Quantification of Changes in Visual Function During Disease Development in a Mouse Model of Pigmentary Glaucoma

Abstract

Purpose: We investigated the relationship between visual parameters that are commonly affected during glaucomatous disease progression with functional measures of retina physiology using electroretinography and behavioral measures of visual function in a mouse model of glaucoma. Electroretinogram components measuring retinal ganglion cell (RGC) responses were determined using the non-invasive Ganzfeld flash electroretinography (fERG) to assess RGC loss in a mouse model of glaucoma.

Methods: Intraocular pressure (IOP), behaviorally assessed measures of visual function, namely visual acuity and contrast sensitivity as well as fERG responses were recorded in 4- and 11-month-old male DBA/2 mice. Scotopic threshold response (STR) and photopic negative response components as well as oscillatory potentials (OPs) were isolated from fERG responses and correlated with IOP, optomotor reflex measurements, and RGC counts.

Results: The 11-month-old DBA/2 mice had significantly elevated IOP, reduced visual performance, as assessed behaviorally, significant RGC loss, deficits in standardized fERG responses, reduced STRs, and differences in OP amplitudes and latencies, when compared with 4-month-old mice of the same strain. STRs and OPs correlated with some visual and physiological parameters. In addition, elevated IOP and RGC loss correlated positively with measures of visual function, specifically with surrogate measures of RGC function derived from fERG.

Conclusions: Our data suggest that RGC function as well as interactions of RGCs with other retinal cell types is impaired during glaucoma. In addition, a later OP wavelet denoted as OP4 in this study was identified as a very reproducible indicator of loss of visual function in the glaucoma mouse model.

Figure 1:

Eleven-month old DBA/2 mice have increased IOP (A), reduced visual function [measured as reduced visual acuity (B) and increased contrast sensitivity threshold (C)] and significant RGC loss (Brn3a-positive cells in D [red label], G) indicative of glaucoma disease progression and when compared to young, non-glaucomatous control mice. Photoreceptor (E, G) and bipolar cell numbers (F, G) and retinal layer thickness (H) in 11-month old glaucomatous mice are not significantly different from 4 month-old non-glaucomatous mice; data are expressed as the number of cells per 1-mm retinal length in a vertical section or the thickness of the retina or layer in µm. In E and F, rhodopsin (Rho) immunoreactivity was found in the outer segments of the rod photoreceptors (red); immunoreactivity for Glycogen Phosphorylase (GP) labelled cone photoreceptors, cone bipolar cells and their terminals (green), and for Protein Kinase α (PKCα) labelled rod bipolar cells and their terminals (purple) for Brn3a labelled RGCs. Nuclei were stained with DAPI (blue). BV= Blood vessel. Data are expressed as mean ± SEM.

Figure 2:

Example traces from A) rod, standard rod–cone, and Hi-intensity rod–cone ISCEV (1–3) protocol response. Following light-adaption (10 minute) with background illumination B) cone and Hi-intensity cone ISCEV (4–5) protocol response and C) standard and Hi-intensity flicker flicker at 30 Hz ISCEV (6–7) protocol response. D) Scotopic and E) Photopic fERG intensity series in 4 month-old (black line) and 11-month old (gray line) DBA/2 mice. Flash intensities are expressed in log cd·s/m².

Figure 3:

Implicit times of a-waves (A) and b-waves (B) and their respective amplitudes (C, D) and the subsequent b/a ratio of the a- and b-wave amplitudes (E) were determined for 4 month-old (closed circles) and 11-month old (open circles) DBA/2 mice.

Figure 4:

11-month old glaucomatous (open circles) DBA/2 mice show deficits in scotopic (A) and photopic amplitudes (B) compared to young, 4 month-old non- glaucomatous mice (closed circles), however, the scotopic (C) and photopic (D) a- and b-wave implicit times remained largely unchanged. Data are expressed as mean ± SEM.

Figure 5:

pSTR (A, C), nSTR (A, D), STR amplitudes (A, E) and PhNR (B, F) were measured from responses to flashes below −4.5 - −3.5 log cd·s/m² for scotopic and 0–1.5 log cd·s/m² for photopic responses of 4 month-old (closed circles) and 11-month old (open circles) DBA/2 mice. In responses to flash stimuli below −3.6 log cd·s/m², where STRs indicate RGC function, 11-month old DBA/2 mice had significantly reduced STRs at −4 log cd·s/m² when compared to 4 month-old DBA/2s. The latency of the STRs (G) and PhNR (H) was not significantly different between ages. Data are expressed as mean ± SEM.

Figure 6:

Scotopic oscillatory potential amplitudes, a measure of RGC function, were analyzed with regard to latency and amplitude at 4 peaks / local maxima termed OP1–4 (A, B) and were measured from the baseline (black dashed horizontal line) to the local maximal of the positive peak (grey dotted vertical line; B). There was no significant difference in OP1–4 amplitudes between the two age groups (C), however, 11-month old DBA/2 mice showed increased amplitudes at OP1 (D), unchanged amplitudes at OP2 (F) and OP3 (H) and decreased amplitudes at OP4 (J). Latencies were slower at OP1 (E) and at lower flash intensities for OP2 (G), OP3 (I) and OP4 (K). Data are expressed as mean ± SEM.

Figure 7:

STRs recorded at a flash intensity of −4 log cd·s/m² were correlated with Visual Acuity, Contrast Sensitivity Threshold, IOP and RGC counts, respectively. Specific Pearson product-moment correlation coefficient r and respective p-values are listed directly in panels, where significant correlations were identified.

Figure 8:

OP amplitudes recorded at flash intensities of 1.5 (OP1) or −1.5 log cd·s/m² (OP2–4) were correlated with Visual Acuity, Contrast Sensitivity Threshold, IOP and RGC number. Specific Pearson product-moment correlation coefficient r and respective p-values are listed directly in panels, where significant correlations were identified.