1Department of Radiology, Stanford University, 2Center for In Vivo Microscopy, Duke University Medical Center, 3Department of Biomedical Engineering, Duke University
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Howles, G. P., Qi, Y., Rosenzweig, S. J., Nightingale, K. R., Johnson, G. A. Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI. J. Vis. Exp. (65), e4055, doi:10.3791/4055 (2012).
Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.
1. Assemble and Calibrate Ultrasound System
2. Prepare the Reagents
3. Animal Preparation
4. Blood-brain Barrier Opening with Microbubbles and Ultra Sound (BOMUS)
5. Neuronal Stimulation
6. Magnetic Resonance Imaging
7. Image Analysis
8. Representative Results
The method presented here has two fundamental steps: (1) BBB Opening with Microbubbles and UltraSound (BOMUS) and (2) Activation-Induced Manganese-enhanced MRI (AIM MRI). Because the latter step depends on the former, it is important to verify successful implementation BOMUS.
Disruption of the blood-brain barrier after administration of a T1-shortening contrast agent (such as manganese or a gadolinium-based agent) results in a signal increase in the brain parenchyma on T1-weighted imaging when compared to brains in which BOMUS was not performed (Figure 4). The distribution of this manganese enhancement is not completely uniform, although it is fairly consistent between animals. The distribution reflects not only inhomogeneity in BBB opening, but also is the intrinsic non-uniform distribution of Mn within the brain 19. The spatial and temporal dynamics of the BBB opening have been further described previously 12.
Once BOMUS has been successfully implemented, the next step is to perform AIM MRI. Many experimental paradigms are possible; however, because there are many potential confounds, the controls and analysis must be carefully designed. Confounding effects include inhomogeneous BBB opening, inhomogeneous accumulation of Mn in the brain, temporal dynamics of Mn diffusion, and nonspecific neuronal activity. In this demonstration, the neuronal response to unilateral stimulation of the vibrissae was mapped. To account for the inhomogeneities and Mn flux, the unstimulated side of each brain was used as an internal control. To account for nonspecific neuronal activity that might vary between animals, the analysis used statistical testing to identify regions that were consistently different among the animals (Figure 2). The results were a three-dimensional difference map and a three-dimensional p-value map (Figure 3), the right side of which indicated regions of higher signal contralateral to the stimulated vibrissae. The left side of the map indicated which regions had significantly higher signal ipsilateral to the stimulated vibrissae. The p-value map identified a broad region of elevated signal contralateral to the stimulated vibrissae which corresponded to the barrel field of the primary sensory cortex, whose response to vibrissae stimulation has been extensively documented by electrophysiology 20,21 and 2-deoxyglucose studies. A more complete discussion of these results has been published previously 13.
Figure 1. Protocol timeline for functional neuroimaging with BOMUS and AIM MRI (Adapted from Howles et al. 13).
Figure 2. Analysis scheme for identifying regions of different intensity between the stimulated and unstimulated sides of each brain. To compare the stimulated side of each brain to its contralateral unstimulated side, a duplicated and mirrored left-unstimulated image set is created. These images are registered, filtered, and normalized. Finally, a t test compares the left-stimulated and left-unstimulated images. The t test is "paired" so that the stimulated side of each brain is only compared to the unstimulated side of the same brain. The t test is "single-tailed" so that one side of the p-value map indicates significantly higher signal on the stimulated side of the brain, while the other side of the p-value map indicates significantly higher signal on unstimulated side of the brain (Adapted from Howles et al. 13).
Figure 3. Results of pooled analysis of 7 animals at two different axial positions. The first column shows the mean of all registered images aligned, so that effectively all mice had their left vibrissae stimulated. These images are overlaid with a color map indicating the average percent increase in signal at each voxel relative to the contralateral hemisphere, as indicated by the color bar. Colored regions on the right side of the image show where the hemisphere contralateral to the stimulation had higher signal. Colored regions on the left side of the image show where the hemisphere ipsilateral to the stimulation had higher signal. The second column shows the same images overlaid with the p-value map indicating the statistical significance of the increase in signal. The third column shows the same p-value map overlaid on the corresponding figures from the Paxinos stereotaxic atlas16 with the barrel fields of the sensory cortex shaded (Adapted from Howles et al. 13).
Figure 4. Spatial distribution of Mn2+ in the brain. Images were acquired 170 min after 0.5 mmol/kg IP MnCl2 from BOMUS-treated (n=5) and control (n=4) mice. After normalization, mean and standard deviation maps were calculated (left panel). Enhancement was greater in the BOMUS-treated mice. Though this enhancement was not uniform across the brain, it was fairly consistent, except near the edges of the brain and ventricles. Using regions of interest (ROIs) drawn around various structures, the mean SNR (+ 1 SD) was calculated across each group (right panel). BOMUS-treated animals showed greater SNR but also greater variance between structures and between animals (Adapted from Howles et al. 13).
Figure 5. To examine tissue effects of BOMUS, brains from BOMUS-treated mice were fixed, sectioned at 500- μm intervals, and stained with hematoxylin and eosin. The mean number of red blood cell extravasations seen in each section of the brain is shown for acoustic pressures of 0.36 MPa (n = 3), 0.52 MPa (n = 4), and 5.0 MPa (n = 1). Error bars show standard error. The second panel shows an example of severe red blood cell extravasation from the brain exposed to 5.0 MPa (Adapted from Howles et al. 12).
Figure 6. Quantitative behavioral testing was used to assess activity, arousal, and responsiveness before anesthesia, and 3 and 24 hours after recovery from anesthesia. The scoring system, described previously 12, was based on the well-established quantitative mouse behavioral assessment developed by Irwin in 196822. The average behavior (± SEM) score for control (n = 3) and BOMUS (0.36 MPa) treated (n = 8) animals is shown. Relative to the pre-anesthesia baseline, all animals show a decrease in behavior score 3 hours after anesthesia, but they largely recover by the next day. At each time point, no difference was seen between the two groups, indicating that BOMUS did not measurably affect animal behavior (Adapted from Howles et al. 12).
Here, a method was presented for noninvasively opening the BBB throughout the whole mouse brain with ultrasound and microbubbles (BOMUS). With the BBB open, Mn2+ was administered and activation-induced manganese-enhanced MRI (AIM MRI) was used to image neuronal response to short-duration stimulation in lightly sedated mice.
Adequate BBB opening was achieved with a peak-negative acoustic pressure of 0.36 MPa. Note, this is the pressure at scalp surface at the center of the ultrasound beam. Measurements of the single-element transducer's beam profile indicate that the acoustic pressure at the beam edge is only about 0.12 MPa. Subsequently, attenuation through the skull reduces the pressure reaching the brain by an estimated 25% (derating based on Choi et al. 23 and adjusted for frequency). This indicates that BBB disruption occurred at peak-negative acoustic pressures of 0.09 MPa (at the center of our beam) to 0.03 MPa (at the edge). These pressures are lower than the levels (typically 0.4 to 0.5 MPa) reported elsewhere 24. This reduced pressure threshold may be due to the higher dose of lipid microbubbles used in this work (approximately 1.2 mL/kg) compared to others. While the dose of microbubbles used was higher than the recommended diagnostic human dose (10 μL/kg), negative effects were not observed.
As specified here, the BOMUS technique is noninvasive and reversible; however, it has the potential to cause damage. In previous work 12, mice treated with BOMUS were evaluated for histologic damage (Figure 5) and behavioral changes (Figure 6). Peak-negative acoustic pressures of 0.36 MPa were associated with no observed negative effects (Figure 6). However, 0.52-MPa BOMUS were associated with a small number of intracerebral red blood cell extravasations in a subset of the animals (Figure 5), and some animals did not recover completely after the procedure. We recommend that an acoustic pressure that does not cause extravasation should be used for AIM MRI experiments.
Just as the BOMUS technique is potentially damaging, the manganese also has well-known toxicity 25. Mn2+ is known to have toxic effects on the neuromuscular junction 26 and nervous system 27. This toxicity is likely responsible for the somnolence of the mice after administration, though the mechanism for this effect is unknown. For approximately the first 60 minutes of stimulation, the mouse remains somewhat somnolent but still responsive to painful stimuli such as a toe pinch. This allows the mouse to tolerate the stimulation without the need for physical restraint. In our experience, this somnolence is adequate for about 60 minutes after which the animal may become restless. Additional chemical restraint can be achieved as needed with about 15 seconds of 5% isoflurane via nosecone. In this demonstration, the somnolence facilitated the stimulation of the vibrissae; however, it may also have reduced the neuronal response in the barrel cortex.
In addition to simply administering Mn2+, the BOMUS technique can be used to globally administer other diagnostic or therapeutic agents. In prior work, BOMUS has been used to administer Gd-DTPA, an MRI contrast agent, to the brain. 12 Nevertheless, many questions remain about the nature of the BBB permeability achieved with BOMUS. First, it is not clear what size agents are able to cross the BBB after BOMUS. Both Mn2+ and Gd-DTPA (500 Da) are fairly small molecules. Second, it is not clear how much the permeability of the BBB varies over the brain. Third, it is not clear whether the BBB opening is a relatively binary effect, or if certain opening parameters can affect the size or rate of material permeation. Though Gd-DTPA distributed fairly evenly through the brain in the above study, it may have been too small and too diffusible to reveal any differences in permeability.
Despite these uncertainties regarding the BOMUS, the method is effective for quickly administering Mn2+ for the purpose of AIM-MRI. AIM-MRI has been used in mice to map neuronal response to long-term (1-2 days) stimulation in mice 28-30, but with this new approach, short-term stimulation experiments are now possible. Previously, rapid administration of Mn2+ was only possible with osmotic BBB disruption using an intracarotid infusion of hypertonic mannitol. This approach was only practical in rats and larger animal models, but even in rats, these studies were limited by the invasiveness and unilaterality of the technique. Because BOMUS can be performed noninvasively, awake stimulation and longitudinal studies should now be possible. Furthermore, because Mn2+ can be administered to both cerebral hemispheres, a wider range of stimulation paradigms are possible. In the above demonstration, the bilateral administration Mn2+ allowed the unstimulated hemisphere to act as an internal control, so that neuronal response to non-specific background stimulation could be separated from the response to the unilateral vibrissae stimulation.
In addition to controlling for non-specific background stimulation, the unstimulated hemisphere also was used to control for homogeneity and consistency of the manganese administration. As seen in other manganese-enhanced MRI experiments19, the distribution data (Figure 4) indicate that the BOMUS technique does not provide a homogeneous enhancement of the brain. Thus, without adequate controls (control animals or an unstimulated hemisphere), regions with higher baseline enhancement are difficult to distinguish from regions whose elevated signal is due to neuronal activity.
Though the baseline Mn2+ enhancement is not homogeneous, the pattern is fairly consistent among individuals. Nevertheless, minor variations in this baseline enhancement could obscure the AIM-MRI signal. In this demonstration, we addressed this potential problem by averaging the AIM-MRI signal over several animals. Alternatively, differences in baseline enhancement could be accounted for by acquiring pre-stimulation images.
The method presented here requires substantial statistical image analysis, which in turn, requires high-fidelity image registration. Of course, such registration is only meaningful if the source data is acquired with a resolution (in all three dimensions) that is sufficiently finer than the structures of interest. In this demonstration, 3D images were acquired with nearly isotropic voxels approximately 160 microns in each dimension, which allowed for excellent anatomical registration. Nevertheless, image registration may limit the spatial resolution of this method-a slight misregistration could average-out very small regions of enhancement. The cerebellum and olfactory bulbs can be particularly difficult to register, because they have a finely layered enhancement and are often out of alignment with the cerebrum.
Here, we have presented a method for mapping neuronal response to short-duration stimuli in awake mice. Though not simple, the method is relatively practical and accessible. This detailed discussion of the limitations and subtleties should hopefully enable the reader to apply the technique to their own experimental questions.
No conflicts of interest declared.
All work was performed at the Duke Center for In Vivo Microscopy, an NIH/NIBIB national Biomedical Technology Resource Center (P41 EB015897) and NCI Small Animal Imaging Resource Program (U24 CA092656). Additional support was provided from NSF Graduate Research Fellowship (2003014921).
|Hydrophone||Sonora Medical Systems, Longmont, CA||SN S4-251|
|Translation stage||Newport Corporation, Irvine, CA|
|Ultrasound transducer||Olympus NDT, Inc., Waltham MA||A306S-SU||Review the manufacturer’s test sheet that accompanies the transducer to find the exact center frequency of that particular transducer, which may differ from the nominal frequency listed in the catalog. (e.g., the nominal frequency of our transducer was 2.25 MHz, but the actual center frequency was 2.15 MHz.)|
|Vevo Imaging Station||VisualSonics, Inc. Toronto, Canada|
|50 dB power amplifier||E&I, Rochester, NY||model 240L|
|Signal generator||Agilent Technologies, Santa Clara, CA||model 33220A|
|MnCl2-(H2O)4||Sigma||Molecular weight varies by batch, call manufacturer for exact measurement|
|Perflutren lipid microspheres||Lantheus Medical Imaging, N. Billerica, MA||DEFINITY|
|Microsphere agitator||Lantheus Medical Imaging, N. Billerica, MA||VIALMIX|
|MR imaging coil||m2m Imaging Corp., Hillcrest, OH||35 mm diameter quadrature transmit/receive volume coil|
|MRI system||GE Healthcare, Milwaukee, WI||GE EXCITE console operating a 7-T horizontal bore magnet|
|Image analysis environment||Visage Imaging, San Diego, CA, MathWorks, Natick MA||Amira MATLAB|