Retinal ganglion cell ablation in guinea pigs.

Guinea pigs are a common model of human ocular conditions; however, their visual function has not been fully characterized. The purpose of this study was to determine the contributions of retinal ganglion cells to structural and functional measures in guinea pigs. Healthy adult guinea pigs (n = 12) underwent unilateral optic nerve crush. Retinal structure was assessed with spectral domain optical coherence tomography (OCT), and thickness of the ganglion cell/nerve fiber layer (GC/NFL) was determined. Visual function was assessed with optomotor tracking of a drifting grating and light adapted electroretinograms (ERGs). From flash ERGs, a-wave, b-wave, oscillatory potentials (OPs), and photopic negative response (PhNR) were analyzed. From pattern ERGs, N1P1 and P1N2 were analyzed. Histological studies were done at various time points for ganglion cell quantification. Optomotor tracking was absent in optic nerve crush eyes following optic nerve crush. Significant thinning of the GC/NFL was evident four weeks following the crush. Flash ERGs revealed a significant reduction in the OP1 amplitude two weeks following crush (P < 0.01) and in the PhNR amplitude six weeks following crush (P < 0.01). There were no significant changes in a-wave, b-wave, or pattern ERG responses (P > 0.05 for all). In vivo OCT imaging showed progressive thinning of inner retinal layers. Ganglion cell density, quantified histologically, was significantly reduced by 75% in the optic nerve crush eye compared to the control eye at four weeks following crush. These findings indicate that retinal ganglion cells contribute to the PhNR and OP1 components of the full field flash ERG, but not significantly to the pattern ERG in guinea pigs. This study demonstrates that OCT imaging and full field flash ERGs are valuable in assessing retinal ganglion cell loss in vivo in guinea pigs and will help to further establish the guinea pig as a model of human ocular pathologies.

Retinal Ganglion Cell Loss and Microglial Activation in a SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) affects the spinal cord, brain stem, and cerebral cortex. In this pathology, both neurons and glial cells are affected. However, few studies have analyzed retinal microglia in ALS models. In this study, we quantified the signs of microglial activation and the number of retinal ganglion cells (RGCs) in an SOD1G93A transgenic mouse model at 120 days (advanced stage of the disease) in retinal whole-mounts. For SOD1G93A animals (compared to the wild-type), we found, in microglial cells, (i) a significant increase in the area occupied by each microglial cell in the total area of the retina; (ii) a significant increase in the arbor area in the outer plexiform layer (OPL) inferior sector; (iii) the presence of cells with retracted processes; (iv) areas of cell groupings in some sectors; (v) no significant increase in the number of microglial cells; (vi) the expression of IFN-γ and IL-1ß; and (vii) the non-expression of IL-10 and arginase-I. For the RGCs, we found a decrease in their number. In conclusion, in the SOD1G93A model (at 120 days), retinal microglial activation occurred, taking a pro-inflammatory phenotype M1, which affected the OPL and inner retinal layers and could be related to RGC loss.

Retinal Topography in Two Species of Baleen Whale (Cetacea: Mysticeti).

Little is known about the visual systems of large baleen whales (Mysticeti: Cetacea). In this study, we investigate eye morphology and the topographic distribution of retinal ganglion cells (RGCs) in two species of mysticete, Bryde's whale (Balaenoptera edeni) and the humpback whale (Megaptera novaeanglia). Both species have large eyes characterised by a thickened cornea, a heavily thickened sclera, a highly vascularised fibro-adipose bundle surrounding the optic nerve at the back of the eye, and a reflective blue-green tapetum fibrosum. Using stereology and retinal whole mounts, we estimate a total of 274,268 and 161,371 RGCs in the Bryde's whale and humpback whale retinas, respectively. Both species have a similar retinal topography, consisting of nasal and temporal areas of high RGC density, suggesting that having higher visual acuity in the anterior and latero-caudal visual fields is particularly important in these animals. The temporal area is larger in both species and contains the peak RGC densities (160 cells mm-2 in the humpback whale and 200 cells mm-2 in Bryde's whale). In the Bryde's whale retina, the two high-density areas are connected by a weak centro-ventral visual streak, but such a specialisation is not evident in the humpback whale. Measurements of RGC soma area reveal that although the RGCs in both species vary substantially in size, RGC soma area is inversely proportional to RGC density, with cells in the nasal and temporal high-density areas being relatively more homogeneous in size compared to the RGCs in the central retina and the dorsal and ventral retinal periphery. Some of the RGCs were very large, with soma areas of over 2,000 µm2. Using peak RGC density and eye axial diameter (Bryde's whale: 63.5 mm; humpback whale: 48.5 mm), we estimated the peak anatomical spatial resolving power in water to be 4.8 cycles/degree and 3.3 cycles/degree in the Bryde's whale and the humpback whale, respectively. Overall, our findings for these two species are very similar to those reported for other species of cetaceans. This indicates that, irrespective of the significant differences in body size and shape, behavioural ecology and feeding strategy between mysticetes and odontocetes (toothed whales), cetacean eyes are adapted to vision in dim light and adhere to a common "bauplan" that evolved prior to the divergence of the two cetacean parvorders (Odontoceti and Mysticeti) over 30 million years ago.

RetFM-J, an ImageJ-based module for automated counting and quantifying features of nuclei in retinal whole-mounts.

The present article introduces RetFM-J, a semi-automated ImageJ-based module that detects, counts, and collects quantitative data on nuclei of the inner retina from H&E-stained whole-mounted retinas. To illustrate performance, computer-derived outputs were analyzed in inbred C57BL/6J mice. Automated characterization yielded computer-derived outputs that closely matched manual counts. As a method using open-source software that is freely available, inexpensive staining reagents that are robust, and imaging equipment that is routine to most laboratories, RetFM-J could be utilized in a wide variety of experiments benefiting from high-throughput, quantitative, uniform analyses of total cellularity in the inner retina.

Cell-Specific Markers for the Identification of Retinal Cells and Subcellular Organelles by Immunofluorescence Microscopy.

Identification of specific cells and subcellular structures in the retina is fundamental for understanding the visual process, retinal development, disease progression, and therapeutic intervention. The increased use of knockout, transgenic, and naturally occurring mutant mice has further underlined the need for retinal cell-specific imaging. Immunofluorescence microscopy of retinal cryosections and whole-mount tissue labeled with cell-specific markers has emerged as the method of choice for identifying and quantifying specific cell populations and mapping their distribution within the retina. Immunofluorescence microscopy has also been important in localizing proteins to specific compartments of retinal cells. In most cases indirect labeling methods are employed in which lightly fixed retinal samples are first labeled with a primary antibody targeted against a cell-specific protein of interest and then labeled with a fluorescent dye-tagged secondary antibody that recognizes the primary antibody. The localization and relative abundance of the protein can be readily imaged under a conventional fluorescent or confocal scanning microscope. Immunofluorescence labeling can be adapted for imaging more than one antigen through the use of multiple antibodies and different, non-overlapping fluorescent dyes. A number of well-characterized immunochemical markers are now available for detecting photoreceptors, bipolar cells, amacrine cells, horizontal cells, Müller cells, and retinal pigment epithelial cells in the retina of mice and other mammals. Immunochemical markers are also available for visualizing the distribution of specific proteins within cells with most studies directed toward photoreceptor cells.