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).

1. Assemble and Calibrate Ultrasound System
<ol>
	<li>The ultrasound system begins with a single-element ultrasound transducer with a diameter wide enough to cover the mouse brain and a center frequency in the range of 2 MHz. The transducer is driven by a 50-dB-power amplifier, which is connected to a signal generator that produces the ultrasound pulse sequence.</li>
	<li>To calibrate the acoustic pressure of the ultrasound system, use a hydrophone to relate the applied voltage to the resultant acoustic pressure. Place the transducer in a water tank over the hydrophone. Apply a simple pulse (e.g., a 10-cycle sinusoid at the transducer's frequency with pulse repetition frequency of 10 Hz) to the transducer. Use a 3-axis translation stage to find the peak response, which should be at the center of the ultrasound beam at the transducer's natural focus (approximately 60 mm for our 13 mm diameter 2.15 MHz transducer).</li>
	<li>Make several measurements over a range of input voltages (e.g., 50-400 mVpp) to verify the linearity of the system. Use a simple linear regression to estimate the relationship between input voltage and acoustic pressure. In our system, input voltages of 258 and 167 mVpp corresponded to peak-negative acoustic pressures of 0.52 and 0.36 MPa.</li>
	<li>Program the signal generator to produce an ultrasound pulse sequence consisting of bursts of sinusoidal pulses at the transducer frequency with 50000 cycles per burst and a burst period of 64 ms. Based on the calibration measurements, set the pulse amplitude to generate peak-negative acoustic pressures of 0.36 MPa at the center of the transducer's natural focus.</li>
</ol>
2. Prepare the Reagents
<ol>
	<li>Dissolve Manganese Chloride tetrahydrate (MnCl2&middot;4H2O) in sterile water at a concentration of 100 mM (300 mOsM) and filter sterilize.</li>
	<li>Produce the perflutren lipid microspheres by "activating" the vial in the manufacturer-supplied agitator for 45 s. For a day of experiments, a single vial can be activated once at the start of the day and used without reactivation for the rest of the day.</li>
	<li>Immediately prior to microsphere administration, agitate the vial by hand for 1 min to resuspend the microspheres. When withdrawing microbubbles from the vial, do not inject room air into the vial, as this degrades the remaining microbubbles. Leave the vial out until the last use of the day, and then store it in a refrigerator. Maintained in this fashion, a single vial can last several days. Re-activate the stored vial in the agitator, prior to the first use on subsequent days.</li>
</ol>
3. Animal Preparation
<ol>
	<li>Anesthetize the animals with isoflurane, delivered by nose cone. The nose cone apparatus should be designed to fix the animal's head precisely and reliably in the same position each time. Our device 15 maintains the head in the "skull-flat" position (i.e., the dorsal skull surface is horizontal) used in the Paxinos brain atlas 16. Titrate the anesthetic to maintain a respiratory rate between 85 and 125 breaths-per-minute. Maintain body temperature using a heat lamp or blown air. Protect the eyes with lubricant.</li>
	<li>Remove the hair from the mouse scalp using an electric trimmer.</li>
	<li>Place a tail vein catheter and an intraperitoneal (IP) catheter. For survival studies, be sure to use appropriate sterile technique; IP catheter placement demonstrated in the video for this article is appropriate only for non-survival experiments.</li>
	<li>Make any additional preparations required for the neuronal stimulation experiment. For mapping of vibrissae stimulation of the barrel field cortex, use a dissecting microscope and microsurgical scissors to cut the vibrissae as close as possible to the skin surface without irritating the follicle or surrounding skin.</li>
	<li>Place ultrasound gel on the scalp, and then lower a water column contained by a thin plastic sheet (e.g., a 7.6 &mu;m trash bin liner) onto the head. Reach through with water column with a cotton-tipped swab to push out any air bubbles that get trapped in the ultrasound gel. Position the ultrasound transducer at its natural focal distance (58 mm) directly over the mouse brain in the column of water, and wipe the transducer with a finger tip to remove any trapped air bubbles.</li>
</ol>
4. Blood-brain Barrier Opening with Microbubbles and Ultra Sound (BOMUS)
<ol>
	<li>Give an intraperitoneal injection of the manganese solution at a dose of 0.5 mmol/kg IP. Cap the intraperitoneal catheter so the manganese does not flow out, and wait 10 minutes to allow it to distribute (Figure 1).</li>
	<li>To open the BBB, administer 30 &mu;L of perflutren lipid microspheres (activated DEFINITY) through the tail vein catheter, and simultaneously, initiate the ultrasound pulse sequence. Continue insonification for 3 minutes.</li>
</ol>
5. Neuronal Stimulation
<ol>
	<li>Allow approximately 40 minutes for brain levels of Mn2+ to stabilize before beginning neuronal stimulation. In this way, the dramatic baseline enhancement due to Mn2+ diffusion across the BBB can be distinguished from the subtle differential enhancement due to the stimulation. Then, begin stimulation with your paradigm of choice (Figure 1).</li>
	<li>For vibrissae stimulation, turn off the isoflurane and remove the nose cone. Move a soft artist's paintbrush manually in a circular motion (1-5 Hz) through the vibrissae array at a distance of approximately 2-5 mm from the skin. Continue the stimulation for 90 min. The manganese has a sedative effect which allows unrestrained stimulation of the animal. If the animal becomes restless, administer 5% isoflurane via nose cone for approximately 15 seconds.</li>
</ol>
6. Magnetic Resonance Imaging
<ol>
	<li>After stimulation, resume anesthesia via nose cone. Continue maintaining the body temperature and titrating the isoflurane level to a respiratory rate of 85 to 125 breaths-per-minute.</li>
	<li>Place the mouse in an MR imaging coil and transfer to the MRI system. Acquire high-resolution 3D T1-weighted MR images. For example, use a 3D spoiled gradient recalled echo (SPGR) sequence with the following parameters: repetition time of 25 ms; echo time of 2 ms; flip angle of 30 degrees; bandwidth of 15.63 kHz; field of view of 20 &times; 20 &times; 12 mm; matrix of 128 &times; 128 &times; 60.</li>
</ol>
7. Image Analysis
<ol>
	<li>Image analysis is specific to the stimulation paradigm used. For the vibrissae stimulation experiment (Figure 2), import the images from several animals into a suitable analysis environment. If some animals were stimulated on the right while others were stimulated on the left, flip some so that all images are effectively "left-stimulated." Then, to compare the stimulated side of each brain to its contralateral unstimulated side, create a duplicated and mirrored left-unstimulated image set is created. Register all images to a common space and then smooth them with a 3 &times; 3 &times; 3 pixel Gaussian kernel.</li>
	<li>Export the data to a mathematical analysis environment, such as MATLAB. Optionally, mask irrelevant anatomy in the datasets. Intensity-normalize the images by the iterative method of Venot et al. 17,18.</li>
	<li>Use a paired, single-tailed t test to compare each voxel of the stimulated sides of each brain to the corresponding contralateral voxel on the unstimulated sides of each brain.</li>
	<li>Display the resultant p-value map to identify regions of differential activity (Figure 3).</li>
</ol>
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.
<img alt="Figure 1" src="/files/ftp_upload/4055/4055fig1.jpg" /> 
Figure 1. Protocol timeline for functional neuroimaging with BOMUS and AIM MRI (Adapted from Howles et al. 13).
<img alt="Figure 2" src="/files/ftp_upload/4055/4055fig2.jpg" /> 
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).
<img alt="Figure 3" src="/files/ftp_upload/4055/4055fig3.jpg" /> 
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).
<img alt="Figure 4" src="/files/ftp_upload/4055/4055fig4.jpg" /> 
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).
<img alt="Figure 5" src="/files/ftp_upload/4055/4055fig5.jpg" /> 
Figure 5. To examine tissue effects of BOMUS, brains from BOMUS-treated mice were fixed, sectioned at 500- &mu;m 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).
<img alt="Figure 6" src="/files/ftp_upload/4055/4055fig6.jpg" /> 
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 (&plusmn; 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).

<ol>
	<li>Aoki, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+the+anoxic+depolarization+of+focal+ischemia+using+manganese-enhanced+MRI.">Detection of the anoxic depolarization of focal ischemia using manganese-enhanced MRI.</a> Magnet. Reson. Med. 50, 7-12 (2003).</li><li>Aoki, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+activity-induced+manganese-dependent+contrast+magnetic+resonance+imaging.">Dynamic activity-induced manganese-dependent contrast magnetic resonance imaging.</a> DAIM MRI). Magnet. Reson. Med. 48, 927-933 (2002).</li><li>Duong, T. Q., Silva, A. C., Lee, S. P., Kim, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+MRI+of+calcium-dependent+synaptic+activity:+Cross+correlation+with+CBF+and+BOLD+measurements.">Functional MRI of calcium-dependent synaptic activity: Cross correlation with CBF and BOLD measurements.</a> Magnet. Reson. Med. 43, 383-392 (2000).</li><li>Lin, Y. J., Koretsky, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+ion+enhances+T-1-weighted+MRI+during+brain+activation:+An+approach+to+direct+imaging+of+brain+function.">Manganese ion enhances T-1-weighted MRI during brain activation: An approach to direct imaging of brain function.</a> Magnet. Reson. Med. 38, 378-388 (1997).</li><li>Lu, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine-induced+brain+activation+detected+by+dynamic+manganese-enhanced+magnetic+resonance+imaging+(MEMRI).">Cocaine-induced brain activation detected by dynamic manganese-enhanced magnetic resonance imaging (MEMRI).</a> P. Natl. Acad. Sci. U.S.A. 104, 2489-2494 (2007).</li><li>Drapeau, P., Nachshen, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+fluxes+and+manganese-dependent+neurotransmitter+release+in+presynaptic+nerve-endings+isolated+from+rat-brain.">Manganese fluxes and manganese-dependent neurotransmitter release in presynaptic nerve-endings isolated from rat-brain.</a> J. Physiol-London. 348, 493-510 (1984).</li><li>Narita, K., Kawasaki, F., Kita, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mn+and+Mg+influxes+through+Ca+channels+of+motor-nerve+Terminals+are+prevented+by+verapamil+in+Frogs.">Mn and Mg influxes through Ca channels of motor-nerve Terminals are prevented by verapamil in Frogs.</a> Brain Res. 510, 289-295 (1990).</li><li>Hynynen, K., McDannold, N., Vykhodtseva, N., Jolesz, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+MR+imaging-guided+focal+opening+of+the+blood-brain+barrier+in+rabbits.">Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits.</a> Radiology. 220, 640-644 (2001).</li><li>Sheikov, N., McDannold, N., Vykhodtseva, N., Jolesz, F., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+the+blood-brain+barrier+opening+induced+by+ultrasound+in+presence+of+microbubbles.">Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles.</a> Ultrasound Med. Biol. 30, 979-989 (2004).</li><li>McDannold, N., Vykhodtseva, N., Raymond, S., Jolesz, F. A., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-guided+targeted+blood-brain+barrier+disruption+with+focused+ultrasound:+Histological+findings+in+rabbits.">MRI-guided targeted blood-brain barrier disruption with focused ultrasound: Histological findings in rabbits.</a> Ultrasound Med. Biol. 31, 1527-1537 (2005).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+blood-brain+barrier+with+focused+ultrasound:+association+with+cavitation+activity.">Targeted disruption of the blood-brain barrier with focused ultrasound: association with cavitation activity.</a> Phys. Med. Biol. 51, 793-807 (2006).</li><li>Howles, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast-enhanced+in+vivo+magnetic+resonance+microscopy+of+the+mouse+brain+enabled+by+noninvasive+opening+of+the+blood-brain+barrier+with+ultrasound.">Contrast-enhanced in vivo magnetic resonance microscopy of the mouse brain enabled by noninvasive opening of the blood-brain barrier with ultrasound.</a> Magnet. Reson. Med. 64, 995-1004 (2010).</li><li>Howles, G. P., Qi, Y., Johnson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic+disruption+of+the+blood-brain+barrier+enables+in+vivo+functional+mapping+of+the+mouse+barrel+field+cortex+with+manganese-enhanced+MRI.">Ultrasonic disruption of the blood-brain barrier enables in vivo functional mapping of the mouse barrel field cortex with manganese-enhanced MRI.</a> Neuroimage. 50, 1464-1471 (2010).</li><li>Woolsey, T. A., Welker, C., Schwartz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomical+studies+of+sml+face+cortex+with+special+reference+to+occurrence+of+barrels+in+layer-4.">Comparative anatomical studies of sml face cortex with special reference to occurrence of barrels in layer-4.</a> J. Comp. Neurol. 164, 79-94 (1975).</li><li>Howles, G. P., Nouls, J. C., Qi, Y., Johnson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+production+of+specialized+animal+handling+devices+using+computer-aided+design+and+solid+freeform+fabrication.">Rapid production of specialized animal handling devices using computer-aided design and solid freeform fabrication.</a> J. Magnet. Reson. Imag. 30, 466-471 (2009).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2001).</li><li>Cross, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+mapping+of+functional+olfactory+connections+of+the+rat+brain+in+vivo.">Statistical mapping of functional olfactory connections of the rat brain in vivo.</a> Neuroimage. 23, 1326-1335 (2004).</li><li>Venot, A., Lebruchec, J. F., Golmard, J. L., Roucayrol, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated-method+for+the+normalization+of+scintigraphic+images.">An automated-method for the normalization of scintigraphic images.</a> J. Nucl. Med. 24, 529-531 (1983).</li><li>Aoki, I., Naruse, S., Tanaka, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+magnetic+resonance+imaging+(MEMRI)+of+brain+activity+and+applications+to+early+detection+of+brain+ischemia.">Manganese-enhanced magnetic resonance imaging (MEMRI) of brain activity and applications to early detection of brain ischemia.</a> Nmr. Biomed. 17, 569-580 (2004).</li><li>Welker, E., Vanderloos, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+correlation+between+barrel-field+size+and+the+sensory+innervation+of+the+whiskerpad+-+a+comparative-study+in+6+strains+of+mice+bred+for+different+patterns+of+mystacial+vibrissae.">Quantitative correlation between barrel-field size and the sensory innervation of the whiskerpad - a comparative-study in 6 strains of mice bred for different patterns of mystacial vibrissae.</a> J. Neurosci. 6, 3355-3373 (1986).</li><li>McCasland, J. S., Woolsey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+2-deoxyglucose+mapping+of+functional+cortical+columns+in+mouse+barrel+cortex.">High-resolution 2-deoxyglucose mapping of functional cortical columns in mouse barrel cortex.</a> J. Comp. Neurol. 278, 555-569 (1988).</li><li>Irwin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+observational+assessment+:+A+systematic+quantitative+procedure+for+assessing+behavioral+and+physiologic+state+of+mouse.">Comprehensive observational assessment : A systematic quantitative procedure for assessing behavioral and physiologic state of mouse.</a> Psychopharmacologia. 13, 222-257 (1968).</li><li>Choi, J. J., Pernot, M., Small, S. A., Konofagou, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive,+transcranial+and+localized+opening+of+the+blood-brain+barrier+using+focused+ultrasound+in+mice.">Noninvasive, transcranial and localized opening of the blood-brain barrier using focused ultrasound in mice.</a> Ultrasound Med. Biol. 33, 95-104 (2007).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+ultrasound+pulses+combined+with+definity+for+targeted+blood-brain+barrier+disruption:+A+feasibility+study.">Use of ultrasound pulses combined with definity for targeted blood-brain barrier disruption: A feasibility study.</a> Ultrasound Med. Biol. 33, 584-590 (2007).</li><li>Silva, A. C., Lee, J. H., Aoki, L., Koretsky, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+magnetic+resonance+imaging+(MEMRI):+methodological+and+practical+considerations.">Manganese-enhanced magnetic resonance imaging (MEMRI): methodological and practical considerations.</a> Nmr. Biomed. 17, 532-543 (2004).</li><li>Meiri, U., Rahamimoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+transmission+-+inhibition+by+manganese+ions.">Neuromuscular transmission - inhibition by manganese ions.</a> Science. 176, 308 (1972).</li><li>Aschner, M., Guilarte, T. R., Schneider, J. S., Zheng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese:+Recent+advances+in+understanding+its+transport+and+neurotoxicity.">Manganese: Recent advances in understanding its transport and neurotoxicity.</a> Toxicol. Appl. Pharm. 221, 131-147 (2007).</li><li>Watanabe, T., Frahm, J., Michaelis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+MRI+of+the+mouse+auditory+pathway.">Manganese-enhanced MRI of the mouse auditory pathway.</a> Magnet. Reson. Med. 60, 210-212 (2008).</li><li>Yu, X., Wadghiri, Y. Z., Sanes, D. H., Turnbull, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+auditory+brain+mapping+in+mice+with+Mn-enhanced+MRI.">In vivo auditory brain mapping in mice with Mn-enhanced MRI.</a> Nat. Neurosci. 8, 961-968 (2005).</li><li>Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+mapping+of+sound-evoked+activity+in+the+mouse+auditory+midbrain+using+Mn-enhanced+MRI.">Statistical mapping of sound-evoked activity in the mouse auditory midbrain using Mn-enhanced MRI.</a> Neuroimage. 39, 223-230 (2008).</li></ol>

No conflicts of interest declared.

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...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Hydrophone</td>
 <td>Sonora Medical Systems, Longmont, CA</td>
 <td>SN S4-251</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Translation stage</td>
 <td>Newport Corporation, Irvine, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ultrasound transducer</td>
 <td>Olympus NDT, Inc., Waltham MA</td>
 <td>A306S-SU</td>
 <td>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.)</td>
 </tr>
 <tr>
 <td>Vevo Imaging Station</td>
 <td>VisualSonics, Inc. Toronto, Canada</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 dB power amplifier</td>
 <td>E&amp;I, Rochester, NY</td>
 <td>model 240L</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Signal generator</td>
 <td>Agilent Technologies, Santa Clara, CA</td>
 <td>model 33220A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MnCl2-(H2O)4</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 <td>Molecular weight varies by batch, call manufacturer for exact measurement</td>
 </tr>
 <tr>
 <td>Perflutren lipid microspheres</td>
 <td>Lantheus Medical Imaging, N. Billerica, MA</td>
 <td>DEFINITY</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microsphere agitator</td>
 <td>Lantheus Medical Imaging, N. Billerica, MA</td>
 <td>VIALMIX</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MR imaging coil</td>
 <td>m2m Imaging Corp., Hillcrest, OH</td>
 <td>&nbsp;</td>
 <td>35 mm diameter quadrature transmit/receive volume coil</td>
 </tr>
 <tr>
 <td>MRI system</td>
 <td>GE Healthcare, Milwaukee, WI</td>
 <td>&nbsp;</td>
 <td>GE EXCITE console operating a 7-T horizontal bore magnet</td>
 </tr>
 <tr>
 <td>Image analysis environment</td>
 <td>Visage Imaging, San Diego, CA, MathWorks, Natick MA</td>
 <td>Amira 
 MATLAB</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

functional neuroimaging using ultrasonic blood-brain barrier disruption and manganese-enhanced mri

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.

Watch this Scientific Journal Video about Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI at JoVE.com

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

functional-neuroimaging-using-ultrasonic-blood-brain-barrier

Research

JoVE Journal

Neuroscience

14.9K Views.  Stanford University. A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Video: Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition termed "risky". In other cases when probabilities cannot be estimated, this is a condition described as "ambiguous". While most people are averse to both risk and ambiguity1,2, the degree of those aversions vary substantially across individuals, such that the subjective value of the same risky or ambiguous option can be very different for different individuals. We combine functional MRI (fMRI) with an experimental economics-based method3 to assess the neural representation of the subjective values of risky and ambiguous options4. This technique can be now used to study these neural representations in different populations, such as different age groups and different patient populations.
In our experiment, subjects make consequential choices between two alternatives while their neural activation is tracked using fMRI. On each trial subjects choose between lotteries that vary in their monetary amount and in either the probability of winning that amount or the ambiguity level associated with winning. Our parametric design allows us to use each individual's choice behavior to estimate their attitudes towards risk and ambiguity, and thus to estimate the subjective values that each option held for them. Another important feature of the design is that the outcome of the chosen lottery is not revealed during the experiment, so that no learning can take place, and thus the ambiguous options remain ambiguous and risk attitudes are stable. Instead, at the end of the scanning session one or few trials are randomly selected and played for real money. Since subjects do not know beforehand which trials will be selected, they must treat each and every trial as if it and it alone was the one trial on which they will be paid. This design ensures that we can estimate the true subjective value of each option to each subject. We then look for areas in the brain whose activation is correlated with the subjective value of risky options and for areas whose activation is correlated with the subjective value of ambiguous options.

Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain.

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition ...

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in remote locations. This paper shows the development of field functional Near Infrared Spectroscopy (fNIRS) imaging to the study of child language, reading, and cognitive development in a rural village setting of C&#244;te d&#39;Ivoire. Innovation in methods and the development of culturally appropriate neuroimaging protocols allow&#160;a first-time look into the brain&#39;s development and children&#39;s learning outcomes in understudied environments. This paper demonstrates protocols for transporting and setting up a mobile laboratory, discusses considerations for field versus laboratory neuroimaging, and presents a guide for developing neuroimaging consent procedures and building meaningful long-term collaborations with local government and science partners. Portable neuroimaging methods can be used to study complex child development contexts, including the impact of significant poverty and adversity on brain development. The protocol presented here has been developed for use in C&#244;te d&#39;Ivoire, the world&#39;s primary source of cocoa, and where reports of child labor in the cocoa sector are common. Yet, little is known about the impact of child labor on brain development and learning. Field neuroimaging methods have the potential to yield new insights into such urgent issues, and the development of children globally.

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy NIRS Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches (functional Near Infrared Spectroscopy) provide advances to the study of the brain in previously inaccessible regions; here, rural C&#244;te d&#39;Ivoire. Innovation in methods and development of culturally-appropriate neuroimaging protocols permits novel study of the brain&#39;s development and children&#39;s learning outcomes in environments with significant poverty and adversity.

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Role of Diffusion MRI Tractography in Endoscopic Endonasal Skull Base Surgery

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of benign and malignant lesions through a natural anatomical extra-cranial pathway, represented by the nasal cavities, avoiding brain retraction and neurovascular manipulation. This is reflected by the patients' prompt clinical recovery and the low risk of permanent neurological sequelae, representing the main caveat of conventional skull base surgery. This surgery must be tailored to each specific case, considering its features and relationship with surrounding neural structures, mostly based on preoperative neuroimaging. Advanced MRI techniques, such as tractography, have been rarely adopted in skull base surgery due to technical issues: lengthy and complicated processes to generate reliable reconstructions for inclusion in the neuronavigation system. 
This paper aims to present the protocol implemented in the institution and highlights the synergistic collaboration and teamwork between neurosurgeons and the neuroimaging team (neurologists, neuroradiologists, neuropsychologists, physicists, and bioengineers) with the final goal of selecting the optimal treatment for each patient, improving the surgical results and pursuing the advancement of personalized medicine in this field.

We present a protocol to integrate diffusion MRI tractography in patient work-up to endoscopic endonasal surgery for a skull base tumor. The methods for adopting these neuroimaging studies in the pre- and intra-operative phases are described.

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of ...

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...