Table 1.
Summary of parameters used in previous preclinical retinal stimulation studies.
Table 2.
Summary of study design.
Figure 1.
(A) Schematic Drawing of the suprachoroidal electrode array used in the present study: (i) fourteen individually insulated platinum (Pt) – iridium (Ir) wires (25 µm diameter) are coiled into a helix and encased within a medical grade silicone tube; (ii) two 2 mm diameter Pt disc electrodes; (iii) twelve 600 µm diameter Pt disc electrodes welded to Pt/Ir wires; iv, spherically conformable medical grade silicone base; viii. (Inset) shows an ∼1∶1 scale photograph of an array on a 1 mm grid. Visible in the photograph is the 25 µm paralyne-insulated Pt-Ir wires which were resistance welded to each electrode. Also visible are two silicone-coated Polyethylene terephthalate (Dacron) patches (arrows) used for fixation at the sclera and orbital bone. (B) Study timeline for a single subject. Abbreviations: e-phys, electrophysiology; stim, stimulation. Individual alterations to timeline are described in Table 3.
Table 3.
Summary of chronic stimulation cohort.
Figure 2.
Method for measuring retinal and fibrous thickness.
Low power micrograph illustrating the histology following chronic implantation and electrical stimulation of a suprachoroidal electrode array. The sclera (double arrowhead) and the space occupied by the electrode array (*) are clearly visible. Note that the electrode array is removed prior to histology. Retinal thickness was measured adjacent to each electrode – as located by the presence of tissue dye (“a” and “b”), as well as approximately 500 µm distal to each electrode (“c”; in a randomly selected direction). Measurements were made from the ‘retinal pigmented epithelium – tapetum junction’ to the ‘retinal nerve fibre layer – vitreous junction’ (inclusive; black boxed inset). Similarly, fibrous thickness was measured on the choroidal and the scleral sides of the implant in the same locations as the retinal thickness measurements (blue and orange oval insets for choroidal and scleral locations, respectively). Control measurements were made for retinal thickness in a paired location on the contralateral control eye. Finally, implanted eye measurements of retinal thickness and fibrosis were correlated post-hoc with lab records of which electrodes were chronically stimulated and which were not [32]. Main panel scale bar = 1000 µm. Solid box scale bar = 500 µm. Blue and orange oval inset scale bars = 10 µm.
Figure 3.
Long-term mechanical stability of the suprachoridal electrode array taken from longitudinal fundus images.
Digitised fundus images recorded immediately following surgery, and over the 15 week chronic implantation period, illustrating the mechanical stability of the implant with respect to the optic nerve and retinal vasculature. Each line depicts the edge of the suprachoroidal electrode array visualised through the fundus image (supplementary Figure S1). An initial settling of the implant was observed during the first weeks with a displacement of 1–2 optic disc diameters (optic disc diameter is approximately 1.5 mm) in the implant outline. The implant location remained stable after 8 weeks of implantation. Note that data from all time points were not always available.
Figure 4.
Representative optical coherence tomography.
(A) Infrared fundus image illustrating the typical location of the electrode array in the superior retina. Orange dashed lines indicates the inferonasal extent of electrode array. Asterisk denotes hyperpigmentation near the tip of array caused by minor insertion trauma (as reported previously [26]). (B) Optical coherence tomography (OCT): en face scan of region delineated by blue square, showing individual 600 µm platinum (Pt) electrodes (arrows) within the body of the array. Orange dashed line and asterisk are as described in (A). (C) Cross-section through the green line in (A) revealing normal retinal morphology and vasculature. Arrows show two 600 µm Pt disc electrodes. Double-ended arrows show measurements used for determining retinal (i) and ‘retina plus choroid’ thickness (ii). Overall, the OCT scans suggested all subject exhibited healthy eye tissue throughout the course of the study.
Figure 5.
Optical coherence tomography (OCT) measured retinal and choroidal thicknesses before and after chronic electrical stimulation.
There was no evidence of a change in either the retinal or retinal plus choroid thickness as a result of the chronic stimulation program either directly underlying or distal to the electrode array. The thickness of the retina, and the retina plus choroid (including the feline tapetal layer) was measured (µm) for each subject (refer to Figure 4). Measurements were made: at the tip of the implanted array, at a location overlying the middle of the array, and at a location directly adjacent to the array (within 500 µm). Measurements were made before the commencement of chronic stimulation (Pre) and at the completion of the chronic stimulation period (Post). Box plots show median (midline), 1st and 3rd quartiles (box edges), whiskers have a maximum length 1.5 times the interquartile range and open circles represent individual points. There are between 5 and 7 data points (subjects) contributing to each boxplot. There was substantial overlap of the pre- and post-stimulation measures for retinal, or retinal plus choroidal, thickness in each of the locations. The estimated mean differences were small, as were the limits of the confidence intervals; none of the comparisons were statistically significant (Table 4).
Table 4.
Mean difference in OCT measured thickness (‘before’ minus ‘after’ chronic stimulation).
Figure 6.
Combined rod-cone maximal full-field electroretinogram (ERG) responses at the completion of the chronic stimulation period.
Examples of the ERG waveforms are shown in the inset with the a- and b-waves indicated. Dashed horizontal line indicates recording baseline. a-wave amplitude is taken from baseline to a-wave trough; b-wave amplitude is taken from a-wave trough to b-wave peak. The median and interquartile range of ERG responses from n = 7 subjects are presented in the box plots. Box plots show median (midline), 1st and 3rd quartiles (box edges), whiskers have a maximum length 1.5 times the interquartile range and open circles represent individual points. There was substantial overlap between the measured response amplitude of the a- and b-waves between the implanted and control eyes. The paired t procedures showed the mean difference (implanted minus non-implanted) for the a-wave was −14.3 µV (95% confidence interval: –50.5, 21.8, p = 0.37), and for the b-wave was −66.6 µV (95% confidence interval: –135.9, 2.8, p = 0.057). Minor differences between eyes can be attributed to electrode placement or normal biological variation.
Figure 7.
Longitudinal changes in electrode impedance.
Electrode impedance (kΩ) across all subjects recorded periodically over the duration of the chronic implant period. Box plots show median (midline), 1st and 3rd quartiles (box edges), whiskers have a maximum length 1.5 times the interquartile range and open circles denote outliers. Pre-implantation, post-explantation and post-cleaning impedance measurements were performed in normal saline. The number of individual electrode measurements that comprise each box plot ranged from 21 to 67 (maximum possible = 72; subject 505 was excluded due to a damaged lead).
Figure 8.
Longitudinal stability of electrically-evoked visual cortex potentials (eEVCP).
(A) Average cortical responses to increasing current are shown on the left. The stimulus is repeated twice at each current level and both response traces are displayed. The amplitude (mV) of the evoked response (within the region indicated by the blue shaded region) as a function of current (mA) is plotted on the right. In this case, evoked cortical potential threshold was 100 µA (as denoted by “T”). (B) eEVCP thresholds for each subject, recorded monthly, starting immediately before the initiation of chronic stimulation (0 months). Box plots show median (midline), 1st and 3rd quartiles (box edges), whiskers have a maximum length 1.5 times the interquartile range and open circles represent individual points. No eEVCP data was available for subject 505 due to a damaged lead. (C) The change in threshold, on a per-electrode basis. The changes were calculated for each electrode separately, in monthly increments, and these data were combined. Box plots show median (midline), 1st and 3rd quartiles (box edges), whiskers have a maximum length 1.5 times the interquartile range and open circles denote outliers. There was little variation in median change in threshold over the three time points. The 95% confidence interval comparing switch on to three months was −119.5 to 383.9 µA; this shows a range of plausible values for the overall change over time, including zero.
Table 5.
Mean differences in eEVCP thresholds in adjacent months and overall.
Figure 9.
Example Spike Recordings and Multi-Unit thresholds.
(A) Typical multiunit recording from one cortical channel in response to stimulation of a single suprachoroidal electrode. (B) Input-output function from the same cortical channel. Threshold was defined as the current required to elicit 50% of the maximum spike rate. (C) Peri-stimulus time histogram (1 ms bin width) across multiple repetitions of all currents presented, indicating two waves of spiking activity post stimulation. Dashed lines in (A) and (C) indicate stimulus onset. Abbreviation: AP, action potential.
Figure 10.
(A) Photomicrograph of a representative retina following chronic electrical stimulation. The red and green arrows indicate histological dye used to mark the sclera at the site of individual electrodes within the implanted array. In this case, red dye was used to indicate non-stimulated electrodes and green dye to indicate stimulated electrodes. Boxes show magnified tissue regions adjacent to the non-stimulated (red box) and stimulated (green box) platinum (Pt) electrodes as well as 500 µm distal to a Pt electrode (blue box). Scale bar = 1 mm. Asterisk denotes the space occupied by the electrode array. (B) Representative example of retina from paired, non-implanted, control eye. The scale bar in panel B = 50 µm, and applies to all magnified boxed regions. In all cases, the inner and outer retina as well as the tapetum, choroid and sclera did not show any significant histomorphological abnormality. There were no observable differences in overall retinal histopathology between samples. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; P, photoreceptors; T, tapetum, C: choroid. Subject ID: 502.
Figure 11.
Retina and fibrous tissue thickness measured from histological sections.
(A) Retinal thickness (µm) was measured in retina adjacent to individual platinum stimulated electrodes, non-stimulated control electrodes, regions approximately 500 µm distal to each electrode (refer to Figure 2), as well as in matched sites from the non-implanted contralateral control eye. There was little variation in the median retinal thickness between measured regions. However, when a linear mixed model accounting for the pairing of observations was used to estimate mean differences in retinal thickness for each condition, a statistically significant difference between stimulated and control conditions was observed. This small estimated mean increase in stimulated retinal thickness (Table 6) was not regarded as being clinically relevant. (B) Fibrosis tissue thickness measurements (µm) were made in retina adjacent to stimulated electrodes, non-stimulated electrodes and in a region approximately 500 µm distal to each electrode. Measurements were repeated for the scleral and choroidal sides of the space occupied by the array. There was no fibrosis in the contralateral eye. In both panels, box plots show median (midline), 1st and 3rd quartiles (box edges), whiskers have a maximum length 1.5 times the interquartile range and open circles denote outliers. There was substantial overlap between the fibrosis distributions in the different locations measured, and between the 95% confidence intervals for the mean fibrosis thickness in each condition (Table 7). Note that the sample sizes reflect the number of unique histological measurements performed for each group. Typically, a single measurement is made at each sample or sample-adjacent site. However, in some cases, due to artifactual damage (or because the histology wasn’t retrieved), a particular site was not measured. As a result, the no-stimulation sample size was n = 7 for panel A, and n = 10 for panel B. Both of these samples are a subset of the maximum possible (i.e. n = 14) if all the samples had been retrieved with no histological artifacts.
Table 6.
Mean difference in histologically measured retinal thickness (comparisons with non-implanted eye).
Table 7.
Mean fibrosis encapsulation thickness (µm) on retinal and scleral sides of the space occupied by the electrode array (with 95% confidence intervals).
Figure 12.
Representative examples of specific histological staining from both stimulated and control eyes.
(A, B) Perls’ Prussian blue; at the site of haemorrhage, the formation of haemosiderin from degraded red blood cells and release of iron complexes would produce a purple color while the addition of neutral red stain colors lysosomes red. No haemorrhage was noted in any of the samples. (C, D) Gram; Gram stain can be used to detect evidence of bacterial infection. None was observed in any of the samples. (E, F) Periodic acid-Schiff (PAS); glycoprotein components of basement membrane and connective tissue components are stained purple, while the haematoxylin counterstains cell nuclei purple. PAS stain can be used to detect evidence of fungal infection. None was observed in any of the samples. Scale bar in panel F = 50 µm, and applies to all panels. The inner retina is shown at the bottom of each image; the outer retina is shown at the top of each image. Asterisks denote the space occupied by the electrode array. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; P, photoreceptors; T, tapetum, C: choroid. Subject ID: 502.
Figure 13.
Representative immunohistochemical stains of the chronically stimulated and control feline retina.
(A, B) anti-glial fibrillary acidic acid (GFAP; red); Müller cells and astrocytes (supporting glial cells of the retina) can be seen in the ganglion cell layer and nerve fibre layer. No evidence of gliosis was noted in any eye in the study. (C, D) Glutamine synthetase (GS; green); Müller cells are evident extending through the retinal layers. (E, F) Neurofilament (NF-200; green); large and small ganglion cells (arrow) can be seen in the ganglion cell layer with their axons forming bundles in the nerve fibre layer (double arrowhead). Horizontal cells found in the inner nuclear layer are also stained in the feline retina. Sections are counterstained with DAPI nuclear stain (blue; panels: A, B, E and F). Variations in intensity of DAPI staining are artifactual. In all cases, normal retinal architecture was observed indicating retinal cell viability. Scale bar in panel F = 50 µm and applies to all panels. The inner retina is shown at the bottom of each image; the outer retina is shown at the top of each image. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Subject ID: 507.