Localized RPE Removal with a Novel Instrument Aided by Viscoelastics in Rabbits

Citation: Thieltges F, Liu Z, Brinken R, Braun N, Wongsawad W, Somboonthanakij S, Herwig M, Holz FG, Stanzel BV. Localized RPE removal with a novel instrument aided by viscoelastics in rabbits. Trans Vis Sci Tech. 2016;5(3):11, doi:10.1167/tvst. 5.3.11 Purpose: We developed a surgical method for localized and atraumatic removal of the retinal pigment epithelium (RPE) with a novel instrument. Methods: Bleb retinal detachments (bRD) were raised with balanced salt solution (BSS) following vitrectomy in 27 rabbits. The RPE was scraped with 3 loop variants (polypropylene [PP], 0.1 mm; PP, 0.06 mm; metal, 0.1 mm) of a custom-made instrument. Stabilization of bRDs with BSS or various concentrations (0.1%-0.5%) of hyaluronic acid (HA) was video analyzed. Perfusion-fixed samples of scraped areas and controls were studied by light and transmission electron microscopy. Results: The bRDs were sufficiently stabilized by 0.25% HA. Using the PP 0.1 mm loop with a single forward/backward stroke, an area of ca. 2.5 3 1.5 mm was nearly devoid of RPE, yet did show occasional Bruch's membrane (BM) defects combined with choriocapillaris hemorrhages in 13% of the bRDs. A single scrape with PP 0.06 mm resulted in unsatisfactory RPE denudement, while repeated scraping maneuvers caused more BM defects and hemorrhages. The metal loop resulted in incomplete RPE removal and massive intraoperative subretinal hemorrhages. Histologically, intact photoreceptor outer segments (POS) were observed above the RPE wounds in bRDs. Controls with bRDs alone showed an intact RPE monolayer with microvilli, with few engulfed remains of POS. Conclusions: Localized removal of RPE in HA stabilized bRD can be achieved by a PP 0.1 mm loop instrument. Translational Relevance: Removal of degenerated RPE may aid RPE cell replacement strategies

Ultrathin Polyimide Membrane as Cell Carrier for Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelium

In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation.Public Library of Science open acces

A Nanofibrillar Surface Promotes Superior Growth Characteristics in Cultured Human Retinal Pigment Epithelium

10.1159/000324045OPHTHALMIC RESEARCH463133-14

Localized RPE Removal with a Novel Instrument Aided by Viscoelastics in Rabbits

10.1167/tvst.5.3.11TRANSLATIONAL VISION SCIENCE & TECHNOLOGY5

A Step by Step Protocol for Subretinal Surgery in Rabbits

10.3791/53927JOVE-JOURNAL OF VISUALIZED EXPERIMENTS201611

Implanted eyes of dystrophic RCS rats.

<p>Immunohistochemical staining showing labeling of injected cells 41 days post-injection with antibodies against human antigen TRA-1-85 (A1-2), RPE marker CRALBP (B1-2, antibody recognizes also rat CRALBP), macrophage marker CD68 (C), and T-cell marker CD3 (D). Large pigmented cells detected 105 days post-injection were positive for CD68 antibody (E). Scale bar 100 μm in A-D and 50 μm in E.</p

In vivo follow-up of hESC-RPE-PI implanted rabbit eyes.

<p>OCT scans showing hESC-RPE-PI in three different rabbit eyes three months after transplantation (A-C). Representative fundus photographs showing hESC-RPE-PI (marked with dotted line) of one rabbit one, two and three months after transplantation (D).</p

Dark-adapted ERG responses following subretinal injections in dystrophic and non-dystrophic RCS rats.

<p>Representative and averaged (2 to 20 responses) ERG recordings from dystrophic and non-dystrophic rats 37 days after subretinal injection of hESC-RPE or medium alone compared to the non-operated fellow eye.</p

Histological analyses of rabbit retinas three months post-transplantation.

<p>HE staining of surgical control (A), PI alone (B) and hESC-RPE-PI (C and D). ONL thicknesses measured from HE samples, surgical control (n = 1), PI alone (n = 3), hESC-PI (n = 5) (E). Measurements were performed with ImageJ software. Error bars show standard deviation.</p