ARVO 2014 Annual Meeting Abstracts 220 Nanotherapy

Transcription

ARVO 2014 Annual Meeting Abstracts
220 Nanotherapy, nanodiagnostics and nanoimaging
Monday, May 05, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 1435–1457/B0037–B0059
Organizing Section: Nanotechnology and Regenerative Medicine
Program Number: 1435 Poster Board Number: B0037
Presentation Time: 8:30 AM–10:15 AM
Measure of the intraocular pressure with three sensors during
manual Perfluorocarbon Liquid injection in UV photocrosslinkable polymer model of the human eye
Marco Dal Vecchio1, Edoardo Grosso1, Antonio M. Fea1, Davide
Casalena2, Simone Marasso2, Matteo Cocuzza2, 4, Giancarlo
Canavese3, Luciano Scaltrito2, 5. 1Clinica Oculistica, Università
di Torino, Torino, Italy; 2Chilab, Materials and Microsystems
Laboratory, Politecnico di Torino, Torino, Italy; 3Center for Space
Human Robotics, IIT, Politecnico di Torino, Torino, Italy; 4Istituto
dei Materiali per l’Elettronica ed il Magnetismo, CNR, Parma, Italy;
5
Microla Optoelectronics srl, Chivasso, Italy.
Purpose: To measure the intraocular pressure (IOP) during the
manual Perfluorocarbon Liquid (PFCL) injection in a simulated pars
plana vitrectomy (PPV) using 23 and 25 Gauge infusion ports in a
model of human eye
Methods: A UV photo-crosslinkable polymer model of the human
eye was employed to measure the absolute pressure from three
sensors during the manual injection of PFCL in a simulated 23 and
25 Gauge PPV. The IOP has been collected by 3 sensors, both in 23
and 25 G, in three experimental settings: in the controlled Venting
Gas Forced Infusion (VGFI) of Balanced Salt Solution (BSS) at 30
mmHg with closed eyeball, in VGFI of F-Decalin at 8 PSI and 12
mmHg of preset BSS infusion, and finally in the manual injection
of PFCL at 12 mmHg preset BSS infusion. Two experienced
vitreoretinal surgeons have repeated twice the manual injection
of PFCL (F-Decalin, Fluoron Gmbh, Ulm, Germany) in order to
simulate the real situation during PPV with a Stellaris PC vitrectomy
machine (Bausch+Lomb, Rochester, NY, USA)
Results: In the VGFI of BSS in closed eyeball at 30 mmHg, the IOP
resulted higher than the preset, but constant (23G: mean 43.70 ± 0,65
mmHg; 25G: mean 47.89 ± 0.13 mmHg). In the VGFI injection of
PFCL at 8 PSI and 12 mmHg of BSS infusion, the IOP shown a little
variation (23G: mean 29,03 ± 0,31 mmHg; 25G: mean 27,56 ± 0,20
mmHg) whereas a wide range in IOP values has been detected during
the manual F-Decalin injection at 12 mmHg of BSS infusion (23G:
min 17.64, max. 120.70, mean 55.25 ± 13.14 mmHg; 25G: min.
22.67, max. 73.31, mean 53.49 ± 13.25 mmHg)
Conclusions: During a 25 or 23 Gauge PPV, the surgeon must
temporally inject a certain amount of PFCL to flatten the retina and
perform various maneuvers (laser treatment, membranes peeling,
etc.). Since usually the injection is performed manually by a syringe,
the pressure at the tip’s is unknown. In our experiments, the IOP
measured by the probe in the macular site has been significantly
higher than the other two, both in 23 G and in 25 G experiments,
suggesting that the direct injection of fluid from the sclerotomy
opposite to the macula may add a certain role in a iatrogenic damage.
Our study’s outcome has shown very high and unsteady IOP values
for manual injection, therefore seems safer to rely on the VFGI
control for the PFCL injection
Commercial Relationships: Marco Dal Vecchio, None; Edoardo
Grosso, None; Antonio M. Fea, None; Davide Casalena, None;
Simone Marasso, None; Matteo Cocuzza, None; Giancarlo
Canavese, None; Luciano Scaltrito, None
RPE cells differentiated from human induced pluripotent
stem cells show production of the visual chromophore and
phagocytosis ability
Akiko Maeda1, 2, Grazyna Palczewska3, Hiroshi Maeno1, Krzysztof
Palczewski2, Masayo Takahashi4, Tadao Maeda1. 1Ophthalmology,
Case Western Reserve Univ, Cleveland, OH; 2Pharmacology, Case
Western Reserve University, Cleveland, OH; 3Polgenix, Cleveland,
OH; 4RIKEN, Kobe, Japan.
Purpose: The visual (retinoid) cycle activity in human iPS derived
RPE (hiPS-RPE) cells has not been fully examined. We examined the
ability for production of visual chromophore, 11-cis-retinal, in hiPSRPE cells. We also examined their phagocytosis ability.
Methods: hiPS-RPE cells and mouse primary RPE (mpRPE) cells
were examined their expression of visual cycle enzymes and ability
for generation of 11-cis-retinal in vitro and in vivo. In addition to
hiPS-RPE and mpRPE cells, ARPE19 cells and macrophage cell line
were also used for study of phagocytosis.
Results: hiPS-RPE cells appeared morphologically similar to mpRPE
cells. Expression of certain visual cycle proteins was maintained
during cell culture of hiPS-RPE cells, whereas expression of these
same molecules rapidly decreased in mpRPE cells. Production of
11-cis-retinal and retinosome formation were documented in hiPSRPE cells in vitro. Transplantation of mpRPE into blind Lrat-/- and
Rpe65-/- mice resulted in the recovery of visual function, including
increased electrographic signaling and endogenous 11-cis-retinal
production. When hiPS-RPE cells were transplanted into the
subretinal space of Lrat-/- and Rpe65-/- mice, their vision improved
as well. Moreover, histological analyses of these eyes displayed
replacement of dysfunctional RPE cells by hiPS-RPE cells.
hiPS-RPE cells also display good expression of phagocytosis markers
including MERTK and some phagocytosis ability.
Conclusions: Our results show that hiPS-RPE cells can exhibit a
functional visual cycle and phagocytosis. These cells could provide
potential treatment options for certain blinding retinal degenerative
diseases.
Commercial Relationships: Akiko Maeda, None; Grazyna
Palczewska, None; Hiroshi Maeno, None; Krzysztof Palczewski,
None; Masayo Takahashi, None; Tadao Maeda, None
Support: EY022658, R44AG043645, P30EY011373, Midwest EyeBanks
Evaluation of Bio-Immune Compatibility of Nanofibrous Scaffold
for Corneal Tissue Engineering
Sahar Salehi1, 2, Thomas Bahners1, Jochen S. Gutmann1, 3, Bernhard
Singer4, Thomas A. Fuchsluger2. 1Deutsches Textil forschungzentrum
Nord-West gGmbH, Krefeld, Germany; 2Oththalmplogy, Düsseldorf
University Hospital, Heinrich-Heine-University, Düsseldorf,
Germany; 3Physikalische Chemie, Universität Duisburg-Essen,
Essen, Germany; 4Institute of Anatomy, Essen University Hospital,
University of Duisburg-Essen, Essen, Germany.
Purpose: An estimated 10 million people suffer worldwide from
vision loss caused by corneal damage. For the worst cases, the only
available treatment is transplantation with human donor corneal
tissue. However, in numerous countries there is a considerable
shortage of corneal tissue of good quality, leading to various
efforts to develop tissue substitutes.The present study aims to
introduce nanofibrous scaffold of poly (glycerol sebacate) PGS as a
biodegradable implant, for the corneal tissue engineering.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
Methods: Nanofibrous scaffolds were produced by electrospinning
method and different compositions of PGS and poly(ε-caprolactone)
(PCL).The biocompatibility of the material was tested in vitro
by MTT assay on days 1,3,5, and 7 to test cell viability of human
corneal endothelium cells(HCEC). Cells treated with medium
only were served as a negative control group. Density of the
grown cells was studied by determining the absorbance intensity.
To examine a potential immunological reaction of the scaffolds,
samples were exposed to mononuclear cells derived from peripheral
blood(PBMCs). As positive controls for induction of proinflammatory
mediators, PBMCs activated by CD3 and IL2 in contact and
without contact to scaffolds were used. After an incubation period
of 3 days, supernatants were assayed for apoptotic assessment and
immunogenic potentials by Annexin FITC V and FACS analysis.
Results: We could successfully demonstrate that cultivation of
HCECs on PGS/PCL scaffolds was possible. Compared to day 3, cell
density determined by microplate absorbance was significantly higher
after 7 days of cultivation(p<0.0001). According to the MTT data,
none of the samples showed toxicity. However, cell proliferation rates
differed amongst samples.
Apoptotic assessments by FACS analysis showed that no composition
stimulated apoptosis or activated PBMCs. All the compositions were
inert for the T-cells. After 3 days the highest viability percentages
comparable with negative control are belonged to un-stimulated
PBMCs and PBMCs stimulated by IL2 in contact to the scaffolds.
Conclusions: PGS/PCL scaffolds produced with different component
compositions are well suitable for cultivation of conjunctival and
corneal endothelial cells. The scaffolds do not promote premature
cell death and do not trigger certain immune reactions tested in vitro.
Further studies are under way to gain more insight into cell-scaffold
interaction.
Commercial Relationships: Sahar Salehi, None; Thomas Bahners,
None; Jochen S. Gutmann, None; Bernhard Singer, None;
Thomas A. Fuchsluger, None
Nanostructured Photopolymers as Stem Cell Scaffolds for
Photoreceptor Development and Transplantation
Kristan Worthington1, 3, Alexandra Bartlett3, Edwin M. Stone1, 2,
Aliasger K. Salem4, 3, Allan Guymon3, Budd A. Tucker1, 2. 1Stephen
A. Wynn Institute for Vision Research, The University of Iowa,
Iowa City, IA; 2Ophthalmology and Visual Sciences, University of
Iowa, Iowa City, IA; 3Chemical and Biochemical Engineering, The
University of Iowa, Iowa City, IA; 4Pharmaceutical Science and
Translational Therapeutics, The University of Iowa, Iowa City, IA.
Purpose: Patients with advanced stages of retinal degenerative
disease would benefit greatly if a means could be devised for
transplanting functional cells into the injured retina. Although bolus
sub-retinal cell injections have been shown to restore function to
retinas with early stages of disease, this success translates poorly to
more advanced disease in which there is a lack of structural support
for the integrating cells. Stem cell scaffolds can be used to provide
this support, but the effects of the physical properties of these
scaffolds (i.e. their nanostructure) on the transplanted cells is poorly
understood.
Methods: Nano-porous cell scaffolds were synthesized by
lyotropic liquid crystalline (LLC) templating of photopolymerizable
poly(ethylene glycol) pre-polymers. Morphology was characterized
using scanning electron microscopy (SEM), small-angle x-ray
scattering (SAXS), and polarized light microscopy (PLM). Surfactant
removal and swelling were both examined gravimetrically. The effect
of nanostructure on the interaction between the cells and the scaffolds
was investigated by seeding the materials with murine induced
pluripotent stem (MiPS) cells, and monitoring their growth and
differentiation with SEM and immunohistochemsitry.
Results: Polymers templated with 30 wt% DTAB, an ionic
surfactant, manifested scattering peaks, birefringence, and porosity
when examined using small-angle x-ray scattering, polarized light
microscopy, and SEM, respectively, all of which are indicators
of nanostructure. This presence of nanostructure improved the
diffusion properties of the material and also influenced the growth
and differentiation of cells. The optimized materials produced were
shown to support the differentiation of induced pluripotent stem cells
to mature retinal cell types.
Conclusions: Nanostructure of photopolymers plays an important
role in cell/material interactions and can be successfully manipulated
to meet the needs of retinal transplant applications. Development of
a material that is biocompatible, implantable, and able to encourage
growth and differentiation of mature retinal cell types would be a
major step toward the successful replacement of lost photoreceptor
cells and restoration of retinal function in patients suffering from
retinal degenerative disease.
Commercial Relationships: Kristan Worthington, None;
Alexandra Bartlett, None; Edwin M. Stone, None; Aliasger K.
Salem, None; Allan Guymon, None; Budd A. Tucker, None
Support: NIH New Innovator Award (DP2OD007483), Stephen A.
Wynn Foundation, NSF (CBET - 0933450)
B-RAF signaling promotes optic nerve regeneration
Jian Zhong1, Kaijie Ma1, Zhigang He2, Kevin J. O’Donovan1. 1Weill
Medical College of Cornell University, Burke Cornell Medical
Research Institute, White Plains, NY; 2Harvard Medical School,
Children, Boston, MA.
Purpose: To investigate the role of intracellular B-RAF signaling in
promoting regeneration in the optic nerve after injury.
Methods: We genetically engineered mice to study gain of B-RAF
function in promoting axon regeneration. These mice bear in the
endogenous B-RAF locus a construct that expresses a kinaseactivated B-RAF (kaB-RAF) upon Cre recombination. Adult mice
were injected with AAV2-Cre into the vitreous of the eye to activate
B-RAF signaling in the RGCs, and 14 days later were subjected to
proximal crush lesion of the optic nerve. Outgrowing axons were
labeled by CTB-Alexa anterograde tracers. Optic nerves were
dissected, sectioned and imaged two weeks after the injury. The same
procedures were performed with mice lacking the canonical B-RAF
effectors MEK1 and MEK2, as well as with mice homozygous for the
conditional deletion of PTEN (Park et al., 2008).
Results: In mice expressing kaB-RAF in RGCs we observed robust
axon regeneration up to 3.5 mm past the lesion site. Comparing to
the PTEN loss-of-function model, the number of fibers growing past
the lesion was almost 4fold higher in the kaB-RAF model. Average
length of regeneration was similar in both models. Blocking of the
canonical B-RAF signaling pathway by conditional elimination of
MEK1 and MEK2 completely abolished axon regeneration induced
by kaB-RAF. Concomitant activation of both B-RAF and the PI3
kinase-mTOR pathway (by removal of PTEN) drove axon growth
beyond a simple additive effect.
Conclusions: We conclude that activation of B-RAF drives
substantial axon regeneration in the crushed optic nerve via canonical
B-RAF – MEK signaling.
Activation of B-RAF enables regenerative axon growth in the crushlesioned optic nerve via the canonical effectors MEK1/MEK2. (A)
Experimental time course. (B) Intravitreal injection of AAV-Cre
induces expression of TdTomato reporter in RGCs. (C) Upper, whole
mount crushed Bax -/- optic nerve. Crush site is indicated by a red
asterisk. Bax null background was used to prevent RGC death after
optic nerve crush. Lower, confocal fluorescence image of the same
nerve. Green, axons anterogradely labeled with CTB-Alexa488. (D)
Regenerative growth in lesioned kaB-RAF expressing optic nerve.
Inset, axons at approx. 3.5 mm from the crush site. (E) Loss of MEK1
and MEK2 abolishes the regeneration driven by kaB-RAF. (F) Optic
nerve regeneration in absence of PTEN. (G) Quantification. Scale bar
= 0.5 mm. N=3 for each group.
Commercial Relationships: Jian Zhong, None; Kaijie Ma, None;
Zhigang He, None; Kevin J. O’Donovan, None
Support: NIH grant R01EY022409
Retinal regeneration by Lgr5+ amacrine cells in adult mammals
Hongjun Liu1, Shenghe Tian1, Nathan Glasgow2, Gregory Gibson3,
Christen Shiber2, Igor Nasonkin1, James L. Funderburgh1,
Simon Watkins3, Joel S. Schuman1, Jon Johnson2. 1Department
of Ophthalmology, University of Pittsburgh School of Medicine,
Pittsburgh, PA; 2Department of Neuroscience, University of
Pittsburgh, Pittsburgh, PA; 3Center for Biologic Imaging, University
of Pittsburgh School of Medicine, Pittsburgh, PA.
Purpose: Like other regions of the central nervous system, the retina
is subject to degenerative diseases. Current common knowledge
suggests that the retina of adult mammals lacks regenerative capacity.
However, the retina of non-mammalian vertebrates, like fish and
amphibians, possesses remarkable capacity of regeneration. Two
major cell types in these lower vertebrates have been demonstrated
to contribute to continuous retinal regeneration: cells of the ciliary
marginal zone (CMZ) and Müller glial cells within the neuroretina.
However, the CMZ is evolutionarily lost in mammals. Müller cells
in mammalian retinas do not proliferate under normal physiological
conditions, and they only possess limited regenerative potential in
response to injury. Based on the anatomical similarity between the
mammalian ciliary body and the lower vertebrate CMZ, a population
of pigmented epithelial cells from the ciliary body was identified as
the mammalian retinal stem cell a few years ago. Initial observations
suggested these cells possess retinal stem cell properties, yet further
analysis demonstrated that they could not differentiate into retinal
neurons in vitro and in vivo. Therefore, whether the mammalian
retina possesses regenerative capacity under normal physiological
conditions still remains undetermined and if so, the cellular source
that serves as the precursors for retinal regeneration in adulthood
is also unknown. The purpose of the current study is to identify
retinal cells in adult mammals that possess regenerative capacity and
function as the putative adult retinal stem cell.
Methods: To identify the putative adult retinal stem cell, we used a
genetic lineage tracing approach.
Results: We demonstrated that Lgr5, an adult stem cell marker in
high turnover tissues and organs, is expressed in a subgroup of retinal
cells in adult mice. Despite exhibiting features of differentiated
retinal amacrine interneurons, these Lgr5+ retinal cells can reenter the cell cycle, proliferate and generate other retinal lineages,
beginning in early adulthood and continuing as the animal ages.
Conclusions: These findings suggest the retina in adult mammals is
not devoid of regeneration as previously thought. Rather, it is plastic
and Lgr5+ amacrine cells contribute to its homeostatic maintenance,
functioning as the putative mammalian adult retinal stem cell. The
identification of such cells provides new therapeutic strategies for
degenerative retinal diseases.
Commercial Relationships: Hongjun Liu, None; Shenghe Tian,
None; Nathan Glasgow, None; Gregory Gibson, None; Christen
Shiber, None; Igor Nasonkin, None; James L. Funderburgh,
None; Simon Watkins, None; Joel S. Schuman, None; Jon
Johnson, None
Support: This research is supported by NIH grant R00AG032356
Isolation of Goblet Cells from Conjunctival Epithelium by
Density-Gradient Centrifugation
Nicole Qiaozhi Lu1, 2, Michael P. Grant3, Jennifer Elisseeff1.
1
Translational Tissue Engineering Center - Wilmer Eye Institute,
Johns Hopkins School of Medicine, Baltimore, MD; 2Materials
Science and Engineering, Johns Hopkins University, Baltimore, MD;
3
Oculoplastics Division - Ocular and Orbital Trauma Center - Wilmer
Eye Institute, Johns Hopkins School of Medicine, Baltimore, MD.
Purpose: Primary conjunctival epithelial cells were subject to
Percoll® density-gradient centrifugation, in order to isolate goblet
cells for the treatment of conjunctival diseases.
Methods: Rabbit conjunctiva tissue was digested with dispase II and
trypsin/EDTA to obtain single cell suspension. Six densities (1.03 –
1.08 g/mL) were prepared by adjusting Percoll with 10× PBS solution
and H2O. The discontinuous gradients were created by layering 2
mL of each Percoll solution in a centrifuge tube, with decreasing
density. The cell suspension was added on top of the gradients, and
centrifuged at 400×g for 20 min. Fractions from each interface were
collected and washed with PBS. Primary cells without centrifugation
were used as control. To identify cell phenotypes, qRT-PCR
(comparative ΔΔCT; gapdh as the reference) was used to analyze gene
expression (ck4, non-goblet cell epithelium; muc5ac, goblet cells;
abcg2, stem/progenitor cells). Flow cytometry experiments were also
carried out to quantify the percent of goblet cells in the fractions with
CK4 and MUC5AC labeling.
Results: After centrifugation, cell suspensions were fractionated into
6 layers (one on each interface), and they were labeled as L1-6 from
top to bottom. The qRT-PCR values were interpreted as muc5ac/
ck4 ratio. With increasing density, the relative expression of muc5ac/
ck4 decreased, and L1 had a higher ratio than control. With respect
to abcg2, L2 had the highest expression level, while both L1 and
L2 expressed more abcg2 transcripts than control. Flow cytometry
quantification further implicated that the MUC5AC-positive
population became smaller from L1 to L6. When compared to control
(15%), L1-L3 contained more MUC5AC-positive cells (18-20%).
At the same time the CK4-positive population became bigger from
L1 to L6. Therefore, with both results combined, L1 and L2 could be
considered to contain the majority of goblet cells and more stem cells
than other layers and the control.
Conclusions: Goblet cell-rich population can be separated from the
primary conjunctival cell mixture, by simple centrifugation with
discontinuous Percoll gradients. Both goblet and stem cells were
enriched in the top 2 layers after centrifugation. Potentially, this
isolation method could be applied to increase the mucin production
and proliferation capacity of harvested cells before transplantation,
which may improve outcomes in conjunctival reconstruction.
Commercial Relationships: Nicole Qiaozhi Lu, None; Michael
P. Grant, Stryker CMF (C), Synthes CMF (C); Jennifer Elisseeff,
Collagen Vitrigel (P)
Support: DoD Eye Patch Grant W81XWH-09-2-0173; Research to
Prevent Blindness
Characterization of Magnetic Nanoparticle Loaded Corneal
Endothelial Cells
David O. Zamora1, Mauris N. DeSilva2, Lauren E. Cornell1,
Randolph D. Glickman3, Heuy-Ching H. Wang1, Anthony J. Johnson1.
1
Ocular Trauma & Vision Restoration, U.S. Army Institute of Surgical
Research, Ft. Sam Houston, TX; 2Maxillofacial Injury & Disease
Department, Naval Medical Research Unit San Antonio-JBSA, Ft.
Sam Houston, TX; 3Ophthalmology, University of Texas Health
Science Center, San Antonio, TX.
Purpose: Current methods to replace diseased or traumatized
endothelium involve a full thickness keratoplasty or a Descemet’s
Stripping Automated Endothelial Keratoplasty procedure to replace
this targeted region of the cornea. We hypothesize magnetic
nanoparticles (MNPs) can be pre-loaded into endothelial cells and
used to deliver them to the posterior of the cornea after injection into
the anterior chamber, while in the presence of a magnetic field.
Methods: Bovine corneal endothelial cells (Astarte Biologics)
were cultured in DMEM medium, containing 10% FBS. Twentythousand cells per well were seeded into 48 well plates (Passage
6-8) and maintained in culture for 48 hrs. Nanomag –D-Spio (biotin
coated; Micromod, Rostock, Germany) were added to the cells in
the following bead to cell ratios: 0:1, 100:1, 1,000:1, and 10,000:1.
Phase contrast and fluorescence microscopy were used to evaluate
the cell morphology of the treated cells. MTT viability assays were
used to evaluate the effects of the MNPs on endothelial cells after
being exposed to them for up to a 48 hr period. Assays were each
performed in triplicate.
Results: Exposure of the MNPs to the endothelium, at any of the
concentrations analyzed, did not appear to affect the confluency
or morphology of the monolayer of cells, as determined by phase
contrast microscopy. Furthermore, the F-actin cytoskeleton of
the cells did not appear to re-arrange or disassemble after these
treatments. Interestingly, endothelial cell MTT metabolism OD
values slightly decreased (0.95, 0.92, 0.89, 0.89) as MNP bead:cell
ratios increased (0:1, 100:1, 1000:1, 10000:1), but was not
statistically significant.
Conclusions: MNPs are an FDA-approved technology for use in
medical procedures in humans. Here we have demonstrated that
bovine endothelial cells can be cultured in the presence of MNPs and
incorporated into these cultures apparently with minimal toxicity. The
initial proof of concept data described here will be used to further
develop this technology using human corneal endothelial cells, in
order to target these cells to the back of the cornea for repairing
injuries to the endothelium.
Commercial Relationships: David O. Zamora, None; Mauris N.
DeSilva, None; Lauren E. Cornell, None; Randolph D. Glickman,
None; Heuy-Ching H. Wang, None; Anthony J. Johnson, None
Retinal Regeneration in Zebrafish: A New Model for Focal Injury
to the Outer Retina
Alex Yuan, Brent A. Bell, Rose M. DiCicco, Charlie Kaul, Joe G.
Hollyfield, Brian D. Perkins, Bela Anand-Apte, Yuankai K. Tao.
Ophthalmology, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: To develop a novel laser-induced injury model to study
retinal regeneration in zebrafish.
Methods: Adult zebrafish were imaged by optical coherence
tomography (OCT) and confocal scanning laser ophthalmoscopy
(cSLO) in room air through a contact lens. Using a custom designed
beam splitter, 532nm laser photocoagulation was applied using the
OCT C-scan image for targeting. Calibrated laser spots of 42-47mW
were delivered to the retina. At multiple intervals post-lesion, fish
were re-imaged using both OCT and cSLO to follow the progression
of each laser burn. Histologic sections were performed at various
intervals to monitor the injury response. Lesion size was measured
using cSLO images and ImageJ software.
Results: Retinal lesions were visible by OCT and cSLO immediately
after laser application. Early OCT changes include an increase in
hyper-reflectance and loss of the outer retina banding pattern. Lesions
were best detected using infrared darkfield imaging on cSLO, but
they were also visible with autofluorescence imaging. The average
lesion size on day 1 was 26057 ± 397μm2. After 1 week the lesion
size and intensity began to decrease and at 15 weeks, lesions were
barely detectable above background and the average size was
9404μm2. Histologic sections showed focal areas of damage localized
to the outer retina. At 15 weeks, the damaged retina had regenerated
and re-established a laminated structure. However, subtle changes can
still be detected by cSLO and OCT imaging.
Conclusions: OCT guided laser photocoagulation can be used to
induce focal retinal injury in zebrafish. Histologic analysis confirmed
focal and selective damage to the outer retina. By week 15, the
normal lamination and architecture of the retina was re-established.
This laser model allows for focal destruction of the retina, leaving
the surrounding retina intact, allowing for improved study of cellular
proliferation, migration, and differentiation during the regenerative
process in zebrafish.
Lesion size decreases over time. (Top) Plot of average lesion size
over time. (Bottom) Representative cSLO images 0-9 weeks after
laser. An outline of the superior lesion is shown in black.
Comparison of lesions 2 days and 15 weeks post-laser. (Top) H&E,
(middle) OCT, (bottom) cSLO. Each set of images were obtained
from a single animal. Black arrowheads point to the corresponding
lesions.
Commercial Relationships: Alex Yuan, None; Brent A. Bell, None;
Rose M. DiCicco, None; Charlie Kaul, None; Joe G. Hollyfield,
None; Brian D. Perkins, None; Bela Anand-Apte, None; Yuankai
K. Tao, None
Support: NEI 1K08EY023608-01 (AY), Research Preventing
Blindness unrestricted grant to Cleveland Clinic Lerner College of
Medicine, and Foundation Fighting Blindness center grant (JGH)
Stem/progenitor properties are preserved during ex vivo
expansion of human oral mucosal epithelial cell sheet by
collagenase but not dispase
Hung-Chi Chen1, David H. Ma1, Hsiang-Fu Huang2, Jesseica Ma1,
Yi-Jen Hsueh1. 1Ophthalmology, Chang Gung Memorial Hospital,
Taoyuan, Taiwan; 2Otolargyngology, Chang Gung Memorial
Hospital, Taoyuan, Taiwan.
Purpose: Previously, in the Phase I trial of cultivated oral mucosal
epithelial transplantation (COMET), we have substantiated efficacy
of such cell therapy in promoting wound healing in patients with
severe ocular surface burns, identified long-term persistence of
transplanted oral mucosal epithelial cells (OMEC) in the cornea,
and concluded that COMET in suitable in promoting corneal
wound healing due to its inferior inhibitory effect on corneal
neovascularization. Recently, collagenase, but not dispase, has been
reported to isolate cell spheres mimicking stem/progenitor cells
from the corneal endothelium or epithelium. In this Phase II trial, we
aimed to investigate the safety and efficacy of isolating OMEC with
collagenase.
Methods: Human oral mucosal remnants removed during oral
surgeries were digested either by 1% collagenase A in PBS overnight
or 1.2 U dispase II/0.25% trypsin EDTA at room temperature for 1.5
hours followed by scrapping. The yielded cell pellets were seeded
on denuded amniotic membrane (DAM) with or without coculture
with 3T3 cells. BrdU incorporation assay, colony formation assay
(CFA), immunostaining and Western blot for stem/progenitor cell
markers (p63 and p75) and differentiation markers (keratins 3 and
13) were performed. Scanning electron microscopy (SEM) was
used to compare surface structures. Finally, Western blot for nuclear
β-catenin was performed.
Results: Collagenase treatment group generated cell sheets that
retained regular cell morphology, and could be maintained in
serum-free media, and even without 3T3 coculture. In contrast, 3T3
coculture was indispensible in the initial culture stage using dispase
II/trypsin. BrdU label index and CFA were both higher in collagenase
group, and so was a prominent p63 expression by Western blot in
that group. Finally, more prominent nuclear β-catenin expression by
Western blot was also noted in the collagenase group.
Conclusions: Collagenase treatment is superior to dispase II/trypsin
treatment in generating human OMEC sheet, and is free of animal
serum and 3T3 cells, which better conforms with contemporary
regulations of cell therapy. Therefore, we suggest adopting such a
cell isolation method in the Phase II clinical trial of COMET. Future
goals are to investigate the molecular mechanisms through which
collagenase treatment can better preserve the stem/progenitor cells of
OMEC.
Commercial Relationships: Hung-Chi Chen, None; David H. Ma,
None; Hsiang-Fu Huang, None; Jesseica Ma, None; Yi-Jen Hsueh,
None
Support: NMRPG 101-2325-B-182A-010
Chitosan Coated Nanostructured Lipid Carriers Containing
Dexamethasone Acetate
Edson Santos-Neto1, Márcia Spuri-Ferreira2, Milton Ruiz-Alves1,
Nádia Bou-Chacra2. 1Oftalmologia, Medicina USP, São Paulo,
Brazil; 2Farmacologia, Farmácia USP, São Paulo, Brazil.
Purpose: The Chitosan, a polycationic biopolymer with
mucoadhesive properties, presents a promising future in the
prolonged ocular delivery of drugs. It also presents biodegradability,
biocompatibility, nontoxicity and ability to increase paracellular
transport of drugs. These properties allow their use as vehicles
for ophthalmic formulations.The aim of this study was to develop
and characterize NLC coated with chitosan (NLC-C) containing
dexamethasone acetate.
Methods: The NLC was formulated using 6.0% (w/w) cetyl
palmitate (CP), 4.0% (w/w) capric/caprylic triglycerides (CCP),1.5%
(w/w) tween® 80, 2.5% (w/w) lutrol®, 0.25% (w/w) soy lecithin
(SL), 0.5% (w/w) sodium dodecyl sulphate (SDS) and 0.05%
(w/w) dexamethasone acetate and obtained by high pressure
homogenization (HPH). The dexamethasone acetate was incorporated
in the lipid phase.CS- NLC was obtained by dissolving 5.0 mL of
chitosan dispersion (CS) 0.75% w/v, in 5.0 mL of the nanoparticle
suspension under magnetic stirring of 200 rpm for two hours.
Measures of mean diameter, polydispersity index (PI) and zeta
potential (ZP) were performed on Zetasizer Nano ZS90®. The NLC
and CS-NLC were maintained at temperature of 6oC ± 2,0 and were
performed in order to evaluate their stability.
Results: The nanoparticles presented mean diameter of 170.5 ±
2.19 nm, PI equal to 0.141 ± 0.01 and ZP of -40.9 ± 0.42 mV. After
the coating procedure, the mean diameter, the PI and the ZP were
changed to respectively 642.7± 6.08 nm, 0.269 ± 0.02 and +50.4 ±
0.63mV. As expected, the NLC-C presented larger mean diameter
and an inversion of the surface charge, which indicated the coating
efficiency. The CS- NLC did not show any increase during a 30-day
period. It seems that the coating was important to CS-NLC stability.
Conclusions: The values obtained for zeta potential after the coating
procedure supports the conclusion that chitosan was adsorbed
on the nanoparticle surface. The coating also contributed to the
maintenance of CS-NLC stability. The developed system in this study
showed interesting properties and therefore it can be proposed as
promising drug delivery system to improve the therapeutic efficacy of
ophthalmic preparations containing dexamethasone acetate.
Commercial Relationships: Edson Santos-Neto, None; Márcia
Spuri-Ferreira, None; Milton Ruiz-Alves, None; Nádia BouChacra, None
Support: Nenhum
Transplantation of Ex-Vivo Genetically Modified Photoreceptor
Precursors
Alona O. Cramer1, Mandeep S. Singh1, Michelle McClements1,
Robert E. MacLaren1, 2. 1Nuffield Laboratory of Ophthalmology,
University of Oxford, Oxford, United Kingdom; 2Oxford Eye
Hospital, Oxford, United Kingdom.
Purpose: Retinitis pigmentosa (RP) is one of the primary causes of
inherited retinal blindness, affecting more than 15 million people
worldwide. In this disease an initial loss of rod photoreceptors leads
to a cellular cascade that promotes a secondary degeneration of
cone photoreceptors. Pluripotent stem cells and rod photoreceptor
progenitors have hence been widely researched as candidates for
cell replacement in RP. Patient-specific induced pluripotent stem
cells (iPSc) could provide an autologous expandable source of
cells for transplantation. However, the use of patient-derived iPSc
would require that the disease-causing mutation be repaired in vitro
before cells are transplanted. Genetic modification and subretinal
transplantation of photoreceptor precursor cells (PPC) are here
studied in an endeavour to develop ex-vivo gene therapy for cell
replacement in retinal degeneration.
Methods: By optimizing early postnatal retinal culture conditions,
rod PPC dissociated from Nrl.GFP+ mice were maintained in
vitro. In this model, green fluorescent protein (GFP) is expressed
specifically in rod photoreceptors. Cultured PPCs were transfected
using DsRed florescent reporter adeno-associated virus (AAV)
vector carrying the human Rhodopsin gene. Magnetic assisted cell
sorting was performed to enrich rod PPC via the cell surface antigen
CD73 and repeated washing steps were performed to remove free
AAV particles. Cells were transplanted into adult mice with retinal
degeneration (Rho-/- and Rd1).
Results: A reliable cell culture system was achieved by use of
supplemented neuronal growth media and incubation at 34 degrees
Celsius. Rod PPC numbers significantly increased ex-vivo in the
first 14 days of culture and maintained high viability for 31 days.
Transfection of PPC was achieved by a florescent reporter AAV8 and
confirmed ex-vivo. Dissociated retinal cell cultures were enriched to
over 85% Nrl.GFP+ positive rod PPC prior to transplantation, and
survival of transplanted Nrl.GFP+ PPCs in the subretinal space was
observed within 14 days.
Conclusions: Prolonged survival of rod PPC in vitro will allow for
a sufficient period of time for ex vivo assessment of gene therapies
before transplantation. Our results show successful transplantation
of genetically modified, rod cells in murine models of retinal
disease and afford a foundation for ex vivo gene therapy in human
photoreceptor precursors derived from autologous iPSc.
Commercial Relationships: Alona O. Cramer, None; Mandeep S.
Singh, None; Michelle McClements, None; Robert E. MacLaren,
None
Support: NIHR Biomedical Research Centre, the Wellcome Trust,
Medical Research Council, UK Department of Health, Special
Trustees of Moorfields Eye Hospital, the Royal College of Surgeons
of Edinburgh, Clarendon Fund Scholarships
The Krüppel-Like Factor Gene Target Dusp14 Regulates Axon
Growth and Regeneration
Keiichiro Iwao1, 2, Akintomide Apara1, Noelia J. Kunzevitzky1, 3, Yan
Wang1, 3, Darcie Moore1, Murray Blackmore4, Jeffrey L. Goldberg1,
3 1
. Bascom Palmer Eye Institute, Miami, FL; 2Ophthalmology,
Kumamoto University, Kumamoto, Japan; 3Shiley Eye Center, La
Jolla, CA; 4Marquette University, Milwaukee, WI.
Purpose: Krüppel-like transcription factor (KLF) family members
regulate intrinsic axon growth ability in vitro and axon regeneration
in vivo in retinal ganglion cells (RGCs) and other CNS neurons. Here
we propose to discover new KLF gene targets that regulate axon
growth and regeneration in central nervous system (CNS) neurons.
Methods: To determine through what gene targets KLFs regulate
axon growth, we examined genes regulated by KLFs in rat RGCs
based on microarray data after KLF7, -9, -11 and -16 expression
in RGCs by viral transduction. KLF candidate gene targets were
screened by quantification of neurite growth in vitro. qRT-PCR,
protein extraction and western blot analysis after gene expression
using primary RGCs were performed to characterize candidate genes.
RGC axon regeneration in a rat optic nerve crush model using shRNA
knock down strategy against a candidate gene was used to examine
physiology in vivo.
Results: We identified Dual specificity phosphatase 14 (Dusp14)
as a KLF gene target in RGCs, induced four-fold after transduction
with KLF9, a KLF family member that significantly suppresses
neurite outgrowth. Dusp14 strongly suppressed neurite outgrowth
of RGCs in vitro, with no effect on neurite number. In P0 RGCs,
which do not express detectable levels of Dusp14 and express very
low levels of KLF9 mRNA, two different Dusp14-specific siRNAs
and a pharmacological inhibitor, PTP inhibitor IV, fully rescued P0
RGC neurite growth after KLF9 overexpression. In P8 RGCs, which
endogenously express KLF9 and Dusp14, the siRNAs and PTP
inhibitor IV not only rescued neurite growth from KLF9-induced
suppression, but also significantly promoted neurite elongation
in control-transfected neurons. Dusp14 and KLF9 inhibited the
activation of mitogen-activated protein kinases (MAPK) including
ERK1/2, JNK and P38. Finally, knocking down Dusp14 expression
in RGCs in vivo enhanced regenerative axon growth after optic nerve
injury.
Conclusions: KLF gene target Dusp14 is a key player limiting axon
growth and regenerative ability downstream of KLFs’ ability to
suppress axon growth in RCGs. Developing strategies to modulate
Dusp14 for enhancement of MAPK signaling may lead to efficient
promotion of axon regeneration after CNS injury.
Commercial Relationships: Keiichiro Iwao, None; Akintomide
Apara, None; Noelia J. Kunzevitzky, None; Yan Wang, None;
Darcie Moore, None; Murray Blackmore, None; Jeffrey L.
Goldberg, None
Support: NEI EY022129, NEI P30-EY022589 and NEI P30EY014801, and Research to Prevent Blindness, Inc (to JLG) and the
Uehara Memorial Foundation in Japan (to KI)
Transport and microglia uptake of dendrimers in normal and
ischemia/reperfusion injury retina
Imran A. Bhutto, Alexander J. Clunies-Ross, Siva P. Kambhampati,
Manoj K. Mishra, Scott D. McLeod, Rangaramanujam Kannan,
Gerard A. Lutty. Ophthalmology, Johns Hopkins Hosp Wilmer Eye
Inst, Baltimore, MD.
Purpose: Microglial activation in retina is a common response
to various neurodegenerative diseases. A hydroxyl-terminated
polyamidoamine (PAMAM) dendrimer-drug conjugate has been used
to target microglia and attenuate neuroinflammation in degenerated
rat retina when injected intravitreally. The aim of this study was to
develop a dendrimer that targets activated microglia in retina and can
be administered intravitreally as well as intravenously.
Methods: The dendrimer-Cy5 (D-Cy5) was evaluated in normal
mouse and in a mouse model of retinal ischemia/reperfusion (I/R)
injury. Transient ischemia was induced in one eye of Balb/c mice by
raising intraocular pressure to 90 mm Hg for 90 minutes followed by
retinal reperfusion which restores normal pressure. The fellow eye
served as a non-I/R control eye. After 6 days post I/R, D-Cy5, free
Cy5 or PBS was administered intravitreally or intravenously. Mice
were then sacrificed at 24 hours, 3 days, and 21 days post injection.
Eyecups were cryopreserved and 8um thick sections used for IHC
using rabbit IBA-1 antibody (macrophage/microglia specific) and
GSA lectin-FITC. D-Cy5 or Cy5 was assessed on the Zeiss 710
confocal microscope and autofluorescence was assessed in the
sections of PBS injected eyes.
Results: In I/R eyes at 24 hours after intravitreal injection, there was
uptake of D-Cy5 at the level of ILM and co-localization with IBA-1+
microglia cells in sensory retina. Where as free Cy5 was diffuse and
intense in retinal blood vessels and appeared to cleared within 72hr.
This distinctive D-Cy5 pattern declined over 21 days in I/R eyes, less
D-Cy5 and more IBA-1+ microglia cells in retina. On the other hand,
intravenous administration showed continuous uptake of D-Cy5 and
more colocalization with IBA-1+ microglia cells in retina and choroid
even at 21 days post injection in I/R eyes. The pattern of enhanced
uptake of D-Cy5 was not seen in the non-I/R retinas and choroids
after either injection.
Conclusions: The nontoxic PAMAM dendrimers appear to be an
excellent drug delivery system to activated microglia. In eyes with
activated microglia after I/R injury, intravenous and intravitreal
D-Cy5 was retained by microglia (IBA+). This approach may yield
a dendrimer-based therapy to decrease inflammation in diseases
associated with microglia activation.
Commercial Relationships: Imran A. Bhutto, None; Alexander
J. Clunies-Ross, None; Siva P. Kambhampati, None; Manoj
K. Mishra, None; Scott D. McLeod, None; Rangaramanujam
Kannan, None; Gerard A. Lutty, None
Support: NH Grant EY01615; EY001765; Research to Prevent
Blindness (Wilmer)
Local Delivery of an Engineered Epithelial Monolayer by
Micropatterned Polymeric Nanosheets
Hirokazu Kaji1, Toshinori Fujie2, Nobuhiro Nagai1, Toshiaki Abe1.
1
Tohoku University, Sendai, Japan; 2Waseda University, Tokyo,
Japan.
Purpose: Age-related macular degeneration (AMD) is the leading
cause of visual impairment and blindness in the elderly population,
whose main complication is the development of subretinal choroidal
neovascularization and degeneration of retinal pigment epithelial
(RPE) cells. In this regard, subretinal transplantation of RPE cells
to the degenerated site has attracted a great deal of attention as an
innovative therapeutics for the treatment of AMD. However, poor
viability, distribution and integration of the transplanted cells in
suspension to the narrow subretinal space have limited this approach.
Therefore, development of cell delivery devices would bring
significant benefits for the AMD treatment. Here, we developed
micropatterned nanosheets consisting of biodegradable poly(lacticco-glycolic acid) (PLGA), which can deliver RPE monolayer to the
subretinal space in a minimally invasive way.
Methods: Micropatterned nanosheets of PLGA were prepared by
combination of spincoating and microcontact printing technique.
RPE cells were cultured on the nanosheets and the cell activity on
the nanosheets was evaluated using LIVE/DEAD staining assay.
Moreover, syringe injection of the micropatterned nanosheet was
examined using swine ocular globes ex vivo.
Results: Despite the mechanical share stress during aspiration and
injection by the syringe needle, the RPE monolayer on the nanosheet
retained the original shape without any fracture, and kept the viability
over 80% regardless of the sheet diameter. On the other hand, the
micropatterned sheet with micrometric thickness hardly retained the
cells (less than 30%). Finally, a micropatterned nanosheet of 1 mm
in diameter was injected into the subretinal space of a swine ocular
globe by a 24G intravenous catheter. The nanosheet was successfully
released into the subretinal place, and fixed on the macula after
removing the prefilled saline without structural distortion.
Conclusions: We developed micropatterned nanosheets consisting of
biodegradable polymers, on which stable monolayer of the RPE cells
were engineered. Owing to the high flexibility of the nanosheets, RPE
cells were injected through the syringe needle without significant
loss of the cellular viability. The flexible micropatterned nanosheets
injectable by the syringe needle hold great promise to transplant
organized RPE cells in a minimum invasive way.
Commercial Relationships: Hirokazu Kaji, None; Toshinori
Fujie, None; Nobuhiro Nagai, None; Toshiaki Abe, None
Support: JSPS KAKENHI (23681027 and 24651156) from MEXT,
Japan
Silk Fibroin Membranes as a Carrier for the Treatment of
Corneal Lesions
Alvaro Meana1, 4, Jose L. Cenis2, Salvador D. Aznar-Cervantes2,
Manuel Chacón1, 4, Natalia Vázquez1, 4, David Cereijo3, Jose
Antonio Rodríguez-Cortés3, David Álvarez3, Jesus Merayo-Lloves1,
4 1
. Superficie Ocular, Fundacion de Investigacion Oftalmologica,
Oviedo, Spain; 2Instituto Murciano de Investigación y Desarrollo
Agrario y Alimentario, Murcia, Spain; 3Fundacion Prodintec, Gijon,
Spain; 4Universidad de Oviedo, Oviedo, Spain.
Purpose: We analyze the viability of silk fibroin (SF) membranes
as a carrier of corneal cells and their potential use as a treatment of
corneal lesions.
Methods: Cocoons of Bombyx mori were degummed (boiling in
0.02N Na2CO3) and
SF was dissolved in 9.3M LiBr and dialyzed against distilled water
into dialysis bags (3,500Da MWCO) for 3 days. Then, 580mL of the
resultant 5% w/v SF solution was cast upon plastic Petri dish 5,8cm
in diameter to give a 10 microns thickness film. Once dried at room
temperature, the water-annealing was performed by placing the SF
films in a water-filled desiccator in vacuum conditions for 24h.
Cells were obtained from normal New Zealand white rabbits and
normal human corneoscleral rims obtained during surgery after
8mm trephination of the graft. Limbal stem cells were obtained
from explants of 2-3mm in diameter of the limbal region. Stromal
and endothelial cells were obtained by digesting the stromal tissue
and the dissected endothelium with Tripsin/EDTA and Collagenase
I respectively. Cells were cultured in different culture media onto
SF membranes using a handling device designed by Prodintec
Foundation for our laboratory.
Corneal cells growing on the SF membranes were examined by
phase contrast microscopy, scanning electron microscopy (SEM) and
immunocytochemistry for antibodies: Cytokeratin High Molecular
Weight and p.63, Vimentin and ZO-1.
Limbal stem cell deficiency (LSCD) and Descemet Membrane and
Endothelial Keratoplasty (DMEK) are currently being performed in
order to test the capabilities of SF membranes in vivo.
Results: Stromal and limbal stem cells showed a high proliferation
capacity in human and rabbit primary cell cultures and were able
to attach and grow onto SF membranes while maintaining their
morphology and cellular markers (Vimentin and Cytokeratin) while
showing a highly population of p.63 positive cells in the limbal stem
cell cultures. Rabbit endothelial cells were effectively cultured on
SF membrane while maintaining a positive stain of ZO-1, however,
human endothelial cells were unable to attach and proliferate in vitro
on this type of membrane.
Conclusions: SF membranes could be improved and studied for their
use as a carrier for the treatment of corneal diseases.
Commercial Relationships: Alvaro Meana, None; Jose L. Cenis,
None; Salvador D. Aznar-Cervantes, None; Manuel Chacón,
None; Natalia Vázquez, None; David Cereijo, None; Jose Antonio
Rodríguez-Cortés, None; David Álvarez, None; Jesus MerayoLloves, None
Support: IPT-2012-1029-010000
Hybrid Gold Nanoparticles: Cellular and Molecular Toxicity to
Human Corneal Fibroblasts
Brandie Morgan1, 2, Ajay Sharma1, 2, Rachel A. Waller1, 2, Nishant
R. Sinha1, 2, Prashant R. Sinha1, 2, Audra N. Stallard1, 2, Saad
Siddiqui1, 2, Rajiv R. Mohan1, 2. 1Harry S. Truman Memorial Veteran
Hospital, University of Missouri, Columbia, MO; 2Departments
of Ophthalmology, School of Medicine and College of Veterinary
Medicine, University of Missouri, Columbia, MO.
Purpose: We showed hybrid PEI gold nanoparticles (PEI2-GNPs)
are highly potent in delivering genes into rabbit cornea in vivo
and human cornea ex vivo (Nanomedicine. 2011, 7:505-13). The
cytotoxicity profiling of PEI2-GNPs is necessary for the clinical
usefulness of this potent nanoparticle vector for developing gene
therapy for corneal diseases. The goal of the present study was to
characterize the effects of PEI2-GNPs on human corneal fibroblasts
proliferation, differentiation, migration, mitochondrial function, and
free radical stress modulation.
Methods: Human donor corneas were used to generate corneal
fibroblast (HCF) cultures. The cultures were exposed to two
identified doses of PEI2-GNPs that showed significant gene transfer
to the cornea in vivo (30-50%; p<0.001). The HCFs were exposed
to these two doses of PEI2-GNPs for 6, 12 and 24 hours. Cellular
migration was analyzed by scratch assay and HCF differentiation
to myofibroblast was determined by smooth muscle actin
immunofluorescence. Cellular proliferation, mitochondrial membrane
potential, reactive oxygen species production, superoxide dismutase
and glutathione peroxidase activity were quantified using commercial
kits. H2O2 exposed HCFs were used as a positive control.
Results: The PEI2-GNPs treatment to HCFs did not alter HCF
migration, differentiation to myofibroblasts, total reactive oxygen
species stress, mitochondrial membrane potential or cellular viability.
PEI2-GNPs showed an insignificant 6-10% decrease in HCF
proliferation and caused a moderate inhibition (7-15%, p<0.05) of
superoxide dismutase.
Conclusions: PEI2-GNPs appear safe for corneal gene therapy. In
vivo studies are warranted to validate safety and tolerability of PEI2GNPs.
Commercial Relationships: Brandie Morgan, None; Ajay
Sharma, None; Rachel A. Waller, None; Nishant R. Sinha,
None; Prashant R. Sinha, None; Audra N. Stallard, None; Saad
Siddiqui, None; Rajiv R. Mohan, None
Support: : National Eye Institute, NIH, Bethesda, Maryland, USA
RO1EY17294 (RRM) Veteran Health Affairs, Washington DC USA
1I01BX00035701 (RRM). Ruth M. Kraeuchi Missouri Endowment
Fund
Revisiting corneal storage using an innovative bioreactor
Gilles Thuret1, 2, Bernard Aurélien1, Nicolas Naigeon1, Tanguy
Nangoum-Fosso1, Zhiguo He1, Campolmi Nelly1, Acquart Sophie3,
Fabien Forest1, 4, Olivier Garraud3, Gain Philippe1. 1Corneal Graft
Biology, Engineering and Imaging Laboratory, EA2521, Federative
Institute of Research in Sciences and Health Engineering, Faculty of
Medicine, Jean Monnet University, Saint-Etienne, France; 2Institut
Universitaire de France, Paris, France; 3Eye Bank, French Blood
Center, Saint-Etienne, France; 4Pathology dpt, University Hospital,
Saint-Etienne, France.
Purpose: Be it at 4°C in sealed vials or viewing chambers, or in
organ-culture at 31°C in sealed vials, two techniques no revisited
since 70’, corneal storage is mandatorily accompanied by a
significant endothelial cell (EC) death (600x more than during
lifetime) and stromal swelling responsible for endothelial folds
triggering additional EC death. The absence of intra ocular pressure
that constitutes one of the main forces opposed to the naturally
hydrophilic stroma plays an important role in this vicious circle
that begins with donor death and is prolonged during storage. The
restoration of pressure gradient with circulation of fluids during
long-term storage may reduce stromal swelling, posterior folding and
consequently improve EC survival. Aim: to present an innovative
corneal bioreactor that restores both parameters.
Methods: Review of the literature and of patents on methods
available to achieve ex vivo restoration of the corneal physiology and
personal works of our laboratory of bioengineering with a patented
corneal bioreactor (N°1250832, U25-B-30444 FR).
Results: Several devices have been published or patented previously
but none with the aim of improving storage (generally for short-term
toxicologic studies) and none using the technical solutions that we
developed. Restoration of an increased pressure at the endothelial
side associated with a continuous renewal of storage medium
allowed rapidly reduction of stromal swelling and improvement of
EC viability, compared to the rustic immersion in a sealed flask or
viewing chambers.
Conclusions: Our innovative corneal bioreactor can be used for
research studies as well as for corneal storage in eye banks.
Commercial Relationships: Gilles Thuret, University Jean Monnet
(P); Bernard Aurélien, None; Nicolas Naigeon, None; Tanguy
Nangoum-Fosso, None; Zhiguo He, None; Campolmi Nelly, None;
Acquart Sophie, University Jean Monnet (P); Fabien Forest, None;
Olivier Garraud, None; Gain Philippe, University Jean Monnet (P)
Support: Appels a projet recherche Université Jean Monnet 2011,
2012 et 2013, Etablissement Français du Sang 2011, Agence
de la BioMédecine 2012, Agence Nationale pour la Sécurité du
Médicament 2012
A Relationship Between the Biofilm on Silicone Tube
and Outcome of Silicone Tube Intubation in Patient with
Nasolacrimal Duct Obstruction
Jae Rock Do, Sung Hyun Kim, Minwook Chang. Ophthalmology,
dongguk university, Goyang, Republic of Korea.
Purpose: The aim of this study was to investigate the correlation
between the optical density of biofilm on silicone tube and
postoperative clinical results.
Methods: The study was prospectively reviewed patients who
underwent silicone tube intubation. The silicone tubes removed
at post-operative 6 months and surgical success was evaluated at
post-operative 12 months. The removed silicone tube from the
nasal cavity after cutting the loop between the upper and lower
puncta were divided 2 piece. One piece was cultured, and the other
piece was measured optical density of biofilm. Each piece divided
3-piece according to average normal nasolacrimal anatomy, part
1 piece(segment of upper 20mm) was located in from puncta to
lacrimal sac, part 2 piece(segment of next 20mm) in nasolacrimal
duct, part 3 piece(other remain segment) in nasal cavity. Part 3 pieces
were excluded in this study. The relationship between the biofilm on
each part of silicone tube, isolation of culture and clinical outcome of
silicone tube intubation was evaluated.
Results: Forty one silicone tubes removed from 31 patients were
enrolled. Thirty cases were surgical success at post-operative
12 months(73.2%), and 11 cases(26.8%) were surgical failure
with persistent epiphora. Bacteria were isolated in 38 silicone
tubes(92.7%). Pseudomonas aeruginosa was isolated most
frequently(10 cases) in part 1 piece, in case of part 2 piece,
Pseudomonas aeruginosa and Staphylococcus aureus(MSSA) were
isolated most frequently(10 cases). Surgical failure was related
with isolation of pseudomonas aeruginosa(fisher’s exact test,
P=0.041). Optical density in part 1 piece showed no statistically
difference between cases of surgical success and failure group(MannWhitney U test, P=0.919). But optical density in part 2 of failure
group(0.41±0.228nm) showed increased optical density statistically,
compared with that of success group(0.25±0.190nm)(Mann-Whitney
U test, P = 0.006).
Conclusions: Optical density of biofilm on silicone tube at
nasolacrimal duct may have effect on clinical outcome of silicone
tube intubation. And isolation of Pseudomonas aeruginosa may
correlation with surgical failure.
Commercial Relationships: Jae Rock Do, None; Sung Hyun Kim,
None; Minwook Chang, None
Development and characterization of nanoplex topical ocular
delivery of moxifloxacin to treat bacterial keratitis
Jayabalan Nirmal1, Gaurav K. Jain2, Vaidehi Garg2, Musarrat
H. Warsi2, Farhan J. Ahmad2, Roop K. Khar3. 1Department of
Pharmaceutical Sciences, Dr. H.S Gour University, Sagar, India;
2
Department of Pharmaceutics, Jamia Hamdard University, New
Delhi, India; 3B. S. Anangpuria Institute of Pharmacy, Faridabad,
India.
Purpose: Moxifloxacin (Mox) eye drops to treat bacterial keratitis
require more frequency of administration due to less corneal
residence time and poor transcorneal penetration. In the present study
developed and evaluated the Polylactic-co-glycolic Acid (PLGA)chitosan Nanoplex system as a carrier for ocular delivery of Mox
to improve ocular penetration and bioavailability by prolonging the
ocular residence time.
Methods: PLGA-chitosan Nanoplexes were prepared by formation
of complex between PLGA and chitosan using 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide as chemical crosslinker.
Double emulsification solvent evaporation method followed by
homogenization of primary emulsion was used to produce Mox
loaded PLGA-chitosan Nanoplexes. Nanoplexes were evaluated
for drug content, particle size (PS), zeta potential (ZP) and invitro
release. Male albino New Zealand rabbits were used as invivo model
for the studies.
Results: Formulation with 0.22% chitosan and pH 5.8 was optimized
using response surface methodology and has PS of 120.3 nm, ZP
of +29.99 mV, encapsulation efficiency of 63.4% and burst release
of 25.7%. Interaction between PLGA and chitosan was confirmed
by Fourier Transform Infrared and Nuclear Magnetic Resonance
spectroscopy. The Near Infrared chemical imaging revealed Nanoplex
was a matrix-type system. Transmission Electron Microscopy,
Scanning Electron Microscopy and Atomic Force Microscopy images
demonstrated nanoplexes were round and mono dispersed. Ability
of Nanoplexes to retain over cornea was demonstrated by gamma
scintigraphy. Ex vivo evaluation using developed OcuFlow apparatus
show sustained release of Mox from the Nanoplex. Five-fold higher
ocular bioavailability was observed for Mox nanoplexes compared
to Mox solution. Histopathology studies show nanoplexes caused no
epithelial damage. Pharmacodynamic studies to induce keratitis using
Staphylococcus aureus in rabbit eyes show significantly less clinical
scores with Mox Nanoplexes as compare to Mox solution treated
eyes.
Conclusions: Mox nanoplex is capable of significantly extending
the corneal residence time and increase transcorneal penetration.
Physico-chemical characteristics, together with in vitro and in vivo
studies show PLGA-chitosan Nanoplexes were efficient carriers
for delivery of Mox to treat bacterial keratitis with improved ocular
bioavailability.
Commercial Relationships: Jayabalan Nirmal, None; Gaurav
K. Jain, None; Vaidehi Garg, None; Musarrat H. Warsi, None;
Farhan J. Ahmad, None; Roop K. Khar, None
The feasibility of vibration motors for a tactile display for the
blind
H Christiaan Stronks1, 2, Paulette Lieby3, Daniel Parker1, Janine
Walker1, 4, Nick Barnes1, 3. 1Computer Vision Research Group, NICTA,
Canberra, ACT, Australia; 2Department of Neuroscience, John
Curtin School of Medical Research, Australian National University,
Canberra, ACT, Australia; 3College of Engineering and Computer
Science, Australian National University, Canberra, ACT, Australia;
4
Centre for Mental Health Research, Australian National University,
Canberra, ACT, Australia.
Purpose: We are developing a low-vision aid based on visual-totactile sensory substitution. In this study, we have determined the
feasibility of vibration motors for use in a tactile display. A suitable
motor should generate multiple discernible intensity levels in order to
convey contrast information to the user.
Methods: Perceptual detection threshold and just-noticeabledifference (JND) for three different vibration motors (Precision
Microdrives™, Ltd) were determined on the skin of the back of 7
healthy, normally-sighted subjects (3 females, 4 males; age range: 2456). The motors differed in size (diameter 8, 10, and 12 mm), which
in turn affected multiple parameters, including output energy (more
for a larger motor) and acceleration time (longer for a larger motor).
Vibrational stimuli were delivered in single 200-ms bursts. Perceptual
threshold and JND, expressed as percentage of maximum duty cycle
(%DC), were determined using adaptive methods based on a staircase
procedure. The total number of available JNDs was estimated using
Weber’s law. Overall effects of motor type on threshold and JND
were tested by repeated measures ANOVA; differences between
motors were tested for significance by Tukey’s multiple comparisons
post hoc test.
Results: The number of available JNDs was significantly different
between motors (P< 0.05). The largest motor had the largest number
of JNDs (mean: 19), followed by the middle (12), and the smallest
motor (9). The number of JNDs was as high as 34 in one subject for
the largest motor. Post hoc multiple comparisons showed a significant
difference between the smallest and largest motor (P< 0.05).
Perceptual thresholds differed markedly between motors (P< 0.001).
The largest motor had a significantly lower threshold (15 %DC) than
the middle motor (23 %DC), which in turn had a lower threshold than
the smallest (26 %DC) (P< 0.01). In contrast, the Weber constant did
not differ significantly between motors (P= 0.1).
Conclusions: We conclude that vibration motors are able to represent
multiple intensity levels, making them suitable for use in a tactile
display. The estimated average number of available JNDs increased
with motor size, and one subject had nearly three dozen available
JNDs when using the largest motor. The most important factor
determining the total number of available JNDs was the detection
threshold.
Commercial Relationships: H Christiaan Stronks, None; Paulette
Lieby, None; Daniel Parker, None; Janine Walker, None; Nick
Barnes, None
Photoreceptor regeneration in transgenic mice
Shu-Zhen Wang, Run-Tao Yan. Ophthalmology, Univ of Alabama at
Birmingham, Birmingham, AL.
Purpose: Photoreceptor regeneration in the mammalian eye remains
an elusive goal. Our laboratory is investigating the possibility of
tweaking the RPE, a tissue with naturally occurring wound healing
ability, for photoreceptor regeneration in situ in the eye. The
underlying theme is to use a regulatory gene with pro-photoreceptor
activity to reprogram RPE cells to initiate photoreceptor
differentiation, so as to channel RPE’s well-known capacities of
proliferation and plasticity towards photoreceptor production inside
the mammalian eye. Previous study showed that transgenic mice
(generated with DNA that would express ngn1 or ngn3 in RPE cells
driven by PRPE65 or PVMD2) contained photoreceptor-like cells in
the subretinal space. This study examines whether new photoreceptor
cells could be born in adult transgenic mice.
Methods: Adult transgenic mice (Tg) and Tg/rd1 mice (generated
by crossing Tg mice with rd1/rd1) received daily intraperitoneal
injection of BrdU for 2 weeks. Eyes were then fixed and analyzed
with double-immunohistochemistry for BrdU incorporation and
photoreceptor protein recoverin.
Results: BrdU+/recoverin+ cells were detected in transgenic mice
aging between 7 weeks and 1 year. Most of the double-labeled cells
localized within the outer nuclear layer of the periphery retina.
Occasionally, double-labeled cells were found to be attached to the
RPE or containing dark pigment granules typically present in RPE
cells, reminiscent of being in a RPE-to-photoreceptor transitional
stage. BrdU+/recoverin+ cells were also detected in 7 week-old and 5
month-old Tg/rd1 mice.
Conclusions: The presence of BrdU+/recoverin+ cells suggests that
new photoreceptor cells could be produced in adult transgenic mice,
conventional and Tg/rd1.
Commercial Relationships: Shu-Zhen Wang, None; Run-Tao Yan,
None
Support: NIH/NEI grant EY011640, EyeSight Foundation of
Alabama Research Grant FY2011-12-276, Research to Prevent
Blindness, and NIH/NEI core grant P30 EY003039
ERG changes after suprachoroideal graft of autologous cells in
AMD
Paolo G. Limoli1, Enzo M. Vingolo2, Sergio Zaccaria Scalinci3,
Marco U. Morales4, Celeste Limoli1. 1Ophthalmology, Centro Studi
Ipovisione, Milan, Italy; 2Ophthalmology, Ospedale S. Maria Goretti,
Latina, Italy; 3Ophthalmology, Università di Bologna, Bologna, Italy;
4
Optical and Visual Sciences, Nottingham University, Nottingham,
United Kingdom.
Purpose: Dry AMD has not a standard therapy yet.Since the 90’s,
however, a series of studies about growth factors’s (GF) use designed
to limit the retinal atrophy advance has begun.
One of the suitable GF autologous sources is represented by adipose
tissue.Other useful autologous sources are: platelets, derivable from
Platelet Rich Plasma (PRP) and adipose derived stem-cells (ADSCs),
included in tissue adipose’s stromal vascular fractions (SVF).
They, surgically grafted into suprachoroidal space, might delay
retinal cell apoptosis through an almost pharmacological modulation
of their secretions.The aim of the work is to demonstrate, by ERG’s
objectivity, an achievable therapeutic effect of the suprachoroidal
graft of adipocytes, together ADSCs in SVF and PRP, able to
generate growth factors.This technique is defined for convenience
LMPT (Limoli modified Pelaez Technique).
Methods: We included retrospectively in the research 7 patients
(12 eyes) suffering from dry AMD (medium age: 71,25 Range
62-80 years). Diagnosis has been verified for each patient by SDOCT (Zeiss Cirrus) and microperimetry (Maia or MP1).Before the
graft (t0), BCVA (Logmar) and close up (pts), maximal, scotopic
and photopic ERG (μV), according to standard ISCEV, have been
considered.Each eye has got an autologous cell graft through LMPT.
One month later (t30), BCVA for distant and near vision, maximal,
scotopic, photopic ERG have been reevaluated.
Results: Functional changes following LMPT are:
Scotopic ERG: from 41,26 to 60,83 μV (47,44% P<0,0005)
Maximal ERG: from 112,22 to 129,68 μV (15,56% P<0,005)
Photopic ERG: from 41,61 to 47,03 μV (13% P<0,05)
BCVA: 0,78 logMAR (non-significant variation P>0,5)
Close-up visus: from 29,5 to 28,42 pts (3,67% P>0,5).
Conclusions: During dry AMD foveal areas become atrophic with a
big reduction of cones; extrafoveal areas, generally better preserved,
keep a big number of rods.The decreased cones’s presence may
clarify the non-significant increase of photopic ERG. The preserved
number of rods may elucidate the significant increase of scotopic
ERG. LMPT seems to have positive effects on ERG data increase,
probably due to paracrine secretions properties of grafted autologous
cells, some of which may be GF.In conclusion, ERG might be
employed for monitoring trophic cell-teraphies’s effect objectively.
LMPT seems to have positive effects on ERG data increase, probably
due to GF secreted from grafted autologous cells.
Commercial Relationships: Paolo G. Limoli, None; Enzo M.
Vingolo, None; Sergio Zaccaria Scalinci, None; Marco U.
Morales, None; Celeste Limoli, None

ARVO 2014 Annual Meeting Abstracts 220 Nanotherapy

Transcription

Similar documents

Practice Brochure - Arizona Retina Institute

Bionic Eyes - Hansma Lab

Retinal Toxicity - Healthcare Professionals

Part 1d

Madagascan tomato frog

A New Treatment for Chronic Central Serous Retinopathy

Retinal Manifestations in Familial Lipoprotein Lipase Deficiency

Bull`s Eye Maculopathy - SUNY Downstate Medical Center

LECTURE 9 Posterior Pole retina pathology_HTN_DM retinopathy

Things about the Eye that every Internist should know COLD!!!