Evidence for Diffuse Central Retinal Edema In Vivo in Diabetic Male Sprague Dawley Rats

Background: Investigations into the mechanism of diffuse retinal edema in diabetic subjects have been limited by a lack of animal models and techniques that co-localized retinal thickness and hydration in vivo. In this study we test the hypothesis that a previously reported supernormal central retinal thickness on MRI measured in experimental diabetic retinopathy in vivo represents a persistent and diffuse edema. Methodology/Principal Findings: In diabetic and age-matched control rats, and in rats experiencing dilutional hyponatremia (as a positive edema control), whole central retinal thickness, intraretinal water content and apparent diffusion coefficients (ADC, ‘water mobility’) were measured in vivo using quantitative MRI methods. Glycated hemoglobin and retinal thickness ex vivo (histology) were also measured in control and diabetic groups. In the dilutional hyponatremia model, central retinal thickness and water content were supernormal by quantitative MRI, and intraretinal water mobility profiles changed in a manner consistent with intracellular edema. Groups of diabetic (2, 3, 4, 6, and 9 mo of diabetes), and age-matched controls were then investigated with MRI and all diabetic rats showed supernormal whole central retinal thickness. In a separate study in 4 mo diabetic rats (and controls), MRI retinal thickness and water content metrics were significantly greater than normal, and ADC was subnormal in the outer retina; the increase in retinal thickness was not detected histologically on sections of fixed and dehydrated retinas from these rats

Summary of central retinal thickness.

<p>Data are presented for the diabetic (D, diabetes duration given) and age-matched controls (C) male SD groups. These results strongly support our previous observations of diabetes-induced thickening in this model <i>in vivo </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#pone.0029619-Berkowitz2" target="_blank">[10]</a>. *, significant compared to age-matched control, <i>P</i><0.05. Error bars represent SEM, and numbers above the bars represent number of animals studied.</p

Representative ADC images.

<p><i>Top (grayscale images)</i>: Representative images from a control rat (the same as featured in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#pone-0029619-g002" target="_blank">Figure 2</a>) with diffusion weighting applied parallel to the optic nerve (top/bottom of page in this image orientation). These representative images were collected at three different b-values (from left to right, 0, 250, and 990 s/mm²). Note that, while signal is lost with mild diffusion weighting in vitreous and anterior chamber – signal there being just above background levels at 250 s/mm² – the retina, with more restricted diffusion, retains signal through b = 990 s/mm². Preferential signal loss in lateral (relative to the anterior and posterior) lens cortex at higher b-values is due to the preferential movement of water parallel to the lens surface. Yellow arrows in the b = 0 image indicate the region of retina linearized and shown below (measuring from the optic nerve head, ∼±30% of the hemiretinal extent). <i>Bottom (color images)</i>: Average ADC maps from each group are presented for visualization purposes only since spatial normalization had not yet been applied (see Legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#pone-0029619-g002" target="_blank">Figure 2</a> for additional discussion). Arrows to the left and right of the color maps indicate the group average location of the retina/choroid border. We also note formal qualitative analysis of ADC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#pone-0029619-g005" target="_blank">Figure 5</a>) was performed on the linearized and group averaged retinal profile.</p

Summary of retinal water content.

<p><i>Top</i>: Absolute intraretinal water content across the central retina in control rats. The y-axis scaling in this Figure was set to help visualize the variations in intraretinal water content. With this scaling, values <74% are not shown. Due to partial volume averaging and the non-linear effects of the very fast flow in the choroidal circulation (relative to that in the inner retina), water content estimates at depths beyond 88% are considered inaccurate. This region, as well as the tissue near the vitreoretinal border (at 0% thickness), are gray-shaded to indicate that they are excluded from further analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#pone.0029619.s008" target="_blank">Appendix S1</a> for more detail). Error bars represent SEM. <i>Bottom</i>: Grand means of relative (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#s4" target="_blank">methods</a>) central intraretinal water content (i.e., from 12–88% thickness) for control rats (C & preDH data, here, “C”), dilutional hyponatremia group (postDH data, here, “DH”), and the diabetic group (D). In both graphs, error bars represent SEM. Numbers above the bars represent number of animals studied.</p

Group comparisons of intraretinal ADC_‖‖ and ADC_⊥ (i.e., water mobility).

<p>Water mobility profiles for control (black; average of groups C, earlyC, and and preDH; n = 17), dilutional hyponatremia (turquoise; ‘postDH’ group; n = 5), and diabetic (red; group ‘D’; n = 7) retinas. Data points in gray-shaded areas near the vitreoretinal border (0% thickness) and retina/choroid border (100% thickness) are excluded to minimize partial volume averaging with non-retinal tissue (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#pone.0029619.s008" target="_blank">Appendix S1</a> for more detail). The solid lines and shaded areas of profiles represent mean and SEM's, respectively. Note that ADC data from the DH group are plotted with the group average control data (groups C, earlyC, and preDH), but statistical findings are based on paired comparisons: Locations where differences between preDH and postDH significantly greater than zero are shown, as are locations where preDH-to-postDH changes were statistically different than the (non-significant) earlyC-to-laterC changes. In either case, we find a significant, heterogeneous, influence of DH on retinal ADC.</p

Representative structural and water content images.

<p>Structural (<i>top</i>; unmodified spin-echo) and water content (<i>bottom</i>; turbo-FLASH) images from a diabetic rat (left column), and a second rat both before (control; middle column) and after (right column) dilutional hyponatremia. <i>Top</i>: Directly under each image, the central retina (±1 mm from the optic nerve head; yellow arrows in top center) is shown after linearization. Colored arrows to the left and right of those animals' linearized retinas indicate the location of the retina/choroid border for each; red – diabetic, blue – dilutional hyponatremia, green – control (before dilutional hyponatremia). Those arrows also indicate the outer border used for the central retinal thickness measurement, since the linearized retinas are shown after co-alignment of the vitreoretinal border. Consistent with the appearance of the structural images, the diabetic and dilutional hyponatremia retinas are visibly thicker than the control retina. The same pattern is visible in group average images, which are shown below each representative subject's linearized retina. The reduced clarity of the retina/choroid border in group average images is due to inter-individual differences in retinal thickness: Since linearized retinas are co-aligned to the vitreoretinal border in this figure, group average images (without spatial normalization) will average retina from one subject with non-retina of another at the retina/choroid border due to the range of retinal thicknesses in each group (diabetic: 199–244 µm; control: 149–203 µm; dilutional hyponatremia 198–227 µm). Such considerations motivated our resampling of retinal profiles onto a %thickness (rather than µm) scale prior to formal analysis (e.g. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#pone-0029619-g003" target="_blank">Figs. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#pone-0029619-g005" target="_blank">5</a>). <i>Bottom</i>: As above, the group average is presented directly beneath the linearized central retina for representative subjects. Here, though, the color-maps shows %water content (%v/v) values after setting vitreous to 99% (see 3^rd paragraph of the ‘MRI Image Analysis’ section within <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029619#s4" target="_blank">Methods</a>). Note that both retinal thickness and %water content will determine the total water content of a retina.</p