Spectral domain optical coherence tomography in mouse models of retinal degeneration

Abstract

Purpose: Spectral domain optical coherence tomography (SD-OCT) allows cross-sectional visualization of retinal structures in vivo. Here, the authors report the efficacy of a commercially available SD-OCT device to study mouse models of retinal degeneration.

Methods: C57BL/6 and BALB/c wild-type mice and three different mouse models of hereditary retinal degeneration (Rho(-/-), rd1, RPE65(-/-)) were investigated using confocal scanning laser ophthalmoscopy (cSLO) for en face visualization and SD-OCT for cross-sectional imaging of retinal structures. Histology was performed to correlate structural findings in SD-OCT with light microscopic data.

Results: In C57BL/6 and BALB/c mice, cSLO and SD-OCT imaging provided structural details of frequently used control animals (central retinal thickness, CRT(C57BL/6) = 237 +/- 2 microm and CRT(BALB/c) = 211 +/- 10 microm). RPE65(-/-) mice at 11 months of age showed a significant reduction of retinal thickness (CRT(RPE65) = 193 +/- 2 microm) with thinning of the outer nuclear layer. Rho(-/-) mice at P28 demonstrated degenerative changes mainly in the outer retinal layers (CRT(Rho) = 193 +/- 2 microm). Examining rd1 animals before and after the onset of retinal degeneration allowed monitoring of disease progression (CRT(rd1 P11) = 246 +/- 4 microm, CRT(rd1 P28) = 143 +/- 4 microm). Correlation of CRT assessed by histology and SD-OCT was high (r(2) = 0.897).

Conclusions: The authors demonstrated cross-sectional visualization of retinal structures in wild-type mice and mouse models for retinal degeneration in vivo using a commercially available SD-OCT device. This method will help to reduce numbers of animals needed per study by allowing longitudinal study designs and will facilitate characterization of disease dynamics and evaluation of putative therapeutic effects after experimental interventions.

Figure 1

Retinal SLO imaging and OCT in C57BL/6 mice (PW4) with a regular retinal structure. (A)-(D): Representative example of en-face imaging using cSLO. (A) native infrared (830 nm), (B) red free (513 nm), and (C) autofluorescence (AF) mode. (D) Fluorescein angiography (FLA) confirming an intact retinal vasculature (arrowhead = artery, arrow = vein). (E)-(H): Corresponding OCT data. (E) Fundus picture with indicated orientation of cross-sectional SD-OCT scans. (F) Corresponding B-Scan at the optic nerve head displaying a Bergmeister’s papilla characterized by a structural remnant of the developmental hyaloid vascular system (asterisk) including blow-up section. (G) Light microscopic data of an age matched C57BL/6 animal. (H) Representative B-Scan with retinal layers labeled. GC/IPL: Ganglion cell / Inner plexiform layer, INL: Inner nuclear layer, OPL: Outer plexiform layer, ONL: Outer nuclear layer, OLM: Outer limiting membrane, I/OS: Inner-/outer segment border, RPE/CC: RPE/Choriocapillary complex.

Figure 2

Retinal SLO imaging and OCT in BALB/c mice (PW10). Typical results of cSLO en-face imaging in the presence of non-pigmented retinal structures in (A) native infrared (830 nm), (B) red free (513 nm), and (C) autofluorescence (AF) modes. (D) Fluorescein angiography (FLA) in BALB/c mice displayed both retinal as well as choroidal vasculature (asterisk), as lack of pigment allows light at λ = 488 nm to better penetrate the RPE/Choriocapillary complex. (E) Fundus image with indicated orientation of (F/H) corresponding B-Scans. (G) Representative histological data of an age-matched BALB/c mouse. (H) Virtual cross section with designation of the different retinal layers. Note the altered signal composition in the outer retina due to lack of pigmentation in BALB/c mice ((F)blow-up). There are four highly reflective layers (HRLs) possibly demarcating the inner/outer segment border (IOS) and RPE, the non pigmented choriocapillary and choroidal structures. GC/IPL: Ganglion cell / Inner plexiform layer, INL: Inner nuclear layer, OPL: Outer plexiform layer, ONL: Outer nuclear layer, OLM: Outer limiting membrane, I/OS: Inner-/outer segment border, RPE/CC: RPE/Choriocapillary complex, Cho: Choroid.

Figure 3

Retinal degeneration and RPE irregularities in the rho^-/- mouse model (PW4). (A) Native infrared (830 nm) and (B) autofluorescence (AF) mode cSLO data. (C) SD-OCT section and blow-up, revealing a complete lack of rod outer segments (ROS) together with an apparent thinning of the outer nuclear layer (ONL). (D) Ex vivo histology for comparison, confirming the observed reduction in ONL thickness and the lack of ROS. GC/IPL: Ganglion cell / Inner plexiform layer, INL: Inner nuclear layer, OPL: Outer plexiform layer, OLM: Outer limiting membrane, IS: Photoreceptor inner segments, RPE/CC: RPE/Choriocapillary complex.

Figure 4

Retinal degeneration and RPE irregularities in the Rpe65^-/- mouse model (PM11). (A) Histology showing lipid droplets from stored retinyl esters (arrows) in the RPE, together with a reduced retinal thickness. (B) Spots of hyperfluorescence in the AF mode, suggesting the presence of photoreceptor debris. (C) OCT cross sectional image confirming a reduction of ONL thickness, while laminar organization (blow-up) was not as clearly delineated as in wt mice. GC/IPL: Ganglion cell / Inner plexiform layer, INL: Inner nuclear layer, OPL: Outer plexiform layer, ONL: Outer nuclear layer, OLM: Outer limiting membrane, I/OS: Inner-/outer segment border, RPE/CC: RPE/Choriocapillary complex.

Figure 5

En-face and SD-OCT imaging in rd1 and corresponding wild type control mice at P11 and P28. At P11, no signs of retinal degeneration were evident in (A/D) cSLO or (B/E) SD-OCT imaging. (C/F) Histology confirmed normal outer nuclear layer thickness. In contrast, rd1 mice at P28 revealed significant retinal degeneration (G). This pattern was also evident in the SD-OCT cross sectional images (H) and corresponding histology (I), where inner retinal layers appeared intact, while the inner nuclear layer seemed to border the RPE almost directly. Outer plexiform/nuclear layers and photoreceptor segments were virtually non-existent. (J-L) Wild type controls at P28 showed no signs of retinal degeneration. All scale bars = 100μm.

Figure 6

Evaluation of inner and outer retinal thickness in wildtype mice and animal models of hereditary retinal degeneration using either SD-OCT in vivo imaging or conventional morphometric assessment by histology. (A-C) Bar graphs indicate inner (dark) and outer (light) retinal thickness. (A) Note statistically significant reduction of outer but not inner retinal thickness in BALB/c, Rho^-/- and RPE65^-/- compared to C57/BL6 mice. (B) There is no statistical significant difference between rd1 and wt mice at P11. While outer retina remained unchanged in the wt mice at P28, there is complete loss of outer retina in the age matched rd1 mice. (C) Conventional morphometric assessment of retinal thickness in histological sections with similar changes as reported by SD-OCT (A-B). Significance was calculated using Student’s T-test. Abbreviations: ns = not significant (p > 0.01), * = p ≤ 0.01, ** = p ≤ 0.001, *** = p ≤ 0.0001. (D) Scatter plot shows the correlation of histological vs. OCT data on total (dots) and outer retinal thickness (error bars only) from C57/BL6, BALB/c, Rho^-/-, RPE65^-/-, rd1-wt P11, rd1 P11, rd1-wt P28 and rd1 P28 mice. Pearson’s correlation coefficients (r²) are displayed for total retinal thickness (y = 0.975x) and outer retinal thickness (y = 1.018x) separately. All data are reported as mean values ± standard deviation (error bars).