Macromolecular markers in normal human retina and applications to human retinal disease.

Macromolecular cell markers are essential for the classification and characterization of the highly complex and cellularly diverse vertebrate retina. Although a plethora of markers are described in the current literature, the immunoreactivity of these markers in normal human tissue has not been fully determined. This is problematic as they are quintessential to the characterization of morphological changes associated with human retinal disease. This review provides an overview of the macromolecular markers currently available to assess human retinal cell types. We draw on immunohistochemical studies conducted in our laboratories to describe marker immunoreactivity in human retina alongside comparative descriptions in non-human tissues. Considering the growing number of eye banks services offering healthy and diseased human retinal tissue, this review provides a point of reference for future human retina studies and highlights key species specific disease applications of some macromolecular markers.

Amino acid immunoreactivity in normal human retina and after brachytherapy.

We localised amino acids in the mid-peripheral aged human retina and a retina that had undergone radiation treatment 10 years earlier. The distribution pattern of glutamate, γ-amino butyric acid (GABA), glycine, glutamine and taurine, reflected patterns established in the primate retina. The retina that had undergone radiation exposure displayed both anatomical and neurochemical remodelling. The proximal retina comprised around 40 to 45 per cent of the total retina and neuronal kinesis and aberrant neuronal projections were also present. Amino acid neurochemistry was strikingly different with Müller cells displaying GABA loading, glycinergic neurons displaced and displaying a very high level of glycine labelling. We conclude that radiation exposure triggered these changes in the human retina and likely reflects general remodelling of structure and function following ischaemic damage to endothelial cells.

Creatine transporter immunolocalization in aged human and detached retinas.

PURPOSE: To identify the distribution of creatine transporter (CRT) in the aged human retina and how this expression pattern is modified after retinal detachment. METHODS: An affinity-purified antibody raised against the CRT was used in the immunohistochemical investigation. The anti-CRT antibody was colocalized with neuronal markers (calbindin, parvalbumin, Islet-1, calretinin, GAD67, Go-alpha), glia markers (glutamine synthetase, glial fibrillary acid protein), and a blood vessel basal membrane marker (laminin). Confocal microscopy was used to visualize the labeling patterns in retinal sections. The level of CRT expression was determined in the retina using a semiquantification method. RESULTS: Immunohistochemical assessment of CRT expression in the normal aged retina revealed strong labeling in photoreceptor synaptic terminals and in inner and outer segments. Labeling was also observed on subpopulations of amacrine cells and ganglion cells as well as in the outer and inner plexiform layers. CRT labeling was observed in blood vessels, although was absent in glial cells. In retinal detachment, the CRT labeling pattern was maintained, although there was an apparent decrease in labeling in inner retina and an increase in CRT expression in photoreceptors. CONCLUSIONS: CRT is expressed in areas of intense metabolic activity, such as photoreceptors, selected cells in the inner retina, and sites of creatine transport into the retina (inner retinal blood vessels and retinal pigment epithelium). The absence of CRT to Müller cells harmonizes with the concept that glial cells are a biosynthetic source of creatine but not a source of creatine to other retinal neurons. The increased immunolabeling of CRT localized to the outer retina in retinal detachment suggests a regional metabolic remodeling occurring in photoreceptor cells.

Functional and anatomical remodeling in human retinal detachment.

Rhegmatogenous retinal detachment is by far the most common indication for retinal surgery and a major cause of severe vision loss. Increased levels of glutamate found in the vitreous of human patients and persistent remodeling, even after reattachment, suggest substantial neurochemical, functional and anatomical changes have occurred in the detached retina. Therefore, this study was designed to characterize the morphological changes and glutamate receptor functionality in human rhegmatogenous retinal detachment. A cation channel permeating probe, agmatine (1-amino-4-guanidobutane; AGB), was employed to track endogenous and kainate (KA) driven channel functionality combined with immunocytochemical characterization of cellular remodeling. In the detached retina increased AGB permeability was identified in the outer retina while there was a decrease in the inner retina in basal conditions. KA receptors exhibited increased AGB permeability in ON bipolar cells and decreased permeability in calbindin labeled inner retinal cells. All retinal detachment samples demonstrated ectopic synaptic protein expression, photoreceptor processes extending toward the inner retina, and other remodeling features of retinal degeneration. These anatomical changes have been demonstrated in animal studies and are novel features unreported in primary cases of human retinal detachment. We conclude that deafferentation in retinal detachment leads to alteration of the glutamatergic pathway.

Functional activation of glutamate ionotropic receptors in the human peripheral retina.

Glutamate is the major neurotransmitter in the vertebrate retina. Neurons involved in the glutamate pathway express α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) receptors. Functional characterization of these ionotropic glutamate receptors can be achieved by using a cation channel permeating probe named agmatine (1-amino-4-guanidobutane; AGB). Retinal mapping using this guanidinium analog has certain advantages including the immunocytochemical identification of a whole population of neurons expressing functional glutamate gated receptor channels. We have extended AGB studies into the functionality of ionotropic receptors in peripheral aged human retina to serve as a comparison for functional analysis of retinopathies such as retinal detachment. We probed the human retina with AGB after activation with AMPA, KA and NMDA. The results showed patterns of AGB entry into neurons consistent with those previously observed in subunit localization studies in adult mammalian retinae including primates. Application of 30 µM AMPA activated receptors in virtually all calretinin immunoreactive AII amacrine cells in the mid-peripheral human retina. About half of the AII amacrine cells showed AGB permeation after incubation with 50 µM KA. Some bipolar cells including DB3 OFF bipolar cells displayed functional KA receptors. Colocalization of AGB with parvalbumin labeled horizontal cells revealed functional KA and AMPA receptors with no responsiveness to NMDA activation. NMDA activation resulted in AGB labeling of ganglion cells and amacrine cells. The present study provides a description of functional ionotropic glutamate receptors in the aged mid-peripheral human retina.

Immunolocalization of gap junction protein connexin43 (GJA1) in the human retina and optic nerve.

PURPOSE: Gap junctions are intercellular channels that have been implicated in the pathogenesis of neuronal death after central nervous system injury. This study determines the expression pattern of gap junction protein connexin43 in the human retina and optic nerve. METHODS: An affinity-isolated polyclonal antibody to the C-terminal segment of the cytoplasmic domain of human connexin43 was used to determine connexin43 localization. Postmortem human eyes were examined by immunohistochemical staining of frozen sections using antibodies to connexin43. Antibody binding was detected using confocal microscopy and fluorochrome-conjugated secondary antibodies. Double-label immunohistochemistry identified the cell types expressing connexin43. RESULTS: Connexin43 immunoreactivity was detected in the human retina on glial fibrillary acidic protein (GFAP)-positive astrocytes in the retinal ganglion cell layer and, to a lesser extent, on the processes of glutamine synthetase-labeled Müller cells. The retinal and choroidal circulations showed strong connexin43 immunolabeling. Dense connexin43 immunoreactivity was present between adjacent cells of the retinal pigment epithelium, and there was diffuse connexin43 immunoreactivity on GFAP-positive astrocytes in the optic nerve. CONCLUSIONS: In the human retina and optic nerve, connexin43 is present on glia, blood vessels, and epithelial cells. An understanding of the distribution of connexin43 in the normal retina and optic nerve may be used to evaluate changes associated with retinal and optic nerve disease.