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Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles
Ultrasound in Med. & Biol., Vol. 30, No. 7, pp. 979 –989, 2004 Copyright © 2004 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/04/$–see front matter
doi:10.1016/j.ultrasmedbio.2004.04.010
● Original Contribution CELLULAR MECHANISMS OF THE BLOOD-BRAIN BARRIER OPENING INDUCED BY ULTRASOUND IN PRESENCE OF MICROBUBBLES NICKOLAI SHEIKOV, NATHAN MCDANNOLD, NATALIA VYKHODTSEVA, FERENC JOLESZ and KULLERVO HYNYNEN Department of Radiology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (Received 3 September 2003; revised 15 April 2004; in final form 29 April 2004)
Abstract—Local blood-brain barrier (BBB) opening is an advantageous approach for targeted drug delivery to the brain. Recently, it has been shown that focused ultrasound (US) exposures (sonications), when applied in the presence of preformed gas bubbles, caused magnetic-resonance (MR) proven reversible opening of the BBB in targeted locations. The cellular mechanisms of such transient barrier disruption are largely unknown. We investigated US-induced changes in endothelial cell fine morphology that resulted in the BBB opening in rabbits. To obtain evidence for the passage of blood-borne macromolecules through the opened transvascular routes, an immunocytochemical procedure for endogenous immunoglobulinG (IgG) was performed, in addition to the routine electron microscopy. An increased number of vesicles and vacuoles, fenestration and channel formation, as well as opening of some tight junctions, were seen in capillaries after low-power (0.55 W) sonication. Immunosignals presented in some of the vesicles and vacuoles, in the cytoplasmic channels and, so rarely, in intercellular clefts; immunosignals could also be seen in neuropil around the blood vessels. Damage to the cellular ultrastructure was not seen in these areas. However, cell destruction and leakage of IgG through defects of the endothelial lining took place at 3 W sonications. The data reveals that several mechanisms of transcapillary passage are possible after such sonications: 1. transcytosis; 2. endothelial cell cytoplasmic openings—fenestration and channel formation; 3. opening of a part of tight junctions; and 4. free passage through the injured endothelium (with the higher power sonications). These findings could be considered in further development of the strategy for drug delivery to brain parenchyma. (E-mail: [email protected]) © 2004 World Federation for Ultrasound in Medicine & Biology. Key Words: Endothelium, Blood-brain barrier, Ultrasound, Immunoelectron microscopy.
Intravenous administration of inert hypertonic solutions (mannitol, arabinose) causes shrinkage of the EC and opening of the tight junctions (Lossinsky et al. 1995; Pan et al. 2000; Rapoport 2000). Some vasoactive amines (bradykinin, histamine) also enhance blood-brain or blood-tumor barrier permeability (Abbott 2000; Inamura et al. 1994; Nomura et al. 1994). A growing number of vesicles and vacuoles in the EC have been described after the application of these agents as ultrastructural evidence for augmented transcellular transport (d’Avella et al. 1998; Grange-Messent et al. 1999; Struzynska et al. 1997). Fenestration and channel formation in the endothelium have also been found and were suggested as possible routes for molecular passage (Dobrogowska et al. 1998; Wijsman and Shivers 1993). Both transmission electron microscopic and immunocytochemical data confirm that tight junctions can be widened and can serve as intercellular routes for transport (Ghabriel et al. 2000, 2002; Lossinsky et al.
INTRODUCTION Site-directed delivery of neurotherapeutics to the brain is generally difficult, due to the presence of the blood-brain barrier (BBB). The BBB is located in a monolayer of endothelial cells (EC) that cover the luminal surface of capillaries. Individual cells are connected to each other with tight junctions that seal the intercellular space. Together with the underlying basement membrane and with the pericytes and astrocyte endfeet that almost entirely cover the capillary’s abluminal surface, a morphofunctional interface is formed that controls the passage of compounds to the brain and the efflux from the brain. Numerous attempts have been made for opening the BBB to facilitate the delivery of drugs to the brain. Address correspondence to: Nickolai Sheikov, Dept. of Radiology, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis St., Boston, MA 02115 USA. E-mail: nsheikov@ bwh.harvard.edu 979
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1995). Some authors, however, have found no ultrastructural changes in tight junctions after BBB opening (d’Avella et al. 1998; Grange-Messent et al. 1999). The ability of US to permeabilize blood-tissue barriers makes it a promising tool for targeting drug delivery (Ng and Liu 2002; Tachibana and Tachibana 2001). Recent studies have demonstrated that large molecules and genes can cross the plasma membrane of cultured cells after different methods of acoustic energy application (Bao et al. 1998; Bao et al. 1997; Taniyama et al. 2002). An electron microscopy-seen US-induced membrane porosity has been reported in experiments in vitro and in vivo (Kerr et al. 1989; Ogawa et al. 2001), but no evidence for passage of macromolecules through these pores was shown. Although most investigators agree that cavitation effects (the interaction of US with gas microbubbles) play the main role in these processes (Koch et al. 2000; Liu et al. 1998; Sundaram et al. 2003), the mechanism of membrane permeabilization and bloodtissue barrier opening by US is still not entirely clear. Earlier, Vykhodtseva et al. (1995) observed in the rabbit brain that short, highpower focused US pulses were sometimes followed by localized BBB opening without apparent damage to the brain parenchyma. However, the US parameters (power, burst length, etc.) that could produce this effect consistently were not found. Mesiwala et al. (2002) also reported that high-intensity focused US is sometimes capable of a selective and nondestructive disruption of the BBB in rats, suggesting, on the basis of their electron microscopy data, that an opening of interendothelial tight junctions could be the mechanism of the BBB disruption. However, no tracer molecules were used in this study to prove a real passage through this possible route. Recently, Hynynen et al. (2001) showed that, if preformed gas bubbles were introduced to the blood stream prior to the focused US exposure, the BBB could be transiently opened at the US focus with no apparent acute damage to the neurons. Later work using the same method also indicated that it does not result in delayed ischemic or apoptotic neuronal damage (Vykhodtseva et al. 2003). The results of those studies indicate that the introduction of the cavitation sites into the blood stream both confined the US effects mostly to the vasculature and reduced the power needed to produce the BBB opening by a factor of two orders of magnitude, which diminishes the risk of tissue damage and makes this technique more easily applied through the intact skull (Hynynen and Jolesz 1998). This technique, when combined with an imaging device that can detect the BBB opening online (such as a magnetic resonance imaging (MRI) scanner), represents a noninvasive means to open the BBB at targeted locations as a possible way for the delivery of drugs or other large-molecular substances to
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the brain. The exact cellular mechanisms of this BBB opening, however, are not well understood. The aim of the present work was to examine the brain capillary ultrastructural changes after the US exposures in order to elucidate the routes through which materials are passed through the US-induced BBB disruption, using electron microscopy and immunoelectron microscopy. MATERIALS AND METHODS Ultrasound A spherically curved transducer with a 100-mm diameter and an 80-mm radius of curvature generated the US field. Two transducers were used over the course of the experiments, with frequencies of 1.63 and 1.50 MHz, respectively. The 1.63-MHz transducer was a 16-channel phased-array transducer with all elements operated in phase, mimicking a single-focus transducer. The acoustic power output and the focal pressure amplitude as a function of applied radiofrequency (RF) power were measured as described earlier (Hynynen et al. 2001). The sonications were performed under MRI guidance and monitoring. The transducer was mounted in an experimental positioning device. The transducer and MRIcompatible positioning hardware were submerged in deionized, degassed water. For these experiments, the system was used only to manually move the transducer. Animal preparation Ten adult male New Zealand white rabbits, weighing 3.5– 4.5 kg, were used. The animals were anesthetized using a mixture of xylazine (10 mg/kg body weight; Lloyd Laboratories, Shenandoah, IA, USA) and ketamine (40 mg/kg body weight; Aveco Co., Inc., Fort Dodge, IA, USA). To allow for US propagation into the brain, a piece of bone about 2 cm ⫻ 2 cm was surgically removed from the parietal surface of the skull of each animal (approximately 1.5–2.0 mm anterior from Bregma to 1.0 mm anterior from Lambda; 10 mm to the left and 10 mm to the right from the midline). The skin was sutured back in place, the animals were allowed to recover and the wound to heal for at least 2 weeks. Before the experiments, the animals were anesthetized again and the hair over the skull was removed with an electric trimmer and depilatory lotion. Our institutional animal committee approved these experiments. MRI The MRI scanner was a standard 1.5 T Signa system (General Electric Medical Systems, Milwaukee, WI, USA). A 7.5-cm diameter surface coil was placed under the head to improve the signal-to-noise ratio (SNR). During each sonication, a time series (20 time points, 4 s/image) of fast spoiled gradient echo images were ac-
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Fig. 1. Experimental setup.
quired (TR:TE 40.9:19.9 ms, flip angle 30°, band width 3.57 kHz, matrix (frequency ⫻ phase) 256 ⫻ 128, fieldof-view (FOV) 10 ⫻ 7.5 cm, slice thickness 3 mm). A complex reconstruction of these images was used to calculate temperature maps based on the temperature dependence of the proton resonant frequency (Ishihara et al. 1995). The scan plane was located across the focus (perpendicular to the beam axis) at the expected focal depth. T2-weighted fast spin echo (FSE) images (TR:TE 2000:85 ms, echo train length 8, FOV 10 cm, slice thickness 1.5 mm, two-signal averages) were obtained to localize the bone window and the target depth in the brain. After the sonications, T1-weighted FSE images (TR:TE 500:17 ms, echo train length 4, three signal averages, FOV 10 cm, matrix size 256 ⫻ 256, slice thickness 1.5 mm) were obtained. These scans were repeated after a bolus of MRI contrast agent (gadopentetate dimeglumine, Magnevist®, Berlex Laboratories Inc, Wayne, NJ, USA) to confirm the focal BBB opening. Experimental protocol The animals were placed on their backs on a plate above the sonication system (Fig. 1). The skin on the top of the head was coupled with a water bag to the water tank that held the transducer. The free water surface of the bag coupled the sound into the skin. Before the experiment, low-power continuous-wave sonications (1.2 W, no US contrast agent) below the level for tissue damage (Hynynen et al. 1997) were delivered at the first sonicated location to ensure that the focus was at the correct location. Acoustic powers of 0.55 or 3 W during the acoustic bursts were used in the experimental sonications, which translated to acoustic pressure amplitudes of 1–3 MPa at
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the focus (in situ estimate). The sonications were pulsed with a burst length of 100 ms and a repetition frequency of 1 Hz. The duration of the whole sonication was 20 s. The 0.55 W power level was chosen based on our previous work and aimed to open the BBB without damage to the neurons. The 3-W sonications aimed to cause BBB opening that was accompanied with damage. Approximately 10 s prior to the start of each sonication, a bolus of US contrast agent (Optison®, Mallinckrodt, Inc., St. Louis, MO, USA) that contains perfluorocarbon gas microbubbles (mean diameter ⫽ 2.0 – 4.5 ⫻ 10⫺6 m; concentration ⫽ 5– 8 ⫻ 108 bubbles/mL) was injected in the ear vein. The injected volume was selected to be 0.05 mL/kg. The bolus was flushed from tubing that extended out of the magnet bore by injecting approximately 1–2 mL of saline. A 5–10 min delay between sonications allowed the bubbles to clear from the circulation. Two to four locations were sonicated in each animal through the bone window in the skull. A total of 25 sonications were targeted at nonoverlapping locations for all 10 rabbits. Nine and eight locations were sonicated at acoustic powers of 0.55 and 3 W, respectively, for the standard electron microscopy examination. Four locations used in the immunoelectron microscopy were at an acoustic power level of 0.55 W; four were at 3 W. The coordinates of the locations were determined by MRI guidance and monitoring and were as follows: 8 mm anterior from the dorsal pole of brain hemispheres, 2 mm lateral from brain midline, 10 mm depth from brain surface. If the second sonication was made in the same hemisphere, it was focused at 3 mm posterior from the first one, at the same depth and lateral distance from the midline. If four locations were sonicated, the first two were mirrored in the contralateral hemisphere. Anatomically, the foci of sonications were in the thalamus. All materials for the ultrastructural study were taken 1 to 2 h after the application of US. Electron microscopy All 25 sonicated locations were examined “blindly” (17 for conventional electron microscopy and eight for immunoelectron microscopy). To make zones of the compromised BBB visible, 2% trypan blue in saline (1.5 mL/kg) was injected IV after the sonications and postultrasound MR scans. To avoid flushing out the contents of the brain vasculature during perfusion fixation, we used immersion fixation. The anesthesia was prolonged and a wide craniotomy was performed, exposing the brain. The animal was euthanized with an IV injection of solution containing pentobarbital sodium and phenytoin sodium (Euthasol® 0.25 mL/kg, Delmarva Laboratories Inc., Midlothian, VA, USA), and the brain was rapidly removed. Using a rabbit brain matrix (ASI instruments, Warren, MI, USA) and under continuous rinsing with
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fixative (2.5% glutaraldehyde in 0.1M sodium cacodylate buffer, pH ⫽ 7.2), horizontal slices from the brain were obtained and pieces of about 1 mm3 were extracted from the blue spots and from nonsonicated (control) areas (n ⫽ 12). The pieces were immersed in the same fixative for 2 h, postfixed for 1 h in 1% osmium tetraoxide, stained in uranylacetate, and embedded in EponAraldite. Ultrathin sections were obtained with Reichert Ultracut-S microtome, then were stained with lead citrate and examined with a Jeol 1200EX electron microscope. Immunoelectron microscopy Tissue pieces from eight sonicated and five nonsonicated locations were fixed in 4% paraformaldehyde ⫹ 0.01% glutaraldehyde in 0.1M phosphate buffer (pH ⫽ 7.4) for 2 h. The samples were washed in 0.12% glycine in phosphate-buffered saline (PBS) for 15 min (to quench free aldehyde groups), then infiltrated with 2.3 M sucrose in PBS for 15 min and frozen in liquid nitrogen. The frozen samples were sectioned at ⫺120°C. The sections were transferred to formvar/carbon-coated copper grids and floated on PBS. The immunogold labeling was carried out at room temperature on a piece of parafilm. Grids were floated on drops of 1% bovine serum albumin (BSA) for 10 min to block unspecific labeling, transferred to 5 L drops of goat antirabbit immunoglobulin G (IgG) conjugated to 10 nm gold (Sigma), diluted 1:10 in 1% BSA in PBS and incubated for 1 h at room temperature. After the incubation, the grids were washed in drops of PBS for 15 min followed by washing in drops of double distilled water. Contrasting/embedding of the labeled grids was carried out on ice in 0.3% uranyl acetate in 2% methyl cellulose for 10 min. Controls for the specificity of the immunostaining consisted of sections preincubated with nonconjugated with gold goat antirabbit IgG antiserum, diluted 1:5 in 1% BSA in PBS overnight at 4°C. RESULTS The BBB disruption was clearly identified in MRI for each location (Fig. 2). The peak temperature measured at the focus during the 3-W sonication was 2.0°C, consistent with our previous results and below the threshold for thermal damage (Hynynen et al. 1997). The focal BBB opening was also visually detected by the presence of the trypan blue selectively at the sonicated locations. In the brain samples obtained from the nonsonicated areas, no damage to the vessel morphology or to the neutropil was found (Fig. 3a). An increased number of vesicles and vacuoles were observed in many EC in areas sonicated with 0.55 W. Groups of vesicles were also seen in the cytoplasm of several pericytes (Fig. 3b). The
Fig. 2. Contrast-enhanced MR image verifying focal opening of the BBB at two sonicated locations. The enhancing zones at the focal coordinates (arrows) indicated a locally disrupted BBB.
luminal surface of the EC appeared with foldings, some of which formed deep pocket-like invaginations that caused the lumen and basement membrane to be separated by a thin layer of cytoplasm or by only luminal and abluminal membranes of the cells. In addition to such fenestrae-like formations, cytoplasmic channels were occasionally seen as free routes between lumen and the basement membrane (Fig. 3c). In many vascular profiles, the tight junctional complexes appeared intact, but some interendothelial clefts were lightened and widened with missing zonulae occludentes. The basement membrane rarely displayed irregularities or splittings. Numerous caveolae attached to its endothelial surface or attached to its both surfaces were seen in some vessels. Free-lying caveolae also presented in the EC and pericytes (Fig. 4a). There were small extravasates of red blood cells (1 to 3–7 cells in three from nine locations), and 35– 40 cells (one from nine locations) in neuropil. Moderate to severe damage to the brain vasculature was found in locations sonicated with acoustic power of 3 W. The EC cytoplasm appeared dark with poorly distinguishable organelles. Spastic arterioles with obturation of the lumen were observed. In some vessels, the endothelial lining was detached, the basement membrane was tortuous, with blurred outlines and, sometimes, membrane discontinuities occurred (Fig. 4b). Single or groups of red blood cells were found in close proximity to some vessels. The pericytes looked damaged and the
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Fig. 3. Photomicrographs of brain microvessels from (a) nonsonicated control and (b)–(d) sonicated with 0.55W areas, showing: (a) scarce vesicles (arrows) and a vacuole (v) in the endothelial cell cytoplasm (EC), a part of a pericyte (P) and neuropil (NP); R ⫽ red blood cell in lumen (L); b ⫽ basement membrane. (b) Numerous vesicles (arrows) in an endothelial cell cytoplasm (EC) and in a pericyte (P, curved arrows). (c) A transendothelial channel (arrow) that exposes the basement membrane, b, to the lumen, L; (*) 2 plasmalemmal pits at the luminal surface of the endothelial cell are shown. (d) Deep channel-like invagination (*) in an edematous-looking endothelial cell (EC, right); the interendothelial cleft (arrowheads) near the invagination does not appear to be widened.
astrocytes appeared edematous, with agglomerated nuclear chromatin (Fig. 4b). In the frozen sections used for immune electron microscopy, labeled IgG was present in the lumina of the vessels in both sonicated and nonsonicated areas. Lack of immunosignals could be seen outside of the vessel lumina in the sections from nonsonicated areas (Fig. 5a). Preincubation of the specimens with goat antirabbit IgG antiserum resulted in no labeling when incubated in gold-labeled IgG antiserum, confirming the specificity of the IgG immunodetection. In capillaries obtained from sonicated areas, gold particles were observed in the EC cytoplasm and in the subendothelial space: in the basement membrane, in pericytes and in neuropil (Fig. 5b). Labeled molecules presented in vacuoles and in caveolae inside the EC (Figs. 6 and 7a). Formation of plasmalemmal pits at luminal surface of the EC and presence of caveolae containing gold particles at both luminal and abluminal fronts of the EC were seen; some signal-bearing
caveolae looked opened to the basement membrane (Fig. 6a, b, c, d). Groups of gold particles were seen inside the pocket-like invaginations of the endothelial cell cytoplasm in close proximity to the basement membrane; passage of labeled IgG through cytoplasmic channels could be seen as well (Fig. 7b). In a few cases, gold particles appeared in interendothelial clefts where the tight junctions seemed lightened and widened (Fig. 7c). In locations sonicated with 3 W, the particles in the neuropil were more numerous in the vicinity of the vessels showing damaged endothelial lining. DISCUSSION The BBB disruption in our experiments was confirmed by both MRI monitoring for the contrast-matter enhancement and trypan blue leakage in the sonicated locations. Both acoustic powers applied (0.55 W and 3 W) resulted in dye and contrast matter leakage. The
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electron microscopy study revealed that barrier opening by focused US in the presence of preformed gas bubbles has its expression in a group of characteristic ultrastructural changes of the brain microvasculature. These ultrastructural changes were found in the sonicated locations, but neither in the nonsonicated contralateral hemispheres nor in nonsonicated areas of the ipsilateral hemispheres. The data obtained suggest that at least four mechanisms of passage of materials through the BBB are possible after such sonications: 1, transcytosis, 2, transendothelial openings—fenestration and channel formation, 3, widening of interendothelial clefts and opening of a part of tight junctions, and 4, free passage through the injured endothelial lining (in locations with 3 W sonication). It is known that there are few cytoplasmic vesicles in the EC in the brain microvasculature (Reese and Karnovsky 1967; Sedlakova et al. 1999). In various experimental or pathological conditions, the number of these vesicles increases and this fact is regarded as an expression of enhanced endo- and transcytosis (Castejon 1998; Chaudhuri 2000; Claudio et al. 1989; Struzynska et al. 1997). A disagreement exists about the mechanism of this vesicular transport. Some authors accept that membrane-bound vesicles take in plasma protein from the lumen, then separate from the luminal plasma membrane, pass across the endothelial cytoplasm and fuse with the abluminal plasma membrane, where they discharge their contents into the underlying basal membrane (Claudio et al. 1989). According to another opinion, a process of channel formation by fusion rather than actual movement of vesicles occurs (Lossinsky et al. 1999). The enhanced number of cytoplasmic vesicles that we found in EC after the sonication suggests an activation of transcellular transport by US. This is confirmed by the EC luminal infoldings and the formation of vacuoles capable of internalizing portions of the blood plasma, indicated by the presence of labeled IgG inside the vacuoles. The appearance of many flask-shaped caveolae on the luminal surface of the EC, the abundant supply of free-lying caveolae in the cytoplasm, as well as the caveolae open to subendothelial space, suggest that US triggers mechanisms that lead to an enhanced formation and trafficking of these vesicular organelles.
Fig. 4. (a) Ultrastructural view of a part of a microvessel from sonicated (0.55 W) area. Numerous caveolae (arrows) attached to the basement membrane, b, and also free caveolae (thick arrow) present in the endothelial cell cytoplasm (EC); caveolae formation is seen on the luminal surface of the cell (arrowheads). A lightened interendothelial cleft with not apparent tight-junctional complexes is shown (double arrows). (b) A damaged blood vessel from the central part of
a location sonicated with 3 W. Very dark, infolded endothelial cell cytoplasm, (EC), tortuous basement membrane, (b), with blurred outlines (arrow) and leakage of blood plasma outside the membrane (*) are shown. Edematous astrocytes (A) and damaged neuropil (NP) are present, and two astrocyte nuclei (N) with agglomerated chromatin are seen at the bottom of the microphotograph; L ⫽ lumen; R ⫽ red blood cell in the lumen; P ⫽ pericyte.
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Fig. 5. (a) Immunoelectron microscopy of specimen obtained from nonsonicated brain area (control). Immunosignals are seen only in the lumen (L) of the vessel, except for one occasional particle (upper right). EC ⫽ endothelial cell cytoplasm; b ⫽ basement membrane; P ⫽ pericyte; NP ⫽ neuropil; R ⫽ red blood cell in the lumen. A sealed interendothelial cleft is shown with arrows. (b) A section from a location sonicated with 0.55 W. In addition to immunosignals in lumen (L), gold particles are present in endothelial cell cytoplasm (EC), basement membrane (b, arrows) and neuropil (NP, arrowheads). A gold particle is likely in an interendothelial cleft (white arrow); R ⫽ red blood cell in lumen.
These findings are of particular interest in the light of the specific functions of the caveolar system in cell physiology. It has been shown that caveolae are directly involved in the internalization of some macromolecules, bacterial toxins and viral pathogens and in delivering them to specific locations in different kinds of mammalian cells (Anderson et al. 1992; Stang et al. 1997; Pelkmans and Helenius 2002). These properties have made caveolae a promising tool for specific binding and intracellular delivery of bioactive molecules, genes and anticancer drugs (McIntosh et al. 2002; Schnitzer 2001).
Although many further investigations are needed, the effect of the low-power US pulses on the vesicle population and on the functional activity of the brain capillary caveolar system could be of importance in the field of targeted pharmacodelivery research. With very limited exceptions, fenestrae are not present in brain vessels and the BBB is functional in normal conditions. In contrast, many kinds of brain tumors display endothelial fenestrations and a limited barrier functionality (Groothius 2000; Vajkoczy and Menger 2000). Several nonneoplastic diseases, such as
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Fig. 6. Transport of labeled IgG molecules by caveolae is shown on photomicrographs obtained from locations sonicated with 0.55 W. (a) A gold particle attached to endothelial cell luminal surface; the fossette seems to indicate the beginning of a caveola formation. (b) A completely formed caveola containing a gold particle at the luminal front of the endothelial cell. (c) A caveola containing labeled IgG at abluminal part of an endothelial cell, close to the basement membrane, b. (d) Caveolae (arrows) with gold particles at abluminal front of an endothelial cell; one of the caveolae is attached and opened to the basement membrane. No gold particles are present in the intercellular cleft (arrowheads). EC ⫽ endothelial cell; L ⫽ lumen; b ⫽ basement membrane.
allergic encephalomyelitis, delayed radiation necrosis and experimental intoxications (lead, soman), can cause fenestration of brain vessels (Hirano et al. 1994). Fenestrae also appear after osmotic opening of the BBB or after intracerebral infusion of vascular endothelial growth factor (Dobrogowska et al. 1998; Roberts and Palade 1995). Attempts to induce fenestration in the brain vascular endothelium by means of chemical agents (retinoic acid, phorbol ester) show that the process develops slowly, taking at least 28 days (Kaya et al. 1996). Comparatively, the US induction of fenestration appeared to be rapid; fenestrae-like openings were seen on electron microscopy on samples obtained 1 and 2 h after the sonication. The cytoplasmic channels that we observed in the EC seem also to be formations for transendothelial passage. Similar cytoplasmic channels have been described in muscle capillaries (Simionescu et al. 1975) and in mouse brain capillaries in response to heat
Fig. 7. Photomicrographs of microvessels from sonicated (0.55 W) areas. (a) Vacuoles (arrowheads) containing gold particles are seen in the endothelial cell cytoplasm. (b) Passage of labeled molecule through a cytoplasmic channel is shown in this capillary profile (inset). (c) A gold particle (arrow) in an interendothelial cleft and a few particles in neuropil (arrow heads). EC ⫽ endothelial cell cytoplasm; L ⫽ lumen; R ⫽ red blood cells; b ⫽ basement membrane; NP ⫽ neuropil.
stress (Wijsman and Shivers 1993). It is widely accepted that the transendothelial channels are formed by fusion of vesicles opened simultaneously to both cell fronts (Bendayan 2002). Our findings showing that labeled IgG molecules escaped through the channels confirm the role of these formations as routes for protein passage through the capillary wall after the sonication in the parameters applied.
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In a compromised BBB, interendothelial clefts are the only extracellular route for transport of macromolecules from blood to brain. In some experiments using hyperosmotic solutions, it was reported that the tracer within cerebral capillaries permeates between adjacent EC, and that the mechanism of this opening might be explained by cell shrinkage and junctional deformation (Lossinsky et al. 1995). On the other hand, some investigators have reported that no apparent gaps in endothelium were observed in analogous experiments with hyperosmolar solutions (Farrell and Shivers 1984; Shivers and Harris 1984). A possible explanation of these differences is that the tight junctions may have opened briefly and then resealed before tissue fixation (Wisniewski and Lossinsky 1991). We observed apparently widened and lightened tight junctions next to fully sealed, not widened clefts in the same sections and in the same capillaries. It is possible to hypothesize that such a different response was due to the nature of the sonication itself; for example, perhaps the forces associated with the bubble oscillations and collapse within the US field are not uniform. We obtained the samples 1 to 2 h after sonications, so it is possible that some of the junctions opened and closed before the samples were retrieved. Only a few gold particles were found in the intercellular clefts; we did not observe a line of several labeled molecules passing through clefts. Evidently, the extracellular route is less accessible for IgG in the conditions of our experiment, and the tortuosity of the clefts could make it difficult for blood protein molecules to pass across the capillary wall. An intriguing finding in our experiments was the insertion of many plasmalemmal pits and vesicles on both the endothelial and perycite sides of the basement membrane. Insertion of numerous caveolae on the basement membrane in brain capillaries has been shown to occur as a result of the enhanced transport of serum albumin in diabetic rats (Bouchard et al. 2002). In that study, however, the vesicles were found predominantly on the endothelial surface of the basement membrane. It is more likely, in our study, that the vesicles were produced as a response to the increased two-way transport after BBB disruption by US, although it is difficult to explain why the transport was so pronounced in both directions. The tight junctions opening, on the other hand, could be considered to be a temporary modification of, or an easily restored damage to zonulae occludentes proteins as it happens, for example, in the process of transvascular white blood cell migration during immunological surveillance of the normal central nervous system or in osmotic opening of the barrier (Rapoport 2000). The lightening and widening of the tight junctional complexes we found were fully comparable with the ultrastructural picture described by Mesiwala et al. (2002) in their work on selective and temporal BBB tight
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junction opening after high-intensity focused US application in rats. Our electron microscopic data show that the sonication with low US acoustic power of 0.55 W during the sound burst (pressure amplitude 1.0 MPa) effectively opens the BBB, preserving the tissue ultrastructure better than the 3-W acoustic power does. The small red blood cell extravasates we found in four from nine locations sonicated with 1 MPa (0.55 W) are comparable with the histological findings reported previously, and correspond to category 1–2 of tissue damage, as described there (Hynynen et al. 2003). Although hemoglobin is known to contribute to nerve cell injury when released into the brain after hemorrhages, it is unlikely that this is the case with these small extravasates in our experiments. We never did see neurons demonstrating electron microscopic signs of acute cell death in 0.55 W locations up to 2 h after the sonications in the present study. A recently completed light microscopy examination for cell necrosis and apoptosis, at the frequency used in the present study, showed that the BBB opening by US, when applied at the same parameters, does not result in delayed damage to neurons up to 1 month after the sonication (Vykhodtseva et al. 2003). Nevertheless, further investigations are necessary in studying these effects. The exact mechanism of how US influences the BBB is not clear. Based on our previous experiments, it can be concluded that the BBB opening is mainly the result of interaction between the US wave and the microbubbles. There are several potential ways that this interaction can produce this bioeffect. First, the US wave causes the bubbles to expand and contract in the capillaries. The expansion of larger bubbles can fill the whole capillary lumen, resulting in a mechanical stretching of the vessel wall. This may result in the opening of the tight junctions. This change in the pressure in the capillary may also evoke biochemical reactions that trigger the opening of the BBB; as is well known, an increase or change in blood pressure can affect the BBB (Kongstad and Grande 2001). The bubble oscillation may also reduce the local blood flow and induce transient ischemia, which can trigger BBB opening (Abraham et al. 2002). Finally, the bubbles can collapse during the sonication, causing highly localized shock waves and fluid jets. These mechanical effects can also be responsible for the opening of the BBB, and almost certainly play an important role in tissue damage induced at higher-pressure amplitude values. CONCLUSION The ultrastructural changes that we observed in the brain microvessels indicate that macromolecules passed
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through the US-induced BBB disruption via several routes. In addition to the tight junction opening, we also observed enhanced vesicular transcellular trafficking, endothelial cell fenestration and channel formation—all pathways for permeation of the endogenous IgG that we used in our experiments as a representative of macromolecular solutes. These findings are ultrastructural evidence for low-power focused US-induced consistent BBB opening in targeted locations. To our knowledge, this is the first demonstration of IgG (i.e, antibodies) delivery at targeted brain areas and also the first description of the routes for macromolecular passage through the US compromised BBB. Each of these routes should be considered in further investigations on US-mediated BBB disruption, as well as in the development of new strategies for antibody, gene or drug delivery to brain parenchyma. Acknowledgments—The authors thank Drs. Roderick Bronson and Peter Vassilev for the valuable discussions and comments on the manuscript. The technical assistance of Ms. Heather Martin in animal preparation is also greatly appreciated. This study was supported by NCI research (grants CA76550 and CA089017) and a grant from CIMIT.
REFERENCES Abbott NJ. Inflammatory mediators and modulation of blood-brain barrier permeability. Cell Mol Neurobiol 2000;20:131–147. Abraham CS, Harada N, Deli MA, Niwa M. Transient forebrain ischemia increases the blood-brain barrier permeability for albumin in stroke-prone spontaneously hypertensive rats. Cell Mol Neurobiol 2002;22:455–462. Anderson RG, Kamen BA, Rothberg KG, Lacey SW. Potocytosis: Sequestration and transport of small molecules by caveolae. Science 1992;255:410–411. Bao S, Thrall BD, Gies RA, Miller DL. In vivo transfection of melanoma cells by lithotripter shock waves. Cancer Res 1998;58:219– 221. Bao S, Thrall BD, Miller DL. Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol 1997; 23:953–959. Bendayan M. Morphological and cytochemical aspects of capillary permeability. Microsc Res Tech 2002;57:327–349. Bouchard P, Ghitescu LD, Bendayan M. Morpho-functional studies of the blood-brain barrier in streptozotocin-induced diabetic rats. Diabetologia 2002;45:1017–1025. Castejon OJ. Electron microscopic analysis of cortical biopsies in patients with traumatic brain injuries and dysfunction of neurobehavioural system. J Submicrosc Cytol Pathol 1998;30:145–156. Chaudhuri JD. Blood brain barrier and infection. Med Sci Monit 2000;6:1213–1222. Claudio L, Kress Y, Norton WT, Brosnan CF. Increased vesicular transport and decreased mitochondrial content in blood-brain barrier endothelial cells during experimental autoimmune encephalomyelitis. Am J Pathol 1989;135:1157–1168. d’Avella D, Cicciarello R, Angileri FF, et al. Radiation-induced bloodbrain barrier changes: Pathophysiological mechanisms and clinical implications. Acta Neurochir Suppl (Wien) 1998;71:282–284. Dobrogowska DH, Lossinsky AS, Tarnawski M, Vorbrodt AW. Increased blood-brain barrier permeability and endothelial abnormalities induced by vascular endothelial growth factor. J Neurocytol 1998;27:163–173.
Volume 30, Number 7, 2004 Farrell CL, Shivers RR. Capillary junctions of the rat are not affected by osmotic opening of the blood-brain barrier. Acta Neuropathol (Berl) 1984;63:179–189. Ghabriel MN, Zhu C, Hermanis G, Allt G. Immunological targeting of the endothelial barrier antigen (EBA) in vivo leads to opening of the blood-brain barrier. Brain Res 2000;878:127–135. Ghabriel MN, Zhu C, Leigh C. Electron microscope study of bloodbrain barrier opening induced by immunological targeting of the endothelial barrier antigen. Brain Res 2002;934:140–151. Grange-Messent V, Bouchaud C, Jamme M, et al. Seizure-related opening of the blood-brain barrier produced by the anticholinesterase compound, soman: New ultrastructural observations. Cell Mol Biol (Noisy-le-grand) 1999;45:1–14. Groothius DR. The blood-brain and blood-tumor barriers: A review of strategies for increasing drug delivery. Neurooncol 2000;2:45–59. Hirano A, Kawanami T, Llena JF. Electron microscopy of the bloodbrain barrier in disease. Microsc Res Tech 1994;27:543–556. Hynynen K, Jolesz FA. Demonstration of potential noninvasive ultrasound brain therapy through an intact skull. Ultrasound Med Biol 1998;24:275–283. Hynynen K, McDannold N, Martin H, Jolesz F, Vykhodtseva N. The threshold for brain damage in rabbits induced by bursts of ultrasound in the presence of an ultrasound contrast agent (Optison). Ultrasound Med Biol 2003;29:473–481. Hynynen K, McDannold N, Vykhodtseva N, Jolesz FA. Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits. Radiology 2001;220:640–646. Hynynen K, Vykhodtseva NI, Chung AH, et al. Thermal effects of focused ultrasound on the brain: Determination with MR imaging. Radiology 1997;204:247–253. Inamura T, Nomura T, Ikezaki K, et al. Intracarotid histamine infusion increases blood tumour permeability in RG2 glioma. Neurol Res 1994;16:125–128. Ishihara Y, Calderon A, Watanabe H, et al. A precise and fast temperature mapping using water proton chemical shift. Magn Reson Med 1995;34:814–823. Kaya M, Chang L, Truong A, Brightman MW. Chemical induction of fenestrae in vessels of the blood-brain barrier. Exp Neurol 1996; 142:6–13. Kerr CL, Gregory DW, Chan KK, Watmough DJ, Wheatley DN. Ultrasound-induced damage of veins in pig cars, as revealed by scanning electron microscopy. Ultrasound Med Biol 1989;15:45– 52. Koch S, Pohl P, Cobet U, Rainov NG. Ultrasound enhancement of liposome-mediated cell transfection is caused by cavitation effects. Ultrasound Med Biol 2000;26:897–903. Kongstad L, Grande PO. Arterial hypertension increases intracranial pressure in cat after opening of the blood-brain barrier. J Trauma 2001;51:490–496. Liu J, Lewis TN, Prausnitz MR. Non-invasive assessment and control of ultrasound-mediated membrane permeabilization. Pharm Res 1998;15:918–924. Lossinsky AS, Buttle KF, Pluta R, Mossakowski MJ, Wisniewski HM. Immunoultrastructural expression of intercellular adhesion molecule-1 in endothelial cell vesiculotubular structures and vesiculovacuolar organelles in blood-brain barrier development and injury. Cell Tissue Res 1999;295:77–88. Lossinsky AS, Vorbrodt AW, Wisniewski HM. Scanning and transmission electron microscopic studies of microvascular pathology in the osmotically impaired blood-brain barrier. J Neurocytol 1995; 24:795–806. McIntosh DP, Tan XY, Oh P, Schnitzer JE. Targeting endothelium and its dynamic caveolae for tissue-specific transcytosis in vivo: A pathway to overcome cell barriers to drug and gene delivery. Proc Natl Acad Sci USA 2002;99:1996–2001. Mesiwala AH, Farrell L, Wenzel HJ, et al. High-intensity focused ultrasound selectively disrupts the blood-brain barrier in vivo. Ultrasound Med Biol 2002;28:389–400. Ng KY, Liu Y. Therapeutic ultrasound: Its application in drug delivery. Med Res Rev 2002;22:204–223.
US and BBB opening: cellular mechanisms ● N. SHEIKOV et al. Nomura T, Inamura T, Black KL. Intracarotid infusion of bradykinin selectively increases blood-tumor permeability in 9L and C6 brain tumors. Brain Res 1994;659:62–66. Ogawa K, Tachibana K, Uchida T, et al. High-resolution scanning electron microscopic evaluation of cell-membrane porosity by ultrasound. Med Electron Microsc 2001;34:249–253. Pan GY, Liu XD, Liu GQ. Intracarotid infusion of hypertonic mannitol changes permeability of blood-brain barrier to methotrexate in rats. Acta Pharmacol Sin 2000;21:613–616. Pelkmans L, Helenius A. Endocytosis via caveolae. Traffic 2002;3: 311–320. Rapoport SI. Functional brain imaging to identify affected subjects genetically at risk for Alzheimer’s disease. Proc Natl Acad Sci USA 2000;97:5696–5698. Reese TS, Karnovsky MJ. Fine structural localization of a blood-brain barrier to exogenous peroxidase. J Cell Biol 1967;34:207–217. Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J Cell Sci 1995;108:2369 –2379. Schnitzer JE. Caveolae: From basic trafficking mechanisms to targeting transcytosis for tissue-specific drug and gene delivery in vivo. Adv Drug Deliv Rev 2001;49:265–280. Sedlakova R, Shivers RR, Del Maestro RF. Ultrastructure of the blood-brain barrier in the rabbit. J Submicrosc Cytol Pathol 1999; 31:149–161. Shivers RR, Harris RJ. Opening of the blood-brain barrier in Anolis carolinensis. A high voltage electron microscope protein tracer study. Neuropathol Appl Neurobiol 1984;10:343–356. Simionescu N, Siminoescu M, Palade GE. Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels. J Cell Biol 1975;64:586–607.
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Stang E, Kartenbeck J, Parton RG. Major histocompatibility complex class I molecules mediate association of SV40 with caveolae. Mol Biol Cell 1997;8:47–57. Struzynska L, Walski M, Gadamski R, Dabrowska-Bouta B, Rafalowska U. Lead-induced abnormalities in blood-brain barrier permeability in experimental chronic toxicity. Mol Chem Neuropathol 1997;31:207–224. Sundaram J, Mellein BR, Mitragotri S. An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes. Biophys J 2003;84:3087–3101. Tachibana K, Tachibana S. The use of ultrasound for drug delivery. Echocardiography 2001;18:323–328. Taniyama Y, Tachibana K, Hiraoka K, et al. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 2002;105: 1233–1239. Vajkoczy P, Menger MD. Vascular microenvironment in gliomas. J Neurooncol 2000;50:99–108. Vykhodtseva NI, Hynynen K, Damianou C. Histologic effects of highintensity pulsed ultrasound exposure with subharmonic emission in rabbit brain in vivo. Ultrasound Med Biol 1995;21:969–979. Vykhodtseva N, McDannold N, Agabian S, Hynynen K. Histology findings after ultrasound exposure of the brain with ultrasound contrast agent—role of ischemia and apoptosis. 3rd International Symposium on Therapeutic Ultrasound, Lyon, France, Inserm, France, 2003;80 – 85. Wijsman JA, Shivers RR. Heat stress affects blood-brain barrier permeability to horseradish peroxidase in mice. Acta Neuropathol (Berl) 1993;86:49–54. Wisniewski HM, Lossinsky AS. Structural and functional aspects of the interaction of inflammatory cells with the blood-brain barrier in experimental brain inflammation. Brain Pathol 1991;1:89–96.
doi:10.1016/j.ultrasmedbio.2004.04.010
● Original Contribution CELLULAR MECHANISMS OF THE BLOOD-BRAIN BARRIER OPENING INDUCED BY ULTRASOUND IN PRESENCE OF MICROBUBBLES NICKOLAI SHEIKOV, NATHAN MCDANNOLD, NATALIA VYKHODTSEVA, FERENC JOLESZ and KULLERVO HYNYNEN Department of Radiology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA (Received 3 September 2003; revised 15 April 2004; in final form 29 April 2004)
Abstract—Local blood-brain barrier (BBB) opening is an advantageous approach for targeted drug delivery to the brain. Recently, it has been shown that focused ultrasound (US) exposures (sonications), when applied in the presence of preformed gas bubbles, caused magnetic-resonance (MR) proven reversible opening of the BBB in targeted locations. The cellular mechanisms of such transient barrier disruption are largely unknown. We investigated US-induced changes in endothelial cell fine morphology that resulted in the BBB opening in rabbits. To obtain evidence for the passage of blood-borne macromolecules through the opened transvascular routes, an immunocytochemical procedure for endogenous immunoglobulinG (IgG) was performed, in addition to the routine electron microscopy. An increased number of vesicles and vacuoles, fenestration and channel formation, as well as opening of some tight junctions, were seen in capillaries after low-power (0.55 W) sonication. Immunosignals presented in some of the vesicles and vacuoles, in the cytoplasmic channels and, so rarely, in intercellular clefts; immunosignals could also be seen in neuropil around the blood vessels. Damage to the cellular ultrastructure was not seen in these areas. However, cell destruction and leakage of IgG through defects of the endothelial lining took place at 3 W sonications. The data reveals that several mechanisms of transcapillary passage are possible after such sonications: 1. transcytosis; 2. endothelial cell cytoplasmic openings—fenestration and channel formation; 3. opening of a part of tight junctions; and 4. free passage through the injured endothelium (with the higher power sonications). These findings could be considered in further development of the strategy for drug delivery to brain parenchyma. (E-mail: [email protected]) © 2004 World Federation for Ultrasound in Medicine & Biology. Key Words: Endothelium, Blood-brain barrier, Ultrasound, Immunoelectron microscopy.
Intravenous administration of inert hypertonic solutions (mannitol, arabinose) causes shrinkage of the EC and opening of the tight junctions (Lossinsky et al. 1995; Pan et al. 2000; Rapoport 2000). Some vasoactive amines (bradykinin, histamine) also enhance blood-brain or blood-tumor barrier permeability (Abbott 2000; Inamura et al. 1994; Nomura et al. 1994). A growing number of vesicles and vacuoles in the EC have been described after the application of these agents as ultrastructural evidence for augmented transcellular transport (d’Avella et al. 1998; Grange-Messent et al. 1999; Struzynska et al. 1997). Fenestration and channel formation in the endothelium have also been found and were suggested as possible routes for molecular passage (Dobrogowska et al. 1998; Wijsman and Shivers 1993). Both transmission electron microscopic and immunocytochemical data confirm that tight junctions can be widened and can serve as intercellular routes for transport (Ghabriel et al. 2000, 2002; Lossinsky et al.
INTRODUCTION Site-directed delivery of neurotherapeutics to the brain is generally difficult, due to the presence of the blood-brain barrier (BBB). The BBB is located in a monolayer of endothelial cells (EC) that cover the luminal surface of capillaries. Individual cells are connected to each other with tight junctions that seal the intercellular space. Together with the underlying basement membrane and with the pericytes and astrocyte endfeet that almost entirely cover the capillary’s abluminal surface, a morphofunctional interface is formed that controls the passage of compounds to the brain and the efflux from the brain. Numerous attempts have been made for opening the BBB to facilitate the delivery of drugs to the brain. Address correspondence to: Nickolai Sheikov, Dept. of Radiology, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis St., Boston, MA 02115 USA. E-mail: nsheikov@ bwh.harvard.edu 979
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1995). Some authors, however, have found no ultrastructural changes in tight junctions after BBB opening (d’Avella et al. 1998; Grange-Messent et al. 1999). The ability of US to permeabilize blood-tissue barriers makes it a promising tool for targeting drug delivery (Ng and Liu 2002; Tachibana and Tachibana 2001). Recent studies have demonstrated that large molecules and genes can cross the plasma membrane of cultured cells after different methods of acoustic energy application (Bao et al. 1998; Bao et al. 1997; Taniyama et al. 2002). An electron microscopy-seen US-induced membrane porosity has been reported in experiments in vitro and in vivo (Kerr et al. 1989; Ogawa et al. 2001), but no evidence for passage of macromolecules through these pores was shown. Although most investigators agree that cavitation effects (the interaction of US with gas microbubbles) play the main role in these processes (Koch et al. 2000; Liu et al. 1998; Sundaram et al. 2003), the mechanism of membrane permeabilization and bloodtissue barrier opening by US is still not entirely clear. Earlier, Vykhodtseva et al. (1995) observed in the rabbit brain that short, highpower focused US pulses were sometimes followed by localized BBB opening without apparent damage to the brain parenchyma. However, the US parameters (power, burst length, etc.) that could produce this effect consistently were not found. Mesiwala et al. (2002) also reported that high-intensity focused US is sometimes capable of a selective and nondestructive disruption of the BBB in rats, suggesting, on the basis of their electron microscopy data, that an opening of interendothelial tight junctions could be the mechanism of the BBB disruption. However, no tracer molecules were used in this study to prove a real passage through this possible route. Recently, Hynynen et al. (2001) showed that, if preformed gas bubbles were introduced to the blood stream prior to the focused US exposure, the BBB could be transiently opened at the US focus with no apparent acute damage to the neurons. Later work using the same method also indicated that it does not result in delayed ischemic or apoptotic neuronal damage (Vykhodtseva et al. 2003). The results of those studies indicate that the introduction of the cavitation sites into the blood stream both confined the US effects mostly to the vasculature and reduced the power needed to produce the BBB opening by a factor of two orders of magnitude, which diminishes the risk of tissue damage and makes this technique more easily applied through the intact skull (Hynynen and Jolesz 1998). This technique, when combined with an imaging device that can detect the BBB opening online (such as a magnetic resonance imaging (MRI) scanner), represents a noninvasive means to open the BBB at targeted locations as a possible way for the delivery of drugs or other large-molecular substances to
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the brain. The exact cellular mechanisms of this BBB opening, however, are not well understood. The aim of the present work was to examine the brain capillary ultrastructural changes after the US exposures in order to elucidate the routes through which materials are passed through the US-induced BBB disruption, using electron microscopy and immunoelectron microscopy. MATERIALS AND METHODS Ultrasound A spherically curved transducer with a 100-mm diameter and an 80-mm radius of curvature generated the US field. Two transducers were used over the course of the experiments, with frequencies of 1.63 and 1.50 MHz, respectively. The 1.63-MHz transducer was a 16-channel phased-array transducer with all elements operated in phase, mimicking a single-focus transducer. The acoustic power output and the focal pressure amplitude as a function of applied radiofrequency (RF) power were measured as described earlier (Hynynen et al. 2001). The sonications were performed under MRI guidance and monitoring. The transducer was mounted in an experimental positioning device. The transducer and MRIcompatible positioning hardware were submerged in deionized, degassed water. For these experiments, the system was used only to manually move the transducer. Animal preparation Ten adult male New Zealand white rabbits, weighing 3.5– 4.5 kg, were used. The animals were anesthetized using a mixture of xylazine (10 mg/kg body weight; Lloyd Laboratories, Shenandoah, IA, USA) and ketamine (40 mg/kg body weight; Aveco Co., Inc., Fort Dodge, IA, USA). To allow for US propagation into the brain, a piece of bone about 2 cm ⫻ 2 cm was surgically removed from the parietal surface of the skull of each animal (approximately 1.5–2.0 mm anterior from Bregma to 1.0 mm anterior from Lambda; 10 mm to the left and 10 mm to the right from the midline). The skin was sutured back in place, the animals were allowed to recover and the wound to heal for at least 2 weeks. Before the experiments, the animals were anesthetized again and the hair over the skull was removed with an electric trimmer and depilatory lotion. Our institutional animal committee approved these experiments. MRI The MRI scanner was a standard 1.5 T Signa system (General Electric Medical Systems, Milwaukee, WI, USA). A 7.5-cm diameter surface coil was placed under the head to improve the signal-to-noise ratio (SNR). During each sonication, a time series (20 time points, 4 s/image) of fast spoiled gradient echo images were ac-
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Fig. 1. Experimental setup.
quired (TR:TE 40.9:19.9 ms, flip angle 30°, band width 3.57 kHz, matrix (frequency ⫻ phase) 256 ⫻ 128, fieldof-view (FOV) 10 ⫻ 7.5 cm, slice thickness 3 mm). A complex reconstruction of these images was used to calculate temperature maps based on the temperature dependence of the proton resonant frequency (Ishihara et al. 1995). The scan plane was located across the focus (perpendicular to the beam axis) at the expected focal depth. T2-weighted fast spin echo (FSE) images (TR:TE 2000:85 ms, echo train length 8, FOV 10 cm, slice thickness 1.5 mm, two-signal averages) were obtained to localize the bone window and the target depth in the brain. After the sonications, T1-weighted FSE images (TR:TE 500:17 ms, echo train length 4, three signal averages, FOV 10 cm, matrix size 256 ⫻ 256, slice thickness 1.5 mm) were obtained. These scans were repeated after a bolus of MRI contrast agent (gadopentetate dimeglumine, Magnevist®, Berlex Laboratories Inc, Wayne, NJ, USA) to confirm the focal BBB opening. Experimental protocol The animals were placed on their backs on a plate above the sonication system (Fig. 1). The skin on the top of the head was coupled with a water bag to the water tank that held the transducer. The free water surface of the bag coupled the sound into the skin. Before the experiment, low-power continuous-wave sonications (1.2 W, no US contrast agent) below the level for tissue damage (Hynynen et al. 1997) were delivered at the first sonicated location to ensure that the focus was at the correct location. Acoustic powers of 0.55 or 3 W during the acoustic bursts were used in the experimental sonications, which translated to acoustic pressure amplitudes of 1–3 MPa at
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the focus (in situ estimate). The sonications were pulsed with a burst length of 100 ms and a repetition frequency of 1 Hz. The duration of the whole sonication was 20 s. The 0.55 W power level was chosen based on our previous work and aimed to open the BBB without damage to the neurons. The 3-W sonications aimed to cause BBB opening that was accompanied with damage. Approximately 10 s prior to the start of each sonication, a bolus of US contrast agent (Optison®, Mallinckrodt, Inc., St. Louis, MO, USA) that contains perfluorocarbon gas microbubbles (mean diameter ⫽ 2.0 – 4.5 ⫻ 10⫺6 m; concentration ⫽ 5– 8 ⫻ 108 bubbles/mL) was injected in the ear vein. The injected volume was selected to be 0.05 mL/kg. The bolus was flushed from tubing that extended out of the magnet bore by injecting approximately 1–2 mL of saline. A 5–10 min delay between sonications allowed the bubbles to clear from the circulation. Two to four locations were sonicated in each animal through the bone window in the skull. A total of 25 sonications were targeted at nonoverlapping locations for all 10 rabbits. Nine and eight locations were sonicated at acoustic powers of 0.55 and 3 W, respectively, for the standard electron microscopy examination. Four locations used in the immunoelectron microscopy were at an acoustic power level of 0.55 W; four were at 3 W. The coordinates of the locations were determined by MRI guidance and monitoring and were as follows: 8 mm anterior from the dorsal pole of brain hemispheres, 2 mm lateral from brain midline, 10 mm depth from brain surface. If the second sonication was made in the same hemisphere, it was focused at 3 mm posterior from the first one, at the same depth and lateral distance from the midline. If four locations were sonicated, the first two were mirrored in the contralateral hemisphere. Anatomically, the foci of sonications were in the thalamus. All materials for the ultrastructural study were taken 1 to 2 h after the application of US. Electron microscopy All 25 sonicated locations were examined “blindly” (17 for conventional electron microscopy and eight for immunoelectron microscopy). To make zones of the compromised BBB visible, 2% trypan blue in saline (1.5 mL/kg) was injected IV after the sonications and postultrasound MR scans. To avoid flushing out the contents of the brain vasculature during perfusion fixation, we used immersion fixation. The anesthesia was prolonged and a wide craniotomy was performed, exposing the brain. The animal was euthanized with an IV injection of solution containing pentobarbital sodium and phenytoin sodium (Euthasol® 0.25 mL/kg, Delmarva Laboratories Inc., Midlothian, VA, USA), and the brain was rapidly removed. Using a rabbit brain matrix (ASI instruments, Warren, MI, USA) and under continuous rinsing with
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fixative (2.5% glutaraldehyde in 0.1M sodium cacodylate buffer, pH ⫽ 7.2), horizontal slices from the brain were obtained and pieces of about 1 mm3 were extracted from the blue spots and from nonsonicated (control) areas (n ⫽ 12). The pieces were immersed in the same fixative for 2 h, postfixed for 1 h in 1% osmium tetraoxide, stained in uranylacetate, and embedded in EponAraldite. Ultrathin sections were obtained with Reichert Ultracut-S microtome, then were stained with lead citrate and examined with a Jeol 1200EX electron microscope. Immunoelectron microscopy Tissue pieces from eight sonicated and five nonsonicated locations were fixed in 4% paraformaldehyde ⫹ 0.01% glutaraldehyde in 0.1M phosphate buffer (pH ⫽ 7.4) for 2 h. The samples were washed in 0.12% glycine in phosphate-buffered saline (PBS) for 15 min (to quench free aldehyde groups), then infiltrated with 2.3 M sucrose in PBS for 15 min and frozen in liquid nitrogen. The frozen samples were sectioned at ⫺120°C. The sections were transferred to formvar/carbon-coated copper grids and floated on PBS. The immunogold labeling was carried out at room temperature on a piece of parafilm. Grids were floated on drops of 1% bovine serum albumin (BSA) for 10 min to block unspecific labeling, transferred to 5 L drops of goat antirabbit immunoglobulin G (IgG) conjugated to 10 nm gold (Sigma), diluted 1:10 in 1% BSA in PBS and incubated for 1 h at room temperature. After the incubation, the grids were washed in drops of PBS for 15 min followed by washing in drops of double distilled water. Contrasting/embedding of the labeled grids was carried out on ice in 0.3% uranyl acetate in 2% methyl cellulose for 10 min. Controls for the specificity of the immunostaining consisted of sections preincubated with nonconjugated with gold goat antirabbit IgG antiserum, diluted 1:5 in 1% BSA in PBS overnight at 4°C. RESULTS The BBB disruption was clearly identified in MRI for each location (Fig. 2). The peak temperature measured at the focus during the 3-W sonication was 2.0°C, consistent with our previous results and below the threshold for thermal damage (Hynynen et al. 1997). The focal BBB opening was also visually detected by the presence of the trypan blue selectively at the sonicated locations. In the brain samples obtained from the nonsonicated areas, no damage to the vessel morphology or to the neutropil was found (Fig. 3a). An increased number of vesicles and vacuoles were observed in many EC in areas sonicated with 0.55 W. Groups of vesicles were also seen in the cytoplasm of several pericytes (Fig. 3b). The
Fig. 2. Contrast-enhanced MR image verifying focal opening of the BBB at two sonicated locations. The enhancing zones at the focal coordinates (arrows) indicated a locally disrupted BBB.
luminal surface of the EC appeared with foldings, some of which formed deep pocket-like invaginations that caused the lumen and basement membrane to be separated by a thin layer of cytoplasm or by only luminal and abluminal membranes of the cells. In addition to such fenestrae-like formations, cytoplasmic channels were occasionally seen as free routes between lumen and the basement membrane (Fig. 3c). In many vascular profiles, the tight junctional complexes appeared intact, but some interendothelial clefts were lightened and widened with missing zonulae occludentes. The basement membrane rarely displayed irregularities or splittings. Numerous caveolae attached to its endothelial surface or attached to its both surfaces were seen in some vessels. Free-lying caveolae also presented in the EC and pericytes (Fig. 4a). There were small extravasates of red blood cells (1 to 3–7 cells in three from nine locations), and 35– 40 cells (one from nine locations) in neuropil. Moderate to severe damage to the brain vasculature was found in locations sonicated with acoustic power of 3 W. The EC cytoplasm appeared dark with poorly distinguishable organelles. Spastic arterioles with obturation of the lumen were observed. In some vessels, the endothelial lining was detached, the basement membrane was tortuous, with blurred outlines and, sometimes, membrane discontinuities occurred (Fig. 4b). Single or groups of red blood cells were found in close proximity to some vessels. The pericytes looked damaged and the
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Fig. 3. Photomicrographs of brain microvessels from (a) nonsonicated control and (b)–(d) sonicated with 0.55W areas, showing: (a) scarce vesicles (arrows) and a vacuole (v) in the endothelial cell cytoplasm (EC), a part of a pericyte (P) and neuropil (NP); R ⫽ red blood cell in lumen (L); b ⫽ basement membrane. (b) Numerous vesicles (arrows) in an endothelial cell cytoplasm (EC) and in a pericyte (P, curved arrows). (c) A transendothelial channel (arrow) that exposes the basement membrane, b, to the lumen, L; (*) 2 plasmalemmal pits at the luminal surface of the endothelial cell are shown. (d) Deep channel-like invagination (*) in an edematous-looking endothelial cell (EC, right); the interendothelial cleft (arrowheads) near the invagination does not appear to be widened.
astrocytes appeared edematous, with agglomerated nuclear chromatin (Fig. 4b). In the frozen sections used for immune electron microscopy, labeled IgG was present in the lumina of the vessels in both sonicated and nonsonicated areas. Lack of immunosignals could be seen outside of the vessel lumina in the sections from nonsonicated areas (Fig. 5a). Preincubation of the specimens with goat antirabbit IgG antiserum resulted in no labeling when incubated in gold-labeled IgG antiserum, confirming the specificity of the IgG immunodetection. In capillaries obtained from sonicated areas, gold particles were observed in the EC cytoplasm and in the subendothelial space: in the basement membrane, in pericytes and in neuropil (Fig. 5b). Labeled molecules presented in vacuoles and in caveolae inside the EC (Figs. 6 and 7a). Formation of plasmalemmal pits at luminal surface of the EC and presence of caveolae containing gold particles at both luminal and abluminal fronts of the EC were seen; some signal-bearing
caveolae looked opened to the basement membrane (Fig. 6a, b, c, d). Groups of gold particles were seen inside the pocket-like invaginations of the endothelial cell cytoplasm in close proximity to the basement membrane; passage of labeled IgG through cytoplasmic channels could be seen as well (Fig. 7b). In a few cases, gold particles appeared in interendothelial clefts where the tight junctions seemed lightened and widened (Fig. 7c). In locations sonicated with 3 W, the particles in the neuropil were more numerous in the vicinity of the vessels showing damaged endothelial lining. DISCUSSION The BBB disruption in our experiments was confirmed by both MRI monitoring for the contrast-matter enhancement and trypan blue leakage in the sonicated locations. Both acoustic powers applied (0.55 W and 3 W) resulted in dye and contrast matter leakage. The
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electron microscopy study revealed that barrier opening by focused US in the presence of preformed gas bubbles has its expression in a group of characteristic ultrastructural changes of the brain microvasculature. These ultrastructural changes were found in the sonicated locations, but neither in the nonsonicated contralateral hemispheres nor in nonsonicated areas of the ipsilateral hemispheres. The data obtained suggest that at least four mechanisms of passage of materials through the BBB are possible after such sonications: 1, transcytosis, 2, transendothelial openings—fenestration and channel formation, 3, widening of interendothelial clefts and opening of a part of tight junctions, and 4, free passage through the injured endothelial lining (in locations with 3 W sonication). It is known that there are few cytoplasmic vesicles in the EC in the brain microvasculature (Reese and Karnovsky 1967; Sedlakova et al. 1999). In various experimental or pathological conditions, the number of these vesicles increases and this fact is regarded as an expression of enhanced endo- and transcytosis (Castejon 1998; Chaudhuri 2000; Claudio et al. 1989; Struzynska et al. 1997). A disagreement exists about the mechanism of this vesicular transport. Some authors accept that membrane-bound vesicles take in plasma protein from the lumen, then separate from the luminal plasma membrane, pass across the endothelial cytoplasm and fuse with the abluminal plasma membrane, where they discharge their contents into the underlying basal membrane (Claudio et al. 1989). According to another opinion, a process of channel formation by fusion rather than actual movement of vesicles occurs (Lossinsky et al. 1999). The enhanced number of cytoplasmic vesicles that we found in EC after the sonication suggests an activation of transcellular transport by US. This is confirmed by the EC luminal infoldings and the formation of vacuoles capable of internalizing portions of the blood plasma, indicated by the presence of labeled IgG inside the vacuoles. The appearance of many flask-shaped caveolae on the luminal surface of the EC, the abundant supply of free-lying caveolae in the cytoplasm, as well as the caveolae open to subendothelial space, suggest that US triggers mechanisms that lead to an enhanced formation and trafficking of these vesicular organelles.
Fig. 4. (a) Ultrastructural view of a part of a microvessel from sonicated (0.55 W) area. Numerous caveolae (arrows) attached to the basement membrane, b, and also free caveolae (thick arrow) present in the endothelial cell cytoplasm (EC); caveolae formation is seen on the luminal surface of the cell (arrowheads). A lightened interendothelial cleft with not apparent tight-junctional complexes is shown (double arrows). (b) A damaged blood vessel from the central part of
a location sonicated with 3 W. Very dark, infolded endothelial cell cytoplasm, (EC), tortuous basement membrane, (b), with blurred outlines (arrow) and leakage of blood plasma outside the membrane (*) are shown. Edematous astrocytes (A) and damaged neuropil (NP) are present, and two astrocyte nuclei (N) with agglomerated chromatin are seen at the bottom of the microphotograph; L ⫽ lumen; R ⫽ red blood cell in the lumen; P ⫽ pericyte.
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Fig. 5. (a) Immunoelectron microscopy of specimen obtained from nonsonicated brain area (control). Immunosignals are seen only in the lumen (L) of the vessel, except for one occasional particle (upper right). EC ⫽ endothelial cell cytoplasm; b ⫽ basement membrane; P ⫽ pericyte; NP ⫽ neuropil; R ⫽ red blood cell in the lumen. A sealed interendothelial cleft is shown with arrows. (b) A section from a location sonicated with 0.55 W. In addition to immunosignals in lumen (L), gold particles are present in endothelial cell cytoplasm (EC), basement membrane (b, arrows) and neuropil (NP, arrowheads). A gold particle is likely in an interendothelial cleft (white arrow); R ⫽ red blood cell in lumen.
These findings are of particular interest in the light of the specific functions of the caveolar system in cell physiology. It has been shown that caveolae are directly involved in the internalization of some macromolecules, bacterial toxins and viral pathogens and in delivering them to specific locations in different kinds of mammalian cells (Anderson et al. 1992; Stang et al. 1997; Pelkmans and Helenius 2002). These properties have made caveolae a promising tool for specific binding and intracellular delivery of bioactive molecules, genes and anticancer drugs (McIntosh et al. 2002; Schnitzer 2001).
Although many further investigations are needed, the effect of the low-power US pulses on the vesicle population and on the functional activity of the brain capillary caveolar system could be of importance in the field of targeted pharmacodelivery research. With very limited exceptions, fenestrae are not present in brain vessels and the BBB is functional in normal conditions. In contrast, many kinds of brain tumors display endothelial fenestrations and a limited barrier functionality (Groothius 2000; Vajkoczy and Menger 2000). Several nonneoplastic diseases, such as
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Fig. 6. Transport of labeled IgG molecules by caveolae is shown on photomicrographs obtained from locations sonicated with 0.55 W. (a) A gold particle attached to endothelial cell luminal surface; the fossette seems to indicate the beginning of a caveola formation. (b) A completely formed caveola containing a gold particle at the luminal front of the endothelial cell. (c) A caveola containing labeled IgG at abluminal part of an endothelial cell, close to the basement membrane, b. (d) Caveolae (arrows) with gold particles at abluminal front of an endothelial cell; one of the caveolae is attached and opened to the basement membrane. No gold particles are present in the intercellular cleft (arrowheads). EC ⫽ endothelial cell; L ⫽ lumen; b ⫽ basement membrane.
allergic encephalomyelitis, delayed radiation necrosis and experimental intoxications (lead, soman), can cause fenestration of brain vessels (Hirano et al. 1994). Fenestrae also appear after osmotic opening of the BBB or after intracerebral infusion of vascular endothelial growth factor (Dobrogowska et al. 1998; Roberts and Palade 1995). Attempts to induce fenestration in the brain vascular endothelium by means of chemical agents (retinoic acid, phorbol ester) show that the process develops slowly, taking at least 28 days (Kaya et al. 1996). Comparatively, the US induction of fenestration appeared to be rapid; fenestrae-like openings were seen on electron microscopy on samples obtained 1 and 2 h after the sonication. The cytoplasmic channels that we observed in the EC seem also to be formations for transendothelial passage. Similar cytoplasmic channels have been described in muscle capillaries (Simionescu et al. 1975) and in mouse brain capillaries in response to heat
Fig. 7. Photomicrographs of microvessels from sonicated (0.55 W) areas. (a) Vacuoles (arrowheads) containing gold particles are seen in the endothelial cell cytoplasm. (b) Passage of labeled molecule through a cytoplasmic channel is shown in this capillary profile (inset). (c) A gold particle (arrow) in an interendothelial cleft and a few particles in neuropil (arrow heads). EC ⫽ endothelial cell cytoplasm; L ⫽ lumen; R ⫽ red blood cells; b ⫽ basement membrane; NP ⫽ neuropil.
stress (Wijsman and Shivers 1993). It is widely accepted that the transendothelial channels are formed by fusion of vesicles opened simultaneously to both cell fronts (Bendayan 2002). Our findings showing that labeled IgG molecules escaped through the channels confirm the role of these formations as routes for protein passage through the capillary wall after the sonication in the parameters applied.
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In a compromised BBB, interendothelial clefts are the only extracellular route for transport of macromolecules from blood to brain. In some experiments using hyperosmotic solutions, it was reported that the tracer within cerebral capillaries permeates between adjacent EC, and that the mechanism of this opening might be explained by cell shrinkage and junctional deformation (Lossinsky et al. 1995). On the other hand, some investigators have reported that no apparent gaps in endothelium were observed in analogous experiments with hyperosmolar solutions (Farrell and Shivers 1984; Shivers and Harris 1984). A possible explanation of these differences is that the tight junctions may have opened briefly and then resealed before tissue fixation (Wisniewski and Lossinsky 1991). We observed apparently widened and lightened tight junctions next to fully sealed, not widened clefts in the same sections and in the same capillaries. It is possible to hypothesize that such a different response was due to the nature of the sonication itself; for example, perhaps the forces associated with the bubble oscillations and collapse within the US field are not uniform. We obtained the samples 1 to 2 h after sonications, so it is possible that some of the junctions opened and closed before the samples were retrieved. Only a few gold particles were found in the intercellular clefts; we did not observe a line of several labeled molecules passing through clefts. Evidently, the extracellular route is less accessible for IgG in the conditions of our experiment, and the tortuosity of the clefts could make it difficult for blood protein molecules to pass across the capillary wall. An intriguing finding in our experiments was the insertion of many plasmalemmal pits and vesicles on both the endothelial and perycite sides of the basement membrane. Insertion of numerous caveolae on the basement membrane in brain capillaries has been shown to occur as a result of the enhanced transport of serum albumin in diabetic rats (Bouchard et al. 2002). In that study, however, the vesicles were found predominantly on the endothelial surface of the basement membrane. It is more likely, in our study, that the vesicles were produced as a response to the increased two-way transport after BBB disruption by US, although it is difficult to explain why the transport was so pronounced in both directions. The tight junctions opening, on the other hand, could be considered to be a temporary modification of, or an easily restored damage to zonulae occludentes proteins as it happens, for example, in the process of transvascular white blood cell migration during immunological surveillance of the normal central nervous system or in osmotic opening of the barrier (Rapoport 2000). The lightening and widening of the tight junctional complexes we found were fully comparable with the ultrastructural picture described by Mesiwala et al. (2002) in their work on selective and temporal BBB tight
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junction opening after high-intensity focused US application in rats. Our electron microscopic data show that the sonication with low US acoustic power of 0.55 W during the sound burst (pressure amplitude 1.0 MPa) effectively opens the BBB, preserving the tissue ultrastructure better than the 3-W acoustic power does. The small red blood cell extravasates we found in four from nine locations sonicated with 1 MPa (0.55 W) are comparable with the histological findings reported previously, and correspond to category 1–2 of tissue damage, as described there (Hynynen et al. 2003). Although hemoglobin is known to contribute to nerve cell injury when released into the brain after hemorrhages, it is unlikely that this is the case with these small extravasates in our experiments. We never did see neurons demonstrating electron microscopic signs of acute cell death in 0.55 W locations up to 2 h after the sonications in the present study. A recently completed light microscopy examination for cell necrosis and apoptosis, at the frequency used in the present study, showed that the BBB opening by US, when applied at the same parameters, does not result in delayed damage to neurons up to 1 month after the sonication (Vykhodtseva et al. 2003). Nevertheless, further investigations are necessary in studying these effects. The exact mechanism of how US influences the BBB is not clear. Based on our previous experiments, it can be concluded that the BBB opening is mainly the result of interaction between the US wave and the microbubbles. There are several potential ways that this interaction can produce this bioeffect. First, the US wave causes the bubbles to expand and contract in the capillaries. The expansion of larger bubbles can fill the whole capillary lumen, resulting in a mechanical stretching of the vessel wall. This may result in the opening of the tight junctions. This change in the pressure in the capillary may also evoke biochemical reactions that trigger the opening of the BBB; as is well known, an increase or change in blood pressure can affect the BBB (Kongstad and Grande 2001). The bubble oscillation may also reduce the local blood flow and induce transient ischemia, which can trigger BBB opening (Abraham et al. 2002). Finally, the bubbles can collapse during the sonication, causing highly localized shock waves and fluid jets. These mechanical effects can also be responsible for the opening of the BBB, and almost certainly play an important role in tissue damage induced at higher-pressure amplitude values. CONCLUSION The ultrastructural changes that we observed in the brain microvessels indicate that macromolecules passed
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through the US-induced BBB disruption via several routes. In addition to the tight junction opening, we also observed enhanced vesicular transcellular trafficking, endothelial cell fenestration and channel formation—all pathways for permeation of the endogenous IgG that we used in our experiments as a representative of macromolecular solutes. These findings are ultrastructural evidence for low-power focused US-induced consistent BBB opening in targeted locations. To our knowledge, this is the first demonstration of IgG (i.e, antibodies) delivery at targeted brain areas and also the first description of the routes for macromolecular passage through the US compromised BBB. Each of these routes should be considered in further investigations on US-mediated BBB disruption, as well as in the development of new strategies for antibody, gene or drug delivery to brain parenchyma. Acknowledgments—The authors thank Drs. Roderick Bronson and Peter Vassilev for the valuable discussions and comments on the manuscript. The technical assistance of Ms. Heather Martin in animal preparation is also greatly appreciated. This study was supported by NCI research (grants CA76550 and CA089017) and a grant from CIMIT.
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