We recently established a new technique to study the three-dimensional relationship between retinal glial cells, neurons, and microvasculature in our laboratory. This technique builds upon the isolated arterially perfused rat eye preparation used in our laboratory for several decades (Su et al., 1995; Yu et al., 2001, 2003). Subsequent immersion staining and overnight clearing of the retinal wholemount using RapiClear®, enables detailed investigation of the cellular structures in 3D using confocal microscopy. This approach offers a shorter and reversible clearing protocol than CLARITY or CUBIC methods (Lu et al., 2021) and enables further investigation into the inter-relationship of cellular structures throughout the full thickness of the retina. We have applied this technique to investigate the structural association between GFAP positive glial cell processes, alpha-smooth muscle actin (α-SMA) surrounding the retinal microvasculature and the retinal ganglion axon through neurofilament medium labelling.

The human brain contains more than a billion neurons. The activities of the neurons are supported by neuroglia. Glial cells are ten times more numerous than neurons and occupy more than half of the brain volume (Hyden, 1961). After more than six decades of experimental studies we know that the behavior of glial cells is at least as complex as neurons and their membrane properties. Glial cells play an important role in signal processing, cellular metabolism, nervous system development, and the pathophysiology of neurological diseases (Ransom, 2012).

Heinrich Müller (1851) provided a detailed description of the radial fibers which we now know to be the principal glial cell of the vertebrate retina. Kölliker (1854) observed similar structures in the human retina and ascribed to them eponymously the name by which they have come to be known: the Müller cells (Sarthy, 2001). Many properties of Müller cells have been studied; such as their electrical, immunochemical, metabolic activities, and cytoplasmic content, displaying similarities to those found in protoplasmic astrocytes (Ransom, 2012). The Müller cell comprise 90 % of the retinal glia and their processes interdigitate with the perikaryon, axons, and dendrites of neurons throughout the retina (Hernández et al., 2010; Sarthy and Ripps, 2001). This close association likely allows glial cells to promote synapse formation and maintain neuronal function by supplying nerve terminals with energy substrates and neurotransmitter precursors (Pfrieger and Barres, 1996). Additionally, Müller cells closely interact with blood vessels of all sizes and locations within the retinal layer. In the nerve fiber layer (NFL) and ganglion cell layer (GCL), large vessels often appear to lie within a hammock of Müller cell processes, while deeper capillaries are surrounded by knob-liked structures and filamentous Müller cell processes (Distler and Dreher, 1996). No doubt Müller cells need to work closely with retinal neurons and microvasculature as well as integrate with the other two glial types: astrocytes, and microglia. Previous studies of the glial-vascular relationship have primarily focused on the interaction of astrocytes with vessels of the superior vascular plexus (SVP) (Zahs and Wu, 2001). Notably lacking from past Müller glia comparative anatomy studies, is a detailed description of the glial-vascular-neuron inter-relationship through the thickness of the retina, encompassing all retinal vascular plexuses (Dreher et al., 1992; Wang et al., 2017). Our proposed technique can help address this gap and facilitate our understanding of Müller cell's role in retinal physiology and pathology.

The specific technique described in this manuscript is based on the isolated arterially perfused rat eye preparation. The rat retina is a vascular retina supplied by a central retinal artery, the same as human and primate retinas (Yu et al., 2003). This micro-perfusion, fixation and labelling technique allows us to preserve the three-dimensional cellular morphology and label the intact retinal microvasculature. Confocal imaging after use of RapiClear® (Yang et al., 2015, 2018; Yu et al., 2017, 2023b) allows us to obtain a volume image to visualize the whole length of the GFAP-positive Müller cell processes (Wang et al., 2017) in relation to the surrounding neurons and microvasculature. More interestingly, this combination of techniques could be used on human donor eyes to investigate the pathophysiology of macular oedema, a manifestation in many retinal diseases.