Amelioration of the nigrostriatal pathway facilitated by ultrasound-mediated neurotrophic delivery in early Parkinson's disease.

The blood-brain barrier (BBB) prevents most drugs from gaining access to the brain parenchyma, which is a recognized impediment to the treatment of neurodegenerative disorders like Parkinson's disease (PD). Focused ultrasound (FUS), in conjunction with systemically administered microbubbles, opens the BBB locally, reversibly and non-invasively. Herein, we show that neither FUS applied over both the striatum and the ventral midbrain, without neurotrophic factors, nor intravenous administration of neurotrophic factors (either through protein or gene delivery) without FUS, ameliorates the damage to the nigrostriatal dopaminergic pathway in the sub-acute MPTP mouse model of early-stage PD. Conversely, the combination of FUS and intravenous neurotrophic (protein or gene) delivery attenuates the damage to the nigrostriatal dopaminergic pathway, by allowing the entry of these agents into the brain parenchyma. Our findings provide evidence that the application of FUS at the early stages of PD facilitates critical neurotrophic delivery that can curb the rapid progression of neurodegeneration while improving the neuronal function, seemingly opening new therapeutic avenues for the early treatment of diseases of the central nervous system.

Targeting Effects on the Volume of the Focused Ultrasound-Induced Blood-Brain Barrier Opening in Nonhuman Primates In Vivo.

Drug delivery to subcortical regions is susceptible to the blood-brain barrier (BBB) impeding the molecular exchange between the blood stream and the brain parenchyma. Focused ultrasound (FUS) coupled with the administration of microbubbles has been proved to open the BBB locally, transiently, and noninvasively both in rodents and in nonhuman-primates (NHPs). The development of this disruption technique independent of MRI monitoring is of primordial importance yet restrained to the targeting optimization. This paper establishes the linear relationship of the incidence angle with the volume of BBB opening ( VBBB ) and the peak negative pressure when sonicating the caudate nucleus and the putamen region of five NHPs. In addition, the effect of central nervous system structures on the opening morphology is evaluated by identification of the gray-to-white-matter ratio at the opening site. Finally, the targeting accuracy is assessed through the estimation of the geometric and angle shift of the opening from the targeted region. Interestingly, results prove a monotonic increase of the opening volume with close to normal incidence angles. Moreover, 80.35% of the opening lies on gray-matter regions compared with only 19.41% attributed to the white matter. The opening was found to be shifted axially, toward the transducer, and laterally with an average angle shift of 4.5°. Finally, we were capable of showing reproducibility of targeting accuracy with the same stereotactic and ultrasonic parameters. This paper documents the a priori prediction of the opening volume through manipulation of the angle and pressure as well as establishing the predictability, accuracy, and safety of FUS-induced BBB opening in NHPs.

Pharmacokinetic analysis and drug delivery efficiency of the focused ultrasound-induced blood-brain barrier opening in non-human primates.

PURPOSE: Focused Ultrasound (FUS) in conjunction with systemically administered microbubbles has been shown to open the Blood-Brain Barrier (BBB) locally, non-invasively and reversibly in rodents and non-human primates (NHP), suggesting the immense potential of this technique. The objective of this study entailed the investigation of the physiologic changes in the brain following the FUS-induced BBB opening and their relationship with the underlying anatomy. MATERIALS AND METHODS: Pharmacokinetic analysis was implemented in NHP's that received FUS at various acoustic pressures. Relaxivity mapping enabled the robust quantitative detection of the BBB opening as well as grey and white matter segmentation. Drug delivery efficiency was measured for pre-clinical validation of the technique. RESULTS: Based on our results, the opening volume and the amount of the gadolinium delivered were found mostly contained in the grey matter, while FUS-induced permeability and drug concentration varied depending upon the underlying brain inhomogeneity, and increased with the acoustic pressure. CONCLUSIONS: Overall, apart from the in vivo protocols for BBB analysis developed here, this study also suggests the important role that FUS can have in efficient drug delivery via localized and transient BBB opening.

Characterizing Focused-Ultrasound Mediated Drug Delivery to the Heterogeneous Primate Brain In Vivo with Acoustic Monitoring.

Focused ultrasound with microbubbles has been used to noninvasively and selectively deliver pharmacological agents across the blood-brain barrier (BBB) for treating brain diseases. Acoustic cavitation monitoring could serve as an on-line tool to assess and control the treatment. While it demonstrated a strong correlation in small animals, its translation to primates remains in question due to the anatomically different and highly heterogeneous brain structures with gray and white matteras well as dense vasculature. In addition, the drug delivery efficiency and the BBB opening volume have never been shown to be predictable through cavitation monitoring in primates. This study aimed at determining how cavitation activity is correlated with the amount and concentration of gadolinium delivered through the BBB and its associated delivery efficiency as well as the BBB opening volume in non-human primates. Another important finding entails the effect of heterogeneous brain anatomy and vasculature of a primate brain, i.e., presence of large cerebral vessels, gray and white matter that will also affect the cavitation activity associated with variation of BBB opening in different tissue types, which is not typically observed in small animals. Both these new findings are critical in the primate brain and provide essential information for clinical applications.

Longitudinal Motor and Behavioral Assessment of Blood-Brain Barrier Opening with Transcranial Focused Ultrasound.

Focused ultrasound (FUS), in combination with microbubbles, has been found to open the blood-brain barrier (BBB) non-invasively. When this technique is used for drug delivery, repeated drug administration and BBB opening are likely required. Therefore, it is worth investigating the long-term effects of FUS-induced BBB opening. In this study, we focused on the assessment of potential behavior changes in mice that could be attributed to repeated BBB opening for up to 6 months. The striatum of animals was unilaterally sonicated either monthly or biweekly throughout the monitoring period. Behavioral assessments were conducted using open-field and rotarod performance tests. Upon completion of each sonication, mice underwent magnetic resonance imaging (MRI) to confirm and assess the volume of the BBB opening. No differences in locomotor activity between BBB-opened and control groups in both biweekly and monthly treated mice were evident up to 6 months. Similarly, there was no affinity for a particular turn angle in the sonicated mice compared with the control animals. However, the positive control group exhibited a significant decrease in locomotor activity, as well as rotation ipsilateral to the sonicated hemisphere. Our results based on the assessment using open-field and rotarod tests indicated that repeated opening of the BBB in the striatum using FUS in conjunction with microbubbles over a period of 6 mo and under the parameters used here did not cause motor impairment, behavioral changes or morphologic alterations. This reinforces the tolerability of repeated and long-term drug delivery using FUS-induced BBB opening.

Acoustic cavitation-based monitoring of the reversibility and permeability of ultrasound-induced blood-brain barrier opening.

Cavitation events seeded by microbubbles have been previously reported to be associated with MR- or fluorescent-contrast enhancement after focused ultrasound (FUS)-induced blood-brain barrier (BBB) opening. However, it is still unknown whether bubble activity can be correlated with the reversibility (the duration of opening and the likelihood of safe reinstatement) and the permeability of opened BBB, which is critical for the clinical translation of using passive cavitation detection to monitor, predict and control the opening. In this study, the dependence of acoustic cavitation on the BBB opening duration, permeability coefficient and histological damage occurrence were thus investigated. Transcranial pulsed FUS at 1.5 MHz in the presence of systemically circulating microbubbles was applied in the mouse hippocampi (n = 60). The stable and inertial cavitation activities were monitored during sonication. Contrast-enhanced MRI was performed immediately after sonication and every 24 h up to 6 d thereafter, to assess BBB opening, brain tissue permeability and potential edema. Histological evaluations were used to assess the occurrence of neurovascular damages. It was found that stable cavitation was well correlated with: (1) the duration of the BBB opening (r(2) = 0.77); (2) the permeability of the opened BBB (r(2) = 0.82); (3) the likelihood of safe opening (P < 0.05, safe opening compared to cases of damage; P < 0.0001, no opening compared to safe opening). The inertial cavitation dose was correlated with the resulting BBB permeability (r(2) = 0.72). Stable cavitation was found to be more reliable than inertial cavitation at assessing the BBB opening within the pressure range used in this study. This study demonstrates that the stable cavitation response during BBB opening holds promise for predicting and controlling the restoration and pharmacokinetics of FUS-opened BBB. The stable cavitation response therefore showed great promise in predicting the BBB opening duration, enabling thus control of opening according to the drug circulation time. In addition, avoiding adverse effects in the brain and assessing the pharmacokinetics of the compounds delivered can also be achieved by monitoring and controlling the stable cavitation emissions.

Enhanced delivery and bioactivity of the neurturin neurotrophic factor through focused ultrasound-mediated blood--brain barrier opening in vivo.

The blood-brain barrier (BBB) constitutes a major obstacle in brain drug delivery. Focused ultrasound (FUS) in conjunction with microbubbles has been shown to open the BBB noninvasively, locally, and transiently to allow large molecules diffusion. Neurturin (NTN), a member of the glial-derived neurotrophic factor (GDNF) family, has been demonstrated to have neuroprotective and regenerative effects on dopaminergic neurons in vivo using invasive drug delivery methods. The brain's ascending nigrostriatal pathway is severely damaged in Parkinson's disease (PD), and therefore the substantia nigra (SN) and striatal caudoputamen (CP) were selected as the target areas. The objective of the study was to investigate whether safe and efficient NTN delivery can be achieved through FUS-induced BBB opening via intravenous administration, and thus trigger the neuroregeneration cascade in the nigrostriatal pathway. After the optimization of FUS parameters and target locations in the murine brain, NTN bioavailability and downstream signaling were detected and characterized through immunostaining. FUS significantly enhanced the delivery of NTN compared with the direct injection technique, whereas triggering of the signaling cascade was detected downstream to the neuronal nuclei. These findings thus indicate the potential of the FUS method to mediate transport of proteins through the blood-brain barrier in a PD animal model.

Microbubble type and distribution dependence of focused ultrasound-induced blood-brain barrier opening.

Focused ultrasound, in the presence of microbubbles, has been used non-invasively to induce reversible blood-brain barrier (BBB) opening in both rodents and non-human primates. This study was aimed at identifying the dependence of BBB opening properties on polydisperse microbubble (all clinically approved microbubbles are polydisperse) type and distribution by using a clinically approved ultrasound contrast agent (Definity microbubbles) and in-house prepared polydisperse (IHP) microbubbles in mice. A total of 18 C57 BL/6 mice (n = 3) were used in this study, and each mouse was injected with either Definity or IHP microbubbles via the tail vein. The concentration and size distribution of activated Definity and IHP microbubbles were measured, and the microbubbles were diluted to 6 × 10(8)/mL before injection. Immediately after microbubble administration, mice were subjected to focused ultrasound with the following parameters: frequency = 1.5 MHz, pulse repetition frequency = 10 Hz, 1000 cycles, in situ peak rarefactional acoustic pressures = 0.3, 0.45 and 0.6 MPa for a sonication duration of 60 s. Contrast-enhanced magnetic resonance imaging was used to confirm BBB opening and allowed for image-based analysis. Permeability of the treated region and volume of BBB opening did not significantly differ between the two types of microbubbles (p > 0.05) at peak rarefractional acoustic pressures of 0.45 and 0.6 MPa, whereas IHP microbubbles had significantly higher permeability and opening volume (p < 0.05) at the relatively lower pressure of 0.3 MPa. The results from this study indicate that microbubble type and distribution could have significant effects on focused ultrasound-induced BBB opening at lower pressures, but less important effects at higher pressures, possibly because of the stable cavitation that governs the former. This difference may have become less significant at higher pressures, where inertial cavitation typically occurs.

Dependence of the reversibility of focused- ultrasound-induced blood-brain barrier opening on pressure and pulse length in vivo.

The most challenging aspect of intravenously-administered drugs currently developed to treat central nervous system (CNS) diseases is their impermeability through the blood-brain barrier (BBB), a specialized vasculature system protecting the brain microenvironment. Focused ultrasound (FUS) in conjunction with systemically administered microbubbles has been shown to open the BBB locally, noninvasively, and reversibly. The objective of this study was to investigate the effect of FUS (center frequency: 1.5 MHz) pulse length (PL), ranging here from 67 µs to 6.7 ms, on the physiology of the FUS-induced BBB opening. Dynamic contrast-enhanced (DCE) and T1-weighted magnetic resonance imaging (MRI) were used to quantify the permeability changes using transfer rate (Ktrans) mapping, the volume of BBB opening (VBBB) and the reversibility timeline of the FUS-induced BBB opening, with the systemic administration of microbubbles at different acoustic pressures, ranging from 0.30 to 0.60 MPa. Permeability and volume of opening were both found to increase with the acoustic pressure and pulse length. At 67-µs PL, the opening pressure threshold was 0.45 MPa, with BBB opening characteristics similar to those induced with 0.60 MPa at the same PL, as well as with 0.67-ms PL/0.30 MPa. On average, these cases had Ktrans = 0.0049 ± 0.0014 min-1 and VBBB = 3.7 ± 4.3 mm(3), and closing occurred within 8 h. The 6.7-ms PL/0.30 MPa induced similar opening with 0.67-ms PL/0.45 MPa, and a closing timeline of 24 to 48 h. On average, Ktrans was 0.0091 ± 0.0029 min-1 and VBBB was 14.13 ± 7.7 mm(3) in these cases. Also, there were no significant differences between the 6.7-ms PL/0.45 MPa, 0.67-ms PL/0.60 MPa and 6.7-ms PL/0.60 MPa cases, yielding on average a Ktrans of 0.0100 ± 0.0023 min-1 and VBBB equal to 20.1 ± 5.7 mm(3). Closing occurred within 48 to 72 h in these cases. Stacked histograms of the Ktrans provided further insight to the nonuniform spatial distribution of permeability changes and revealed a correlation with the closing timeline. These results also suggest a beneficial complementary relationship between the elongation of the PL and the decrease of the peak negative acoustic pressures, and vice versa. Linear regression between Ktrans and VBBB showed a good correlation fit. Also, the time required for closing linearly increased with VBBB. The volume rate of decrease was measured to be 11.4 ± 4.0 mm3 per day, suggesting that the closing timeline could be predicted from the initial volume of opening. Finally, no histological damage was detected in any of the cases 7 d post-FUS, indicating the safety of the methodology and parameters used.

Activation of signaling pathways following localized delivery of systemically administered neurotrophic factors across the blood-brain barrier using focused ultrasound and microbubbles.

The brain-derived neurotrophic factor (BDNF) has been shown to have broad neuroprotective effects in addition to its therapeutic role in neurodegenerative disease. In this study, the efficacy of delivering exogenous BDNF to the left hippocampus is demonstrated in wild-type mice (n = 7) through the noninvasively disrupted blood-brain barrier (BBB) using focused ultrasound (FUS). The BDNF bioactivity was found to be preserved following delivery as assessed quantitatively by immunohistochemical detection of the pTrkB receptor and activated pAkt, pMAPK, and pCREB in the hippocampal neurons. It was therefore shown for the first time that systemically administered neurotrophic factors can cross the noninvasively disrupted BBB and trigger neuronal downstream signaling effects in a highly localized region in the brain. This is the first time that the administered molecule is tracked through the BBB and localized in the neuron triggering molecular effects. Additional preliminary findings are shown in wild-type mice with two additional neurotrophic factors such as the glia-derived neurotrophic factor (n = 12) and neurturin (n = 2). This further demonstrates the impact of FUS for the early treatment of CNS diseases at the cellular and molecular level and strengthens its premise for FUS-assisted drug delivery and efficacy.

A quantitative pressure and microbubble-size dependence study of focused ultrasound-induced blood-brain barrier opening reversibility in vivo using MRI.

Focused ultrasound in conjunction with the systemic administration of microbubbles has been shown to open the blood-brain barrier (BBB) selectively, noninvasively and reversibly. In this study, we investigate the dependence of the BBB opening's reversibility on the peak-rarefactional pressure (0.30-0.60 MPa) as well as the microbubble size (diameters of 1-2, 4-5, or 6-8 µm) in mice using contrast-enhanced T(1)-weighted (CE-T(1)) MR images (9.4 T). Volumetric measurements of the diffusion of Gd-DTPA-BMA into the brain parenchyma were used for the quantification of the BBB-opened region on the day of sonication and up to 5 days thereafter. The volume of opening was found to increase with both pressure and microbubble diameter. The duration required for closing was found to be proportional to the volume of opening on the day of opening, and ranged from 24 h, for the smaller microbubbles, to 5 days at high peak-rarefactional pressures. Overall, larger bubbles did not show significant differences. Also, the extent of BBB opening decreased radially towards the focal region until the BBB's integrity was restored. In the cases where histological damage was detected, it was found to be highly correlated with hyperintensity on the precontrast T(1) images.

Noninvasive and localized blood-brain barrier disruption using focused ultrasound can be achieved at short pulse lengths and low pulse repetition frequencies.

Ultrasound methods in conjunction with microbubbles have been used for brain drug delivery, treatment of stroke, and imaging of cerebral blood flow. Despite advances in these areas, questions remain regarding the range of ultrasound parameters that disrupt the blood-brain barrier (BBB). In this study, several conditions were investigated to either enhance or reduce the likelihood of BBB disruption. Pulsed focused ultrasound (frequency: 1.5 MHz, pressure: 0.46 MPa, pulse repetition frequency (PRF): 0.1 to 25 Hz, pulse length (PL): 0.03 to 30 milliseconds) was noninvasively and locally administered to a predetermined region in the left hemisphere in the presence of circulating preformed microbubbles (Definity, Lantheus Medical Imaging, N. Billerica, MA, USA; 0.01, 0.05, 0.25 µL/g). Trans-BBB delivery of 3-kDa dextran was observed at PRFs as low as 1 Hz, whereas consistent delivery was observed at 5 Hz and above. Delivery was demonstrated at a PL as low as 33 microseconds. Although the delivered dextran concentration increased with the PL, this also increased the heterogeneity of the resulting distribution. In conclusion, key parameters that disrupt the BBB were identified out of a wide range of conditions. Reducing the total number of emitted acoustic cycles by shortening the PL, or decreasing the PRF, was also found to facilitate a more spatially uniform distribution of delivered dextran.