Integration of Perforated Subretinal Prostheses With Retinal Tissue

Abstract

Purpose: To investigate the integration of subretinal implants containing full-depth perforations of various widths with rat and pig retina across weeks of implantation.

Methods: In transgenic P23H rhodopsin line 1 (TgP23H-1) rats and wild-type (WT) pigs, we examined four subretinal implant designs: solid inactive polymer arrays (IPA), IPAs with 5- or 10-μm wide perforations, and active bipolar photovoltaic arrays (bPVA) with 5-μm perforations. We surgically placed the implants into the subretinal space using an external approach in rats or a vitreoretinal approach in pigs. Implant placement in the subretinal space was verified with optical coherence tomography and retinal perfusion was characterized with fluorescein angiography. Rats were sacrificed 8 or 16 weeks post-implantation (wpi) and pigs 2, 4, or 8 wpi, and retinas evaluated at the light microscopic level.

Results: Regardless of implant design, retinas of both species showed normal vasculature. In TgP23H-1 retinas implanted with 10-μm perforated IPAs, inner nuclear layer (INL) cells migrated through the perforations by 8 wpi, resulting in significant INL thinning by 16 wpi. Additionally, these retinas showed greater pseudo-rosette formation and fibrosis compared with retinas with solid or 5-μm perforated IPAs. TgP23H-1 retinas with bPVAs showed similar INL migration to retinas with 5-μm perforated IPAs, with less fibrosis and rosette formation. WT pig retina with perforated IPAs maintained photoreceptors, showed no migration, and less pseudo-rosette formation, but more fibrosis compared with implanted TgP23H-1 rat retinas.

Conclusions: In retinas with photoreceptor degeneration, solid implants, or those with 5-μm perforations lead to the best biocompatibility.

Keywords: biocompatibility; migration; prosthetic; retina; retinitis pigmentosa.

Figure 1

Representative IPA and bPVA implants. (A) IPA device with 70-μm pixels separated by 5-μm perforations. (B) bPVA implants with 70-μm pixels separated by 5-μm perforations. Insets show magnified region of the implant. Scale bar: 100 μm.

Figure 2

Representative fundus imaging (A, C), fluorescein angiography (B), and OCT imaging (D, E) of implanted TgP23H-1 rats (A, B, D) and WT pig (C, E). (A) At 2 weeks after implantation with an active bPVA with 5-μm perforations, the position of the implant superior to the optic nerve (white arrow) was confirmed by fundus imaging in TgP23H-1 rats. (B) Fluorescein angiography showed normal retinal macro- and microvasculature overlying the implant (black arrows indicate implant border) in the same retina. (C) The position of the active bPVA with 5-μm perforations was confirmed in a WT pig via fundus imaging (white arrow indicates optic nerve head; black arrow indicates subretinal implant). (D) OCT image of a TgP23H-1 rat implanted with a 5-μm perforated IPA device (white arrow) for 8 weeks (black arrow indicates INL). (E) OCT image of WT pig implanted with a 5-μm perforated bPVA (white arrow indicates subretinal implant; black arrow indicates INL). Due to the higher optical density of crystalline silicon relative to ocular tissue, the bPVA implant appears about 2× thicker than it is in reality. Asterisk indicates vitreous side of OCT image. For a reference scale, the implant devices are 0.8 × 1.2 mm, and 30 μm in thickness.

Figure 3

Retinal cell migration depends on perforation width and duration of implantation. Representative histological images of TgP23H-1 rat retinas implanted for 8 weeks with (A) solid IPAs (B) IPAs with 5- or (C) 10-μm perforations or (D) bPVAs implants having 5-μm perforations. The second row shows TgP23H-1 rat retinas implanted with 5- (E, F) or 10-μm (G, H) perforations for 8 (E, G) or 16 (F, H) wpi. Perforated devices show INL cells on the RPE side of the implant (arrows in B, C, E–H) suggesting migration of the INL. (C) The double arrows indicate significantly more INL nuclei on the RPE side in retinas with 10-μm perforated IPAs. (H) By 16 wpi, the INL layer on the RGC side of the retinas implanted with 10-μm perforated IPAs was significantly thinner than retinas implanted with 5-μm perforated IPAs for a similar duration or retinas examined at 8 wpi. The black artifacts in the active bPVA section (D) are remnants of the bPVA device. Arrowhead: INL nuclei within the perforations. Scale bar: 50 μm. GCL, ganglion cell layer; ONL, outer nuclear layer.

Figure 4

Cell migration depends on perforation width and duration post-implantation. INL thickness on the RPE side of the implant (A, D), INL thickness of the RGC side of implant (B, E) and total INL thickness (C, F) of TgP23H-1 rats after implantation with various subretinal perforated devices after 8 weeks (A–C) or comparing 8 and 16 weeks post-implantation (D–F). (A) Retinas implanted with 10-μm perforated IPA devices showed significant migration of INL to the RPE side of the implant (one-way ANOVA F(3, 14) = 19.72, P < 0.001) compared with solid devices or 5-μm perforated IPAs. (B) No significant differences in INL thickness on the RGC side of the implant were found. (C) Total INL thickness (both retinal and RPE sides of the device) was significantly greater for retinas implanted with 10-μm perforated IPAs (one-way ANOVA F(3, 14) = 6.70, P < 0.01). (D) INL thickness in retinas with 10-μm IPA devices was significantly greater on the RPE side of the implant only at 8 wpi (two-way ANOVA F(1, 14) = 7.28, P < 0.05). (E) By 16 wpi, retinas implanted with 10-μm perforated IPAs showed significantly thinner INL on the RGC side (two-way ANOVA F(1, 14) = 7.18, P < 0.05). (F) The migration of the INL neurons increased total retinal thickness with the 10-μm perforated IPAs at 8 wpi (two-way ANOVA F(1, 14) = 19.81, P < 0.001. Error bars: represent SEM. Multiple comparisons *** P < 0.001, **P < 0.01, * P < 0.05.

Figure 5

Representative pseudo-rosettes and fibrosis observed in a TgP23H-1 rat implanted with 10-μm perforated IPA device. Pseudo-rosettes of photoreceptor nuclei were observed in the ONL (asterisks). Fibrosis was observed around the implant (arrow).

Figure 6

Representative histological images of TgP23H-1 rat (A) and WT pig retinas (B) implanted with active bPVAs having 5-μm wide perforations. Active bPVA showed close proximity of the INL due to cell migration across the implant without significant retinal disruption when implanted using an external (A) or vitreoretinal (B) approach. The black artifacts are remnants of the bPVA device after histological sectioning.