NEUROGLIAL DYSFUNCTION IN DIABETIC RETINOPATHY

Key Words: Retina; Neuron; Retinal ganglion cell; Microglia; Macroglia; Astrocyte; Müller cell; Neurodegeneration; Visual function; Electroretinogram.

THE RETINA IS A MULTICELLULAR PHOTON

SENSOR AND PHOTOMULTIPLIER

The retina is a unique component of the central nervous system (CNS), containing a variety of neurons and glial cells. This elegant and complex structure converts visual stimuli into chemical and electrical signals that are transmitted to the brain via the optic nerve. Clinicians and neuroscientists view the retina from distinct perspectives, and both disciplines provide instructive lessons about retinal composition and function. Clinicians view the fundus of the retina as the sum of the visible structures – retinal blood vessels, pigment epithelium, choroid and, to some extent, the vitreous humor. By contrast, neuroscientists concentrate on the cellular organization that is largely invisible to clinicians. This brief overview of retinal structure and function emphasizes the critical role of the invisible neurosensory retina in normal vision, to illustrate how these characteristics may predispose it to compromise the metabolic stresses of diabetes. These relationships are essential to the interpretation of the clinical features of diabetic retinopathy and the processes that disrupt vision.

The cellular anatomy of the mammalian retina was revealed in great detail by Ramón y Cajal more than 100 years ago, when he defined the neuronal subclasses as well as the Müller cells and astrocytes, using Golgi silver staining methods. He recognized the laminar structure of the retina and the fact that neurons comprise the majority of retinal cells. This structure, highly conserved throughout vertebrate evolution from fish to humans, is comprised of three layers of neurons and two layers of synapses, which are minute structures that facilitate interneuronal communication through the release of, and response to, chemical transmitter substances (1).

The neurons are categorized as follows:

First-order: photoreceptors, which produce a neurochemical response to photons Second-order: bipolar, horizontal, and amacrine (“without axons”) cells, which conduct

and regulate signals from photoreceptors

Third-order: ganglion cells, which collect and integrate information from second-order neurons that are organized into opposing on/off receptive fields and transmit electrical impulses via axons which terminate primarily in the lateral geniculate body of the thalamus.

THE NEURONS OF THE RETINA

The histological configuration of the retina implies that communication between neurons can be broken down into at least three distinct stages. Retinal neurons are organized in three layers separated by two plexuses of densely packed synaptic connections that enable different types of neurons to communicate with each other. The two layers of synapses (plexiform layers) mediate signal transduction from the photoreceptors (firstorder neurons) through bipolar interneurons (second-order neurons) to the ganglion cells (third-order neurons). The axons of the retinal ganglion cells cofasciculate to form

The photoreceptors absorb photons of light and begin a cascade of signal transduction. Rod photoreceptors are extremely sensitive to light, and maintain vision in low illumination (scotopic conditions), while cone photoreceptors mediate color vision in bright light (photopic conditions), and afford a high degree of visual acuity. The primate retina contains a fovea, which has a high concentration of cone photoreceptors to augment acuity. Signals are transmitted from photoreceptors by synaptic connections in the outer plexiform layer to bipolar cells and horizontal cells. Horizontal cells provide modulatory input to photoreceptor terminals, and also perform pan-retinal signal averaging through a network of inter-horizontal gap junctions to refine visual information. Bipolar cells, subtypes of which selectively connect with rod or cone photoreceptors, transmit signals from the photoreceptors to the ganglion cells, while amacrine cells refine both bipolar and ganglion cell output. Together, these vertical and lateral interactions allow the retina to function over a wide dynamic range.

Synaptic communication in the retina is mediated by a variety of neurotransmitters. Photoreceptors and bipolar cells release the excitatory neurotransmitter, glutamate, while horizontal and amacrine cells release the inhibitory neurotransmitter, gammaaminobutyric acid (GABA). In addition, neurotransmitters, including dopamine, acetylcholine, and norepinephrine, are also involved in neurotransmission. Glutamate and GABA, however, are the predominant signaling molecules and contribute to synaptic communication among almost all retinal neurons. These substances act on neurons to generate an intraneuronal electrical potential that subsequently results in the release of chemical neurotransmitters from the receiving cell, enabling transduction of visual information through multiple types of retinal neurons. In the complex signal processing pathway of the retina, the outer plexiform layer (OPL) contains connections between photoreceptors, horizontal cells, and bipolar cells, while the inner plexiform layer (IPL) comprises connections between the bipolar, amacrine, and ganglion cells. Each plexiform layer achieves one step in the vertical transmission of visual signals through the retina, as well as additional horizontal signal modification mediated by horizontal and amacrine cells, which sharpen signals by emphasizing pertinent information, while minimizing interference and extraneous noise. Although a high degree of redundancy may preserve basic neurotransmission in many pathological states, each neuronal subtype fulfills a specific and fundamental role in signal processing that is critical to various aspects of normal vision. The functional aspects of various retinal neurons, such as the constitutively active dark current of the photoreceptors and the necessity for ganglion cells to maintain large, complex dendritic branches, impose a tremendous metabolic requirement that the limited retinal vasculature and retinal pigment epithelium must meet without interfering with the phototransduction by the photoreceptor outer segments. To meet this requirement, retinal neurons rely heavily on effective vascular and supporting glial cell function for waste removal and trophic support (4–8).

THE GLIAL CELLS OF THE RETINA

The glial cells of the retina form three main populations: astrocytes, Müller cells, and microglia (Fig. 2). Astrocytes originate in the optic nerve and migrate into the maturing retina to form a monolayer apposed to the inner limiting membrane, where they interact with neurons and vascular cells (7, 9), and contribute to the maintenance of the inner

Fig. 2. Localization of retinal glia. Glial cells are differentially distributed throughout the neural retina. Müller cells are large radial glia that span the entire thickness of the retina, with endfeet contacting the inner limiting membrane and microvilli projecting through the outer limiting membrane to the subretinal space. Astrocytes are located primarily in the inner retina along the nerve fiber layer, where they appose the inner limiting membrane. Microglia reside in the inner retina, typically in the inner plexiform layer/ganglion cell layer and inner plexiform layer/inner nuclear layer interfaces.

part of the blood–retina barrier (10). Müller cells are unique radial glia cells that closely interact with retinal neurons, providing nutritional support and regulating neuronal microenvironments by removing GABA and glutamate from synapses (6). Microglia are monocyte-derived cells with immunological functions in the immuno-privileged CNS. As such, microglial activation is implicated in retinal immune responses that often contribute to neurodegeneration and inflammation. These cells produce inflammatory cytokines, but also provide neuroprotective substances that aid in the support of neuronal and vascular cells (11). Together, these three types of glial cells support and influence neuronal and vascular function. Healthy functioning of glial and vascular cells is required to provide nutrients, waste disposal and trophic support to the neural retina, but it is the neurons and glia themselves that induce and maintain the vascular phenotype optimal for their own survival. Gross pathological abnormalities have been identified in retinal vasculature with diabetes, including pericyte ghosts, acellular capillaries, leukostasis, and tight junction breakdown, all of which have the potential to contribute to vascular leakage and nonperfusion (12–14). These events may induce glial-cell changes, such as astrocyte and Müller-cell reactivity and microglial activation, which can further exacerbate vascular dysfunction through the release of cytokines and vascular endothelial