Planum

The implant, protected by a long steel tube, was advanced through a retroauricular incision to the lateral orbital rim and guided inside the orbit to the surface of the eyeball ([21 (link)]; figure 2a,b,e). The silicone cable (figure 2a) was implanted subperiostally beneath the temporal muscle. The polyimide foil was then protected by a silicone tube and guided from the lateral orbital rim, where it was fixed, to the equator of the eye. Subsequently pars plana vitrectomy was performed. A localized retinal detachment was created by saline injection in the upper temporal quadrant above the planned scleral and choroidal incision area. After preparation of a scleral flap, the implant was advanced ab externo transchoroidally along a guiding foil into the subretinal space until it reached the preoperatively defined position ([22 ]; see electronic supplementary material, chapter 2d). Although putting a chip directly under the fovea has not turned out to be a surgical problem we had abstained in initial patients from placing the chip under the macula, but asked to place the chip closer and closer to the foveola as the surgical learning curve improved. Silicone oil was then injected into the vitreous cavity to support retinal reattachment. No serious adverse events were noted during the course of the study. For post-operative observations and consideration on surgical safety see electronic supplementary material, chapter 2f).
Figure 2.

Implant position in the body. (a) The cable from the implanted chip in the eye leads under the temporal muscle to the exit behind the ear, and connects with a wirelessly operated power control unit. (b) Position of the implant under the transparent retina. (c) MPDA photodiodes, amplifiers and electrodes in relation to retinal neurons and pigment epithelium. (d) Patient with wireless control unit attached to a neckband. (e) Route of the polyimide foil (red) and cable (green) in the orbit in a three-dimensional reconstruction of CT scans. (f) Photograph of the subretinal implant's tip at the posterior eye pole through a patient's pupil.

All animal procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statements on the care and used of animals in ophthalmic research. Adult male rd1 mice (>6 weeks of age) were anaesthetised by intraperitoneal injection of ketamine (72 mg/kg) and xylazine (16 mg/kg). The pupils were fully dilated with 1% tropicamide and 2.5% phenylephrine eye drops, and a custom-made ultrafine needle (Hamilton RN needle, 34 gauge) was attached to a 5 μl Hamilton glass syringe and passed at 45 degrees through the pars plana into the vitreous cavity (for intravitreal delivery) or into the subretinal space without retinal perforation (for subretinal delivery). Injections were performed under direct visualisation of the needle tip using an operating microscope (Leica Microsystems), avoiding lenticular contact and blood vessels. Each eye received a total intravitreal dose of 1E+14 genome copies in a 3 μl volume or 2 μl subretinal bleb (6 eyes per vector).

Vitreous samples were collected at the Ophthalmology Service of Leiria-Pombal Hospital, Portugal, as previously described (35 (link)), according to a protocol approved by the hospital ethics committee (Code: CHL-15481). All patients included in this study gave their informed consent, which adhered to the tenets of the Declaration of Helsinki. Vitreous samples were collected in sterile cryogenic vials at the beginning of pars plana vitrectomy by aspiration into a 2 mL syringe attached to the vitreous cutter. Upon collection, vitreous samples were placed immediately on ice and frozen at -80°C until further analysis. The medical history of the patients was assessed to confirm the diagnosis, baseline characteristics, and associated diseases. Demographic characteristics, including age and gender, and the description of corresponding vitreous samples are summarized in Table 1 (more details in Supplementary Table 1). Samples from patients subjected to intraocular surgeries or intravitreal drug treatments in the previous 3 months were excluded from the study. Most patients underwent surgery for ERM removal due to the marked decrease in visual acuity. For label-free proteomic analysis, 12 patients (7 women and 5 men) diagnosed with PDR (n=4), dry AMD (n=4), and ERM (n=4) were selected. Older patients or with other serious illnesses associated (e.g., neoplasia) were removed from the study. For MRM, a larger cohort (n=65) was used, including some of the samples previously analyzed in the LFQ experiment. MRM experiments were performed on vitreous samples from patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and rhegmatogenous retinal detachment (RRD) with and without proliferative vitreoretinopathy (PVR) (n=13). Finally, 27 patients were selected for Western Blot (WB) analysis, including patients with ERM (n=5), PDR (n=9), AMD (n=6), and RRD (n=7), from which 3 patients have PVR.

The animals were euthanized with intramuscular ketamine 40 mg/kg and xylazine 10 mg/kg before and at 14 and 28 days after GM treatment. The collected left temporal bones were fixed in 4% paraformaldehyde/phosphate–buffered saline (PBS) (pH 7.4) for 2 h, and then the CAs were harvested under a microscope (SZX9, Olympus, Japan). The fixed specimens were immersed in PBS with 30% sucrose for 6 h, embedded in 5% agarose (type IX–A, Sigma–Aldrich, St. Louis, MO, USA) and 20% sucrose in PBS, then frozen in n–hexane (−60 °C). These specimens were cut vertically into 15 µm thick sections from the planum semilunatum to the center of the crista on a cryostat (Tissue–Tek Cryo3, Sakura Finetek, Tokyo, Japan) [41 (link)]. At intervals of 45 µm, five sections, including the center of CA, were immunostained.