Structural and functional MRI reveals multiple retinal layers

Abstract

MRI is a noninvasive diagnostic modality that reveals anatomy, physiology, and function in vivo without depth limitation or optical interference. MRI application to the retina, however, remains challenging. We improved spatial resolution to resolve layer-specific structure and functional responses in the retina and confirmed the laminar resolution in an established animal model of retinal degeneration. Structural MRI of normal rat retinas revealed three bands corresponding histologically to (i) the combined ganglion cell layer/inner nuclear layer plus the embedded retinal vessels, (ii) the avascular outer nuclear (photoreceptor) layer and its photoreceptor segments, and (iii) the choroidal vascular layer. Imaging with an intravascular contrast agent (gadolinium-diethylene-tri-amine-pentaacetic acid) enhanced the retinal and choroidal vascular layers bounding the retina, but not the avascular outer nuclear layer and the vitreous. Similarly, blood-oxygen-level-dependent (BOLD) functional MRI revealed layer-specific responses to hyperoxia and hypercapnia. Importantly, layer-specific BOLD responses in the two vascular layers were divergent, suggesting the two vasculatures are differentially regulated. To corroborate sensitivity and specificity, we applied layer-specific MRI to document photoreceptor degeneration in Royal College of Surgeons rats. Consistent with histology, layer-specific MRI detected degeneration of the outer nuclear layer. Surprisingly, MRI revealed increased thickness in the choroidal vascular layer and diminished BOLD responses to hyperoxia and hypercapnia in the Royal College of Surgeons rat retinas, suggesting perturbation of vascular reactivity secondary to photoreceptor loss. We conclude that MRI is a powerful investigative tool capable of resolving lamina-specific structures and functional responses in the retina as well as probing lamina-specific changes in retinal diseases.

Fig. 1.

Layer-specific anatomical imaging of the retina. (A) A bar depicting a 0.5-mm-thick MRI slice overlaid on an edge-enhancement processed eye image illustrating the potential partial-volume effect. (B) Anatomical images at 60 × 60 μm resolution from a normal adult rat. Three distinct layers (solid arrows) of alternating bright, dark, and bright bands are evident. Sclera (dashed arrow) appears hypointense.

Fig. 2.

Contrast-enhanced MRI delineating two vascular layers bounding the retina. (A and B) Contrast-enhanced images at 60 × 60 μm before (A) and after (B) Gd-DTPA administration. (C) The subtracted image. The two arrows in the expanded views indicate the inner and outer bands of the retina corresponding to the two vascular layers bounding the retina. The dark lines across the lens arose from magnetic and radiofrequency heterogeneity. Three arrowheads indicate signal enhancement of extraocular tissues supplied by Gd-DTPA permeable vessels.

Fig. 3.

Automated determination of MRI retinal thicknesses. (A) The retina was segmented by using an edge-detection technique (green trace). Retinal thickness was quantified from point a to point b, cognizant of the irregular retinal thickness from the posterior pole to the pars plana. (B) Intensity profiles of two animals delineate the outer, middle, and inner bands. The vertical dashed arrows indicate the vitreous boundary. Values in micrometers indicate the band thicknesses of the solid trace.

Fig. 4.

Simulations to validate MRI thickness determination. (Upper) Simulated retinas were constructed based on parameters in Fig. 3 and sampled at 60 μm per pixel. (Lower Left) Two noise levels were evaluated (Gaussian noise of 1 and 8 times that of vitreous). The retina was segmented (green trace), and an intensity profile was obtained. (Lower Right) Comparison of the input and simulated thicknesses showed accurate width determination given the contrast, signal-to-noise ratio, and band separations. Note that the midpoint intensities are not at the same height.

Fig. 5.

Histological section of a normal adult Sprague–Dawley rat retina stained with toluidine blue. Three vertical bars on the left show the assignments of the three MRI-derived layers. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS+OS, inner and outer photoreceptor segment; CH, choroidal vascular layer.

Fig. 6.

Differential layer-specific BOLD fMRI of the retina. Lamina-specific BOLD fMRI responses to hyperoxia (100% O₂) (A) and hypercapnia (5% CO₂ in air) (B) from a normal rat at 90 × 90 μm in-plane resolution. BOLD percent-change maps are overlaid on echoplanar images. The color bar indicates 1–20% BOLD changes.

Fig. 7.

Anatomical MRI and histology of retinal degeneration. (A–C) Anatomical images at 60 × 60 μm resolution of P16 RCS retina before photoreceptor degeneration (control) (A), and degenerated P120 RCS retina before (B) and after (C) i.v. administration of Gd-DTPA. The arrowheads in C indicate signal enhancement of extraocular tissues. (D and E) Intensity profiles (D) and histological sections (E) show thinning of the P120 compared with the P16 RCS retina. The dashed blue and red arrows in D indicate the vitreous boundaries.