- Meeting abstracts
- Open Access
- Published:
5th International Symposium on Focused Ultrasound
North Bethesda, MD, USA. 28 August- 1 September 2016
Journal of Therapeutic Ultrasound volume 4, Article number: 31 (2016)
A1 Treatment of essential tremor and Parkinson’s disease tremor by MRI guided Focused Ultrasound: a report of 38 consecutive cases in a single center
Menashe Zaaroor, Alon Sinai, Dorit Goldsher, Ayelet Eran, Maria Nassar, Ilana Schlesinger
Rambam Health Care Campus, Haifa, Israel
Objectives
Thalamotomy of the ventral intermediate nucleus (VIM) is effective in alleviating medication resistant tremor in patients with essential tremor (ET) and Parkinson’s disease (PD). MRI guided Focused Ultrasound (MRgFUS) is an innovative technology that enables non-invasive thalamotomy via thermal ablation.
Methods
Thirty eight ET and PD patients with severe medication resistant tremor underwent MRgFUS underwent unilateral VIM thalamotomy using MRgFUS. Effect was evaluated using clinical Rating Scale of Tremor (CRST) in ET patients and Unified PD Rating Scale motor part (UPDRS) in PD patients. Quality of life was assessed by Quality of life in ET Questionnaire (QUEST) and PD Questionaire (PDQ-39).
Results
Tremor stopped in the treated hand in 37 patients immediately following the treatment. In one patients tremor was modified but not abolished. At one month post-treatment, the ET patients’ CRST score decreased from 38.6 ± 12.0 to 9.3 ± 7.7 (p < 0.001) and QUEST scores decreased from 44.8 ± 17.8 to 13.1 ± 15.9 (p < 0.001). In PD patients UPDRS-motor part decreased from 26.2 ± 8.7 to 16.3 ± 11.0 (p = 0.0087) and PDQ39 decreased from 40.8 ± 18.2 to 26.5 ± 15.1 (p = 0.027). During follow up of 1-24 months (mean 10.9 ± 8.1 months) tremor reappeared in seven of the patients, but in all but three, to a lesser degree than before the procedure.
Adverse events that transiently occurred during sonication included: Headache (n = 11), short lasting vertigo (n = 17) and dizziness (n = 4), nausea (n = 4), burning scalp sensation (n = 3), vomiting (n = 3) and lip paresthesia (n = 2). Adverse events that lasted after the procedure included gait ataxia (n = 5), unsteady feeling when walking (n = 4,) unilateral taste disturbances (n = 3) and hand ataxia (n = 3). All adverse events were transient and none lasted beyond 3 months.
Conclusions
MRgFUS VIM thalamotomy to relieve medication resistant tremor was safe and effective in ET, and PD. Current results emphasize its low adverse events profile and high efficacy in treating tremor. Large randomized studies are needed to assess prolonged efficacy and safety.
A2 Focused Ultrasound likely dominates deep brain stimulation and stereotactic radiosurgery for medically-refractory essential tremor: an initial decision and cost-effectiveness analysis
Jonathon Parker1, Vinod Ravikumar1, Pejman Ghanouni1, Sherman Stein2, Casey Halpern1
1Stanford University, Stanford, California, USA; 2University of Pennsylvania, Philadelphia, Pennsylvania, USA
Objectives
Essential Tremor (ET) is one of the most common neurologic conditions, and conservative measures are frequently suboptimal. Recent data from a multi-institution, randomized controlled clinical trial demonstrated that Magnetic Resonance-guided Focused Ultrasound (MRgFUS) thalamotomy improves upper limb tremor in medically refractory ET. This study assesses the cost-effectiveness of this novel therapy in comparison to existing procedural options.
Methods
PubMed and Cochrane Library searches were performed for studies of MRgFUS, Deep Brain Stimulation (DBS), and Stereotactic Radiosurgery (SRS) for ET. Pre- and post-operative tremor-related disability scores were collected from 32 studies involving 83 MRgFUS, 615 DBS, and 260 SRS cases. Utility (defined as percent change in functional disability) was calculated, and Medicare reimbursements were collected as a proxy for societal cost – costs of MRgFUS for ET were derived from a combination of available costs of approved indications and SRS costs where appropriate. A decision and cost-effectiveness analysis was then constructed, implementing meta-analytic techniques.
Results
MRgFUS thalamotomy resulted in significantly higher utility scores compared with DBS and SRS based on estimates of Medicare reimbursement (p < 0.001). MRgFUS was also the most inexpensive procedure out of the three (p < 0.001).
Conclusions
Preliminary experience with MRgFUS for ET suggests that this novel therapeutic may be more effective than available alternatives and potentially less costly for society. It thus will likely “dominate” DBS and SRS as a more cost-effective option for medically refractory ET. Our findings support further investigation of MRgFUS for ET and broad adoption.
A3 Tractography-based VIM identification for Focused Ultrasound thalamotomy: initial results
Vibhor Krishna, Amelia Hargrove, Punit Agrawal, Barbara Changizi, Eric Bourekas, Michael Knopp, Ali Rezai
The Ohio State University, Columbus, Ohio, USA
Objectives
The ventral intermediate nucleus (VIM) is not visible on conventional Magnetic Resonance Imaging (MRI). A novel method for tractography-based VIM identification has recently been described. We report the short-term clinical results of prospective VIM targeting with tractography in a cohort of patients undergoing Focused Ultrasound thalamotomy.
Methods
All patients underwent structural and diffusion weighted imaging (60 diffusion directions, 2 mm isovoxel) with 3 Tesla MRI scanner (Philips Ingenia CX). The images were processed using streamline tractography (Stealth Viz, Medtronic Inc.). The lateral and posterior borders of VIM were defined by tracking the pyramidal tract and medial lemniscus respectively. A VIM region of interest (ROI) was placed 3 mm away from these borders (Figs. 1, 2 and 3). The structural connectivity of this VIM ROI was confirmed to the motor cortex (M1) and cerebellum. The coordinates of tractography-based VIM in relation to posterior commissure were noted for surgical targeting. The parameters analyzed include a clinical tremor scale (pre-, intraoperative, and post operative), operative time, and number of sonications.
Results
Tractography-based VIM targeting was successful in 7 out of 8 patients. The coordinates of tractography-based VIM were significantly different from the standard coordinates (3-D distance 3.9 ± 2.4 mm). Therapeutic sonication (>55 °C temperature, 10 seconds) at the tractography target resulted in >50 % tremor improvement with intraoperative objective tremor assessment without any motor or sensory side-effects. The mean operative time was 78 ± 3.3 minutes with 12.8 ± 3.9 average sonications. Overall the tremor scores significantly improved one month after surgery (preop CRST total 62.1 ± 15.5 versus 30.3 ± 14.1, two tailed t-test p = 0.006). None of the patients experienced sensory deficits or motor weakness during follow-up.
Conclusions
We report that prospective tractography-based VIM targeting is safe and feasible. The short-term clinical results are satisfactory. Long-term tremor efficacy outcomes are desirable to further assess the usefulness of this technique.
Axial T1 projection showing the relation of VIM target 3 mm medial and anterior to pyramidal tract and medial lemniscus respectively
Postoperative sagittal T1 projection demonstrating the relationship between pyramidal tract and medial lemniscus in relation to thalamotomy lesion
Postoperative axial T1 projection demonstrating the relationship between pyramidal tract and medial lemniscus in relation to thalamotomy lesion
A4 Targeted delivery of brain-penetrating non-viral GDNF gene vectors to the striatum with MRI-guided Focused Ultrasound reverses neurodegeneration in a Parkinson’s disease model
Brian Mead1, Namho Kim2, Panagiotis Mastorakos2, Jung Soo Suk2, Wilson Miller1, Alexander Klibanov1, Justin Hanes2, Richard Price1
1University of Virginia, Charlottesville, Virginia, USA; 2Center for Nanomedicine/Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, USA
Objectives
Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the motor control pathways of the brain. Gene therapy using glial cell derived neurotrophic factor (GDNF) has shown some limited promise for treating PD; however, we hypothesize that outcomes could be further improved by enhancing gene vector distribution. We previously developed a gene therapy approach that entails delivering systemically administered non-viral gene-bearing nanoparticles (BPN) across the Blood-Brain Barrier with MRI-guided Focused Ultrasound (FUS). BPN rapidly penetrate brain tissue due to a dense coat of polyethylene glycol, and this approach mediates efficient and localized transgene expression in the brain of healthy rats. Here, we tested whether the FUS-mediated delivery of GDNF plasmid-bearing BPN (GDNF-BPN) reverses neurodegeneration in the rat 6-OHDA PD model.
Methods
6-OHDA rats were ultrasonically coupled to a 1.15 MHz MRI-compatible FUS transducer. T2 and T2* pre-treatment scans were obtained to allow FUS targeting of striatum. Microbubbles (2x105/g) and 100 μg of ~50 nm non-viral GDNF plasmid-bearing BPN (polyethylene glycol/polyethylenimine) were co-injected i.v. and FUS was applied at 0.6 MPa, with a 0.5 % duty cycle, for 2 min. Contrast T1 and T2* images allowed semi-real time confirmation of BBB disruption and safety, respectively. Efficacy was assessed using an ELISA for GDNF, tyrosine hydroxylase (TH) and VMAT2 immunolabeling for neural degeneration, HPLC for dopamine, and behavioral analysis (i.e. apomorphine-induced rotational asymmetry and forepaw use bias in 6-OHDA rats).
Results
Striatum-targeted delivery of GDNF plasmid-bearing BPN with FUS led to an ~80 % reduction in apomorphine-induced rotational asymmetry, eliminated forepaw use bias (Fig. 4a,b), and fully restored TH+ dopaminergic neuron density in both the substantia nigra pars compacta (SNpc) and striatum compared to untreated 6-OHDA rats (Fig. 4c,d). T2* MRI confirmed safety of the BBB opening approach.
Conclusions
FUS-mediated delivery of systemically circulating non-viral GDNF-BPN to the striatum of 6-OHDA rats confers a significant behavioral benefit as well as a restoration of TH+ cell number in the nigrostriatal pathway, indicating cessation and/or reversal of neurodegeneration. Our studies indicate that delivery of GDNF-BPN with FUS may provide a powerful, non-invasive and highly tailorable gene therapy approach to slow or stop the neurodegenerative process in PD.
Graphs of rotational bias (a) or forepaw use bias (b) following 6-OHDA injection. (c) Representative images of TH-immunlolabeled sections through the SNpc. Graphs represent TH+ cell number in SNpc (d) and staining intensity in the striatum (e). * p < 0.05
A5 Focused ultrasound facilitated gene delivery for neuro-restoration in Parkinson’s disease mice
Shutao Wang, Oluyemi Olumolade, Tara Kugelman, Vernice Jackson-Lewis, Maria Eleni (Marilena) Karakatsani, Yang Han, Serge Przedborski, Elisa Konofagou
Columbia University, New York, New York, USA
Objectives
Not released for publication
Methods
Not released for publication
Results
Not released for publication
Conclusions
Not released for publication
A6 MRI-g-FUS for the treatment of Alzheimer’s disease
Kullervo Hynynen1, Isabelle Aubert2
1Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada; 2Sunnybrook Research Institute, Toronto, Ontario, Canada
Objectives
Over the past ten years consistent effort has been put forward at the University of Toronto to develop Focused Ultrasound methods for the treatment of AD. This talk will review the progress made so far.
Methods
The first studies demonstrated safe antibody delivery in AD mouse model with significant reduction in the plaque load. A follow up studies with two-photon microscopy showed that blood vessels with plague deposits showed a different type of opening than vessels in normal brain but large molecule delivery into the brain was still possible in these animals. Another study demonstrated that plaque reduction can be achieved by just opening the BBB with microbubbles.
Results
The histology revealed stimulation of neurogenesis. Multiple treatments of old mice resulted in memory rescue without any observable side-effects. A follow up study demonstrated that this neurogenesis was not induced with exposures that did not cause observable BBB opening even with the presence of the microbubbles. An ongoing study in large animals has shown that half-brain BBB opening can be safely and repeatable performed indicating the feasibility of clinical translation.
A7 Scanning Focused Ultrasound disruption of the blood-brain barrier as an Alzheimer’s disease therapy
Gerhard Leinenga1, Rebecca Nisbet2, Robert Hatch1, Anneke Van der Jeugd3, Harrison Evans2, Jürgen Götz2
1Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, St Lucia, Queensland, Australia; 2The University of Queensland, Brisbane (St Lucia Campus), QLD, Australia; 3University of Leuven, Leuven, Belgium, Belgium
Objectives
Alzheimer’s disease is the most common form of dementia. Pathological abnormalities in the Alzheimer’s disease brain includes the presence of amyloid-beta plaques, hyperphosphorylation and intracellular aggregation of tau and synaptic degeneration. Focused ultrasound combined with intravenous injection of microbubbles has been shown to reversibly open the blood-brain-barrier (BBB). By moving the focus in scanning mode we are able to open the BBB throughout the brain of a mouse. Here we tested the effects of repeated scanning ultrasound (SUS) in APP23 amyloid plaque-bearing mice, pR5 tau mice and wild-type mice to determine the effects of SUS on amyloid, tau and dendritic spines.
Methods
The device used was the Therapy Imaging Probe System (TIPS, Philips Research), which has an eight-element annular array transducer with a focal length of 80 mm a radius of curvature of 80 mm, a 33 mm central opening, and a motorized 3D positioning system. The focus 6 dB size was 1.5 mm x 1.5 mm x 12 mm at 1 MHz. Settings that were applied were 1 MHz centre frequency, 0.7 MPa peak rarefactional pressure applied outside the skull, 10 Hz pulse repetition frequency, 10 ms pulse length and 10 % duty cycle immediately after retroorbital injection of in-house made microbubbles. APP23 mice that accumulate amyloid beta, pR5 mice that overexpress FTD-mutant tau, and wild-type C57Bl/6 mice were treated weekly by scanning ultrasound (SUS) for periods of 4 to 7 weeks.
Results
In APP23 mice we used repeated scanning ultrasound (SUS) treatments of the mouse brain to remove amyloid-beta. Spinning disk confocal microscopy revealed extensive internalization of Abeta into the lysosomes of activated microglia in mouse brains subjected to SUS. Plaque burden was reduced in SUS-treated AD mice compared to sham-treated animals. Treated AD mice also displayed improved performance on three memory tasks.
In PR5 mice we investigated the efficacy of a novel tau isoform-specific single chain antibody fragment, RNX, delivered by passive immunization in the P301L human tau transgenic pR5 mouse model. When administration of RNX was combined with scanning ultrasound (SUS), RNX delivery into the brain and uptake by neurons were markedly increased, as were reductions in tau phosphorylation and anxiety-like behavior.
In wild-type mice we investigated the effects of SUS on neuronal excitability and morphology. We performed patch-clamp recordings from hippocampal CA1 pyramidal neurons in wild-type mice 2 and 24 hours after a single SUS treatment, and one-week and three months after six weekly SUS treatments. No change in CA1 neuronal excitability was observed compared to sham-treated neurons at any time-point. Multiple SUS treatments had the effect of preventing the loss of CA1 synapses that occurred in sham-treated neurons.
Conclusions
We show that scanning Focused Ultrasound disruption of the BBB has multiple biological effects in the brain which make it an attractive candidate for an Alzheimer disease therapy. SUS reduced plaque burden and amyloid-beta levels in APP23 amyloid mice, through activation of microglia, and improved performance on tests of memory function. In pR5 tau mice SUS alone reduced hyperphosphorylation of tau, and enhanced the delivery of anti-tau antibodies resulting in improved reductions in pathology and behavioral abnormalities. In wild-type mice SUS was shown to have no effect on the firing of hippocampal neurons or their morphology, but prevented spine loss at 3 months after six weekly SUS treatments. If these effects on Abeta and tau pathology, and dendritic morphology are recapitulated in human patients SUS may emerge as a promising AD therapy.
A8 Scanning ultrasound as a treatment tool of proteinopathies including Alzheimer’s disease
Jürgen Götz1, Rebecca Nisbet1, Ann Van der Jeugd1, Harrison Evans1, Gerhard Leinenga2
1The University of Queensland, Brisbane (St Lucia Campus), QLD, Australia; 2Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, St Lucia, Queensland, Australia
Objectives
Neurological disorders constitute a substantial social and economic burden, as they cause considerable ill health but few direct deaths. Treatment strategies for neurodegenerative diseases are hampered by the fact that the Blood-Brain Barrier (BBB) establishes an efficient barrier for therapeutic agents (Leinenga et al., Nature Reviews Neurology 2016). We have recently shown that scanning ultrasound (SUS) allows microglial-mediated clearance of extracellularly deposited amyloid-beta in APP mutant APP23 mice and restores memory functions in three cognitive tests to wild-type levels, in the absence of overt damage to the brain (Leinenga and Götz, Science Translational Medicine 2015) However, it had not been determined whether SUS treatment reduces the intracellular tau pathology that together with amyloid deposition characterizes Alzheimer’s disease.
Methods
We investigated the efficacy of a novel tau-specific single chain antibody fragment, delivered by passive immunization in the human tau transgenic pR5 mouse model, a model of the tau pathology of Alzheimer’s disease (Götz et al., Science 2001). To further assess the efficacy and drug-delivering ability of SUS, we established four experimental groups, using the novel anti-tau antibody that was injected weekly over four weeks, either on its own, or together with SUS. A third group used SUS only, and a fourth was the anaesthesia control group. The mice were analysed on the elevated plus maze, histologically and biochemically. Furthermore, uptake of the antibody by the brain was determine using fluorescently labelled single-chain antibody fragments.
Results
A histological and biochemical analysis of the pR5 tau transgenic mice revealed that SUS as well as the employed antibody ameliorated the tau pathology that characterizes the pR5 mice. In addition, the anxiety-like behaviour that characterizes pR5 mice was significantly reduced. We furthermore found enhanced delivery of the antibody using SUS yielding a synergistic therapeutic effect as determined by histology and using the elevated plus maze.
Conclusions
Our study suggests that SUS is a method that benefits diseases with protein aggregates more generally, whether they are intra- or extracellular. The therapeutic delivery combined with SUS could offer significant clinical benefits for the treatment of patients with Alzheimer’s disease and related tauopathies. Considering that the yearly costs of passive immunotherapy for AD is expected to exceed $25,000 per patient, combining SUS with antibody delivery could drastically reduce these costs.
A9 Enhancement of FUS mediated delivery of stem cells to the brain
Paul Fishman1, Paul Yarowsky2, Victor Frenkel3, Shen Wei-Bin3, Ben Nguyen3
1University of Maryland School of Medicine/Baltimore VAMC, Baltimore, Maryland, USA; 2Research & Development Service, VA Maryland Healthcare System and University of Maryland School of Medicine, Baltimore, Maryland, USA; 3University of Maryland School of Medicine, Baltimore, Maryland, USA
Objectives
FUS mediated Blood-Brain Barrier disruption (BBBD) can enable even large therapeutics such as stem cells to enter brain from the bloodstream and could be a major advance in cell delivery over current invasive methods of brain injection. The efficiency of cellular entry after FUS mediated BBBD alone however is low. We hypothesized that this process could be enhanced by combining it with a complementary strategy termed magnetic targeting. Stem cells can be safely loaded with super-paramagnetic iron oxide nanoparticles (SPION) in culture, allowing cells to be attracted by an external magnet. Our previous study showed SPION loaded stem cells to have enhanced brain retention near a magnet on the skull in a rat model of traumatic brain injury, where BBBD also occurs. The goal of our current project was to determine if magnetic attraction of SPION loaded stem cells would also enhance their delivery to brain after FUS mediated BBBD.
Methods
With a small animal MRI guided FUS device (Image Guided Therapy, IGT and 7 T Bruker MRI), we sonicated young adult rats (~120 g) with both radiologic (enhancement of the target region with gadolinium on post-sonication TI MRI), and histologic (staining with Evans’ blue dye) evidence of BBBD, without tissue damage or hemorrhage. Confirmation of the cells within brain as those injected was performed by staining with Perl’s reagent for iron and by immuno-histochemistry with a human specific antigen. The procedure was then combined with the application of a powerful magnet to the head directly after IV injection of hNPCs.
Results
With BBBD alone human neuro-progenitor cells (hNPCs) loaded with SPION were observed in rat brain after intravenous (IV) injection directly after sonication only within the treated regions. To demonstrate the effect of magnetic attraction, we injected equal numbers of SPION and non-SPION labeled cells, where each cell type was labeled with a different fluorophore. In animals that had FUS mediated BBBD followed by a magnet applied to the head, significantly greater numbers of SPION labeled cells were observed compared to the non-labeled cells. This result was most pronounced in regions of the brain close to the skull (cerebral cortex) and magnet surface. More powerful magnets including magnetic arrays resulted in more effective retention of SPION labeled cells in even deeper brain regions such as the striatum. There, 90 % of hNPCs observed contained SPIONs compared to 60-70 % with a less powerful magnet.
Conclusions
These results demonstrate that the use of magnetic attraction can substantially enhance delivery of stem cells after BBBD. In prior published work, stem cells were delivered to brain after FUS mediated BBBD using cells injected directly into the carotid artery. In an effort to accomplish this goal in a safer and less invasive manner, our study utilized IV cell injection (tail vein), supporting the view that the combination of FUS mediated BBBD and magnetic attraction can allow stem cells to enter brain with a minimally invasive strategy.
A10 Fluorescent lipid microbubbles for targeted brain drug delivery through the Focused Ultrasound-induced blood-brain barrier opening in vivo
Carlos Sierra Sanchez, Camilo Acosta, Cherry Chen, Shih-Ying Wu, Maria Eleni (Marilena) Karakatsani, Elisa Konofagou
Columbia University, New York, New York, USA
Objectives
Focused ultrasound (FUS) in the presence of lipid microbubbles can induce non-invasive, transient and reversible Blood-Brain Barrier (BBB) opening. This study entailed assessment of the feasibility of fluorescently loaded microbubbles, labeled with the fluorophore 5-dodecanoylaminfluorescein (C-12), as a vector for targeted brain drug delivery. Compared to prior studies by our group, where fluorescently-labeled dextrans were co-administered with microbubbles, this new methodology improves the safety and allows a more targeted drug delivery with potentially lower toxicity, avoiding systemic exposure. The main objective was thus to determine feasibility and safety of using the loaded microbubbles as carriers towards targeted brain drug delivery with simultaneous cavitation monitoring.
Methods
A spherical, single-element, FUS transducer (center frequency 1.5 MHz) was used. A pulse-echo transducer (center frequency 10 MHz), confocally mounted at the center of the FUS transducer, was utilized for passive cavitation detection (PCD). FUS (pulse length 10,000 cycles; pulse repetition frequency 5 Hz; duration 5 minutes; acoustic pressure 450-750 kPa) targeted mouse brains in vivo, in combination with fluorescent microbubbles for C-12 delivery, which was evaluated by in vivo transcranial PCD, through the quantification of inertial (ICD) and stable harmonic (SCDh) and ultraharmonic (SCDu) cavitation doses at 30, 60 and 300 s; together with ex vivo fluorescence imaging. The BBB opening was verified using in vivo T1-w Magnetic Resonance Imaging (MRI). The safety of this technique was assessed through ex vivo hematoxylin & eosin staining for microhemorrhage detection and immunohistochemistry (Iba-1 for microglial activation) together with in vivo T2-w MRI for edema assessment.
Results
Successful targeted C-12 delivery was achieved at 600 and 750 kPa in six out of 14 cases (Fig. 5). Comparison of ICD, SCDh and SCDu between successful and unsuccessful cases yielded a statistically significant linear relationship between the successful targeted drug delivery and CD and specific thresholds for efficient delivery were identified.
No edema was detected in mice sacrificed on Day 0 but edema appeared on Day 1 on mice sacrificed on Day 7. In all cases cases (except one) it was repaired within a week. Microhemorrhages were observed after sonication in some cases but were also cleared within the first week. However, a higher number of cell nuclei was observed in the sonicated region compared to the unsonicated side in some mice survived up to one week after opening. Iba-1 immunohistochemistry also showed microglial activation.
Conclusions
FUS was applied in conjunction with fluorescent microbubbles and, for the first time, the existence of CD thresholds for assessing successful drug delivery was defined. For CD above these thresholds, significant fluorescent enhancement was observed, demonstrating C-12 targeted delivery. One week after sonication, edema was cleared out but microglial activation was observed in certain cases. Therefore, this study indicates the feasibility and safety of a new methodology of FUS-induced BBB opening for targeted albeit potentially riskier brain drug delivery and provides a platform for predicting successful delivery via PCD.
BBB opening and fluorescence delivery: T1-w MRI showing BBB opening at pressures (a) 450, (b) 600 and (c) 750 kPa. Fluorescence delivery (green) and DAPI (blue) in two horizontal sections of mouse brains sonicated at (d) 600 and (e) 750 kPa
A11 Ultrasound-mediated delivery of gadolinium and fluorescent–labelled liposomes through the Blood-Brain Barrier
Muna Aryal1,2, Iason T Papademetriou3, Yong-Zhi Zhang2, Chanikarn Power1,2, Nathan McDannold1,2, Tyrone Porter3
1Brigham and Women’s Hospital, Boston, Massachusetts, USA; 2Harvard Medical School, Boston, Massachusetts, USA; 3Boston University, Boston, Massachusetts, USA
Objectives
The main objectives of this study were: 1) to examine whether gadolinium and fluorescent labelled liposomes can extravasate into the brain parenchyma after ultrasound mediated Blood-Brain Barrier disruptions; and 2) to test whether extravasated liposomes were size dependent or not. The liposomes were labelled with gadolinium (Gd) and fluorophore, thus enabling detection of extravasated liposomes via MRI in vivo and fluorescence methods in tissue, respectively.
Methods
Liposomes labelled with gadolinium and fluorophore were prepared using lipid film hydration and extrusion to two different sizes; ~70-85 nm and ~ 130-150 nm. Animals were divided into two different groups based on the use of particle sizes; group A (~70-85 nm) and group B (~130-150 nm). Focused ultrasound mediated Blood-Brain Barrier disruption (BBBD) was produced in one hemisphere in 15 mice. Particles were injected before sonication. Sonications (0.69 MHz at 0.42 MPa) were performed in two locations combine with Definity (10 μl/kg). Acoustic emissions were recorded during FUS. T1-weighted contrast enhanced and T2*-weighted MRI were used to confirm Gd leakage and damage detection respectively. Mice were euthanized 5-24 hours after FUS and post-process for fluorescence measurement.
Results
In T1-weighted contrast enhanced MRI, gadolinium-leakage was able to detect on sonicated area at 5-24 after FUS but not on non-sonicated area (control). Detection of fluorescence signal from brain tissue homogenates confirm the liposomal particles extravasation on sonicated locations. On group A, gadolinium and fluorescence signal intensities on sonicated locations were increased by 26 % and 62 % respectively as compared with control and signal enhancement were statistically significant compared with control ( p = 0.017 and p = 0.02 respectively, two-tailed, paired ttest). On group B, gadolinium and fluorescence signal intensities on sonicated locations were increased by 24 % and 40 % respectively as compared with control. Comparison of fluorescence signal intensities between two groups on sonicated location was statically significant whereas it was not significant on controls (p < 0.05 and p = 0.07 respectively, one-tailed unpaired ttest).
Conclusions
Overall, this work demonstrates that ultrasound can deliver upto ~ 150 nm liposomes that labelled with gadolinium and fluorophore through the Blood-Brain Barrier. The results indicate that the extravasation of liposomes were size dependent.
A12 Sterile inflammatory response (SIR) in the brain following exposure to low intensity pulsed Focused Ultrasound and microbubble infusion
Zsofia Kovacs1, Saejeong Kim1, Neekita Jikaria1, Farhan Qureshi2, Michele Bresler2, Joseph Frank2
1National Institutes of Health, Bethesda, Maryland, USA; 2National Institutes of Health Clinical Center, Bethesda, Maryland, USA
Objectives
Magnetic Resonance Imaging (MRI)-guided pulsed Focused Ultrasound (pFUS) in combination with systemic injection of microbubbles (MB) is being advocated to increase drug or gene delivery by causing localized Blood-Brain Barrier (BBB) disruption (D). The objective of this study is to investigate the molecular and cellular responses following pFUS + MB associated with BBBD in the rat brain.
Methods
Female Sprague Dawley rats (<200 g) were sonicated at 0.3 MPa acoustic pressures with 10 ms burst length and 1 % duty cycle (9 focal points, 120 sec/9 focal points) using a single-element spherical FUS transducer (589.636 kHz; FUS Instruments). 100 μl Optison™ MB (GE Healthcare) was administered intravenously. Gadofosveset-enhanced T1w images were obtained with a 3.0 T MRI (Phillips). Proteomic and mRNA expression in the brain following pFUS + MB were analyzed with ELISA, Western blot, quantitative real-time PCR or immunofluorescent staining (Fig. 6a). Proteomics were normalized to sham and statistical analysis was performed by one-way ANOVA corrected for multiple comparisons. Rats were also injected with 8 mg/kg Rhodamine encapsulated magnetic polymers (MicroTRACK™; BioPal) 3 days prior to sonication to label splenic macrophages (CD68) to monitor tropism to the brain.
Results
Post contrast T1w MRI and histology showed open BBB without evidence of microhemorrhage. Within 5 minutes following sonication, increased expression of pro-inflammatory and anti-inflammatory cytokines, chemokines and trophic factors (CCTF) was detected in the parenchyma lasting up to 24 hours (Fig. 6b). Increases in heat shock protein 70 (HSP70), tumor necrosis alpha (TNFa), and interleukin (IL) 1a, 1b and 18 consistent with damage associated molecular patterns (DMAP)1 and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) inflammatory pathways were observed with SIR to injury2 (Fig. 6b). NFkB pathway-related gene activation along with anti-apoptotic genes, immune cell chemoattractants, selectins, and cell adhesion molecules showed significant (>2fold) increases in mRNA expression (Fig. 6c). Histological analysis showed significant increases (p < 0.05) in the following: number of TUNEL positive cells within 6 hrs, GFAP and Iba1 staining for activated astrocytes and microglia (1-24 hrs) and increase of ICAM up to 24 hrs post sonication. We also detected a >4 fold greater (p < 0.05) CD68 positive cells on day 6 post sonication containing intracellular fluorescent beads within the pFUS + MB treated hemisphere compared to contralateral hemisphere.
Conclusions
The temporal molecular response to pFUS + MB is indicative of SIR2 originating from the parenchyma. The pattern of cytokines immediately after pFUS + MB is initiated by cellular release of DAMPs and TNFa observed with mild cerebral trauma or ischemia [1,2]. Increases in monocyte chemoattractant protein (MCP-1), vascular endothelial growth factors (VEGF), stromal derived factor 1 (SDF-1), erythropoietin (EPO) and brain derived neurotropic factor (BDNF) are associated with BBBD stimulating angiogenesis, neurogenesis and stem cell migration observed with ischemia and trauma. These results indicate that pFUS + MB rapidly affects the cerebral vasculature as evident by BBBD in addition to the shockwave from MB collapse that induces mild stress within various cellular elements in the parenchyma inducing a SIR.
References
1. Chen, G. Y., et al. 2010 Nat Rev Immunol 10(12):826-37.
2. Gadani, S. P., et al. 2015 Neuron 87(1):47-62.
pFUS + MB elicits a transient microenvironmental response in the brain that reflects a sterile inflammation
A13 Volumetric MR thermometry in a clinical transcranial MR guided Focused Ultrasound system
Henrik Odéen1, George Chiou2,3, John Snell4, Nick Todd2, Bruno Madore2,3, Dennis Parker1
1University of Utah, Salt Lake City, Utah, USA; 2Brigham and Women’s Hospital, Boston, Massachusetts, USA; 3Harvard Medical School, Boston, Massachusetts, USA; 4Focused Ultrasound Foundation, Charlottesville, Virginia, USA
Objectives
Transcranial MR-guided Focused Ultrasound (tcMRgFUS) applied within a small central brain volume has achieved excellent outcomes in treatment of movement disorders (Elias NEJM 2013). Although transducers used in tcMRgFUS have been designed with large apertures to spread the beam energy over as much skull as possible to reduce skull and cortex heating, currently used MR temperature imaging (MRTI) methods cannot monitor the temperature increase over the entire insonified brain volume. Instead, temperatures are typically measured in one 2D slice, leaving the majority of the insonified brain volume unmonitored. We have previously developed and published methods to achieve fully 3D MRTI covering the entire insonified brain with good spatial and temporal resolution (Todd MRM 2009/2010) but the techniques have not been evaluated on clinical tcMRgFUS systems. In this work-in-progress study we demonstrate the value of volumetric MRTI with two different pulse sequences applied during heating on a clinical tcMRgFUS system.
Methods
PRF MRTI was performed with a product 3D gradient recalled echo (GRE) pulse sequence and a custom-implemented 3D GRE segmented echo planar imaging (EPI) pulse sequence on a 3 T MRI (Discovery 750 T, GEMS). tcFUS heatings were performed in an ex vivo human skull filled with tissue-mimicking gel (ATS Laboratories) in a clinical tcMRgFUS system (ExAblate Neuro, Insightec).
Pulse sequences parameters are listed in Table 1. All data were zero filled interpolated (ZFI) to 1-mm isotropic spacing (GRE data additionally ZFI to 0.5-mm spacing). The skull was physically positioned in the FUS system, and the focus electronically steered, to target deep brain-structures located outside the normal treatment envelope. FUS sonications were applied at 940 W for 30/60 s while imaging with the GRE/EPI sequences, respectively. The EPI sequence used an echotrain length of 16 with bi-polar readout gradient, sampling 192 phase encodings in each direction, so that full “positive only” and “negative only” images could be reconstructed.
k-space data were retrospectively down sampled by a factor of R = 4 and 8 (split into multiple time-frames without throwing any data away) for the GRE and EPI experiments, respectively, giving acquisitions times of 3.6 and 7.8 s, and reconstructed with a temporally constrained reconstruction algorithm (Todd MRM 2009).
Results
Figure