Enabling visualization of GFAP-positive retinal glial cells, neurons and microvasculature in three-dimensions

Glial cells are one of the most numerous cell types in the vertebrate retina and they serve to support neurovascular function. The principal glial cell in the retina is the Müller cell, accounting for approximately 90 % of all retinal glial cells. Müller cells are phenotypically elongated in shape and were first described as 'radial fibers' by Heinrich Müller in 1851. Their structure spans the entire thickness of the retina, through all retinal layers from the internal to external limiting membrane. This unique three-dimensional spatial arrangement enables Müller cells' direct contact with almost all cell types in the retina to perform its function. Despite this, the current study of Müller cells has largely been limited to thin sections or in culture, which provide limited detail about its spatial arrangement and interconnection with other cell types. The novel technique described here enables the three-dimensional visualization of GFAP-positive Müller cell processes in rodent retina and is based on the isolated arterially perfused rat eye preparation. Our micro perfusion technique utilizes the microvasculature as the delivery channel to quickly and effectively preserve all retinal elements. Intravascular labelling enables visualization of the intact three-dimensional retinal microvasculature within its normal neuronal and glial confines. Additional immersion immunolabeling and subsequent clearing with RapiClear® enables the three-dimensional visualization of different retinal elements and their physical interaction. Volume rendering of confocal image stacks acquired from these specimens can facilitate the study of such interactions in normal and disease models to further our understanding. This technique may be replicated in human donor retinae for future investigations to provide insight into Müller cell form and spatial relationship with other cell types.