Layer-Specific Manganese-Enhanced MRI of the Diabetic Rat Retina in Light and Dark Adaptation at 11.7 Tesla.

PURPOSE: To employ high-resolution manganese-enhanced MRI (MEMRI) to study abnormal calcium activity in different cell layers in streptozotocin-induced diabetic rat retinas, and to determine whether MEMRI detects changes at earlier time points than previously reported. METHODS: Sprague-Dawley rats were studied 14 days (n = 8) and 30 days (n = 5) after streptozotocin (STZ) or vehicle (n = 7) injection. Manganese-enhanced MRI at 20 × 20 × 700 µm, in which contrast is based on manganese as a calcium analogue and an MRI contrast agent, was obtained in light and dark adaptation of the retina in the same animals in which one eye was covered and the fellow eye was not. The MEMRI activity encoding of the light and dark adaptation was achieved in awake conditions and imaged under anesthesia. RESULTS: Manganese-enhanced MRI showed three layers, corresponding to the inner retina, outer retina, and the choroid. In normal animals, the outer retina showed higher MEMRI activity in dark compared to light; the inner retina displayed lower activity in dark compared to light; and the choroid showed no difference in activity. Manganese-enhanced MRI activity changed as early as 14 days after hyperglycemia and decreased with duration of hyperglycemia in the outer retina in dark relative to light adaptation. The choroid also had altered MEMRI activity at 14 days, which returned to normal by 30 days. No differences in MEMRI activity were detected in the inner retina. CONCLUSIONS: Manganese-enhanced MRI detects progressive reduction in calcium activity with duration of hyperglycemia in the outer retina as early as 14 days after hyperglycemia, earlier than any other time point reported in the literature.

Comparison of retinal and cerebral blood flow between continuous arterial spin labeling MRI and fluorescent microsphere techniques.

PURPOSE: To compare basal retinal and cerebral blood flow (BF) values using continuous arterial spin labeling (CASL) MRI and fluorescent microspheres. MATERIALS AND METHODS: A total of 41 animals were used. BF was measured using an established microsphere technique (a mixture of 2.5 million 8 µm green and 0.5 million 10 µm blue fluorescent microspheres) and CASL MRI blood flow measurement in the rat retina and brain at 7 Tesla (T) and 11.7T, respectively. RESULTS: Retinal BF by MRI was 1.18 ± 0.57 mL/g/min and choroidal BF was 8.14 ± 1.8 mL/g/min (n = 6). Microsphere retinal BF was 9.12 ± 2.8 µL/min per tissue and choroidal BF was 73.38 ± 44 µL/min per tissue (n = 18), corresponding to a retinal BF value of 1.22 ± 0.36 mL/g/min by means of a wet weight conversion. The wet-weight of the choroid could not be determined. To corroborate our findings, cerebral BF (CBF) by MRI was also analyzed. In the cerebral cortices, CBF was 0.91 ± 0.29 mL/g/min (n = 14) by CASL MRI and 1.09 ± 0.37 mL/g/min (n = 6) by microspheres. There were no significant differences found between MRI and microsphere blood flow in the retina and brain. CONCLUSION: BF values in the rat retina and cerebral cortex by MRI are in agreement with those obtained by the microsphere technique.

Postocclusive reactive hyperemia occurs in the rat retinal circulation but not in the choroid.

PURPOSE: We tested the hypothesis that retinal blood flow has a postocclusive reactive hyperemia response modulated by occlusion duration and metabolic activity, and that choroidal blood flow does not. METHODS: Anesthetized and paralyzed rats (n = 34) were studied. Retinal and choroidal blood flow was measured by laser speckle imaging and laser Doppler flowmetry, respectively. Blood oxygenation level-dependent functional magnetic resonance imaging (BOLD fMRI) was used to measure changes in relative blood oxygenation of the retinal and choroidal circulations. Transient carotid occlusion was elicited with a hydraulic occluder on the common carotid artery. Several occlusion durations were tested during dark, constant light, and flicker light conditions to modulate metabolic demand. The hyperemia response magnitude was quantified by integrating the area above the blood flow baseline for the 3 minutes after release of the occlusion. RESULTS: Systemic arterial pressure (108.2 ± 1.4 mm Hg) was unaffected by the carotid occlusions, and was similar among animals and conditions. Retinal blood flow had a reactive hyperemia, but choroidal blood flow did not (e.g., 14 ± 2%.sec versus 0.5 ± 4%. sec after 60-second occlusion). The hyperemia magnitude increased as a nonlinear function of occlusion duration and reached a plateau at occlusion durations < 60 second. The hyperemia magnitude was not altered by different lighting conditions at occlusion durations of 15 and 60 seconds. BOLD fMRI results were similar to the laser-based blood flow measurements. CONCLUSIONS: The results indicate that metabolic local control has a negligible role in choroidal blood flow regulation and only partially accounts for the blood flow behavior in the retinal circulation.

MRI study of cerebral, retinal and choroidal blood flow responses to acute hypertension.

Blood flow (BF) in many tissues is stable during significant fluctuations in systemic arterial blood pressure or perfusion pressure under normal conditions. The regulatory mechanisms responsible for this non-passive BF behavior include both local and neural control mechanisms. This study evaluated cerebral BF (CBF), retinal BF (RBF) and choroidal BF (ChBF) responses to acute blood pressure increases in rats using magnetic resonance imaging (MRI). A transient increase in blood pressure inside the MRI scanner was achieved by mechanically inflating a balloon catheter to occlude the descending aorta near the diaphragm. We verified the rat model of mechanical occlusion and MRI approach by first measuring blood-flow regulatory responses to changing BP in the brain under normoxia and hypercapnia where the phenomenon is well documented. Retinal and choroidal blood-flow responses to transient increased arterial pressure were then investigated. In response to an acute increase in blood pressure, RBF exhibited autoregulatory behavior and ChBF exhibited baroregulation similar to that seen in the cerebral circulation. This approach may prove useful to investigate retinal and choroidal vascular dysregulation in rat models of retinal diseases with suspected vascular etiology.

Quantitative retinal and choroidal blood flow during light, dark adaptation and flicker light stimulation in rats using fluorescent microspheres.

PURPOSE: The present study aimed to quantify retinal and choroidal blood flow (BF) during light, dark adaptation and flicker light stimulation using the microsphere technique. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were anesthetized with isoflurane. Eyes were dark (Group I, n = 8), light (Group II, n = 8) adapted or stimulated with 10 Hz flicker light (Group III, n = 10). Retinal and choroidal BF were measured by a previously established method, using a mixture of 8 µm yellow-green and 10 µm red fluorescent microspheres. The microspheres were counted ex vivo in the dissected retina and choroid and in the reference arterial blood under a fluorescent microscope. RESULTS: The choroidal BF was 64.8 ± 29 µl/min (mean ± SD) during dark adaptation, not significantly different from that during light adaptation (66.0 ± 17.8 µl/min). The retinal BF was 13.5 ± 3.2 µl/min during 10 Hz flickering light stimulation, significantly higher than that during dark adaptation in the control fellow eyes (9.9 ± 2.9 µl/min). The choroidal BF values were not statistically different between flicker stimulation and dark adaptation. Retinal BF was 11.6 ± 2.9 µl/min during light adaptation. Dark adaptation did not increase retinal BF (Group I, 8.2 ± 2.4 µl/min; Group II, 9.9 ± 2.9 µl/min). CONCLUSIONS: These findings argue against a dark-induced or flicker-induced functional hyperemia in the choroid as a result of the demands of the outer retina. Retinal BF was not higher during dark adaptation. Our data support the conclusion that the inner retina has a higher energy demand in flicker conditions relative to dark.

Layer-specific manganese-enhanced MRI of the retina in light and dark adaptation.

PURPOSE: To employ functional manganese-enhanced MRI (MEMRI) to image layer-specific changes in calcium-dependent activities in the rat retina during light versus dark adaptation. METHODS: Functional MEMRI at 20 × 20 × 700 µm was used to study light and dark adaptation in the same animals (N = 10) in which one eye was covered and the fellow eye was not. The activity encoding of the light and dark adaptation was achieved in awake conditions and imaged under anesthesia. T(1)-weighted MRI at 11.7 tesla (T) was performed using two identical radiofrequency transceiver coils to allow interleaved MRI acquisitions of the two eyes. An intravascular contrast agent was also used to verify layer assignments. RESULTS: MEMRI detected contrasts among the inner retina, outer retina, and choroid. Independent confirmation of the vascular layers and boundaries between layers was documented with an intravascular contrast agent. The retinal layer thicknesses agreed with published data. The outer retina had lower MEMRI activity in light compared with dark adaption (P < 0.001), consistent with the increased metabolic demand associated with the "dark current." The inner retina had higher MEMRI activity in light compared with dark adaption (P < 0.05). The choroid MEMRI activity was not statistically different between light and dark adaptation (P > 0.05). CONCLUSIONS: This study demonstrated a high-resolution MEMRI protocol to image functional activities among different layers of the retinas in awake animals during light and dark adaptation. This approach could have potential applications in animal models of retinal dysfunction.

High-resolution 3D MR microangiography of the rat ocular circulation.

PURPOSE: To develop high-spatial-resolution magnetic resonance (MR) microangiography techniques to image the rat ocular circulation. MATERIALS AND METHODS: Animal experiments were performed with institutional Animal Care Committee approval. MR microangiography (resolution, 84×84×84 µm or 42×42×84 µm) of the rat eye (eight rats) was performed by using a custom-made small circular surface coil with an 11.7-T MR unit before and after monocrystalline iron oxide nanoparticle (MION) injection. MR microangiography measurements were made during air, oxygen, and carbogen inhalation. From three-dimensional MR microangiography, the retina was virtually flattened to enable en face views of various retinal depths, including the retinal and choroidal vascular layers. Signal intensity changes within the retinal or choroidal arteries and veins associated with gas challenges were analyzed. Statistical analysis was performed by using paired t tests, with P<.05 considered to indicate a significant difference. Bonferroni correction was used to adjust for multiple comparisons. RESULTS: The central retinal artery, long posterior ciliary arteries, and choroidal vasculature could be distinguished on MR microangiograms of the eye. With MR microangiography, retinal arteries and veins could be distinguished on the basis of blood oxygen level-dependent contrast. Carbogen inhalation-enhanced MR microangiography signal intensity in both the retina (P=.001) and choroid (P=.027) compared with oxygen inhalation. Carbogen inhalation showed significantly higher signal intensity changes in the retinal arteries (P=.001, compared with oxygen inhalation), but not in the veins (P=.549). With MION administration, MR microangiography depicted retinal arterial vasoconstriction when the animals were breathing oxygen (P=.02, compared with animals breathing air). CONCLUSION: MR microangiography of the eye allows depth-resolved imaging of small angiographic details of the ocular circulation. This approach may prove useful in studying microvascular pathologic findings and neurovascular dysfunction in the eye and retina.

3D magnetic resonance microscopy of the ex vivo retina.

3D-MR microscopy at 11.7T and 20 × 20 × 57 µm resolution was performed on formalin-fixed rat eyes with: (I) no contrast agent and (II) Gadodiamide (Omniscan(®) ) added to the fixative. Group I data showed generally poor contrast among layers. Group II data showed markedly better lamina-specific contrast with the nerve fiber + ganglion cell layer and inner nuclear layer being hypointense, and the inner plexiform, outer plexiform, outer nuclear layer, and the segments being hyperintense. The signal-to-noise ratio in group II was higher than group I, consistent with Gadodiamide acting as a T(1) -contrast agent. All major retinal layers were assigned and their thicknesses quantified with corroboration by histology. MR microscopy allows nondestructive examination of valuable specimens and could have applications in disease and in vivo.

Pharmacological MRI of the choroid and retina: blood flow and BOLD responses during nitroprusside infusion.

Nitroprusside, a vasodilatory nitric oxide donor, is clinically used during vascular surgery and to lower blood pressure in acute hypertension. This article reports a novel application of blood flow (BF) and blood oxygenation level dependent (BOLD) MRI on an 11.7T scanner to image the rat chorioretinal BF and BOLD changes associated with graded nitroprusside infusion. At low doses (1 or 2 µg/kg/min), nitroprusside increased BF as expected but decreased BOLD signals, showing an intriguing BF-BOLD uncoupling. At high doses (3-5 µg/kg/min), nitroprusside decreased BF and markedly decreased BOLD signals. To our knowledge, this is the first pharmacological MRI application of the retina. This approach has potential to open up new avenues to study the drug-related hemodynamic functions and to evaluate the effects of novel therapeutic interventions on BOLD and BF in the normal and diseased retinas.

Anatomical, blood oxygenation level-dependent, and blood flow MRI of nonhuman primate (baboon) retina.

The goal of this study was to demonstrate high-resolution anatomical, blood oxygenation level-dependent, and blood flow MRI on large nonhuman primate retinas using a 3-Tesla clinical scanner as a first step toward translation. Baboon was chosen because of its evolutionary similarity to human. Anesthetized preparation, free of eye-movement artifacts, was used to evaluate clinical scanner hardware feasibility and optimize multimodal protocols for retinal MRI. Anatomical MRI (0.1×0.2×2.0 mm3) before contrast-agent injection detected three alternating bright-dark-bright layers. The hyperintense inner strip nearest to the vitreous was enhanced by an intravascular contrast agent, which likely included the ganglion and bipolar cell layer and the embedded retinal vessels. The hypointense middle strip showed no contrast enhancement, which likely included the avascular outer unclear layer and photoreceptor segments. The hyperintense outer strip showed contrast enhancement, which likely corresponded to the choroid vascular layer. In the posterior retina, the total thickness including the choroid was 617±101 µm (±standard deviation, n=7). Blood oxygenation level-dependent functional MRI (0.3×0.6×2.0 mm3) of oxygen inhalation relative to air increased the signals by 6.5±1.4%. Basal blood flow (2×2×2 mm3) was 83±30 mL/100 g/min (air), and hypercapnia increased blood flow by 25±9% (P<0.05). This study demonstrates multimodal MRI to image anatomy, physiology, and function on large nonhuman primate retinas using a clinical scanner, offering encouraging data to explore human applications.

Lamina-specific functional MRI of retinal and choroidal responses to visual stimuli.

PURPOSE: To demonstrate lamina-specific functional magnetic resonance imaging (MRI) of retinal and choroidal responses to visual stimulation of graded luminance, wavelength, and frequency. MATERIALS AND METHODS: High-resolution (60 × 60 µm) MRI was achieved using the blood-pool contrast agent, monocrystalline iron oxide nanoparticles (MION) and a high-magnetic-field (11.7 T) scanner to image functional changes in the normal rat retina associated with various visual stimulations. MION functional MRI measured stimulus-evoked blood-volume (BV) changes. Graded luminance, wavelength, and frequency were investigated. Stimulus-evoked fMRI signal changes from the retinal and choroidal vascular layers were analyzed. RESULTS: MRI revealed two distinct laminar signals that corresponded to the retinal and choroidal vascular layers bounding the retina and were separated by the avascular layer in between. The baseline outer layer BV index was 2-4 times greater than the inner layer BV, consistent with higher choroidal vascular density. During visual stimulation, BV responses to flickering light of different luminance, frequency, and wavelength in the inner layer were greater than those in the outer layer. The inner layer responses were dependent on luminance, frequency, and wavelength, whereas the outer layer responses were not, suggesting differential neurovascular coupling between the two vasculatures. CONCLUSIONS: This is the first report of simultaneous resolution of layer-specific functional responses of the retinal and choroid vascular layers to visual stimulation in the retina. This imaging approach could have applications in early detection and longitudinal monitoring of retinal diseases where retinal and choroidal hemodynamics may be differentially perturbed at various stages of the diseases.

Blood oxygenation level-dependent (BOLD) functional MRI of visual stimulation in the rat retina at 11.7 T.

Although optically based imaging techniques provide valuable functional and physiological information of the retina, they are mostly limited to the probing of the retinal surface and require an unobstructed light path. MRI, in contrast, could offer physiological and functional data without depth limitation. Blood oxygenation level-dependent functional MRI (BOLD fMRI) of the thin rat retina is, however, challenging because of the need for high spatial resolution, and the potential presence of eye movement and susceptibility artifacts. This study reports a novel application of high-resolution (111 × 111 × 1000 µm(3)) BOLD fMRI of visual stimulation in the anesthetized rat retina at 11.7 T. A high-field MRI scanner was utilized to improve the signal-to-noise ratio, spatial resolution and BOLD sensitivity. Visual stimuli (8 Hz diffuse achromatic light) robustly increased BOLD responses in the retina [5.0 ± 0.8% from activated pixels and 3.1 ± 1.1% from the whole-retina region of interest (mean ± SD), n = 12 trials on six rats, p < 0.05 compared with baseline]. Some activated pixels were detected surrounding the pupil and ciliary muscle because of accommodation reflex to visual stimuli, and were reduced with atropine and phenylephrine eye drops. BOLD fMRI scans without visual stimulations showed no significantly activated pixels (whole-retina BOLD changes were 0.08 ± 0.34%, n = 6 trials on five rats, not statistically different from baseline, p > 0.05). BOLD fMRI of visual stimulation has the potential to provide clinically relevant data to probe hemodynamic neurovascular coupling and dysfunction of the retina with depth resolution.

Striatal and cortical BOLD, blood flow, blood volume, oxygen consumption, and glucose consumption changes in noxious forepaw electrical stimulation.

Recent reports showed noxious forepaw stimulation in rats evoked an unexpected sustained decrease in cerebral blood volume (CBV) in the bilateral striatum, whereas increases in spike activity and Fos-immunoreactive cells were observed. This study aimed to further evaluate the hemodynamic and metabolic needs in this model and the sources of negative functional magnetic resonance imaging (fMRI) signals by measuring blood oxygenation-level-dependent (BOLD), cerebral-blood-flow (CBF), CBV, and oxygen-consumption (i.e., cerebral metabolic rate of oxygen (CMRO(2))) changes using an 11.7-T MRI scanner, and glucose-consumption (i.e., cerebral metabolic rate of glucose (CMRglc)) changes using micro-positron emission tomography. In the contralateral somatosensory cortex, BOLD, CBF, CBV, CMRO(2) (n=7, P<0.05), and CMRglc (n=5, P<0.05) increased. In contrast, in the bilateral striatum, BOLD, CBF, and CBV decreased (P<0.05), CMRO(2) decreased slightly, although not significantly from baseline, and CMRglc was not statistically significant from baseline (P>0.05). These multimodal functional imaging findings corroborate the unexpected negative hemodynamic changes in the striatum during noxious forepaw stimulation, and support the hypothesis that striatal hemodynamic response is dominated by neurotransmitter-mediated vasoconstriction, overriding the stimulus-evoked fMRI signal increases commonly accompany elevated neuronal activity. Multimodal functional imaging approach offers a means to probe the unique attributes of the striatum, providing novel insights into the neurovascular coupling in the striatum. These findings may have strong implications in fMRI studies of pain.