Gerald J. & Dorothy R. Freidman Foundation Symposium 2008 FUS-mediated reversible modulation of region-specific brain function in animal model Jong-Hwan Lee*, Yongzhi Zhang*, Wonhye Lee*, Krisztina Fischer*, Alexander Bystritsky*, Nathan McDannold*, and Ferenc A. Jolesz*, and Seung-Schik Yoo*, *Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School Presented to Gerald J. & Dorothy R. Freidman Foundation Symposium 2008

Reversible and non-invasive regional modulation of cortical function Needed for short-term/long-term modulation of the brain function. Ultimately, it may provide non-pharmaceutical intervention of appetite control ! Transcranial magnetic stimulation (TMS) suffers from limited penetration depth and lacks spatial specificity. Transcranial direct current stimulation (tDCS) lacks spatial specificity. Deep brain stimulation (DBS) is invasive. www.biomag.hus.fi/tms/Thesis/dt.html The main objective of the study was to examine if it is possible to induce reversible regional modulation of cortical function non-invasively.

Idea: Pulsed FUS (FUP) Motivation and Earlier work Things to avoid; Trans-skull delivery of FUS (Hynynen et al. 2004) Reversible modulation of activity in ex vivo brain tissue (Bachtold et al. 1998) Previous works on animal sciatic nerves. Things to avoid; Irreversible tissue damage or seizure induction. BBB disruption. Excessive heating. Proposed method: Instead of continuous application of HIFU, apply the low intensity FUS stimulation with sufficient inter-stimulation intervals as a train of pulses. Motivation of the work is indeed started from the earlier work. First, trans-skull deliver of FUS was demonstrated by Hynynen and colleagues at BWH Second, the reversible modulation of activity in ex vivo hippocampal brain tissue has been demonstrated. And more importantly, the potential modulation of evoked potential on animal sciatic nerves indicated that FUS can modulate the neuronal function; however, the state of the brain function that are modulated by the FUS, in vivo, has not been demonstrated. Given that, we had to avoid ….

Initial excursion: Real-time fMRI during FUP Why real-time fMRI? To monitor the blood-oxygenated-level-dependent (BOLD) MRI signals that are associated with the activation in real-time, non-invasively. Allows the adjustment of the FUP parameters on-fly. No stimulation stimulation 21 s 12 s T-1 signal equl A Detected activation at p<0.001 level and approximate location of the FUP application in yellow circle. Although we originally proposed to use the ex vivo brain tissue to examine the range of parameters to be used for the experiment, we decided to go right ahead to animal study. And using real-time fMRI helped us to conduct this initial excursion. rtfMRI can monitor the BOLD fMRI signal that are associated with the activation in real-time, non-invasively. Thus allows the adjustment of FUP parameters on-fly. This figures shows one of the initial example from our study. Block-based fMRI stimulation paradigm was used to detect the rabbits visual cortex induced by the light stimulaiton. Detected activation at p<0.001 level and approximate location of the FUP application in yellow circle. Then using real-time fMRI, the BOLD signal that are originating from the brain area of interest was monitored, in regard to the duration of the given FUP parameters. This slide shows the example of the such method. Where the period between the green lines indicate the duration of visual stimulation. The rtfMRI allowed us to change different FUP parameters and it is evident that BOLD signal is reduced upon the FUP while the activity seems to recovered after about 5 second of stimuation. 50 W/cm2 Recovery TRIAL EXAMPLE No stimulation stimulation 15 s 45s 12 s T-1 signal equl 15s FUP application and intensity adjustment

Experimental flow Spotting of the FU ‘hot spot’ using phantom 690 KHz ultrasound transducer with 8 cm focal depth Anatomical localization Functional localization via fMRI BOLD-sensitive EPI sequence to detect level of activation Adjustment of positioning system Application of FUP with fMRI fMRI without FUP to monitor MRI Thermometry Fast SPGR sequence; phase Dependent thermometry [Group#1] Perfusion of Trypan blue to examine BBB disruption, followed by the histological analysis. [Group #2] Survival for 1 week with behavior observation, followed by the histological analysis Transducer Ultrasound beam Water tank Water bag Rabbit Skull Plastic plate MRI Coil Brain Sonication target Positioning system Control Computer Function Generator Amplifier Power Meter MRI Room Matching Network This slide shows the experimental flow First, this figure illustrates the experimental setup where the transducer with 8cm focal depth was used to sonicate the rabbit brain in the magnet. the FUS hot spot was spotted using the phantom via continuous sonication at 400W/cm2. Then the rabbit underwent anatomical scan, and fMRI was used to locate the visual areas. After locating the activation, the positioning stage was used to move the FUP focus to the site of visual area. FUP was applied and fMRI was used to monitor the state of brain activity with and without the FUP. We have two groups of animals, one was sacrificed immediately after the experiment and infused with trypan blue to examine the presence of BBB disruption and to examine any tissue damage. The other group was allowed to survive the FUP application and examined about 1 week later with behavior observation followed by the histological analysis.

Experimental setup 3T MRI rtfMRI Processor MRI Console Pulse Regulator Pulse Monitor Power Monitor Function Generator Power Amplifier

Event-related response and FUP In order to capture the effect of FUP during short-trial based visual stimulation, event-related fMRI design was adapted. FUP (500 μs duration and 10 msec inter-pulse-interval) at 50W/cm2 intensity was applied for 9, 18, and 27 sec. Temporal progression of activation No stimulation stimulation 15 s 9 s 12 s 45 s FUP application L R C PreFUP FUP BOLD Contrast (%) measured from each session 3min In order to capture the effect of FUP during short-trial based visual stimulation, event-related fMRI design was adapted. FUP (500 μs duration and 10 msec inter-pulse-interval) at 50W/cm2 intensity was applied for 9, 18, and 27 sec, that are synchronized with the duration of the visual stimulation. The BOLD signal amplitude was calculated along with the state of activation using general linear model. The figures show the example obtained from the 9s FUP stimulation and recovery after the FUP. This time, interestingly, we observed slightly elevated signal level during FUP. Immediately after the FUP the BOLD signal was reduced and slowly recovered more than 10 min time period. 5min 7min 11min

Presence of BBB disruption or tissue damage? Trypan Blue perfusion after FUP (within 30min) showed no apparent BBB disruption. Histological analysis (H&E stain) showed no tissue damage even at 350W/cm2 pulse (10 msec inter-pulse-interval) application of the FUP for 27 sec. Survival experiment showed normal post-FUP animal behavior and normal histology results. McDannold et al. BWH, PNAS 2007 A B C D E

MR Thermometry No temperature rise during 27s application of FUP at 500 μs duration and 10 msec inter-pulse-interval at 50W/cm2 intensity. – Virtually no temperature change Even at higher level of energy at 350W/cm2 intensity, only 0.95 C rise of temperature was detected after 27s FUP application. 27s 18s 9s We were motivated to examine the presence of any temperature modulation, which have been implicated in previous studies on ex vivo studies. MRI thermometry was used, and as you can see, there was no alternation of the temperature, which may rule out the temperature-sensitive modulation of the observed effects

Conclusions Reversible modulation of region-specific brain function was demonstrated via pulsed application of focused ultrasound energy. The given FUP parameter appears to be safe to be used, both short-term and long-term. The BOLD signal showed that the visual-stimulation-induced activation was enhanced during the FUP application, followed by the period of non-responsive stages, which suggest the ‘relative refractory period’.– More confirmatory study is needed using different stimulation parameters. Potential mechanism ? Temperature-mediation was ruled out. Possibly via mechanical modulation. Electrophysiological confirmation will be followed.

Future Direction Application of the FUP to modulate neural circuitries in hypothalamus (targeting ventromedial nucleus), and subsequent appetite control. Testing on obese ob/ob Leptin deficient mice Effect of down-regulation ? Effect of up-regulation ? Challenges Size of region-of-interest in mouse is small. Potential induction of modulation in other neuro-endocrine systems. From Harlan catalogue

Acknowledgement Magdalini C. Pilatou, Ph.D. Lisa Treat, M.S. Jason White, Ph.D. and with the financial support from Gerald J. & Dorothy R. Freidman Foundation Focused Ultrasound Surgery Foundation Bystritsky Family Foundation , and all anonymous rabbits… Disclaimer: This rabbit was NOT used in the experiment