Carboxygenated ames medium

SEM imaging was performed post-mortem on retinas explanted and fixed from RCS rats 6 months after implant surgery and on the relative dissected prosthesis. CO₂ anesthesia and cervical dislocation were performed to sacrifice the animals. The eye and retina dissection was realized under dim red light. Eyes were enucleated and transferred to carboxygenated Ames’ medium (Sigma Aldrich), where the cornea, lens and part of the vitreous were removed. The tissues were then fixed in 1.25 % glutaraldehyde in 0.1 mM sodium cacodylate buffer, post-fixed in 1 % OsO4, 0.1 M sodium cacodylate, dehydrated, and cut into sections under a stereomicroscope. Imaging of the full and sham silk-only devices explanted from the animals and freed of tissue was also performed. The post-mortem preparations were coated with a 10 nm evaporated Au layer and observed with a JEOL-7500 electron microscope (Jeol, Tokyo, Japan).

Retinas from New Zealand White rabbits ranging between 3–5 kg of weight were used in this study. These animals were anesthetized with a mixture of ketamine/xylazine (40/5 mg/kg) by IM injection and euthanized with an overdose of sodium pentobarbital (100 mg/kg) following protocols approved by the Institutional Animal Care and Use Committee and conforming to National Institutes of Health (NIH) guidelines. The eyes were enucleated and the anterior segment removed along the ora serrata, followed by removal of vitreous humor. The resulting retina-sclera preparation was cut into 4 to 5 sections and the tissue pieces maintained in carboxygenated Ames’ medium (Sigma-Aldrich, St. Louis, MO) at ambient temperature. For light microscopy, tissue pieces were immersion fixed in either 4% (w/v) paraformaldehyde (PFA) or 4% N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC; Sigma-Aldrich) in 0.1M phosphate buffer (PB; pH 7.4). We found 20 minutes in 4% EDAC fixation was the best condition to visualize synaptic proteins. Retinal pieces were cryoprotected in 30% sucrose in PB overnight at 4°C, sectioned vertically at 12µm on a cryostat and collected on slides. For whole mount preparations, retinas were isolated from eyecup, flattened on a black nitrocellulose filter paper (Millipore, Billerica, MA) and fixed in 4% EDAC for 1 hour to preserve tissue integrity.