ARVO 2016 Annual Meeting Abstracts
347 Retinal Prosthesis and Stem Cells
Tuesday, May 03, 2016 11:00 AM–12:45 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 3713–3742/D0174–D0203
Organizing Section: Retina
Program Number: 3713 Poster Board Number: D0174
Presentation Time: 11:00 AM–12:45 PM
Evaluation of electrical stimulation of the retina with optical
coherence tomography
Alejandra Gonzalez Calle, James D. Weiland. Biomedical
Engineering, University of Southern California, Los Angeles, CA.
Purpose: Retinal prostheses have demonstrated the capability to
elicit the sensation of light and to give test subjects the ability to
detect motion and the location of large objects. To create more
complex shape perceptions, each electrode should be perceived as a
small, focal spot, which when combined together can form a complex
shape. Long pulse stimulation can be used to achieve focal percepts
in patients with RD (Retinal Degeneration) diseases; however, safety
is a concern because long pulses require more charge than short
pulses to elicit a response from neural tissue. To assess safety of
stimulation, high charge density and long-pulse duration stimuli were
applied to rabbit retina and OCT images acquired before, during, and
after stimulation.
Methods: Pigmented rabbits (N=7) were anesthetized and OCT
images were taken as control before stimulation. Left eye was used
in every rabbit. A return electrode was placed on the rabbit’s head
and the stimulating electrode (Platinum/Iridium needle electrode)
was placed inside the eye 56 26 away from the retina. Different
pulse widths, pulse amplitudes, frequencies, stimulation time and
size electrodes were used during these experiments (Table 1). OCT
images were acquired every 2 min during and after stimulation. At
the end of the experiment FA images were taken to evaluate any
vessel leakage, then the eye was enucleated and taken for histology
evaluation.
Results: Retinal thickness was measured to assess retinal damage. 7
animals were used during the experiments and different parameters
points were evaluated using the same animal. When using 250 in
diameter electrode and stimulating for 30 min (Frequency: 333 Hz,
Charge Density: 1.63 mC/cm2) we could see an increase in retinal
thickness with an initial value of 160.3 2.05 and a final value of 190
0.81 showing an increase on retinal thickness of 19.52 % 1.86%
compared to the baseline. A paired t-test revealed a statistically
significant increase in thickness (p=0.004). This damage expanded
laterally for 1.27 mm 0.19 mm. For the other parameters evaluated,
there was not increase of retinal thickness after stimulation.
Conclusions: Preliminary results show that electrical stimulation at
very high charge densities cause swelling of the retina within minutes
of stimulation. The stimulus level at which swelling was noted is
significantly higher than that used for stimulation in humans.
Commercial Relationships: Alejandra Gonzalez Calle;
James D. Weiland, None
Support: NH Grant EY022931 and Research to Prevent Blindness
Program Number: 3714 Poster Board Number: D0175
Presentation Time: 11:00 AM–12:45 PM
Focal electrical stimulation of the retina
Andrew C. Weitz1, 2, Devyani Nanduri2, Robert J. Greenberg3,
Mark S. Humayun1, 2, Robert H. Chow4, 2, James D. Weiland1, 2.
1
Ophthalmology, University of Southern California, Los Angeles,
CA; 2Biomedical Engineering, University of Southern California,
Los Angeles, CA; 3Second Sight Medical Products, Inc., Sylmar, CA;
4
Physiology & Biophysics, University of Southern California, Los
Angeles, CA.
Purpose: Epiretinal implants have restored partial vision to the blind
by electrically stimulating surviving retinal neurons. Unintended
stimulation of retinal ganglion cell (RGC) axons causes patients to
see large, elongated visual phosphenes, making it difficult for them
to perceive forms. This study tested whether alternative electrical
stimulus waveforms could produce focal retinal activation.
Methods: The GCaMP5G calcium indicator was virally expressed
in RGCs of wild-type (Long Evans) and retinal degenerate (S334terline-3) rats. Transduced retinas were isolated and mounted RGCside-down on a multielectrode array. Calcium imaging was used to
map the patterns of cells activated by different electrical stimulation
protocols. Electrode diameter (30–200 µm) and pulse duration
(0.06–100 ms/phase) were varied. Synaptic blockers were used in
some experiments to pharmacologically isolate RGCs. The effect of
pulse width on phosphene shape was tested in an epiretinal implant
(Argus I) subject, blind with retinitis pigmentosa.
Results: In rats, pulse durations 16 ms/phase and shorter stimulated
axons, activating a streak of RGCs that extended to the retina’s edge.
Pulses 25 ms/phase and longer stimulated inner retinal neurons
while avoiding RGC axons, producing focal responses. Threshold
charge density became higher as pulse width increased (e.g., for
200-µm electrodes, stimulus thresholds were 1.8 and 22.4 µC/cm2
for 0.1- and 25-ms/phase pulses, respectively). Response shapes in
wild-type and degenerate retinas were similar, but stimulus thresholds
of inner retinal cells were elevated by 3× in retinal degenerate rats.
Multielectrode stimulation with 25-ms/phase pulses was used to
pattern letters on the retina. The letter V, for example, was generated
with seven 30-µm electrodes (see figure). The height of the V on the
retina corresponds to 0.78° of human visual angle and is equivalent
to viewing 5-mm-tall text from typical reading distance (40 cm).
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ARVO 2016 Annual Meeting Abstracts
Psychophysical testing in an epiretinal implant patient demonstrated
that short pulses (0.46 ms/phase) produce elongated phosphenes,
while long pulses (25 ms/phase) evoke focal spots of light.
Conclusions: Our results demonstrate that stimulus pulse durations
two orders of magnitude longer than those typically used in existing
retinal implants avoid RGC axons and activate the retina focally. If
validated through further human testing, these findings may lead to
improved visual acuity for epiretinal implant users.
Commercial Relationships: Andrew C. Weitz, None;
Devyani Nanduri, None; Robert J. Greenberg, Second Sight
Medical Products, Inc.; Mark S. Humayun, Second Sight Medical
Products, Inc. (P), Second Sight Medical Products, Inc. (I),
Second Sight Medical Products, Inc. (C); Robert H. Chow, None;
James D. Weiland, None
Support: NSF Grant EEC-0310723, NIH Grant 1R01EY022931,
NIH Grant R01 GM85791, Research to Prevent Blindness
Clinical Trial: NCT00279500
Program Number: 3715 Poster Board Number: D0176
Presentation Time: 11:00 AM–12:45 PM
Effect of stimulus pulse on evoked potential amplitude in
the superior colliculus of rats by electrical stimulation with
Suprachoroidal-Transretinal Stimulation (STS) retinal prosthesis
Yukari Nakano1, Yasuo Terasawa1, 2, Hiroyuki Kanda3, Koji Osawa1,
Motoki Ozawa4, Tomomitsu Miyoshi5, Hajime Sawai6. 1Research and
Development Division, Nidek Co., Ltd., Gamagori, Japan; 2Graduate
School of Materials Science, Nara Institute of Science & Technology,
Ikoma, Japan; 3Department of Applied Visual Science, Osaka
University Graduate School of Medicine, Suita, Japan; 4Nidek Co.,
Ltd., Gamagori, Japan; 5Department of Integrative Physiology, Osaka
University Graduate School of Medicine, Suita, Japan; 6Department
of Health Sciences, School of Nursing, Osaka Prefecture University,
Habikino, Japan.
Purpose: Electrical stimulation with rectangular waveform is
generally used for retinal prosthesis irrespective of the place of
electrode implantation, but to find out more effective electrical
stimulation parameter is one of the important elements for the
development of retinal prosthesis.
In order to find out more effective electrical stimulation parameter,
in this study, we examined the effect of waveforms on evoked
potential by electrical stimulation with Suprachoroidal-Transretinal
Stimulation (STS).
Methods: Platinum stimulation electrode (D: 0.5 mm, H: 0.3 mm)
was set onto the region of partial scleral resection the eyeball of
normal rats (Long-Evans, N=10) and retinal degenerated rats (Royal
College of Surgeons; RCS, N=10) under anesthesia.
Current pulses (inward current-first) were applied between the
stimulation electrode and the return electrode (platiniridium wire, D:
0.2 mm) that was placed inside the eye.
Three types of waveforms (rectangular, triangular and sinusoidal
wave) were used as current pulse.
The pulse duration of the three waveforms was fixed at 0.5 ms
in order for them to have the same quantity of electric charge by
adjusting the amplitude of electric current.
Evoked potential by electrical stimulation was recorded with a needle
electrode (tungsten: 0.5 MΩ) in the superior colliculus.
Results: Distinct responses were observed at approximately 5.0 ms
(P1) and 10 ms (N1) after stimulation in all the rats irrespective of
waveforms.
In both normal and RCS rats, evoked potential amplitude had
significant differences between rectangular and triangular, rectangular
and sinusoidal wave (p<0.01, paired t-test).
Rectangular wave elicited the lowest response.
The evoked potential amplitudes of triangular and sinusoidal wave
were an average of 1.5 and 1.9 times, respectively, as high as that of
rectangular wave.
Conclusions: Sinusoidal wave output was the highest in both normal
and RCS rats among rectangular, triangular and sinusoidal wave, and
rectangular wave output was the lowest (Fig.1).
This result corresponded to previous studies in vitro [Goo, Yong Sook
et. al., TJSMBE Vol. 51, 2013].
This suggested that electrical stimulation could be applied with a
smaller quantity of electric charge by using sinusoidal wave output.
Fig1. Evoked potential peak-to-peak amplitudes. RCS (N=10). Error
bars: SD.
Commercial Relationships: Yukari Nakano, NIDEK Co.,Ltd.;
Yasuo Terasawa, NIDEK Co.,Ltd.; Hiroyuki Kanda, NIDEK
Co.,Ltd. (F); Koji Osawa, NIDEK Co.,Ltd.; Motoki Ozawa, NIDEK
Co.,Ltd.; Tomomitsu Miyoshi, NIDEK Co.,Ltd. (F); Hajime Sawai,
NIDEK Co.,Ltd. (C)
Support: NIDEK Co., Ltd.
Program Number: 3716 Poster Board Number: D0177
Presentation Time: 11:00 AM–12:45 PM
Development of an alpha retinal ganglion cell model for epiretinal
electrode stimulation
Ethan D. Cohen1, Esra Neufeld2, Hazael Montanaro2,
Maria I. Iacono1, Leonardo M. Angelone1, Wolfgang Kainz1. 1Div.
Biomedical Physics, Office of Sci. and Eng. Labs, Ctr. for Dev. &
Rad. Health, FDA, Silver Spring, MD; 2Computational Life Sciences,
IT’IS Research Foundation, Zurich, Switzerland.
Purpose: Using whole-cell recording and an epiretinal stimulus
electrode similar in size to clinical electrodes, we investigated how
the action potential thresholds of OFF-alpha retinal ganglion cells
varied with stimulus electrode distance from the cell body, to develop
a NEURON computational model of the alpha ganglion cell.
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ARVO 2016 Annual Meeting Abstracts
Methods: Under anesthesia, the retina from rabbits was isolated
and perfused in Ames Ringer. Large ganglion cells were selected for
whole-cell recording using dim red or IR Nomarski illumination.
A small hole was made in the inner limiting membrane to allow
pipette access. The frequency vs. current firing properties of the
ganglion cell were studied using 100pA current steps. Next, a thin
insulated 100µm diameter Pt stimulus electrode was positioned at
a 30 degree angle near the axon hillock/soma approximately 50µm
above the retina. A series of 0.5msec ascending cathodic, followed
by anodic current pulses (20msec apart) were used to examine how
firing threshold varied laterally with soma proximity in 100µm steps.
After recording, the morphology of the dye-filled ganglion cell was
imaged and processed with anti-lucifer yellow antibodies for confocal
microscopic reconstruction. The morphology of selected cells were
entered into Neurolucida for segmentation, fitted with ion channels
in NEURON, and imported into a novel electromagnetic-neuronal
dynamics modeling program to simulate extracellular stimulation.
Results: When current steps were injected into the alpha cell
body, a large pipette current was needed to elicit action potential
threshold averaging 500±170pA (n=5 cells; mean ± std. dev). Some
spike adaptation to firing was observed. Using epiretinal stimulus
electrodes, action potentials were only generated by cathodic current
pulses. When the electrode was placed adjacent to the cell body at the
axon hillock region, thresholds for activation averaged 10.8±11.3nC
(n= 5 cells; mean ± std. dev.). The spatial threshold for extracellular
stimulation of the ganglion cells by the Pt electrode was localized to
a region ~100µm around the initial segment/axon hillock of the cell
body.
Conclusions: Large thinly insulated epiretinal stimulus electrodes
activate action potentials in alpha ganglion cells in a spatially
localized manner near the initial segment/soma region of the retinal
ganglion cell. Synaptic activity may alter spike threshold.
Commercial Relationships: Ethan D. Cohen, None;
Esra Neufeld; Hazael Montanaro, None; Maria I. Iacono, None;
Leonardo M. Angelone, None; Wolfgang Kainz, None
Program Number: 3717 Poster Board Number: D0178
Presentation Time: 11:00 AM–12:45 PM
Electric stimulus duration alters network-mediated responses
depending on retinal ganglion cell type
Maesoon Im1, 2, Shelley I. Fried2, 3. 1Ophthalmology, Henry Ford
Hospital, Northville, MI; 2Boston VA Healthcare System, Boston,
MA; 3Neurosurgery, Massachusetts General Hospital/HMS, Boston,
MA.
Purpose: Despite some recent progress with retinal prosthetics the
optimal duration for the electric stimulus remains unknown. Recently,
we reported that an identical electric pulse elicits distinct responses
in ON and OFF types of retinal ganglion cells (RGCs). The temporal
properties to repetitive stimuli were also different in the two types.
Thus, we hypothesize that varying the duration of the stimulus
would alter the responses in different ways across different types of
RGCs, thus raising a possibility to selectively activate one type over
the other. Here, we systematically investigated network-mediated
responses in various types of RGCs as a function of stimulus
duration.
Methods: Cell-attached patch clamp was used to record spikes from
RGCs in the rabbit retina explant. RGCs were classified as ON or
OFF cells by their response to stationary flashes and further classified
as Brisk Transient (BT) or Brisk Sustained (BS) subtypes by their
electric responses. After cell type classification, monophasic halfsinusoidal stimuli with durations of 5-100ms and amplitudes adjusted
to keep total charge constant were presented to targeted RGCs. Each
stimulus was repeated 7 times. We recorded the spiking activity in 7
ON BT, 10 ON BS, 8 OFF BT, and 14 OFF BS cells.
Results: The pattern of network-mediated responses was unique for
each type of RGC across a wide range of stimulus durations. We
found that: 1) In ON cells, there was a distinct difference between
BT and BS subtypes in number of spikes elicited by same stimulus
duration but the distinction was less clear across OFF subtypes. 2)
Across the durations tested, both types of ON cells showed dramatic
changes in the number of evoked spikes while OFF cells showed
smaller changes in their responses. 3) This disparity between ON and
OFF cells resulted in the ratio of spikes elicited by the two types to be
maximized at a stimulus duration of ~10 ms.
Conclusions: ON and OFF types of RGCs exhibited fundamental
differences in responses to a wide range of durations of the electric
stimulus, resulting in variable levels of ON vs. OFF selectivity at
different durations. Together with our previous report that an electric
stimulus evokes more physiological responses from ON cells than
OFF cells, the finding here of a stimulus duration that maximizes the
ON/OFF response ratio may offer enhanced clinical guidelines to
retinal prosthetic community.
Commercial Relationships: Maesoon Im; Shelley I. Fried, None
Support: Boston VA Healthcare System (1I01RX000350-01A1) and
NIH (R01EY023651)
Program Number: 3718 Poster Board Number: D0179
Presentation Time: 11:00 AM–12:45 PM
Relative power consumption at the electrode-retina interface
during retinal stimulation with voltage versus current controlled
stimulus pulses
Kiran Nimmagadda2, 3, Navya Davuluri4, James D. Weiland1, 4.
1
Ophthalmology, USC, Los Angeles, CA; 2Neuroscience Graduate
Program, University of Southern California, Los Angeles, CA;
3
USC - Caltech MD/PhD Program, Los Angeles, CA; 4Biomedical
Engineering, University of Southern California, Los Angeles, CA.
Purpose: The purpose of this study is to compare power consumed at
the electrode-retina interface between rectangular current controlled
and voltage controlled stimulus pulses.
Methods: Eleven Long Evans female rats under anesthesia were used
for this in-vivo study. A 75 um diameter cylindrical Pt-Ir electrode
was inserted into the left eye, and placed 50-100 um from the retina.
Charge balanced biphasic voltage-controlled and current-controlled
stimulus pulses of varying pulse width and amplitude were delivered
to the retina. The pulse widths were 0.3 ms, 0.5 ms, 1 ms, and 2
ms. For each pulse width, voltage and current-controlled pulse
trains with charge levels from 10 nC to 60 nC were delivered to the
retina. An oscilloscope was used to measure and record the voltage
waveform of the stimulus pulses at the electrode-retina interface. A
sense resistor in the stimulus current path was used to measure and
record the current waveform delivered to the retina. As previously
reported, electrically evoked responses (EERs) were recorded from
the superior colliculus (SC) in response to the retinal stimulation.
Results: The voltage and current waveforms delivered to the retina
were used to measure the power consumed at the electrode-retina
interface during the cathodic phase for each stimulus condition. For
stimulus pulse widths of 0.3 ms, 0.5 ms, and 1 ms, there was no
statistically significant difference between the power consumed by
the voltage-controlled pulses versus the current-controlled pulses
(student t-test, p > 0.05) at all charge levels tested. For stimulus pulse
width of 2 ms, there was no statistically significant difference in
the power consumed for stimulus charge levels of 20 nC and 30 nC
(student t-test, p > 0.05), while voltage pulses consumed significantly
more power than current pulses for charge levels of 40 nC and 50 nC
(student t-test, p < 0.05).
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
Conclusions: In general, there is no significant difference in the
power consumed at the electrode-retina interface between voltage
and current controlled stimulus pulses, except for long pulse widths
like 2ms and charge levels higher than 40 nC. This needs to be taken
into consideration in combination with our previously reported results
comparing the EERs in response to voltage versus current stimulus
pulses when determining the most efficient stimulus parameters for
electronic retinal prostheses.
Commercial Relationships: Kiran Nimmagadda; Navya Davuluri,
None; James D. Weiland, None
Support: NSF CBET-1343193 and Research to Prevent Blindness
Program Number: 3719 Poster Board Number: D0180
Presentation Time: 11:00 AM–12:45 PM
Surgical feasibility of wide-field dual-array suprachoroidal–
transretinal stimulation (STS) prosthesis in middle-sized animals
Takeshi Morimoto2, Hiroyuki Kanda2, Tomomitsu Miyoshi3,
Takao Endo1, Tibor K. Lohmann4, 2, Kohji Nishida1,
Takashi Fujikado2. 1Ophthalmology, Osaka Univ Graduate Sch
of Med, Suita, Japan; 2Applied Visual Science, Osaka University
Graduate School of Medicine, Suita, Japan; 3Integrative Physiology,
Osaka University Graduate School of Medicine, Suita, Japan;
4
Ophthatmology, Aachen RWTH Aachen University, Aachen,
Germany.
Purpose: To investigate the feasibility of implanting a newly
developed wide-field dual-array suprachoroidal–transretinal
stimulation (STS) prosthesis in healthy dogs and cats.
Methods: Three healthy dogs and three healthy cats were used in this
study. The STS dual array consisted of two arrays with 25 electrodes.
The arrays were implanted into a scleral pocket of each of three
healthy beagle dogs and three healthy cats under general anesthesia.
Color fundus photography and Optical coherence tomography (OCT)
were performed postoperatively. The animals were euthanatized after
the experimental period and the retinas were evaluated histologically.
Results: All the prostheses were successfully implanted without
complications, and no serious complications occurred during
the experimental period. The fixation of the implant was stable
throughout the experimental period. Fundus photographs and OCT
revealed no serious damage in the retina and choroid around the
arrays.
Histologic evaluations showed good preservation of the retina over
the electrode array.
Conclusions: Implantation of a newly developed wide-field dualarray STS retinal prosthesis into a scleral pocket of animals is
surgically feasible and can be performed without significant damage
to the retina or the animal. These findings indicate that it might be
possible to implant more STS electrode arrays to cover a larger area
of the retina to activate a larger visual field.
Commercial Relationships: Takeshi Morimoto, None;
Hiroyuki Kanda, None; Tomomitsu Miyoshi; Takao Endo,
None; Tibor K. Lohmann, None; Kohji Nishida, None;
Takashi Fujikado, None
Program Number: 3720 Poster Board Number: D0181
Presentation Time: 11:00 AM–12:45 PM
Evaluation of the spatial resolution of electrode arrays for
suprachoroidal retinal prosthesis by recording single-unit
activities in the lateral geniculate nucleus
Hiroyuki Kanda1, Tomomitsu Miyoshi2, Takeshi Morimoto1,
Takashi Fujikado1. 1Applied Visual Science, Osaka University
Graduate School of Medicine, Suita, Japan; 2Integrative Physiology,
Osaka University Graduate School of Medicine, Suita, Japan.
Purpose: To evaluate the spatial resolution of an electrode array for
retinal prosthesis by suprachoroidal–transretinal stimulation (STS),
we recorded the single-unit activities of relay cells in the lateral
geniculate nucleus (LGN).
Methods: Implantation surgeries were performed in cats (n = 4)
under general anesthesia. The electrode array was chronically
implanted into the scleral pocket of eyes. The electrode array had the
same specifications as that used in the 2nd generation device of STS
retinal prosthesis. This electrode array comprised 49 bullet-shaped
electrodes that were 0.5 mm in diameter and 0.3 mm in height. The
center-to-center distance of the electrodes was 0.75 mm. Under
general anesthesia, acute experiments for the evaluation of spatial
resolution of the electrode array were performed in 2–4 weeks after
the implantation surgeries. The electrode position was identified by
optical coherence tomography. Charge-balanced biphasic pulses were
applied (pulse duration, 0.5 mm; current intensity, 0.1–1.0 mA) to
the retina via each electrode independently. Stimulating trials were
repeated 10 or 40 times, and response probabilities of single-unit
activities in LGN relay neurons were analyzed. Their receptive fields
were identified by visual stimulation, and the relationship between
response probabilities and distances from the center of the receptive
fields to the stimulating sites were evaluated.
Results: The response probabilities decreased as the distance
between the stimulating site and center of its receptive field
increased. With 0.5 mA of stimulus intensity, the responsive area
exhibiting a response probability of more than 50% was identified
within a range of 1 mm from the stimulating site.
Conclusions: These findings demonstrate that this electrode array
is able to achieve localized stimulation. Moreover, spatial resolution
by the STS approach appears to be acceptable, although the distance
between the electrode and retina in this approach is larger than that in
epi- or sub-retinal approaches.
Commercial Relationships: Hiroyuki Kanda, NIDEK (P);
Tomomitsu Miyoshi, None; Takeshi Morimoto, None;
Takashi Fujikado, NIDEK (P)
Support: coordination, support and training program for
transnational research, MEXT, Japan
Program Number: 3721 Poster Board Number: D0182
Presentation Time: 11:00 AM–12:45 PM
Spatio-temporal characteristics of retinal responses to subretinal
photovoltaic stimulation
Richard Smith1, Elton Ho2, Georges A. Goetz2, 3, Xin Lei3,
Theodore Kamins3, Jim Harris3, Keith Mathieson4,
Daniel V. Palanker2, 5, Alexander Sher1. 1Santa Cruz Institute
for Particle Physics, UC Santa Cruz, Santa Cruz, CA; 2Hansen
Experimental Physics Lab, Stanford, Palo Alto, CA; 3Electrical
Engineering, Stanford, Palo Alto, CA; 4Institute of Photonics,
University of Strathclyde, Glasgow, United Kingdom;
5
Ophthalmology, Stanford, Palo Alto, CA.
Purpose: To measure the spatio-temporal receptive fields of the
retinal ganglion cells activated via subretinal photovoltaic prostheses
in degenerate and wild-type rat retinas, and compare them to the
natural light responses in healthy retinas.
Methods: Activity of the RGCs was measured in-vitro with a 512
channel multielectrode array. Photovoltaic array with 70µm pixels
was placed on the photoreceptor side of healthy and degenerate
(RCS) rat retinas. Prosthetic stimulation was performed with 880nm
light pulsed at 20Hz and modulated by an LCD displaying binary
white noise movies at frame rates of 10Hz and 20Hz. For stimulation
of the healthy retina, the same LCD was used to display white noise
movies at 30Hz frame rate with continuous visible light illumination.
Spatio-temporal receptive fields of the RGCs were measured by
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
calculating the cells’ spiked triggered average (STA) responses to the
stimulus.
Results: Significant STA responses to prosthetic stimulation were
detected for 10Hz and 20Hz movie frame rates. Prosthetic responses
in RCS retinas were classified based on their STAs into two types:
eON and eOFF, similar to classification into ON-center and OFFcenter types in the healthy retina. The RCS STAs had an average
receptive field diameter of 141±11µm and response latency of
~100ms. ON- and OFF-center RGCs in the healthy retina, classified
according to their responses to visible light, had response latency of
~180ms. While both ON- and OFF-center RGCs had eON responses
to prosthetic stimulation, OFF-center RGCs had half the latency and
higher degree of transiency in prosthetic response compared to the
ON-center cells. Average receptive field diameter was 239±11µm for
visible light and 204±9µm for the prosthetic responses.
Conclusions: Significant prosthetic STA response to white noise
movies at 20Hz frame rate indicates that prosthetic vision can operate
within a standard range of video frame rates. Spatial receptive fields
with prosthetic stimulation in degenerate retinas were smaller than
in normal retinas, while these, in turn, were smaller than with natural
visual responses in healthy RGCs. Response kinetics were faster
with prosthetic stimulation, as expected from the absence of the
phototransduction step. Presence of the eON and eOFF responses in
the RCS retina demonstrates that prosthetic vision can exhibit some
parallel retinal processing even in degenerate retinas.
Commercial Relationships: Richard Smith, None; Elton Ho;
Georges A. Goetz, None; Xin Lei, None; Theodore Kamins,
Pixium Vision (C); Jim Harris, None; Keith Mathieson, None;
Daniel V. Palanker, Pixium Vision (P), Pixium Vision (C);
Alexander Sher, None
Support: NIH R01–EY-018608 (DP)
Program Number: 3722 Poster Board Number: D0183
Presentation Time: 11:00 AM–12:45 PM
Spatial aspects of electrical desensitization in mouse retina
Archana Jalligampala1, 2, Eberhart Zrenner1, Daniel L. Rathbun1.
1
Institute for Ophthalmic Research & Center for Integrative
Neuroscience, University of Tuebingen, Tuebingen, Germany;
2
Graduate Training Center of Neuroscience, University of Tuebingen,
Tuebingen, Germany.
Purpose: Repetitive electrical stimulation of the retina causes
desensitization during which retinal network responses to further
stimulation are reduced. This has been shown to be a potential
neural correlate of visual percept fading often reported by human
visual prosthesis subjects. Earlier studies have investigated the
temporal aspects of desensitization however the lesser known spatial
component is poorly understood. Here we further investigate the
above in healthy mouse retinas
Methods: Retinal ganglion cell (RGC) spiking responses were
recorded invitro from adult wt mouse retinas (c57BL/6,P56-100)
using multi-electrode array(MEA). Cathodal-first, biphasic,
rectangular current pulses with varying stimulus amplitude (0.5-10µA
at 1ms) were delivered epiretinally to obtain RGC thresholds. On
obtaining the average threshold(mean ± SD) a series of pulse pairs
(at 2x threshold) with different interpulse intervals(10-600ms) and
varied spatial separation(200-1000µm) between the two pulses were
presented to study the spatiotemporal effects of desensitization on
RGC responses. Additionally, a high-density MEA(60 electrodes,
40µm pitch, 10µmØ) were used for dense spatial sampling(40200µm). To examine multiple stimulus pulse effect, trains of ten
stimuli(2- 62.5Hz) were also applied.
Results: We found that the pulse paradigms required for effective
stimulation of the retinal network werein agreement with previously
published studies (n=20). Additionally, the RGC responses exhibited
paired-pulse depression with decreasing interpulse interval and
decreased responses with increasing frequency (n=15)suggesting
quick adaption of the RGC responses. No desensitization was
observed at interpulse distances>800µm. With decreasing distances
the response of the second pulse with respect to first(priming) pulse
decreased significantly until 100µm (n=14). For distances<100µm the
responses to the second pulse were indiscernible.
Conclusions: Desensitization follows a nonlinear interaction of
time and distance. This nonlinearity may be caused by various
mechanisms (receptor desensitization, inhibitory feedback) which
could discern the spatial spread of desensitization into local and
global component, thereby providing a better understanding of
fading. The minimal interelectrode distance to avoid desensitizing
interactions is 800µm. Therefore a strategy of interleaved activation
of electrodes separated by at least 800µm is recommended to avoid
desensitization effects.
Commercial Relationships: Archana Jalligampala, None;
Eberhart Zrenner, Retinal Implant AG (R), Retinal Implant AG (I),
Retinal Implant AG (F), Retinal Implant AG (P), Retinal Implant AG
(S); Daniel L. Rathbun, None
Support: BMBF SysRetPro FKZ: 031A308; BMBF FKZ:
01GQ1002; DFG EXC307, Kerstan Foundation, ProRetina
Foundation
Program Number: 3723 Poster Board Number: D0184
Presentation Time: 11:00 AM–12:45 PM
Implications of low prosthetic contrast sensitivity for delivery of
visual information
Georges A. Goetz1, 2, Richard Smith3, Xin Lei4, Ludwig Galambos4,
Theodore Kamins4, Keith Mathieson5, Alexander Sher3,
Daniel V. Palanker2, 6. 1Neurosurgery, Stanford University, Stanford,
CA; 2Hansen Experimental Physics Laboratory, Stanford University,
Stanford, CA; 3Santa Cruz Institute for Particle Physics, University
of California Santa Cruz, Santa Cruz, CA; 4Electrical Engineering,
Stanford University, Stanford, CA; 5Institute of Photonics, University
of Strathclyde, Glasgow, United Kingdom; 6Ophthalmology, Stanford
University, Stanford, CA.
Purpose: To measure contrast sensitivity of the retinal response to
subretinal photovoltaic stimulation, and assess its impact on delivery
of visual information.
Methods: We measure ex-vivo the full-field contrast sensitivity of
healthy rat retina stimulated with white light, and of degenerate rat
retina stimulated with a subretinal prosthesis above the flicker fusion
frequency (20Hz). We model the effects of fixational eye movements
on retinal response by decomposing simulated images on the retina
into a static and a dynamic component. We use the measured contrast
sensitivity function to predict retinal ganglion cell activity elicited by
the dynamic component of the image.
Results: Prosthetic vision exhibits significantly reduced contrast
sensitivity and no OFF responses in degenerate retina, with 65%
contrast changes required to elicit responses, as compared to 3-7%
changes required with visible light. The maximum number of action
potentials elicited is, at most, half of its natural counterpart for the
ON pathway. Our model predicts that for the majority of natural
images the contrast sensitivity of prosthetic vision is insufficient
for triggering RGC activity by fixational eye movements. For these
images, increasing the contrast alone is insufficient. Therefore, image
processing should locally enhance both contrast and sparsity, or
stimulation frequencies should be reduced to below the flicker fusion
to elicit retinal response.
Conclusions: The low contrast sensitivity and lack of OFF responses
hamper delivery of visual information via a subretinal prosthesis.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
Fixational eye movements, which normally prevent static images
from fading, are likely to be insufficient for eliciting responses
to natural scenes with subretinal stimulation above flicker fusion
frequency. Image pre-processing might alleviate this limitation.
Alternatively, subretinal implants may need to rely on lower
stimulation frequencies.
Commercial Relationships: Georges A. Goetz; Richard Smith,
None; Xin Lei, None; Ludwig Galambos, None; Theodore Kamins,
None; Keith Mathieson, None; Alexander Sher, None;
Daniel V. Palanker, Stanford University (P), Pixium Vision (P),
Pixium Vision (C)
Support: NIH grant R01-EY-018608, DOD grant
W81XWH-15-1-0009, Stanford Spectrum fund, BWF CASI, Pew
Charitable Trusts Scholarship
Program Number: 3725 Poster Board Number: D0186
Presentation Time: 11:00 AM–12:45 PM
Apoptosis reduction by a photoelectric dye used for Okayama
University-type retinal prosthesis (OURePTM)
Shihui Liu1, Toshihiko Matsuo1, Osamu Hosoya2, Tetsuya Uchida3.
1
Ophthalmology, Okayama University Graduate School of
Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan;
2
Neurogenomics, Okayama University Graduate School of Medicine,
Dentistry, and Pharmaceutical Sciences, Okayama, Japan; 3Polymer
Materials Science, Okayama University Graduate School of Natural
Science and Technology, Okayama, Japan.
Purpose: We previously demonstrated that the photoelectric dyecoupled polyethylene film (OURePTM), which was implanted in
subretinal space of Royal College of Surgeons (RCS) rat’s eyes,
prevented retinal neurons from apoptotic death. In addition, the
photoelectric dye was proven safe in several biological tests for
cytotoxicity and for systemic and immunological toxicities. In this
study, we verified whether the photoelectric dye, in itself, would
protect retinal neurons from apoptosis using RCS rats, a model of
retinitis pigmentosa.
Methods: Twenty RCS rats at 4 weeks of age were randomly
assigned to 5 groups, with each group comprising 4 rats. The rats
had intravitreous injection of 3 μl solution at different concentrations
of the dye, a 10-fold dilution series from 0.0082 μg/ml to 82 μg/ml,
in the left eyes of each group. Saline injection at the same volume
served as control in the right eyes of each group. The rats were
housed under a 12-hour light/dark cycle for 14 days. As an additional
experiment, another group of 3 rats with dye injection at 82 μg/
ml were covered with a lightproof box to keep them in 24-hour
daily dark condition. Two weeks after the dye injection, rats were
sacrificed. Apoptosis was detected in frozen sections of the eyes, and
also stained immunohistochemically for glial fibrillary acidic protein
(GFAP) and protein kinase C alpha (PKC-α). The immunreactive
areas were measured by the Image J software.
Results: The number of apoptotic cells significantly decreased in
the retina of dye-injected eyes, compared with saline-injected eyes
(P<0.05, two-way analysis of variance, ANOVA), under 12-hour
light/dark cycle. Significant decrease of apoptotic cells was also
noted in the retina of rats with dye injection, kept under 24-hour
dark condition. The area immunoreactive for GFAP was significantly
decreased in the retina of dye-injected eyes when compared with
controls (P<0.05, two-way analysis of variance, ANOVA). No
significant difference was seen in PKC-α immunostaining between
the dye-injected and saline-injected eyes.
Conclusions: Photoelectric dye injection exerts an inhibiting effect
on apoptotic death of photoreceptors in RCS rats and also suppresses
GFAP-expression in the retinal Müller cell. The photoelectric dye is a
promising molecule for developing new medications used for retinitis
pigmentosa.
Commercial Relationships: Shihui Liu, None; Toshihiko Matsuo,
None; Osamu Hosoya, None; Tetsuya Uchida, None
Program Number: 3726 Poster Board Number: D0187
Presentation Time: 11:00 AM–12:45 PM
Retinal safety of near infrared radiation in photovoltaic
restoration of sight
Henri Lorach1, 2, Jenny Wang1, 2, Dae Yeong Lee1, 3, Roopa Dalal1,
Philip Huie3, Daniel V. Palanker1, 2. 1Ophthalmology, Stanford
University, Stanford, CA; 2HEPL, Stanford University, Stanford, CA;
3
Ophthalmology, Gachon University, Incheon, Korea (the Republic
of).
Purpose: Photovoltaic restoration of sight requires intense near
infrared (NIR) light to effectively stimulate retinal neurons. We
assess the retinal safety of such irradiation in the rabbit eye with and
without the presence of a retinal implant.
Methods: Retinal damage threshold was determined in pigmented
rabbits exposed to 880nm laser radiation. A computational model
of the rabbit eye was used to assess the temperature increase with
and without the subretinal silicon implant. The model was validated
by comparison of the computed temperature with (a) previously
published experimental data in pigmented rabbits and (b) with the
damage thresholds in intact and in implanted rabbits.
Results: In pigmented rabbits, the 50% probability (ED50) of retinal
damage during 100s long exposures with 1.2mm diameter beam
occurred at 175mW. According to computational model of the rabbit
eye, it corresponded to the temperature rise of 12.5°C, matching the
published data. In presence of an implant, the same temperature rise
was expected at 78mW of power. In implanted animals, no visible
damage was observed up to 60mW, and ED50 corresponded to
71mW. In the normal use conditions of the photovoltaic subretinal
prosthesis (5mW/mm2, 5ms, 40Hz), the retinal temperature rise is not
expected to exceed 0.43°C.
Conclusions: The NIR light intensity required for activation of the
photovoltaic retinal prosthesis is well within the safety limits for
chronic use (< 1°C).
Commercial Relationships: Henri Lorach, Pixium Vision (C);
Jenny Wang, None; Dae Yeong Lee, None; Roopa Dalal, None;
Philip Huie, None; Daniel V. Palanker, Pixium Vision (C)
Support: NIH Grant R01–EY-018608, Department of Defense Grant
W81XWH–15–1–0009, Pixium Vision Grant 1170660-200-UDERL
Program Number: 3727 Poster Board Number: D0188
Presentation Time: 11:00 AM–12:45 PM
Fabrication and in-vitro characterization of novel hydrogel-based
electrodes for visual prostheses
Yasuo Terasawa1, 3, Hiroyuki Tashiro2, Yukari Nakano1, Koji Osawa1,
Motoki Ozawa4, Toshihiko Noda3, Takashi Tokuda3, Jun Ohta3,
Kazutoshi Haraguchi5. 1Vision Institute, R&D Div, NIDEK Co
Ltd, Gamagori, Japan; 2Department of Health Sciences, Faculty of
Medical Sciences, Kyushu University, Fukuoka, Japan; 3Materials
Science, Nara Institute of Science and Technology, Ikoma, Japan;
4
Nidek Co.,Ltd., Gamagori, Japan; 5College of Industrial Technology,
Nihon University, Narashino, Japan.
Purpose: Charge injection capacity (CIC), the maximum injectable
charge without causing irreversible electrochemical reactions, is
important characteristic of stimulating electrode for visual prostheses.
Introducing hydrogel onto electrode surface is expected to enhance
in-vivo CIC, but fabrication of such electrode is difficult because of
poor mechanical properties of hydrogel. The purpose of this study is
to fabricate and characterize the novel hydrogel-based electrodes.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
Methods: Electrode arrays were fabricated using
polydimethylsiloxane (PDMS) and platinum foil. After forming
0.4mm-thick PDMS base layer, 0.02mm-thick platinum foil was
stuck on the base layer. The platinum foil was then cut to form
electrodes and conductive lines by femtosecond laser. Next, the
electordes were coated with polyethylene glycol. After applying
PDMS top layer, electrode surfaces were exposed by dissolving
the polyethylene glycol with water. Nanocomposite hydrogel,
composed of poly (N,N-dimethylacrylamide) and synthetic hectorite,
was injected into the electrode openings to fill 0.5mm-diameter
0.4mm-height spaces above the platinum foil electrodes. CICs were
measured in room-temperature PBS.
Results: Fabrication of hydrogel-coated electrodes was successfully
performed by applying 90°C heat to the hydrogel in DI water after
hydrogel injection into the electrode openings (see Fig.1).
The average CIC of non-hydrogel electrodes and hydrogel electrodes
were 182µC/cm2 and 160µC/cm2 respectively. No statistically
significant difference was observed between non-hydrogel electrodes
and hydrogel electrodes (paired t-test, N=3).
Conclusions: The abovementioned results indicated that hydrogel
had enough permeability of water and ions to pass the current
of electrical stimulation. This suggested that the hydrogel-based
electrode had a potential to enhance the in-vivo CIC because the
application of hydrogel onto metal electrode intervened to deteriorate
in-vivo CIC of conventional electrodes such as protein absorption and
low counterion availability. We will study in-vivo performance of the
hydrogel electrodes in the next step.
Fig.1 Electrode with hydrogel (right) and without hydrogel (left).
Commercial Relationships: Yasuo Terasawa, Nidek Co.,Ltd.;
Hiroyuki Tashiro, Nidek Co.,Ltd. (F); Yukari Nakano, Nidek
Co.,Ltd.; Koji Osawa, Nidek Co.,Ltd.; Motoki Ozawa, Nidek
Co.,Ltd.; Toshihiko Noda, Nidek Co.,Ltd. (F); Takashi Tokuda,
Nidek Co.,Ltd. (F); Jun Ohta, Nidek Co.,Ltd. (F);
Kazutoshi Haraguchi, None
Program Number: 3728 Poster Board Number: D0189
Presentation Time: 11:00 AM–12:45 PM
Focal activation of retinal ganglion cells with epiretinal implants:
The AIS as a key component
Paul Werginz1, Alex Hadjinicolaou2, 3, Shelley I. Fried2, 3,
Frank Rattay1. 1Institute for Analysis and Scientific Computing,
Vienna University of Technology, Vienna, Austria; 2Vision Research
Laboratory, Boston VA Medical Center, Boston, MA; 3Department of
Neurosurgery, Massachusetts General Hospital & Harvard Medical
School, Boston, MA.
Purpose: A modeling study was conducted to examine how the
low-threshold region of axon initial segments (AIS) can be utilized
to focally stimulate the ganglion cell (GC) layer with epiretinal
implants.
Methods: The model neuron was derived from the morphology of
a mouse OFF GC. Transmembrane currents were computed with
a Hodgkin-Huxley type model using a backward Euler integrator.
Low- and high-threshold sodium channels were incorporated with
specific distributions along the axon. External potentials evoked by
a monopolar disc electrode were calculated by the method of finite
elements.
Results: The response of multiple closely neighbored GCs elicited
by 0.1ms cathodic pulses was computed. Activation thresholds were
compared to the threshold of a passing axon from a GC located
400μm away. Thresholds for multiple neurons having their AIS close
to the stimulating electrode were lower than threshold for the passing
axon (3.18μA for a 20μm disc electrode diameter variation of passing
axon (0.5,1,2μm caused 30,18,11 focally activated GCs). In contrast,
the region which can be focally activated is rather independent of size
of small electrodes (10,20,40μm disc diameter resulted in 18,19,20
focally activated GCs).
Conclusions: According to the model assumption of about 2500 GCs
per mm2 the low-threshold region in the proximity of the AIS can be
utilized for direct stimulation of about 20 GCs close to the electrode
without eliciting spikes in bypassing axons. Focal stimulation is
not sensitive to electrode size but rather to the GC’s axon diameter.
Further investigations of GC properties will be needed to explore
how threshold variations depend on different GC types.
A: Top view of multiple densely packed GCs and one far distant GC
are activated at different thresholds by a disc electrode (diameter
20μm,filled red circle). Spacing between the cells was 20μm in
x-direction and GC columns were shifted in 5μm steps vertically.
Inset: Model GC in 3D view. B: Activation threshold for each GC
location is marked in the somas of the GCs. One passing axon with
its soma 400μm distant to the electrode has a spiking threshold of
3.18μA. 18 GCs (red) have lower thresholds than the passing axon
and can therefore be focally activated. The grey shaded region
indicates GC locations with thresholds lower than of the passing
axon. The AIS region is indicated by purple bars. Pulse length:
0.1ms; electrode 30μm above the axon.
Commercial Relationships: Paul Werginz; Alex Hadjinicolaou,
None; Shelley I. Fried, None; Frank Rattay, None
Support: Austrian Science Fund (FWF), Grant No. P 27335-B23
Program Number: 3729 Poster Board Number: D0190
Presentation Time: 11:00 AM–12:45 PM
Extraction of directly-evoked Retinal Ganglion Cell response
from stimulus artifact using three artifact subtraction algorithms
Jungryul Ahn1, Myounghwan Choi2, DaeJin Park1, Kyo-In Koo2,
Yongsook Goo1. 1Department of Physiology, School of Medicine,
Chungbuk National University, Cheong-ju, Korea (the Republic of);
2
Department of Biomedical Engineering, University of Ulsan, Ulsan,
Korea (the Republic of).
Purpose: By electrical stimulation on retina, there are two - early
(directly-evoked) and late (indirectly-evoked) - retinal ganglion cell
(RGC) responses evoked. Direct RGC response which is desirable
for the retinal prosthesis to recreate the appropriate spike pattern
is likely to be obscured by stimulus artifact. In this study, we aim
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
to isolate the directly-evoked RGC response from stimulus artifact
using three algorithms; Subtraction of Artifacts by Local Polynomial
Approximation (SALPA), Moving Average Filter (MAF), and
Forward Reverse (FR) filter. SALPA is a well-established algorithm
and the other two are new custom-made algorithms for artifact
subtraction.
Methods: Rd1 mice after postnatal 10 week were used (n=4).
The RGC responses were extracellularly recorded with 8 × 8
multi-electrode array (MEA) in which one electrode was used as
stimulating electrode and all other electrodes as recording electrode.
Fifty cathodic phase-1st biphasic current pulses (duration 500 μs,
amplitude 5~60 μA) were applied at every 1 sec. Three artifact
subtraction algorithms were used: Subtraction of Artifacts by Local
Polynomial Approximation (SALPA) is based on curve fitting of
polynomial function. For Moving Average Filter (MAF), fitting
number of 23 was used. For forward reverse (FR) filter, 100 Hz
high pass and 500 Hz low pass was used for cut-off frequency. The
latencies of first spikes following 50 stimuli were averaged in each
electrode and the latencies acquired from three algorithms were
statistically compared. False positive error (algorithm mistakes the
artifact as the spike) and missing error (missing early response as
residual artifact) were calculated.
Results: SALPA, MAF, and FR filter detected latency of early RGC
spikes as 8 ± 3.6 ms, 13.1 ± 5.2 ms and 13.2 ± 4.7 ms, respectively
(p < 0.001). False positive error and missing error for SALPA,
MAF, and FR filter were 4%, 5%, and 2%, and 2%, 26%, and 21%,
respectively.
Conclusions: In terms of latency of early RGC response, SALPA
detects latency earlier than two other algorithms (p < 0.001). For
false-positive error, FR filter shows best performance. For missing
error, SALPA shows best performance. Since MAF and FR filter’s
performance depends on fitting number and cut-off frequency, in
future study, we try to minimize the false-positive and missing error
by fine-tuning of both MAF and FR filter.
Commercial Relationships: Jungryul Ahn, None;
Myounghwan Choi, None; DaeJin Park, None; Kyo-In Koo, None;
Yongsook Goo, None
Support: the MEST (NRF-2010-0020852, NRF2013R1A1A3009574) of Korea
Program Number: 3730 Poster Board Number: D0191
Presentation Time: 11:00 AM–12:45 PM
Oscillatory Rhythm in Degenerate Mouse (rd10) Retina
Modulates Retinal Ganglion Cell Response to Electrical Stimulus
Dae-jin Park1, JungRyul Ahn1, Seong Kwang Cha1, Yong Sook Goo1,
Solomon Senok2. 1Department of Physiology, School of Medicine,
Chungbuk National University, Cheongju, Korea (the Republic of);
2
Neuroscience, College of Medicine, Alfaisal University, Riyadh,
Saudi Arabia.
Purpose: Appearance of aberrant spiking activity coupled with 5
~15 Hz oscillatory local field potential (LFP) has been reported in
degenerate mouse retinas. These LFPs have been regarded as noise
which reduced the efficacy of prosthetic stimulation. Since LFP
varies with postnatal ages in rd10 mice, here, we investigated if the
LFP might affect RGC responses to electrical stimulation in rd10
retina according to degeneration process.
Methods: We recorded electrically-evoked RGC spikes using 8×8
multi-electrode array (MEA) in rd10 mice at postnatal week (PNW)
2 ~34. Retinal patches were placed RGC layer down on MEA.
Fifty cathodic phase-1st biphasic square pulses (duration: 500 µs,
pulse amplitude: 5 ~60 μA) were applied at every 1 sec. Previously,
1st peak in post-stimulus time histogram (PSTH) showed better
modulation efficacy than subsequently appeared remnant peaks. Thus,
stimulus efficacy of PSTH 1st peak and remnant peaks was compared
among different age groups to clarify potential information-carrying
role of multiple peaks. Also, correlation between evoked spike
numbers in PSTH peaks and LFP was calculated.
Results: Evoked spike number of PSTH 1st peak is significantly
higher at PNW 6.5 (p<0.05), PNW 20 (p<0.001) and lower at PNW
10 (p<0.05) than that of all other ages except PNW 2. Modulation
range of RGC response to electrical stimulus is narrower at these
ages, when power of ~ 5 Hz oscillation is much higher (PNW 6.5
and 20) or lower (PNW 10) than those in other age groups. Remnant
peaks show broader modulation range at PNW 8&26 than those at
other ages. Power of ~5 Hz oscillation shows good correlation with
evoked spike number of PSTH 1st peak in group of PNW 6.5&8
(R=0.7, p<0.001), but it has weak correlation with 1st peak in group
of PNW 15 ~34 (R=0.4, p<0.05). There is no correlation between
power of oscillation and evoked spike number of remnant peaks
across all ages (R=0.2, p<0.05).
Conclusions: Power of ~5 Hz oscillation in rd 10 retinas modulates
RGC response to electrical stimulus in PSTH 1st peak at PNW 6.5
~8. At PNW 15 ~34, ~5 Hz oscillation has only a limited effect on
PSTH 1st peak. These results suggest that power of oscillation may
be an important factor determining the efficacy of electrical stimulus.
Furthermore, high modulation efficacy of PSTH remnant peaks at
PNW 8&26 may support the idea of information-carrying role of
remnant peaks.
Commercial Relationships: Dae-jin Park, None; JungRyul Ahn,
None; Seong Kwang Cha, None; Yong Sook Goo, None;
Solomon Senok, None
Support: NRF-2010-0020852, NRF-2013R1A1A3009574
Program Number: 3731 Poster Board Number: D0192
Presentation Time: 11:00 AM–12:45 PM
Towards an improvement of the visual function preservation in
RCS rats by transplantation of human stem-cell-derived RPE
and photoreceptor precursor cells
Laura Fontrodona1, Laia Miquel2, Yolanda Muñoz2, Diana Mora
Ramírez1, Anna Salas Torras1, Barbara Ferreira-de-Souza1,
Marina Riera1, Silvia Albert2, Anna Veiga2, Jose Garcia-Arumi1.
1
Ophthalmology, Vall d’Hebron Research Institute (VHIR),
Barcelona, Spain; 2Stem Cell Bank, Center of Regenerative Medicine
in Barcelona (CMRB), Barcelona, Spain.
Purpose: Cell therapy for retinal degenerative diseases is a newborn
reality that should be constantly subjected to refinement. We used the
Royal College of Surgeons (RCS) rat model to test the hypothesis
that subretinal injection of a combination of photoreceptor precursor
cells (PPC) and Retinal Pigmented Epithelium (RPE) cells derived
from human stem cells would provide better benefits than a cell
therapy uniquely using RPE cells.
Methods: We differentiated and obtained RPE and PPC from two
human stem cell lines of distinct origins: embryonic stem cells
(hESC) and induced pluripotent stem cells from cord blood (hiPSC).
Following characterization of these cell-derived populations, five
groups of p21 RCS rats received a subretinal transplant of: (i, ii) a
combination of PPC and RPE from hESC or hiPSC; (iii, iv) RPE
alone from hESC or hiPSC; (v) cell culture media as controls. The
visual function was assessed through Ganzfeld electroretinography
at several time points up to four months after injection. After
determining the survival of the grafts in the host eye, we studied their
capacity to integrate and function in the rat retina through histological
and immunohistochemical techniques.
Results: Eyes injected with any type of cell therapy boost a
significant slowdown effect on the progression of retinal degeneration
compared to control eyes. Interestingly, while during the first 2
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to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
months no differences were observed between the groups that
received the combined or the RPE alone treatments; upon 3 months
of transplantation, eyes injected with a combination of PPC and RPE
responded significantly better to light stimuli than eyes that had only
received RPE cells. Such effect was observed up to 4 months after
treatment and in concordance with it, at that time eyes treated with
the combination of PPC and RPE displayed higher number of ONL
(outer nuclear layer) cell rows than RPE alone treated eyes.
Conclusions: Our preliminary results might be pointing towards an
upgraded version of the generally used RPE cell therapy if combined
with PPC. We are currently characterizing the degree of integration of
the grafts and to which extent these therapies are able to protect the
host retinas against the degenerative process.
Commercial Relationships: Laura Fontrodona; Laia Miquel,
None; Yolanda Muñoz, None; Diana Mora Ramírez, None;
Anna Salas Torras, None; Barbara Ferreira-de-Souza, None;
Marina Riera, None; Silvia Albert, None; Anna Veiga, None;
Jose Garcia-Arumi, None
Program Number: 3732 Poster Board Number: D0193
Presentation Time: 11:00 AM–12:45 PM
Acute Synaptic and Neuronal Anatomical Changes Following
Insertion of Sub-Retinal Electrode Array in an Enucleated
Human Eye
Jinghua Chen1, Gloria DeWalt2, William Eldred2, Marcus Gingerich3,
Douglas Shire3, Joseph F. Rizzo4, Henry J. Kaplan1. 1Ophthalmology
and Visual Sciences, University of Louisville, Louisville, KY;
2
Department of Biology, Boston University, Boston, MA; 3Bionic Eye
Technologies, Inc., Fishkill, NY; 4Ophthalmology, Harvard Medical
School, Boston, MA.
Purpose: This work is related to the efforts of the Boston Retinal
Implant Project to develop a sub-retinal prosthesis to restore basic
vision following photoreceptor degeneration. A sub-retinal prosthesis
was implanted into an enucleated human eye, after pars plana
vitrectomy and a focal retinal detachment, to evaluate acute synaptic
and neuronal anatomical changes in response to the surgery and
implant.
Methods: The inactive sub-retinal implant included secondary power
and data coils, a hermetic case and a parylene coated polyimide
electrode array. A pars plana vitrectomy was performed and a focal
retinal detachment was created. The coil and the hermetic case were
sutured onto sclera and the electrode array was placed into the subretinal space ab externally after enucleation. The array remained in
situ for about 30 minutes prior to fixation in paraformaldehyde. The
retina close to versus further away from the array were cryoprotected,
and embedded before being frozen. Cross sections were obtained.
Normal donkey serum was used to minimize non-specific binding
prior to incubation in various antisera: calretinin, parvalbumin,
and synaptotagmin at 4°C. Following incubation in fluorescently
conjugated secondary antibodies slides were coverslipped in
Vectashield Antifade Mounting Medium with DAPI. Confocal
microscopy was used for visualization.
Results: There was a reduction in calretinin immunoreactivity in the
outer plexiform layer, reduced somatic labeling in the inner nuclear
layer, and a loss of punctate labeling in the inner plexiform layer near
the array compared to further away. Similarly, there was a decrease
in parvalbumin labeling in horizontal cell somata in the inner nuclear
layer near the array and there were extensive arborizations into the
outer and inner nuclear layers compared to regions further away. And,
there was a reduction in synaptotagmin positive punctate labeling in
both the inner and outer plexiform layers near the array compared to
retina further away.
Conclusions: Neuronal and synaptic changes were present near the
implant. Additional work will be necessary to determine whether
changes are transient or long lasting and to evaluate how these
cellular changes may affect vision restoration. These results are of
uncertain significance given that this is a singular case and that there
was no control eye for comparison. Control studies are underway.
Commercial Relationships: Jinghua Chen, None; Gloria DeWalt,
None; William Eldred, None; Marcus Gingerich, Bionic Eye
Technologies, Inc.; Douglas Shire, Bionic Eye Technologies, Inc.;
Joseph F. Rizzo, None; Henry J. Kaplan, None
Support: Department of Veterans Affairs (VA Grant C4266C,
VA Grant A7078R, VA Grant C7055L, VA Grant I01 RX00117801A2), NIH Grant 1-R01-HL090856-01A1, Massachusetts Lions
Eye Research Fund, Inc., NSF (Cornell NanoScale Science and
Technology Facility); an unrestricted grant from Research to
Blindness, New York, NY
Program Number: 3733 Poster Board Number: D0194
Presentation Time: 11:00 AM–12:45 PM
ARGUS II® ELECTRONIC EPIRETINAL PROSTHESIS IN
ADVANCED DRY AMD: SAFETY AND FEASIBILITY STUDY
AND PRELIMINARY FUNCTIONAL RESULTS
Paulo E. Stanga1, 2, Assad Jalil1, EMMANOUIL TSAMIS1, 2,
ALESSANDRO PAPAYANNIS1, Jessy D. Dorn3, Robert J. Greenberg3,
WILL MCGUIRE3. 1Manchester Vision Regeneration (MVR) Lab at
Manchester Royal Eye Hospital & NIHR/Wellcome Trust Manchester
CRF and Manchester Royal Eye Hospital, Central Manchester
University Hospitals NHS Foundation Trust, Manchester, United
Kingdom; 2Institute of Human Development, Faculty of Medical
and Human Sciences, University of Manchester, Manchester, United
Kingdom; 3Second Sight Medical Products, Inc, Sylmar, CA.
Purpose: Evaluate the safety and benefit of the Argus II® electronic
epiretinal prosthesis in late stage “dry” age-related macular
degeneration (AMD) (d-AMD) with severely affected central vision.
Methods: Non-randomised, non-controlled, prospective, singlecentre study. Four d-AMD patients aged between 69 and 81 years,
with logMAR 1.0 (6/60) or worse ETDRS vision in both eyes,
subfoveal geographic atrophy (GA) confirmed on ultra-wide
field colour and fundus autofluorescence (UWF-FAF) imaging
and scotoma within the central 20° visual field confirmed by
microperimetry and 60° visual field testing, were included and
implanted in the worst eye. The positioning of the array of electrodes
over the area of GA and the distance between them and the
neuroretina was assessed on UWF-FAF (Fig. 1) and Swept SourceOCT. Patients underwent pre and post-operative visual function
testing.
Results: All implants were placed within the area of GA. As of
November 30, 2015, four patients have been implanted and followedup for a period ranging from 0.2 to 5.6 months. All patients showed
central visual function elicited by the Argus II over the area of GA.
No patient reported confusion with the system ON and both eyes
open. Surgical results were reproducible across the four patients. On
the first post-operative day, the first patient presented with a retinal
detachment under the cable, which responded to air tamponade. At 3
months post-implant, this same patient performed significantly better
with the System ON than OFF (with both eyes open) on direction
of motion testing; and achieved 1.9 logMAR visual acuity with the
System ON, and better than 1.6 logMAR with the System OFF. With
the system ON and both eyes open, this patient reported being able
recognize the outline of faces and some facial characteristics, such as
open/closed mouth within their central vision.
Conclusions: We report on the first ever successful integration of
artificial (central-acquired) and natural (peripheral-residual) vision in
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ARVO 2016 Annual Meeting Abstracts
humans. We also report on the first ever implantation of an electronic
retinal prosthesis in AMD. The Argus II electronic epiretinal
prosthesis seems to be well tolerated and able to restore some central
visual function in late stage “dry” AMD.
Array of electrodes over GA. Red arrow: compression retinal fold.
Green arrow: scleral indentation induced by the episcleral electronics
package.
Commercial Relationships: Paulo E. Stanga, Second Sight
Medical Products, Inc (R), Second Sight Medical Products, Inc
(C), Second Sight Medical Products, Inc (F); Assad Jalil, None;
EMMANOUIL TSAMIS, None; ALESSANDRO PAPAYANNIS,
None; Jessy D. Dorn, Second Sight Medical Products, Inc;
Robert J. Greenberg, Second Sight Medical Products, Inc. (P),
Second Sight Medical Products, Inc., Second Sight Medical Products,
Inc. (I); WILL MCGUIRE, Second Sight Medical Products, Inc,
Second Sight Medical Products, Inc (I)
Clinical Trial: NCT02227498
Program Number: 3734 Poster Board Number: D0195
Presentation Time: 11:00 AM–12:45 PM
Local and systemic immune responses associated with rejection
of iPS-derived RPE allografts transplanted into rhesus macaques
Trevor J. McGill1, 2, Jonathan W. Stoddard2, Lauren M. Renner2,
Robert Bonnah2, Emily Johnson2, Ilhem Messaoudi3,
Steven T. Bailey1, Andreas Lauer1, Shoukhrat Mitalipov3,
David J. Wilson1, Martha Neuringer2, 1. 1Ophthalmology, Casey Eye
Institute, OHSU, Portland, OR; 2Neuroscience, Oregon National
Primate Research Center, Beaverton, OR; 3Oregon National Primate
Research Center, Beaverton, OR.
Purpose: To determine the viability of allogeneic iPS-derived RPE
cells transplanted into the subretinal space of rhesus macaques, and to
examine the resulting local and systemic immune response.
Methods: GFP-labelled allogeneic iPS-derived RPE cells were
transplanted into the subretinal space of one eye of 4 monkeys,
followed by transplantation into the contralateral eye 4-5 weeks later.
No immunosuppression was used. Retinas were examined pre- and
post-surgery by color fundus photography, fundus autofluorescence,
and OCT imaging. Peripheral blood mononuclear cells were
monitored weekly for changes in frequency of circulating naïve and
memory T- and B-cell subsets. Animals were sacrificed 3 weeks
following the second transplantation and retinas were examined
morphologically and immunohistochemically.
Results: Ophthalmic imaging revealed successful delivery of the
cells in all eyes. In general, GFP fluorescence faded to nonvisible
levels by ~3 weeks post-transplantation in the first eye, and by ~2
weeks post-transplantation in the second eye. In all eyes, infiltration
of mononuclear cells into the choroid and subretinal space resulted
in ablation of grafted cells and disruption of host RPE and choroid
in the immediate vicinity of the graft. In one animal, a few
residual transplanted cells were observed in the center of a bolus
of mononuclear cells. The mononuclear cells stained positively
primarily for B-cell and T-cell antibodies. Microglial cell activation
was evident in all cell-transplanted eyes. All animals showed
systemic immune activation; T and B cell proliferation increased 2- to
3-fold starting ~14 days after the initial surgery, often accompanied
by increased frequency of memory T and B cells.
Conclusions: Our data demonstrate that transplantation of
allogeneic iPS-derived RPE cells into the subretinal space without
immune suppression induces an immune response that could
compromise survival of the cells, and this response is accelerated
after injection in the second eye. Similar results were observed
after allogeneic embryonic-derived RPE transplantation into the
same species (Neuringer et al ARVO, 2014). These findings have
important implications for clinical application of cell-based therapy,
underscoring the need for evaluation of autologous cell sources
and/or adequate T- and B-cell immune-suppression to ensure graft
survival.
Commercial Relationships: Trevor J. McGill;
Jonathan W. Stoddard, None; Lauren M. Renner, None;
Robert Bonnah, None; Emily Johnson, None; Ilhem Messaoudi,
None; Steven T. Bailey, None; Andreas Lauer, None;
Shoukhrat Mitalipov, None; David J. Wilson, None;
Martha Neuringer, None
Support: NIH Grants R01EY021214, P51OD011092, CEI
P30EY010572, and an unrestricted grant from RPB.
Program Number: 3735 Poster Board Number: D0196
Presentation Time: 11:00 AM–12:45 PM
A New Immunodeficient Dystrophic RCS Rat Model for
Transplantation Studies Using Human Derived Cells
Biju Thomas2, Cindy H. Shih2, Danhong Zhu1, Juan C. Martinez2,
David R. Hinton2, 1, Mark S. Humayun2. 1Pathology, University of
Southern California Keck School of Medicine, Los Angeles, CA;
2
Ophthalmology, University of Southern California Keck School of
Medicine, Los Angeles, CA.
Purpose: To introduce the mutation of Mertk gene present in
dystrophic Royal College of Surgeon (RCS) rat into non-dystrophic
Athymic nude rat to develop an immunodeficient RCS rat model with
retinal degeneration (nude dystrophic RCS rats). This model can be
used to minimize the adverse effects of xenograft transplantation.
Methods: Female homozygous Pigmented dystrophic RCS rats
(RCS-p+/ RCS-p+) were crossed with male pigmented nondystrophic Athymic nude rats (Hsd:RH-Fox1^rnu/Fox1^rnu, no
T-cells). Genotyping for Fox1 and RCS-p+ gene were determined by
PCR analysis. Retinal degenerative disease condition was assessed
by OCT imaging and optokinetic (OKN) visual behavioral testing.
Morphological analysis of the retina was based on haematoxylin
and eosin (H&E) staining. Age-matched normal pigmented nondystrophic Athymic Nude rats were used as controls for the above
experiments.
Results: The homozygous Fox1 gene was identified based on the
hairless phenotype. The homozygous RCS-p+ gene was identified
based on PCR analysis. Approximately 6% of the F2 pups (11/172)
were homozygous for both RCS-p+ gene and Foxn1 gene (nude
dystrophic RCS rats). OCT imaging at 12 weeks of age demonstrated
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ARVO 2016 Annual Meeting Abstracts
significant loss of retinal thickness in double homozygous RCS
rats (p<0.0001, 254.10±3.06 µm non-dystrophic nude retina vs
197.20±2.61 µm nude dystrophic retina). This was further confirmed
by histological evaluation of the eye. Based on H&E staining,
photoreceptor layer thickness in 12 week old nude dystrophic RCS
rats was limited to two to three layers in the peripheral region of the
retina and to a single layer in the central region of the retina. Visual
functional deficit in nude dystrophic RCS rats was evidenced by
severe loss of OKN visual acuity.
Conclusions: A new immunodeficient nude dystrophic RCS rat
model is developed by crossing female homozygous Pigmented
dystrophic RCS rats with male pigmented non-dystrophic Athymic
nude rats. This newly created rat model can be extremely useful
to test the effect of human cell transplantation studies without
interference of immunosuppression.
Commercial Relationships: Biju Thomas; Cindy H. Shih, None;
Danhong Zhu, None; Juan C. Martinez, None; David R. Hinton,
None; Mark S. Humayun, None
Support: 5R01 EY020796
Program Number: 3736 Poster Board Number: D0197
Presentation Time: 11:00 AM–12:45 PM
Dissociation protocol and optimal conditions for clinical-grade
induced pluripotent stem cell-derived retinal precursor cells
Brittni Scruggs, Budd A. Tucker, Chunhua Jiao, Janet Riley,
Edwin M. Stone, Robert F. Mullins, Elliott H. Sohn. Wynn Institute
for Vision Research, University of Iowa, Iowa City, IA.
Purpose: There is no defined dissociation protocol for GMP-grade
induced pluripotent stem cell-derived (iPSC) retinal precursor cells
(RPCs), and there are few data defining the effects of retinal cell
transplantation conditions on cell viability. We tested the hypotheses
that human iPSC-derived RPCs could be isolated after dissociation
from neurospheres and that cell viability could be improved by
optimizing the temperature, storage conditions, and injection
conditions.
Methods: Skin fibroblasts were obtained from patients with known
retinal disease to generate iPSCs, which were then grown to produce
eyecup-like structures. These neurospheres at early (60 days),
intermediate (90 days), and later (>150 days) stages were dissociated
in the presence of either dispase or papain solutions. The dissociation
protocol was optimized for cell yield, and isolated cells were
evaluated using immunocytochemical analyses to detect cell surface
markers. RPCs were injected through needle cannulas of different
gauges (31G vs. 41G) using different storage temperatures (room
temp vs. 0 C) after varying lengths of storage time (10 min vs. 2 hr).
Cell viabilities were determined using a tetrazolium (MTS) assay
and an automatic cell counter. All conditions were tested in three
independent experiments in triplicate using cells isolated from five
patients. Statistical analysis of three or more groups were performed
using one-way ANOVA with post-hoc testing.
Results: RPCs were isolated from iPSC-derived neurospheres using
various dissociation conditions; the papain solution at 37 C yielded
the highest cell count and viability when compared to the conditions
using dispase. Data suggest that the neurospheres and isolated cells
stain positively for multiple retinal cell surface markers (e.g., PAX6,
MITF). There was a significant decrease in cell viability when
dissociated iPSC-derived RPCs were injected in vitro using the 41G
cannula compared to no cannula or to the 31G cannula. Specifically,
the MTS assay showed that 79.33+/-6.52% of cells survived the 31G
injection compared to 53.32+/-3.12% of cells using the 41G cannula
(p<0.01). Our data also suggest that neither temperature nor transport
time affect the RPC viability.
Conclusions: RPCs can be isolated from iPSC-derived neurospheres,
and the viability of these cells is improved with a larger sized needle
cannula.
Commercial Relationships: Brittni Scruggs, None;
Budd A. Tucker, None; Chunhua Jiao; Janet Riley, None;
Edwin M. Stone, None; Robert F. Mullins, None; Elliott H. Sohn,
Regeneron (F), GSK (F), Oxford Biomedica (F)
Support: Wynn Institute Endowment for Vision Research
Program Number: 3737 Poster Board Number: D0198
Presentation Time: 11:00 AM–12:45 PM
Human retinal progenitor sheet transplants in immunodeficient
retinal degenerate (RD) rats
Magdalene J. Seiler1, 2, Bryce T. McLelland2, Anuradha Mathur2,
Robert Lin2, De Guzman T. Alexander2, Parth N. Patel2,
Brian Cummings1, 2, Robert B. Aramant2. 1Physical Medicine &
Rehabilitation, University of California, Irvine, Irvine, CA; 2Sue
& Bill Gross Stem Cell Research Center, University of California,
Irvine, Irvine, CA.
Purpose: To follow the development of human fetal retinal sheet
transplants in vivo after transplantation to the subretinal space of
immunodeficient RD rats (SD-Foxn1 Tg{S334ter}3Lav = RD nude
rats; cross of NIH nude and S334ter-3 rats). Previously, rat retinal
sheet transplants restored visual responses.
Methods: Fetal human eyes (11-15.7 weeks gestation, 5 different
samples) obtained from an hSCRO-approved supplier were shipped
overnight in cold hibernation medium. After dispase treatment,
retina with RPE was dissected; and tissue sheets (size 0.8 x 1.3 mm)
transplanted on the same day to the subretinal space of 24-31 d old
RD nude rats (n=22). Transplanted eyes were imaged by highresolution spectral-domain optical coherence tomography (SD-OCT;
Bioptigen Envisu R2200 SD-OIS) at 1 wk, 1 mo, and up to 7 mo.
after transplant at several time points. Four rats with large transplants
were recorded for visual responses in the superior colliculus (SC) at
6.7-8.6 mo. post surgery. Rats were euthanized between 0.5 and 8.6
months post surgery for histological evaluation.
Results: Seven of 22 rats developed corneal ulcers which cleared up
later upon treatment with betadyne in 3 of 7 rats. SD-OCT scan analysis
showed transplant placement in the subretinal space, presence or absence
of surgical trauma, and development of laminated areas or rosettes. In 6
rats, transplants had slipped out at the insertion site. 5 rats contained only
degenerating transplants. The four 15.7 week gestation transplants all
degenerated within 3 months of transplant. In 10 of 18 transplants from
11-12 week donors, large transplants developed, with clear development
of plexiform layers first seen in OCT at 3 months post surgery. At 6-8
months post surgery, transplant photoreceptors developed short outer
segments. Immunohistochemistry confirmed the formation of retinal
layers and photoreceptor maturation of the human grafts. However, all
SC recorded rats showed either no or weak responses to bright light.
Transplant cells extended processes into the host retina, and transplanted
cells migrated into the host retina.
Conclusions: Human fetal retina (11-12 wks gestation) transplanted
as sheets to the subretinal space of RD nude rats can develop mature
photoreceptors and begin to integrate with the host retina. In the 5
animals with SC recording, such integration did not have a significant
effect on functional vision improvement in this RD rat model.
Commercial Relationships: Magdalene J. Seiler,
Ocular Transplantation LLC (P); Bryce T. McLelland,
None; Anuradha Mathur, None; Robert lin, None; De
Guzman T. Alexander, None; Parth N. Patel, None;
Brian Cummings, None; Robert B. Aramant, Ocular
Transplantation LLC (P), Ocular Transplantation LLC
Support: CIRM TR4-06648
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ARVO 2016 Annual Meeting Abstracts
Program Number: 3738 Poster Board Number: D0199
Presentation Time: 11:00 AM–12:45 PM
Optimizing subretinal injection conditions for retinal gene and
stem cell therapy
Elliott H. Sohn, Chunhua Jiao, Brittni Scruggs, Diana Brack,
Edwin M. Stone, Robert F. Mullins, Budd A. Tucker. University of
Iowa Dept of Ophthalmology, Wynn Institute for Vision Research,
Iowa City, IA.
Purpose: There is an increasing number of human gene therapy and
stem cell surgery trials that require subretinal injection to achieve
therapeutic effect. However, there is scant data to support the surgical
techniques and instrumentation used in these studies. Moreover, it is
unknown whether higher injection rates can cause retinal damage; it
is also unclear whether blebs from larger injection cannulas can still
result in retinal reattachment without air-fluid exchange. We sought to
analyze outcomes from several key components of surgery including:
1) retinal reattachment with variably sized needle-cannulas and 2)
effect of injection rate on retinal morphology.
Methods: Yucatan mini pigs underwent vitrectomy and subretinal
injection (by a surgical assistant) of 300uL of viral vector, iPSderived RPE/photoreceptor precursor cells, or control buffers using
a 31G or 41G polyamide cannula. Air-fluid exchange was not done.
Indirect ophthalmology was performed at sacrifice (POW1-POM3)
to detect retinal reattachment and RPE changes. Morphology of
eyes was assessed on H&E stained paraffin sections using light
microscopy. Immunofluorescence staining with anti-RPE65 antibody
was used to detect RPE changes in a subset of eyes with pseudoGA. Additionally, cadaveric pig eyes underwent vitrectomy and
subretinal injection of 300uL of buffer at 1.8mL/min or 0.18mL/
min using an automated injector. Eyes were immediately fixed in
4% paraformaldehyde then embedded in paraffin for morphologic
analysis.
Results: For the survival surgeries, 100% had spontaneous retinal
reattachment using both 31G (n=32 eyes) and 41G (n=32) cannulas.
RPE changes or pseudo-GA was seen in 12/32 eyes with the 41G
cannula and 22/32 eyes with the 31G cannula. Histologically, of 11
eyes with pseudo-GA seen on exam, RPE was relatively intact but
there was depigmentation of the apical villi in 6 eyes; loss of RPE65
expression was seen in 8. Of cadaveric eyes injected at a speed
of 1.8mL/min, there was significant loss of RPE cells in 5/6 eyes
compared to 0/2 injected at 0.18mL/min (p<0.05).
Conclusions: Both 31G and 41G cannulas can be used to
successfully administer subretinal injections that will spontaneously
reattach without need for air fluid exchange in pigs. Higher
bleb injection speeds may result in RPE changes or pseudo-GA
seen post-operatively on ophthalmoscopy that correlates to RPE
depigmentation and loss of RPE65 expression.
Commercial Relationships: Elliott H. Sohn, Regeneron (F),
GlaxoSmithKline (F), Oxford Biomedica (F); Chunhua Jiao, None;
Brittni Scruggs, None; Diana Brack, None; Edwin M. Stone,
None; Robert F. Mullins; Budd A. Tucker, None
Support: Wynn Institute Endowment for Vision Research
Program Number: 3739 Poster Board Number: D0200
Presentation Time: 11:00 AM–12:45 PM
Optimizing a Biodegradable Scaffold for Clinical Applications of
iPS Cell Derived RPE Tissue
Vladimir Khristov, Nathan Hotaling, Yichao Li, Haohua Qian,
Qin Wan, Ruchi Sharma, Arvydas Maminishkis, Juan Amaral,
Sheldon S. Miller, Janine Davis, Kapil Bharti. National Eye Institute,
Bethesda, MD.
Purpose: Previous work suggests replacing diseased RPE with a
healthy autologous RPE sheet can provide vision rescue for AMD
patients. Here we evaluate biodegradable and non-biodegradable
substrates for RPE sheet maturation, biocompatibility, and
degradation in vivo and in vitro.
Methods: Induced pluripotent stem (iPS) cells are differentiated
into RPE using a directed-differentiation protocol with three phases:
neuroectoderm/RPE progenitor, committed and immature RPE.
Immature RPE cells are seeded onto electrospun poly(lactic-coglycolic acid) (PLGA) or non-biodegradable polyester scaffolds.
RPE on these two scaffolds are evaluated in vitro using molecular
and electrophysiological assays and in vivo for biodegradability,
mechanical properties, and biocompatibility. Mature tissues are
implanted in a laser-injured pig eye subretinal space and evaluated
using optical coherence tomography, multifocal ERG, and histology.
Results: PLGA and polyester substrates produce functionally
similar tissues in vitro. Electrophysiological and gene expression
experiments show that RPE cells grown on PLGA scaffolds and
polyester membranes form single cell layer, develop transepithelial
resistance more than 200 ohms/cm2, and show electrophysiological
responses to [K+] and ATP changes that are similar to responses
previously observed in native human RPE. In vitro evaluation of
PLGA scaffolds showed complete degradation over 8-10 weeks,
without affecting RPE monolayer integrity. Following transplantation
into pig subretinal space, PLGA scaffold continued to degrade
without causing toxicity to the RPE monolayer. RPE monolayer on
biodegradable scaffold rescued laser-induced RPE and retina injury.
These PLGA scaffolds showed comparable integration results to
polyester implants.
Conclusions: We confirm that biodegradable substrates are suitable
for RPE transplantation in degenerative eye diseases. RPE cells
behave similar on biodegradable and non-biodegradable substrates,
forming a tissue similar to native RPE in its molecular and functional
properties. Biodegradability and biocompatibility data from a pig
model demonstrates that PLGA scaffold is safe, with the added
benefit of being resorbed by the surrounding tissue over time, leaving
no foreign material in the eye.
Commercial Relationships: Vladimir Khristov, None;
Nathan Hotaling, None; Yichao Li, None; Haohua Qian, None;
Qin Wan, None; Ruchi Sharma, None; Arvydas Maminishkis,
None; Juan Amaral, None; Sheldon S. Miller, None; Janine Davis,
None; Kapil Bharti, None
Support: NIH/NEI, NIH/NIA Intramural Research Program and
Common Fund Therapeutic Challenge Award
Program Number: 3740 Poster Board Number: D0201
Presentation Time: 11:00 AM–12:45 PM
SURVIVABILITY AND SURFACE COVERAGE OF HUMAN
RETINAL PIGMENT EPITHELIUM (RPE) ON AGED
HUMAN BRUCH’S MEMBRANE (BM) CAN BE RESTORED
USING PATCH GRAFTS of YOUNG BM
Qun Zeng1, Alex Del Priore1, Ahmet Hondur1, Lucian V. Del Priore2,
Tongalp H. Tezel1. 1Department of Ophthalmology, The Edward
Harkness Eye Institute, Columbia University, College of Physicians
and Surgeons, New York, NY; 2Department of Ophthalmology, Storm
Eye Institute, Medical University of South Carolina, Charleston, SC.
Purpose: To test whether the survivability and surface coverage of
human RPE on aged BM can be restored by repairing the age-related
defects with patch grafting BM harvested from young donors.
Methods: BM explants were harvested from freshly enucleated
(<24 hours) three aged (>65) and three young (<50) adult human
eyes. Defects in age-related macular degeneration were mimicked by
flattening the old BM on a HTTP membrane and stripping away the
RPE basal lamina mechanically. Explants were then stabilized on 4%
agarose in 30 mm petri dishes exposing the aged inner collagenous
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ARVO 2016 Annual Meeting Abstracts
layer onto which two 8-mm patch grafts of young donor BM was
attached using a biocompatible photopolymerizable gel composed of
100 mg/mL poly(ethylene glycol) diacrylate (PEG-DA) with 10 mg/
mL hyaluronic acid (HA, viscosity 5200 mP.sec). After sterilization
of the construct, 500,000 viable and synchronized ARPE-19 cells
(700 cell/mm2/22% surface coverage) were seeded. RPE cells were
fed with Fluoresbrite microsphere beads and surface coverage was
calculated by image analyses of the fluorescence microphotographs
taken at regular intervals for up to 2 weeks. RPE plated onto treated
culture plates served as control. Results were also confirmed with
light microscopy.
Results: ARPE-19 cells did not attach to agarose. Cells attached
initially onto aged BM but remained round, eventually detach from
the surface and failed to cover the aged BM. RPE cells that fell onto
grafted young donor BM revealed the high attachment rates and form
confluent sheets over the young BM patch graft on day 12. Surface
coverage on aged BM, young BM patch grafts and tissue culture
plate on days 4 (4.8±0.5%, 11.3±0.2%, 14.9±0.3%), 6 (0.1±0.01%,
57.8±0.2%, 99.9±3.6%) and 12 (0.0±0.0, 98.7±1.5%, 100±4.3%)
indicated that cells plated on young BM patches attach and cover
the surface comparable to tissue culture plastic. However, after
confluence RPE cells do not cross the boundaries between the aged
and young grafts to cover the aged BM. Grafted BM patches remain
flat and attached to aged-BM throughout RPE proliferation and
surface coverage.
Conclusions: RPE transplantation strategies should involve methods
to repair aged BM for graft survival. Age-related defects of BM
can be repaired by young BM patch grafts. These patch grafts can
increase survival and surface coverage of transplanted RPE.
Commercial Relationships: Qun Zeng, None; Alex Del Priore,
None; Ahmet Hondur, None; Lucian V. Del Priore, None;
Tongalp H. Tezel, None
Support: Supported by an unrestricted grant from Research to
Prevent Blindness, Inc, NYC; TUBITAK, Ankara, Turkey and
Sylvian Foundation New York, NY
Program Number: 3741 Poster Board Number: D0202
Presentation Time: 11:00 AM–12:45 PM
Intravitreal Autologous Bone Marrow Derived Stem Cells in
Ischemic Maculopathy Results after 12 Months Follow-up
Felipe P. Borges, Rubens C. Siqueira, Murilo W. Rodrigues,
Amanda J. Dos Santos, Andre Messias, Rodrigo Jorge.
Ophthalmology, University of Sao Paulo, Ribeirao Preto, Brazil.
Purpose: To evaluate the effects of a single intravitreal injection of
autologous bone-marrow stem cells (ABMSC) in in retinal vascular
diseases with ischemic maculophaty
Methods: Prospective, single blind, phase I/II non-randomized
clinical trial (ClinicalTrials.gov Identifier: NCT01518842),
including 15 patients (15 eyes) with ischemic maculopathy due to
diabetic retinopathy (DR), central retinal vein occlusion (CRVO)
or central retinal artery occlusion (CRAO) showing best-corrected
visual acuity (BCVA) of 0.4 logMAR or worse, absence of
active neovascularization, media opacity, prior vitrectomy and
ocular pathologies. Patients underwent intravitreal injection of
approximately 106(0,1mL) autologous bone marrow-derived
mononuclear cells (containing CD 34+ cells) and were evaluated
before injection (baseline), and monthly during follow-up. A
comprehensive ophthalmological evaluation was performed,
including BCVA measurement, full field electroretinography,
multifocalelectroretinography, microperimetry, fluorescein
angiography, SD-OCT and adaptive optics imaging.
Results: So far, 16 eyes were treated, 9 completed the 12-month
follow-up, 2 were excluded for diabetic medical complications. From
the treated eyes, 2 had macular ischemia due to arterial occlusion, 5
due to venous occlusion and 9 due to DR. Parameters were analyzed
for months 1,3, 6 and 12. No significant changes were observed for
all parameters. No significant ocular or systemic adverse effects
were observed. There was a small but no significant improvement on
BCVA in logMAR, P = 0.3655, P = 0.3655 and P = 0.3743 (1, 3 and
12 months respectively).
Conclusions: Intravitreal injection of autologous bone-marrow stem
cells (ABMSC) seem to be safe in eyes with ischemic maculopathy,
and in this small cohort was associated with no significant changes in
twelve months. A larger number of cases and a longer follow-up are
needed to confirm these findings.
Commercial Relationships: Felipe P. Borges, None;
Rubens C. Siqueira, None; Murilo W. Rodrigues, None;
Amanda J. Dos Santos, None; Andre Messias, None;
Rodrigo Jorge, None
Clinical Trial: NCT01518842
Program Number: 3742 Poster Board Number: D0203
Presentation Time: 11:00 AM–12:45 PM
Clincal trial: subretinal transplantation of CTS hESC derived
RPE in the treatment of wet Age-related Macular Degeneration
(wAMD)
Zheng Qin Yin1, Yong Liu1, Shiying Li1, Hai Wei Xu1, Yi Wang1,
Chen Qian2, Qi Zhou3. 1Ophthalmology, Southwest Hospital, Third
Military Medical University, Chongqing, China; 2Southwest Hospital,
Third Military Medical University, Chongqing, China; 3Institute of
zoology, Chinese Acadamy of Sciences, Beijing, China.
Purpose: To evaluate the safety and efficacy of Retinal pigment
epithelium (RPE) transplantation for retinal degenerative diseases
affecting the RPE-photoreceptor complex, clinically to treat the AMD
and Stargardt’s disease.
Methods: The xeno-free clinical-grade human embryonic stem
cell line (CTS hESC) was induced to RPE cells by spontaneous
differentiation method. A serious of safety assessments were
finished for potential bacteria, mycoplasma, murine viruses
according to standard protocols. The characteristics of this
CTS hESC derived RPE-specific attributes including purity,
differentiation and phagocytosis by quantitative PCR, FACS and
immunohistochemistry staining. The purified CTS hESC derived
RPE at passage 3 were used for clinical purpose. After registered
with ClinicalTrials.gov (ChiCTR-OCB-15006423), we performed
clinical study of subretinal transplantation of CTS hESC derived
RPE in treatment of wet Age-related Macular Degeneration
(wAMD).
Results: The first three patients underwent vitrectomy and removal of
choroidal neovascularization (CNV) membrane successfully. During
the three months follow-up till now, there were no rejection showed
in the clinical tests, interestingly all three patients had improved their
vision 7, 15 to 41 letters respectively in ETDRS visual acuity test
and the patients’ previous feeling of central vision blurry due to CNV
disappeared.
Conclusions: As we know this is the first study of subretinal
transplantation of hESC derived RPE in the treatment of wet
Age-related Macular Degeneration, which opened a new approach
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ARVO 2016 Annual Meeting Abstracts
for the treatment of retinal degeneration diseases by the human
embryonicstem cells in clinics.
Commercial Relationships: Zheng Qin Yin; Yong Liu; Shiying Li,
None; Hai Wei Xu, None; Yi Wang, None; Chen Qian, None;
Qi Zhou, None
Support: National Basic Research Program of China
No.2013CB967002
Clinical Trial: ChiCTR-OCB-15006423
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