Local and reversible blood brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications

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1 NeuroImage 24 (2005) Local and reversible blood brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications Kullervo Hynynen,* Nathan McDannold, Nickolai A. Sheikov, Ferenc A. Jolesz, and Natalia Vykhodtseva Department of Radiology, Brigham and Women s Hospital, and Harvard Medical School, Boston, MA 02115, USA Received 20 November 2003; revised 4 June 2004; accepted 11 June 2004 The purpose of this study was to test the hypothesis that burst ultrasound in the presence of an ultrasound contrast agent can disrupt the blood brain barrier (BBB) with acoustic parameters suitable for completely noninvasive exposure through the skull. The 10-ms exposures were targeted in the brains of 22 rabbits with a frequency of 690 khz, a repetition frequency of 1 Hz, and peak rarefactional pressure amplitudes up to 3.1 MPa. The total exposure (sonication) time was 20 s. Prior to each sonication, a bolus of ultrasound contrast agent was injected intravenously. Contrast-enhanced MR images were obtained after the sonications to detect localized BBB disruption via local enhancement in the brain. Brain sections were stained with H&E, TUNEL, and vanadium acid fuchsin (VAF) toluidine blue staining. In addition, horseradish peroxidase (HRP) was injected into four rabbits prior to sonications and transmission electron microscopy was performed. The MRI contrast enhancement demonstrated BBB disruption at pressure amplitudes starting at 0.4 MPa with approximately 50%; at 0.8 MPa, 90%; and at 1.4 MPa, 100% of the sonicated locations showed enhancement. The histology findings following 4 h survival indicated that brain tissue necrosis was induced in approximately 70 80% of the sonicated locations at a pressure amplitude level of 2.3 MPa or higher. At lower pressure amplitudes, however, small areas of erythrocyte extravasation were seen. The electron microscopy findings demonstrated HRP passage through vessel walls via both transendothelial and paraendothelial routes. These results demonstrate that completely noninvasive focal disruption of the BBB is possible. D 2004 Elsevier Inc. All rights reserved. Keywords: Ultrasound; Bioeffects; Cavitation; Blood brain barrier; Apoptosis; Ischemia * Corresponding author. Department of Radiology, Brigham and Women s Hospital, and Harvard Medical School, 75 Francis Street, Boston, MA Fax: address: kullervo@bwh.harvard.edu (K. Hynynen). Available online on ScienceDirect ( Introduction Advances in neuroscience have resulted in the development of new diagnostic and therapeutic agents and genes that may be used to study and treat many central nervous system (CNS) diseases (Pardridge, 2002a). However, the use of these agents is often limited by their access to the CNS via the blood supply because the blood brain barrier (BBB) protects the brain from foreign molecules (Abbott and Romero, 1996; Kroll and Neuwelt, 1998; Nag, 2003b; Pardridge, 2002a). Several methods have been tested to circumvent the BBB including chemical modification of drugs to make them lipophilic or the use of carriers to aid propagation through the barrier (Pardridge, 2002a,b, 2003). Another option is to utilize an intraarterial infusion of hypertonic solution that initiates a BBB disruption lasting from a few minutes to a few hours (Doolittle et al., 2000). Each of the methods described above requires intraarterial catheterization and produces diffuse, nonfocal BBB disruption within the entire tissue volume supplied by the injected artery branch (Abbott and Romero, 1996; Kroll and Neuwelt, 1998). The BBB disruption over a large volume, however, may prove a disadvantage for many applications since the agents may have undesired, often dose-limiting side-effects due to their spread within the CNS. To prevent this, a localized and reversible imageguided disruption of the BBB would provide anatomically or functionally targeted drug delivery while preserving an intact BBB to protect the nontargeted regions. Today, such localized drug delivery can only be accomplished by a direct injection of the agent into the targeted brain volume (Kroll and Neuwelt, 1998). Such direct injections are limited by the slow diffusion of molecules in the brain, the need to open the skull and penetrate nontargeted brain tissue, and the attendant risk of neurological damage, bleeding, and infection. There is experimental evidence that focused ultrasound can selectively disrupt the BBB locally in the brain (Mesiwala et al., 2002; Patrick et al., 1990; Vykhodtseva et al., 1995). However, the disruption is often associated with damage to the exposed brain tissue. Recently, a method has been developed wherein the ultrasound exposures are performed in the presence /$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi: /j.neuroimage

2 K. Hynynen et al. / NeuroImage 24 (2005) of a circulating agent consisting of preformed gas bubbles (Hynynen et al., 2001). The MRI and histology results have shown reproducible and reversible BBB disruption at the frequency of 1.63 MHz. Since the bubbles are restricted to the vasculature, the injury to the surrounding brain tissue has proven minimal. One major shortcoming of ultrasound is that it is strongly attenuated by bone; hence, for brain applications (according to conventional practice), an acoustic window must be constructed by a craniotomy. Recent experimental and theoretical papers (Hynynen and Jolesz, 1998; Sun and Hynynen, 1998; Thomas and Fink, 1996) have shown that focal, trans-skull ultrasound exposure of brain tissue may be accomplished by using large surface area phased arrays and information derived from modern imaging methods to correct the ultrasound wave distortion produced by the skull (Clement and Hynynen, 2002; Sun and Hynynen, 1998). These studies indicate, moreover, that optimal transcranial focusing can be achieved at frequencies below 1 MHz. However, since the behavior of gas bubbles in an ultrasound field is highly dependant on the frequency; it is not known whether the BBB disruption can be safely induced at frequencies sufficiently low to allow propagation through the skull bone. The purpose of this paper is to explore the disruption of the BBB in a frequency range feasible for trans-skull sonications and to determine the biological route for the material transport into the brain tissue. Materials and methods Ultrasound equipment The ultrasound fields were generated by an in-house manufactured, focused, piezoelectric transducer with a 100-mm diameter, an 80-mm radius of curvature, and a resonant frequency of 0.69 MHz. The half-maximum pressure amplitude diameter and length of the produced focal spot were 2.3 and 14 mm, respectively. The transducer driving equipment were similar to those described in (Hynynen et al., 2001). Animals Twenty-two (22) New Zealand white rabbits (approximately 3 4 kg, males) were anesthetized by a mix of 40 mg/kg ketamine (Aveco Co., Inc., Fort Dodge, IA) and 10 mg/kg of Fig. 1. A diagram showing the sonication arrangement. Table 1 Summary of the experiments Experiment Determine thresholds for the BBB disruption/ tissue damage Verify closing of the BBB Electron microscopy xylazine (Lloyd Laboratories, Shenandoah, IA). An ultrasound window was created by removing a piece of skull (approximately mm) and replacing the skin over the bone window. This opening allowed for a more accurate estimation of the peak focal pressure amplitude in the tissue than is achievable with sonications through the skull. The experiments were performed no sooner than 10 days postsurgery. The head was shaved and any residual hair was removed with hair remover lotion. The head was fixed in the treatment position by a plastic holder on top of the water tank (Fig. 1). The sonications were performed through the skull opening such that each of the focal spots were aimed into a different location of the brain (at least 3 mm apart) so that the focal spots were not overlapping. All of these experiments were approved by our institutional animal committee. Sonications No. of rabbits No. of locations Pressure amplitude (MPa) Average time between sonication and contrast injection 14/13 a 52/48 a min (n = 14) 81 min (n =4) , min (n =4) 3h(n =4) 5h(n =1) (n =4) a One brain was lost before histology preparation. The animals were prepared for the experiments as described above and placed on the system. T2-weighted images were obtained to determine the extent of the bone window and to aid in the selection of target locations in the brain. The sonications were delivered to between one and six locations in the gray matter with the center of the focal spot at approximately 10 mm deep in each brain. The sonications were pulsed with a burst length of 10 ms and a repetition frequency of 1 Hz. The duration of the whole sonication was 20 s. The peak acoustic power levels were kept constant over the duration of each sonication but ranged from 0.08 to 8 W depending on the sonication. The acoustic power output and the focal pressure amplitude as a function of applied radiofrequency power was measured as described earlier (Hynynen et al., 1997). Approximately 10 s before each sonication, a bolus (0.05 ml/kg) of ultrasound contrast agent (Optison, Mallinckrodt Inc., St. Louis, MO) that contains albumin coated microbubbles (mean diameter = m; concentration = bubbles/ml) was injected into the ear vein (Hynynen et al., 2001). Five- to ten-minute intervals between sonications allowed the bubbles to mostly clear from the circulation before the next sonication. Three sets of experiments were performed (Table 1). In the first, 14 rabbits were sonicated over the full range of pressure amplitudes ( MPa). These locations were examined for signal intensity

3 14 K. Hynynen et al. / NeuroImage 24 (2005) Table 2 The MRI parameters used in the study Sequence Purpose Repetition time (ms) Echo time (ms) Matrix Flip angle (8) Bandwidth (khz) FSE T1-W Contrast Enhancement FSE T2-W Tissue Anatomy Targeting FSE T1-W = fast spin echo T1-weighted sequence; FSE T2-W = fast spin echo T2-weighted sequence. Number of acquisitions enhancement in MRI and with light microscopy to establish the thresholds for BBB disruption and tissue damage. In these experiments, sonications were performed at locations in both hemispheres to maximize the number of locations. In the second set of experiments, an additional four rabbits were sonicated at pressure amplitudes of 0.8 and 1.0 MPa, which is below the threshold for tissue damage established in the first set of experiments (see below). In these animals, one to two locations were sonicated in one hemisphere; the contralateral hemisphere served as a control. These rabbits were followed with MR imaging for several hours to establish the amount of time needed for repair of the BBB disruption. The MR contrast agent injection was repeated at desired time points after the sonication. These animals were not part of the light microscopy study. In the third set of experiments, another four rabbits were sonicated at pressure amplitudes of 0.8 MPa and were used for the electron microscopy study described below. These animals were not examined in light microscopy. MRI The MRI scanner was a standard 1.5 T Signa system (General Electric Medical Systems, Milwaukee, WI). A 7.5-cm diameter surface coil was placed under the head. The sonications were performed through the hole of the coil that was filled with the bag containing degassed water (Fig. 1). A T2-weighted fast spin echo (FSE) sequence was used to aim the beam at the brain. Following the sonications, T1-weighted FSE images were obtained and repeated after injection of an intravenous (iv) bolus (0.125 mmol/kg) of gadopentetate dimeglumine MR contrast agent (Magnevist, Berlex Laboratories Inc., Wayne, NJ) to detect and evaluate the opening of the blood brain barrier (BBB). The parameters for the MRI scans are given in Table 2. The time between the sonications and the contrast agent are listed in Table 1. Signal analysis The MRI contrast enhancement was evaluated at each target location by averaging the signal intensity over a 3 3 voxel ( mm) region of interest (ROI). The signal was normalized to the baseline value in the ROI before the contrast injection. ROIs of nonsonicated brain tissue proximal to sonicated locations served as controls. The average, normalized contrast enhancement observed in these control locations was subtracted from that in the targeted locations to estimate the impact of the sonications. In addition, a larger 6 6 voxel control area was selected outside of the sonicated regions, and the average standard deviation of the signal intensities of the voxels in that area was calculated over the entire time series of images. If the contrast enhancement at the focal area was higher than the average standard deviation value in the control area, then the sonication was judged to have induced disruption of the BBB. In the four rabbits sonicated in one hemisphere only, the signal enhancement at the focal coordinate was compared to that in the same structure in the contralateral hemisphere. This method allowed for an internal control that reduced any uncertainties that might be associated with selecting the control regions in different structures when targets were sonicated in both hemispheres. Light microscopy The animals were sacrificed approximately 4 h after the last sonication (in one case, at 48 h). The brains were immediately removed and fixed in 10% buffered neutral formalin. They were embedded in paraffin and serially sectioned at 6 Am (across the beam direction; parallel to the MRI slices). Every 50th section (interval of 0.3 mm) was stained with hematoxylin and eosin (H&E) for histologic examination. The sonication locations in the MR images were correlated with the histology slides by measuring Fig. 2. A contrast-enhanced FSE T1-weighted image of a rabbit brain. (A) Image plane across the focus after sonications at pressure amplitudes of 0.4, 0.5, 0.8, and 1.4 MPa (subtraction: before after injection) and (B) image oriented along the ultrasound beam of two of the locations.

4 K. Hynynen et al. / NeuroImage 24 (2005) their distance from anatomical structures and then identifying the same structures in the histology. Stains to detect apoptotic cells and ischemic neurons were used on the neighboring sections of the focal plane. These stains were employed because a possible vascular injury can induce a microischemic impact and result in ischemic neuronal injury. The apoptotic staining method utilized in this study was first described by Gavrieli et al. (1992). TUNEL staining (ApoptTag kit, Intergen Company, Purhase, NY) was used for the detection of DNA fragmentation and apoptotic bodies in the cells. The sections were counter stained with 0.5% Methyl Green. To visualize ischemic neurons in the sonicated areas, vanadium acid fuchsin (VAF) staining and toluidine blue counter staining were employed (Victorov et al., 2000). The total number of ischemic (acidophilic and hyperchromatic) and the total number of normal neurons were counted in 10 nonoverlapping microscopic fields (1000) chosen in the regions exhibiting the most pronounced morphological changes in each sonicated area. Some ischemic neurons represent postmortem histological artifacts due to the mechanical damage that may occur during removal and dissection of the brain (Cammermeyer, 1978); hence, as a control, the total number of ischemic and normal neurons was also counted in 10 nonoverlapping microscopic fields outside of each sonicated region close to its boundary. The author who performed the histology evaluation was blinded to the ultrasound parameters but had knowledge of the targeted locations. Electron microscopy Horseradish peroxidase (Molecular weight 40,000 Da and diameter of 5 nm), used extensively as a marker for transmission electron microscopy (Nag, 2003a), was selected to evaluate the Fig. 4. The average normalized signal intensity change measured in the T1- weighted FSE images after sonication as a function of the time between the sonication and the MRI contrast agent injection. This value was obtained for each sonication location from the enhancement curves such as is presented in Fig. 3 by averaging the signal intensity changes in the six to eight images after the injection of the contrast agent. Thus, it is approximately the maximum enhancement induced by the contrast agent injection. Open circles: four animals from the threshold study (group 1 in Table 1); closed circles: animals from the follow up study (group 2 in Table 1) (mean F SD). The pressure amplitude of these sonications was 0.8 MPa. ultrastructural basis of BBB disruption. Four rabbits were anesthetized as described above and given 1% promethazine (1 ml/kg ip) (Sigma) to block histamine release induced by horseradish peroxidase (HRP) administration. Trypan blue (1.5 ml/kg of Fig. 3. The normalized signal intensity measured in FSE T1-weighed images as a function of time after injection of contrast agent for a sonicated location (solid circle) and a nonsonicated location in the contralateral hemisphere (open circle) (mean F SD). The data are from one animal from the second group in Table 1 sonicated at 0.8 MPa. Fig. 5. Top: The normalized signal intensity change measured in FSE T1- weighed images after injection of contrast agent as a function of the applied pressure amplitude (mean F SD). Bottom: The percentage of sonicated locations that demonstrated BBB disruption and tissue necrosis (bgrade 3Q effects) as a function of the applied pressure amplitude.

5 16 K. Hynynen et al. / NeuroImage 24 (2005) % solution in saline) was administered (through the ear vein) to macroscopically indicate the degree of BBB disruption (Nag, 2003a). HRP type VI (Sigma) dissolved in saline (300 mg/kg) was injected immediately after the trypan blue through the same vein in two rabbits. In the other two animals, HRP type II (Sigma) was administered in the same doses. Two to three locations in each brain were then sonicated; the animals were sacrificed at deliberately selected intervals of approximately 1, 5, 20, and 60 min following each sonication. The animals were euthanized (EuthasolR, Delmara Laboratories Inc., Midlothian, VA, 1 ml iv), and the brains were fixed either by perfusion through the aorta (one animal) or by immersion fixation using 2.5% paraformaldehyde + 1.5% glutaraldehyde in 0.1 M phosphate buffer (ph 7.2). The two methods were used to eliminate potential problems associated with each method. Pieces of about 0.5 mm 3 from the sonicated areas (blue spots) and from nonsonicated areas (controls) were fixed for 2 h in the same fixative. Distance measurements from the MR images were used to correlate the sample locations and the MR image analysis. They were then washed in 0.1 M TRIS buffer (ph 7.4) and transferred to an incubation medium containing 3,3Vdiaminobenzidine and hydrogen peroxide for 45 min at room temperature. After postfixation in 2% osmium tetraoxide in PBS for 2 h, the pieces were dehydrated in ethanol, passed through propylene oxide, and embedded in Epon-Araldite. Ultrathin sections unstained or stained with uranyl acetate and lead citrate were observed with a JEM-1200EX electron microscope at 80 kv. Results MRI observations The BBB opening was observed in the sonicated locations in the T1-weighted contrast-enhanced scans (Fig. 2). Fig. 3 shows Fig. 6. Examples of the histology observations in VAF toluidine sections. (A) Grade 0: no damage was observed in the sonicated location. (B) Grade 1: a few extravasated erythrocytes were seen within the focal region. (C) Grade 2: microscopic areas of perivascular extravasations over the whole sonicated area, some of them associated with evident damage to the brain parenchyma (insert magnification of an ischemic cell). (D) Grade 3: extensive extravasation; hemorrhagic lesions (infarct).

6 K. Hynynen et al. / NeuroImage 24 (2005) Table 3 The histology grading of the sonicated tissues Grade Description 0 No detected damage 1 One to a few tiny red blood cell extravasations 2 Petechial hemorrhages; mild damage to the brain parenchyma 3 Hemorrhagic or nonhemorrhagic local lesions the signal intensity in T1-weighted imaging in one sonicated location and likewise in the same anatomical location on the opposite side of the brain. Following sonication, the signal intensity at the targeted location increased by 15 20% compared to the control location. BBB disruption was not detected after an additional MRI contrast injection 5 h following the sonication. The signal intensity change was greatest immediately after the sonications and decreased as a function of time (Fig. 4). Three hours following the sonications, the signal intensity increase was only approximately 10 20% of that measured initially. The signal intensity enhancement due to BBB disruption increased as a function of the pressure amplitude and was evident at the lowest value tested (0.4 MPa) (Fig. 5, top). Sixty percent of the locations had focal contrast enhancement greater than the standard deviation of the signal in the normal brain at 0.4 MPa. By 1.4 MPa, all locations showed evidence of BBB disruption (Fig. 5, bottom). Histology Forty-eight locations from among 13 brains were histologically evaluated in H&E-stained serial sections. In many cases, the sonicated areas differed little from the nonsonicated regions. The most noticeable effect observed in other cases was the areas of extravasation (42/48 locations), ranging from a few scattered erythrocytes in the mildest cases to extensive extravasation at the highest pressure amplitude levels (Fig. 6). These extravasations were limited to the focal volume of the ultrasound beam. These histological observations were divided into four grades ranging from no damage (grade 0) to tissue necrosis (grade 3) (Table 3). The degree of these vascular effects increased as a function of pressure amplitude when necrosis was induced at or above 2.3 MPa (Fig. 5B). At pressure amplitudes below 2.3 MPa, the extravasations were observed in the absence of obvious parenchymal damage. In the mildest cases (grade 1), the effects were limited to one or a few small erythrocyte extravasations, which was the dominant effect at pressure amplitudes up to approximately 0.5 MPa. In more severe cases (grade 2), there were multiple areas of erythrocyte extravasations, light vacuolation of the neuropil adjacent to some of affected microvessels and alteration in the shape and stainability of a few neurons and astrocytes. Mild neutrophil infiltration was seen in the affected regions 48 h following sonication. Grade 2 damage dominated at exposure levels of between 0.5 and 1.4 MPa. In the VAF toluidine-blue-stained sections with the largest apparent effect, the entire sonicated area contained on average five or fewer injured neurons when the pressure amplitude was below 1.4 MPa. Most of these neurons appeared dark stained (hyperchromatic) and thus might represent reversibly injured neurons. At a pressure amplitude of 2.3 MPa, the number of ischemic neurons increased (Table 4). For tissue effects less than grade 3, the only cells that were positive for ischemia were neurons. TUNEL-stained sections showed a few apoptotic cells in both in the vessel walls and at the brain parenchyma at pressure amplitude values lower than 2.3 MPa (Fig. 7). With TUNEL technique, using counterstaining, some cell types could be clearly identified according to their size, shape, and specific locations as endothelial cells, intravascular erythrocytes, or leukocytes. Overall, the TUNEL technique showed very few positive-stained cells, and these cells were primarily located in blood vessels and glia. However, it is possible that some individual cells were small neurons and could not be easily distinguished from glial cells and extravascular leukocytes by light microscopy. The average number of apoptotic cells increased with pressure amplitude. At pressure amplitudes of 2.3 MPa and higher, the tissue damage was always associated with multiple apoptotic cells. A few scattered apoptotic cells were also found outside the sonicated areas (Table 4). Electron microscopy In the samples obtained from the nonsonicated areas, HRP filled the vessel s lumina in brains fixed by immersion, while in the cases of the perfusion fixation, the vessels appeared to be empty. HRP did not penetrate the interendothelial clefts. Vesicles containing HRP were present in only a few endothelial cells (ECs). No HRP was seen in the subendothelial space: the basement membrane, pericytes, or neuropil. In all of the sonicated areas, passage of HRP through the vessel walls could be seen. Capillaries, arterioles, and venules were involved in this process, which showed a dependence on the circulation time of the tracer. For animals sacrificed approximately 1 min after sonication, the HRP reaction product was seen in small vacuoles and vesicles located at the luminal front of the ECs. Some of the vesicles were in the process of formation and taking HRP from the lumen (Fig. 8). This process was more apparent in the samples obtained from the immersion-fixed brains. No HRP was Table 4 The number of ischemic and apoptotic cells in the sonicated areas Pressure amplitude (MPa) Number of apoptotic cells Number of apoptotic cells in vessels Number of ischemic cells Number of sonicated locations F F F F F F F F F F F F F F F F F F Many Many 14 F Many Many 12 F The number of cells was counted in 10 nonoverlapping microscopic fields chosen in each sonicated area. These counts were averaged for each pressure amplitude value (mean F SD shown). The pressure amplitude value 0 MPa represents the average value for all control areas in all of the brains. When the sonicated areas were compared with the control areas (t test), the number of apoptotic cells was statistically higher ( P b 0.05) at pressure amplitude values at or above 0.8 MPa. The number of cells on the vessel walls was statistically higher than in the control locations in all sonicated areas at or above 0.5 MPa. The number of ischemic cells was statistically higher than the control locations at or above 0.8 MPa.

7 18 K. Hynynen et al. / NeuroImage 24 (2005) Fig. 7. Examples of apoptotic cells after sonications at 1.4 MPa (A) and 3.1 MPa (B). In (A), only a single small cell, probably a glia cell (arrow), next to intact neurons was seen to be undergoing apoptosis. In (B), many of the cells within this lesion appeared apoptotic (left: low magnification; right: high magnification). present in the interendothelial clefts, basement membrane, pericytes, or neuropil (Fig. 8). For animals sacrificed approximately 5 min after sonication, HRP-positive reaction was seen in caveolae, vacuoles, and lysosome-like profiles in the ECs cytoplasm. The tight junctions in some of the vessels appeared to be open and permitted HRP to pass through the intercellular cleft, penetrating into the basement membrane and the perivascular space. In the samples acquired in animals sacrificed approximately 20 min after sonication, a positive reaction for HRP was observed in the abluminal zones of the EC, in the basal membrane, and in the interstitium of the neuropil (Figs. 9A and B). For a sacrifice time of 1 h following sonication, the distribution of the HRP was similar. In some sonicated locations, mild to moderate perivascular and interstitial edema was found. Small groups of extravasated red blood cells were seen in three of eleven locations, and a few ruptures of the microvessel walls were found. Discussion Fig. 8. Photomicrograph showing a cross section of a capillary 1 min after sonication in the presence of circulating HRP. HRP fills the lumen (L) but is not present in the basement membrane (b), pericyte (P), or in the neuropil (NP). Formation of a caveola (long arrow) incorporating HRP is seen at the luminal surface of the endothelial cell, while the caveolae at the abluminal front of the cell are free from HRP (arrowheads). An interendothelial cleft shows the penetration of HRP into its luminal opening, but the tracer seems to be stopped at the tight junction (short arrows). RBC red blood cell in the lumen; NEC nucleus of the endothelial cell (immersion fixation of the brain). The results showed that BBB disruption is possible at a frequency of 0.69 MHz with minimal damage to the exposed brain parenchyma cells. The frequency used is suitable for highly localized sonication through human skull as has been reported earlier (Clement and Hynynen, 2002). This noninvasive method may be useful in delivering therapeutic or diagnostic agents in the central nervous system (Pardridge, 2002a). The electron microscopy findings confirm the effective disruption of the BBB by ultrasound under the conditions applied. The cellular mechanisms of HRP passage through vessel wall included both transendothelial (via caveolae and cytoplasmic vacuolar structures) and paraendothelial (via intercellular clefts) routes. These mechanisms are similar to those observed in other conditions resulting in BBB disruption (such as hypertension, injection of bradykinin, hyperosmotic agents, etc.) (Nag, 2003c). The BBB disruption took place in capillaries, arterioles, and venules, demonstrating similar mechanisms. The data obtained show that blood-circulating macromolecules (at least with molecular weight 40,000) invade the brain interstitial space and thus demonstrate that many therapeutic and diagnostic agents may be used in the brain.

8 K. Hynynen et al. / NeuroImage 24 (2005) Fig. 9. (A and B) Vessel and perivascular neuropil 19 min after sonication in the presence of HRP (perfusion fixation of the brain). (A) Numerous caveolae containing peroxidase (arrowheads) have moved to abluminal front of the endothelial cell (EC) and into the pericyte (P). The tracer has infiltrated the basement membrane (b) and the interstitium of the neuropil (arrows). (B) The passage of HRP through interendothelial clefts with evidently opened tight junctions is shown with arrows. Peroxidase has reached the middle and abluminal part of the cleft and has penetrated the basement membrane (b). Staining for HRP also is seen outside the vessel s wall in the neuropil (asterisks). L lumen; E red blood cell in the lumen; NP neuropil. The BBB disruption occurred in this study at a lower pressure amplitude (0.4 MPa) than was found in the earlier study using the same animal model and the same ultrasound contrast agent (0.7 MPa in that case) (Hynynen et al., 2001). This is consistent with the threshold of inertial cavitation that have been shown to occur at a lower pressure amplitude level when the frequency is decreased (Apfel and Holland, 1991). In this study, we used more sensitive histological techniques than in the earlier study (Hynynen et al., 2001) to investigate the role of apoptosis or ischemia following ultrasound disruption of the BBB. In addition, the focal spot size was larger and increased the likelihood of detecting any adverse events associated with the sonications. Thus, these results increase the confidence that ultrasound can be used to disrupt the BBB for delivering agents to the brain. Although found relatively rarely (3 of 11 cases), the endothelial damage observed with electron microscopy suggests the need for further investigations of endothelial and nerve cell ultrastructure. More work is needed to verify that there are no longterm effects and that the exposed brain tissue remains fully functional. Similarly, additional experiments must demonstrate that an adequate amount of a specific agent can be delivered through the BBB to achieve the desired therapeutic or diagnostic goal. In addition, there can be a difference in sensitivity of the cell to ultrasound exposure between white and gray matter. We did not investigate this matter in the present study; all locations were targeted in gray matter. The parameters used in this study are suitable for noninvasive sonications through human skull. The maximum acoustic power over the 20-s duration averaged 80 mw distributed over the focal spot area, which however was not strong enough to induce measurable temperature elevation in the tissue (Hynynen et al., 1997) and should therefore be safe for trans-skull sonications. At pressure amplitudes up to approximately 0.5 MPa, the BBB was disrupted in most sonicated locations with only a few extravasated erythrocytes and zero to two ischemic neurons or apoptotic cells in the entire area. These results suggest that these exposures can be used to deliver therapeutic or imaging agents with an acceptable impact on the brain tissue. At higher pressure amplitudes up to 1.4 MPa, there were more extravasated erythrocytes, and the total number of the ischemic and apoptotic cells was increased on average to five to six in the section. The importance of these effects is not clear, though they appear minimal and would most likely be acceptable for therapeutic interventions such as the delivery of genes or chemotherapy agents for tumor or other serious disease treatments. In conclusion, this study has demonstrated that for the first time, focused ultrasound can produce focal BBB disruption deep within the brain at a frequency that can be focused through human skull bone while sustaining little damage to the brain tissue. This finding may have considerable impact on future brain research, diagnostics, and therapy. Acknowledgments This research was supported by NIH research grant no. CA76550 and CA and a grant from CIMIT. References Abbott, N.J., Romero, I.A., Transporting therapeutics across the blood brain barrier. Mol. Med. Today 2, Apfel, R.E., Holland, C.K., Gauging the likelihood of cavitation from short-pulse, low duty cycle diagnostic ultrasound. Ultrasound Med. 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