Novel Porcine Retina Cultivation Techniques Provide Improved Photoreceptor Preservation

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in industrialized countries among people over 60 years. It has multiple triggers and risk factors, but despite intense research efforts, its pathomechanisms are currently not completely understood. AMD pathogenesis is characterized by soft drusen in Bruch’s membrane and involves the retinal pigment epithelium–Bruch’s membrane-choroid complex and adjacent structures, like photoreceptors. This study explores the potential of novel cultivation techniques to preserve photoreceptors in retinal explants to gain better insights in AMD pathology. The porcine retina explants were cultured for 4 and 8 days using three different explantation techniques, namely, control (photoreceptors facing down, touching the filter), filter (photoreceptors facing up, turned sample using a filter), and tweezers (photoreceptors facing up, turned sample using tweezers). Optical coherence tomography revealed that the tweezers method had the best capacity to limit thinning of the retinal explants. Both novel methods displayed advantages in maintaining outer segment thickness. Additionally, immunofluorescence evaluation revealed a better preservation of opsin + cells and rhodopsin signal intensity in both novel methods, especially the tweezers method. Furthermore, RT-qPCR analysis demonstrated an upregulation of OPSIN and RHODOPSIN mRNA expression in tweezers samples at 8 days. Amacrine and bipolar cell numbers were not altered at day 4 of cultivation, while cultivation until 8 days led to reduced bipolar cell numbers. At 4 days, CALRETININ mRNA was upregulated in filter samples, but protein kinase C alpha expression was downregulated. Retinal ganglion cells were diminished in both novel techniques due to a direct physical contact with the insert. Remarkably, no difference in TUBB3 mRNA expression was detected among the techniques. Nevertheless, both novel methods exhibited an improved retention of photoreceptor cells. In conclusion, the tweezers technique was the most promising one. Due to the high homology of the porcine to the human retina, it provides a reasonable alternative to in vivo rodent models. Consequently, an adapted coculture system based on the current findings may serve as an ex vivo model suitable to analyze AMD pathomechanisms and novel therapeutic approaches.

Hypoxic Processes Induce Complement Activation via Classical Pathway in Porcine Neuroretinas

“…SD-OCT analysis of porcine retinas was performed on day two and four using an SD-OCT (Spectralis, Heidelberg Engineering, Heidelberg, Germany). As described before, a special adaptor was used with a customized mounting device, which fits the filter inserts (Ø 12 mm) [ 38 ]. For analysis of retinal thickness, three 30°-line scans (ART:100)-images of each retina were taken.…”

Complement Factor H Loss in RPE Cells Causes Retinal Degeneration in a Human RPE-Porcine Retinal Explant Co-Culture Model

“…The local institutional ethics committee approved all the experiments conducted with porcine retinal explants. The explants were prepared from 6-month-old pigs weighing on an average 100 kg within 2–3 h of enucleation as previously described [ 27 ]. After electrocution euthanization, the enucleated eyes were transported in ice directly to the research facility.…”

FH loss in RPE cells causes retinal degeneration in a human RPE-porcine retinal explant co-culture model

“…All the experiments conducted with porcine retinal explants were approved by the local institutional ethics committee. The explants were prepared from 6-month-old pigs weighing on an average 100kg within 2-3 hours of enucleation as previousely described [26]. They were euthanized by electrocution and the enucleated eyes were transported in ice directly to the research facility.…”