This study elucidates the relevance of FUS-induced BBB opening as a method for understanding biological processes and for testing the therapeutic effectiveness of drugs. Our method neither requires an MRI system, nor a craniotomy. It is spatially accurate, and is simple enough to implement in most laboratories. A single, one-minute FUS application combined with microbubbles was sufficient to noninvasively and selectively open the BBB and deliver molecules larger than the BBB's natural size exclusion threshold of 400 Da. As our model tracers, we chose 3, 70, and 2000 kDa dextrans that were fluorescently tagged. The 3 and 70 kDa dextrans were measured by dynamic light scattering to have a hydrodynamic diameter of 2.33±0.38 and 10.2±1.4 nm, respectively. In one study, 3 and 70 kDa dextrans were found to be 3 and 14 nm in diameter (Thorne and Nicholson 2006
), and, in another, the 70 and 2000 kDa dextrans were found to be 14.8 and 54.4 nm in diameter, respectively (Lebrun and Junter 1994
This study has determined that the molecular weight threshold for dextran traversing the FUS-induced BBB opening was between 70 and 2000 kDa and that the 2000 kDa dextran could not be delivered. This suggests that there is a size limit associated with the technique. Large molecules and other constituents within the circulatory system, such as microbubbles (1-10 μm) and red blood cells (8 μm), cannot cross the FUS-opened BBB. According to prior literature, the largest molecule delivered through using FUS was Herceptin at 148 kDa (Kinoshita et al. 2006
), which is within the threshold range reported here. Despite the fact that Herceptin has different chemical properties than dextran, it may suggest that the molecular weight threshold, beyond which a molecule cannot be delivered via the FUS-induced BBB opening, may lie between 148 and 2000 kDa.
Both 3 and 70 kDa dextrans were shown to traverse the BBB after 1-min FUS sonication at a single location (i.e., onto the hippocampus) by an increase in the fluorescence throughout the targeted region (Figs. and ). When comparing these two molecules, the 3 kDa dextran was distributed throughout the sonicated hippocampus at a higher overall relative concentration (). The 70 kDa dextran was spatially distributed in two distinct patterns: a low level of fluorescence that was diffuse throughout the hippocampus, and high levels of fluorescence consisting of punctated regions near, or along, larger vessels such as the internal and external transverse hippocampal vessels, the longitudinal hippocampal artery, and the PCA. In the case of the punctate regions, fluorescence did not extend further than a few microns away. The 70 kDa dextran also accumulated along the longitudinal hippocampal arterial wall, suggesting that the markers were either contained within, or attached to, the membranes of cells that constitute the vascular wall such as endothelial and smooth muscle cells (). Both 3 and 70 kDa dextrans were most concentrated at or near the internal and external hippocampal vessels and the vessels within the thalamus (Figs. and ).
In order to determine what proportion of the hippocampus could be treated and whether the permeated area would vary with the molecular weight of the molecules delivered, the percent area of the hippocampus exhibiting fluorescence was calculated. Dextrans were delivered to a significant percentage of the total targeted area (i.e., hippocampus): 54.2 ± 24.6% for 3 kDa dextran and 45.6 ± 12.6% for 70 kDa dextran in the cross-section analyzed (). The center of the acoustic focal volume (i.e., the focal point) was positioned to overlap with the medial portion of the hippocampus and avoid overlap of the beamwidth with the lateral side of the skull. This placement away from the lateral hippocampal region limited the spatial extent of the BBB opening to the hippocampus. In future studies, a larger, or smaller, area of BBB opening could be induced by adjusting the pressure amplitude, altering the FUS transducer geometry, or sonicating multiple locations.
The acoustic parameters and microbubbles described in this study have been used in many of our previous and concurrent experiments. The spatial deposition patterns of 3 kDa dextran () obtained in this study correlated with our previous high-field T1
-weighted spin-echo MRI study () (Choi et al. 2008
) that quantified the in vivo
deposition of a 573-Da MRI contrast agent (gadolinium) through the FUS-induced BBB opening of separate mice (Choi et al. 2007a
). Similar to the results shown in Figs. and , the gadolinium molecules accumulated in the parenchyma through the FUS-induced BBB opening. However, they were distributed in a non-gaussian and heterogeneous pattern (). This observation helped contribute to the rationale for performing the present study. Preliminary assessment of the safety of FUS-induced BBB opening using the same acoustic parameters reported in this paper is part of a concurrent study. Hematoxylin and eosin-stained sections of FUS-induced BBB opened brains revealed petechial red blood cell extravasations, but no neuronal damage (Konofagou et al. 2009
). The in vivo
assessment of the spatial distribution of MRI contrast agents and the comprehensive assessment of safety are some of our ongoing studies that, together with the results from this paper, will help elucidate the greater potential of FUS-induced BBB opening.
Fig. 6 Comparison of results obtained using MRI and fluorescence microscopy. (A) MR images acquired on the same day as BBB opening reveal MR contrast agent (574 Da) leakage throughout the left (targeted) hippocampus and the surrounding regions. (B) In the same (more ...)
Four major findings were reported in this paper. First, dextrans of 3 and 70 kDa were delivered trans-BBB while 2000 kDa dextran was not. Second, compared to 70 kDa dextran, a higher concentration of 3 kDa dextran was delivered through the opened BBB. Third, the 3 and 70 kDa dextrans were both diffusely distributed throughout the targeted brain region. However, the 70 kDa dextran was diffuse at a lower concentration while having numerous punctate regions of higher concentration, indicating a more heterogeneous overall distribution. Finally, dextrans were deposited at larger amounts proximal to larger vessel branches such as the internal and external transverse hippocampal vessels, and the vessels within the thalamus, when compared to other regions in the targeted hippocampus. These results suggest that FUS and microbubbles may deliver 70 kDa agents across the BBB, but that 3 kDa and smaller agents may be delivered at a more effective concentration. A broader applicability for delivering inhibitors, growth factors, peptides, and other biomarkers and therapeutic agents to a volume on the order of cubic millimeters was thus shown. FUS and microbubbles constitute a non-contact, noninvasive, localized, and transient delivery method for cortical and subcortical structures with the critical capability of depositing agents at sizes relevant for biomarkers and neurologically potent drugs.