Subretinal surgery: functional and histological consequences of entry into the subretinal space

Abstract

Background and objectives: Gene-therapy, stem-cell transplantation and surgical robots hold the potential for treatment of currently untreatable retinal degenerative diseases. All of the techniques require entry into the subretinal space, which is a potential space located between the retina and the retinal pigment epithelium (RPE). Knowledge about obstacles and critical steps in relation to subretinal procedures is therefore needed. This thesis explores the functional and histological consequences of separation of the retina from the RPE, extensive RPE damage, a large cut in the retina (retinotomy) and RPE phagocytosis in a porcine model.

Methods: Experiments were performed in 106 female domestic pigs of Danish landrace distributed over five studies. Under general anesthesia, different procedures for expansion of the subretinal space were conducted. Outcomes were visual function measured electrophysiologically with multifocal electroretinogram (mfERG) and retinal morphology examined histologically. Study I: The effect of anesthesia on mfERG was examined by repeated recordings for 3 hr in isoflurane or propofol anesthesia. Outcome was mfERG amplitude. Study II: Consequences of a large separation of the photoreceptors from the RPE were examined by injecting a perfluorocarbon-liquid (decalin) into the subretinal space. Two weeks after, in a second surgery, decalin was withdrawn. Outcomes were mfERG and histology 4 weeks after decalin injection. Study III: Extensive RPE damage was examined by expanding the subretinal space with saline and removing large sheets of RPE-cells through a retinotomy. Outcomes were mfERG and histology 2, 4 and 6 weeks after the procedure. Study IV: Consequences of a large retinotomy were examined by similar procedures as in Study III, but in study IV only a few RPE cells were removed. Outcomes were mfERG and histology 2 and 6 weeks after surgery. Study V: Clearance of the subretinal space was examined by injecting fluorescent latex beads of various sizes into the subretinal space. Outcome was histologic location of the beads at different time intervals after the procedure.

Results: Study I: MfERG amplitudes decreased linearly as a function of time in propofol or isoflurane anesthesia. Duration of mfERG recording could be decreased without compromising quality, and thereby could time in anesthesia be reduced. Study II: MfERG and histology remained normal after reattachment of a large and 2-week long separation of the photoreceptors and RPE. Repeated entry into the subretinal space was well tolerated. Fluid injection into the subretinal space constitutes a risk of RPE-damage. Study III: Removal of large sheets of retinal pigment epithelial cells triggered a widespread rhegmatogenous-like retinal detachment resulting in visual loss. Study IV: A large retinotomy with limited damage of the RPE was well tolerated, and visual function was preserved. Study V: Subretinal latex beads up to 4 μm were phagocytosed by the RPE and passed into the sub-RPE space. Beads up to 2 μm travelled further through the Bruch's membrane and were found in the choroid, sclera and inside blood vessels.

Conclusion: A large expansion of the subretinal space, repeated entry, a large retinotomy and limited RPE damage is well tolerated and retinal function is preserved. Subretinal injection of fluid can damage the RPE and extensive RPE damage can induce a rhegmatogenous-like retinal detachment with loss of visual function. Foreign substances exit the subretinal space and can reach the systemic circulation.