Manganese‐enhanced MRI reveals multiple cellular and vascular layers in normal and degenerated retinas

The neural retina can be characterized histologically into six major distinct layers (1). From the vitreo-retinal interface, these layers are the nerve fiber layer/ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, and photoreceptor-segment layer. The interspersed plexiform layers are synaptic links between adjacent nuclear layers. Two flanking blood supplies nourish the retina. The retinal vasculature, located closest to the vitreous, exists predominantly within the ganglion cell layer but also projects capillaries deep into the inner plexiform layer and inner nuclear layer. In some species, including rats, deep capillaries are also sparsely present in the proximal outer plexiform layer (2). The choroidal vasculature, on the other hand, is external to the neural retina and is sandwiched between the retinal pigment epithelium (RPE) and the sclera. If considered with the six histologically defined layers, the choroidal vasculature constitutes an additional (seventh) layer. The outer nuclear layer and the photoreceptor-segment layer are avascular. Most retinal imaging modalities, such as fundus photography and optical coherence tomography for structural imaging as well as fluroscein angiography and laser Doppler flowmetry for blood flow imaging, are based on optics. These techniques are generally limited to probing the inner retinal surface and/or large surface vessels and the signals are confounded by tissue absorption and optical scattering. Signals arising from multiple retinal layers are generally difficult to separate. Moreover, disease-induced opacity of the vitreous humor and/or lens precludes the use of optically based imaging techniques to study the retina. In contrast, magnetic resonance imaging (MRI) is widely used for noninvasive imaging of brain anatomy, physiology, and function in vivo. The nascent capacity of MRI to distinguish various tissue types can be further enhanced by using exogenous contrast agents. Manganese (Mn), for example, is both an MRI contrast agent and a calcium analog. Mn is trapped in the intracellular space with a half-life on the order of days (3) and thus enhances intracellular MRI signal. Mn appears to preferentially accumulate in neuronal cell bodies compared to dendritic processes by cell volume (4–6) and thus Mn-enhanced MRI (MEMRI) in the retina could in principle be used to resolve the interdispersed nuclear and plexiform layers. MEMRI of the retina following intraperitoneal administration of Mn has been used as a measure of retinal function after prolonged light and dark adaptation (7). Gd-DTPA (gadolinium-diethylene-triamine-pentaacetic acid) is another contrast agent widely used to selectively enhance the vascular compartment in the brain because normal blood–brain and blood–retinal barriers as well as the RPE are impermeable to Gd-DTPA (8). In this study we explored high-contrast MEMRI to resolve rat retinal layers in vivo at 25 × 25 μm in-plane resolution. Mn was injected directly into the vitreous. Seven distinct bands of alternating signal intensities were consistently detected. Three additional sets of experiments were performed to corroborate layer assignments. First, Gd-DTPA was injected intravenously to Mn-injected rats to visualize the two vascular layers bounding the retina. Second, standard histology was performed and histological layer thicknesses were obtained for cross-validation of MRI-derived layer assignments and laminar thicknesses. Finally, we investigated an established animal model of photoreceptor degeneration, the Royal College of Surgeons (RCS) rats (9). RCS rats have a genetic defect in the RPE that prevents proper phagocytosis of the photoreceptor segments, resulting in a spontaneous and complete degeneration of the photoreceptors by postnatal day 90 (P90). MEMRI detected the expected loss of the outer plexiform layer, outer nuclear layer, and photoreceptor-segment layer, consistent with histology. This study demonstrates laminar structures in the in vivo retina at very high spatial resolution and contrast without depth limitation.