Neuro-regeneration by ultrasonic activation of neural stem cells using a novel MRI guided focused ultrasound system

Abstract

Embodiments disclose systems and apparatus that provide neurogenesis activation by FUS stimulation including activating quiescent NSCs in the subventricular zone of the lateral ventricles and promoting the proliferation of NSCs and subsequent differentiation into newly regenerated neurons. Also disclosed is a conventional MRI guided tFUS system programmed for use with ultrasonic activation of NSCs. A wireless electronic driving configuration for transcranial use under MRI guidance is provided and a cylindrically distributed ultrasound transducer array design to provide increased steering range in at least two axes. A close integration of tFUS with MRI navigation, achieved by a novel wearable thin-film MRI coil array tightly integrated with the transducer array, enables full navigation performance of MRI in real time during FUS treatment.

Description

Docket No.102489.0002PCT -1- NEURO-REGENERATION BY ULTRASONIC ACTIVATION OF NEURAL STEM CELLS USING A NOVEL MRI GUIDED FOCUSED ULTRASOUND SYSTEM CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority on U.S. Application No.18 / 828,341 filed September 9, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] This invention relates to magnetic resonance imaging (MRI) guided transcranial focused ultrasound (tFUS) therapeutic systems and methods, and more specifically to an innovative technology of ultrasonic activation of neural stem cells to replenish damaged neurons for the treatment of various neural disorders. This invention is also related to a novel wireless FUS system configuration under MRI guidance, in a tight integration of a thin-film MRI coil array with a cylindrically distributed tFUS transducer array, for transcranial therapeutic applications ranging from oncological as well as functional ablations with higher intensity to neuromodulations with lower intensity. BACKGROUND OF THE INVENTION

[0003] High intensity focused ultrasound (HIFU) had been proposed and demonstrated as early as in 1957 to deliver focused ultrasound energy from outside of the body to a focal spot in the targeted area inside the body for thermal treatment. The temperature of the target lesion can be raised to above 60 degrees Celsius (℃) in a few seconds causing tissue to undergo necrosis. Thus, HIFU has been used as one of several thermal ablation modalities for various tumors, even though it was initially applied to brain for neurological disorders. Compared to other conventional surgical operations, HIFU offers a minimum invasive, outpatient alternative with no incision or exposure to ionization radiation, minimum side effect and thus fast recovery. However, the applications of HIFU had not been widely accepted clinically until the use of Magnetic Resonance Imaging (MRI) to guide and monitor tissue temperature and degree of damage.

[0004] Additionally, in recent years, focused ultrasound (FUS), under MRI guidance, has been used to treat various neurological and psychiatric disorders in a transcranial 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -2- focused ultrasound (tFUS) configuration. Placed on top of the patient’s head, such a tFUS transducer device is equipped with multiple transducer elements, distributed over the surface of a hemispherical shape, transmitting multiple ultrasound beams through a water bath to penetrate the skull. The phase or delay time of the multiple transducer elements can be adjusted in various ways to enable the multiple ultrasound beams to focus on a pre- defined set of successive focal spots of pre-defined size to cause biological effect thermally or acoustically. An MRI guided tFUS system was approved by FDA for the treatment of essential tremor in 2016, and Parkinson’s disease with main symptom of tremor in 2018. Such a tFUS system directs the focused ultrasonic beams to a tiny target spot in the brain called ventral intermediate nucleus (VIM) of the thalamus and raises the temperature to above 60 degrees centigrade to remove aberrant synaptic junctions in the brain fiber circuitry through functional ablation.

[0005] The system was developed initially for high intensity focused ultrasound (HIFU) brain tumor surgery in which ultrasound is used to ablate irreversibly to cause necrosis on pathologic tissue. The ultrasonic intensity employed for tumor ablation is in the range from 10W / cm2to as high as 10,000W / cm2. Similar energy level is used for functional ablation described above for treating essential tremor. Another milder application was to apply focused ultrasound to disrupt blood brain barrier (BBB) for improved drug delivery in the brain. It is important to attribute certain technological breakthroughs in the development of methods for accurate focusing of ultrasound waves down to mm scale through the skull using extensive numerical simulation. In summary, in order to pass ultrasound beams through the skull structure with high acoustic reflectance and focus on the target over a finite focal spot, a combination of lower ultrasonic frequency, significant number of independently driven transducer elements, and numerical simulation based on known cranium structure and composition must be employed together.

[0006] The advancements in tFUS technology for higher intensity applications such as ablation and functional ablation, in ultrasound phased array systems and associated numerical targeting and beamforming algorithms to accurately and non-invasively target millimeter-scale neural structures within human cranium, have also paved way for a broader research activity in recent years in various neuromodulation experiments, in which, much lower ultrasonic intensity is used. The so-called low intensity focused ultrasound (LOFU) employs an intensity less than 10W / cm2, lower by two to three orders of magnitude than HIFU. It is widely expected these targeting solutions can be readily applied for 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -3- neuromodulation to achieve similar accuracy and resolution by attenuating the transmit power and adjusting the waveform to achieve a desired non-invasive and spatially confined effect on both deep and superficial neural structures. While underlying biophysics continues to be studied for possible mechanisms of action, empirical results have demonstrated causal neural stimulation and inhibition using LOFU.

[0007] Specifically, aging populations not only suffer from many diseases (e.g., dementia) but also cause huge social impacts. Both current situations and future trends of aging directly concern all of us. The most common form of dementia is Alzheimer’s disease (AD), contributing to up to ~70% of cases. According to WHO, dementia is a syndrome in which deterioration in cognitive functions occurs on patients beyond what is expected from usual biological aging. It affects memory, thinking, orientation, comprehension, calculation, learning capacity, language and judgement. To help the journey of healthy aging, novel regenerative medicine is necessary to deal with these health issues. The goal of regenerative medicine is to restore the functionality of tissues or organs damaged by aging. In this study, five groups of experiments are performed and compared the cell numbers of different cell lineages after traumatic brain injury (TBI) and / or focused ultrasound (FUS) stimulation.

[0008] The impairment in AD is commonly accompanied or preceded by changes in mood, emotional control, behavior, or motivation. It has physical, psychological, social and economic impacts, not only for patients but also for their caretakers, families and society at large. The last AD drug development pipeline showed that disease-modifying therapies (DMT) are currently the most frequently investigated agents, being 82.5% of the total number of considered agents. The meta-analysis of PET-SUVR showed an overall significant effect of monoclonal antibodies (mAbs) in reducing amyloid (SMD –0.88), but when considering clinical efficacy, data on CDR-SB showed that treated patients had a clinically non-relevant lower worsening (MD –0.15). PET-SUVR stands for the Standardized uptake value ratio (SUVR) of PET scanning. The clinical dementia rating (CDR) scale is commonly used to diagnose dementia due to AD. The sum of boxes of the CDR (CDR-SB) has been applied for tracing the progression of cognitive impairment in the early stages of AD. There is currently no treatment available to cure or prevent dementia. Anti-dementia medicines and DMT developed to date have limited efficacy and are primarily labeled for AD, while numerous new treatments are being investigated in various stages of clinical trials. Most efforts have been put into supporting and improving 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -4- the lives of patients and their caretakers and families, including early diagnosis, optimal management, and controlling known risk factors for prevention.

[0009] AD is a progressive neurodegenerative disorder with a poor prognosis. AD is associated with dysregulation of several biologic pathways due to genetic, epigenetic, and environmental causes. The main culprits of AD are the accumulation of amyloid-beta peptides (Abeta) into amyloid plaques. In multiple AD research models, aggregated Abeta damages neurons and disrupts neurotransmission, induces neuroinflammation, and promotes neurodegeneration. Other aggregates like Tau and TAR DNA-binding protein 43 (TDP43) could result in similar damaged neurons in AD / dementia. Therefore, the therapy to prevent or effectively treat AD might be targeting these aggregates using mAbs for Abeta, Tau, or TDP43. However, clinical trials using the mAbs approach have been discouraging. The most recent trial using lecanemab, although moderately slowing the deterioration rate, was also associated with adverse events. It is most likely that the neurons in the AD brains are irreversibly damaged by the aggregates, thus dissolving or neutralizing the aggregates might not be the most favorable approach.

[0010] Adult neurogenesis persists in the mammalian brain throughout life, occurring mainly in two regions: the subventricular zone (SVZ) of lateral ventricles (LV) and the dentate gyrus (DG) of hippocampus. Although neurogenesis declines in the adult brain, it can be marginally stimulated in response to ischemia and trauma. Since the endogenously regenerative capacity of damaged brain is limited, enhancement of its quiescent regenerative potential presents a goal as a therapeutic option for neural diseases. Therapeutic effects of neural stem cells (NSCs) for damaged neurons such as cerebral infarction, spinal cord injury, and sciatic nerve injury have been reported. This invention has shown that the brain-specific FGF1 gene promoter is active in the SVZ of cerebral ventricles. Further, it is confirmed that intranasal delivery of nanoparticle-encapsulated FGF1 can restore cognitive, memory, and learning function of the APP-PSEN1 transgenic mice of the AD animal model.

[0011] Several clinical studies have shown the promise of stem cells (SCs), including MSCs and NSCs, in repairing neurological damage. The use of SC-derived exosomes for therapy has also been contemplated. However, SCs also present challenges such as availability of SCs, complications and compatibility of cell implantation and the possibility of ectopic tissue formation. Consequently, noninvasive methods such as transcranial 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -5- focused ultrasound (FUS) may reduce many of the safety concerns related to the use of SCs, and other reagents such as exosomes and growth factors that require injection. Accumulating studies indicated ultrasound therapy is a safe and efficient treatment. The ultrasound therapy is widely applied in models of traumatic brain injuries [Controlled cortical impact (Su et al., 2017a, b) and ischemic stroke (Chen et al., 2018; Ichijo et al., 2021)] and neurodegeneration diseases [Dementia (Eguchi et al., 2018) and AD (Lin et al., 2015)]. The study showed ultrasound stimulation protective effects through the enhancement of neurotrophic factors in brain disorders. Ultrasound could further reduce apoptosis and inflammation, promote neuroregeneration, and restore behavioral activities on aluminum-induced cerebral damages in rats (Lin et al., 2015). SUMMARY OF THE INVENTION

[0012] This invention discloses new methods applied to significantly increase the numbers of NSCs and proliferating young neurons by administrating FUS in the lateral ventricles (LV) of mouse brains. This invention demonstrated that the FUS stimulation on AD mice improves their cognitive and memory / learning capabilities when analyzed using Novel Object Recognition Test (NORT) and Morris water maze (MWM) analyses. The approach disclosed in this invention is a non-invasive and effective way to replenish the damaged neurons. Studies administering FUS onto the hippocampus, the area of the brain where cognition, spatial learning and memory occur, of AD mice demonstrated increased regenerated neurons and improved cognition and memory / learning capacities.

[0013] While the above invention of activation of NSC is administered using a set of specific FUS parameters in ultrasound frequency, pulse repetition frequency, duty cycle and intensity, on tFUS systems that are programmed to perform the combination of FUS parameters, a novel MRI guided tFUS is disclosed in this invention that employs a cylindrically distributed transducer array, which is tightly integrated with a wearable thin- film MRI coil array, and a FUS system that is driven by a wireless or cableless multi- channel electronic driving scheme. The system so disclosed is further equipped with high resolution brain fiber pathway structure MRI for targeting and follow-up assessment, as well as high spatial / temporal / thermometry resolution for real-time localization and monitoring. The system is further able to program beam-forming control algorithms to produce low level acoustic focus coverage over a pre-defined treatment region. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -6-

[0014] This invention discloses the proliferating cells (BrdU+ cells) were increased after traumatic brain injury (TBI); however, young neurons (DCX+ cells) were significantly decreased. Furthermore, the number of young neurons could be enhanced by FUS stimulation for 1 day or 4 days after TBI, in a cumulative manner. In addition, even without TBI, the cell numbers of BrdU+ cells, DCX+ cells and BrdU+ / DCX+ cells were dramatically increased after FUS stimulation for 4 days. The results indicated the neurogenesis was activated by FUS stimulation. In addition, it is demonstrated that FUS could enhance the increase of F1B-GFP+ neural stem cells (NSCs). Our results demonstrate that FUS could activate quiescent NSCs in the subventricular zone of the lateral ventricles and promote the proliferation of NSCs and subsequent differentiation into newly regenerated neurons. Further, it is demonstrated that FUS could improve the memory / learning capabilities in both undamaged, healthy mice and Camk2A / DTA Alzheimer’s disease mice. This technique could help the rejuvenation of damaged neurons not only in neurodegenerative diseases, including Dementia, Mild Cognitive Impairment, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and multiple sclerosis, but also in other neural diseases, including stroke, traumatic brain injury, concussion and spinal cord injury. It might also be able to enhance the cognition and memory / learning skills of healthy individuals.

[0015] Furthermore, this invention has disclosed a conventional MRI guided tFUS system, designed for higher-intensity oncological and functional ablation, can be programmed for use with the disclosed invention of ultrasonic activation of NSCs for the treatment of various neurodegenerative diseases. In view of a lower-intensity application of FUS, this invention discloses a novel wireless electronic FUS driving configuration under MRI guidance for, but not limited to, transcranial use. In addition, this invention discloses a cylindrically distributed ultrasound transducer array design to provide increased steering range in at least two axes. Further, a close integration of tFUS with the MRI navigation, achieved with a novel wearable thin-film MRI coil array tightly integrated with the transducer array, enables full navigation performance of MRI in real time during FUS treatment to meet higher demand in thermometry resolution. Also disclosed is the use of mathematic techniques to derive beamforming control signals to yield an ultrasound focusing target with arbitrary shape or size within a pre-defined treatment region coverage. BRIEF DESCRIPTION OF THE DRAWINGS 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -7-

[0016] Various embodiments are described and illustrated herein with reference to the drawing in which like items are indicated by the same reference numeral, and in which:

[0017] FIG.1 shows a system architectural configuration of a conventional MRI guided FUS system

[0018] FIG.2 shows a system architectural configuration of a new and improved MRI guided wireless FUS system.

[0019] FIG.3A illustrates a cylindrically distributed tFUS transducer configuration.

[0020] FIG. 3B illustrates a cylindrically distributed tFUS transducer housed in an enclosure.

[0021] FIG. 4A illustrates the translational mechanical degree of freedom in one axis on an exemplary sliding mechanism.

[0022] FIG. 4B illustrates the rotational degree of freedom pivoting in one axis (patient’s left-right) for the cylindrically distributed tFUS transducer array relative to patient’s head wearing the thin-film MRI head coil array.

[0023] FIG.5 illustrates the Cylindrically Distributed tFUS Transducer Array Tightly Integrated in the MRI Guided tFUS System with A Thin-film MRI Coil Array.

[0024] FIGS.6A-6D illustrate the transcranial ultrasound stimulated the LVs of mice.

[0025] FIGS.7A-7B illustrate the safety of FUS stimulation onto mouse brains.

[0026] FIGS. 8A-8C illustrate the needle puncture wounding increased the proliferating cells but decreased the immature neurons.

[0027] FIGS.9A-9C illustrate the BrdU+and DCX+cells were highly increased after FUS stimulation in the injured brain.

[0028] FIGS.10A-10D illustrate FUS enhanced neurogenesis in LVs of aged mice.

[0029] FIG.11 illustrates the scheme of experimental procedures to test the FUS effect in CamK2A / DTA transgenic mice.

[0030] FIGS.12A-12C illustrate the amelioration of AD phenotype in CamK2A / DTA mice following FUS treatment in hippocampus. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -8- DETAILED DESCRIPTION OF THE INVENTION FUS Activation of Neural Stem Cells

[0031] This invention discloses a new treatment methodology for neural degenerative disorders such as Alzheimer disease, traumatic brain injury, and stroke, by using transcranial focused ultrasound to activate quiescent neural stem cells (NSCs) resident in the brain. The ultrasound intensity level used is in the general range of low intensity focused ultrasound, in conjunction with a certain pulse repetition frequency (PRF) and duty cycle, so as not to cause physiologically significant temperature elevation. In principle, an existing tFUS system that can be attenuated to operate in a low intensity range is most likely programmable to use for the disclosed technique of activation of neural stem cells. Tight Integration of MRI and FUS

[0032] In this transcranial configuration, tight integration of MRI head coil array with tFUS transducer device is critical for the demanding treatment precision to take full advantage of MRI capability for image navigation, including targeting, positioning, aiming, real-time thermal monitoring and dose control, immediate prognosis, and follow up. Not only does advanced MRI technology play a pivotal role in targeting defective neural circuitry to be treated in support of the soft tissue contrast provided by usual anatomic structural images, but thermometry and / or acoustic dosage monitoring have also become increasingly demanding in speed and sensitivity, considering the lower acoustic dosage employed and / or resultant physiologically insignificant thermal elevation required in LOFU applications such as our disclosed technology of activation of NSCs.

[0033] However, during treatment, MRI scans can only be performed with either the large size whole body MRI coil or a simple two-loop MRI coil inserted in the water bath of the tFUS transducer device. A typical MRI multiple-channel head coil is itself a helmet like structure and cannot be placed simultaneously with the tFUS transducer helmet. Thus, MRI images may be obtained prior to ultrasound treatment. However, whereas the images used for targeting or treatment planning are acquired with head coil prior to treatment, the actual position of the head during treatment when tFUS transducer is in place could exhibit a mis-registration on the images used for planning. A stereotactic frame is required and anchored onto patient skull to ensure the precision of the treatment target locations. Additionally, once the tFUS transducer is in place, MRI sensitivity can no longer be fully 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -9- realized using body coil or simple two-loop coil let alone full signal-to-noise ratio (SNR) and parallel acceleration performance.

[0034] In a previously filed application, a novel design of a wearable thin-film MRI coil array was disclosed that can be tightly integrated with a typical tFUS transducer device, enabling full MRI performance to be utilized for real-time tFUS navigation. Wireless FUS System Configuration

[0035] A typical FUS system consists of a control unit to set up beamforming parameters for ultrasonic signal generation as well as pulse parameters appropriate for the planned FUS treatment protocol, a high frequency signal generator, a pulse modulator to shape the ultrasonic driving pulse, a power amplification unit to drive necessary ultrasonic intensity, coaxial cables connecting, through impedance matching circuit, to the transducer or transducer array, all above repeated in multiple channels. FIG.1 shows one exemplary configuration of a conventional MRI guided FUS system, in which, major hardware subsystems or modules can be summarized to perform corresponding software or system functions as below.

[0036] As shown in FIG.1, an FUS Console 100 consists of a CPU with computation capability to provide a user interface module 101 (software) to allow the operator to control the system and manage the overall operation of the FUS system. The user interface module 101 is facilitated with a “graphic interface” for the operator to view and interact using graphic tools on the acquired MRI localization images, imported from the MRI Console 110, to facilitate a “treatment planning functionality” to determine the treatment parameters such as target location, size, and desired treatment intensity. The user interface module 101 outputs the treatment parameters to a beamforming processing module 102 resident in the FUS Console to generate precise control signals, including but not limited to frequency, time delay or phase shift, and amplitude, channel by channel, to drive individual Transducer Elements 106 in the tFUS ultrasound transducer array, via a beamforming controller unit 103 located in the hardware cabinet, so as to shape and steer the ultrasound beams to focus on the desired target location. At the same time, treatment parameters are input to a power control module 103 to generate power control signals to the power amplification and / or attenuation unit 105 for each channel, which in turns drives each transducer element 106 to ensure the power levels are set to transmit ultrasound waves with desired intensity for the prescribed treatment methodology while maintaining safety limits. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -10-

[0037] Once the treatment starts, the FUS Console sends navigation imaging commands to the MRI Console 110 to synchronize the thermal or acoustic monitoring scans with the treatment protocol and to import the images to the FUS Console in pre-determined real-time time intervals. The FUS Console is further facilitated with a “real-time thermal monitoring module” that processes the image data of the monitoring scans and display the MRI thermal or acoustic maps fused onto the localization images used for planning for real- time treatment monitoring. Based on the monitoring data available during treatment, the user interface module 101 further enables the operator to make change or adjustment of the treatment parameters and to pause or stop the treatment accordingly.

[0038] As shown in FIG.1, the control signals for beamforming and power, generated in the FUS Console 100, are sent to the FUS Beamforming Controller unit 104, which is typically a microprocessor or digital signal processor (DSP) furnished with “control algorithms” to enable real-time processing and feedback. The Beamforming Controller 104 further translates control signals into time dependent and channel specific input to the multi-channel Signal Generators 105 to generate pulse modulated radio-frequency signal for each channel at the desired ultrasound frequency. The Signal Generator 105 is in turn connected to the Power Amplifier or Attenuator unit 106 channel by channel. The output FUS driving signals are connected to co-axial cables 108, through the Filter Panel 121 installed on the MRI shielding room 120, to individual Transducer Elements distributed 107 in the FUS transducer device. When tFUS system is integrated within an MRI navigation system for much desired real-time image guidance to ensure treatment safety and precision, cable connections through RF shield room often become one major source of cross interference and large number of connections for large number of channels further causing great technical difficulty and increased cost.

[0039] Considering the application of tFUS toward the activation of NSCs and / or various neuromodulation procedures, a novel system design is disclosed that takes advantage of the lower intensity required. Furthermore, a wireless or cableless design, as shown in FIG. 2, is disclosed in which the electronic signal generation and amplification components 206, together with a certain micro-processor manager 205, are fabricated inside the enclosure of the transducer, or populated on the back of the transducer elements as one exemplary embodiment, wherein the FUS control signals are transmitted via off-the- shelf wire-less signal format device 203, such as but not limited to WIFI or IrDA (Infrared Data Association), and received on the transducer end by corresponding transceiver device 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -11- 204 before relaying to the Micro-processor. The reciprocal feedback signals can be conveyed in the reversed direction along the same wire-less path. In one exemplary embodiment, the signal generation and amplification components can be selected from off- the-shelf FPGA, Piezo Driver, DDS (direct digital synthesis) devices or chips. In another, a multi-channel pulser with integrated beamformer channel can be used. Cylindrical FUS Transducer Array

[0040] Traditional tFUS transducer elements are fabricated to distribute over the inner surface of a hemi-spherical geometry. The steering range of the focal spot is normally quite limited around the natural focus location even though the focus accuracy can achieve mm scale. This invention discloses herein a transcranial FUS transducer array with elements distributed over the inner surface of a cylindrical 3D geometric shape, that, based on numerical simulation, can achieve increased steering range electronically in at least two axes. FIG.3 shows an exemplary design of a cylindrical tFUS transducer array, a) with and b) without enclosure, with treatment target locations of hippocampus and lateral ventricle indicated in the brain for NSCs activation. Such a transducer array comprises up to 1024 elements or more depending on the focusing requirement. In addition to electronic beamforming degrees of freedom, Mechanical positioning device with degree of translational freedom at least in the long axis, as shown in FIG. 4A, and that for tilt or swivel about one or two of the azimuthal directions as shown in FIG.4B, may be devised to provide up to six degrees of mechanical freedom in steering the focal spot in addition to electronic steering with phase shift or time delay. FIG. 5 shows the integration of a cylindrically distributed transducer array in the MRI guided tFUS system with a thin-film MRI coil array.

[0041] The mathematic problem of ultrasonic wave deflection, absorption, or mode transformation upon traveling through mostly the side of the human skull, instead of the top, is formatted in the cylindrical coordinates system and solved with reasonably reduced difficulty. A method is disclosed herein to apply time-reversal mathematic technique to derive phase shift or time delay beamforming control to the multiple transducer elements to produce low level acoustic focus coverage over a pre-defined target region, e.g., the ventricle, larger than the otherwise sharp focal spot. This is to compare with applying FUS on one focal spot at a time and moving to adjacent spots to cover the intended target region. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -12-

[0042] In a preferred embodiment, this invention discloses a treatment method for neural degenerative disorders such as, but not limited to, Alzheimer disease, traumatic brain injury, and stroke, comprising steps of using a transcranial focused ultrasound to activate quiescent neural stem cells (NSCs) resident in a human brain, wherein the ultrasound intensity level employed is in a general range of low intensity focused ultrasound with a certain pulse repetition frequency (PRF) and duty cycle, and without producing physiologically significant temperature elevation.

[0043] In another preferred embodiment, this invention discloses a treatment method of neural degenerative disorders such as, but not limited to, Alzheimer disease, traumatic brain injury, and stroke, with a step of using the transcranial focused ultrasound that further includes a step of employing an existing tFUS system (FIG. 1) with a programmable attenuation for operating in low intensity range for activating the neural stem cells.

[0044] In another preferred embodiment, this invention discloses a treatment method of neural degenerative disorders such as, but not limited to, Alzheimer disease, traumatic brain injury, and stroke, with a step of using the transcranial focused ultrasound system and that further includes a step of employing and integrating with an MRI navigation system (FIG. 2), wherein an MRI coil array is tightly integrated with the tFUS transducer array device (FIG.5).

[0045] In another preferred embodiment, this invention discloses a treatment method of neural degenerative disorders such as, but not limited to, Alzheimer disease, traumatic brain injury, and stroke, with a step of using the transcranial focused ultrasound and that further includes a step of employing a novel FUS system configuring with a multi-channel wireless or cable-less driving electronics (FIG.2).

[0046] In another preferred embodiment, this invention discloses a treatment method of neural degenerative disorders such as, but not limited to, Alzheimer disease, traumatic brain injury, and stroke, with a step of using the transcranial focused ultrasound and that further includes a step of employing a transcranial FUS transducer array with elements distributed over an inner surface of a cylindrical 3D geometric shape (FIG. 3A and 3B) based on numerical simulation enabled to achieve increased steering range in at least two axes.

[0047] In another preferred embodiment, this invention discloses a treatment method of neural degenerative disorders such as Alzheimer disease, traumatic brain injury, and 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -13- stroke, with a step of using the transcranial focused ultrasound and further includes a step of applying a time-reversal mathematic technique to derive phase shift or time delay beamforming control to the multiple transducer elements to produce low level acoustic focus coverage over a pre-defined treatment region, e.g., the ventricle, larger than the otherwise sharply focused spot, to be more efficient comparing with application of FUS on one focal spot at a time and moving to adjacent spots to cover an intended target region.

[0048] In another preferred embodiment, this invention discloses a treatment method of neural degenerative disorders such as, but not limited to, Alzheimer disease, traumatic brain injury, and stroke, with a step of using the transcranial focused ultrasound under MRI guidance and further includes a step of applying a magnetic resonance imaging (MRI) receiver coil device. The MRI receiver coil device comprises a) a thin-film substrate layer configured in a dome shape; b) a coil array positioned around a circumference of an exterior surface of the thin-film substrate layer; c) a thin-film cover layer positioned over the exterior surface of the thin-film substrate layer such that the coil array is positioned between the thin-film cover layer and the thin-film substrate layer; and d) an end ring engaging the thin-film substrate layer and the thin-film cover layer such that a watertight seal is formed around the coil array between the thin-film substrate layer, the thin-film cover layer, and the end ring.

[0049] In another preferred embodiment, this invention discloses an apparatus for treatment of neural degenerative disorders such as, but not limited to, Alzheimer disease, traumatic brain injury, and stroke. The device comprises a magnetic resonance imaging (MRI) receiver coil wherein the MRI receiver coil device further comprises a) thin-film coil array wearable over the patient’s head, b) a gel-pad lining inside of the thin-film coil array and tightly fitted over patient head, c) an end-ring with mechanism sealable to the tFUS transducer device, and that the thin-film coil device enables the ultrasonic beams emitted by the transducer array to go through the thin-film coil and enter patient’s skull without much impeding or attenuation.

[0050] Material and Methods-Animals: Different animal tests using various materials are conducted to confirm the effectiveness of the therapeutic methods disclosed in this invention. All animal experiments were performed in accordance with the animal protocol approved by the Institutional Animal Care and Use Committee of the National Health Research Institutes. F1B-GFP transgenic mice were on FVB background and generated in 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -14- our lab, which expressed GFP reporter under control of human F1B promoter as described previously (Chen et al., 2015). The mice were housed in IVC cages with normal diet and freshwater. The light cycle of housing was controlled for 12 hours from 7 AM to 7 PM.

[0051] Focused ultrasound (FUS) Transducer: The in-house transducer was made of a spherical bowl piezoceramic with an aperture diameter of 20 mm and the focal length of 16 mm. The PZT4 ceramic chips (Ceramic Transducer Design, Taiwan) with resonant frequency of 2.0 MHz were used as the material of the resonator in this study due to its high depoling voltage and low dielectric losses under a high electric drive. The front end of the transducer was a hollow cone coupler, and the cone was filled with degassed water and sealed with a 0.03-mm thick Mylar film. The ellipsoidal focal zone of the transducer was located at a site 2.5 mm beneath the Mylar film, and the focal width and depth were 1.5 mm and 5.9 mm, respectively (FIG. 6A and 6B). The 30-AWG coaxial cables (D1370115BT, Wellshow Technology, Taiwan) were used for electrical connection. The impedance of the transducer was measured by an impedance analyzer (Impedance Analyzers 6500B, Wayne Kerr Electronics, UK). The matching circuits were designed by the software Smith Chart (Smith V3.10, Bern University of Applied Sciences, Switzerland) to fit the requirements of the impedance phase close to 0°, and the impedance magnitude close to 50 Ω at the frequency of 2.0 MHz The transducer was driven by a function generator (33521A, Agilent, Santa Clara, CA, USA) and a radiofrequency power amplifier (1040 L, Electronics and Innovation, Rochester, NY, USA) in all experiments.

[0052] Ultrasound Parameters: Ultrasonic parameters, including a pulsed wave, pulse width of 10 ms, pulse repetition frequency of 10 Hz, duty cycle of 10%, focal spatial-peak pulse-average intensity of 32 mW / cm2, and a sonication time of 5 min, were obtained by controlling the function generator and power amplifier. The acoustic pressure of the intensity was measured by the hydrophone (0.2 mm, Precision Acoustics, Dorset, UK).

[0053] Ultrasound Stimulations: Mice (6-9 months old) were anesthetized by using isoflurane (Halocarbon, USA) and hair was removed using depilatory cream. The mouse and the ultrasonic transducer were fixed on the Stoelting 51500D digital stereotaxic instrument. For non-invasive procedure, bregma was identified by observing the midline between two eyes and the horizontal line 0.5 cm behind the outer corner of the eyes (FIG. 6C). The ultrasound transducer was placed on the scalp and targeted at LV (bregma 0.5 mm 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -15- and lateral 0.75 mm) and driven for 5 min (FIG. 6D). Each mouse was stimulated by ultrasound with the same parameters for 4 consecutive days.

[0054] Referring to FIGS. 6A-6D, the FUS impact areas were shown as yellow oval shapes in coronal section in FIG. 6A and sagittal section in FIG. 6B. FIG. 6C illustrates, for non-invasive ultrasound stimulation, the bregma was identified as the crossing of midline between the two eyes and 0.5 cm behind the outer corner of the eyes (red dot). FIG. 6D illustrates the ultrasound transducer was placed onto the mouse brain.

[0055] Needle puncture Injury: Adult mice were deeply anesthetized, and hair removed using depilatory cream. Before surgery, mice were IM injected ketoprofen 5 mg / kg (Safestar, Taiwan) and Baytril 5mg / kg (Bayer, Germany) to relieve pain and reduce infection. The scalp was sterilized with 70% alcohol and povidone-iodine and cut by scalpels to identify the landmark of the skull. A 28-gauge needle was applied to penetrate the brain into LVs (bregma 0.5 mm, lateral ± 0.75 mm, and deep 3.75 mm) to create a needle puncture injury. Then ultrasound immediately stimulated the injury site for 5 min. After surgery, mice were SC injected 1 ml Coforta (Bayer, Germany) and 1 ml Ringer’s solution (Nang Kuang Pharmaceutical, Taiwan) of each. The mice were put on a heating blanket during recovery after surgery.

[0056] BrdU labeling and tissue Processing: All the mice were IP injected BrdU 50 mg / kg (MedChem Express, USA) for 4 consecutive days. One week after ultrasound stimulation or BrdU labeling, mice were sacrificed. Mice were deeply anesthetized by isoflurane and intracardially perfused with cold PBS, then fixed by 4% paraformaldehyde (PFA, Electron Microscopy Sciences, USA). Brains were fixed in 4% PFA at 4˚C overnight for the postfixation. Free-floating of coronal brain sections (40 ^^m) were sliced using the vibratome (Leica, VT1200S Microtome, Germany) and preserved in 0.5% sodium azide (Sigma, USA) at 4˚C.

[0057] Immunohistochemistry: For BrdU staining, free-floating brain sections were washed three times with 1x PBS for 5 min then incubated in 1 M HCl at 45°C for 45 min. Sections were rinsed in 0.1 M boric acid (pH 8.5) for 10 min and then washed three times with 1x PBS. Incubation of brain sections in primary antibodies: rat anti-BrdU 1:500 (abcam, ab6326), rabbit anti-DCX 1:500 (abcam, ab18723) and goat anti-GFP 1:500 (Rockland, 600-101-215) at 4°C overnight with PBS containing 0.3% Triton X-100. After PBS washing, sections were incubated in secondary antibodies: Alexa 594 donkey anti-rat 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -16- 1:500 (Invitrogen, A21209), Alexa 647 donkey anti-rabbit 1:500 (Jackson ImmunoResearch Laboratories, 711-605-152) and Alexa 488 donkey anti-goat 1:500 (Jackson ImmunoResearch Laboratories, 705-545-147) with 1x PBS / 0.3% Triton X-100, at room temperature for 1 h. Sections were mounted with Fluoroshied mounting medium with DAPI (GeneTex, Taiwan).

[0058] Image and Statistics: To quantify the cell number of LV, three brain sections (bregma: 0.98 mm, 0.5 mm, and 0.14 mm) of each mouse were selected and analyzed. The LVs were photographed using a 63x objective lens of Leica TCS SP5 confocal microscope (Leica, Germany). The Z-stacking images from individual sections were captured every 0.6 mm thick. At least 8 images were recorded and analyzed from each LV. The cell numbers from confocal images were determined by using MetaMorph image analysis software. The positive cells of F1B-GFP, BrdU, DCX and BrdU / DCX were normalized with DAPI to quantify the percentage ratio. All data were presented as mean ± SD. The Student’s t-test was used to analyze the statistical significance of ratios. The p values were shown as < 0.05 (*), < 0.01 (**) and < 0.001(***).

[0059] Camk2A / DTA Alzheimer’s disease transgenic Mice: To confirm the effectiveness of this invention, Camk2a-tTA / tetO-DTA (abb. Camk2a / DTA) mice are generated, which have doxycycline-repressed diphtheria toxin A (DTA) expression under the control of a promoter of Ca2+ / calmodulin-dependent protein kinase II (Camk2a), as described in our publication (Chen et al., 2020). Since the Camk2a promoter is only active in hippocampus, the toxin only will be expressed in hippocampus to cause hippocampal atrophy once doxycycline (Dox) is withdrawn from mouse diet. Our data show that hippocampal atrophy was induced in 6-week-old Camk2a / DTA mouse brains by 30-day Dox-withdrawal and the numbers of neurons, NeuN+-cells, were significantly decreased in the hippocampi. Mice were subjected to behavioral analysis (eg, Novel Object Recognition Test [NORT], Novel Location Recognition Test [NLRT], Morris Water Maze [MWM]) at the time points indicated, and then mice were sacrificed, and the brains were collected for further analysis.

[0060] Animal behavior Analyses: All cognition, learning, and memory tests were performed as described previously (Chen et al., 2020). The numbers of animals for each behavioral group are indicated in the figure legends or Brief Description of Drawings. Each mouse received only one behavioral test per day. In NORT and NLRT, each mouse was 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -17- allowed to explore the objects for 5 minutes (exploratory phase) and then was returned to the cage for another 5 minutes. After the 5-minute interval in the cage, the mouse was returned to (a) the chamber with the previously exposed object and a novel object (NORT) or (b) the chamber in which one of the two objects was displaced from its original position (NLRT), for a 3-minute test phase. Exploration counted as positive if the mouse's head was within one inch of the object with neck extended and vibrissae moving. The exploratory phase and test phase were videotaped to measure (time for exploring novel object or location) / (time for total exploring). In the MWM test, the learning trials were performed at the same time on D1 to D5. The trial began from a different quadrant of the pool for each day. Each trial ended when the mouse arrived at the platform, or after 60 seconds had passed. The mice were immediately removed from the pool at the end of the trial. All tracks from all trials were recorded and analyzed using the Videotrack software (Viewpoint). On day six, the resting platform was removed from the water maize, and the duration mice staying within the quadrant and frequency mice returning to the quadrant were measured.

[0061] Results: The FUS treatment did not affect body weight: In this study, an ultrasound transducer is designed and implemented which created a small focal zone to cover the lateral ventricle (LV) of the mouse brain (FIG.6A - 6B). To examine the safety of this focus ultrasound (FUS) stimulation onto the mouse brain, two groups of mice are set to evaluate the effect on body weight after FUS stimulation. Mice in the FUS group were treated with FUS 5 min for 4 days. The Sham mice were applied with the same anesthesia procedures but without the FUS stimulation (FIG. 7A). As shown in FIG.7B, there was no difference in body weight between the two groups of mice with or without FUS treatment during the four-day treatment period. Further, mice exhibited normal food- intake and behavior after FUS stimulation.

[0062] Referring to FIGS.7A-7B, an illustration of the safety of FUS stimulation onto mouse brains is provided. FIG. 7A illustrates the timeline of FUS treatment. In the FUS group, mice were treated with FUS 5 min for 4 days (in the Sham group, mice were anesthetized by using isoflurane for 20 min without any treatment to mimic the duration of the FUS group anesthetized) and with FIG. 7B, the FUS group showed no difference in body weight compared with the Sham group (the white bars show the body weight of Sham group (without FUS treatment) and the black bars show the body weight of FUS group after being treated with FUS stimulation for 4 days (FUS group), where n = 5 per group). 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -18-

[0063] Results: Brain injury stimulated the increase of proliferating cells but decreased the number of immature neurons: First, the effect is investigated, after trauma, on the microenvironment of brain. To prevent high cell death via traumatic brain injury, the needle puncture into the LVs is applied to create mild brain injury (Akamatsu and Hanafy, 2020). In the group of Injury w / o FUS, 6–9-month-old mice received penetrating brain injuries in both left and right LVs on Day 1. BrdU, a thymidine homolog, can incorporate into the newly synthesized DNA of proliferating cells (Zhang et al., 2001). Doublecortin (DCX) is a young neuron marker (Brown et al., 2003; Zhang and Jiao, 2015). The localization of BrdU- and DCX-positive cells could track the cell fate of neurogenesis. To investigate the development from labeled proliferating cells to young neurons, mice in both the Sham group and the FUS group were rested for one week after BrdU administration (FIG.8A). The BrdU-labeling for four consecutive days is used to track the development of proliferating cells (FIG.8A and 8B). The cell counting results showed that the number of BrdU+cells was increased after the brain suffered the injury of needle puncture (p = 0.0048, FIG.8C). However, the number of DCX+new neuronal cells substantially decreased after brain injury (p = 0.0038, FIG.8C). The neurogenesis index of BrdU+ / DCX+cells was not affected between the two groups. These results demonstrated that brain injury facilitated the increasing of newborn cells, but the newborn cells did not contribute to neuronal development. This invention therefore showed that these newborn cells are largely astroglial cells (data not shown) and are likely to be glial scars.

[0064] FIG. 8A illustrates that, in the Sham group (n =7), mice have received BrdU injection for 4 days and, in the Injury w / o FUS group (n = 8), mice were needle-penetrated into LVs at day 1 and injected BrdU from day 1 to day 4. FIG. 8B provides schematic illustrations of coronal brain sections showing the treatment in the brains. The red lines indicated the needle puncture injuries. FIG. 8C illustrates the percentage of F1B-GFP+, BrdU+, DCX+and BrdU+ / DCX+(B / D) cells after brain injury by needle puncture. White bars indicated the Sham group. Blue bars indicated the Injury w / o FUS group. **p < 0.01.

[0065] Results: FUS stimulation activated quiescent NSCs to become proliferating cells and rejuvenated neurogenesis after brain injury: The next step is to understand whether FUS drives the neurogenesis in the injured mouse brain. The localization of BrdU- and DCX- positive cells could track the cell fate of neurogenesis after ultrasound stimulation. For non-invasive ultrasound stimulation, the bregma was identified as shown in FIG.6C, therefore focal zones might slightly deviate during the 4 days of FUS stimulation (FIG. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -19- 9B). Experiments are conducted to elucidate whether FUS stimulation onto the specific focal zone after brain injury could promote the process of neurogenesis. In the group of Injury + FUS, the FUS focused onto the injury sites right after brain injury for four consecutive days accompanying BrdU administration (FIG. 9A and 9B). In the group of Injury w / o FUS, mice received needle puncture with IP injected BrdU for four days without FUS stimulation. One week after FUS treatment (Injury + FUS group) or BrdU injection (Injury w / o FUS group), mice were sacrificed and IHC was used to investigate the cell numbers and distribution of proliferating cells and immature neurons in vivo. Our results showed injured brains treated with FUS further increased the percentage of F1B-GFP+cells, BrdU+cells, DCX+cells, and BrdU+ / DCX+cells in a statistically significant manner (p = 0.00006, p = 0.000077, p = 0.0000022 and p = 0.000012, respectively; FIG.9C). These results demonstrated FUS focusing on LV could promote neurogenesis progression through increased proliferating cells and driving the development of young neurons in the injured brain.

[0066] Referring now to FIGS.9A-9C, the figures illustrate that the BrdU+and DCX+cells were highly increased after FUS stimulation in the injured brain. FIG.9A illustrates that the mice of the Injury w / o FUS group (n = 8) were administered needle puncture into LVs and injected with BrdU for 4 days, and the group of Injury + FUS (n = 9) was needle penetrated into LVs at day 1 and treated with FUS stimulation from day 1 to day 4. FIG. 9B is schematic illustrations of coronal brain sections showing the treatment in the brain. The red lines indicated the needle puncture injuries. The yellow oval shapes indicated the FUS impact areas. Since the scalps were not opened from Day 2 to Day 4, the FUS impact areas might slightly deviate compared with Day 1. FIG. 9C illustrates the percentage of F1B-GFP+, BrdU+, DCX+and BrdU+ / DCX+(B / D) cells after brain injury with or without FUS treatment. White bars indicated the Injury w / o FUS group. Blue bars indicated the group of Injury + FUS. ***p < 0.001.

[0067] Results: In mice without brain injury, both proliferating NSCs and neurogenesis were enhanced by FUS alone: To evaluate the effect of ultrasound onto the LV and track the neurogenesis in mice without brain injury, mice were used to receive the FUS stimulation for 5 min onto the LVs and BrdU injections during four days of FUS stimulation (FIG.10A). To investigate the development from BrdU-labeled proliferating cells to young neurons, mice in both the Sham group and the FUS group were rested for one week after BrdU administration (FIG.10A). The IHC results showed the accumulation of BrdU+(red), 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -20- DCX+(green), and BrdU+ / DCX+cells expressed in the LVs of aged mice after FUS stimulation for 4 days (FIG. 10C). The high expression of DCX+cells indicated the neurogenesis was activated in mice after ultrasound stimulation (FIG.10C-e and 10D). The statistical results indicated the BrdU+and DCX+cells were significantly increased in the FUS group compared with the Sham group (p = 0.008 and p = 0.039, FIG. 10D). Furthermore, the BrdU+ / DCX+cells were dramatically increased in FUS group (p = 0.00093), showing the neurogenesis in LVs was increased in mice during 4 days FUS stimulation by focusing the impact zone in a specific area that is known to be abundant for NSCs. Most importantly, F1B-GFP+-NSCs did not decrease in number, even though a portion of the NSCs pool was destined to differentiate into newly formed neurons. Thus, it is demonstrated that FUS activated the quiescent NSCs and replenished the pool of NSCs, while the activated NSCs are programmed to differentiate into new neurons.

[0068] Referring now to FIGS. 10A-10D, the figures illustrate FUS enhanced neurogenesis in LVs of aged mice. FIG. 10A illustrates that the mice in the Sham group (n=7) received BrdU injection for 4 days, and in the FUS group (n = 6), mice were treated with FUS 5 min and BrdU injection for 4 days. FIG. 10B is schematic illustrations of coronal brain sections showed the treatment in the brains. The yellow oval shapes indicated the FUS impact areas. Due to non-invasive ultrasound treatment, the FUS impact areas might slightly move during the 4 days stimulations. FIG.10C illustrates the double-labeling IHC of the LVs from Sham mice and FUS mice. Panels a - c, the Sham group. Panels d - f, the FUS group. The BrdU+cells and DCX+cells were represented as red and green, respectively. scale bar = 50 ^^m. LV: lateral ventricle. FIG.10D illustrates the percentage of F1B-GFP+, BrdU+, DCX+and BrdU+ / DCX+(B / D) cells after FUS treatment for 4 days. White bars indicated the Sham group. Yellow bars were the FUS group. *p < 0.05, **p < 0.01, and ***p < 0.001.

[0069] Results: FUS increased the cognitive / learning capacities of the CamK2A / DTA mice that exhibited AD Phenotype: The test results demonstrating that ultrasonic wave with focusing energy and micro-heat could activate the quiescent F1B-GFP+-NSCs in the subventricular zone of F1B-GFP transgenic mice, prompted us to test if FUS could alleviate the AD phenotype of the Camk2A / DTA transgenic mice. In subsequent experiments, similar approaches are carried out to determine the effects of FUS on the amelioration of AD phenotype in Camk2a / DTA mice. Thus, doxycycline was removed from the diet of 6- week-old DTA mice and Camk2a / DTA mice for 30 days, as described in our publication 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -21- (Chen et al., 2020). On the 31st day, doxycycline was returned to the mouse chow. Mice were maintained on doxycycline-enriched chow, except for the 30-day Dox-free period to incur hippocampal atrophy. After the Dox-free period, mice were treated with FUS 5 min for each hippocampus half. BrdU was injected intraperitoneally to label the newly proliferating cells. The experimental procedures were schematized in FIG.11.

[0070] Referring now to FIG. 11, the figure illustrates the scheme of experimental procedures to test the FUS effect in CamK2A / DTA transgenic mice. Doxycycline was removed from the diet of 6-week-old DTA mice and Camk2a / DTA mice for 30 days, as described in our publication (Chen et al., 2021). On the 31st day, doxycycline was returned to the mouse chow. Mice were maintained on doxycycline-enriched chow, except for the 30-day Dox-free period to incur hippocampal atrophy in Camk2a / DTA mice, but not in DTA mice, which served as the undamaged group. After the Dox-free period, mice were treated with FUS 5 min for each hippocampus half. BrdU was injected intraperitoneally to label the newly proliferating cells. Novel Object Recognition Test (NORT), Novel Location Recognition Test (NLRT), and Morris Water Maze (MWM) were tested on the mice as scheduled.

[0071] After the FUS treatment (FUS), both damaged Camk2a / DTA mice (D, doxycycline withdrawn) and undamaged (U, continued feeding doxycycline) mice were subject to behavioral analyses, in comparison with the Control groups (C, without FUS treatment) as described in our publication (Chen et al., 2020). Both Novel object recognition test (NORT) (FIG. 12A) and Morris water maze (MWM) on Day 6 without resting platform (as determined by Duration and Frequency) (FIGS. 12B-12C) could demonstrate that the FUS stimulation on AD mice improves their cognitive and memory / learning capabilities. Our results showed that FUS could improve the memory / learning capabilities in both undamaged, healthy mice and Camk2A / DTA AD mice.

[0072] Referring to FIGS. 12A-12C, the figures illustrate the amelioration of AD phenotype in CamK2A / DTA mice following FUS treatment in hippocampus. FIG. 12A illustrates the results of Novel Object Recognition Test (NORT). After training for five days with the resting platform, the measured duration is shown in FIG.12B and measured frequency in FIG.12C in Morris Water Maze (MWM) on Day 6 without resting platform. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -22-

[0073] This invention applies the discoveries that a cell proliferation in the brain is enhanced in response to ischemia and trauma, even though neurogenesis declines as the animal aged. In this study, a BrdU-labeling is used to evidence that the newborn cells were generated. To recapitulate cell proliferation in traumatic brain injury, needle puncture injuries near the LV were conducted. It is found that the needle puncture wounding led to the increase of proliferating cells (BrdU+) but to the decrease of the cell number of young neurons (DCX+) (FIG. 8C). The results indicated that the most activated-dividing cells caused by injury did not preferentially develop into neurons, implicating that the milieu of brain injury was not beneficial to neurogenesis. It is likely that the BrdU+-proliferating cells detected following brain injury here are astroglial cells resulting in glial scar, which is known to hamper neurogenesis instead.

[0074] This invention discloses a FUS transducer that targeted a small focal zone and stimulated the specific brain area in LV adjacent to puncturing needle wounds to explore if BrdU-labeling newborn cells were increased via ultrasound stimulation. The results showed that 5-minute FUS stimulation each day for four consecutive days after brain injury, significantly increased the newborn neurons 4-fold compared with mice without FUS stimulation (Injury w / o FUS group vs. Injury + FUS group: DCX+cells, 10.0%. vs.40.4%). In addition, the neurogenesis progressing was dramatically improved by ultrasound stimulation more than 4-fold (Injury w / o FUS group vs. Injury + FUS group: BrdU+ / DCX+cells, 2.8% vs. 11.4%) (FIG.9C). FUS treatment for just one day demonstrated a similar trend albeit to a lesser degree (data not shown). The data indicated that the suppression of neurogenesis observed in brain injury was reversed through the activation of neuronal differentiation via ultrasound stimulation. Our results demonstrated this ultrasound device could be safely used onto the aged-mouse brain and promote adult neurogenesis. Moreover, FUS could drive the cell fate of proliferating cells into differentiated neurons in the environment of the damaged brain.

[0075] The test results further demonstrated that FUS alone could increase the number of newly generated young neurons without the challenge of needle puncture wounding. The high number of BrdU+ / DCX+cells showed ultrasound stimulation directly induced the cell fate of newborn cells to differentiate into neurons (Sham group vs. FUS group: BrdU+cells, 8.5% vs.15.0%; DCX+cells, 14.6% vs.20.4%; BrdU+ / DCX+cells, 2.4% vs.7.0%). Thus, the results demonstrated FUS alone without brain injury can improve neurogenesis in the LV nearly 3-fold after 5-minute FUS stimulation each day for four consecutive days. It is 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -23- important to note that, with the recruitment of existing NSCs to differentiate into neurons, the number of F1B-GFP+cells did not decrease but actually increased slightly (FIG. 9C and 10D), demonstrating that the quiescent NSCs are activated and regenerated following FUS stimulation in order to maintain, or slightly increase, the total number of the pool of NSCs.

[0076] FGF1 has been demonstrated to involve neuroprotective function to reduce the lesion size and promote functional recovery in the CNS injury rodent model (Ghazavi et al., 2017; Li et al., 2018; Tsai et al., 2015). Our previous studies showed human FGF1B promoter is activated in the ependymal cells and neurons in F1B-GFP transgenic mouse brain (Chen et al., 2015). It is also showed that these F1B-GFP+ cells are multipotent NSCs, and can functionally repair sciatic nerve injury in rats, mice and mini-pigs. The statistical results showed that F1B-GFP+-cells were more highly expressed in forebrain ventricles (LV, D3V, and 3V) than midbrain (Aq) and hindbrain (4V) ventricles. Importantly, previous studies indicated the quiescent SVZ ependymal cells transiently transformed into neuroblast in response to stroke (Carlen et al., 2009; Gregoire et al., 2015; Zhang et al., 2007; Zhao et al., 2009). Investigation of the cell number and distribution of F1B-GFP+- cells in different treatments found F1B-GFP+-cells were increased in the group of Injury + FUS. However, the process did not find F1B-GFP+ / BrdU+cell or ectopic distribution of F1B-GFP+cells. Therefore, when committing the fate of F1B-GFP+-ependymal cells to neurogenesis, the FGF1B promoter was inactivated. A new method to label and track the F1B-GFP+-ependymal cells will need to be established to identify those cells after transforming. Further study is to cross F1B-Cre transgenic mice and Rosa26 transgenic mice to generate F1B-Cre / Rosa26 double transgenic mice. These F1B-Cre / Rosa26 mice, together with F1B-GFP mice, will enable the tracking of the migratory paths of the newly differentiated neurons, and to substantiate the mechanism of action. Moreover, Notch signaling maintains the integrity of ependymal cells and controls the maturation of neurons that give rise from ependymal cells after stroke (Gregoire et al., 2015; Zhao et al., 2009). This will be likely a key point to further investigate the correlation of Notch signaling, NSCs, and ultrasound stimulation after brain injury.

[0077] Since FUS transducer could directly promote the SVZ neurogenesis in both normal-condition brain and injured brain, subsequent studies are carried out to apply FUS in the animal models of aging-related neurodegeneration diseases, i.e., AD, to more specifically understand the effect of FUS. Here, the Camk2A / DTA transgenic AD mouse 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -24- model is used wherein doxycycline was withdrawn from the chow and diphtheria toxin A was expressed to destroy hippocampal neurons. These transgenic mice, without doxycycline, exhibited hippocampal atrophy and cognitive deficiency. After FUS treatment, NORT, NLRT and MWM were used to gauge the improvement of the cognition. As shown in FIG.12A, it is obvious that NORT is improved when FUS is administered in both the undamaged mice and damaged AD mice. In MWM analyses after five days of training, then the resting platform was removed on Day 6, the AD mice with FUS treatment could improve their learning / memory skill to the level of the healthy mice as reflected by the duration the mice remained in the quadrant where the resting platform once was. Remarkably, when healthy mice were treated with FUS, their learning / memory capabilities are further improved (FIG. 12B). With the frequency test, the healthy mice could further improve their cognition, but the AD mice could not (FIG.12C). Thus, the results showed that FUS could improve the memory / learning capabilities in both undamaged, healthy mice and the Camk2A / DTA AD mice in different ways.

[0078] Further efforts will be continued to establish MRI-guided ultrasound stimulation equipment and preform on the animals and human. Through the MRI-guided ultrasound stimulation, the FUS will be more precisely applied onto specific brain areas, e.g., LV, DG of the hippocampus, hypothalamus, olfactory bulb, or cochlea, in order to determine the FUS effect on neurogenesis in neurodegenerative disease, including but not limited to Dementia, Mild Cognitive Impairment, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis, as well as other neural diseases, including but not limited to stroke, traumatic brain injury, concussion and spinal cord injury. Further, the method disclosed in this invention could be employed as a follow- up or adjunct treatment after surgical and / or interventional procedures for stroke, brain tumors, and other neurological disorders. The method could be developed as a prevention or delay of onset for neurodegenerative disorders including Dementia, Mild Cognitive Impairment, AD, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis, or even applied as an improvement of cognition, memory, and / or learning capabilities for the healthy people and other vertebrate species, comprising primates.

[0079] According to the disclosures made above, this invention discloses in the preferred embodiments, a system for treating neural degenerative disorders, comprising Alzheimer's disease, comprising a transcranial focused ultrasound system for propagating and steering a transcranial focused ultrasound beam to activate quiescent neural stem cells 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -25- in a human brain, using an ultrasound bio-effect, to replenish neurons and to achieve improvements in cognition, memory, and / or learning capabilities of the patients. In another embodiment, this invention further discloses a system that further comprises a programmable ultrasound intensity controller to control and apply the transcranial focused ultrasound beam at an intensity range of 10 W / cm2or lower for activating the neural stem cells without causing a physiologically significant temperature elevation. In another embodiment, this invention further discloses a system that further comprises a beamforming controller to set the phase shifts or time delays for the transducer elements, a wireless electronic driving apparatus to deliver signals to the ultrasound transducer elements in the transcranial focused ultrasound transducer device, and a transcranial focused ultrasound transducer array comprising the transducer elements distributed over an inner surface of a three-dimensional cylinder to achieve an increased steering range and focusing resolution along at least two axes of a target region. In another embodiment, the system further includes an MRI navigation system to integrate and navigate the transcranial focused ultrasound beam to precisely activate the quiescent neural stem cells resident in target regions in the human brain. In another embodiment, the system further includes a computation device for applying time-reversal mathematic techniques based on the real- time MRI navigation images, to derive phase shifts or time delays for inputting to a beamforming controller to produce a low-level acoustic focus on pre-determined target locations over a pre-defined treatment region coverage. In another embodiment, the system further includes an MRI coil array tightly integrated with a transcranial therapeutic ultrasound transducer array device. In another embodiment, the system further includes a thin-film coil array device wearable over a patient's head, a gel-pad lining between inside of the thin-film coil array and the patient head, and an end-ring with a mechanism sealable to the transducer device, and wherein the thin-film coil device enables the ultrasonic beams emitted by the transducer array to pass through and enter a patient's skull without much impeding or attenuation. Another embodiment, the transcranial focused ultrasound system is employed for use as a follow-up or adjunct treatment after surgical and / or interventional procedures for stroke, brain tumors, and other neurological disorders. In another embodiment, the transcranial focused ultrasound system is employed for use to prevent or delay the onset for neurodegenerative disorders comprising Dementia, Mild Cognitive Impairment, Alzheimer’s disease, and Parkinson’s disease. In another embodiment, the transcranial focused ultrasound system is employed for use to improve cognition, memory, and / or learning capabilities for healthy people. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -26-

[0080] According to the disclosures made above, this invention discloses in the preferred embodiments, a method for treating neural degenerative disorders comprising Alzheimer's disease, the method comprising a step of applying a transcranial focused ultrasound beam and controlling ultrasound parameters to activate quiescent neural stem cells resident in a human brain, to replenish neurons, and to improve a patient’s cognition, memory, and / or learning capabilities. In another embodiment, the method further includes a step of applying the transcranial focused ultrasound beam without causing a physiologically significant temperature elevation in the human brain. In another embodiment, the step of activating the quiescent neural stem cells further includes a step of applying an ultrasound transducer device having a plurality of transducer elements outside a human skull to generate and propagate the transcranial focused ultrasound beams through various media including water, ultrasonic gel, skin, bone, and soft tissues to focus into a converged target location in the human brain. In another embodiment, the step of propagating the transcranial focused ultrasound beam further includes a step of steering the transcranial focused ultrasound beam to a target region, comprising a subventricular zone of lateral ventricles, third ventricle and fourth ventricle, and subgranular zone of hippocampus, wherein neural stem cells located, therein to inflict heat and / or pressure to activate the quiescent neural stem cells to proliferate. In another embodiment, the method further includes a step of applying the transcranial focused ultrasound beam to rejuvenate neural stem cells to replace damaged neurons caused by neurological diseases comprising traumatic brain injury and stroke. In another embodiment, the method further includes a step of applying the method as a follow-up or adjunct treatment after surgical and / or interventional procedures for treatments of stroke, brain tumors, and other neurological disorders. In another embodiment, the method further comprises a step of applying the method to prevent or delay the onset for neurodegenerative disorders comprising Dementia, Mild Cognitive Impairment, Alzheimer’s disease, and Parkinson’s disease. In another embodiment, the method further comprises a step of applying the method to improve cognition, memory, and / or learning capabilities for healthy people. In another embodiment, the method further includes a step of applying the transcranial focused ultrasound beam to improve cognition, memory, and / or learning capabilities in both undamaged, healthy animals and Alzheimer’s disease animal models, including Camk2A / DTA mice.

[0081] In another embodiment, the step of applying the transcranial focused ultrasound beam further includes a step of programming and controlling an ultrasound intensity level 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -27- in a range of 10 W / cm2or lower, and programming and controlling an ultrasound pulse repetition frequency and duty cycle, for activating the quiescent neural stem cells without causing a physiologically significant temperature elevation in the brain. In another embodiment, the step of applying the transcranial focused ultrasound beam further includes a step of employing a transcranial focused ultrasound system equipped with a wireless electronic driving apparatus to deliver signals to the transcranial focused ultrasound transducer elements. In another embodiment, the step of applying the transcranial focused ultrasound beam further includes a step of employing a transcranial focused ultrasound transducer array with the plurality of transducer elements distributed over an inner surface of a three-dimensional cylinder to achieve an increased steering range and focusing resolution along at least two axes of the target region in the human brain. In another embodiment, the method further includes a step of employing an MRI navigation system to integrate and navigate the transcranial focused ultrasound beam to precisely activate the quiescent neural stem cells resident in the target region in the human brain, In another embodiment, the step of employing an MRI navigation system further includes a step of applying time-reversal mathematic techniques, based on real-time MRI navigation images, to derive phase shifts or time delays in a multiple channel beamforming control to drive multiple transducer elements to produce a low-level acoustic focus on pre-determined target locations over a pre-defined treatment region coverage. In another embodiment, the step of employing the MRI navigation system further comprises a step of implementing an MRI coil array to tightly integrate with a transcranial therapeutic ultrasound transducer array device. In another embodiment, step of employing the MRI navigation system further comprising a step of employing an MRI coil array tightly integrated with the transcranial focused ultrasound therapeutic transducer device and the MRI coil array comprising a thin- film coil array wearable over a patient's head, having a gel-pad lining between inside of the thin-film coil array and the patient head, and further having an end-ring with a mechanism sealable to the transducer device, and wherein the thin-film coil array enables the ultrasonic beams emitted by the transducer array to pass through and enter patient's skull without much impeding or attenuation.

[0082] Therefore, it is to be understood that the present invention is not to be limited to the specific examples illustrated and that modifications and other examples are intended to be included within the scope of the appended claims. Moreover, although the foregoing description and the associated drawings describe examples of the present invention in the 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -28- context of certain illustrative combinations of elements and / or functions, it should be appreciated that different combinations of elements and / or functions may be provided by alternative implementations without departing from the scope of the appended claims. Accordingly, parenthetical reference numerals in the appended claims are presented for illustrative purposes only and are not intended to limit the scope of the claimed subject matter to the specific examples provided in the present invention. 22756482.1 a09 / 09 / 25

Claims

Docket No.102489.0002PCT -29- CLAIMS What is claimed is:

1. A method for treating neural degenerative disorders comprising Alzheimer's disease of a patient, the method comprising: applying a transcranial focused ultrasound beam; and controlling ultrasound parameters resulting in activation of quiescent neural stem cells resident in a brain of the patient, replenishing neurons in the brain of the patient, and improving cognition, memory, or learning capabilities of the patient.

2. The method of claim 1, further comprising: applying the transcranial focused ultrasound beam without causing a physiologically significant temperature elevation in the brain of the patient.

3. The method of claim 1, wherein the step of activating the quiescent neural stem cells further includes applying an ultrasound transducer device having a plurality of transducer elements on an exterior of a skull of the patient to generate and propagate the transcranial focused ultrasound beams through various media including water, ultrasonic gel, skin, bone, or soft tissues resulting in the transcranial focused ultrasound beam being focus to a converged target location in the brain of the patient.

4. The method of claim 3, wherein the step of propagating the transcranial focused ultrasound beam further includes steering the transcranial focused ultrasound beam to a target region, wherein the target region comprises a subventricular zone of lateral ventricles, a third ventricle, a fourth ventricle, and a subgranular zone of a hippocampus of the brain of the patient, wherein neural stem cells located, therein to inflict heat or pressure to activate the quiescent neural stem cells to proliferate.

5. The method of claim 1, further comprising: applying the transcranial focused ultrasound beam resulting in rejuvenation of neural stem cells and replace damaged neurons caused by neurological diseases comprising traumatic brain injury and stroke. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -30- 6. The method of claim 5, wherein operations of applying the transcranial focused ultrasound beam and controlling the ultrasound parameters are performed as a follow-up or an adjunct treatment after surgical or an interventional procedure configured to treat stroke or brain tumors.

7. The method of claim 1 wherein applying the transcranial focused ultrasound beam and controlling the ultrasound parameters is configured to prevent or delay onset for neurodegenerative disorders including any of Dementia, Mild Cognitive Impairment, Alzheimer’s disease, or Parkinson’s disease.

8. The method of claim 1 further comprising: wherein applying the transcranial focused ultrasound beam and controlling the ultrasound parameters is performed on both undamaged, healthy animals and Alzheimer’s disease animals including Camk2A / DTA mice.

9. The method of claim 1, wherein applying the transcranial focused ultrasound beam further includes: programming and controlling an ultrasound intensity level in a range of 10 W / cm2or lower, and programming and controlling an ultrasound pulse repetition frequency and duty cycle, wherein programming and controlling the ultrasound intensity level and the ultrasound pulse repetition frequency and duty cycle result is activation of the quiescent neural stem cells without causing a physiologically significant temperature elevation in the brain of the patient.

10. The method of claim 1, wherein applying the transcranial focused ultrasound beam further includes delivering signals to the transcranial focused ultrasound transducer elements by employing a transcranial focused ultrasound system equipped with a wireless electronic driving apparatus.

11. The method of claim 1, wherein applying the transcranial focused ultrasound beam further includes employing a transcranial focused ultrasound transducer array with the plurality of transducer elements distributed over an inner surface of a three-dimensional 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -31- cylinder resulting in an increased steering range and focusing resolution along at least two axes of a target region in the brain of the patient.

12. The method of claim 1 further comprising: precisely activating the quiescent neural stem cells resident in the target region in the human brain by employing an MRI navigation system resulting in integrating and navigating the transcranial focused ultrasound beam.

13. The method of claim 13, wherein employing the MRI navigation system further includes applying time-reversal mathematic techniques, based on real-time MRI navigation images, resulting in derivation of phase shifts or time delays in a multiple channel beamforming control to drive multiple transducer elements to produce a low-level acoustic focus on pre-determined target locations over a pre-defined treatment region coverage.

14. The method of claim 13, wherein employing the MRI navigation system further comprises implementing an MRI coil array to tightly integrate with a transcranial therapeutic ultrasound transducer array device.

15. The method of claim 14, wherein employing the MRI navigation system further comprising employing the MRI coil array tightly integrated with the transcranial focused ultrasound therapeutic transducer device and, wherein the MRI coil array comprises a thin- film coil array wearable over a head of the patient and having a gel-pad lining between inside of the thin-film coil array and the patient head, and further having an end-ring with a mechanism sealable to the transducer device, and wherein the thin-film coil array enables the ultrasonic beams emitted by the transducer array to pass through a skull of the patient.

16. A system for treating neural degenerative disorders, comprising Alzheimer's disease, comprising a transcranial focused ultrasound system for propagating and steering a transcranial focused ultrasound beam to activate quiescent neural stem cells in a human brain, using an ultrasound bio-effect, to replenish neurons and to achieve improvements in cognition, memory, and / or learning capabilities of the patients.

17. The system of claim 17 further comprising: a programmable ultrasound intensity controller to control and apply the transcranial focused ultrasound beam at an intensity range of 10 W / cm2or lower for 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -32- activating the neural stem cells without causing a physiologically significant temperature elevation, a wireless electronic driving apparatus to deliver signals to the ultrasound transducer elements in the transcranial focused ultrasound transducer device, a beamforming controller to set the phase shifts or time delays for the transducer elements, and a transcranial focused ultrasound transducer array comprising the transducer elements distributed over an inner surface of a three-dimensional cylinder to achieve an increased steering range and focusing resolution along at least two axes of a target region.

18. The system of claim 17 further comprising: an MRI navigation system to integrate and navigate the transcranial focused ultrasound beam to precisely activate the quiescent neural stem cells resident in target regions in the human brain.

19. The system of claim 19, further comprising: a computation device for applying time-reversal mathematic techniques based on the real-time MRI navigation images, to derive phase shifts or time delays for inputting to a beamforming controller to produce a low-level acoustic focus on pre-determined target locations over a pre-defined treatment region coverage.

20. The system of claim 19, further comprising an MRI coil array tightly integrated with a transcranial therapeutic ultrasound transducer array device.

21. The system of claim 21, wherein the MRI coil array includes: a thin-film coil array device wearable over a patient's head, a gel-pad lining between inside of the thin-film coil array and the patient head, and an end-ring with a mechanism sealable to the transducer device, and wherein the thin-film coil device enables the ultrasonic beams emitted by the transducer array to pass through and enter a patient's skull without much impeding or attenuation. 22756482.1 a09 / 09 / 25Docket No.102489.0002PCT -33- 22. The system of claim 17, wherein the transcranial focused ultrasound system is employed for use as a follow-up or adjunct treatment after surgical and / or interventional procedures for stroke, brain tumors, and other neurological disorders.

23. The system of claim 17, wherein the transcranial focused ultrasound system is employed for use to prevent or delay the onset for neurodegenerative disorders comprising Dementia, Mild Cognitive Impairment, Alzheimer’s disease, and Parkinson’s disease.

24. The system of claim 17, wherein the transcranial focused ultrasound system is employed for use to improve cognition, memory, and / or learning capabilities for the healthy people. 22756482.1 a09 / 09 / 25

Images