The very large electrode array for retinal stimulation (VLARS)-A concept study

Abstract

Background:
The restoration of vision in blind patients suffering from degenerative retinal diseases like retinitis pigmentosa (RP) may be obtained by local electrical stimulation with retinal implants.
In this study, a very large electrode array for retinal stimulation (VLARS) was introduced and tested regarding its safety in implantation and biocompatibility. Further, the array's stimulation capabilities were tested in an acute setting.
Material and Method: The polyimide-based implants have a diameter of 12 mm, cover approximately 110 mm² of the retinal surface and carrying 250 iridium oxide coated gold electrodes.
The implantation surgery was established in cadaveric porcine eyes. To analyze biocompatibility, ten rabbits were implanted with the VLARS device, and observed for 12 weeks using slit lamp examination, fundus photography, optical coherence tomography (OCT) as well as ultrasound imaging. After enucleation, histological examinations were performed.
In acute stimulation experiments, electrodes recorded cortical field potentials upon retinal stimulation in the visual cortex in rabbits.
Results: Implantation studies in rabbits showed that the implantation surgery is safe but difficult. Retinal detachment induced by retinal tears was observed in five animals in varying severity. In five cases, corneal edema reduced the quality of the follow-up examinations. Findings in OCT-imaging and funduscopy suggested that peripheral fixation was insufficient in various animals. Results of the acute stimulation demonstrated the array's ability to elicit cortical responses.
Conclusion: Overall, it was possible to implant very large epiretinal arrays. The VLARS elicits cortical answers corresponding to the position of the retinal stimulation. The VLARS device offers the opportunity to restore a much larger field of visual perception when compared to current available retinal implants.&#13.

Full text

Journal of Neural Engineering
ACCEPTED MANUSCRIPT • OPEN ACCESS
The very large electrode array for retinal stimulation (VLARS) – a
concept study
To cite this article before publication: Tibor Karl Lohmann et al 2019 J. Neural Eng. in press https://doi.org/10.1088/1741-2552/ab4113
Manuscript version: Accepted Manuscript
Accepted Manuscript is “the version of the article accepted for publication including all changes made as a result of the peer review process,
and which may also include the addition to the article by IOP Publishing of a header, an article ID, a cover sheet and/or an ‘Accepted
Manuscript’ watermark, but excluding any other editing, typesetting or other changes made by IOP Publishing and/or its licensors”
This Accepted Manuscript is © 2019 IOP Publishing Ltd.
As the Version of Record of this article is going to be / has been published on a gold open access basis under a CC BY 3.0 licence, this Accepted
Manuscript is available for reuse under a CC BY 3.0 licence immediately.
Everyone is permitted to use all or part of the original content in this article, provided that they adhere to all the terms of the licence
https://creativecommons.org/licences/by/3.0
Although reasonable endeavours have been taken to obtain all necessary permissions from third parties to include their copyrighted content
within this article, their full citation and copyright line may not be present in this Accepted Manuscript version. Before using any content from this
article, please refer to the Version of Record on IOPscience once published for full citation and copyright details, as permissions may be required.
All third party content is fully copyright protected and is not published on a gold open access basis under a CC BY licence, unless that is
specifically stated in the figure caption in the Version of Record.
View the article online for updates and enhancements.
This content was downloaded from IP address 157.99.138.180 on 25/09/2019 at 12:14

1
The Very Large Electrode Array for Retinal Stimulation (VLARS) – A concept study
1
2
Lohmann, Tibor Karl 1*, Haiss, Florent 4,5, Schaffrath, Kim 1, Schnitzler, Anne-Christine 1,
3
Waschkowski, Florian 2, , Barz, Claudia 1,4, van der Meer, Anna-Marina 1, Werner, Claudia 1,
4
Johnen, Sandra 1, Laube, Thomas 4, Bornfeld, Norbert 4, Mazinani, Babak Ebrahim 1, Rößler, Gernot
5
1, Mokwa, Wilfried 2, Walter, Peter 1
6
7
8
1. Department of Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
9
2. Institute for Materials in Electric Engineering I, RWTH Aachen University, Aachen, Germany
10
3. Institute of Neuroscience and Medicine, INM-10, Research Centre Jlich, Jlich, Germany
11
4. Interdisciplinary Center of Clinical Research IZKF, Neuroscience Group, RWTH Aachen
12
University
13
5. Unit of Neural Circuit Dynamics and Decision Making, Institut Pasteur, Paris, France
14
6. Department of Ophthalmology, University Hospital Essen, Essen, Germany
15
16
17
18
19
20
21
*Corresponding Author: Lohmann, Dr. med. Tibor Karl, Department of Ophthalmology, University
22
Hospital RWTH Aachen, Pauwelsstraße 30, 52074 Aachen
23
E-mail: tlohmann@ukaachen.de
24
25
26
27
Keywords:
28
Retinal stimulation, retina implant, multielectrode array, cortical activation, local field potentials,
29
vitreoretinal surgery, biocompatibility
30
31
32
33
34
35
36
Page 1 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

2
37
38
Abstract:
39
Background: The restoration of vision in blind patients suffering from degenerative retinal diseases
40
like retinitis pigmentosa (RP) may be obtained by local electrical stimulation with retinal implants.
41
In this study, a very large electrode array for retinal stimulation (VLARS) was introduced and tested
42
regarding its safety in implantation and biocompatibility. Further, the array’s stimulation capabilities
43
were tested in an acute setting.
44
Material and Method: The polyimide-based implants have a diameter of 12 mm, cover approximately
45
110 mm² of the retinal surface and carrying 250 iridium oxide coated gold electrodes.
46
The implantation surgery was established in cadaveric porcine eyes. To analyze biocompatibility, ten
47
rabbits were implanted with the VLARS device, and observed for 12 weeks using slit lamp examination,
48
fundus photography, optical coherence tomography (OCT) as well as ultrasound imaging. After
49
enucleation, histological examinations were performed.
50
In acute stimulation experiments, electrodes recorded cortical field potentials upon retinal stimulation
51
in the visual cortex in rabbits.
52
Results: Implantation studies in rabbits showed that the implantation surgery is safe but difficult. Retinal
53
detachment induced by retinal tears was observed in five animals in varying severity. In five cases,
54
corneal edema reduced the quality of the follow-up examinations. Findings in OCT-imaging and
55
funduscopy suggested that peripheral fixation was insufficient in various animals. Results of the acute
56
stimulation demonstrated the array’s ability to elicit cortical responses.
57
Conclusion: Overall, it was possible to implant very large epiretinal arrays. On retinal stimulation with
58
the VLARS responses in the visual cortex were recorded. The VLARS device offers the opportunity to
59
restore a much larger field of visual perception when compared to current available retinal implants.
60
61
62
63
64
65
66
67
68
69
70
71
72
Page 2 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

3
73
1. Introduction.
74
Retinitis pigmentosa (RP) is a retinal dystrophy leading to blindness due to several mutations in genes
75
encoding for key proteins involved in the basic visual processes (1). The disease remains a leading
76
course of blindness in developed countries (2). To this day, no adequate therapy is available for the very
77
advanced stages of complete or near complete blindness. Through the course of the disease,
78
photoreceptor cell degeneration leads to progressive visual impairment, although neural cells of the
79
inner retina remain functional (3, 4). Thus, targeting these cellular structures using retinal implant
80
systems for electrical stimulation can restore meaningful visual perception in patients (5-7).
81
Targeting the retina has shown benefits in terms of surgical feasibility and a relatively low risk profile
82
compared to stimulation of the cortex or the optic nerve (8, 9). Also, the inherent retinotopy and neuronal
83
modulation of elicited phosphenes can be used to further improve the visual perception of subjects
84
implanted with retinal stimulator systems.
85
The epiretinal Argus II and the subretinal Alpha IMS resp. AMS systems are approved for clinical use in
86
the EU and the US. Currently approximately 350 patients received an Argus II (Second Sight, Sylmar,
87
CA, USA) implant and approximately 100 received the Alpha IMS resp. AMS implant (Retina Implant
88
AG, Reutlingen, Germany) (10, 11). However, using these prostheses, visual rehabilitation is limited.
89
Patients with RP are suffering from a progressive narrowing of the visual field (12). The currently
90
available systems only restore a visual field of approximately 10° visual angle requiring scanning of the
91
area either with eye or head movements (13). Studies assume that a visual field of about 27° and
92
256 electrodes are needed to obtain a visual field sufficiently granting orientation and movement, which
93
exceeds the capability of the currently available systems (13). Thus, to restore a wider visual field, larger
94
implants are necessary. We developed a flexible and thin retinal implant consisting of a multielectrode
95
array approximately three times the size of the comparable epiretinal Argus II device and mounting
96
250 electrodes for epiretinal stimulation, which we called the Very Large Array Retinal Stimulator
97
(VLARS) (14).
98
The conducted concept study was focused on the surgical feasibility and biocompatibility of the VLARS
99
structures, as well as preliminary testing its efficacy.
100
Firstly, cadaveric porcine eyes were used to establish an implantation procedure. Secondly,
101
implantations in rabbits were conducted. During a 12-week follow-up, the biocompatibility of the large
102
multielectrode array implants was examined. Thirdly, acute stimulation experiments were performed to
103
evaluate cortical responses induced by electrical stimulation of the retina.
104
105
106
107
108
Page 3 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

4
2. Materials and Methods.
109
2.1. Fabrication of the device and device parameters
110
Design, fabrication and electrochemical functionality testings of the VLARS were previously published
111
(14).
112
In brief, the VLARS multielectrode arrays (MEA) were designed using the simulation software
113
COMSOL (COMSOL AB, Stockholm, Sweden). A base layer of polyimide was spin coated on a
114
metalized silicon wafer and polymerized at 400°C. The gold leads and stimulating electrodes were
115
electroplated on the polyimide layer with a thickness of 2.5 µm and covered with another layer of
116
polyimide. The entire structure was encapsulated with the hydrophobic and biocompatible polyimide
117
Parylene C for isolation and durability. The gold electrodes were coated with platinum and reactively
118
sputtered with iridium oxide for low electrode impedance resulting in lower voltages during stimulation.
119
Figure 1 shows four schematic designs from an early stage of development: a circular, a spiral, a star,
120
and a globe shaped array.
121
Subsequent altering of the thickness of the Parylene C layer on one side of the array and introducing the
122
array to thermal contact treatment of 125°C to 155°C for five minutes resulted in an inherent curvature
123
of the devices. Depending on the annealing temperature, the time of temperature exposition, the
124
thickness of the Parylene C coating and the parameters of the deposition process, the contraction, thus
125
the inherent curvature can be modified (15). Two arrays were curved following this technique prior to
126
implantation in rabbit eyes.
127
During the implantation in cadaveric porcine eyes the star and the globe shaped structures were tested.
128
During the biocompatibility study in rabbit eyes, solely the star shaped array was implanted. Both
129
designs have a central aperture and apertures on the peripheral edges (figure 6 B, star shaped array)
130
suitable for epiretinal fixation with retinal tacks (Geuder AG, Heidelberg, Germany).
131
The arrays measure 12 mm in diameter and cover approximately 110 mm² resulting in a visual angle of
132
37.6° or a visual field of 18.8°, respectively. Both designs mount 250 individual electrodes, each having
133
a diameter of 100 µm except the larger return electrode at the base of the array’s connecting lead that
134
has a diameter of 1 mm. The electrodes’ density is higher towards the array’s center with a pitch of
135
300 µm, thus mimicking the distribution of photoreceptor and postsynaptic cells in the retina (figure 6
136
B).
137
138
Page 4 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

5
Figure 1: Schematic design of the VLARS early in development. A: early star shaped design. B early
globe shaped design. C: spiral shaped design. D: circular shaped design.
139
140
2.2. Device handling: implantation in cadaveric porcine eyes
141
To establish a safe and feasible surgery process, the implantation was initially tested in cadaveric porcine
142
eyes, that were obtained from a local abattoir. For the implantation surgery, the eyes were fixated on an
143
implantation socket. The surgery was performed using a surgical microscope (Zeiss Model OPMI 6-
144
CFR XY, S5 Tripod, Carl Zeiss AG, Jena, Germany).
145
Lensectomy was performed via a 2.5 mm corneal incision using a standard phacoemulsification
146
technique (OMNI, Fritz Ruck Ophthalmologische Systeme, Eschweiler, Germany) (Figure 2 A and B).
147
For pars plana vitrectomy, three 20 G scleral incisions were placed for the infusion, the light source and
148
vitreous surgery instruments (Fritz Ruck Ophthalmologische Systeme, Eschweiler, Germany). For
149
infusion (Figure 2 C), buffered saline solution (BSS, Alcon, Fort Worth, USA) was used. The posterior
150
capsulorhexis was performed with the 20 G cutter to eventually protrude the VLARS structure from the
151
anterior chamber towards the posterior pole of the retina after completing the vitrectomy. To support
152
retinal attachment and safely lower the VLARS on the posterior pole, perfluocarbon liquid (PFCL) (F-
153
Decalin 1.93 g/cm³ Fluoron GmbH, Ulm, Germany) was injected into the eye. The VLARS was pushed
154
into the anterior chamber using the implantation cone positioned in the enlarged corneal opening
155
(enlarged to approx. 5 to 6 mm) (Figure 2 E). The implantation cone was a modified pipette tip
156
(Eppendorf AG, Hamburg, Germany). To reduce adhesion of the array to the cone’s inner surface it was
157
filled with a cohesive viscoelastic fluid (Healon, Johnsen and Johnson, New Brunswick, USA). The
158
corneal incision was sutured with Nylon 10-0 threads (Alcon, Fort Worth, USA). Once inside the eye,
159
the array was unfolded, displaced from the anterior chamber towards the vitreous cavity and sunk on
160
the PFCL bubble placed covering the posterior pole (Figure 2 F). Removing of the PFCL with a 32 G
161
cannula led to the positioning of the VLARS. The VLARS was fixated on the posterior retinal pole by
162
inserting a titanium retinal tack (Fa. Geuder, Heidelberg, Germany) through the array’s central aperture
163
(Figure 2 J). Once the intraocular manipulators and the infusion were removed, the pars-plana incisions
164
were sutured with Vicryl 7-0 threads (Alcon, Wort Worth, USA). The final position of the retinal tack
165
and the VLARS was reassured using a 60 D lens (Kilp lens 60D, 20°, Geuder AG, Heidelberg, Germany)
166
(Figure 2 K).
167
168
Page 5 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

6
Figure 2: Implantation procedure of the VLARS in a cadaveric porcine eye. Note that D to F show
the implantation of the globe shaped array in comparison to the implantation of the star shaped
array shown in G to L.
A and B: Phacoemulsification of the lens; C: Fixation of the 20 Ga infusion;
D to F: Insertion of the globe shaped VLARS using the implantation cone.
D: The globe shaped array on top of the implantation cone. E: Pushing the array forward with the
retinal tack holder (arrow). F: Poor positioning of the array in the anterior chamber (arrow shows
overlapping peripheral apertures).
G to I: Insertion of the star shaped VLARS using the implantation cone.
G: The star shaped array on top of the implantation cone. The retinal tack is pushed through the
aperture to move the array through the cone (arrow). H: Pushing the array forward with the retinal
tack holder (arrow). Note the concentric folding of the VLARS.
Page 6 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

7
I: Good positioning of the array in the anterior chamber. A wing of the star shaped array is clearly
observable on the right side (arrow).
J: Inserting the retinal tack; K: Retinal tack in central aperture fixating the VLARS on the retinal
pole (as seen through a 60D Kilp’s lens), L: VLARS in position on the posterior retinal pole.
169
2.3. Implantation in rabbit eyes
170
All animal experiments, both in the semi-chronic implantation and the acute stimulation experiments,
171
were performed according to the ARVO declaration for the use of animals in research and adhered to
172
the “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985), the OPRR Public
173
Health Service Policy on the Human Care and Use of Laboratory Animals (revised 1986) and the U.S.
174
Animal Welfare Act, as well as according to the German Law for the Protection of Animals and after
175
obtaining approval by local authorities and ethics committee.
176
Inactive arrays were used to test the surgical implantation and biocompatibility in-vivo. The stimulators
177
were implanted into the right eye of ten rabbits (four Chinchilla Bastard, six New Zealand White). The
178
animals were housed under standard conditions with an even twelve-hour light/dark cycle and had
179
access to food and water ad libidum. Implantation was performed following the protocol established
180
during the implantation experiments in cadaveric porcine eyes, as described above.
181
Prior to the surgery, the rabbits received topical anesthesia by applying proxymetacaine hydrochloride
182
0.5% eye drops (Proparakain-POS, Ursapharm, Saarbrücken, Germany), as well as dilating eye drops
183
containing phenylephrine hydrochloride 2.5% and tropicamide 0.5% (MS-mydriatic eye drops,
184
Pharmacy of the RWTH Aachen University, Germany).
185
During the surgery in Chinchilla Bastard rabbits, 4 to 5 mg per kg bodyweight xylazine (Xylazin 2%
186
Bernburg®, Medistar, Ascheberg, Germany) and 50 to 70 mg per kg bodyweight ketamine (Ketamin
187
10%, Ceva Tiergesundheit GmbH, Düsseldorf, Germany) were used as anesthetics. For the New Zealand
188
White rabbits, 0.1 mg per kg bodyweight xylazine and 20 mg per kg bodyweight ketamine were used.
189
The surgical field was treated with 10% povidone-iodine solution (Betaisodonna, Mundipharma GmbH,
190
Limburg, Germany) for disinfection.
191
To adjust the eye position during surgery, incisions in the conjunctiva were performed, two opposing
192
straight ocular muscles were hooked and looped with Ethilon 5-0 threads (Alcon, Fort Worth, USA).
193
The sclerotomies were done in 1.5 mm distance to the limbus. During 20 G vitrectomy,
194
triamcinoloneacetonide 10 mg/ml (Volon A 10 mg-Kristallsuspension, Dermapharm AG, Grünwald,
195
Germany) was injected to facilitate vitreous removal. The array was advanced into the anterior chamber
196
via the implantation cone shown above, as well as advanced directly through the corneal incision using
197
various surgical devices: a tear duct probe, a push-pull manipulator, as well as several types of surgical
198
forceps (Geuder AG, Heidelberg, Germany). After placing the VLARS on top of the PFCL bubble on
199
Page 7 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

8
the posterior retinal pole, the array was fixated using a titanium retinal tack (Fa. Geuder, Heidelberg,
200
Germany), as described above. To complete the surgery the eye was filled with air.
201
At the end of the surgery, the animals received an intracameral injection of 750 mg cefuroxime
202
(Cefuroxim Fresenius 750 mg, Fresenius Kabi DE, Bad Homburg, Germany) and 4 mg
203
dexamethasondihydrogenphosphat-Dinatrion (Fortecortin Inject 4 mg, Merck KGaA, Darmstadt,
204
Germany), as well as subconjunctival injection of gentamicin 8 mg (Gentamicin 8 mg Rotexmedica,
205
ROTEXMEDICA GmbH Arzneimittelwerk) and 50 mg prednisolon-21-succinat (Prednisolon H
206
50 mg, Merck KGaA, Darmstadt, Germany) to prevent infection and reduce inflammation.
207
Clinical examinations evaluating appearance and behaviour of the animals were conducted daily. 20 mg
208
of the nonsteroidal anti-inflammatory drug carpofen (Rimadyl 20 mg, Zoetis GmbH, Berlin, Germany)
209
was given once a day for three days after the surgery. Antibiotic and anti-inflammatory ointment and
210
eyedrops (Isopto-Max Augensalbe, Dexamethason 1mg/g, Neomycin 3500IE/g, Polymyxin-B-sulfat
211
6000IE/g, Novartis Pharma, Basel, Switzerland) as well as mydriatic eyedrops (MS-mydriatic eye drops,
212
Pharmacy of the RWTH Aachen University, Germany) to prevent synechia were applied twice daily for
213
at least seven days, and in some cases longer depending on presence of inflammation.
214
215
2.4. Follow-up in the semi-chronic implantations
216
The follow-up was conducted for twelve weeks, using slit lamp examination, funduscopy, ultrasound
217
imaging, spectral domain optical coherence tomography (SD-OCT) and fundus photography. They were
218
conducted in general anaesthesia using ketamine and xylazine as described above. The animals
219
underwent these follow-up diagnostics 1, 2, 4, 6, 8 and 12 weeks after implantation.
220
2.4.1. Slit lamp examination and funduscopy
221
For a clinical evaluation of the anterior segment a manual slit lamp was used (Bonnoskop II Mod. 66,
222
hand piece: Carl Zeiss AG, Jena, Germany). Funduscopy was achieved using a 20D lens (Volk Optical
223
Inc., Mentor, USA). Main points of interest were signs of inflammation or infection around the
224
implanted eye, corneal clarity, signs of inflammation in the anterior chamber, presence of hyphema as
225
well as vitreal blood, fundus visibility, position and fixation of the VLARS.
226
2.4.2. Fundus photography
227
For fundus photography, a Zeiss FF450Plus camera system (Carl Zeiss AG, Jena, Germany) with a
228
Canon EOS 5D digital camera capturing system (Canon Inc., Tokyo, Japan) was used. In cases of good
229
visibility, the array’s position was determined by the fixation of the retinal tack and the displacement of
230
the array’s wings.
231
2.4.3. Optical coherence tomography
232
SD-OCT was performed with a Spectralis OCT system (Heidelberg Engineering, Heidelberg,
233
Germany). Cross-sectional images were taken in the periphery as well as in the center of the device, if
234
possible. En-face images were taken using confocal infrared imaging with a wavelength of 715 nm.
235
Page 8 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

9
2.4.4. Ultrasound imaging
236
Ultrasound imaging was performed with a 10 Mhz B-scanning probe (Aviso S, Quantel Medical,
237
Cournon d'Auvergne Cedex, France). It was used in cases of severe corneal edema or vitreal
238
haemorrhage to evaluate the position of the VLARS and possibly identify retinal detachment and
239
tearing, respectively.
240
241
After the last follow-up examination, euthanasia was induced using an overdose of a 2 mg/kg dose of a
242
barbiturate (Narcoren, Merial GmbH, Hallbergmoos, Germany). Death was confirmed both clinically
243
and by electrocardiographic monitoring.
244
245
2.5. Histology
246
2.5.1. Hematoxylin and eosin staining
247
Histology was performed as described by Rösch et. al. (16). After enucleation, the eyes were fixated,
248
punctured and immersion fixated for 30 minutes using 4% paraformaldehyde (PA) in 0,1 M phosphate
249
buffer (PB) at room temperature. A tissue dehydration automat (mtm, Slee, Mainz, Germany) was used
250
to dehydrate the ocular tissue using a series of increasing ethanol concentrations (twice with 70%, twice
251
with 96%, three times with 100%, one hour each), followed by xylene (three times in one hour) and
252
paraffin (four times in one hour). Prior to the next steps of histological preparation, the VLARS and the
253
retinal tack were carefully removed after performing a circular incision in the anterior segment of the
254
eye. The ocular tissue was embedded in paraffin and cut in sections of 5 µm thickness using a microtome
255
(R. Jung GMBH, Heidelberg, Germany). The position was chosen to capture tissue beneath the VLARS.
256
After deparaffinization and rehydration, standard hematoxylin and eosin staining ensued. The images
257
were taken using a microscope with an integrated image capturing system (Leica DMRX microscope,
258
Leica Biosystems, Wetzlar, Germany).
259
2.5.2. Resin embedding and thin-grind cutting
260
Five eyes were prepared for an embedding in the plastic Technovit 7200 VLC for thin-grind cutting at
261
the location of retinal tack protrusion. Technovit 7200 VLC (Kulzer GmbH, Hanau, Germany) is a one-
262
component adhesive based on glycolmethacrylat. Fixation of the eyes was performed as described
263
above. For infiltration of the tissue a 1:1 mixture of alcohol and Technovit 7200 (Kulzer GmbH, Hanau,
264
Germany), followed by pure Technovit 7200 was used in a light-proof container. The posterior segment
265
of the eye including the array and retinal tack were embedded parallel to the plane of interest into the
266
embedding mould of the Technovit system. The area to be examined was facing downwards, the entirety
267
of the tissue was covered in Technovit 7200 without trapping air or elevating the tissue from the base of
268
the mould. Polymerization was done using low light intensity and temperatures below 40°C, while light
269
with a higher intensity was applied during the second stage to cause polymerization in the infiltrated
270
tissue. Cutting and grinding of the embedded tissue was performed with the EXAKT tissue preparation
271
Page 9 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

10
devices (EXAKT 300 CP, EXAKT 400 CS, EXAKT Advanced Technologies GmbH, Norderstedt,
272
Germany). Each slice of embedded tissue was fixated to an acrylic glass holder using the precision
273
adhesive Technovit 7210 VLC. Each slice had a thickness of 100 µm. Images were taken with a Leica
274
DMRX microscope (Leica Biosystems, Wetzlar, Germany).
275
276
2.6. Surgery for acute stimulation experiments in rabbit eyes
277
The acute stimulation experiments were conducted in two pigmented Chinchilla Bastard rabbits.
278
General anesthesia was obtained by isoflurane (Abbott GmbH & Co. KG, Wiesbaden, Germany) at 4%
279
and maintained at 2% to 2.5%. The animals were intubated with a 2.5mm tracheal tube (Rüsch,
280
Rüschelit® Super Safety Clear, Sulzbach, Germany). For analgesia, a perfusion of fentanyl 0.5 mg/ml
281
(fentanyl 0.5 mg-rotexmedica, ROTEXMEDICA GmbH Arzneimittelwerk) at 0.1 ml/h to 0 7ml/h was
282
preserved.
283
During surgery the animals were fixated in a stereotactic frame. The points of contact of the frame and
284
the animal’s head were anesthetized with a subcutaneous injection of bupivacainehydrochloride 0.5%
285
(Bupivacain-RPR-Actavis 0.5, Actavis, Luxemburg). To assess the VLARS’ capability to elicit cortical
286
responses upon electrical epiretinal stimulation, a 32-channel silicone probe for cortical recording -
287
consisting of four shanks with eight electrodes arranged linearly on each shank (Figure 6 A) - was
288
carefully advanced into the V1 visual cortex (E32-150-S4-L10-10.5-1400-1200, ATLAS
289
Neuroengineering, Leuven, Belgium) after a craniotomy over the area of interest was performed. The
290
iridium oxide recording electrodes used for cortical recordings have a diameter of 35 µm. The length of
291
the shafts is 10.5 mm and 10 mm for the inner two and outer two respectively. The pitch of the shafts is
292
1.2 mm and 1.4 mm for the inner two and outer two respectively. Each shaft has eight electrodes starting
293
from the tip with an inter-electrode distance of 150 µm. The four shafts have a width of 140 µm and a
294
thickness of 50 µm. Reference and ground of the recording system were connected to a silver wire that
295
was placed at the edge of the craniotomy. The silver wire and the craniotomy were covered with low
296
melting point agarose (4% in Ringer solution, Sigma Aldrich, Darmstadt, Germany) to prevent
297
dehydration of the neocortex.
298
After obtaining cortical responses upon visual stimulation by a light flash, the cortical recording
299
electrode was temporarily removed. The animals were then rotated out of the recording position so that
300
the eye was positioned suitable for vitreoretinal surgery underneath a microscope (Zeiss Model OPMI
301
6-CFR XY, S5 Tripod, Carl Zeiss AG, Jena, Germany).
302
The vitreoretinal surgery was performed following the protocol established for the semi-chronic
303
implantation as described above, except changing the 20 G ports to a 23 G port system.
304
Prior to inserting the active VLARS, the anterior chamber was filled with a viscoelastic fluid (Healon,
305
Johnsen and Johnson, New Brunswick, USA) and the corneal incision was enlarged to 5 mm. Then, the
306
array was advanced into the anterior chamber with the cable connector for the stimulator resting in the
307
Page 10 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

11
incision. The incision was sutured with two Vicryl 6-0 sutures (Alcon, Wort Worth, USA).
308
Subsequently, the active VLARS was advanced on top the PFCL phase on the posterior retinal pole.
309
The PFCL was carefully removed and the implant was lowered towards the retinal surface. The residual
310
PFCL was removed to prevent isolating effects. During the acute stimulation experiment, the VLARS
311
was not fixated using retinal tacks to reduce the risk of retinal damage.
312
For the following air tamponade, the air pressure was set to 30 mmHg with the vessels at the optic disk
313
clearly perfused. After fixation of the connector cable at the stereotactic frame the animal was gently
314
rotated back to the recording position. Afterwards, the correct position of the implant was confirmed by
315
indirect fundoscopy with a 20D lens (Volk Optical Inc., Mentor, USA).
316
After finalization of the acute stimulation and recording experiments, the animals were sacrificed by a
317
lethal dose of fentanyl 0.5 mg/ml (fentanyl 0.5 mg-rotexmedica, ROTEXMEDICA GmbH
318
Arzneimittelwerk).
319
320
2.7. Cortical recordings in an acute stimulation setting
321
During stimulation, up to 24 electrodes on the VLARS were active. In the experiment displayed here,
322
electrodes of two clusters were used: the blue cluster (Figure 6 B, blue circle S1) contained four active
323
electrodes, while the red cluster (Figure 6 B, red circle S2) contained nine active electrodes. A biphasic
324
stimulation pulse repeated three times with 90 µA per electrode in the blue cluster and 80 µA per
325
electrode in the red cluster for 200 µs at a frequency of 200 Hz was chosen.
326
Data were acquired with a Multichannel USB recording system (ME32-FAI-µPA-System, Multi
327
Channel Systems, Reutlingen, Germany) and digitized at 25 kHz.
328
To obtain local field potentials (LFPs), data were down-sampled to 5 kHz and low-pass filtered
329
(Butterworth, 2nd order, 300 Hz cutoff frequency) using MC_Rack software (Multichannel Systems
330
MCS GmbH). LFPs were averaged across all trials with Spike2 software (Cambridge Electronic Design).
331
To assess, if LFP responses to the stimulus were significant, baseline activity was measured 100 ms
332
before stimulus onset and compared to responses 0.012 s to 0.15 s after stimulus onset (paired t-test,
333
Bonferroni-corrected; Microsoft XLSTAT). To determine if retinal stimulation elicited differential
334
responses in different recording locations, responses were first normalized to the absolute value of the
335
response amplitude within each data set. Then response amplitudes were determined as the minimum
336
values during a 0.012 s to 0.15 s window after stimulus onset and compared across stimulation
337
conditions (unpaired t-test, Bonferroni-corrected).
338
339
3. Results
340
3.1. Implantation surgery and handling of the device in cadaveric porcine eyes
341
The implantation of the VLARS in cadaveric porcine eyes had the purpose to establish a regimen for
342
the implantation in the in-vivo biocompatibility study with suitable duration and a high safety level. Up
343
Page 11 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

12
to the actual implantation of the VLARS, the techniques applied did not exceed techniques of standard
344
vitreoretinal surgery. Cadaveric porcine eyes beneficially share an equal size with human eyes and in
345
the cadaveric surgery, bleeding and the development of proliferative membranes as seen in porcine eyes
346
after in-vivo surgery have not to be considered (17).
347
Additionally, the best method of safely introducing the large array into the eye had to be discovered and
348
tested. Ideally, the array is folded to temporarily reduce its surface area, thus decreasing the size of the
349
corneal incision to the diameter of the implantation cone’s most anterior segment (approx. 5 mm). The
350
star shaped array folded concentrically, while the globe shaped stimulator was rolled up. The arrays
351
were advanced using the retinal tack holder or surgical forceps with silicone tips suitable for handling
352
electrical devices (Geuder AG, Heidelberg, Germany). During surgery, it became obvious, that the globe
353
shaped array did not properly fold as expected, thus leading to an uncontrolled opening and difficult
354
handling inside the implantation cone and the anterior segment of the eye, respectively. Figure 2 D to F
355
shows the process of introducing the globe shaped array into the anterior chamber. As seen in figure 2 E
356
and F, the globe shaped VLARS does not fold concentrically. These findings lead to abandoning the
357
globe design in later stages of this study. The insertion of the star shaped array by using the implantation
358
cone is depicted in figure 2 G to I. Being able to fold the star shaped structure concentrically proved to
359
be a major advantage over the globe shaped array granting easier handling and a controlled passage into
360
the anterior chamber.
361
In cadaveric porcine eyes, corneal opacity depends on the duration between enucleation and beginning
362
of the surgery. In our study, we did not encounter severe corneal opacity. Lensectomy and pars-plana
363
vitrectomy were conducted in a suitable duration and did not cause adversities. Insufficient suturing and
364
an overly wide corneal incision caused low intraocular pressure in some cases. Reduced composure of
365
the eye can lead to choroidal swelling in an in-vivo setting and to premature contact of the array with
366
the retinal surface.
367
Overall, our group introduced an implantation surgery, which could be transferred to the semi-chronic
368
in-vivo study in rabbits.
369
370
3.2. Feasibility and safety of the implantation procedure in the implantations in rabbits
371
In the in-vivo implantation study in rabbits, safe handling and retinal fixation of the array rendered to be
372
the most challenging aspects. Table 1 collects the results of the implantation surgeries and the results
373
gathered during the follow-up period. The most important findings concerning surgical feasibility and
374
the follow-up examinations are summed up in the next paragraphs.
375
In the first implantation, the corneal incision applied for the phacoemulsification was enlarged to
376
approximately 10 mm. Thus, enabling better mobilization of the cone and the VLARS yet causing a loss
377
of ocular stability. The implantation cone did not prove to be significantly beneficial compared to the
378
use of the surgical instruments directly inserting the array through the corneal incision. By using these
379
Page 12 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

13
instruments, the corneal incision did not have to be further enlarged from the initial 5 mm, thus granting
380
a better ocular composure, shorter time of implantation and easier handling. The implantation cone was
381
ultimately abandoned after the first in-vivo implantation.
382
Positioning and fixation of such large stimulating arrays into rabbit eyes were challenging. In six of the
383
ten rabbits a stable epiretinal position at the posterior pole could be achieved at the end of the surgery
384
using the central aperture for the retinal tack. In three subjects it was not possible to use the central tack
385
aperture (aperture was located directly over the optic disc/aperture was ripped during implantation
386
procedure), which resulted in fixating the array with two peripherally placed tacks in one animal (animal
387
6), and one peripherally placed tack in two animals (animals 4 and 7). In two of these subjects (animals
388
4 and 6), the array was not fixated sufficiently at the end of the implantation surgery, yet the study was
389
continued to examine the effects on the eye. In one animal, the central retinal tack did not grant epiretinal
390
fixation (animal 10). During the fourth week follow-up examination of this animal, severe retinal
391
detachment, as well as a complete dislocation of the array were detected, leading to the immediate
392
termination of the follow-up and euthanasia.
393
Primary retinal detachment and retinal tears were observed as severe adverse events. These occurred in
394
five of ten rabbits in varying severity (table 1). Retinal tears were caused by the sharp edges of the large
395
arrays harming the retina either during positioning of the array at the posterior pole or during phases of
396
hypotony with temporary involution of the eye. Out of these cases, two animals (animals 4 and 10)
397
experienced a larger retinal detachment, while three animals (animals 5, 6 and 7) experienced focal
398
retinal tearing. In one animal, the accidental contact of the infusion with the opposing retinal surface
399
during a period of low intraocular pressure caused severe retinal tearing and eventually retinal
400
detachment (animal 10).
401
Intravitreal bleeding varied in its severity, yet noteworthy bleeding occurred in three eyes (animals 3, 6,
402
and 7). The bleeding was staunched in all cases and did not jeopardize the ongoing implantation. One
403
eye (animal 4) suffered from subretinal hemorrhage. One eye (animal 9) suffered from a choroidal
404
detachment, which vanished over the cause of the follow-up.
405
Using PFCL as a liquid cushion to support the stimulator seemed mandatory in order to avoid
406
uncontrolled movement of the device within the globe although removing the PFCL bubble from
407
underneath the array was difficult and time consuming. Tangential movements of the stimulator put the
408
retina at risk for retinal tears. Overall, the implantation procedure is feasible, yet challenging on different
409
levels. The mean duration for the implantation surgery was approximately two hours, decreasing over
410
the course of the study.
411
412
3.3. Clinical follow-up in the semi-chronic implantation in rabbits
413
Clinical follow-up over twelve weeks could be achieved in eight of ten animals. In two animals, the
414
follow-up phase was terminated after four weeks: one animal died in anaesthesia during the control
415
Page 13 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

14
examination (animal 4), the follow-up of the other animal was terminated because of a severe retinal
416
detachment and dislocation of the VLARS, as mentioned above (animal 10).
417
Throughout the 12-week follow-up period, no significant intraocular inflammation or endophthalmitis
418
was observed. One animal showed a fibrinous reaction and hyphema after implantation, in addition to a
419
severe corneal edema (animal 8). Another animal showed epithelial vascularisation and a severe corneal
420
edema (animal 4).
421
Overall, significant corneal edema with tendency to clear over time occurred in five rabbits (animals
422
1,4,6,7 and 8), with three cases of a severe edema likely due to the relatively large corneal incision
423
(animals 4, 7 and 8). Treatment of the edema consisted of an ointment of 1 g of hydrocortisone-acetate
424
and glucose 70% (HCG-Augensalbe. Pharmacy of the RWTH Aachen University, Germany). The
425
edema disappeared gradually under treatment, yet it highly interfered with funduscopy, fundus
426
photography and OCT-imaging in the follow-up examinations.
427
The quality of OCT-imaging was not only reduced by the opacity of the cornea, but also by diffuse
428
reflection of the array’s surface, the gold wiring and electrodes, respectively. Depending on the
429
condition of the cornea, analysis of epiretinal alignment during the follow-up phase was therefore
430
conducted by using summarized information of all analyzing methods: funduscopy, optical coherence
431
tomography and ultrasound imaging.
432
The overall analysis of epiretinal alignment during the complete clinical follow-up can be categorized
433
in three categories: good (complete to 2/3 contact of the stimulator), medium (2/3 to 1/3 contact of the
434
stimulator) and poor (< 1/3 contact of the stimulator). Good epiretinal contact could be achieved in two
435
animals (animals 2 and 3 [until premature death]), medium in four animals (animals 1,4,7 and 9) and
436
poor in four animals (animals 5,6, 8 and 10). Overall, epiretinal alignment turned out to be good even
437
after surgical complications, as well as be poor due to later complications during the follow-up period.
438
Figure 3 A to D depicts four observations obtained with OCT-imaging in two animals at different time
439
points. While figure 3 A shows proper alignment in the periphery, the array is distant to the retinal
440
surface in figure 3 B. Figure 3 C shows protrusion of the array into the retina in the same animal, while
441
in figure 3 D the retina experiences stress under the radial pressure of the array.
442
In figure 3 E to F, fundus photography 12 weeks after implantation in two animals is shown. The retinal
443
tack is observable in the central aperture. Note the difference in reflectance hinting to varying alignment
444
to the retinal surface.
445
Page 14 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

15
Figure 3: OCT-imaging and fundus photography of the VLARS after implantation.
A to D: En-face infrared (left side) and OCT-imaging (right side) of the implanted VLARS. The
arrow in the infrared image on the left-hand side indicates the position and direction of the OCT-
image.
A (Animal 2): Close alignment of the peripheral segment of the array (arrow) to the retinal surface
(one week after implantation). Note subretinal fluid (asterix) and the artifact casted by the gold edging
of the peripheral aperture (diamond sign).
B (Animal 1): Gap between the retinal surface and the peripheral segment of the array (triangle) (7
weeks after implantation).
C (Animal 1): Protrusion of the array into the retina (arrow) (twelve weeks after implantation).
Page 15 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

16
D (Animal 2): The array’s edge puts pressure on the retinal surface (arrow) (one week after
implantation). Note subretinal fluid (asterix).
E and F: Fundus photography twelve weeks after implanting the VLARS.
E (Animal 2) and F (Animal 5): The VLARS on the retinal pole. Note the difference in reflection of
the VLARS’ surface hinting an elevation of the wings. The retinal tack is highlighted with an arrow.
446
3.4. Open-sky imaging and histological analysis
447
Prior to the histological staining and resin embedding, open sky imaging was performed. This allowed
448
us to determine the retinal fixation of the VLARS and possible damage on the retina. Figure 4 A shows
449
good epiretinal positioning of a star shaped VLARS. The retinal tack is positioned in the central aperture.
450
The results obtained from the open sky examination varied. After twelve weeks, five out of the ten
451
implanted eyes showed insufficient fixation of the VLARS and dislocation of the retinal tacks. In one
452
eye, retinal detachment was observed, which had not been seen during the implantation surgery or the
453
follow-up. However, it is not clear, if dislocation of either tack or the VLARS could possibly be caused
454
by the preceding treatment for histological staining or the traumatic opening of the eye.
455
Figure 4 B shows the result of grinding the embedded eye upon the section of the tack penetrating the
456
retina. In this implantation surgery, the tack was inserted into a peripheral aperture (figure 4 B, top right
457
corner). The tack protrudes intentionally into the eye’s sclera as its desired position. Moderate scar
458
tissue is found around the retinal penetration as indicated by the asterix in figure 4 B. The findings were
459
also observable in the other eyes which underwent resin embedding (data not shown).
460
Figure 4 depicts the hematoxylin and eosin staining 12 weeks after implantation. The sections shown in
461
figure 5 A was taken from underneath a wing of a star shaped array, while the section in figure 5 B is
462
taken from underneath the array’s center.
463
Both sections show moderate vacuolization, predominantly in the outer and inner nuclear layer. In
464
figure 5 A, no signs of inflammation or cellular migration can be observed. The layers of the
465
neurosensory retina seem intact, the shown detachment is likely caused by the preceding fixating
466
treatment. In figure 5 B, the retinal structure seems disturbed, the retinal layers are degenerated,
467
compatible to histological findings in retinal detachment. While in open-sky imaging retinal detachment
468
was not decisively observable, preretinal gliosis was found underneath the array. The findings are
469
consistent in the obtained sections of other implanted eyes (data not shown).
470
471
Page 16 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

17
472
Figure 5: Hematoxylin and eosin staining twelve weeks after implantation.
A (Animal 1): The histological section depicted was located underneath a wing of the star shaped
VLARS. The section shows moderate vacuolization in the inner nuclear layer (dashed arrow) and the
outer nuclear layer (arrow), as well as preretinal gliosis (diamond sign). The layers of the retina are
intact.
Figure 4: Open-sky imaging and epoxy-resin embedding.
A (Animal 1): Open-sky imaging of the VLARS. The white arrow indicates the central position of
the retinal tack. The enucleation took place twelve weeks after the implantation.
B (Animal 7): Epoxy-resin embedding of the retinal tack and the VLARS on the retina. The epoxy-
resin embedding took place twelve weeks after implantation. The retinal tack (black arrow) protrudes
into the sclera. In this eye, a peripheral aperture was used to insert the retinal tack (top right picture,
the white arrow indicates the retinal tack). The diamond sign indicates the space between the VLARS
array above and the retina beneath. The paragraph sign indicates the sclera. The asterixis indicated
moderate scar tissue around the penetrating area of the retinal tack.
Page 17 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

18
B (Animal 1): The histological section depicted was located closely to the center of the star shaped
VLARS. The section shows moderate vacuolization in the inner nuclear layer (dashed arrow) and the
outer nuclear layer (arrow), as well as preretinal gliosis (diamond sign). Note the degeneration of the
retinal structure, presumably caused by a retinal detachment. In both sections, the subretinal space is
marked with the asterixis.
473
Table 1: Results of the semi-chronic implantation of the VLARS in ten rabbits.
474
Animal
Method of
Implant-
ation
Retinal
Tack/Tack
fixation
during
implantatio
n
Adverse events during
surgery
Follow-Up
Fundus imaging
OCT-imaging
Open-sky
imaging
1 CB
Flat
VLARS
cone
central/
stable
moderate (difficulty
retracting PFCL from
underneath the array)
moderate adverse
events (corneal
edema, retinal
penetration of array)
Bad visibility from
weeks 2 to 7
partly distant to retinal
surface, partly
protruding into the
retina (figure 3 B/C)
array fixated, mild
gliosis beneath array
(figure 4 A)
2 A
Flat
VLARS
cannula,
push-pull
manipulator
central/
stable
mild (mild choroidal
bleeding)
mild adverse events
(slight retinal
penetration of array,
gliosis)
good visibility (figure
2 E)
epiretinal position in
week 1 (figure 3 A/D)
array fixated, mild
gliosis beneath array
3 A
Flat
VLARS
cannula,
anatomical
forceps
central/
stable
mild (mild intravitreal
bleeding)
mild adverse events
(slight retinal
penetration of array,
gliosis)
†
good visibility
†
epiretinal alignment
†
array fixated, wings
adjacent to retinal
surface
†
4 A
Flat
VLARS
tear duct
probe
central* and
peripheral/
dislocation
of central
tack,
unstable
fixation
severe
(subretinal/choroidal
bleeding,
phacoemulsification
malfunction causing
corneal damage, retinal
tearing adjacent to
array, dislocation of
central tack due to tear
in central aperture)
severe adverse events
(severe edema with
vascularization,
intravitreal bleeding,
dislocation of retinal
tack, hyphema)
bad visibility over
course of 12 weeks
due to corneal edema
and vitreal bleeding
array partly distant to
retinal surface,
week 12: bubble of
heavy liquid beneath
array
array fixated, retinal
protrusion, gliosis
5 A
Flat
VLARS
rhexis
forceps
central/in
position,
slightly
loose
moderate (retinal tearing
adjacent to array)
mild adverse events
(moderate injection of
conjunctiva)
good visibility (figure
3 F)
low quality
array fixated, wings
distant to surface,
gliosis
6 A
Flat
VLARS
tear duct
probe
two
peripheral
nails/
dislocation
of one tack,
unstable
fixation
moderate (use of two
tacks, mild vitreal
bleeding, peripheral
retinal detachment
Moderate adverse
events (corneal
edema, low IOP,
shallow anterior
chamber)
bad visibility after
first week due to
vitreal bleeding
week 8: array partly
distant to retinal surface
bad fixation, only one
peripheral tack,
gliosis
7 CB
Flat
VLARS
anatomical
forceps
one
peripheral
tack*2/
stable
moderate (vitreal
bleeding after inserting
first peripheral tack,
dislocation of peripheral
tack, retinal tearing
adjacent to array)
moderate adverse
events (severe corneal
edema, slight retinal
penetration of array,
gliosis, retinal
wrinkling)
good visibility
low quality
array fixated,
peripheral tack, 1/3
of wings distant to
retinal surface
8 CB
Flat
VLARS
tear duct
probe
central/
initially
stable
mild (relatively large
corneal incision)
severe adverse events
(severe edema with
vascularization,
fibrinous reaction
anterior chamber,
hyphema, dislocation
bad visibility through
course of 12 weeks
low quality
array not fixated,
gliosis
Page 18 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

19
of retinal, retinal
bleeding, tack
dislocation?)
9 A
Curved
VLARS
anatomical
forceps
central/
stable
mild (choroidal
detachment)
mild adverse events
(slight vitreal
bleeding during the
first week after
implantation)
bad visibility in week
1 due to vitreal
bleeding
low quality, capture part
of the array distant to
retinal surface
array fixated,
coagulated
hemorrhage on array
10 CB
Curved
VLARS
anatomical
forceps
central/
dislocation
of entire
array, yet
tack in
central
aperture
moderate (infusion
caused tearing of
peripheral retina during
period of hypotony,
retinal tack repositioned
multiple times)
severe adverse events
(complete retinal
detachment)
†²
good visibility
†²
low quality, capture part
of the array distant to
retinal surface
array not fixated
†²
CB: Chinchilla bastard rabbit
NW: New Zealand White rabbit
*1: Central tack dislocated during implantation surgery.
*2: Second peripheral tack dislocated during implantation surgery.
†: Animal died during anesthesia at the four week follow-up examination.
†²: Termination of follow-up due to complete dislocation of the epiretinal array.
475
3.5. Findings of the implantation surgery in the acute stimulation
476
The acute stimulation experiments took place after the implantation surgeries of the ten rabbits were
477
concluded, thus the protocol was refined and the surgical tools were established.
478
Switching to a valved 23 Ga port system for the pars-plana vitrectomy did not cause any adverse effects
479
but instead seems to give a greater stability of the eye pressure during the procedure.
480
To grant a stable position of the animal in the acute stimulation experiment the animals are fixated in a
481
stereotactic frame. This set-up allowed us to safely maneuver the animal back and forth from a position
482
suitable for retinal stimulation and cortical recording to a position suitable for surgery. Any movement
483
after implantation of the VLARS had to be done with great caution since the epiretinal VLARS was not
484
fixated with a retinal tack as in the semi-chronic implantation. Lacking any form of physical fixation,
485
close epiretinal alignment was solely granted by the success of the implantation, and especially the
486
careful removal of the PFCL bubble beneath the array. Using any form of heavy liquid (i.e. PFCL,
487
silicone oil) was discarded due to their isolating properties, thus prohibiting retinal stimulation.
488
Another important difference to the semi-chronic implantation surgery was the presence of a connector
489
cable leading to the stimulator unit outside the eye positioned at the corneal incision. A permanent
490
aperture in the anterior segment of the eye would lead to a temporary instability of the eye. Adding
491
multiple Nylon 10-0 sutures (Alcon, Fort Worth, USA) to the corneal incision eventually granted
492
stability.
493
Overall, safely maneuvering the animal as well as the lack of physical fixation added further challenges
494
to the implantation surgery, yet in the two cases a successful implantation was achieved and the acute
495
stimulation and recording was performed.
496
497
Page 19 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

20
3.6. Cortical recordings in acute epiretinal stimulation
498
The cortical recording electrode and one of its shanks are shown in figure 6 A. After the craniotomy,
499
we were able to record cortical potentials elicited by bright light stimulation to verify the electrode’s
500
correct position. Figure 6 B shows the schematic design of the star shaped VLARS illustrating the
501
distribution of active electrodes, as well as giving a detailed insight in the array’s measurements.
502
Figure 6 B shows the active electrode clusters encircled, the active electrodes are graphically enlarged
503
and colored corresponding to the stimulus. The active electrode clusters used in the acute study shown
504
below are highlighted in blue for stimulus 1 (figure 6 B, S1) and red for stimulus 2 (figure 6 B, S1).
505
Figure 6 C to E give an overview of the results obtained during the acute stimulation experiments in one
506
of the two tested rabbits.
507
The normalized response amplitudes in figure 6 C demonstrate a different cortical response pattern
508
corresponding to the varying area of stimulation on the retina. The blue and red asterisks indicate
509
significantly larger responses to stimulus 1 and 2, respectively. Looking at individual electrodes with
510
seemingly high responses the assumption above is further supported. Figure 6 D and E compare
511
electrodes from shank A and C, showing that the recorded local field potentials are significant to both
512
clusters of stimulation, yet significantly higher for shank A when stimulation occurred with electrodes
513
in the blue cluster or in shank C when stimulation occurred with electrodes in red cluster. Thus, we were
514
able to demonstrate the VLARS’ ability to elicit reproducible cortical responses corresponding to
515
epiretinal stimulation in distant areas of the retinal surface.
516
517
Page 20 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

21
Figure 6: Retinal stimulation evokes LFP responses in rabbit primary visual cortex (V1) A: Schematic
of the 32-channel silicone probe inserted in V1 in the rostro-caudal direction (A-D shanks; A, left
side). Each shank comprises eight electrodes spanning cortical layers 1 to 6 (A, right side).
B: Schematic design of the star shaped VLARS-design. The connecting cable has a width of 2 mm,
the radius measured from the central aperture is 6 mm. The apertures in the center and in the periphery
have a diameter of 400 µm. The single return electrode has a diameter of 1 mm (large grey electrode),
the other electrodes have a diameter of 100 µm (not shown in figure). Note that the distance between
the electrodes varies based on their position (approx. 520 µm on the wings versus 300 µm in the
Page 21 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

22
center); The electrode cluster S1 consists of five active electrodes. The electrodes are highlighted in
blue, and represent the corresponding cortical responses to stimulus 1 (blue traces) in C to E. The
electrode cluster S2 consists of nine active electrodes. These electrodes are highlighted in red, and
represent the corresponding cortical responses to stimulus 2 in C to E. The remaining two encircled
electrode clusters consist of 5 active electrodes each.
C: Normalized response amplitudes to stimulus 1 (blue traces) and stimulus 2 (red traces) for each
electrode (horizontal: shanks A-D, vertical: electrodes 1 to 8). Following stimulus onset at time 0 s,
three stimulation artifacts are visible before stimulus-evoked responses emerge as negative
deflections. Blue and red asterisks indicate significantly larger responses to stimulus 1 and 2,
respectively (unpaired t-test, Bonferroni-corrected). These responses were also significantly different
from baseline. Shaded error bars indicate the standard error of the mean (SEM).
D and E: Examples demonstrating the reversal of response magnitudes during stimulation. Stimulus
1 (blue) elicited a greater response at electrode A5 compared to C5, while stimulus 2 (red) evoked a
larger response at C5 than A5 (unpaired t-test, Bonferroni-corrected; panel D). Similarly, stimulus 1
elicited a stronger response at electrode A6 than C6, while stimulus 2 did the reverse (unpaired t-test,
Bonferroni-corrected; panel E).
518
4. Discussion
519
The concept study was aiming at introducing an epiretinal stimulator capable of stimulating a wider
520
aspect of the retinal surface than any other currently available stimulators, thus creating a meaningful
521
visual field. We chose the epiretinal pathway due to our group’s experience in this field as well as the
522
overall positive feedback received from the commercially available epiretinal stimulator system ARGUS
523
II (17-20).
524
Besides the obvious complexity of engineering such microsystems, the major obstacle was a feasible
525
and safe implantation procedure. Altering the method of inserting the array into the anterior chamber by
526
using various standard tools pathed the way for a less timely and safer implantation. In the acute setting
527
we decided to refrain from retinal fixation via a retinal tack to spare the retina from the additional risk
528
of tearing and detachment. This was suitable since the experiment was performed in general anesthesia
529
and in a stereotactic apparatus hindering any uncontrolled movement.
530
Corneal edema, intravitreal bleeding and insufficient fixation of the arrays were the main impairments
531
during and after semi-chronic implantation surgery. Especially retinal detachment and tearing, as well
532
as safe and sufficient fixation of the array were threatening the success in various subjects as detailed
533
above. Over the course of the study, our group was able to improve the implantation procedure, yet
534
using retinal tacks to fixate the array on the retinal pole remains unsuitable. It is crucial to consider the
535
different dimensions of the rabbit’s eye compared to those of humans, or pigs, on which prior
536
implantation experiments were performed (17, 21, 22). The VLARS was developed with the human eye
537
Page 22 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

23
in mind, yet the design of the experiment and the legislation on animal experiments made the use of a
538
smaller animal more suitable. We expect, that the implantation of the VLARS in a human eye would be
539
less traumatic for the retina. The observations gathered during the follow-up period gave a mixed image
540
of success, but also failure as shown above.
541
Calculations have shown, that our VLARS device’s 250 electrodes cover a possible visual field of 18.8°,
542
corresponding to a visual angle of 37.6° (14). According to the work of Dagnelie et al., a visual angle
543
of 27° consisting of a 16x16 pixel matrix is the threshold for navigating safely through a given
544
environment (13). In theory the VLARS structure fulfills this requirement. To do reading tasks
545
efficiently, Cha et al. claim that 625 pixels on a 10 mm by 10 mm matrix are necessary (23). This would
546
theoretically grant a visual acuity of 20/30 far surpassing the capabilities of any current retinal
547
stimulator. The proclaimed necessity of a central visual acuity of 20/80 to perform pattern recognition
548
tasks by Palanker et. al. demands about 18000 individual pixels on an array of 3 mm in diameter (24).
549
This further demonstrates the gap between the requirements of a retinal stimulator and the current
550
technical as well as physiological feasibility. The 250 electrodes mounted on the VLARS do not match
551
those requirements either although surpassing the 60 electrodes of the epiretinal ARGUS II device by a
552
large margin. Additionally, creating a significantly larger epiretinal array did not have the intention of
553
improving central acuity significantly, yet adding meaningful visual field. The changing distribution, as
554
depicted in figure 6, with an electrode pitch of 300 µm in the central 2.5 mm² of the array and a pitch of
555
520 µm on the peripheral wings further underlines this aspect.
556
Implanting particularly large retinal stimulators has been the goal of different other groups as well, yet
557
the focus was mainly laying on suprachoroidal implantation. Villalobos et. al designed a single array for
558
suprachoroidal implantation measuring 19 x 8 mm and mounting 21 electrodes. The array is implanted
559
into a rather large scleral pocket with an opening close to the corneal limbus. Their studies conclude
560
that implantation is safe and feasible, and the array elicits cortical activation via suprachoroidal
561
stimulation (25, 26). This approach was further pursued by Abbott et. al., members of the same group,
562
by increasing the number of electrodes to 44 on a silicone carrier about the same size (17 x 8.5 mm) as
563
the one used by Villalobos (25, 27). Results from a chronic passive implantation showed promising
564
results (27). Comparable to that approach, Lohmann et. al tested a dual-array suprachoroidal stimulator
565
with a size of 5.18 mm x 5.18 mm for each of the arrays, mounting up to 50 electrodes in total
566
theoretically (25 electrodes on each of the two arrays) (28). A safe implantation procedure was shown
567
is their work, as well as chiasma responses and changes in the reflectance caused by trans-choroidal
568
stimulation. Suprachoroidal implantation seems feasible for larger arrays because intraocular surgery is
569
not necessary nor any kind of fixation on the retinal surface. Using a scleral pocket, the suprachoroidal
570
array is aligned to the eye inherent curvature (29). However, compared to epiretinal and subretinal
571
stimulation, suprachoroidal stimulation systems have a major disadvantage concerning the visual acuity.
572
The highest accessible visual acuity of suprachoroidal systems seems significantly inferior to those
573
Page 23 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

24
shown in epiretinal and subretinal systems with an acuity of 20/4451 in the suprachoroidal system
574
developed by Ayton et al versus 20/1262 in the epiretinal ARGUS II or even 20/200 in the subretinal
575
alpha-IMS (20, 30, 31). Especially the Alpha-IMS surpasses other approaches of prosthetic vision in
576
terms of sheer electrode number by a large margin with 1500 individual electrodes, yet ultimately
577
creating a 38 x 40 pixel matrix granting a visual angle of 11° by 11° (30). Thus, surpassing epiretinal
578
systems in theoretical visual acuity, the size of subretinal systems is limited by the method of
579
implantation (24, 32). By implanting multiple subretinal photovoltaic arrays, the PIXIUM Vision SA
580
PRIMA system attempts to combine both high resolution of subretinal implants and the restauration of
581
a large visual field. Currently, an interventional clinical trial including patients suffering from age-
582
related macular degeneration, opposed to retinal dystrophies, is in progress (clinicaltrials.gov identifier:
583
NCT03333954).
584
Sharing similar properties with the VLARS, the POLYRETINA is an approach introduced recently by
585
Ferlauto et. al. (33). The PDMS (polymithelysiloxane) based epiretinal array mounts 2215 photovoltaic
586
stimulating pixels granting a theoretical visual angle of 46.3° and a theoretical restauration of visual
587
acuity of about 20/600. During the implantation procedure the flexibility of the POLYRETINA is used
588
to concentrically fold the array and insert it over a scleral incision of about 6 to 7 mm very much like
589
the implantation procedure shown in this work. Also, the POLYRETINA array takes a hemispherical
590
shape to achieve a close alignment to the retinal surface, conquering a challenge displayed in our work.
591
Curving the VLARS array prior to the implantation did not result in better properties for the retinal
592
fixation in our study, though total surface area of the POLYRETINA surpasses the VLARS by a large
593
margin, resulting in even greater challenges concerning epiretinal alignment. While showing promising
594
results in implantation experiments in dummy and cadaveric eye, as well as demonstrating capabilities
595
of stimulating rd10 mouse retina, the newly developed array has yet to proof itself in an in-vivo setting
596
(33).
597
598
5. Conclusion
599
Overall, our study showed the possibility of combining the beneficial properties of retinal stimulation
600
of epiretinal systems shown in the past, and the possible recovery of meaningful peripheral vision. Along
601
the primary implantation procedure and further testing different challenges occurred which could be
602
dealt with as described above, yet especially retinal fixation of a very large epiretinal array remains an
603
aim for future studies.
604
605
Acknowledgement
606
Data and findings of this manuscript were partially shown at “The Eye and the Chip 2016”. The VLARS
607
project was supported with a grant from the Jackstädt Foundation and with additional funding from the
608
Hans Lamers Foundation. The authors thank Claudia Werner for her outstanding technical support.
609
Page 24 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

25
Rudolph Pivik supported the VLARS team with the rotational stereotactic frame.
610
611
612
613
References.
614
1. Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet (London, England).
615
2006;368(9549):1795-809.
616
2. Kocur I, Resnikoff S. Visual impairment and blindness in Europe and their prevention.
617
Br J Ophthalmol. 2002;86(7):716-22.
618
3. Humayun MS, Prince M, de Juan E, Jr., Barron Y, Moskowitz M, Klock IB, et al.
619
Morphometric analysis of the extramacular retina from postmortem eyes with retinitis
620
pigmentosa. Investigative ophthalmology & visual science. 1999;40(1):143-8.
621
4. Lin TC, Chang HM, Hsu CC, Hung KH, Chen YT, Chen SY, et al. Retinal prostheses in
622
degenerative retinal diseases. Journal of the Chinese Medical Association : JCMA.
623
2015;78(9):501-5.
624
5. Humayun MS, de Juan E, Jr., Weiland JD, Dagnelie G, Katona S, Greenberg R, et al.
625
Pattern electrical stimulation of the human retina. Vision research. 1999;39(15):2569-76.
626
6. Rizzo JF, 3rd, Wyatt J, Loewenstein J, Kelly S, Shire D. Perceptual efficacy of electrical
627
stimulation of human retina with a microelectrode array during short-term surgical trials.
628
Investigative ophthalmology & visual science. 2003;44(12):5362-9.
629
7. Rizzo JF, 3rd, Wyatt J, Loewenstein J, Kelly S, Shire D. Methods and perceptual
630
thresholds for short-term electrical stimulation of human retina with microelectrode arrays.
631
Investigative ophthalmology & visual science. 2003;44(12):5355-61.
632
8. Lewis PM, Ayton LN, Guymer RH, Lowery AJ, Blamey PJ, Allen PJ, et al. Advances in
633
implantable bionic devices for blindness: a review. ANZ journal of surgery. 2016;86(9):654-9.
634
9. Ghezzi D. Retinal prostheses: progress toward the next generation implants. Frontiers
635
in neuroscience. 2015;9:290.
636
10. Rachitskaya AV, Yuan A. Argus II retinal prosthesis system: An update. Ophthalmic
637
genetics. 2016;37(3):260-6.
638
11. Gekeler K, Bartz-Schmidt KU, Sachs H, MacLaren RE, Stingl K, Zrenner E, et al.
639
Implantation, removal and replacement of subretinal electronic implants for restoration of
640
vision in patients with retinitis pigmentosa. Current opinion in ophthalmology.
641
2018;29(3):239-47.
642
12. Geruschat DR, Turano KA, Stahl JW. Traditional measures of mobility performance
643
and retinitis pigmentosa. Optometry and vision science : official publication of the American
644
Academy of Optometry. 1998;75(7):525-37.
645
13. Dagnelie G, Keane P, Narla V, Yang L, Weiland J, Humayun M. Real and virtual
646
mobility performance in simulated prosthetic vision. Journal of neural engineering.
647
2007;4(1):S92-101.
648
14. Waschkowski F, Hesse S, Rieck AC, Lohmann T, Brockmann C, Laube T, et al.
649
Development of very large electrode arrays for epiretinal stimulation (VLARS). Biomedical
650
engineering online. 2014;13(1):11.
651
15. Waschkowski FM, W. Wide Field Epiretinal Stimulator with Adjustable Curvature. SSI
652
2017: International Conference and Exhibition on Integration Issues of Miniaturized Systems;
653
09.03.2017; Cork2017.
654
Page 25 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

26
16. Rosch S, Johnen S, Muller F, Pfarrer C, Walter P. Correlations between ERG, OCT, and
655
Anatomical Findings in the rd10 Mouse. Journal of ophthalmology. 2014;2014:874751.
656
17. Menzel-Severing J, Sellhaus B, Laube T, Brockmann C, Bornfeld N, Walter P, et al.
657
Surgical Results and Microscopic Analysis of the Tissue Reaction following Implantation and
658
Explantation of an Intraocular Implant for Epiretinal Stimulation in Minipigs. Ophthalmic
659
research. 2011;46(4):192-8.
660
18. Roessler G, Laube T, Brockmann C, Kirschkamp T, Mazinani B, Goertz M, et al.
661
Implantation and explantation of a wireless epiretinal retina implant device: observations
662
during the EPIRET3 prospective clinical trial. Investigative ophthalmology & visual science.
663
2009;50(6):3003-8.
664
19. Keseru M, Feucht M, Bornfeld N, Laube T, Walter P, Rossler G, et al. Acute electrical
665
stimulation of the human retina with an epiretinal electrode array. Acta ophthalmologica.
666
2012;90(1):e1-8.
667
20. Ho AC, Humayun MS, Dorn JD, da Cruz L, Dagnelie G, Handa J, et al. Long-Term
668
Results from an Epiretinal Prosthesis to Restore Sight to the Blind. Ophthalmology.
669
2015;122(8):1547-54.
670
21. Bozkir G, Bozkir M, Dogan H, Aycan K, Guler B. Measurements of axial length and
671
radius of corneal curvature in the rabbit eye. Acta medica Okayama. 1997;51(1):9-11.
672
22. Sanchez I, Martin R, Ussa F, Fernandez-Bueno I. The parameters of the porcine
673
eyeball. Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes
674
Archiv fur klinische und experimentelle Ophthalmologie. 2011;249(4):475-82.
675
23. Cha K, Horch K, Normann RA. Simulation of a phosphene-based visual field: visual
676
acuity in a pixelized vision system. Annals of biomedical engineering. 1992;20(4):439-49.
677
24. Palanker D, Vankov A, Huie P, Baccus S. Design of a high-resolution optoelectronic
678
retinal prosthesis. Journal of neural engineering. 2005;2(1):S105-20.
679
25. Villalobos J, Nayagam DA, Allen PJ, McKelvie P, Luu CD, Ayton LN, et al. A wide-field
680
suprachoroidal retinal prosthesis is stable and well tolerated following chronic implantation.
681
Investigative ophthalmology & visual science. 2013;54(5):3751-62.
682
26. Villalobos J, Fallon JB, Nayagam DA, Shivdasani MN, Luu CD, Allen PJ, et al. Cortical
683
activation following chronic passive implantation of a wide-field suprachoroidal retinal
684
prosthesis. Journal of neural engineering. 2014;11(4):046017.
685
27. Abbott CJ, Nayagam DAX, Luu CD, Epp SB, Williams RA, Salinas-LaRosa CM, et al.
686
Safety Studies for a 44-Channel Suprachoroidal Retinal Prosthesis: A Chronic Passive Study.
687
Investigative ophthalmology & visual science. 2018;59(3):1410-24.
688
28. Lohmann TK, Kanda H, Morimoto T, Endo T, Miyoshi T, Nishida K, et al. Surgical
689
feasibility and biocompatibility of wide-field dual-array suprachoroidal-transretinal
690
stimulation prosthesis in middle-sized animals. Graefe's archive for clinical and experimental
691
ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle
692
Ophthalmologie. 2016;254(4):661-73.
693
29. Ameri H, Ratanapakorn T, Ufer S, Eckhardt H, Humayun MS, Weiland JD. Toward a
694
wide-field retinal prosthesis. Journal of neural engineering. 2009;6(3):035002.
695
30. Stingl K, Bartz-Schmidt KU, Besch D, Braun A, Bruckmann A, Gekeler F, et al. Artificial
696
vision with wirelessly powered subretinal electronic implant alpha-IMS. Proceedings
697
Biological sciences. 2013;280(1757):20130077.
698
31. Ayton LN, Blamey PJ, Guymer RH, Luu CD, Nayagam DA, Sinclair NC, et al. First-in-
699
human trial of a novel suprachoroidal retinal prosthesis. PloS one. 2014;9(12):e115239.
700
Page 26 of 27AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript

27
32. Yue L, Weiland JD, Roska B, Humayun MS. Retinal stimulation strategies to restore
701
vision: Fundamentals and systems. Progress in retinal and eye research. 2016;53:21-47.
702
33. Ferlauto L, Airaghi Leccardi MJI, Chenais NAL, Gillieron SCA, Vagni P, Bevilacqua M, et
703
al. Design and validation of a foldable and photovoltaic wide-field epiretinal prosthesis.
704
Nature communications. 2018;9(1):992.
705
706
Page 27 of 27 AUTHOR SUBMITTED MANUSCRIPT - JNE-102885.R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accepted Manuscript