OCT abnormalities in <i>rd16;Nrl^−/−</i> mice.

<p>(A) Upper panels: Representative OCT scans vertically across ∼2 mm of retina (centered at the ONH, optic nerve head) in a WT mouse and in two <i>rd16;Nrl^−/−</i> mice of different ages. Lower panels: Magnified parts of the superior region of the retinal sections with overlaid longitudinal reflectivity profiles (LRPs) to demonstrate the reflective abnormalities in the outer retinal region in <i>rd16;Nrl^−/−</i> mice (b and c) compared with C57BL6 WT (a). (B) Upper two panels: Vertical OCT sections quantified for ONL+ thickness in two age groups of <i>rd16;Nrl^−/−</i> mice. Regions of outer retina with pseudorosettes were excluded in the measurement. ONL+ profiles in the older (P83–89, n = 12 eyes) age group were thinner than those in younger (P31–41, n = 35 eyes) mice; gray bands in the P83–89 plot represent mean±2 SD for ONL+ thickness of the P31–41 mice. For reference, insets at lower right of the upper two plots show original raw data before suppression of pseudorosette regions. Third panel from top: Means of ONL+ data across the vertical meridian in two age groups (error bar: ± SD; P31–41, open circles; P83–89, filled triangles). Lowest panel: Histograms showing average ONL+ fraction across vertical meridian of two age groups (*represents <i>p</i><0.001). (C) Histological sections of <i>rd16;Nrl^−/−</i> retina at 4 different ages from P21 to P80, compared with a WT retinal section. Histograms show ONL fraction (based on the earlier age group) in <i>rd16;Nrl^−/−</i> mice from peripheral retina (n = 6 eyes in each of the two age groups, *represents <i>p</i> = 0.01).</p

Spatial and temporal distribution of pseudorosettes in <i>rd16;Nrl^−/−</i> retina.

<p>(A) Histological sections from peripheral retina of two <i>rd16;Nrl^−/−</i> mice at different ages demonstrating the presence of pseudorosettes (arrows). Calibration = 50 μm. (B) Schematic drawing of the mouse retina indicating the coverage of the central OCT raster scans (red circle). ONH is centered in the drawing. Integrated <i>en face</i> image of the central region of a P41 <i>rd16;Nrl^−/−</i> mouse showing how pseudorosettes appear as white dots (B, right panel, red circle). (C) Pseudorosette distribution within the central retinal region in a young (P31) and an older (P83) <i>rd16;Nrl^−/−</i> eyes. Insets (up and right) show average pseudorosettes as density in different sectors of the central retinal region sampled (n = 8 eyes for both age groups). (D) Upper: Histograms comparing number of pseudorosettes in the central retina by OCT at two different ages (P31, n = 10 eyes; P83, n = 8 eyes). Lower: Pseudorosette counts from histological sections of peripheral retina of two different age groups (P21–40, n = 6 eyes; P60–80, n = 6 eyes). Both data sets in <i>rd16;Nrl^−/−</i> mice indicate that the number of rosettes decreases with age (*represents <i>p</i><0.001 and <i>p = </i>0.01 for the upper and lower graphs, respectively). Error bars, ± SD from the mean.</p

Expression of photoreceptor proteins is reduced over time in <i>rd16;Nrl^−/−</i> mice.

<p>Representative cross sections (20X) from central and peripheral retina of P21, P40, P60 and P80 <i>rd16;Nrl^−/−</i> mice were immunostained for the presence of cone transducin alpha (GNAT2) (A) or S- cone opsin (B) and PNA (A,B). Despite maintenance of cone outer segment sheaths (PNA), both GNAT2 and S-opsin expression are markedly reduced by P60 in both central and peripheral retina. INL- inner nuclear layer, ONL- outer nuclear layer, IS/OS- inner segments/outer segments. Calibration = 35 μm.</p

Structure and function in the <i>rd16;Nrl^−/−</i> mouse retina.

<p>(A) ERG b-wave amplitudes of responses to UV- and M-cone stimuli as a function of age in <i>rd16;Nrl^−/−</i> mice from P34 to P83 (n = 95) with comparisons to data from previously recorded signals in <i>Nrl^−/−</i> (squares <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Cheng1" target="_blank">[11]</a>; square with cross <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Mears1" target="_blank">[19]</a>), and <i>rd16</i> (crosses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Cideciyan3" target="_blank">[7]</a>) mice. Upper: Cone b-wave responses to ultraviolet (UV, 360 nm peak) stimuli in the <i>rd16;Nrl^−/−</i> mice are severely reduced compared with those of <i>Nrl^−/−</i> mice at comparable ages. Amplitudes in <i>rd16</i> mice are low compared to the other mice. It is also notable that ERGs of the <i>Nrl^−/−</i> mice remain relatively stable throughout this age range, while ERGs of the <i>rd16; Nrl^−/−</i> and <i>rd16</i> mice decline in amplitude with age. Lower: Responses to green (510 nm) stimuli are substantially lower in amplitude than those from UV-cone stimuli. Again, <i>Nrl^−/−</i> mice have the largest amplitudes and do not decline with increasing age within this time period. The <i>rd16;Nrl^−/−</i> waveforms are lower in amplitude and there is a reduction with age. Only limited data were available for <i>rd16</i> mice and these fell within the range of <i>rd16;Nrl^−/−</i> amplitudes. Waveforms for representative <i>rd16;Nrl^−/−</i> mice at various ages (grey-filled circles) are illustrated in the panels at right. Grey lines: linear regression fit to log-converted data (dashes) and 95% prediction intervals (solid). Squares with cross at earliest age in graphs: <i>Nrl^−/−</i> data from Mears et al., 2001 (B) Photoreceptor structure (ONL+) as a function of the combined UV- and M-cone ERG b-wave amplitudes. ONL+ remains similar to the value at P31 (youngest age <i>rd16;Nrl^−/−</i> we studied) across various degrees of ERG amplitude reduction. Horizontal dashed line is the reference level for the lower limit of retinal structure thickness at P31 (−2SD from the mean at this age); photoreceptor structure above this lower limit indicates no difference compared to the data of P31 (error bars, +2SD from mean).</p

Structure and function in the central retina of <i>CEP290</i>-LCA patients.

<p>(A) Cross-sectional OCT scans along the horizontal meridian through the fovea in a normal subject, a <i>CEP290</i>-LCA patient, and an RP patient. ONL is highlighted in blue. Inset shows location of scan. (B) Relationship of foveal ONL thickness and visual acuity in <i>CEP290</i>-LCA patients. Bar graph represents the average ±1SD foveal ONL thickness of eyes in the different visual acuity ranges (n = 3, for 0.1–1 LogMAR; n = 5, for 1–2 LogMAR; and n = 11, for 2–NLP). Dashed line is lower limit of normal and emphasizes that despite low acuities, foveal ONL is within normal limits. Inset, data from a series of RP patients plotted similarly to show the more expected relationship between structure and visual acuity in retinal degenerations (n = 3, for 0–0.2 LogMAR; n = 20, for 0.2–1 LogMAR; and n = 3 for 1–2 LogMAR). Dashed line is also lower limit of normal. (C) Relationship in <i>CEP290</i>-LCA patients of width of the ONL in the central retina and patient age at time of examination. ONL width was unable to be defined in a 32-year-old <i>CEP290</i>-LCA patient with maculopathy. Solid line is linear regression. Inset, traced central ONL peaks in representative patients of different ages.</p