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.
Highlights
Vision loss is one of the most dreaded constraints together with cancer and Morbus Alzheimer (Scott et al, 2016)
The SD-OCT enabled an assessment of porcine retina samples during different time points
There are animal models and cell culture approaches available, there is a certain demand for organ culture models or organoids, mimicking the molecular mechanisms contributing to Age-related macular degeneration (AMD)
Summary
Vision loss is one of the most dreaded constraints together with cancer and Morbus Alzheimer (Scott et al, 2016). The early form of AMD is characterized by the presence of lipid-rich deposits, e.g. drusen, and retinal pigment epithelium (RPE) hypopigmentation and hyperpigmentation (Curcio et al, 2013; Klettner et al, 2013). The exudative (wet) form has choroidal neovascularization, resulting in edema and photoreceptor degeneration (Ferris et al, 2013). Several risk factors, such as advanced age, genetic disposition, family history of AMD, race, smoking, obesity, or hypertension, are known to be involved in this multifactorial disease (Mares et al, 2011; Grassmann et al, 2015; Merle et al, 2019). In AMD, characteristic extracellular lipid-rich deposits between outer retinal cells are formed (Buitendijk et al, 2013). The exact AMD pathogenesis is still not fully understood
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