patients with open-angle glaucoma
scheduled for primary trabeculectomy (TE) with mitomycin C
were enrolled in this prospective case-control study. Biomechanical
parameters were assessed with the Corneal Visualization Scheimpflug
Technology (Corvis ST, Oculus, Wetzlar, Germany), immediately
prior to and at least 6 months after uncomplicated TE. Main outcome
measures were 10 parameters out of the 52 parameters of the new
research-software (software version 102R1126): changes in IOP,
pachymetry, maximal deformation amplitude, A2 time, HC time, A1
deflection amplitude, whole eye movement, deflection amplitude 2
ratio, CP-ratio and DA-ratio. SPSS and linear mixed models with IOP
and CCT as covariates were used to compare the parameters before
and after TE (means ± standard error).
Results: IOP and pachymetry changed statistically significantly from
20.00±1.55 to 10.47±1.52 mmHg (P=0.001) respectively 544.0±5.37
to 521.1±1.52 µm (P=0.001).
There was a strong correlation between IOP and pachymetry and
most of the measured parameters. The adjusted maximal deformation
amplitude (1.059±0.038 to 1.200±0.037 mm; P=0.014), the A1
deflection amplitude (0.102±00.3 to 0.089±0.003; P=0.018), the
deflection amplitude 2 ratio (5.357±0.086 to 5.612±0.084; P=0.046)
and the DA-ratio (1.592±0.008 to 1.617±0.008: P=0.034) changed
statistically significantly whereas the CP-ratio (4.611±0.061 to
4.756±0.060; P=0.072) did not change statistically significantly.
A2 time was only correlated with IOP but not with CCT and did not
change statistically significantly (21.098±0.243 to 21.588±0.238 s;
P=0.071).
HC-time and whole eye movement showed no correlation with IOP
and CCT and did not change statistically significantly after TE (HCtime: 16.202±0.189 to 16.234±0.189; P=0.908; whole eye movement:
0.309±0.016 to 0.309±0.016 mm; P=0.996).
Conclusions: Despite a marked IOP reduction and a weakening
of the globe after trabeculectomy there are some biomechanical
parameters as measured with the Corvis ST that change, whereas
some do not change postoperatively. Maybe parameters that change
do reflect ocular rather than corneal or orbital biomechanical
characteristics.
Commercial Relationships: Karin R. Pillunat, None;
Eberhard Spoerl, None; Lutz E. Pillunat, None
Clinical Trial: NCT02287545
Program Number: 3547 Poster Board Number: A0245
Presentation Time: 11:00 AM–12:45 PM
Assessment of residual effects due to topical medication on
corneal stiffness in primary open angle glaucoma
Sushma Tejwani1, Mathew Francis2, Shoruba Dinakaran1,
Rahul Mehta1, Rohit Shetty2, Abhijit Sinha Roy2. 1Glaucoma,
Narayana Nethralaya, Bangalore, India; 2Narayana Nethralaya,
Bangalore, India.
Purpose: We studied whether topical medication in primary open
angle glaucoma (POAG) eyes altered the corneal stiffness using airpuff applanation and a viscoelastic model.
Methods: A total of 48 POAG eyes (60 ± 10 years) on glaucoma
medication and 44 normal eyes (62 ± 10 years) were analyzed
using Corvis-ST (OCULUS Optikgerate Gmbh, Germany), an
air-puff applanation device using high speed scheimpflug imaging
to measure corneal deformation. Patients were grouped into those
using prostaglandin only, beta blocker only and a combination of
prostaglandin plus beta blocker. In the combination group only one
medication from each type was used. A viscoelastic model was
applied to corneal deformation (Figure 1), which performed explicit
quantification of corneal stiffness (kc), extra-ocular tissue stiffness
(kg) and viscosity (µg). These parameters were then analyzed using
multivariate analysis of covariance, using intraocular pressure (IOP)
and central corneal thickness (CCT) as covariates. A p<0.05 was
considered statistically significant. All measurements are reported as
mean ± standard error of the mean.
Results: Mean kc was 108.92 ± 2.90 N/m, 120.87 ± 6.28 N/m,
124.24 ± 5.51 N/m and 105.45 ± 4.94 N/m in the normal,
prostaglandin group, beta blocker group and combination group,
respectively. Multivariate analyses showed that both IOP (p=0.001)
and type of medication (p=0.02) significantly correlated with kc
indicating higher stiffness in the prostaglandin group and beta blocker
group. Eyes in the combination group had kc similar to normal eyes
(p>0.05). CCT (p=0.25), kg (p = 0.81) and µg (p = 0.89) were similar
between the groups.
Conclusions: The study provided clinical evidence of modulated
corneal stiffness in treated POAG eyes. However, the reasons behind
differential modulation of corneal stiffness only in eyes using single
medication are not fully understood. Further investigation with larger
sample size is required.
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to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Holter were comparable. The highest frequency associated with the
heart rate was confirmed in both CSLA and ECG spectra. However,
such a close correspondence was difficult to identify in active time
of a subject where the TF-CLS signal becomes noisier than that
encountered in the resting time or sleep.
Conclusions: Our study revealed that continuous 24-hour CSLA
signals follow a sine-wave pattern typical to other diurnal changes in
physiological measurements. TF-CLS can also be used as a potential
clinical tool to monitor heart rate variability, particularly in a sleep
period.
Commercial Relationships: Monika E. Danielewska,
None; Marek Rekas, None; Anna Byszewska, None;
Joanna Wierzbowska, None; Katarzyna Petz, None; D
Robert Iskander, None
Figure 1
Commercial Relationships: Sushma Tejwani, None;
Mathew Francis, None; Shoruba Dinakaran, None; Rahul Mehta,
None; rohit shetty, Carl zeiss (F), Narayana Nethralaya (P),
Allergan (F); Abhijit Sinha Roy, Carl zeiss (F), Bioptigen (F),
Avedro (F), Topcon (F), Cleveland Clinic Innovations (P), Narayana
Nethralaya (P)
Program Number: 3548 Poster Board Number: A0246
Presentation Time: 11:00 AM–12:45 PM
Estimating diurnal changes in ocular dimension fluctuations
using the Triggerfish Contact Lens Sensor and assessing its
potential in 24-hour monitoring of heart rate variability in
glaucoma patients
Monika E. Danielewska1, Marek Rekas2, Anna Byszewska2,
Joanna Wierzbowska2, Katarzyna Petz2, D Robert Iskander1.
1
Department of Biomedical Engineering, Wroclaw University of
Technology, Wroclaw, Poland; 2Department of Ophthalmology,
Military Institute of Medicine, Warsaw, Poland.
Purpose: To develop an independent algorithm for evaluating
diurnal dimensional changes at the corneoscleral limbus area (CSLA)
measured with the Triggerfish Contact Lens Sensor (TF-CLS) and to
ascertain whether continuous 24-hour acquisition with this device can
be clinically utilized for monitoring heart rate variability (HRV).
Methods: The study involved 10 patients with primary open-angle
glaucoma. 24-hour continuous monitoring of CSLA and ECG signals
was performed with TF-CLS and Holter ECG, respectively. Raw TFCLS and ECG data were numerically analysed in Matlab. Up to 288
CSLA signal packets sampled at 10 Hz for a period of 30 s at 5 min
intervals were extracted, concatenating the median signal levels into
a time series. Detrending of the median signal and best-sine wave
fitting were performed. A corresponding time interval of the ECG
recording was assigned. Using a time-domain-peak-detector a series
of RR intervals and corresponding changes in time intervals between
any two successive maxima of the CSLA packet were determined
and used as the two estimators of the HRV within the packet.
Further, spectral estimation of each pair of CSLA and ECG signals
was performed to assess the correspondence between dimensional
changes at CSLA and heart rate activity.
Results: Estimation of the median signal level in each TF-CLS
packet has been performed in a way to exclude sudden signal changes
corresponding to blinks and other artefacts. The new methodology
showed evident sine-wave diurnal dimensional changes at CSLA
for all the subjects. Diurnal HRVs obtained from TF-CLS and ECG
Program Number: 3549 Poster Board Number: A0247
Presentation Time: 11:00 AM–12:45 PM
Corneal Peak Distance (PD) is Higher in Primary Angle Closure
Glaucoma (PACG) Subjects than in Normal and Primary Open
Angle Glaucoma (POAG) Subjects
Owen Png1, Mani Baskaran2, 3, Tin Tun2, Sourabh Sharma2, Yih
Chung Tham2, Ching-Yu Cheng2, 3, Tin Aung2, 3, Michael J. Girard4.
1
Duke-NUS Graduate Medical School, Singapore, Singapore;
2
Singapore Eye Research Institute, Singapore, Singapore; 3Singapore
National Eye Centre, Singapore, Singapore; 4National University of
Singapore, Singapore, Singapore.
Purpose: To investigate if corneal biomechanical parameters,
measured by the Corvis (CST, Oculus Optikgeräte GmbH, Wetzlar,
Germany), including the widely reported deformation amplitude, are
significantly different amongst normal, PACG, and POAG subjects.
Methods: 194 Chinese subjects, comprising of 80 normal, 63 POAG
and 51 PACG subjects, were recruited in this cross-sectional study.
Patients with previous ocular surgeries or trauma were excluded.
All recruited participants underwent a comprehensive ophthalmic
examination, including visual acuity assessment, IOP measurement
with Goldmann applanation tonometry, and automated refraction
and keratometry. Corneal biomechanical parameters, including
applanation length, time and velocity, deformation amplitude, peak
distance and radius, were analyzed using the Oculus Corvis ST
air-puff tonometer. Mean differences of the parameters amongst the
subgroups were evaluated by statistical analysis of variance and
multiple regression analyses. Sensitivity analysis was performed for
regression after matching for age.
Results: The mean age and proportion of males were higher in
the glaucoma subgroups. Corneal Peak distance (PD) deformation
was lower in normal compared to glaucoma subjects [Normal:3.44
± SD1.24mm vs POAG:4.30±1.22mm vs PACG:4.13 ±1.16mm,
p<0.0001]. PD remained higher in only PACG subjects compared to
normals after correcting for age, gender, spherical equivalent (SE),
intraocular pressure (IOP) and central corneal thickness (OR 1.7,
95% CI 1.2,2.6, p=0.007). Deformation amplitude was not influenced
by the type of glaucoma, but varied with IOP, age, gender and SE.
Conclusions: Corneal peak distance, but not deformation amplitude,
was higher in PACG. Our results may reflect altered corneal
biomechanical properties due to collagen remodeling in PACG eyes.
Further investigations are required to assess the clinical relevance of
biomechanical parameters as a predictor of glaucoma progression.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: Owen Png, None; Mani Baskaran;
Tin Tun, None; Sourabh Sharma, None; Yih Chung Tham, None;
Ching-Yu Cheng, None; Tin Aung, None; Michael J. Girard, None
Support: NUSYIA_FY13_P03/R-397-000-174-133, NMRC/
CSA/033/2012
Program Number: 3550 Poster Board Number: A0248
Presentation Time: 11:00 AM–12:45 PM
Correlating corneal biomechanical properties with lamina
cribosa in healthy subjects
Francisco Pérez Bartolomé, Jose María Martínez de la Casa,
Irene Camacho Bosca, Federico Sáenz Francés, Julián García
Feijoó. Ophthalmology, Hospital Clínico San Carlos, Madrid, Spain.
Purpose: To examine interrelations between corneal biomechanics,
ocular biometric variables and optic disc size (ODS), lamina cribosa
depth (LCD) or thickness (LCT) in a healthy population.
Methods: In a cross sectional case-control study, the following
measurements were made in 56 eyes of 56 participants: axial length,
anterior chamber depth, lens thickness and central corneal thickness
using the optical biometer Lenstar LS900; and corneal hysteresis,
corneal resistance factor (CRF), Goldman-correlated intraocular
pressure (IOPg) and corneal-compensated IOP (IOPcc) using the
ocular response analyzer. Serial horizontal enhanced depth imaging
optical coherence tomography (EDI OCT) B- scans of the optic nerve
head were obtained in each participant. Mean ODS, mean LCD and
mean LCT were measured in 11 equally-spaced horizontal B- scans,
excluding the LC insertion area under Bruch’s membrane and scleral
rim.
Results: Data for 40 eyes were available for statistical analysis. LCD
was greater in men than women by a mean of 45.34 ± 19.88 µm
(95% CI 5.092 - 85.58 µm; P= 0.028; Student’s t). LCT was directly
correlated with ODS (r= 0.331; P= 0.042). Corneal biomechanical
properties and ocular biometrics variables were poorly (nonsignificantly) correlated with LCD, LCT and ODS.
Conclusions: Insufficient evidence was detected to indicate
significant correlation between corneal biomechanical properties or
ocular biometric variables and ODS, LCD or LCT.
Commercial Relationships: Francisco Pérez Bartolomé, None;
Jose María Martínez de la Casa, None; Irene Camacho Bosca,
None; Federico Sáenz Francés, None; Julián García Feijoó, None
Program Number: 3551 Poster Board Number: A0249
Presentation Time: 11:00 AM–12:45 PM
Ocular rigidity: a novel non-invasive clinical method
Diane N. Sayah2, 1, Javier Mazzaferri2, Luke Beaton2,
Félix Lalonde2, 3, Maribel Hidalgo2, 4, Santiago Costantino2, 5,
Mark R. Lesk2, 5. 1School of Optometry, University of Montreal,
Roxboro, QC, Canada; 2Ophthalmology, Maisonneuve-Rosemont
Hospital Research Center, Montreal, QC, Canada; 3Biomedical
Sciences, University of Montreal, Montreal, QC, Canada; 4Institut
National de la Recherche Scientifique, Varennes, QC, Canada;
5
Ophthalmology, University of Montreal, Montreal, QC, Canada.
Purpose: Considerable evidence suggests that ocular rigidity (OR)
plays a key role in the pathogenesis of open angle glaucoma (OAG)
and other ocular diseases. While there is currently no reliable clinical
method to assess OR, our group has recently developed a technique
permitting a direct and non-invasive measurement in humans
(Beaton et al., 2015). The purpose of this clinical study is to compare
correlations between OR and other factors, such as age, axial length
(AL) and ocular pulse amplitude (OPA) to previous studies using
invasive techniques or post-mortem eyes.
Methods: Sixty-two subjects (27 normal, 27 with OAG and 8 with
functional trabeculectomy blebs) were enrolled and measurement
of OR was done using our novel technique. This method is based
on Friedenwald’s equation, involving OPA as measured by DCT
(Dynamic Contour Tonometry, Ziemer group) and pulsatile ocular
volume change (ΔV) as measured with our automated segmentation
method. Video-rate Spectral Domain Optical Coherence Tomography
(SD OCT, Spectralis OCT Plus, Heidelberg Engineering) time series
with Enhanced Depth Imaging was carried out at the macula. Each
video was analyzed using our segmentation algorithm to find the
mean magnitude of choroidal thickness change (ΔCT) and thus derive
ΔV adjusting for AL (IOL Master, Zeiss Meditec). Heart rate was also
measured during the video imaging and served as a validation for the
pulsatile changes measured by OCT.
Results: The mean OR coefficient calculated with our technique was
0.037 ± 0.022 uL-1, a similar value to previous invasive methods. As
anticipated, a negative correlation was found between OR and AL
(r= -0.31, p= 0.01) for all groups combined. A positive correlation
between OR and OPA was found in all groups (r= 0.46, p= 0.0002).
Although the relationship between OR and age (r= 0.16, p= 0.2)
was not significant for all groups, it was positively correlated in the
Normal group (r= 0.41, p=0.04), where the age range was much
wider. A negative correlation was found between ΔCT and OR (r=
-0.58, p= 0.00) and also between ΔV and OR in all groups (r= -0.64,
p=0.000).
Conclusions: We found a negative correlation between OR and AL
and a positive correlation between OPA and OR, as well as between
age and OR, which is the same as expected from older invasive
techniques. This confirms the validity of our novel non-invasive
technique and will permit us to lead further investigation of OR in
ocular disease.
Commercial Relationships: Diane N. Sayah, None;
Javier Mazzaferri; Luke Beaton, None; Félix Lalonde, None;
Maribel Hidalgo, None; Santiago Costantino, Univalor (P);
Mark R. Lesk, Heidelberg Engineering (F), Univalor (P)
Support: Canadian Institutes of Health Research, Natural Sciences
and Engineering Research Council of Canada and Fonds de
Recherche en Ophtalmologie de l’Université de Montréal (S.C. and
M.R.L.), D.N.S. is supported by a Vision Health Research Network
Graduate Student Performance Bursary
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to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 3552 Poster Board Number: A0250
Presentation Time: 11:00 AM–12:45 PM
The Relationships between Corneal Elasticity Measured by
Surface Wave Elastography and other Ocular Variables
Arash Kazemi1, Jay W. McLaren1, Christopher M. Pruet1, ShuaiChun Lin2, 1, Xiaoming Zhang1, Arthur J. Sit1. 1Ophthalmology,
Mayo Clinic Rochester, Rochester, MN; 2Ophthalmology, Wills Eye
Hospital, Philadelphia, PA.
Purpose: Biomechanical properties of the eye are important for
understanding the risk of glaucoma. The relationships between
corneal elasticity and other ocular variables are unknown. In this
study we determined Young’s modulus of elasticity in corneas of
normal subjects by using a novel non-invasive technique based on
surface wave elastography and determined relationships between
corneal elasticity and corneal hysteresis (CH), corneal resistance
factor (CRF), the ocular rigidity coefficient, and other ocular
parameters.
Methods: In 28 eyes of 14 healthy participants (ages 25-63; mean
34.3 years) intraocular pressure (IOP) was measured by using
Goldmann applanation tonometry (GAT) and pneumatonometry.
The ocular rigidity coefficient was calculated from IOP measured
in the supine position with and without a 10-gm weight added to
the tonometer. Central corneal thickness (CCT) was measured from
Scheimpflug images (Pentacam HR, Oculus). CH and CRF were
measured by using the Ocular Response Analyzer (ORA, Reichert).
The elasticity of the cornea was then determined by surface wave
elastography. A spherical-tipped probe (4 mm diameter) was placed
on the closed eyelid and vibrated at 100 Hz for 0.1 second. Surface
waves were recorded by ultrasonography as they propagated around
the eye and Young’s modulus was calculated from the measured
speed of wave propagation. Associations between variables were
explored by Pearson correlation and significances were determined
by using generalized estimating equation models to account for
possible correlation between fellow eyes of the same subject.
Results: Young’s modulus of elasticity was strongly correlated
with IOP (r=0.65, p<0.001 from GAT, and r=0.58, p<0.001 from
pneumatonometry) but not with CRF (r=0.44, p=0.18), CH (r=0.25,
p=0.57), or the ocular rigidity coefficient (r=-0.05, p=0.69).
Conclusions: Young’s modulus of elasticity in normal eyes increases
as IOP increases, consistent with published in-vitro studies. The
lack of correlations of CRF and the ocular rigidity coefficient with
Young’s modulus suggest that these variables may not be surrogate
measures of corneal elasticity. Further work is required to determine
if elasticity is altered in glaucoma patients.
Commercial Relationships: Arash Kazemi, None;
Jay W. McLaren, None; Christopher M. Pruet, None; ShuaiChun Lin, None; Xiaoming Zhang, None; Arthur J. Sit
Support: 1- Research to Prevent Blindness 2- Mayo Foundation for
Medical Education and Research
Program Number: 3553 Poster Board Number: A0251
Presentation Time: 11:00 AM–12:45 PM
Relationships between Axial Length and Ocular Biomechanical
Properties
Christopher M. Pruet, Arash Kazemi, Xiaoming Zhang,
Jay W. McLaren, Arthur J. Sit. Ophthalmology, Mayo Clinic,
Rochester, MN.
Purpose: Glaucoma is associated with increased axial length and
myopia, but it is not known if this is because of unique biomechanical
properties of longer eyes. In this study, we investigated the
relationships between axial length and ocular rigidity, Young’s
modulus of the cornea, intraocular pressure (IOP), and other
biomechanical variables.
Methods: Axial length was measured by ultrasonography in 28 eyes
from 14 healthy participants (ages 34.3±10.7 years, mean±SD), and
IOP was measured by Goldmann applanation tonometry (GAT) in the
sitting position. Ocular rigidity was estimated from the Friedenwald
equation and IOP measured by pneumatometry in the sitting position,
and in the supine position with and without a 10-gram weight added
to the tonometer probe. Corneal hysteresis, the corneal resistance
factor, and cornea-corrected IOP were measured by using the Ocular
Response Analyzer (ORA, Reichert). Central corneal thickness was
measured from Scheimpflug images (Pentacam HR, Oculus). Young’s
modulus was determined by measuring the wave propagation in the
cornea using the surface wave elastography. The wave propagation
in the eye was generated noninvasively for 0.1 seconds at 100 Hz
by using a handled device on the closed eyelids. The relationships
between axial length and other variables were explored by Pearson
correlation for right eyes.
Results: Axial length was 24.2±1.2 and 24.1±1.2 mm for right
and left eyes respectively, ocular rigidity was 0.024±0.005 and
0.025±0.004 mmHg/µL, IOP by GAT was 15.3±2.4 and 15.0±2.4
mmHg, and the corneal Young’s modulus was 752±115 and 713±141
kPa. Axial length was correlated with ocular rigidity (r=-0.70,
p=0.006 right and r=-0.68, p=0.007 left). Axial length was not
correlated with measurements from the ORA, Young’s modulus of
the cornea, IOP, or central corneal thickness (p>0.08).
Conclusions: The strong negative correlation between ocular rigidity
with axial length is consistent with published invasive measurements
of ocular rigidity and estimates of rigidity based on Schiotz
tonometry. In healthy eyes, corneal elasticity is not related to axial
length, suggesting that the increased glaucoma risk associated with
myopia is not related to unique corneal biomechanical properties in
long eyes. Further research is needed to determine if scleral elasticity
is altered with glaucoma.
Table. Results
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ARVO 2016 Annual Meeting Abstracts
changes from baseline occurred in 51.8% and 33.9% of examinations,
respectively. In the subgroup with intraocuar pressure less than 20
mmHg, 16.7% and 5.6% demonstrated progression by axial length
and disc changes, respectively.
Conclusions: In infantile glaucoma patients who have failed angle
surgery and medical therapy, progressive pathologic axial length
changes occur more frequently than optic disc changes. These
changes can occur even when the intraocular pressure is under 20
mmHg.
Commercial Relationships: Kara M. Cavuoto, None;
Korey Jaben, None; Ta C. Chang, None
Ocular rigidity vs. Axial length. p<0.008
Corneal Young’s modulus vs. Axial Length. p>0.08.
Commercial Relationships: Christopher M. Pruet, None;
Arash Kazemi, None; Xiaoming Zhang, None; Jay W. McLaren;
Arthur J. Sit, Sensimed AG (C), Allergan Inc. (C), AcuMEMS, Inc.
(C), Aerie Pharmaceuticals Inc. (C)
Support: Research to Prevent Blindness, Mayo Foundation
Program Number: 3554 Poster Board Number: A0252
Presentation Time: 11:00 AM–12:45 PM
Progression in infantile glaucoma: comparing structural and
biometric changes
Kara M. Cavuoto, Korey Jaben, Ta C. Chang. Univ of Miami Sch of
Medicine, Miami, FL.
Purpose: The detection of progression in infantile glaucoma is based
on clinical impressions of optic nerve and axial length changes.
However, the sensitivity, specificity and relation to intraocular
pressures of these changes are unknown. The purpose of our study
was to evaluate the frequency of optic nerve and axial length changes
in uncontrolled infantile-onset glaucoma.
Methods: Retrospective masked grading of disc photos, review of
axial lengths and review of medical records of children with infantile
glaucoma who failed angle surgery and medical management prior to
age of three years.
Results: 56 examinations of 16 eyes (14 patients) were included.
Axial length changes (greater than 30% of expected value) and disc
Program Number: 3555 Poster Board Number: A0253
Presentation Time: 11:00 AM–12:45 PM
Racioethnic Differences in the Biomechanical Environment of the
Lamina Cribrosa
Jonathan P. Vande Geest1, 5, Sundaresh Ram3, Stephen J. Howerton1,
Forest Danford1, Urs Utzinger4, 5, Jeffrey J. Rodriguez3, 2. 1Aerospace
and Mechanical Engineering, The University of Arizona, Tucson,
AZ; 2Graduate Interdisciplinary Program of Biomedical Engineering,
The University of Arizona, Tucson, AZ; 3Electrical and Computer
Engineering, The University of Arizona, Tucson, AZ; 4BIO5 Institute,
The University of Arizona, Tucson, AZ; 5Biomedical Engineering,
The University of Arizona, Tucson, AZ.
Purpose: It is well established that those of African descent (AD)
and Hispanic ethnicity (HE) are disproportionately affected by
primary open-angle glaucoma compared to those of European descent
(ED). Studies involving the biomechanical environment of the optic
nerve head point to the lamina cribrosa (LC) as the primary region
of mechanical insult. Investigating the biomechanical factors in the
LC among different groups may help elucidate the mechanism for
unbalanced outcomes of glaucoma. We investigated quantitative
differences in 3D micro-strains within the LC among the three
populations.
Methods: We acquired nine pairs of normal human donor poles,
three each from AD, HE, and ED (self-identified racioethnicities).
The LC was imaged using multiphoton microscopy at four pressure
levels: 5, 15, 30, and 45 mmHg. Image stacks of second harmonic
generation were collected to a depth of 350 μm. 3D displacement
fields were obtained between adjacent pressure pairs for each
population, using an automated digital volume correlation technique,
which aims to minimize the difference between two 3D datasets
by applying a displacement field to one dataset and measuring how
closely it matches the other. Three strain invariants were calculated
from Green’s strain tensor obtained from the displacement field.
Results: Fig. 1 shows the mean strain invariant values for each
population for each adjacent pressure pair. A Student’s t-test between
each individual population shows a statistically significant difference
for all three LC strain invariants (Table 1).
Conclusions: Experimental results show that the pressure-dependent
biomechanical response of the LC is different among the three
populations (AD, HE, ED). These quantitative differences should be
further verified with larger sample sizes.
Fig. 1: Mean and standard deviation of the pressure-dependent strain
invariant values for each population.
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ARVO 2016 Annual Meeting Abstracts
eyes was more posteriorly curved following acute elevations in IOP
when compared to that of normal eyes.
Commercial Relationships: Jonathan P. Vande Geest, None;
Sundaresh Ram; Stephen J. Howerton, None; Forest Danford,
None; Urs Utzinger, None; Jeffrey J. Rodriguez, None
Support: NIH Grant 1R01EY020890
Program Number: 3556 Poster Board Number: A0254
Presentation Time: 11:00 AM–12:45 PM
Shape Changes of the Anterior Lamina Cribrosa in Healthy and
Glaucoma Eyes following Acute Intraocular Pressure Elevations
Tin A. Tun1, Owen Png2, Baskaran Mani1, 2, Sourabh Sharma1,
Hla M. Htoon1, 2, Sri Gowtham Thakku1, Ching-Yu Cheng1, 2,
Nicholas G. Strouthidis3, 1, Tin Aung1, 2, Michael J. Girard1.
1
Singapore Eye Research Institute, Singapore, Singapore; 2Duke-NUS
Graduate Medical School, Singapore, Singapore; 3NIHR Biomedical
Research Centre, Moorfields Eye Hospital NHS Foundation Trust and
UCL Institute of Ophthalmology, London, United Kingdom.
Purpose: To estimate and compare changes in anterior lamina
cribrosa (LC) morphology in normal and glaucoma eyes following
acute elevations in intraocular pressure (IOP)
Methods: One optic nerve head (ONH) of 18 ocular hypertension
(OHT), 21 primary open angle glaucoma (POAG), 33 chronic
primary angle closure glaucoma (PACG) and 31 normal subjects
was imaged using spectral-domain optical coherence tomography
(OCT; Spectralis, Heidelberg Engineering, Germany). IOP was raised
twice by gently applying a force (0.64 N then 0.9 N) to the anterior
sclera (through the lower eye lid) using an ophthalmo-dynamometer
(spring-loaded indenter). After each IOP increment, IOP was held
constant, measured with a TonoPen, and each ONH was rescanned
with OCT. In each OCT volume, the anterior LC was enhanced with
adaptive compensation, manually delineated, and its global shape
index (GSI) calculated (Thakku et al., IOVS. 2015 Jun;56(6):360414). GSI is a single index that provides a global measure of LC shape
and that varies from -1 (posteriorly curved LC) to +1 (anteriorly
curved LC). Wilcoxon signed-Rank tests and Linear Mixed Models
were used to assess the differences in GSI across groups.
Results: Of the 103 subjects, 51.5% were males with a mean age
of 64.5±7.1 years. At baseline, IOP was 17.5±3.4 mmHg and was
increased to 37.6±5.9, then 46.2±6.1 mmHg. When considering
the first IOP increment, the GSI median was significantly smaller
than that at baseline in normal (Z=2.73, P=0.006) and in glaucoma
subjects (Z=2.45, P=0.014), but not in OHT subjects (Z=77,
P=0.734). For the second IOP increment, the GSI median was
significantly smaller than that at baseline in normal (Z=3.2, P=0.001)
and in OHT (Z=32, P=0.018). (Figure1) After adjusting for age,
gender and baseline IOP, mixed model analysis showed that the LC
of PACG eyes was significantly more posteriorly curved than that of
normal subjects (Estimated Mean difference -0.15, 95% confidence
interval = -0.299, -0.001, P=0.047), but this was not found for other
diagnoses.
Conclusions: We demonstrated that acute IOP elevations altered
anterior LC shape in living human eyes. The connective tissues of
OHT eyes might be stiffer than those from all other groups because
the LC of OHT eyes only deformed at high IOP. The LC of PACG
Commercial Relationships: Tin A. Tun, None; Owen Png,
None; Baskaran Mani, None; Sourabh Sharma, None;
Hla M. Htoon, None; Sri Gowtham Thakku, None; ChingYu Cheng; Nicholas G. Strouthidis, None; Tin Aung, None;
Michael J. Girard, None
Program Number: 3557 Poster Board Number: A0255
Presentation Time: 11:00 AM–12:45 PM
A Novel Virtual Fields Method to Measure the Biomechanical
Properties of Human Optic Nerve Head Tissues
Liang Zhang1, 2, Mani Baskaran3, 4, Tin Aung3, 5,
Nicholas Strouthidis3, 6, Michael J. Girard2, 3. 1NUS Graduate School
for Integrative Sciences and Engineering, National University of
Singapore, Singapore, Singapore; 2Ophthalmic Engineering &
Innovation Laboratory, Department of Biomedical Engineering,
National University of Singapore, Singapore, Singapore; 3Singapore
Eye Research Institute, Singapore National Eye Centre, Singapore,
Singapore; 4Eye-ACP, Office of Clinical, Academic and Faculty
Affairs, Duke-NUS Medical School, Singapore, Singapore; 5Yong
Loo Lin School of Medicine, National University of Singapore,
Singapore, Singapore; 6NIHR Biomedical Research Centre,
Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of
Ophthalmology, London, United Kingdom.
Purpose: To develop a novel and fast virtual fields method (VFM) to
measure the patient-specific biomechanical properties of optic nerve
head (ONH) tissues.
Methods: Our method was designed to extract the biomechanical
properties of human ONH tissues (choroid, peripapillary sclera,
prelamina, and lamina cribrosa), given their full-field IOP-induced
deformations. The latter can be derived through 3D tracking of in
vivo optical coherence tomography (OCT) images (Girard et al., J
R Soc Interface. 2013; 10:20130459). To verify our technique, we
first generated ‘artificial’ ONH deformation data from predetermined
(known) ONH tissue biomechanical properties using finite element
analysis (Figure 1a-b). Using such deformations, if we are able to
match back the known biomechanical properties, it would indicate
that our VFM (Pierron, F., & Grédiac, M. 2012. Springer Science
& Business Media) technique is accurate. We assumed that the
prelamina, the choroid and the lamina cribrosa can be described
with a single stiffness parameter (elastic moduli: Ep, Ec and El);
the peripapillary sclera can be described with 2 stiffness parameters
representing the stretch-induced stiffening of the collagen fibers (c3
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ARVO 2016 Annual Meeting Abstracts
and c4), and 2 microstructural parameters representing the main
orientation (θp) and the degree of alignment (k) of the collagen
fibers. The ‘artificial’ IOP-induced ONH deformations were fed
into the VFM algorithm (custom written in C++) to extract back the
biomechanical properties. The computational speed of VFM was then
compared to that of a gold standard stiffness measurement method
(inverse finite element).
Results: From the given ONH deformations, VFM successfully
matched back the biomechanical properties of ONH tissues with high
accuracy and efficiency. For all parameters, the percent errors were
less than 1% (Figure 1c). Our method was also able to recover the
highly-aligned circumferential organization of the collagen fibers in
the peripapillary sclera. Finally, VFM was found 60 times faster than
gold-standard stiffness measurement methods.
Conclusions: VFM may be capable of measuring the biomechanical
properties of human ONH tissues with high speed and accuracy.
It has potential in identifying patient-specific ONH biomechanical
properties in the clinic if combined with OCT.
Commercial Relationships: Liang Zhang, None; Mani Baskaran,
None; Tin Aung, None; Nicholas Strouthidis; Michael J. Girard,
None
Support: NUS Young Investigator Award (NUSYIA_FY13_P03,
R-397-000-174-133); Ministry of Education, Academic Research
Funds, Tier 1 (R-397-000-140-133; R-397-000-181-112).
Program Number: 3558 Poster Board Number: A0256
Presentation Time: 11:00 AM–12:45 PM
Influence of tissue viscoelasticity on the optic nerve head
perfusion: a mathematical model
Daniele Prada1, Riccardo Sacco3, Bernardo Cockburn4,
Lorena Bociu5, Justin Webster6, Brent A. Siesky2, Alon Harris2,
Giovanna Guidoboni1, 2. 1Mathematical Sciences, Indiana UniversityPurdue University Indianapolis, Indianapolis, IN; 2Ophthalmology,
Indiana University, Indianapolis, IN; 3Mathematics, Politecnico
di Milano, Milano, Italy; 4Mathematics, University of Minnesota,
Minneapolis, MN; 5Mathematics, North Carolina State University,
Raleigh, NC; 6Mathematics, College of Charleston, Charleston, SC.
Purpose: Proper perfusion of the optic nerve head (ONH) tissue is
vital to visual function. Alterations in material properties of ONH
structures, such as the lamina cribrosa (LC), have been associated
with many pathologies, including glaucoma. We hypothesize that
changes in LC viscoelasticity may compromise LC perfusion in
response to sudden variations of intraocular pressure (IOP), possibly
leading to disc hemorrhages. Due to the difficulty of isolating these
factors experimentally, we utilized a mathematical model as virtual
lab to investigate IOP’s influence on LC perfusion.
Methods: The LC is modeled as a poro-visco-elastic material, where
blood vessels are viewed as pores in a solid matrix. LC vascular
porosity (ratio between blood volume and LC total volume) and LC
permeability (ability of the porous material to allow fluid passing
through it) depend on the local state of stress and strain. Blood flow
is driven by the pressure difference between the short posterior ciliary
arteries and the central retinal vein. The LC is subject to scleral
tension, retrolaminar tissue pressure and IOP. A finite element method
is used to simulate and compare the behavior of the integrated time
rate of change of the blood kinetic energy (W) in response to sudden
temporal IOP variations (Fig1) with and without LC viscoelasticity
(d=1 and d=0, respectively) (Fig2).
Results: If d=0, W exhibits two sharp peaks at the IOP switch-on
and switch off times (Fig2). These peaks represent sharp and acute
local changes in blood flow and pressure, with consequent perfusion
instability and irregularity, in response to sharp changes in IOP. If
d=1, these peaks are not present and W remains bounded at lower
values (Fig2). These findings suggest that the lack of viscoelasticity
may increase the LC susceptibility to localized damage due to peaks
in the integrated time rate of change of the blood kinetic energy as
IOP experiences sudden changes in time, as they normally occur
during the day.
Conclusions: Our theoretical results suggest that even physiological
changes in IOP may induce pathological changes in LC perfusion if
the LC viscoelasticity provided by the collagen fibers is not intact
(due, e.g., to ageing or disease). The clinical implications of LC
perfusion deficits during IOP variation should be further investigated
to elucidate their impact on glaucoma pathophysiology.
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ARVO 2016 Annual Meeting Abstracts
Methods: A finite element model of optic nerve head tissues
(pre- and postlaminar neural tissue, lamina cribrosa, central retinal
vessel, sclera, and pia meter) was constructed. Stresses, strains and
deformations were computered using finite elemnt modeling for a
range of elevated IOP and reduced CSFP. Cup-disc ratio (C/D) were
measured basic on the finite element model.
Results: At an IOP of 20mmHg, C/D averaged from 0.38 to 0.48
with reduced CSFP from 10 mmHg to 2 mmHg. At an CSFP of 10
mmHg, C/D averaged from 0.38 to 0.43 with elevated IOP from 20
mmHg to 28 mmHg. By comparison, at a same trans-lamina cribrosa
pressure of 16 mmHg, C/D is 0.45 caused by elevated IOP while C/D
is 0.42 caused by reduced CSFP.
Conclusions: <!--StartFragment --><!--StartFragment -->Finite
element analysis of strain in the lamina cribrosa caused by acute
elevated IOP are biologically significant and capable of contributing
to the development of glaucomatous optic neuropathy. With mild
elevated IOP and reduced CSFP, it is less biologically significant and
may not result to direct mechanical damage. Moreover, according
to C/D ratio, IOP- and CSFP-induced deformation of the lamina
cribrosa at the same lamina cribrosa pressure is different. This finding
implies that, there may be exists another mechanism of glaucomatous
optic neuropathy with mild elevated IOP and reduced CSFP.
Commercial Relationships: YINGYAN MAO
Commercial Relationships: Daniele Prada, None; Riccardo Sacco;
Bernardo Cockburn, None; Lorena Bociu, None; Justin Webster,
None; Brent A. Siesky, None; Alon Harris, Isama therapeutics
(C), Stemnion Inc. (C), Nano Retina (C), Oxymap (I), Biolight
(C), AdOM (C), Ono (C), Science Based Health (C), AdOM (I);
Giovanna Guidoboni, None
Support: This work has been partially supported by the NSF
DMS-1224195, NIH 1R21EY022101- 01A1, a grant from Research
to Prevent Blindness (RPB, NY, USA), an Indiana University
Collaborative Research Grant of the Office of the Vice President
for Research, the Chair Gutenberg funds of the Cercle Gutenberg
(France) and the Labex IRMIA (University of Strasbourg, France).
Program Number: 3559 Poster Board Number: A0257
Presentation Time: 11:00 AM–12:45 PM
Finite element analysis of pressure effect on biomechanical
response of the optic nerve head in glaucoma
YINGYAN MAO. Beijing Tongren Eye Center, Beijing Institute of
Ophthalmology, Beijing, China.
Purpose: Decreased cerebrospinal fluid pressure (CSFP), parallel
to an elevated intraocular pressure (IOP), is associated with the
pathogenesis of glaucomatous optic neuropathy. An improved
understanding of how pressure effect optic nerve head (ONH)
biomechanical environment, is necessary to understand how pressure
play a role in glaucomatous optic neuropathy.
Program Number: 3560 Poster Board Number: A0258
Presentation Time: 11:00 AM–12:45 PM
Probabilistic Modeling of Intracranial Pressure Effects on Optic
Nerve Biomechanics
C R. Ethier1, Andrew J. Feola1, Julia Raykin1, Jerry G. Myers2,
Emily S. Nelson2, Brian C. Samuels3. 1Biomedical Engineering,
Georgia Institute of Technology, Atlanta, GA; 2NASA Glenn
Research Center, Cleveland, OH; 3Ophthalmology, UAB,
Birmingham, AL.
Purpose: Altered intracranial pressure (ICP) is involved/implicated
in several ocular conditions: papilledema, glaucoma and Visual
Impairment and Intracranial Pressure (VIIP) syndrome. The
biomechanical effects of altered ICP on optic nerve head (ONH)
tissues in these conditions are uncertain but likely important. We
here quantified ICP-induced deformations of ONH tissues, using
finite element (FE) and probabilistic modeling (Latin Hypercube
Simulations (LHS)) to consider a range of tissue properties and
relevant pressures.
Methods: We extended the Sigal et al. (IOVS, 2005) model of
the posterior eye/ONH to include pia mater, dura mater and optic
nerve (ON) extending 10 mm posterior to the globe. We considered
three ICP conditions: (i) elevated (chronic microgravity [VIIP] or
intracranial hypertension); (ii) supine on earth; and (iii) upright on
earth. For each condition, we used LHS to generate a range of ICPs,
tissue mechanical properties, intraocular pressures and mean arterial
pressures as FE model inputs. Sclera, pia and dura were modeled as
a neo-Hookean matrix with embedded collagen fibers; neural tissues
were modeled as isotropic/linear. Outcome measures were peak
principal strains in the lamina cribrosa (LC), ON and retina, since
cells are sensitive to mechanical strain.
Results: Strains in the LC and retina generally decreased as ICP
increased; however, the opposite was true in the retro-laminar ON.
Notably, c. 47% of simulations in the elevated ICP condition showed
peak ON strains exceeding the most extreme strains expected under
terrestrial conditions (Fig). Individual characteristics predisposing for
such “extreme strains” included a soft ON or pia.
Conclusions: Elevated ICP acting directly on the optic nerve
results in extreme strains which may trigger a mechanobiologic
response. Surprisingly, elevated ICP had only a modest effect on
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ARVO 2016 Annual Meeting Abstracts
LC biomechanics. These results are relevant to understanding ON
axoplasmic stasis in intracranial hypertension/papilledema and VIIP.
Cumulative probability distributions for predicted peak tensile and
compressive strains in the optic nerve across a simulated population
of individuals, generated by LHS. Each curve corresponds to one
ICP condition, as indicated. The shaded regions show strain ranges
expected under terrestrial conditions. The red lines identify the
percentage of simulations experiencing “extreme strains” when
exposed to elevated ICP.
Commercial Relationships: C R. Ethier, None; Andrew J. Feola,
None; Julia Raykin, None; Jerry G. Myers, None;
Emily S. Nelson, None; Brian C. Samuels, None
Support: NASA grant NNX13AP91G, Georgia Research Alliance
Program Number: 3561 Poster Board Number: A0259
Presentation Time: 11:00 AM–12:45 PM
In vivo evaluation of optic disc changes and anterior lamina
cribrosa displacement after acute cerebrospinal fluid pressure
reduction in healthy humans
Muriel Poli1, 3, Alain M. Bron4, Eric Sellem2, Philippe Denis3,
Carole Burillon3. 1Centre Ophtalmologique Pole Vision Val d’Ouest,
Lyon, Dominica; 2Centre Ophtalmologique Kleber, Lyon, France;
3
Centre Hospitalier Universitaire, Lyon, France; 4Centre Hospitalier
Universitaire, Dijon, France.
Purpose: Low cerebrospinal fluid pressure (CSF-P) has been
associated with the pathogenesis of glaucomatous optic neuropathy,
in particular in patients with low-tension glaucoma, and glaucoma
has been associated with optic nerve head (ONH) and lamina cribrosa
(LC) positional changes. The aim of this study was to determine
the effect of acute CSF-P reduction on ONH and anterior LC
displacement using swept-source optical coherence tomography (SSOCT) in healthy subjects.
Methods: In this interventional study, 16 eyes of 8 in-patient adults
benefiting from diagnostic lumbar puncture (LP) for non-ocular
diseases were recruited prospectively. All ONH and LC imaging
were performed using a commercially available SS-OCT device
(6mm, 164 overlap) before, 5, 60 and 360 minutes after LP. The
internal limiting membrane (ILM), posterior surface of the Bruch’s
membrane/retinal pigment epithelium complex (BM/RPE), neural
canal opening (NCO), retinal vessels (RV), outer limit of the choroid
(ELC) and anterior LC surface (ALCS) were manually delineated by
3 separate observers and compared.
Results: Four males and four females were recruited with a mean
age, 50.4 ± 11.5 years and a mean body mass index, 22.7 ± 4.6 kg/
m2). Mean volume of CSF collected was 1,63 ± 0,53 mL. At all times
after LP, we did not observe any displacement of ILM, BM/RPE,
NCO, RV, ELC or ALCS, even for two patients that experienced post
LP headache.
Conclusions: While acute and transient elevation of intra-ocular
pressure has been associated with a condensation of neuroretinal
rim, prelaminar tissue, and LC, without major changes in the optic
disc size and position of the anterior LC surface, acute significant
reduction of CSF-P was not associated with any morphological
changing in neither ONH nor LC position.
Commercial Relationships: Muriel Poli; Alain M. Bron, None;
Eric Sellem, None; Philippe Denis, None; Carole Burillon, None
Program Number: 3562 Poster Board Number: A0260
Presentation Time: 11:00 AM–12:45 PM
Asymmetry in anterior lamina cribrosa surface tilting may
contribute to retinal nerve fiber layer defect in myopia
Eun Jung Lee, Jong Chul Han, Changwon Kee. Ophthalmology,
Samsung Medical Center, Sungkyunkwan university, Seoul, Korea
(the Republic of).
Purpose: Factors associated with retinal nerve fiber layer (RNFL)
defect development in myopic eyes are not clearly validated. We
performed a retrospective study to investigate the factors associated
with development of RNFL defect in myopic eyes, including optic
nerve head morphology and anterior lamina cribrosa surface (ALCS)
tilt.
Methods: We retrospectively reviewed medical records of myopic
patients who visited department of Ophthalmology at Samsung
Medical Center for examination of glaucoma. All participants
underwent comprehensive ophthalmologic examinations including
optic disc measurement with Cirrus HD spectral-domain optical
coherence tomography (OCT). Eyes were divided into two groups
of comparable degree of myopia, according to the presence of RNFL
defect. Cup-to-disc ratio, ovality index, and torsion were measured
manually using fundus photography. ALCS tilt angle was calculated
by measuring the acute angle between ALCS and Bruch’s membrane
opening plane in horizontal and vertical B-scans. Angle α was
defined as ALCS tilt angle in horizontal B-scan, and angle β as in
vertical B-scan. Angle δ was the difference between α and β, and we
used absolute value of δ for analysis. Factors including optic nerve
head parameters and tilt angles were compared between two groups.
Results: In total, 34 eyes in group with RNFL defect, and 22 eyes
without RNFL defect were analyzed. Spherical equivalent in two
groups were -5.25 D and -5.07 D, respectively, and axial length
was 26.3 and 25.6 mm, respectively. Age, intraocular pressure,
ovality index and torsion were not significantly different between
two groups, but ratio of peripapillary atrophy to disc area was
significantly larger in group with RNFL defect (p=0.011) than in
group without RNFL defect. Among tilt angles, α, β, and δ did not
show significant difference, but |δ| was significantly larger in group
with RNFL defect than in group without RNFL defect (11.62° and
6.29°, respectively, p=0.007, Table 1, Figure 1). In multivariate
analysis, |δ| was related to the presence of RNFL defect (p=0.046).
Conclusions: Tilted disc morphology on funduscopic examination
does not fully explain the development of RNFL defect. As observed
in this study, deeper structural features as asymmetric ALCS tilt
might have a role in RNFL damage, possibly through its twisted or
distorted shape to strain the nerve axons.
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ARVO 2016 Annual Meeting Abstracts
constant rate to failure during tension recording in a load cell under
physiological temperature and humidity. Finite element analysis
(FEA) using SolidWorks software was used to predict resulting
stress/strain during adduction 0.1 radians beyond ON straightening.
Physical dimensions of the FEA mesh were scaled to human
histological and MRI data.
Results: Young’s modulus, a measure of stiffness of an elastic
isotropic material, was averaged (±SD) for 5 specimens of each
tissue: lowest for ON at 5.2±0.8 MPa and peripapillary sclera at
5.5±1.7 MPa, intermediate for peripheral sclera at 14±5 MPa,
and highest for ON sheath at 45±13 MP. FEA indicated 405 kPa
maximum stress and 17 % maximal strain induced in temporal side
of the ON scleral canal. In the LC, maximum predicted stress was 80
kPa, greatest temporally. Predicted mechanical changes were ~30fold larger than produced by simulated IOP increase to 50 mmHg.
Conclusions: ON sheath traction in adduction concentrates severe
stress in the temporal peripapillary sclera in a distribution identical
both to patterns of peripapillary atrophy and temporal ON head
tilting typical of NTG, and ON deformations in adduction recently
confirmed by OCT to occur in normal subjects and patients with
NTG (Chang et al, ARVO'16). This IOP-independent stress could
plausibly cause ON damage when accumulated over time, and thus
cause NTG. Tractional ON damage may be particularly prevalent in
patients with shorter ONs as in axial myopia. This study supports a
new biomechanical concept of glaucoma as resulting at least in part
from external traction on the ON sheath, rather than exclusively on
pressure exerted within the eye.
Commercial Relationships: Andrew Shin, None; Joseph Park,
None; Joseph L. Demer, None
Support: NIH EY08313
Commercial Relationships: Eun Jung Lee, None; Jong Chul Han,
None; Changwon Kee, None
Program Number: 3563 Poster Board Number: A0261
Presentation Time: 11:00 AM–12:45 PM
Optic Nerve (ON) Sheath Traction on the Temporal Peripapillary
Sclera in Adduction: Biomechanical Evidence for a Role in
Normal Tension Glaucoma (NTG)
Andrew Shin, Joseph Park, Joseph L. Demer. Ophthalmology, Stein
Eye Institute, UCLA, Los Angeles, CA.
Purpose: The historically central role of high intraocular pressure
(IOP) in glaucomatous ON damage has been challenged by the
observation that IOP is not elevated in many patients considered
to have NTG. We propose that NTG may be caused by tractional
deformation of the optic canal and lamina cribrosa (LC) by tautness
of ON sheath in adduction recently recognized to occur in adduction
(Demer, ARVO 2015).
Methods: Uniaxial tensile loading was used to characterize Young’s
modulus in 4 fresh adult bovine tissues: ON, ON sheath, and
peripapillary and peripheral sclera. Specimens were elongated at
Program Number: 3564 Poster Board Number: A0262
Presentation Time: 11:00 AM–12:45 PM
Regional Variations in the Strains of the Human Optic Nerve
Head
Dan Midgett2, Thao D. Nguyen2, Harry A. Quigley1, Mary
Ellen Pease1, Christian Franck3, Mohak Patel3. 1Ophthalmology,
Johns Hopkins, Baltimore, MD; 2Mechanical Engineering, Johns
Hopkins University, Baltimore, MD; 3Engineering, Brown University,
Providence, RI.
Purpose: The mechanical behavior of the lamina cribrosa (LC) plays
an important role in the development glaucomatous optic neuropathy.
We developed an in vitro inflation method to measure global and
regional LC strains caused by controlled pressurization.
Methods: Six human eyes from 5 donors (ages 26-71) obtained
within 48 hours post-mortem had the choroid and retina removed,
the optic nerve removed at the myelin line, and the posterior sclera
mounted on a custom inflation chamber. The pressure was raised
to 5, 10, and 45 mmHg. A Zeiss LSM 710 NLO microscope, tuned
to 790nm with a band pass 390-410nm filter, was used to acquire
two duplicate stacks of the LC structure using second harmonic
generation (SHG) imaging at each pressure. The SHG stacks were
treated with Huygens Essential deconvolution algorithm using a
theoretical point spread function (SVI) and local adaptive histogram
equalization (FIJI) to enhance contrast. The Fast Iterative Digital
Volume Correlation (DVC) algorithm (Bar-Kochba et al. 2014) was
used to post-process the SHG stacks to calculate the 3D displacement
fields and correlation error. The correlation coefficient was used to
remove poorly correlating subsets and Gaussian filtering was used
to remove local error spots. Strains were calculated by fitting a high
order polynomial function to the displacement field and taking the
gradient. The LC was divided into 4 quadrants surrounding the
central retinal artery and vein (inferior-nasal (IS), inferior-temporal
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ARVO 2016 Annual Meeting Abstracts
(IT), superior-nasal (SN), superior-temporal (ST). The principal
strains were averaged and analyzed for regional differences.
Results: Displacements and strains in the LC exhibited substantial
variation in the plane but not through the thickness (Figure 1).
The average shear strains (in plane: -0.15±0.14%, out-of-plane:
-0.22±0.23%, 0.19±0.34%) were significantly smaller than the normal
strains. For nearly all eyes, the strain at 45mmHg along the inferiorsuperior axis (3.02±1.09%) was larger than along the nasal-temporal
axis(2.03±0.75%). The maximum principal strain was largest in the
IT quadrant and smallest in the IN quadrant. In paired t-tests, the
maximum principal strain in IT was significantly larger than in IN
(p=0.026) and nearly significantly larger than in SN (p=0.067) and IT
(p=0.076).
Conclusions: Regional variations in pressure-derived strains in
the LC may help to explain the progression of axonal damage in
glaucoma.
Commercial Relationships: Dan Midgett, None; Thao D. Nguyen,
None; Harry A. Quigley, None; Mary Ellen Pease, None;
Christian Franck, None; Mohak Patel, None
Support: NSF CAREER Award #1253453, PHSR Grant EY021500,
EY02120, EY01765
Program Number: 3565 Poster Board Number: A0263
Presentation Time: 11:00 AM–12:45 PM
In-vivo Modulation of Intraocular and Intracranial Pressures
Causes Nonlinear and Non-monotonic Deformations of The
Lamina Cribrosa
Huong Tran1, 2, Andrew P. Voorhees1, Bo Wang1, 2, Ning-Jiun Jan1, 2,
Elizabeth Tyler-Kabara3, Larry Kagemann1, 2, Hiroshi Ishikawa1, 2,
Joel S. Schuman1, 2, Matthew A. Smith1, 2, Gadi Wollstein1,
Ian A. Sigal1, 2. 1UPMC Eye Center, Eye and Ear Institute,
Ophthalmology and Visual Science Research Center, Department
of Ophthalmology, University of Pittsburgh School of Medicine,
Pittsburgh, PA; 2Department of Bioengineering, University of
Pittsburgh, Pittsburgh, PA; 3Department of Neurosurgery, University
of Pittsburgh, Pittsburgh, PA.
Purpose: Our goal was to measure the in-vivo effects on the optic
nerve head (ONH) of acute modulation of intraocular and intracranial
pressures (IOP and ICP).
Methods: In 4 eyes of 3 monkeys, IOP and ICP were modulated
through cannulas inserted into the eye and ventricle. Each pressure
was set at 4 levels (low, baseline, high, very high), and the ONHs
imaged with a SD-OCT. The anterior lamina cribrosa (ALC) and
scleral canal opening at Bruch membrane (BMO) were manually
marked in 18 radial sections per scan (Fig 1A). Custom code was
used to reconstruct 3D ALC surfaces and register BMOs. ALC depths
were computed relative to the BMO best-fit plane within regions
visible in all scans of an eye (Fig 1B) and normalized to baseline in
each monkey. Hence, ALC depth less than 100% meant more anterior
ALC and vice versa.
Results: IOP and ICP had nonlinear and effects on the median ALC
depth (Fig 2). The ranges for normalized median ALC depth were
78-116%, 36-122%, 65-105%, and 66-104% in eyes 1R, 2R, 3R
and 3L. There were strong interaction between the pressures effects,
meaning that effects of one pressure depended strongly on the other
pressure. Pressure effects were non-monotonic. For example, in eye
3R at 20 mmHg ICP, the LC displaced anteriorly when IOP increased
from 8 to 15mmHg, and posteriorly for IOP from 15 to 30mmHg (Fig
2C). In all 4 eyes, the most anterior LCs occurred with IOPs below
baseline (15mmHg) and very high ICP (20-45mmHg).
Conclusions: Acute modulation of either IOP or ICP above or below
baseline can cause substantial deformations of the ALC, sometimes
anteriorly and other times posteriorly. These deformations were nonlinear and non-monotonic, with strong interactions between IOP and
ICP.
Figure 1. Manual delineation and reconstruction of ONH structures.
(A) Example markings on a radial slice (B) Example heat map of
ALC depth, calculated as distance from ALC surface (reconstructed
red radial markings) to best-fit BMO plane (outline shown in blue).
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ARVO 2016 Annual Meeting Abstracts
Conclusions: Our results indicate that the sclera response to macroscale stretch is highly inhomogeneous at the micro-scale, in both the
stretch level at which fiber bundles uncrimp and their uncrimping
rate. Understanding the micro-scale response to mechanical loading
is important for determining the role of collagen architecture on
the macro-scale tissue biomechanics, and for understanding the
biomechanical environment of scleral cells and their contributions to
tissue growth and remodeling.
Figure 2: Nonlinear and non-monotonic effects of IOP and ICP
on in-vivo LC deformations. All ALC depths were normalized to
baseline values (IOP=15 mmHg, ICP=8-10 mmHg) and median
values (%) were plotted (blue=more anterior, red=more posterior).
Green asterisks (*) corresponded to pressure conditions. Dotted green
line showed an example of non-monotonic effect of increasing IOP at
ICP=20 mmHg in monkey 3-right.
Commercial Relationships: Huong Tran, None;
Andrew P. Voorhees, None; Bo Wang, None; Ning-Jiun Jan,
None; Elizabeth Tyler-Kabara, None; Larry Kagemann, None;
Hiroshi Ishikawa, None; Joel S. Schuman, Carl Zeiss Meditec, Inc.
(P); Matthew A. Smith, None; Gadi Wollstein, None; Ian A. Sigal
Support: Eye and Ear Foundation of Pittsburgh, PA; National
Institutes of Health grants (P30-EY00809, R01EY025011,
R01EY023966, T32-EY017271); Glaucoma Research Foundation
Shaffer Grant
Program Number: 3566 Poster Board Number: A0264
Presentation Time: 11:00 AM–12:45 PM
Novel method reveals heterogeneous micro-scale response of
sclera collagen bundles to homogeneous macro-scale stretch
Ning-Jiun Jan1, 2, Michael Iasella2, Mason Lester2, Danielle Hu1,
Kira L. Lathrop1, Andrew P. Voorhees1, Huong Tran1, 2,
Gadi Wollstein1, Joel S. Schuman1, 2, Ian A. Sigal1, 2. 1Ophthalmology,
University of Pittsburgh School of Medicine, UPMC Eye Center,
Eye and Ear Institute, Ophthalmology and Visual Science Research
Center, Pittsburgh, PA; 2Bioengineering, Swanson School of
Engineering, University of Pittsburgh, Pittsburgh, PA.
Purpose: Collagen has a natural waviness called crimp that largely
determines the nonlinear mechanical behavior of many ocular tissues.
However, almost nothing is known about how this waviness changes
with stretch. We have developed a novel method for observing and
measuring collagen crimp changes during stretch. Our goal was to
quantify the effects of macro-scale stretch on the micro-scale stretch
and waviness of collagen fiber bundles of sclera.
Methods: 3 sheep eyes were acquired from a local abattoir and
processed within 12 hours. The eyes were cryosectioned axially
(30µm). The equatorial sclera was mounted to a uniaxial stretcher
(Fig1A). Six samples were imaged with polarized light microscopy at
various levels of macro-scale stretch and analyzed for collagen fiber
orientation (Fig1B-D). Using manual markings, the local microscale stretch and waviness were tracked through different levels of
stretch for several bundles of each section. Waviness was defined as
the normalized SD of the fiber orientations along the bundle. Linear
mixed effect models were used to test the association between bundle
stretch and loss of waviness (uncrimping).
Results: We tracked the stretch and waviness of 21 bundles over
an average of 9 stretch levels. Waviness decreased significantly
with increasing micro-scale stretch (p<0.0001, Fig2A). Bundles
had variable initial waviness, though all bundles uncrimped with
sufficient stretch. Even within the same sample under homogeneous
macro-scale stretch, bundles varied in responses, uncrimping at
different levels of stretch and at different rates (Fig2B).
Fig 1. Tissue was held by clamps (A) and imaged at different levels
of stretch. Panels B-D shows the micro-scale fiber orientation of a
region (arrow in A) at increasing levels of stretch.
Fig 2. Stretch vs. waviness for all samples pooled (A) and for 3
example collagen bundles from 1 sample of eye 1 (B).
Commercial Relationships: Ning-Jiun Jan, None; Michael Iasella;
Mason Lester, None; Danielle Hu, None; Kira L. Lathrop, None;
Andrew P. Voorhees, None; Huong Tran, None; Gadi Wollstein,
None; Joel S. Schuman, Zeiss (P); Ian A. Sigal, None
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to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Support: NIH: R01 EY023966 (IAS), T32-EY017271, R01
EY013178 (JSS), R01 EY025011 (IAS/JSS), P30 EY008098; Eye
and Ear Foundation (Pittsburgh, PA).
Program Number: 3567 Poster Board Number: A0265
Presentation Time: 11:00 AM–12:45 PM
Collagen crimp waviness, tortuosity and period have different
patterns around the eye
Danielle Hu1, Ning-Jiun Jan1, 2, Michael Iasella1, 2, Huong Tran1, 2,
Yun Ling1, Joel S. Schuman1, 2, Alexandra Judisch1,
Andrew P. Voorhees1, Gadi Wollstein1, 2, Ian A. Sigal1, 2.
1
Opthalmology, University of Pittsburgh, Pittsburgh, PA;
2
Bioengineering, University of Pittsburgh, Pittsburgh, PA.
Purpose: Collagen undulations, or crimp, play a key role in
determining the biomechanical properties of the eye. However, very
little is known about the characteristics of crimp and how they vary
throughout the globe. We measured three characteristics of collagen
fiber crimp in different locations of the globe: period, waviness, and
tortuosity.
Methods: Two lamb eyes and one sheep eye from a local abattoir
were fixed at 0 mmHg, and cryosectioned axially at 30 μm. Sections
were imaged with polarized microscopy and local fiber orientation
determined using a recently described method. Period was measured
manually by marking fiber bundles. Waviness and tortuosity were
quantified along the markings using custom code. Waviness was
defined as the normalized SD of fiber orientation, and tortuosity as
the ratio between path length and end-to-end length of a fiber. Linear
mixed effect models were used to determine regional differences (Fig
1), and a linear model to determine if high parameter values were
associated with higher variability.
Results: More than 2,000 measurements were made. Period,
waviness, and tortuosity varied substantially across the eye
globe (Fig 2). Crimp period (mean±SD) was lowest in the cornea
(14.2±3.4μm) and superior/inferior peripapillary sclera (PPS,
12.8±2.8μm) and highest at the equators (29.1±9.5μm), whereas
waviness and tortuosity were lowest at equators (0.06±0.04 and
1.012±0.002, respectively) and highest in the cornea (0.17±0.13
and 1.018±0.011, respectively). Equatorial sclera and the rest of the
sclera were not significantly different in period (P>0.05), but were
highly significantly different in waviness and tortuosity (P<0.01).
For all parameters, high values were associated with high variability
(P<0.01).
Conclusions: Period, waviness, and tortuosity of the collagen varied
across the globe. Spatial trends of waviness and tortuosity were
similar, whereas period was significantly different, particularly when
comparing equators to other parts of the sclera. Our results evidence
specific spatial patterns in collagen crimp parameters, which likely
reflect the mechanisms that determine local biomechanical properties.
Waviness and tortuosity may relate to tissue stiffness while period
may relate to the level of organization.
Axial sections of sheep eyes were divided into 17 regions for
analysis.
Mean (line) and SE (shading) of collagen parameters across the globe
pooled over all eyes.
Commercial Relationships: Danielle Hu, None; Ning-Jiun Jan,
None; Michael Iasella, None; Huong Tran, None; Yun Ling, None;
Joel S. Schuman; Alexandra Judisch, None; Andrew P. Voorhees,
None; Gadi Wollstein, None; Ian A. Sigal, None
Support: NIH: R01 EY023966 (IAS), T32-EY017271, R01
EY013178 (JSS), P30 EY008098; Eye and Ear Foundation
(Pittsburgh, PA).
Program Number: 3568 Poster Board Number: A0266
Presentation Time: 11:00 AM–12:45 PM
ONH Deformation in Porcine Eyes Using Ultrasound Speckle
Tracking
Elias Pavlatos, Xueliang Pan, Richard T. Hart, Paul A. Weber,
Jun Liu. The Ohio State University, Columbus, OH.
Purpose: The mechanical environment of the optic nerve head
(ONH) is thought to play an important role in the onset of glaucoma.
This study aims to map and quantify the strains within the ONH in
response to intraocular pressure (IOP) elevation.
Methods: Ten porcine globes were tested within 72 hours
postmortem. The optic nerve was trimmed to the outer surface of
the peripapillary sclera. A portion of the cornea was removed using
a 7.5mm trephine along with the intraocular contents, and the ocular
shell was mounted using a custom-built pressurization chamber.
The chamber was connected to a programmable syringe pump
(PHD Ultra, Harvard Apparatus) and pressure sensor (P75, Harvard
Apparatus) to control and monitor IOP. Preconditioning with 20
pressure cycles from 5 to 30 mmHg was followed by equilibration
at 5 mmHg for 30 minutes. The globes were inflated by increasing
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ARVO 2016 Annual Meeting Abstracts
IOP from 5 to 30 mmHg with 0.5 mmHg steps every 15 seconds. 2D
cross-sections of radiofrequency data were obtained along the nasaltemporal meridian of the ONH at each pressure step. An ultrasound
speckle tracking algorithm was used to calculate the strains within the
scanned cross-section (Tang & Liu, J Biomech Eng 2012, 134(9)).
The ONH was divided into two equal thickness layers to compare the
anterior and posterior response (Fig 1a). Histology was obtained for
one eye using periodic acid-Schiff staining (Fig 1b).
Results: The average through-thickness and in-plane strains for
all ten eyes at 30 mmHg were -0.052 ± 0.014 and 0.018 ± 0.008
respectively. The through-thickness compressive strains in the
anterior ONH were 4.6 times higher than in the posterior ONH at 30
mmHg (0.087 vs. 0.019, p<0.001) while the in-plane tensile strains
were slightly but significantly lower in the anterior ONH (0.015 vs.
0.022; p=0.006). The same trend was observed at 15 mmHg (Fig 2).
Conclusions: The anterior ONH (largely the prelaminar neural
tissue) appeared to experience large compression during IOP
elevation. The more posterior region had significantly reduced
compression, likely due to the structural support of the lamina
cribrosa. Experimental characterization of ONH deformation will
help to better understand the biomechanical etiologies of glaucoma.
Fig 1. B-mode ultrasound image (a) and histological section of
porcine ONH (b).
Fig 2. Through-thickness (TT) compressive strains and in-plane (IP)
tensile strains in the ONH of porcine eyes (n=10) and strain maps
from one eye at 30 mmHg.
Commercial Relationships: Elias Pavlatos, None; Xueliang Pan,
None; Richard T. Hart; Paul A. Weber, None; Jun Liu, None
Support: NEI Grant RO1EY020929
Program Number: 3569 Poster Board Number: A0267
Presentation Time: 11:00 AM–12:45 PM
Polarization Sensitive Optical Coherence Tomography (PSOCT)
Demonstrates Strain Dependent Birefringence in Ocular Tissues
Joseph Park, Andrew Shin, Joseph L. Demer. Jules Stein Eye
Institute, University of California - Los Angeles, Los Angeles, CA.
Purpose: Ex vivo, destructive testing has demonstrated that
mechanical properties of the sclera of glaucomatous and myopic
eyes are abnormal. A technique for in vivo measurement of these
properties would be valuable. Many biological tissues containing
regular arrays of collagen exhibit birefringence, an optical property
in which refractive index depends on the light’s polarization and
propagation direction. PSOCT is an interferometric imaging
technique employing orthogonal polarization paths that can image
local birefringence. Since mechanical loading alters the orientation
and geometry of fibrils in collagen, we sought to determine if
birefringence changes can be used as a non-invasive optical method
to infer local mechanical properties of ocular connective tissues.
Methods: An infrared (1300 nm) PSOCT scanner (Thorlabs
PSOCT-1300) was mounted over a uniaxial tensile load cell
consisting of a linear motor and strain gauge. Ten specimens each of
fresh bovine equatorial sclera, extraocular tendon (EOT), and optic
nerve sheath (ONS) were elongated to failure at constant rate of 0.1
mm/s while birefringence images were captured every 117 ms. In
each frame, average birefringence phase retardation was calculated
using MATLAB (Mathworks, Natick, MA), and birefringence values
were synchronized with instantaneous strain and tissue tension. We
then computed the birefringence modulus, defined as the change of
birefringence as a function of strain.
Results: In each tissue, infrared phase retardation was a monotonic
function of both strain and stress up to the point of specimen
rupture. Mean (±SD) birefringence modulus was highest for sclera at
11.3±4.4×10-4, lower for EOT at 5.5±2.0×10-4, and least for ONS at
1.1±0.9×10-4 (P<0.012).
Conclusions: Sclera, EOT, and ONS have distinct but widely
differing birefringence moduli that quantify how birefringence
changes with mechanical strain. Measurement of birefringence
moduli by PSOCT may enable non-invasive optical monitoring
of mechanical strain in these tissues, particularly in sclera where
birefringence is most sensitive to strain. In vivo optical strain
measurement may be valuable for study of eye diseases such as
glaucoma and strabismus.
Commercial Relationships: Joseph Park; Andrew Shin, None;
Joseph L. Demer, None
Support: National Eye Institute EY08313 and Research to Prevent
Blindness
Program Number: 3570 Poster Board Number: A0268
Presentation Time: 11:00 AM–12:45 PM
Finite Element (FE) Modeling of Optic Nerve Head (ONH)
Biomechanics in a Rat Model of Glaucoma
Stephen A. Schwaner1, Marta Pazos4, Hongli Yang2,
Claude F. Burgoyne2, C R. Ethier3, 1. 1Mechanical Engineering,
Georgia Institute of Technology, Atlanta, GA; 2Ophthalmology,
Devers Eye Institute, Portland, OR; 3Biomedical Engineering,
Georgia Institute of Technology, Atlanta, GA; 4Departamento del
Luminotecnia, Hospital Esperanza. Parc de Salut Mar., Barcelona,
Spain.
Purpose: The rat is widely used to study glaucoma. However,
rat ONH anatomy differs from human, likely producing different
biomechanics. Rat ONH biomechanics have not been characterized
but are important to e.g. understand ONH astrocyte mechanobiology
in glaucoma. Digital 3D histomorphometric reconstructions (Pazos+,
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ARVO 2016 Annual Meeting Abstracts
EER, 2015 & in press) are now available to drive FE modeling.
Here we describe a pipeline for creating the first FE models allowing
determination of hard/impossible-to-measure rat ONH biomechanics
under glaucomatous loading conditions.
Methods: Following perfusion fixation, the following landmarks
were manually delineated within 40 digital radial sections through a
normotensive rat ONH reconstruction: neurovascular scleral canal,
nerve, central retinal artery (CRA), central retinal vein (CRV),
perineural vascular plexus (PNVP), and inferior arterial canal
(IAC). From point clouds, we created solid tissue geometries for FE
modeling in ABAQUS. Tissues were modeled as isotropic neoHookean materials with elastic moduli based on values from past
human modeling studies (Sigal+, IOVS, 2004) and Poisson’s ratio
= 0.49 to enforce near-incompressibility. IOP was applied to the
anterior surface of the model, and venous and arterial blood pressures
(BP) were applied to CRA and CRV lumens. Edge boundary
conditions were applied via submodeling: a simplified posterior
eye model was created/solved, and displacements from matching
positions were applied to the scleral edges of a local model (Fig 1).
Results: To establish proof-of-principle, we explored a range of IOP
and BPs in a single ONH. Figure 2 shows exemplar results at IOP
= 20 mmHg and arterial/venous BPs = 61/41 mmHg. ONH stresses
and strains ranged widely, with 95th percentile values of 14.6 kPa and
23.3% (first principal stress and strain), exceeding those expected in
human ONH at equivalent IOPs.
Conclusions: This method for FE model construction of the rat
ONH is a first step in understanding rat ONH biomechanics. Model
enhancements, incorporating rat-specific tissue properties and
modeling additional normal and hypertensive ONHs will allow us to
study biomechanical effects of tissue architecture and map regional
strain variations to biological outcomes. This will improve our
understanding of axonal injury pathogenesis in glaucoma.
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ARVO 2016 Annual Meeting Abstracts
Purpose: To consider if the optic nerve in older rat eyes shows an
age related change in response to acute simultaneous intracranial
pressure (ICP) and intraocular pressure (IOP) modification.
Methods: Young (8 m.o., n=5/group) and old (19 m.o., n=6-8/
group) anesthetised (60:5mg/kg ketamine:xylazine) Long-Evans rats
underwent acute pressure modification. ICP was manometrically set
to low (-5mmHg), normal (5mmHg) or high (25mmHg) levels via a
double-lumen cannula placed into the lateral ventricle (-1.5mm from
bregma, ±2mm from midline). At each ICP level, IOP was elevated
from 10 to 90 mmHg in 10mmHg steps (3 minutes each) via an intravitreal cannula in the ipsilateral eye. The posterior pole was imaged
using spectral domain-optical coherence tomography (Bioptigen)
and analysed for peripapillary retinal surface deformation (100um
from the optic nerve centre), retinal nerve fiber layer (RNFL) and
total retina thickness, the width of Bruch’s membrane opening (BMO
width) and minimum rim thickness (MRT). Thickness is expressed
as a percentage of baseline (IOP 10mmHg), and deformation is
expressed as a difference from baseline (mean±SEM). Data were
compared at each ICP level using a two-way ANOVA (IOP effect
between ages).
Results: There was significantly more surface deformation in young
compared with old rats, particularly at ICP 5 mmHg (p<0.01). The
RNFL was more compressed by IOP in older eyes with low ICP
compared with younger eyes (old: 80%±5% vs. young: 113%±5%,
p<0.01), but not at normal (p=0.99) and high ICP (p=0.18). Total
retinal thickness and BMO width showed no age-related effect at any
ICP level (all p > 0.05). MRT at BMO was compressed significantly
less in older eyes when ICP was -5 (old: 84%±2% vs. young:
77%±5%, p=0.04) and 5 mmHg (old: 86%±3% vs. young: 76±5%,
p=0.01), however no difference was observed at 25 mmHg (p=0.08).
Conclusions: Compared with young rats, the peripapillary retina in
aged rats shows less surface deformation and rim compression with
IOP elevation at all ICP levels. However, with low ICP, IOP-induced
RNFL compression was exacerbated by older age. This data suggests
the optic nerve in old rats is less compliant to pressure changes,
which may produce greater peripapillary RNFL compression.
Commercial Relationships: Da Zhao, None; Christine T. Nguyen,
None; Zheng He, None; Algis J. Vingrys, None; Bang V. Bui, None
Commercial Relationships: Stephen A. Schwaner, None;
Marta Pazos; Hongli Yang, None; Claude F. Burgoyne, None;
C R. Ethier, None
Support: Georgia Research Alliance (CRE). NIH RO1 EY010145
(JCM) and P30 EY010572 (JCM), Research to Prevent Blindness
(JCM), NIH T32 EY007092 (SAS), NIH R01 EY011610 (CFB),
Legacy Good Samaritan Foundation (CFB), the Sears Medical Trust
(CFB), and the Alcon Research Institute (CFB).
Program Number: 3571 Poster Board Number: A0269
Presentation Time: 11:00 AM–12:45 PM
Effects of intracranial and intraocular pressure modification on
the optic nerve of young and old rats
Da Zhao, Christine T. Nguyen, Zheng He, Algis J. Vingrys,
Bang V. Bui. Optometry and Vision Sciences, University of
Melbourne, Melbourne, VIC, Australia.
Program Number: 3572 Poster Board Number: A0270
Presentation Time: 11:00 AM–12:45 PM
A Pilot Study for the Initial Validation of a Tree Shrew Model of
Glaucoma
Brian C. Samuels, Christopher A. Girkin, Lisa Hethcox, J
Crawford C. Downs, Wenjie Zhan. Ophthalmology, University of
Alabama at Birmingham, Birmingham, AL.
Purpose: Retinal ganglion cell injury in glaucoma occurs at the
level of the lamina cribrosa. Thus, there is increasing interest in
understanding the mechanobiology of the optic nerve head. To
study this, researchers need animal models that closely resemble the
relevant human anatomy and develop key pathologic features of the
human disease. While primates and rodents are the most widely used
animal models of glaucoma, each has significant limitations. Tree
shrews (Tupaia belangeri) are a mammalian species closely related to
primates and offer the benefit of being accessible to most researchers
and they have a load-bearing connective tissue lamina resembling
primates. The purpose of this pilot study was to begin validating the
magnetic bead occlusion model of experimental glaucoma in tree
shrews.
Methods: Experimental glaucoma was induced in adult tree shrews
(n=9) using a modified version of the magnetic bead occlusion
method (Samsel et al; 2011). Briefly, magnetic beads were thoroughly
washed and 0.1ml injected into the anterior chamber. Beads were
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ARVO 2016 Annual Meeting Abstracts
directed to the angle using a magnet. All animals were followed for
3 months. Weekly IOP measurements were obtained using the iCare
TonoLab tonometer and biweekly spectral domain optical coherence
tomography (SDOCT) images of the optic nerve head were obtained
using the Heidelberg Spectralis SD-OCT. At the end of the study,
the eyes, optic nerves, and brains were fixed. Axon counts were
completed and three-dimensional reconstructions of the optic nerve
head were created for histomorphometric analysis.
Results: IOP in the experimental glaucoma eye was significantly
elevated compared to the fellow control eye each week (average
weekly IOP = 23±3 vs 9±2 mmHg; p<0.001). Retinal nerve fiber
layer (RNFL) thickness declined at a rate of 5.9um/week in the
experimental glaucoma eyes and was unchanged in the control eye
(Week 12 RNFL thickness = 76±35 vs 135±21 um; p<0.001). In vivo
SD-OCT imaging showed cupping and posterior displacement of the
lamina cribrosa in the glaucomatous eyes, which was confirmed by
3-D histomorphometry. Axon counts were reduced consistent with
IOP elevation.
Conclusions: Additional model validation is required, but this pilot
study confirms our belief that the tree shrews with experimental
glaucoma develop key pathologic features of the human disease
and this model has the potential to help researchers accelerate our
understanding of glaucoma pathophysiology.
Commercial Relationships: Brian C. Samuels, None;
Christopher A. Girkin, None; Lisa Hethcox, None; J
Crawford C. Downs, None; Wenjie Zhan, None
Support: NIH grant K08EY02359403, Research to Prevent
Blindness, EyeSight Foundation of Alabama
cleavage was tested in vitro by incubating the gRNAs, Cas9 protein
and a WT target site PCR fragment. A total of 69 eggs were injected
with the gRNAs/Cas9/donor DNA mix and 21 were implanted. Six
founder pups were born. F1 pups screened by SB resulted in the
expected 2.9 kb (5’ probe), 3.7 kb (3’probe) and 2.1 kb (internal
probe). F2 pups were identified as WT, loxP/+ or loxP/loxP by PCR
with 2 sets of primers yielding 505 bp (5’loxP), 324 bp (3’loxP) and
264 bp (WT) fragments. Mgp-floxed mice are fertile and have normal
life span (6 months at submission)
Conclusions: The CRISPR/CAS9 strategy presented here has been
successful in generating the first reported Mgp-floxed mouse line.
The availability of this mouse is an essential requirement to generate
Mgp conditional KOs in TM and ppSC, and it would be invaluable
for the creation of stiffness mouse models in glaucoma
Commercial Relationships: Terete Borras, None; Kumar Pandya,
None; Dale Cowley, None; Renekia Elliott, None
Support: NIH EY13126, Research to Prevent Blindness (RPB)
Program Number: 3573 Poster Board Number: A0271
Presentation Time: 11:00 AM–12:45 PM
Generation of a viable, fertile Matrix-Gla (Mgp)-floxed mouse by
CRISPR/CAS9 technology. Relevance for the creation of stiffness
mouse models of glaucoma
Terete Borras1, 2, Kumar Pandya3, 4, Dale Cowley3, Renekia Elliott1.
1
Ophthalmology, Universsity of North Carolina at Chapel Hill,
Chapel Hill, NC; 2Gene Therapy Center, Chapel Hill, NC; 3University
of North Carolina at Chapel Hill, Chapel Hill, NC; 4Transviragen,
Raleigh, NC.
Purpose: To create mouse models to study eye stiffness in Glaucoma.
We have previously shown that the inhibitor of calcification/
stiffness Matrix gla (Mgp) gene is highly expressed in the trabecular
meshwork (TM) and peripapillary sclera (ppSC) in a Mgp knock-in
mouse model. Because Mgp knock-out (KO) mice are lethal, here
we set out to generate a Mgp-loxP mouse line which would allow the
creation of TM & ppSC conditional KOs. We further investigated
CRISPR/CAS9 strategies for the generation of floxed mice
Methods: Our CRISPR/CAS9 strategy involved: 1) search for
optimal insertion sites for the loxP sequences in the Mgp gene,
presence of nGG sequences and absence of conserved or repeated
regions 2) generation of 2 guides RNA (gRNA) by in vitro
transcription containing sequences homologous to the insertion site
and binding to Cas9 3) generation of recombinant Cas9 protein 4)
generation of a donor exogenous DNA containing loxP sequences (37
nt) flanked by restriction sites and homologous arms 5) co-injection
of the 2 gRNAs, Cas9 protein and donor vector into one cell embryo
and implantation on the foster mother 6) screening of pups by
Southern Blot (SB)
Results: loxP sites were inserted to flank Mgp exons 3 and 4, at 330
bp 5’ of exon 3 and 893 bp 3’ of exon 4. Predicted excision leads
to a non-functional Mgp protein. To assure better integration and
insertion in cis, the loxP sites were flanked by 5’ 1,100 bp and 3’ 705
bp homology arms (donor vector). The efficiency of gRNA-mediated
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