Structural changes in the retina after implantation of subretinal three-dimensional implants in mini pigs

Abstract

The retinal structural changes after subretinal implantation of three-dimensional (3D) microelectrodes were investigated in a mini pig. Three types of electrode were implanted into the subretinal spaces of nine mini pigs: 75-μm-high 3D electrodes on a 200-μm-thick right-angled polydimethylsiloxane (PDMS) substrate (group 1); a 140-μm-thick sloped PDMS substrate without electrodes (group 2); and a 140-μm-thick sloped PDMS substrate with 20-μm-high 3D electrodes (group 3). One mini pig was used as a control. Spectral domain-optical coherence tomography (SD-OCT) images were obtained at baseline and 2, 6, and 12 weeks post-surgery. Retinal specimens were immunostained using a tissue-clearing method 3 months post-implantation. The 75-μm-high 3D electrodes progressively penetrated the inner nuclear layer (INL) and touched the inner plexiform layer (IPL) 2 weeks post-surgery. At 6 weeks post-operatively, the electrodes were in contact with the nerve-fiber layer, accompanied by a severe fibrous reaction. In the other groups, the implants remained in place without subretinal migration. Immunostaining showed that retinal ganglion and bipolar cells were preserved without fibrosis over the retinal implants in groups 2 and 3 during the 12-week implantation period. In summary, SD-OCT and immunohistology results showed differences in the extent of reactions, such as fibrosis over the implants and penetration of the electrodes into the inner retinal layer depending on different types of electrodes. A sloped substrate performed better than a right-angled substrate in terms of retinal preservation over the implanted electrodes. The 20-μm-high electrodes showed better structural compatibility than the 75-μm-high 3D electrodes. There was no significant difference between the results of sloped implants without electrodes and 20-μm-high 3D electrodes, indicating that the latter had no adverse effects on retinal tissue.

Keywords: implant design; retinal prosthesis; structural retinal change; subretinal implant; three-dimensional microelectrodes.

FIGURE 1

Various subretinal implants used in the assessment of structural retinal changes. (A) 75-μm-high three-dimensional (3D) electrodes on a right-angled polydimethylsiloxane (PDMS) substrate, (B) a sloped PDMS substrate without electrodes, and (C) 20-μm-high 3D electrodes on a sloped PDMS substrate.

FIGURE 2

Spectral domain–optical coherence tomography (SD-OCT) images showing retinal changes after the implantation of 75-μm-high three-dimensional (3D) electrodes (group 1). (A) The edges of the electrode tips progressively penetrated the inner nuclear layer (INL) and had reached the inner plexiform layer (IPL) 2 weeks after surgery. (B) The penetrating 3D electrodes were in contact with the nerve fiber layer (NFL) at 6 weeks. (C) At 12 weeks, the tips of the electrodes nearly penetrated the atrophic NFL. The overlying retina remained flat instead of wavy along the contours of the 3D electrodes.

FIGURE 3

Spectral domain–optical coherence tomography (SD–OCT) images at 2 and 12 weeks after surgery and photographic images after coronal dissection of enucleated eyeballs from groups 2 and 3. (A) The implanted polydimethylsiloxane (PDMS) substrate without three-dimensional (3D) electrodes (group 2) remained stable for 12 weeks. Outer retinal layer degeneration was homogeneous over the substrate. The outer nuclear layer (ONL) and photoreceptor layer (PRL) disappeared from 2 weeks onward. The inner retina was relatively intact. (B) The 20-μm-high 3D electrodes (group 3) penetrated the lower portion of the inner nuclear layer (INL) and did not reach the IPL. The 3D electrodes were stably integrated into the subretinal space by 12 weeks. The ganglion cell layer (GCL), IPL, and INL remained intact over the electrodes and at the edges of the electrodes throughout the follow-up period of 12 weeks.

FIGURE 4

Total retinal layer (TRL) thicknesses over the electrode, over the substrate, and over the substrate edge or slope in each group at 2, 6, and 12 weeks after implantation. On spectral domain–optical coherence tomography (SD–OCT) images, the TRL thicknesses in group 1 showed a trend of constant progressive decrease over time at all measurement points. Although both groups 2 and 3 demonstrated similar trends of decreasing TRL thickness at all measurement points during the 12-week observation period, the degrees of decrease seemed to be less severe than in group 1.

FIGURE 5

Confocal images of immunohistochemical staining using the tissue-clearing method 12 weeks after surgery. 4′,6-diamidino-2-phenylindole (DAPI) staining (scale bar = 500 μm) was used to observe gross cell morphology and the implant’s location, while microtubule associated protein 2 (MAP2) and protein kinase C-α (PKC-α) were used to observe the ganglion cell layer (GCL) and the bipolar cell layer, respectively for (A) the control eye, (B) an eye implanted with sloped polydimethylsiloxane (PDMS) substrate (group 2), and (C) an eye with 20-μm-high electrodes on a sloped PDMS substrate (group 3). In groups 2 and 3, MAP2 staining in the en-face view indicated that ganglion cells were preserved. Moreover, the three-dimensional (3D) electrodes did not contact the GCL directly on vertical and horizontal dissection. PKC-α staining indicated that the bipolar cell layer was also preserved in both groups.

FIGURE 6

Confocal images of glial fibrillary acidic protein (GFAP) staining using the tissue-clearing method 12 weeks after surgery. Compared to that in the control eye (A), retinal fibrosis in the ganglion cell layer (GCL) was not prominent in groups 2 [sloped polydimethylsiloxane (PDMS) substrate] (B) and 3 [20-μm-high three-dimensional (3D) electrodes on the sloped PDMS substrate] (C). Glial proliferation in the inner nuclear layer (INL) was not detected in any group.