Abstract
purpose. This study explored the feasibility of mapping the retina’s responses to visual stimuli noninvasively, by using functional magnetic resonance imaging (fMRI).
methods. fMRI was performed on a 9.4-Tesla scanner to map activity-evoked signal changes of the retina-choroid complex associated with visual stimulation in anesthetized cats (n = 6). Three to 12 1-mm slices were acquired in a single shot using inversion-recovery, echo-planar imaging with a nominal in-plane resolution of 468 × 468 μm2. Visual stimuli were presented to the full visual field and to the upper and lower visual fields. The stimuli were drifting or stationary gratings, which were compared with the dark condition. Activation maps were computed using cross-correlation analysis and overlaid on anatomic images. Multislice activation maps were reconstructed and flattened onto a two-dimensional surface.
results. fMRI activation maps showed robust increased activity in the retina-choroid complex after visual stimulation. The average stimulus-evoked fMRI signal increase associated with drifting-grating stimulus was 1.7% ± 0.5% (P < 10−4, n = 6) compared with dark. Multislice functional images of the retina flattened onto a two-dimensional surface showed relatively uniform activation. No statistically significant activation was observed in and around the optic nerve head. Hemifield stimulation studies demonstrated that stimuli presented to the upper half of the visual field activated the lower part of the retina, and stimuli presented to the lower half of the visual field activated the upper part of the retina, as expected. Signal changes evoked by the stationary gratings compared with the dark basal condition were positive but were approximately half that evoked by the drifting gratings (1.0% ± 0.1% versus 2.1% ± 0.3%, P < 10−4).
conclusions. To the best of our knowledge, this is the first fMRI study of the retina, demonstrating its feasibility in imaging retinal function dynamically in a noninvasive manner and at relatively high spatial resolution.
The retina is more than an array of light sensors. It is a part of the central nervous system, and it generally performs complex signal processing before sending its signals to the lateral geniculate nucleus and the visual cortex.
1 Metabolism and blood flow of the retina are complex and in many aspects unique.
2 For example, being only approximately 250 μm thick, the retina is one of the most vascularized tissues in the body, has one of the highest oxidative metabolic rates per tissue weight,
3 and yet still manages to produce an inordinate amount of lactate under basal conditions. The retina is nourished by the retinal and choroidal blood supplies,
4 which feed the inner and the outer retina, respectively. Oxygen transport into the highly structured layers of cells in the retina relies heavily on diffusion, with oxygen tension midway between the inner and outer retina approaching close to hypoxic levels under normal physiological conditions.
5 Hence, the retina is particularly susceptible to ischemic injuries, as in the case of diabetic retinopathy.
6 7 The choroidal blood flow, which feeds the outer retina including the photoreceptors, is many times higher than the cortical blood flow.
8 9 10 It has been suggested that such high blood flow is necessary for maintaining retinal oxygenation
11 and for dissipating heat produced by the incident light on the retina,
12 13 although these issues remain controversial.
Retinal physiology and function have been studied by using a number of methods, including, but not limited to, standard psychophysical evaluation, standard
14 and multifocal
15 electroretinography, oxygen microelectrodes,
5 laser Doppler flowmetry,
16 17 blue-field entoptic technique,
18 and fluorescein angiography.
19 These techniques can provide valuable functional and physiological information on the retina in research and/or clinical settings. Most of these techniques require an unobstructed pathway of light from the cornea through the lens and to the retina. Disease states with media opacity, such as cataract and some diseases of the vitreous body, preclude the use of many of these techniques. Vitreous humor oxygen tension has been assessed by measuring water relaxation time.
20 This technique, however, has poor sensitivity and is only useful for measuring large and steady-state oxygenation changes in the vitreous humor. Therefore, noninvasive and dynamic imaging of the retina and its functional response to visual stimuli at the tissue level, without depth limitation and in three dimensions by using functional magnetic resonance imaging (fMRI), could have numerous important applications. This method is also expected to complement other techniques mentioned.
fMRI is a powerful, noninvasive imaging method capable of high spatial resolution. It has been widely used in imaging brain processes, ranging from sensory perceptions to cognitive functions. The most commonly used fMRI technique is based on blood oxygenation level-dependent (BOLD) contrast, first described by Ogawa et al.
21 in rat brains and subsequently used for mapping brain functions in humans.
22 23 24 BOLD contrast originates from the intravoxel magnetic field’s inhomogeneticity, which is induced by paramagnetic deoxyhemoglobin in the erythrocytes in blood. Magnetic susceptibility differences between the compartmentalized paramagnetic deoxyhemoglobin in blood and the surrounding tissues generate magnetic field gradients across and near the vascular-tissue boundary. Changes in regional deoxyhemoglobin content can be visualized in susceptibility-sensitized (T
2*- or T
2-weighted) BOLD images. When a specific task (e.g., finger tapping) is performed, regional cerebral blood flow increases disproportionally, overcompensating the stimulus-induced oxygen-consumption rate increase needed to fuel the increased neural activity and thus resulting in a regional reduction in deoxyhemoglobin concentration. Therefore, BOLD signals after elevated neural activity increase compared with basal conditions, making it possible to dynamically and noninvasively map changes in neural activities.
There is considerable evidence that visual stimuli evoke changes in tissue blood flow and tissue oxygen tension in the retina. Riva et al.
25 and Longo et al.
26 demonstrated with laser Doppler that blood flow in the retina increases after visual stimulation, and Scheiner et al.
27 reported stimulus-evoked changes in blood flow using a blue-field simulation technique. Linsenmeier et al.,
5 using oxygen electrode recording, showed that oxygenation in different retinal cell layers also increases after visual stimulation. Therefore, it is not unreasonable to postulate that a BOLD fMRI signal response could be detected, because the BOLD signal derives from changes in tissue oxygenation as a result of blood flow modulation. In this study, we explored the feasibility of extending functional MRI to image the retina’s response to various visual stimuli. The difficulties and challenges of retinal fMRI are discussed, and the solutions to these problems are detailed. The results presented herein demonstrate that fMRI of the retina is a promising method that has the potential to provide valuable information in a noninvasive manner.
fMRI experiments were performed on a 9.4-Tesla, 31-cm horizontal MRI scanner (Magnex Scientific, Abingdon, UK), equipped with a 30-Gauss/cm gradient (11.0 cm inner diameter, 300-μs rise time; Magnex Scientific), and a workstation console ( Unity INOVA; Varian, Palo Alto CA). A custom-built, small-surface coil was placed lateral to the right eye. Anatomic images were acquired using a data acquisition scheme (Turbo-FLASH) with an inversion-recovery contrast to suppress the strong signals from the vitreous humor for clear identification of the retinal-vitreous border. Sagittal anatomic images were acquired using the following parameters: interimage delay (TR), 3.5 seconds; flip angle, 10°; echo time (TE), 3.5 ms; inversion delay (TI), 1.4 seconds; data matrix, 128 × 128; and field of view (FOV), 3 × 3 cm2. Typically, 9 to 12 sequential 1-mm anatomic images were acquired.
BOLD fMRI sagittal images were acquired using a gradient-echo, echo-planar imaging (EPI) data acquisition scheme,
31 with fat suppression and inversion-recovery contrast. EPI is widely used in fMRI studies, because it is more sensitive to stimulus-evoked T
2* and T
2 changes, and is less sensitive to physiological motion than other pulse sequences.
32 The single-shot, EPI sequence parameters were: TR, 3.5 seconds; TI, 1.4 seconds; TE, 12 ms; slice thickness, 1 or 2 mm; data matrix, 64 × 64; and FOV, 3 × 3 cm
2 (nominal in-plane resolution of 468 × 468 μm
2). Shifted-echo acquisition with the center echo at the 20th k-space line was used to achieve an echo time of 12 ms, approximating tissue T
2* for optimal BOLD contrast.
33 Fat suppression was achieved using three chemical-shift-selective (CHESS, 10-ms sinc) pulses and crusher gradients.
34 The strong signals from the vitreous humor were suppressed using a nonspatially selective inversion (10-ms hyperbolic secant) pulse with an inversion delay of 1.4 seconds. Typically, 3 to 12 multislice fMRI images were acquired in an interleaved fashion after a single-inversion pulse (total time for one multislice set was 3.5 seconds). Although there is a spread of TI values (1.4–1.7 seconds) across different slices, the vitreous signal was reasonably suppressed across multiple slices, because of its long T
1 at high field.
For the drifting gratings versus dark stimulus, a single fMRI measurement consisted of a three-epoch paradigm consisting of 20-20-20-20-20-20-20 images (underscore indicates drifting-grating stimulus is on). For the sequence of dark, drifting-, and stationary-grating stimuli, a single fMRI measurement consisted of two repeats of the following scheme: 20 images during dark, 20 images during drifting-grating, and 20 images during stationary-grating stimuli. Typically, approximately 15 repeated fMRI measurements were made on each animal.
Given these spatial resolutions, the term “retina” is used herein to refer to the retina-choroid complex. Although the inversion contrast used for suppression of the vitreous humor could yield a small contribution of blood flow to the fMRI signal, we refer to the fMRI signal as the BOLD signal, because our preliminary data suggested that the BOLD contribution was likely to be dominant (see the Discussion section).
fMRI of the retina is challenging. Major technical difficulties encountered in fMRI studies of the retina include imaging a thin curve tissue, imaging in regions of high-susceptibility distortion, signal contamination from fat and vitreous humor, and saccade-related motion artifacts. The retina is only approximately 250 μm thick; thus, high-resolution imaging must be used, resulting in relatively low signal-to-noise ratio. The use of a high field is helpful, because high fields increase the signal-to-noise and contrast-to-noise ratios. With a spatial resolution of 468 × 468 × 1000 μm3, there are partial-volume effects that reduce functional contrast. Nevertheless, these data indicate that the fMRI signal is sufficiently strong to be readily detectable. The retina is physically located close to the nasal cavity and the frontal sinus, which are regions with large susceptibility differences, increasing the likelihood of image distortion. It is therefore critical to optimize the magnetic field homogeneity. A spin-echo acquisition scheme (instead of the gradient-echo scheme used herein) can be used to reduce signal loss due to intravoxel dephasing, although it is, in general, expected to yield a relatively poorer functional contrast. Major fat signals outlining the back of the eye can be reasonably suppressed using CHESS pulses and crusher gradients. Fat suppression eliminates chemical-shift ghost artifacts due to the resonance offset between the water and the fat signals. The strong signals from the vitreous humor can be efficiently suppressed with an inversion contrast, allowing a clear visualization of the retinal border and minimizing the partial volume effect by the otherwise overwhelming signal from the vitreous humor.
With regard to motion artifacts, saccadic motion is of primary concern. With an EPI acquisition scheme, which acquires the entire k-space data of one image within approximately 20 ms, motion within a single image is expected to be minimal. Saccadic and other potential motion artifacts between images can be significant, even under anesthetics, and can falsely correlate with the stimulus paradigm, as demonstrated in
Figure 1 . This observation is consistent with previous findings that systemic anesthetics are not completely effective in paralyzing the rectus muscles responsible for saccadic motion. However, with the addition of systemic paralytics and topical eye drop anesthetics, saccadic and lens motion were substantially and sufficiently reduced.
Although the BOLD response is likely to be dominant, there could be a small contribution from changes in blood-flow signal. Blood-flow weighting could arise from the use of a small surface coil for magnetization inversion, which could yield partial spatial selectivity, even for a nonspatially selective pulse. Such a blood flow-weighted signal would add constructively to the positive BOLD signal (if blood flow increased, as in activation of neurons in the cerebral cortex), resulting in increased functional contrast. Our preliminary data, however, suggest that blood flow’s contribution under these experimental MR parameters is small (data not shown). Although this blood flow weighting does not compromise the potential of this method to image retinal function, separate measurements of BOLD and blood flow fMRI signals nevertheless should yield valuable information regarding the unique retinal metabolism and hemodynamics under basal and elevated activity conditions.
In an attempt to further understand the fMRI signal response under drifting and stationary stimuli, we measured the fMRI signal responses under drifting and stationary gratings compared with the dark. Both drifting and stationary gratings evoked an increase in the BOLD (oxygenation) response compared with the dark condition. Our observation is consistent with that of Bill and Sperber
39 who used deoxyglucose technique and found that flickering light evokes a higher metabolic rate in the inner retina than steady light. Our observation is also in accordance with that of Stefansson,
40 who used an oxygen electrode to measure preretinal oxygenation changes as a function of the room light intensity and found that oxygen tension increases with increasing room light intensity compared with darkness. It should be noted that a simple illumination compared with dark is generally known to result in hyperpolarization of the photoreceptors and, thus, a decrease in metabolism. Consequently, a decrease in fMRI signal is expected compared with dark. Our data showed, on the contrary, that the fMRI signal change is positive under constant illumination compared with dark, suggesting there is an overall regional metabolic and blood flow increase. One possible explanation for this discrepancy is that hyperpolarization, per se, evokes a net metabolic increase (and commensurately larger blood flow increase) in the photoreceptors and/or in other cell types (such as bipolar and horizontal cells) along signal transduction pathway. Another possibility is that the electrophysiogical and fMRI measurements might not be measuring the same parameters, in that hyperpolarization is recorded from the photoreceptors, whereas the fMRI signal samples the entire retinal tissue. The difference in stimuli used in the electrode recording and our fMRI measurements could be a factor. Further studies are needed to fully understand the fMRI signal changes. Comparison across different modalities under identical or similar experimental conditions is necessary to determine whether there are indeed discrepancies between these measurements.
The retina is nourished by the retinal and choroidal blood supplies.
4 The choroidal blood flow, which nourishes the photoreceptors, is many times higher than the retinal blood flow
8 9 10 (4–10 times, depending on species and regions on the retina). The fMRI signal changes could originate from the choroidal and/or the retinal vessels. At current spatial resolution, it is not yet possible to separate the choroidal and retinal contributions to the fMRI signal. We are working on increasing spatial resolution in an attempt to resolve these contributions. Understanding the sources and mechanisms underpinning the fMRI signal changes in the retina is important for designing better experiments and for making full use of this method for imaging retinal “function.”