Age and intraocular pressure in murine experimental glaucoma.

Age and intraocular pressure (IOP) are the two most important risk factors for the development and progression of open-angle glaucoma. While IOP is commonly considered in models of experimental glaucoma (EG), most studies use juvenile or adult animals and seldom older animals which are representative of the human disease. This paper provides a concise review of how retinal ganglion cell (RGC) loss, the hallmark of glaucoma, can be evaluated in EG with a special emphasis on serial in vivo imaging, a parallel approach used in clinical practice. It appraises the suitability of EG models for the purpose of in vivo imaging and argues for the use of models that provide a sustained elevation of IOP, without compromise of the ocular media. In a study with parallel cohorts of adult (3-month-old, equivalent to 20 human years) and old (2-year-old, equivalent to 70 human years) mice, we compare the effects of elevated IOP on serial ganglion cell complex thickness and individual RGC dendritic morphology changes obtained in vivo. We also evaluate how age modulates the impact of elevated IOP on RGC somal and axonal density in histological analysis as well the density of melanopsin RGCs. We discuss the challenges of using old animals and emphasize the potential of single RGC imaging for understanding the pathobiology of RGC loss and evaluating new therapeutic avenues.

Longitudinal In Vivo Changes in Retinal Ganglion Cell Dendritic Morphology After Acute and Chronic Optic Nerve Injury.

Purpose: To characterize in vivo dendritic changes in retinal ganglion cells (RGCs) after acute (optic nerve transection, ONT) and chronic (experimental glaucoma, EG) optic nerve injury. Methods: ONT and EG (microbead model) were carried out in Thy1-YFP mice in which the entire RGC dendritic arbor was imaged with confocal fluorescence scanning laser ophthalmoscopy over two weeks in the ONT group and over two and six months, respectively, in two (groups 1 and 2) EG groups. Sholl analysis was used to quantify dendritic structure with the parameters: area under the curve (AUC), radius of the dendritic field, peak number of intersections (PI), and distance to the PI (PD). Results: Dendritic changes were observed after three days post-ONT with significant decreases in all parameters at two weeks. In group 1 EG mice, mean (SD) intraocular pressure (IOP) was 15.2 (1.1) and 9.8 (0.3) mmHg in the EG and untreated contralateral eyes, respectively, with a significant corresponding decrease in AUC, PI, and PD, but not radius. In group 2 mice, the respective IOP was 13.1 (1.0) and 8.8 (0.1) mmHg, peaking at two months before trending towards baseline. Over the first two months, AUC, PI, and PD decreased significantly, with no further subsequent changes. The rates of change of the parameters after ONT was 5 to 10 times faster than in EG. Conclusions: Rapid dendritic changes occurred after ONT, while changes in EG were slower and associated with level of IOP increase. The earliest alterations were loss of inner neurites without change in dendritic field.

GCaMP3 expressing cells in the ganglion cell layer of Thy1-GCaMP3 transgenic mice before and after optic nerve injury.

The genetically encoded green fluorescent protein-based calcium sensor, GCaMP, has been used to detect calcium transients and report neuronal activity. We evaluated the specificity of GCaMP3 expression to retinal ganglion cells (RGCs) of the transgenic Thy1-GCaMP3 mouse line in healthy control animals and in those after optic nerve transection (ONT). Retinas from control mice (n = 4) were isolated and stained for RNA-binding protein with multiple splicing (RBPMS) and choline acetyltransferase (ChAT), specific markers for RGCs and cholinergic amacrine cells, respectively. GCaMP3 expression was enhanced with green fluorescent protein (GFP) immunoreactivity. In one subset of animals, ONT was performed 3, 7, or 14 days before sacrifice (n = 4, 4, 4, respectively). Cells positive for GCaMP3, RBPMS, ChAT, as well as the population of co-labeled cells, were quantified. In another subset of animals (n = 4), in vivo confocal scanning laser ophthalmoscope imaging was performed in the same mice at baseline and at 3, 7 and 14 days after ONT. The mean (SD) densities of GCaMP3, RBPMS, and ChAT expressing cells in control retinas were 2663 (110), 3401 (175), and 1041 (47) cells/mm2, respectively. Of the GCaMP3+ cells, 92 (1)% were co-labeled with RBPMS, while 72 (1)% of RBPMS-labeled cells expressed GCaMP3. ChAT expressing cells were not co-labeled with GCaMP3. The number of GCaMP3+ and RBPMS+ cells decreased dramatically after ONT; 78%, 39%, and 18% of GCaMP3+ and 80%, 40%, and 15% of RBPMS+ cells, relative to control retinas, survived at 3, 7, and 14 days after ONT. However, the number of ChAT+ cells did not change. There was a progressive decrease in GCaMP3 fluorescence after ONT in in vivo images. The majority of RGCs in the ganglion cell layer of Thy1-GCaMP3 mice express GCaMP3. There was an expected progressive and specific loss of GCaMP3 expression after ONT. Thy1-GCaMP3 transgenic mice have potential for longitudinal assessment of RGCs in injury models.

Effects of 3D Stratification of Retinal Ganglion Cells in Sholl Analysis.

BACKGROUND: Sholl analysis is used to quantify the dendritic complexity of neurons. Differences between two-dimensional (2D) and three-dimensional (3D) Sholl analysis can exist in neurons with extensive axial stratification of dendrites, however, in retinal ganglion cells (RGCs), only 2D analysis is typically reported despite varying degrees of stratification within the retinal inner plexiform layer. We determined the impact of this stratification by comparing 2D and 3D analysis of the same RGCs. NEW METHOD: Twelve retinas of mice expressing yellow fluorescent protein in RGCs under the control of the Thy1 promotor were whole-mounted. The entire dendritic arbor of 120 RGCs was traced, after which 2D and 3D Sholl analysis was performed. Two parameters describing dendritic complexity; area under the curve (AUC) and peak number of intersections (PNI) were then derived and analyzed. RESULTS AND COMPARISON WITH EXISTING METHODS: The AUC and PNI were significantly higher with 3D analysis compared to 2D analysis with medians of 2805 and 2508 units, and 31 and 27, respectively (P < 0.01). Both 2D and 3D AUC increased with arbor thickness. The discrepancy in AUC between the two methods depended on mean AUC (with every 1 unit increase in mean AUC resulting in a discrepancy of 0.1 unit), but not arbor thickness. CONCLUSION: In RGCs imaged in vitro, there is a difference in AUC and PNI derived with 2D and 3D Sholl analysis. Where possible, 3D Sholl analysis of RGCs should be performed for more accurate quantitative analysis of dendritic structure.

Colocalization of optical coherence tomography angiography with histology in the mouse retina.

Optical coherence tomography angiography (OCT-A) allows in vivo, non-invasive, functional imaging of retinal perfusion. The purpose of this study was to determine the reliability of OCT-A in visualizing the complete retinal vasculature by comparing in vivo OCT-A images to matched ex vivo retinal tissue in mice. Adult female C57BL/6 mice were imaged to obtain OCT-A images of the superficial vascular complex, intermediate capillary plexus and deep capillary plexus. Z-stack fluorescence images of whole-mounted retinas, labeled for vascular endothelial cells by anti-isolectin immunohistochemistry and FITC-dextran perfusion, were generated. The OCT-A and fluorescence images were manually colocalized and vessel length measured for each of the techniques. Mean vessel length among all plexuses showed less than 13% difference between OCT-A and lectin immunohistochemistry and less than 4% difference between OCT-A and FITC-dextran perfusion. The strength of the correlation between OCT-A and lectin immunohistochemistry ranged from 0.46-0.95, while that between OCT-A and FITC-perfusion ranged from 0.67-0.88. OCT-A visualized retinal vasculature in vivo to a similar extent in matched ex vivo histology images. Our results show that OCT-A is a reliable method for acquiring in vivo images of retinal perfusion in mice, with the ability to differentiate each vascular plexus.

Optical Coherence Tomography Angiography in Mice: Quantitative Analysis After Experimental Models of Retinal Damage.

Purpose: We implemented optical coherence tomography angiography (OCT-A) in mice to: (1) develop quantitative parameters from OCT-A images, (2) measure the reproducibility of the parameters, and (3) determine the impact of experimental models of inner and outer retinal damage on OCT-A findings. Methods: OCT-A images were acquired with a customized system (Spectralis Multiline OCT2). To assess reproducibility, imaging was performed five times over 1 month. Inner retinal damage was induced with optic nerve transection, crush, or intravitreal N-methyl-d-aspartic acid injection in transgenic mice with fluorescently labeled retinal ganglion cells (RGCs). Light-induced retinal damage was induced in albino mice. Mice were imaged at baseline and serially post injury. Perfusion density, vessel length, and branch points were computed from OCT-A images of the superficial, intermediate, and deep vascular plexuses. Results: The range of relative differences measured between sessions across the vascular plexuses were: perfusion density (2.8%-7.0%), vessel length (1.9%-4.1%), and branch points (1.9%-5.0%). In mice with progressive RGC loss, imaged serially and culminating in around 70% loss in the fluorescence signal and 18% loss in inner retinal thickness, there were no measurable changes in any OCT-A parameter up to 4 months post injury that exceeded measurement variability. However, light-induced retinal damage elicited a progressive loss of the deep vascular plexus signal, starting as early as 3 days post injury. Conclusions: Vessel length and branch points were generally the most reproducible among the parameters. Injury causing RGC loss in mice did not elicit an early change in the OCT-A signal.

Retinal Characterization of the Thy1-GCaMP3 Transgenic Mouse Line After Optic Nerve Transection.

Purpose: GCaMP3 is a genetically encoded calcium indicator for monitoring intracellular calcium dynamics. We characterized the expression pattern and functional properties of GCaMP3 in the Thy1-GCaMP3 transgenic mouse retina. Methods: To determine the specificity of GCaMP3 expression, Thy1-GCaMP3 (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J) retinas were processed for immunohistochemistry with anti-green fluorescent protein (anti-GFP, to enhance GCaMP3 fluorescence), anti-RBPMS (retinal ganglion cell [RGC]-specific marker), and antibodies against amacrine cell markers (ChAT, GABA, GAD67, syntaxin). Calcium imaging was used to characterize functional responses of GCaMP3-expressing (GCaMP+) cells by recording calcium transients evoked by superfusion of kainic acid (KA; 10, 50, or 100 µM). In a subset of animals, optic nerve transection (ONT) was performed 3, 5, or 7 days prior to calcium imaging. Results: GFP immunoreactivity colocalized with RBPMS but not amacrine cell markers in both ONT and non-ONT (control) groups. Calcium transients evoked by KA were reduced after ONT (50 µM KA; ΔF/F0 [SD]; control: 1.00 [0.67], day 3: 0.50 [0.35], day 5: 0.31 [0.28], day 7: 0.35 [0.36]; P < 0.05 versus control). There was also a decrease in the number of GCaMP3+ cells after ONT (cells/mm2 [SD]; control: 2198 [453], day 3: 2224 [643], day 5: 1383 [375], day 7: 913 [178]; P < 0.05). Furthermore, the proportion of GCaMP3+ cells that responded to KA decreased after ONT (50 µM KA, 97%, 54%, 47%, and 58%; control, 3, 5, and 7 days, respectively). Conclusions: Following ONT, functional RGC responses are lost prior to the loss of RGC somata, suggesting that anatomical markers of RGCs may underestimate the extent of RGC dysfunction.

The retino-retinal projection: Tracing retinal ganglion cells projecting to the contralateral retina.

We investigated the presence of a direct retino-retinal (R-R) projection between the two eyes via the optic chiasm of retinal ganglion cells (RGCs) in adult Long-Evans rats. We also explored the presence of collateral projections originating from these cells to the brain. In the first group of animals, right optic nerves (ONs) were orbitally transected approximately 2mm behind the globe followed by application of fluorochrome (2% Fluorogold [FG]) to the optic nerve stump to retrogradely label the R-R projection RGCs (R-RGCs) on the contralateral side. Animals were then sacrificed after 3, 5, 7, or 21 days. Contralateral retinas were fixed, whole-mounted, and imaged for R-RGCs. In a second group of animals, RGCs were retrogradely labeled with 15% rhodamine-ß-isothiocynate (RITC) at the superior colliculi, where approximately 96% of rat RGCs synapse. Seven days later, the right ONs were transected and 2% FG applied to the proximal and distal ON stumps. Animals were then sacrificed after 5 days. Contralateral retinas were examined for co-labeled (RITC/FG) RGCs. Control rats underwent the same procedures excluding fluorescent tracer application. In the first group of animals, the number of R-RGCs in the contralateral eye ranged from 3 to 25 and did not depend on survival time. The second group of animals revealed evidence of co-labeled contralateral RGCs. Results suggest that a greater number of R-RGCs persist into adulthood than previously reported [M. Müller, H. Holländer, 1988]. Furthermore, the presence of co-labeled RGCs in the contralateral eye indicates that in adult rodents some R-R projections have a collateral projection to the brain, whereas previous reports had only found collateral projections in newborns.