Abstract
Purpose:
The purpose of this study was to study the effects of inner retinal tissue support in adult full-thickness porcine retinal explants.
Methods:
Full-thickness retinal sheets were isolated from adult porcine eyes. 6x6 mm retinal explants were cultured for 5 or 10 days using a previously established protocol with the photoreceptors positioned against the culture membrane (porous polycarbonate) or with a novel approach where the inner limiting membrane (ILM) faced the membrane. The grafts were analyzed morphologically using hematoxylin and eosin staining (H&E), transmission electron microscopy (TEM) and immunohistochemistry.
Results:
Standard cultures (photoreceptors towards the membrane) displayed a progressive loss of retinal lamination, strong immunohistochemical labeling of activated, hypertrophic Müller cells (GFAP) and weak GS labeling consistent with gliosis. Immunohistochemistry revealed few surviving photoreceptors and ganglion cells. TEM of 5 days in vitro (DIV) standard explants displayed extensive cell death, degenerated synapses in both plexiform layers and no intact photoreceptors. Müller cell sprouting was abundant epiretinally and subretinally with processes growing into the culture membrane pores. In contrast, H&E staining of the explants cultured with the ILM facing the culture membrane revealed a retained laminar architecture, with well defined nuclear layers as well as photoreceptor inner and outer segments at 5 and 10 DIV. Immunohistochemistry showed no signs of Müller cell activation (GFAP) and GS labeling of the Müller glia appeared normal at both time points. Rhodopsin labeling displayed strong labeling of rod outer segments at both 5 and 10 DIV. NeuN-labeled ganglion cells were present at both time points. TEM of 5 DIV explants revealed a multitude of intact photoreceptors with inner and outer segments, as well as conventional and ribbon synapses in both plexiform layers. The inner and the outer limiting membranes appeared intact and no Müller cell hypertrophy was present on the inner or outer retinal surface.
Conclusions:
Physical support of the inner retina appears to be an important factor for the preservation of the retinal laminar architecture and cell survival in vitro. Using this novel paradigm, gliotic reactions are attenuated which may prevent the disruption of vital processes such as glutamate metabolism and tissue homeostasis.
Keywords: 426 apoptosis/cell death •
449 cell survival •
540 glia