Systems and devices for enhancing ultrasonic cerebrospinal fluid clearance and methods for use

Abstract

Devices and system configured to deliver ultrasound to the brain may enhance brain waste clearance for a variety of purposes for acute and / or chronic indications. During use, ultrasound may be projected into multiple regions of the brain to increase circulation of cerebrospinal fluid (CSF) and / or interstitial fluid (ISF) within the brain, increase mechanical mixing of CSF and / or ISF in the skull and / or between intracranial fluid compartments, induce mechanosensitive channel activation, and / or shift a microglial phenotype from the diseaseassociated state to the hemeostatic state, which may impact a neuroimmunologic state of the subject's brain. The ultrasound may be projected into the subject's brain in accordance with an ultrasonic cerebrospinal fluid clearance (UCC) treatment protocol that sets parameters for the UCC treatment. On some occasions, the parameters of a UCC treatment protocol may be responsive to subject characteristics and / or may be used to predict an outcome of treatment.

Description

SYSTEMS AND DEVICES FOR ENHANCING ULTRASONIC CEREBROSPINAL FLUID CLEARANCE AND METHODS FOR USECROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 654,789, filed May 31 , 2024, which application is incorporated herein by reference in its entirety.GOVERNMENT RIGHTS

[0002] This invention was made with Government support under contracts MH114252, NS1 14438, and NS115637 awarded by the National Institutes of Health. The Government has certain rights in the invention.TECHNICAL FIELD

[0003] This patent application pertains to medical devices and, in particular to systems, devices, and methods configured to enhancing clearance of waste from the brain.BACKGROUND

[0004] Impairment of cerebrospinal fluid (CSF) circulation has been shown to be both a feature of certain neurologic disorders and a pathogenic mechanism of those diseases. In particular, in the setting of both ischemic and hemorrhagic stroke, neurodegenerative diseases, and traumatic brain injuries, CSF and interstitial fluid clearance has been shown to be both impaired and a potential avenue for treatment. Impaired clearance of blood products in intracranial hemorrhage has been suggested as pathogenic in the setting of hemorrhagic stroke and traumatic brain injury, with increased clearance via the meningeal lymphatics contributing to improved outcomes preclinically. Moreover, prospective upregulation of CSF circulation, whether pharmacologically or through invasive intervention, yields improved outcomes both preclinically and clinically. Indeed, upregulation of meningeal lymphatic clearance through pharmacologic intervention shows improvement in certain stroke models. Additionally, proactive CSF drainage in the setting of subarachnoid hemorrhage (SAH) improves all cause morbidity and mortality and proactive evacuation of the hematoma improves outcomes in intracerebral / intraparenchymal hemorrhage (ICH) . These data suggest that a modality to drive CSF circulation could improve outcomes in varied neurologic diseases, including but not limited to hemorrhagic brain injury.SUMMARY

[0005] The systems and devices disclosed herein include a housing configured to fit over a portion of a subject’s head, house a plurality of ultrasound devices, and orient an active side of each of the plurality of ultrasound devices toward the subject’s head when worn so that the plurality of ultrasound devices may project ultrasound into the subject’s brain. In some embodiments, the device may be configured as an ultrasonic cerebrospinal fluid clearance (UCC) device. In some embodiments the plurality of ultrasound devices may be configured to project ultrasound into to a plurality of regions of a subject’s brain to increase a circulation rate of cerebrospinal fluid (CSF) within the subject’s brain and egress from the subject’s brain. Additionally, or alternatively, the plurality of ultrasound devices may be configured to project ultrasound into a plurality of regions of a subject’s brain to mechanically induce mixing of CSF within different compartments of the subject’s brain, induce removal of waste from the subject’s brain and / or activate mechanosensitive channels within the subject’s brain to increase the circulation rate flow of the CSF within the subject’s brain and egress from the subject’s brain, wherein activation of the mechanosensitive channels may also shift microglia from a disease-associated state to a homeostatic state.

[0006] In some embodiments, each ultrasound device of the device may be configured to emit ultrasound with the same characteristics (e.g., pressure, intensity, frequency, etc.) and in other embodiments, the device may include two or more sets of ultrasound device(s) configured to project ultrasound with different characteristics. In some cases, the ultrasound emitted by one or more of the ultrasound device(s) may be low-intensity focused ultrasound. The ultrasound devices may be configured to emit ultrasound that generates in situ peak negative pressure within the subject’s brain within a range of 0.1 -1 .1 MPa, 0.1 -1 MPa, 0.1 - 0.95MPa, 0.15-3MPa, 0.1 -9MPa, 0.1 -5MPa, 0.15-0.3MPa, or 0.15-0.95MPa

[0007] At times, the devices and / or housing disclosed herein may include and / or be configured to communicate with a heat sensing device configured to sense heat within the subject’s brain and / or on the subject’s scalp. Additionally, or alternatively, the devices and / or housing disclosed herein may include and / or be configured to communicate with a sensor (e.g., a microphone) configured to measure ultrasound reflected from the subject’s head.

[0008] The device of any of the above claims, wherein the plurality of ultrasound devices includes a first ultrasound device arranged within the housing to project ultrasound into a front of the subject’s brain, a second ultrasound device arranged within the housing to project ultrasound into a back of the subject’s brain, a third ultrasound device arranged within the housing to project ultrasound into a first side of the subject’s brain, and a fourth ultrasound device arranged within the housing to project ultrasound into a second side of the subject’s brain.

[0009] In some embodiments, the systems and / or devices disclosed herein may include a first ultrasound device arranged within the housing to project ultrasound into a region proximate to a front left side of the subject’s brain; a second ultrasound device arranged within the housing to project ultrasound into a region proximate to left side of the subject’s brain; a third ultrasound device arranged within the housing to project ultrasound into a region proximate to a rear-left side of the subject’s brain; a fourth ultrasound device arranged within the housing to project ultrasound into a region proximate to an upper-left side of the subject's brain; a fifth ultrasound device arranged within the housing to project ultrasound into a region proximate to a front right side of the subject’s brain; a sixth ultrasound device arranged within the housing to project ultrasound into a region proximate to right side of the subject’s brain; a seventh ultrasound device arranged within the housing to project ultrasound into a region proximate to a rear-right side of the subject’s brain; and an eighth ultrasound device arranged within the housing to project ultrasound into a region proximate to an upper-right side of the subject’s brain.

[0010] The methods disclosed herein may use the systems and / or devices disclosed herein to, for example, enhance waste clearance from the brain and / or treat chronic and / or acute brain injury and / or disease. The treatment may be referred to as ultrasonic CSF clearance (UCC). For example, a neurological diagnosis and / or characteristic for a subject may be received and used to query a UCC treatment protocol database for a UCC treatment protocol that matches the neurological diagnosis and, optionally, one or more subject characteristics and / or clinician preferences. A UCC treatment protocol that matches the neurological diagnosis may be received from the UCC treatment protocol database responsively to the query and used to set up a UCC device and / or system to provide a UCC treatment to a subject. When treating a subject, an indication that a UCC device and / or housing thereof is positioned on the subject’s head, or a portion (e.g., forehead, back of the head, above the left and / or right ear, etc.) and / or is otherwise ready to project ultrasound into the subject’s head may be received and, upon receipt, projection of ultrasound into the subject’s brain via one or more of the ultrasound devices of the plurality of ultrasound devices included in the device positioned on the subject’s head may be initiated and / or performed in accordance with the received UCC treatment protocol. Exemplary indications that the UCC device and / or a housing thereof is in position on the subject’s head include, but are not limited to, a message from a user who visually confirms the UCC device and / or a housing thereof is in a proper position and / or is ready for use that is communicated to a processor, controller, and / or cloud computing platform via, for example, manual interaction with a user interface, an indication that a sensor (e.g., EEG sensor, contact sensor, electrical lead, pressure sensor, etc.) housed by the housing is in contact with the user’s head, skin, and / or scalp, a result of an analysis of an image (ultrasound image or visual image) the subject’s head and / or brain to determine whether thehousing and / or one or more ultrasound devices of a UCC device are properly positioned relative to the subject’s brain, and / or analysis of one or more signals from a receiver (e.g., microphone) configured to detect ultrasound reflected by the subject’s head and / or brain to determine whether or not the ultrasound is properly transmitted into the subject’s brain and / or a portion thereof.

[0011] The UCC treatment protocol database may store a plurality (e.g., 1 ,000-100,000,000) of UCC treatment protocols that are associated, correlated, and / or indexed to one or more neurological diagnoses, subject characteristics, subject symptoms, clinician preferences, prior treatment outcomes, feedback regarding a UCC treatment, a degree of responsiveness of the subject to prior UCC treatments, subject comorbidities, and / or other treatments the subject is receiving. In some cases, one or more of the UCC treatment protocols may be generated by a UCC treatment model and / or include a predicted outcome that, in some embodiments, may be determined by the UCC treatment model.

[0012] In some embodiments, the UCC treatment protocol may include, for example, a schedule for administration of UCC treatments to the subject, a duty cycle for one or more of the ultrasound devices of the device over the duration of the UCC treatment, and / or an intensity of the ultrasound projected into the subject’s brain by one or more of the ultrasound devices of the device over the duration of the UCC treatment. Additionally, or alternatively, a selected and / or queried-for UCC treatment protocol may define, for example, a duration of a UCC treatment session, a set of instructions for a serial projection of ultrasound by the plurality of ultrasound devices, a set of instructions for an alternating projection of ultrasound by the plurality of ultrasound devices, a set of instructions for a simultaneous projection of ultrasound by the plurality of ultrasound devices, and / or a set of instructions for projection of ultrasound by a subset of the plurality of ultrasound devices. For example, when the neurological diagnosis is an acute diagnosis, the UCC treatment protocol provides instructions for administering approximately 30-60 minutes of UCC to at least a portion (e.g., approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the subject's brain using the device over a series of days. In another example, the neurological diagnosis may be a chronic diagnosis and the UCC treatment protocol provides instructions for administering approximately 15-30 minutes of UCC to the portion of the subject’s brain using the device over a series of months.

[0013] In some embodiments, the methods of using the systems and / or devices disclosed herein may include receiving a set of neurological information for a subject, inputting the set neurological information into a UCC treatment model; and receiving an output from the UCC treatment model that may be responsive to the set of neurological information, the output including a UCC treatment protocol that includes a set of instructions for use of the device and / or systems disclosed herein to provide a UCC treatment to the subject. An output UCCtreatment protocol may include, for example, a schedule for administration of UCC treatments to the subject, a duty cycle for one or more of the ultrasound devices of the device over the duration of the UCC treatment, and / or an intensity of the ultrasound projected into the subject's brain by one or more of the ultrasound devices of the device over the duration of the UCC treatment. At times, the set of instructions for use of the UCC device may define, for example, a duration of a UCC treatment session, a set of instructions for a serial projection of ultrasound by the plurality of ultrasound devices, a set of instructions for an alternating projection of ultrasound by the plurality of ultrasound devices, a set of instructions for a simultaneous projection of ultrasound by the plurality of ultrasound devices, and / or a set of instructions for projection of ultrasound by a subset of the plurality of ultrasound devices.

[0014] On some occasions, the output is provided to a display device so that a clinician may view, modify, and / or approve the UCC treatment protocol. Additionally, or alternatively, the output may be provided to one or more systems and / or devices disclosed herein so that the subject may be treated with the output UCC treatment protocol. The set of neurological information for the subject may include, but is not limited to, an image of the subject’s brain, a scan of the subject’s brain, a dimension of the subject’s head, a diagnosis for the subject, an indication when an adverse neurological event occurred for the subject, an indication of the subject’s responsiveness to UCC treatment, an indication of a treatment the subject may be received other than the UCC treatment. On some occasions, the set of neurological information may be used to select and / or optimize a UCC treatment protocol for the subject.

[0015] In some embodiments, the output from the UCC treatment model may further include a predicted outcome of the UCC treatment protocol for the subject that may be provided to a display device. Additionally, or alternatively, the output from the UCC treatment model may include a plurality of UCC treatment protocols and a predicted outcome for each the UCC treatment protocols. An indication of each UCC treatment protocol of the plurality of UCC treatment protocols and / or the predicted outcome of each UCC treatment protocol may then be provided to a display device.

[0016] In some embodiments, feedback regarding the subject following administration of at least one UCC treatment of the UCC treatment protocol may be received while and / or after the subject is treated and updating the UCC treatment model responsively to the feedback. Exemplary feedback includes, but is not limited to a result of a diagnostic test, a result of a blood test for a biomarker of neurological waste, a chemical composition of lymphatic fluid extracted from a cervical lymph node, an indication of heat within the subject’s scalp or brain while the subject may be undergoing at least one UCC treatment, an indication of cavitation within the subject’s scalp or brain while the subject may be undergoing at least one UCC treatment, and an image of the subject’s brain.

[0017] In some embodiments, an image (e.g., MRI, ultrasound, or CT scan image) may be received and analyzed to determine one or more features (e.g., size, position of regions of interest, condition within a region of interest, etc.) thereof. The one or more features may be used to determine and / or select an operational parameter for a UCC device and / or UCC treatment protocol by using and / or querying, for example, a UCC treatment protocol database and / or UCC treatment model. The operational parameter may be provided to a clinician for review, modification, approval, and / or to one or more of the UCC devices and / or systems disclosed herein.

[0018] During use, an indication that the UCC device is positioned on the subject’s head, or a portion thereof (e.g., forehead, above a left and / or right ear, back of the head, etc.) may be received and, upon receipt, projection of ultrasound into the subject’s brain via one or more of the ultrasound devices of the plurality of ultrasound devices in accordance with the operational parameter may be initiated and / or continued in accordance with the operational parameter. Exemplary operational parameters include, but are not limited to, a duration of a UCC treatment session, a set of instructions for a serial projection of ultrasound by the plurality of ultrasound devices, a set of instructions for an alternating projection of ultrasound by the plurality of ultrasound devices, a set of instructions for a simultaneous projection of ultrasound by the plurality of ultrasound devices, and / or a set of instructions for projection of ultrasound by a subset of the plurality of ultrasound devices.

[0019] In some embodiments, feedback may be received and the operational parameter may be adjusted responsively to the feedback. Exemplary feedback includes, but is not limited to, a temperature of the subject’s scalp and / or brain and / or how much ultrasound is backscattered and / or reflected from the subject’s brain or skull. On some occasions, the feedback may be another image(s) of the subject’s brain while the subject may be being treated with the device and / or after the subject is treated. The image may be analyzed to, for example, determine the efficacy and / or safety of the treatment and, on some occasions, the operational parameter may be adjusted responsively to a result of the analysis.

[0020] In some embodiments, provided herein is a noninvasive low intensity transcranial focused ultrasound protocol that facilitates the removal of pathogenic substances from the cerebrospinal fluid (CSF) and the brain interstitium. The treatment may be referred to as ultrasonic CSF clearance (UCC). This protocol yields direct alterations of the neuroimmune environment, shifting microglia from the disease-associated state to the reparative, homeostatic state. There are also associated changes in increasing aquaporin endfeet polarization, a key glymphatic mediator. The treatment is shown to reduce neuroinflammatory and neurocytotoxic profiles, improved behavioral outcomes, decreased morbidity and, importantly, increased survival in an animal model for acute brain injury. The method isperformed in the absence of co-administration of exogenous agents, e.g. nanoparticles, microbubbles, etc.

[0021] The methods of the disclosure are applied to remove pathogenic substances from the cerebrospinal fluid (CSF) and the brain interstitium. Substances may have a broad range of sizes, e.g., from 1 kDa up to about 5 Lim in size, for example from 10 kDa to 5 y.m. The substance may be a large molecular agent having a molecular weight ranging, e.g., from 100 kDa to 5 | m, from 150 kDa to 5 |j.m, from 250 kDa to 5 jim. In some embodiments the pathogenic substances are products of hemorrhage, including without limitation red blood cells and red blood cell debris.

[0022] In some embodiments an individual selected for treatment suffers from a condition in which there has been an acute injury to the brain, including without limitation, traumatic brain injury; hemorrhage, including subarachnoid and intraparenchymal hemorrhage; ischemic injury; and the like. In some embodiment an individual selected for treatment suffers from a condition in which there is a chronic disease or injury of the brain, e.g. autoimmune or infectious encephalitis; neurodegenerative disease; tumor-induced brain injury, edema, and intracranial pressure elevation; concussion, headache, migraine, hydrocephalus including normal pressure hydrocephalus, arachnoid cyst (ruptured or unruptured), intracranial hypertension; epilepsy; sleep and circadian rhythm disorders; affective mental health disorders, and the like. In some embodiment the individual suffers from hemorrhagic brain injury, e.g. hemorrhagic stroke.

[0023] The ultrasound is typically applied as a scanning transcranial focused ultrasound, where the whole brain is targeted. The ultrasound may have a frequency ranging from 100 kHz to 1000 kHz, and may be at least about 100 kHz, at least about 200 kHz, at least about 250 kHz, at least about 350 kHz, at least about 450 kHz, and up to about 1000 kHz, up to about 900 kHz up to about 800 kHz, up to about 700 kHz, up to about 600 kHz, up to about 500 kHz. In some embodiments the ultrasound has a frequency ranging from 200 kHz to 500 kHz; from 250 to 450 kHz.

[0024] The ultrasound may have a pressure between about 0.05 MPa to about 0.6 MPa; from about 0.05 MPa to about 0.2 MPa; from about 0.2 MPa to about 0.5 MPa; from about 0.3 MPa to about 0.5 MPa, and may be about 0.45 MPa.

[0025] The ultrasound pulse width may be from about 5 us to about 100 ms, for example from about 100 [is, from about 500 [is, from about 1 ms, from about 5 ms, up to about 500 ms, up to about 250 ms, up to about 100 ms; and may be from about 500 pis to about 500 ms, from about 1 ms to about 100 ms, and in some embodiments is around 50 ms.

[0026] The ultrasound may be performed with a duty cycle of from about 1 -100%; for example from about 5% to about 75%, from about 10% to about 50%, from about 20% toabout 40%. The ultrasound may be performed for at least about 1 minutes, at least about 5 minutes, at least about 10 minutes, and up to about 60 minutes, up to about 45 minutes, up to about 30 minutes. In some embodiments it is performed from about 10 minutes to about 30 minutes.

[0027] In some embodiments, the composition or method described herein is used in combination with one or more methods of imaging (e.g. fMRI or PET), measuring electrophysiology (e.g. EEG), and / or behavioral assessment of brain function. The efficacy of the method is optionally monitored by determining the clearance of such pathogenic products, e.g. by assessment of cerebrospinal fluid, assessment of lymph nodes for the presence of such products. Alternatively or in combination, a subject may be assessed by clinical indicia of the condition being treated, e.g. hematoma size, intracranial pressure, etc.

[0028] In embodiments involving the treatment of an acute condition, including without limitation traumatic brain injury; hemorrhage, including subarachnoid and intraparenchymal hemorrhage; ischemic injury; and the like, treatment may be performed shortly after the incident occurs or is diagnosed, e.g. within about 3 days, within about 2 days, within about 1 day, and may be performed with 24 hours, within 18 hours, within 12 hours, within 6 hours, within 3 hours of occurrence or diagnosis.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention is illustrated byway of example, and not limitation, in the figures of the accompanying drawings.

[0030] FIGS. 1 A-1 B. 1 A provides a cut-away diagram of a subject with a portion of his / her brain exposed. FIG. 1 B is a detailed view of a portion of subject’s brain showing a skull, lymphatic vessels, and a subarachnoid space.

[0031] FIG. 2 is a block diagram of an exemplary system for delivering ultrasonic CSF clearance (UCC) to a subject, in accordance with some embodiments of the present invention.

[0032] FIGS. 3A-3I. 3A provides a top view of a first exemplary UCC device with four ultrasound devices, in accordance with some embodiments of the present invention; FIG. 3B provides a side view of a second exemplary UCC device being worn by a subject, in accordance with some embodiments of the present invention; FIG. 3C provides a frontperspective view of the UCC device of FIG. 3B, in accordance with some embodiments of the present invention; FIG. 3D provides and underside perspective view of the UCC device of FIG. 3B, in accordance with some embodiments of the present invention; FIG. 3E provides a top view of a third exemplary UCC device, in accordance with some embodiments of the present invention; FIG. 3F provides a front view of the third exemplary UCC device of FIG. 3E, in accordance with some embodiments of the present invention; FIG. 3G provides a back view of the third exemplary UCC device of FIG. 3E, in accordance with some embodiments of thepresent invention; FIG. 3H provides a side view of the third exemplary UCC device of FIG. 3E, in accordance with some embodiments of the present invention; FIG. 31 provides an underside view of the third exemplary UCC device of FIG. 3E, in accordance with some embodiments of the present invention.

[0033] FIG. 4 provides a flowchart illustrating an exemplary method for building and / or training a UCC treatment model and / or evaluating the accuracy of a UCC treatment model, in accordance with some embodiments of the present invention.

[0034] FIG. 5 provides a flowchart illustrating an exemplary method for determining operational and / or therapeutic parameters for a UCC device and / or providing UCC treatment to a subject, in accordance with some embodiments of the present invention.

[0035] FIG. 6 provides a schematic diagram of an exemplary way in which UCC treatment and / or ultrasound may be delivered to subject’s brain, in accordance with some embodiments of the present invention.

[0036] FIGS. 7A-7C. FIG. 7A provides a beam plot of a subject’s brain in the X-Z plane, in accordance with some embodiments of the present invention; FIG. 7B provides a beam plot of the subject’s brain in the X-Y plane, in accordance with some embodiments of the present invention; and FIG. 7C provides a beam plot of the subject’s brain in the Y-Z plane, in accordance with some embodiments of the present invention.

[0037] FIGS. 8A-8E. Ultrasonic CSF clearance (UCC) enhances the clearance of hemorrhage from the CSF and interstitial brain compartments a. Schematic of the focused ultrasound protocol (FUS; 250k Hz, 0.45 MPa, 25% duty-cycle, 50 ms pulse width) efflux experiment, b. Simulated ultrasound pressure beam profiles and effect of skull on the intracranial profile, c. Timeline of subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH) experiments, d. UCC clears CSF dispersed red blood cells (RBCs) compared to sham in a SAH model. There is a greater accumulation of RBCs in the deep cervical lymph nodes (dCLNs) of SAH mice with UCC compared to SAH sham. e. UCC clears interstitial RBCs compared to sham in an ICH model. There is a greater accumulation of RBCs in the deep cervical lymph nodes (dCLNs) of ICH mice with UCC compared to sham. No red blood cells were observed in separate cohorts of healthy control brain or lymph node tissue. PNP: Peak Negative Pressure. Data presented as box plots. Two-tailed Mann-Whitney U-test. *: p < 0.05, **: p < 0.01 . n = 6 for each group.

[0038] FIGS. 9A-9H. UCC reduces neuroinflammation and neurocytotoxicity posthemorrhagic brain injury, a. Schematic of the subarachnoid hemorrhage (SAH) model with the location of interest (LOI) sampled for histological analyses, b. Immunohistological assessment of SAH brains for microglial activation (IBA-1 , purple) and c. gliosis / astrocytic activation (GFAP, green) revealed a decrease in both neuroinflammatory markers with UCC compared to sham. d. Histological assessment of SAH brains for neuronal degeneration (FJ-C, green)revealed a decrease with UCC compared to sham. e. Schematic of the intracerebral hemorrhage (ICH) model with the LOI sampled for histological analyses, f. Immunohistological assessment of ICH brains for microglial activation and g. gliosis / astrocytic activation revealed a decrease in both neuroinflammatory markers with UCC compared to sham. h. Histological assessment of ICH brains for neuronal degeneration revealed a decrease with UCC compared to sham. Data presented as box plots. Healthy control data are presented for reference and are repeated from b-d to f-h. Two-tailed Mann-Whitney U- test. *: p < 0.05, **: p < 0.01 . n = 6 for each group.

[0039] FIGS. 10A-10D. UCC improves behavioral outcomes and reduces morbidity and mortality post-hemorrhagic brain injury, a. Timeline of intracerebral hemorrhage (ICH) outcomes experiment, b. Behavioral / functional assessments (corner turn test and grip strength) revealed an improvement in functional outcomes as early as 6 days posthemorrhage with UCC compared to sham. c. Decreased morbidity outcomes (body weight and brain water content, edema) with UCC compared to sham. d. Increased survival with UCC intervention compared to sham, X2(2) = 4.08, p < 0.05. Time series data points presented as mean ± S.D., two group comparisons presented as box plots; healthy control baseline levels are presented for reference (dashed lines, grey); b-c: Mann-Whitney U-tests with multiple comparisons controlled using the False Discovery Rate (FDR) method (two-stage step-up, Benjamini, Krieger, and Yekutieli); c, left: Kruskal-Wallis H test followed by Dunn’s multiple comparisons test; c, right: two-tailed Mann-Whitney U-test; d: log-rank (Mantel-Cox) test. *: p < 0.05, **: p < 0.01 . n=18 for each group; n=6 for brain edema endpoint.

[0040] FIGS 11 A-1 1 D. Spatial transcriptomic analysis demonstrates UCC mediated changes in inflammatory markers post hemorrhagic stroke, a. Brain region level annotations of leiden- derived spatial clusters of stereotranscriptomic data at bin size of 100. Bins are colored by annotation: Cblm: Cerebellum; ChP: Choroid Plexus; DG: Dentate Gyrus; DL-Ctx: Deep layer cortex; Hyp: Hypothalamus; NAc-Str: Nucleus accumbens; OB: Olfactory bulb; RN-BS-Pons: Reticular nucleus I brain stem; Str: Striatum; Thai: Thalamus; UL-Ctx: Upper layer cortex; WM: White matter. The Striatum and Thalamus regions together constitute the perihematomal regions of the ICH groups, b. Gene group scoring across different brain regions and conditions (Left: Heatmap, Right: Spatial distribution). Inflammation score - G0:0006954; Microglia score - GO:1903978; “disease-associated” and “homeostatic” scores are from manually curated lists, c. Key markers of glymphatic (AQP4), glia (GFAP), microglia (IBA1 , P2YR12) and inflammation (CD68, LGALS3). d. Volcano plots of all genes across 4 different comparisons for data derived from the striatum and thalamus, which for ICH animals constitute the perihematoma region. The number of DEGs are shown in parentheses, determined as | log2FC|>1 ; q-value <0.05.

[0041] FIGS. 12A-12G. UCC promotes blood clearance by modulating neuroinflammatory and glymphatic states a. Intracerebral hemorrhage (ICH) model with the location of interest (LOI) sampled for histological analyses, b. Immunohistological assessment of ICH brains for constitutive astrocytic counts (S100P+) revealed no significant difference with ICH + UCC compared to ICH + Sham. c. Immunohistological assessment with 3D reconstruction of microglia revealed a significant increase in branching, a hallmark of the homeostatic microglial state, with ICH + UCC compared to ICH + Sham. d. Immunohistological assessment of microglial inflammatory states (CD68+, red; GAL-3+, yellow) revealed a decrease in pro- inflammatory disease-associated microglial markers with ICH + UCC compared to ICH + Sham. e. Immunohistological assessment of purinergic receptors (P2RY12+, yellow) that reflect a homeostatic microglial state revealed an increase with ICH + UCC with a trend towards the healthy controls, compared to ICH + Sham. f. Immunohistological assessment with 3D reconstruction of microglia (IBA-1 +, green) revealed increased phagocytosis of red blood cells (TER-199+, red) with ICH + UCC compared to ICH + Sham. g. Immunohistological assessment of astrocytic endfeet water channel localization (AQP4+, green) revealed an increase in polarization to the vasculature with ICH + UCC compared to ICH + Sham. Data presented as box plots; healthy control data are presented for reference. Two-tailed Mann- Whitney U-test. *: p < 0.05, **: p < 0.01 . n = 4 for each group.

[0042] FIGS. 13A-13E. UCC is reversed by a mechanosensitive ion channel blocker, a. Timeline of intracerebral hemorrhage (ICH) experiment with UCC and a mechanosensitive ion channel blocker (GsMTx4). b. Administration of a GsMTx4 reduced the blood product clearance of UCC, as assessed by hematoma volumes across treatment groups, c. Immunohistological analysis of meningeal lymphangiogenesis (LYVE-1 +) revealed no significant difference between control, ICH + Sham, and ICH + UCC during this timeline of injury and treatment, d. In healthy, uninjured mice, immunohistological analysis of purinergic receptors (P2RY12+, yellow) that reflect a homeostatic microglial state revealed an acute increase with only one application of UCC in compared to control, e. In healthy, uninjured mice, immunohistological analysis of aquaporin-4 (AQP4+, green) to astrocytic endfeet revealed no significant difference in polarization with only one application of UCC in compared to control. Data presented as box plots; healthy control data are presented for reference. Kruskal-Wallis with post-hoc Dunn’s multiple comparisons test. *: p < 0.05, **: p < 0.01 , ***: p < 0.001. n = 8 mice for each group in Fig 13b and n = 4 mice for each group in Fig 13c-e.

[0043] FIG. 14. Schematic for the proposed model of the mechanisms underlying the efficacy of UCC. In hemorrhagic stroke, aquaporin expression and disease-associated microglial states are promoted (Fig. 11 ). Disease-associated microglia inhibit the polarization of aquaporins to astrocytic endfeet. UCC acts principally via mechanosensitive ion channel activation (Fig. 13) and potentially also via direct mechanical fluid compartmental intermixing.Via mechanosensitive ion channel activation, UCC shifts microglia from the disease- associated to the homeostatic state (Fig. 12), in coherence with prior literature. This microglial phenotypic shift yields increased RBC phagocytosis (Fig. 12) and disinhibition of astrocytic endfeet aquaporin polarization (Fig. 1 1 ,12). The combination of these effects (mechanical fluid mixing, aquaporin polarization, increased debris phagocytosis) allows for clearance of brain injury debris via the meningeal lymphatics to the deep cervical lymph nodes (Fig. 8).

[0044] FIGS. 15A-15C. Focused ultrasound parameter optimization for UCC. a. Timeline of FUS parameters assessment experiment, b. (Left) Schematic of 650kHz and 250kHz transducer. Each transducer used was f=1 .0 with 30 mm (650 kHz) or 70 or 100 mm (250 kHz) aperture. (Right) Fluorescence intensity following pulsed ultrasound application (0.45 MPa estimated peak in situ negative pressure, 25% duty cycle, 5 Hz PRF, 10 min) at the indicated ultrasound frequency or sham (CNTRL) with quantification of mean fluorescent intensity following intracisternal injection of a 2kDa tracer dye at 60 min post-injection, c. (Left) Representative coronal brain slice and quantification of mean fluorescent intensity following intracisternal injection of a 2kDa tracer dye at 60 min post-injection. (Right) Given the inflection at 0.45 MPa estimated in situ peak negative pressure with saturation of the effect at higher pressures, this pressure was chosen for subsequent analysis, b. Data presented as box plots; n = 4 mice at each frequency; two-tailed Mann-Whitney U-test; **: p < 0.01 . c. Data presented as mean ± S.D.; n = 3 mice at each pressure.

[0045] FIGS. 16A-16C. Focused ultrasound induced thermal effects a. (Left) Simulated heat accumulation for a single cycle of UCC, without consideration of thermoregulation due to brain perfusion. (Right) Experimentally measured temperature via a thermocouple placed intracranially before and after UCC application (thermocouple removed for UCC application with <5 sec time to replace the probe). K-wave 3D heating simulation outputs in coronal (b) and sagittal (c) plane. Outputs include the resulting acoustic pressure amplitude (top left), volume rate of heat deposition (top center), temperature after the 50 ms burst of FUS (top right), and temperature after cooling 150 ms before the next ultrasound burst (bottom left). The remaining outputs show any voxels above the CEM43 temperature threshold (bottom middle) and ablated tissue (bottom right), which were entirely zero for these analyses.

[0046] FIGS. 17A-17C. Pharmacologic mechanosensitive ion channel activation decreases survival outcomes and fails to clear hematoma a. Timeline of intracerebral hemorrhage (ICH) experiment with mechanosensitive ion channel agonist (Yoda-1 ), b. Increased survival with UCC intervention (84.0%) compared to ICH sham (51 .0%) but decreased survival with Yoda- 1 (10%), x2(2) = 13.40, p < 0.05. c. Administration of Yoda-1 did not significantly reduce blood product clearance in contrast to UCC, as assessed by hematoma volumes across treatment groups. Kruskal-Wallis with post-hoc Dunn’s multiple comparisons test. UCC, Sham, andGsMTx4 data repeated from Fig. 5. *: p < 0.05, p < 0.01 , p < 0.001 . n = 8 mice for each group except n=3 for Sham + Yoda-1 due to high mortality.

[0047] Throughout the drawings, the same reference numerals, and characters, unless otherwise stated, are used to denote like features, elements, components, or portions of the illustrated embodiments. Moreover, while the subject invention will now be described in detail with reference to the drawings, the description is done in connection with the illustrative embodiments. It is intended that changes and modifications can be made to the described embodiments without departing from the true scope and spirit of the subject invention as defined by the appended claims.WRITTEN DESCRIPTION

[0048] Clearance of waste from the brain is performed by the brain's glymphatic system via a flow of cerebrospinal fluid (CSF) secreted by the choroid plexuses within the subarachnoid space, communicating with the perivascular spaces (typically around cerebral arteries) where it then contacts and mixes with interstitial fluid (ISF) and waste within the brain parenchyma and exits via the perivascular spaces (typically around cerebral veins) to either enter back into the subarachnoid space or communicate directly with venous blood via arachnoid granulations or with meningeal lymphatic vessels. The debris from the mixed CSF and ISF is eventually delivered to venous blood via arachnoid granulations or to the meningeal lymphatic system for elimination from the brain. The meningeal lymphatic system debris typically transports to the cervical lymph nodes before re-entering the blood circulation by the usual connections of the lymphatic and blood circulation. The debris in the blood circulation, whether via arachnoid granulations directly or via the lymphatic system, distributes in the rest of the systemic circulation prior to eventual elimination from the body. Exemplary waste that may cleared from the brain via this process includes, but is not limited to, blood breakdown products, hemorrhagic debris, inflammatory molecules, (e.g., inflammatory cytokines), beta-amyloid, tau, toxic metabolites, and / or alpha-synuclein, plaque, neurotoxic blood products, breakdown products of the cerebral extracellular matrix, and other compounds that may disrupt neural functioning as well as lead to neuroinflammation.

[0049] Neurologic conditions including, for example, trauma, inflammation, disease, and neurodegeneration share a fundamental pathology: the accumulation of toxic waste products in the brain, due to impaired clearance of brain waste, which fuels neuroinflammation, accelerates neuronal loss, lengthens recovery times, and worsens patient outcomes and the costs associated with treatment of these conditions. Despite decades of research, no noninvasive therapy exists to enhance brain waste clearance, leaving millions of patients vulnerable to long-term disability and cognitive decline. Meanwhile, invasive approaches to proactively drain cerebrospinal fluid, inducing increasing fluid circulation and debris clearance,have shown improvements in all-cause morbidity / mortality, supporting the need to develop systems that can promote cerebrospinal fluid circulation and debris clearance less invasively. While current treatments focus on symptom management, the systems, devices, and methods disclosed herein target an underlying cause (i.e., poor brain waste clearance) of neurologic conditions and / or decline without the use of drugs, contrast agents, or surgical procedures, which makes them broadly applicable across diverse neurologic conditions and patient populations. The systems, devices, and methods disclosed herein improve rates of brain waste clearance by non-invasively delivering ultrasound (e.g., low-intensity focused ultrasound (LIFU)) therapy to the brain, which enhances bulk CSF flow and / or ISF flow throughout the brain and cranium, thereby increasing brain waste clearance rates and reducing pathology caused by the accumulation of waste in the brain. The net effect of this application also modulates glia and microglia of the brain to promote glymphatic system upregulation and a shift of the brain neuroimmune state towards a homeostatic, reparative one, which altogether further promotes debris clearance.

[0050] The systems, devices, and methods disclosed herein may be embodied as housings that include one or more ultrasound devices configured to non-invasively project ultrasonic energy into a subject’s head and brain, in some embodiments, the systems, devices, and methods disclosed herein may be configured to deliver ultrasound to a portion, a large portion, or the entirety, of the brain regardless of the diagnosis, size, and / or location of an injury (e.g., hematoma). The ultrasound projected into a subject’s brain may work to mechanically stimulate flow of CSF and / or ISF to assist with clearance of brain waste caused by, for example, subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), aging, neurodegenerative disease, traumatic brain injury, autoimmune diseases (e.g. multiple sclerosis), a brain tumor, and / or ischemic stroke. In some embodiments, use of the systems, devices, and methods disclosed herein may address a primary injury / cause for neurologic disease and / or reduce, or at times, eliminate secondary injury caused by, for example, inflammation.

[0051] The systems, devices, and methods disclosed herein may be applied to a subject in any neurological condition. Thus, they may be used on subjects who are conscious, unconscious, and / or have recently suffered (or may have suffered) a brain injury or trauma to, for example, improve outcomes, reduce injury, and / or lower mortality, disability, and / or complication rates for subjects. In some embodiments, the systems, devices, and methods disclosed herein may be used repeatedly to progressively clear waste from the brain over time and may be used to clear waste positioned deep within the brain caused by, for example, a deep-brain hematoma, that would otherwise be untreatable / inoperable.

[0052] In some embodiments, the systems, devices, and methods disclosed herein may be configured to limit and / or mitigate neuroinflammatory and / or neurotoxic responses followinghemorrhagic injury. At times, use of the systems, devices, and / or methods disclosed herein may dampen the brain’s inflammatory response to hemorrhage and / or injury by, for example, significantly reducing microglial activation (indicated by IBA-1 ) and / or astrocytic reactivity (indicated by GFAP) via, for instance mechanosensitive channel activation. Additionally, or alternatively, use of the systems, devices, and / or methods disclosed herein may reduce neurotoxicity in the brain following injury, which can confer neuroprotection against secondary injury caused by the toxins (e.g., inflammation) and / or brain edema by shifting the state of neuroimmunologic cells (e.g. microglia) from their disease-associated phenotype to their reparative, homeostatic phenotype via, for example, a mechanosensitive channel mediated mechanism.

[0053] Additionally, or alternatively, the systems, devices, and / or methods disclosed herein may be used to directly and / or indirectly sculpt the neuroimmune environment to, for example, yield a more reparative state of the resident microglia and / or immune cells in the brain. This may, for example, dampen immune responses which may delay and / or reverse progress of autoimmune neurological disease (e.g., multiple sclerosis) and / or an inflammatory response to trauma (e.g., concussion or stroke) that may cause secondary injury. Thus, the systems, devices, and methods disclosed herein may be used to treat autoimmune diseases that affect the brain by administering UCC treatment sessions via one or more of the UCC devices disclosed herein over time (e.g., weeks, months, years, and / or a subject’s lifespan).

[0054] Additionally, or alternatively, use of the systems, devices, and / or methods disclosed herein may be configured to increase CSF flow and / or brain waste clearance via a mechanically mediated effect using mechanotransduction pathways (e.g., mechanosensitive ion channels) rather than and / or in addition to passive convective forces or thermal effects. In some cases, this mechanically mediated effect may directly induce a convection or oscillation of the CSF and / or ISF within the brain and / or or a mechanical change of the extracellular matrix to then drive downstream CSF circulation.

[0055] In some embodiments, the systems, devices, and / or methods disclosed herein may be configured to act as a noninvasive and nonpharmacologic agonist of molecular mechanotransduction pathways to elicit increased CSF and / or ISF circulation and egress and / or induce a prolonged cascade of events lasting, for instance, tens of minutes to hours leading to CSF and / or ISF circulation upregulation and, consequently, brain waste clearance. This may be caused by actuation of mechanosensitive channels, which are known to play a critical role in cellular responses to mechanical stimuli, such as the ultrasound provided by the systems, devices, and methods disclosed herein. Additionally, or alternatively, the systems, devices, and / or methods disclosed herein may be configured to provide a mechanosensitive channel mediated mechanism that does not depend on sensory stimulation or a specific neural activity pattern as mechanism for action for efficient brain waste clearance.

[0056] The systems, devices, and methods disclosed herein may be operative and / or configured to induce mechanosensitive channel activation that may act to directly shift microglial phenotype from a disease-associated state to a hemeostatic state, which may affect neuroimmunologic state, increase phagocytic activity, and / or yield disinhibition and, in some cases, direct induction of aquaporin astrocytic endfeet polarization, which may increase glymphatic waste evacuation efficacy. Additionally, or alternatively, systems, devices, and methods disclosed herein may be operative and / or configured to increase mechanical mixing between intracranial fluid compartments and, over time (e.g., successive applications of UCC treatment), may also be configured to increase meningeal lymphatic sprouting, both of which may improve clearance of waste from the brain and central nervous system. For example, when a hemorrhagic stroke occurs, aquaporin expression and disease-associated microglial states are promoted. The disease-associated microglia inhibit the polarization of aquaporins to astrocytic endfeet. To counter these effects, the systems, devices, and methods disclosed herein provide UCC treatment to the brain, which may activate mechanosensitive ion channels and / or mechanically encourage intermixing of CSF and / or ISF. Activation of the mechanosensitive ion channels may shift microglia from the disease-associated state caused by the hemorrhagic stroke to the homeostatic state. This microglial phenotypic shift may yield increased waste (e.g., red blood cell) phagocytosis and / or disinhibition of astrocytic endfeet aquaporin polarization. One or more of these effects (i.e., (mechanical fluid mixing, aquaporin polarization, increased debris phagocytosis) of UCC treatment may reduce neuroinflammation, enhance bulk flow of CSF and / or ISF, and / or otherwise improve clearance of the waste caused by the hemorrhagic stroke from the subject’s brain via evacuation of the debris from the brain via the meningeal lymphatics to the deep cervical lymph nodes.

[0057] Possible advantages of the systems, devices, and methods disclosed herein include, but are not limited to, improved outcomes and / or accelerated recovery for subjects. Additionally, or alternatively, the systems, devices, and methods disclosed herein may be used in a variety of settings including, but not limited to, subject bedside, rehabilitation (inpatient and / or outpatient), and / or home care settings, thereby increasing access to neurological treatment. Additionally, or alternatively, the systems, devices, and methods disclosed herein may be provided at a lower price point than traditional high-risk and high-cost treatments like neurosurgery (e.g., stereotactic or endoscopic hematoma evacuation, shunt placement, etc.), which often require highly skilled teams of surgeons and prolonged intensive care hospital stays.

[0058] Turning now to the figures, FIG. 1 A provides a cut-away diagram of a subject 100 with a portion of his / her brain 1 10 showing. Also shown in FIG. 1 A are subject’s choroid plexus 1 15, and cervical lymph nodes 120. FIG. 1 B is a detailed view of a portion of subject’s brain 1 10 showing a skull 130, lymphatic vessels 132, and a subarachnoid space 134. In someembodiments, the UCC and / or ultrasound protocols described herein may be configured and / or designed to mechanically stimulate choroid plexus 1 15 to activate mechanosensitive channels therein, which may, in turn, modulate CSF production and resorption.

[0059] FIG. 2 is a block diagram of an exemplary system 200 for delivering UCC to a subject. System 200 includes a computer and / or controller 210, an optional display device and / or user interface 215, an optional electronic medical record database 220, an optional communication network 225, an optional power source 260, a UCC treatment protocol database 265, one or more heat sensing devices 270, and a UCC device 230 that includes a housing 235 configured to house a plurality (first-Nth) of ultrasound transducers 240A-240N, a communication port 250, a sensor 280, an optional contact sensor 285, and an optional power coupling 255.

[0060] Computer and / or controller 210 may be any device configured to digitally process information including, but not limited to, a processor, a laptop computer, a tablet computer, and / or a smart phone. Optional display device and / or user interface 215 may be any display device 215 (e.g., speaker, screen, touch screen, series of lights, etc.) configured to provide information to a user and optional user interface device(s) 215 include, but are not limited to, keyboards, trackpads, and microphones configured to receive input from a user. Optional electronic medical record database 220 may be any database storing medical information and / or records for a subject and / or a set of subjects. In some embodiments, access to optional electronic medical record database 220 may be via a third party computer system (not shown). Power source 260 may be any source of electrical power including, but not limited to, a battery and / or a coupling to a wall outlet. In some cases, power source 260 may be resident within UCC device 230 and, in these embodiments, power source 260 may be embodied as a battery and power coupling 255 may be embodied as an electrical coupling to the battery.

[0061] In some embodiments, computer and / or controller 210 may be configured to act as a communication terminal to cloud computing platform 275 via, for example, communication network 225 and may facilitate provision of the results of machine learning and / or artificial intelligence calculations (e.g., training, testing, and / or tuning of a model and / or algorithm) performed on cloud computing platform 275. Exemplary computers 210 include desktop and laptop computers, servers, tablet computers, personal electronic devices, mobile devices (e.g., smart phones), and the like. Exemplary display devices 215 are computer monitors, tablet computer devices, and displays provided by one or more of the components of system 200. In some instances, display and / or user interface 215 may be resident in computer and / or controller 210. In some embodiments, UCC treatment protocol database 265 and / or another datastore or memory within and / or in communication with computer and / or controller 210 and / or cloud computing platform 275 may be configured to store inputs and / or rules used for machine learning, training, and / or sets of instructions for computer and / or controller 210 and / or cloud computing platform 275 as, for example, described herein with reference to, for example,method(s) 400 and / or 500 as shown in FIGs. 4 and 5, respectively, and discussed below. In some embodiments, computer and / or controller 210 may be configured as a controller that controls operation (e.g., duty cycle, intensity, etc.) of UCC device 230 and / or one or more ultrasound devices 240A-240N that may, or may not, be in communication with a computer and / or other processing device (e.g., cloud computing platform 275) configured to, for example, execute one or more methods and / or processes described herein. In some embodiments, controller 210 may be embodied as a switch or plurality of switches that turn on and / or off responsively to instructions from a processor like computer 210 when system 200 includes a controller and a computer and / or cloud computing platform 275.

[0062] Cloud computing platform 275 may be any cloud computing platform configured to run a machine learning and / or artificial intelligence software program and / or support a machine learning and / or artificial intelligence architecture such as TensorFlow. Exemplary cloud computing platforms include, but are not limited to, Amazon Web Service (AWS), Rackspace, and Microsoft Azure. Exemplary machine learning and / or artificial intelligence architectures include neural networks, deep neural networks, artificial neural networks, Bayesian networks, and / or software or hardware that utilizes artificial intelligence.

[0063] Heat sensing device 270 may be configured to measure heat within / on the subject's scalp and / or brain to, for example, assist with determining whether the subject is being exposed to too much ultrasound and / or that the UCC device is working properly. In some embodiments, heat sensing device 270 may be embodied as a thermometer configured to measure a scalp temperature of the subject. Additionally, or alternatively, heat sensing device 270 may be embodied as a heat-sensing camera configured to measure a temperature within the subject’s head. Sensor 280 may be configured as, for example, an ultrasound sensor and / or microphone configured to measure ultrasound reflected from the subject, which may be used to, for example, determine efficacy of UCC device 230 and / or whether the ultrasound is causing cavitation within the subject’s brain (which would reflect the ultrasound projected by one or more ultrasound devices 240A-240N of UCC device 130) and / or whether the ultrasound is incident upon an implant within the brain that may reflect the ultrasound. Additionally, or alternatively, sensor 280 may be configured as a tissue oximetry sensor, a functional near-infrared spectrometry (fNIRS) sensor, and / or an electroencephalogram (EEG) sensor.

[0064] Housing 235 may be embodied as, for example, a helmet, a band, and / or a hat that, in some embodiments, may be adjustable and / or configurable to, for example, a size of a subject’s head and / or a type of treatment to be provided to the subject. In some embodiments, housing 235 and / or placement of one or more ultrasound devices 240A-240N may be adjustable to accommodate regions of a subject’s head to which delivery of ultrasound is not desired as may be the case with, for example, external wounds (e.g., bruises, abrasions, orsurgical incisions) and / or brain implants. Adjustments may include removal of one or more ultrasound devices 240A-240N from housing 235 that would otherwise overly a region of the brain, scalp, or subject tissue to which ultrasound is not to be applied and / or movement of one or more ultrasound devices 240A-240N within housing 235. In one embodiment, ultrasound devices 240A-240N may be embodied as an ultrasound transducer with a center frequency of 250kHz, a radius of curvature of, for example, 30-90mm, 50mm and / or 70mm, and an f (i.e., ratio of the radius of curvature to the diameter of the ultrasound device) equal to approximately 1 .

[0065] The UCC devices 230 disclosed herein may be configured to deliver ultrasound to one or more regions of the brain serially and / or simultaneously via a plurality of ultrasound devices 240A-240N. Each ultrasound device 240A-240N may be configured to sonicate an underlying region of the subject’s brain and the ultrasound may propagate through and underlying column of brain tissue that is approximately 4-10mm, 5-8mm, 3-30mm, 2-25mm, 10mm, 7mm, 5mm, and / or 3mm wide at the skull. In some embodiments, a shape of the column of ultrasound may taper, or narrow, as it propagates in the brain to reach an approximate focal point proximate to the center of the subject’s brain. In some instances, ultrasonic devices 240A-240N may be arranged within housing 235 to cause constructive interference between pressure waves caused by the ultrasound for some regions of the brain. Additionally, or alternatively, ultrasound devices 240A-240N may be arranged and / or operated so that foci peaks of ultrasound devices 240A-240N are separated in space (i.e., do not overlap) within the brain to, for example, avoid undesirable (e.g., too intense and / or heatgenerating) levels of ultrasound delivery. This may be accomplished by, for example, a UCC treatment protocol as disclosed herein and / or using a duty cycle of 20%, 25%, 30%, 50% or 75% for one or more of the ultrasound devices included in a UCC device to interleave which ultrasound devices operate at the same time.

[0066] In some embodiments, ultrasound devices 240A-240N may be arranged and / or operated so that approximately the entire brain (with the possible exception of the brain stem or near the skull base) receives approximately the same amount / intensity of ultrasound. Additionally, or alternatively, ultrasound devices 240A-240N may be arranged and / or operated so that central brain regions (e.g., ventricles, thalamus, central subcortical brain, and / or superficial meninges) receive more ultrasound than other brain regions. In some cases, the UCC devices disclosed herein may be configured so that no, or minimal, ultrasound is delivered to the skull base to, for example, provide safe delivery of UCC therapy by UCC device 230. Additionally, or alternatively, ultrasound devices 240A-240N may be arranged and / or operated so that particular regions of interest of the brain receive more ultrasound (e.g., greater intensity) than other regions. For example, if the subject has suffered an ischemic stroke, the location of the ischemic stroke may be a region of interest and ultrasound devices240A-240N proximate to and / or within a line-of-sight of the region of interest may receive project more ultrasound into the subject’s brain than other ultrasound devices 240A-240N buy, for example, projecting ultrasound from ultrasound devices 240A-240N that have foci intersecting with and / or proximate to the region of interest.

[0067] UCC treatment protocol database 265 may be configured to store a plurality (e.g., 1 ,000-100,000,000) UCC treatment protocols and / or aspects and / or features of UCC treatment protocols. Additionally, or alternatively, UCC treatment protocol database 265 may be configured to store instructions for the delivery of UCC by one or more of the UCC devices and / or systems disclosed herein according to one or more of the UCC treatment protocols.

[0068] The UCC treatment protocols stored in UCC treatment protocol database 265 may be associated with and / or indexed to, for example, one or more of a diagnosis, subject characteristic, desired outcome, symptoms, subject comorbidities, additional treatments (e.g., medication, physical therapy, etc.) the subject has and / or is receiving, an indication of whether a neurological condition of the patient is chronic, acute, severe, and / or mild, and / or a time period that has elapsed since an acute incident for the subject’s brain. Additionally, or alternatively, the UCC treatment protocols stored in UCC treatment protocol database 265 may be manually generated and / or generated via execution of one or more machine learning, artificial intelligence, and / or modeling processes as, for example, described herein with regard to FIG. 4 and method 400.

[0069] In some embodiments, various UCC treatment protocols may share some characteristics and, in other embodiments, some UCC treatment protocols may be unique and / or tailored to particular circumstances like subject characteristics, symptoms, and / or diagnoses. For example, in many circumstances a UCC treatment protocol may be configured with features to increase bulk CSF and / or ISF circulation, induce CSF and / or ISF mixing, shift neuroimmunologic state, increase / disinhibit aquaporin polarization, and / or activate mechanosensitive ion channels. These features may adjusted and / or added to for UCC treatment protocols configured for particular circumstances. For example, for acute indications (e.g. ischemic or hemorrhagic stroke) a UCC treatment protocol may be applied frequently in the acute phase of disease (e.g. 3 times within a week or daily) for extended applications (e.g. 30-60 min) to maximize the rate of increase of the ultrasound-induced effects. In some embodiments, for chronic conditions (e.g. sequela of repeated mild traumatic brain injury, chronic traumatic encephalopathy, neurodegenerative diseases) a UCC treatment protocol may be applied less frequently (e.g. once weekly) and for shorter durations (e.g. 10- 30 min).

[0070] In some embodiments, a UCC treatment protocol may have a plurality of phases that change over time as measured from an incident. For example, an initial phase of a UCC treatment protocol for a subject suffering from a hemorrhagic stroke may include dailyapplications of UCC for 60 minutes for three days following the hemorrhagic stroke, then the UCC treatment protocol may be changed to daily applications of UCC for 30 minutes for the next three days (i.e., days 4-6) following the hemorrhagic stroke, then the UCC treatment protocol may be changed again to applications of UCC for 30 minutes every other day for the eighth, tenth, and twelfth days following the hemorrhagic stroke and / or until all waste from the hemorrhagic stroke is cleared from the subject’s brain.

[0071] Contact sensor 285 may be one or more optional sensors configured to provide information and / or feedback to, for example, computer and / or controller 210 and / or cloud computing platform 275 that indicates whether housing 235 and / or one or more ultrasound device(s) 240A-240N are in contact with the subject (e.g., head, scalp, forehead, and / or skin). In some embodiments, contact sensor 285 may be configured to measure signal strength and / or a proportion of ultrasound emitted by one or more of the ultrasound devices 240A-240N that is reflected and / or backscattered from the subject tissue. In these embodiments, the processor 210 and / or cloud computing platform 275 may determine whether a magnitude of the reflected ultrasound is above a threshold and, if so, may provide an error indication (e.g., message to display and / or user interface 215 and / or illumination of an indicator light) to a user and / or operator to inform the user and / or operator that housing 235 may not be properly seated on the subject’s head and / or projecting ultrasound into the subject’s head as intended and / or proscribed by, for example, a UCC treatment protocol as disclosed herein.

[0072] Additionally, or alternatively, contact sensor 285 may be configured as an electrical lead, or electrode, configured to detect direct, or indirect (e.g., via conductive gel) contact with skin (e.g., scalp) and provide information (e.g., an electrical signal) to computer and / or controller 210 and / or cloud computing platform 275. In these embodiments, the processor 210 and / or cloud computing platform 275 may determine whether the electrical signal indicates that housing 235 is properly seated on the subject's head and provide an error indication (e.g., message to display and / or user interface 215 and / or illumination of an indicator light) to a user and / or operator to inform the user and / or operator that housing 235 may not be properly seated on the subject’s head and / or projecting ultrasound into the subject’s head as intended and / or proscribed by, for example, a UCC treatment protocol as disclosed herein.

[0073] FIG. 3A provides a top view of a first exemplary UCC device 230A with four ultrasound devices in a first housing 235A configured to fit over a subject’s head and orient an active side of the ultrasound devices toward the subject’s head and / or brain. Housing 235A includes a first ultrasound device 240A positioned on a front side (as oriented in FIG. 3A) of housing 235A so that it may be positioned over / proximate to a subject’s forehead when worn, a second ultrasound device 240B positioned on a back side of housing 235A so that it may be positioned proximate to the back of subject’s head when worn, a third ultrasound device 240C positioned on a left side (as oriented in FIG. 3A) of housing 235A so that it may be positioned proximateto a left side of the subject’s head when worn, and a fourth ultrasound device 240D positioned on a right side (as oriented in FIG. 3A) of housing 235A so that it may be positioned proximate to a right side of the subject’s head when worn.

[0074] FIGs. 3B-3D provide various views of a second exemplary UCC device 230B that includes a second housing 235B embodied as a helmet configured to fit over a portion of a subject’s head and eight ultrasound devices 240A-240H. In particular, FIG. 3B provides a side view of UCC device 230B being worn by subject 100, FIG. 3C provides a front-perspective view of UCC device 230B, and FIG. 3D provides and underside perspective view of UCC device 230 B.

[0075] Housing 235B includes first ultrasound device 240A positioned on a front right side (as oriented in FIG. 3C) of housing 235B so that it may be positioned over / proximate to a right side of a subject’s forehead when worn as shown in FIG. 3B, second ultrasound device 240B positioned on a lower-right-front side (as oriented in FIG. 3C) of housing 235B so that it may be positioned proximate to (e.g., in front of) a subject's right ear when worn as shown in FIG. 3B, third ultrasound device 240C positioned on a lower-right-back side (as oriented in FIG. 3D) of housing 235B so that it may be positioned proximate to (e.g., behind) a subject’s right ear when worn as shown in FIG. 3B, fourth ultrasound device 240D positioned in the upper middle (as oriented in FIG. 3C) of housing 235B so that it may be positioned proximate to a top-right side of a subject’s head when worn as shown in FIG. 3B, a fifth ultrasound device 240E positioned on a front left side (as oriented in FIG. 3D) of housing 235B so that it may be positioned proximate to (e.g., over) a left side of a subject’s forehead when worn in a manner similar to that of first ultrasound device 240A, a sixth ultrasound device 240F positioned on a lower-left-front side (as oriented in FIG. 3D) of housing 235B so that it may be positioned proximate to (e.g., in front of) a subject’s left ear when worn in a manner similar to that of second ultrasound device 240B, a seventh ultrasound device 240G positioned on a lower-left- back side (as oriented in FIG. 3D) of housing 235B so that it may be positioned proximate to (e.g., behind) a subject’s left ear when worn in a manner similar to that of third ultrasound device 240C, and an eighth ultrasound device 240H positioned on in the upper middle (as oriented in FIG. 3D) of housing 235B so that it may be positioned proximate to a top-left side of a subject’s head when worn in a manner similar to that of fourth ultrasound device 240D.

[0076] FIG. 3E provides a top view, FIG. 3F provides a front view, FIG. 3G provides a back view, FIG. 3H provides a side view, and FIG. 3I provides an underside view of a third exemplary UCC device 230C that includes ultrasound devices of differing sizes and / or diameters that include first-eighth ultrasound devices 240A-240H as well as six additional ultrasound devices 240I-240N that may be of a smaller size than first-eighth ultrasound devices 240A-240H and positioned therebetween in a third housing 235C as shown. First- eighth ultrasound devices 240A-240H and smaller ultrasound devices 240I-240N may bearranged within third housing 235C to, for example, maximize coverage area and / or provide approximately uniform delivery of ultrasound to the subject’s brain and / or regions of the brain.

[0077] In particular, third UCC device 230C includes first ultrasound device 240A positioned on a front right side (as oriented in FIG. 3E) of housing 235C so that it may be positioned over / proximate to a right side of a subject’s forehead when worn, second ultrasound device 240B positioned on a lower-right side (as oriented in FIG. 3E) of housing 235C so that it may be positioned proximate to (e.g., in back of) the subject’s right ear when worn, third ultrasound device 240C positioned on a lower-right-back side (as oriented in FIG. 3E) of housing 235C so that it may be positioned proximate to (e.g., behind) the subject’s right ear when worn, fourth ultrasound device 240D positioned in the upper middle (as oriented in FIG. 3E) of housing 235C so that it may be positioned proximate to a top-right side of the subject’s head when worn, fifth ultrasound device 240E positioned on a front left side (as oriented in FIG. 3E) of housing 235C so that it may be positioned proximate to (e.g., over) a left side of the subject’s forehead when worn, sixth ultrasound device 240F positioned on a lower-left-front side (as oriented in FIG. 3E) of housing 235C so that it may be positioned proximate to (e.g., in front of) the subject’s left ear when worn, seventh ultrasound device 240G positioned on a left-back side (as oriented in FIG. 3E) of housing 235C so that it may be positioned proximate to (e.g., above) the subject’s left ear when worn, eighth ultrasound device 240H positioned on in the upper middle (as oriented in FIG. 3E) of housing 235C so that it may be positioned proximate to a top-left side of the subject’s head when worn, a ninth ultrasound device 2401 positioned above first ultrasound device 240A and between fourth and eighth ultrasound device 240D and 240H so that it may be positioned proximate to an upper left forehead of the subject's head when worn, a tenth ultrasound device 240J triangulated between first, second, and fourth ultrasound device 240A, 240B, and 240D so that it may be positioned proximate to the subject’s left ear when worn, an eleventh ultrasound device 240K positioned on a right lower side of third housing 235C next to third ultrasound device 2400 so that it may be positioned proximate to lower-right-back side of the subject’s head when worn, a twelfth ultrasound device 240L positioned above third ultrasound device 240C and between second and seventh ultrasound device 240B and 240G so that it may be positioned proximate to an upper-back side of the subject’s head when worn, a thirteenth ultrasound device 240M positioned above between eleventh and sixth ultrasound device 240K and 240F so that it may be positioned proximate to a lower right side of the subject’s head when worn, and a fourteenth ultrasound device 240N positioned between fourth, seventh, and eighth ultrasound device 240D, 240G, and 240H so that it may be positioned proximate to top of the subject’s head when worn.

[0078] FIG. 4 provides a flowchart illustrating an exemplary method 400 for building and / or training a UCC treatment model and / or evaluating the accuracy of a UCC treatment model. Method 400 may be executed by, for example, system 200 and / or a component thereof. Insome embodiments, method 400 may be executed by, for example, cloud computing platform 275 and / or a combination of cloud computing platform 275, computer and / or controller 210, UCC treatment protocol database 265, network 225, and / or EMR 220.

[0079] Initially, a plurality of sets of information for a respective plurality of subjects may be received (step 405). The received information may include, but is not limited to, pre- and post- UCC treatment brain scans, brain images, diagnostic assessment results (e.g., cognitive assessment results, motor skill assessment results, etc.), blood tests (e.g., blood tests looking for markers of neurological conditions and / or neuroinflammation), demographic information about each subject, medical record information for each subject, and / or UCC treatment information for each subject. The UCC treatment information for each subject may include, but is not limited to, whether or not a subject received a UCC treatment, parameters of a UCC treatment protocol for each subject, and how long each subject received UCC treatments under a UCC treatment protocol. In some embodiments, the plurality of sets of information may include information for healthy controls. Additionally, or alternatively, in some embodiments, each of the plurality of sets of information may be correlated, or otherwise associated with, a number of different categories and / or characteristics that may be specific to, for example, subject demographics, comorbidities, diagnosis, diagnostic test results, a time period following an event (e.g., stroke, concussion, etc.) that triggered a neurological condition associated with a diagnostic test and / or brain image, parameters of a UCC treatment protocol used to treat the subject, and so on. These categories and / or characteristics may be used, for example, select and / or optimize a UCC treatment protocol for a particular subject during, for example, execution of 500 (explained below with regard to FIG. 5).

[0080] In step 410, the plurality of sets of information may be divided into a training set of information and a test set of information. In step 415, a set of machine learning inputs may be selected and / or received. The set of machine learning inputs may be configured and / or selected to train and / or generate a UCC treatment model and may include, but are not limited to, rules for how the UCC treatment model is to be generated, trained, tuned, and / or iterated upon via, for example, execution of process 400, rules for predicting an outcome of a UCC treatment protocol, and / or safety inputs and / or rules.

[0081] In step 420, the training set of information may be input into the machine learning / AI architecture to generate and / or train a first version of a UCC treatment model. The first version of the UCC treatment model may then be tested using the testing set of information (step 425). In step 430, the first version of the UCC treatment model may be updated, adjusted, and / or otherwise iterated upon responsively to an outcome of the testing to, for example, improve the accuracy, safety, and / or efficacy of the UCC treatment model. A result of the execution of step 430 may be a second version of the UCC treatment model.

[0082] Optionally, in step 435, the second version of the UCC treatment model may be used to generate a plurality (e.g., 1000 - 100,000,000) of UCC treatment protocols that may be associated with, for example, one or more subject characteristics (e.g., age, head size, comorbidities, etc.), diagnoses, UCC treatment responsiveness, diagnosis, diagnostic test results, a time period following an event (e.g., stroke, concussion, etc.), brain dimensions and / or anatomy, a position of a brain pathology, parameters of a UCC treatment protocol used to treat the subject, and so on. On some occasions, the UCC treatment protocols generated via execution of step 435 may be stored in UCC treatment protocol database 265.

[0083] In some embodiments, execution of step 435 may include predicting and / or optimizing one or more intercranial pressure fields that may be created by administration of a UCC treatment on a subject and / or calibrate one or more ultrasound devices of a UCC device to achieve desired pressure and / or acoustic emission characteristics. Additionally, or alternatively, execution of step 435 may include predicting and / or optimizing UCC treatment delivery so that the resulting intercranial pressure field(s) improve brain waste clearance, shift neuroimmunologic state, induce CSF and / or ISF fluid mixing, and / or increasing / disinhibiting aquaporin polarization.

[0084] Optionally, in step 440, a set of neurological information for a subject may be received. The set of the set of neurological information may include, but is not limited to, an image of the subject's brain, a dimension of the subject’s head, a diagnosis for the subject, an indication of when an adverse neurological event occurred for the subject, an indication of the subject's responsiveness to UCC treatment, an indication of a treatment the subject is received other than the UCC treatment. The neurological information received in step 440 and / or other information about the subject, which may be provided by, and / or in response to, a query of, for example, an electronic medical record for the subject which may be stored in EMR database 220 may then be input into the second version of the UCC treatment model (step 445) to generate an output (step 450). The output generated and / or received via execution of step 450 may include, for example, a UCC treatment protocol that includes a set of instructions for use of a UCC device like the UCC devices disclosed herein to enhance brain waste clearance as, for example, described herein. In many instances, the UCC treatment protocol of the output is configured and / or programmed to optimize the UCC treatment for the subject using the set neurological information and / or other information about the subject and / or a diagnosis for the subject. Exemplary UCC treatment protocols generated via execution of step 450 may include, for example, a schedule for administration of UCC treatments to the subject, a duty cycle for one or more of the ultrasound devices of a UCC device providing the treatment (e.g., UCC device 230, UCC device 230A, UCC device 230B, or UCC device 230C) over the duration of the UCC treatment, and a feature (e.g., frequency, intensity, beam width, etc.) ofthe ultrasound projected into the subject’s brain by one or more of the ultrasound devices of the UCC device over the duration of the UCC treatment.

[0085] In some embodiments, the output may further include a predicted outcome of the UCC treatment protocol for the subject and, in these embodiments, the predicted outcome may be provided to a display device (e.g., display and / or user interface 215) so that, for example, it may be evaluated by a clinician. In some embodiments, the output may include a plurality (e.g., 3-15) of UCC treatment protocols and, optionally, a predicted outcome for each the UCC treatment protocol for the subject. In these embodiments, each UCC treatment protocol of the plurality of UCC treatment protocols and (when determined) a predicted outcome of each UCC treatment protocol may be provided to a display device (e.g., display and / or user interface 215) so that, for example, it may be evaluated by a clinician and / or selected for use with a subject.

[0086] Optionally, method 400 may continue with step 455 wherein feedback regarding the subject following administration of at least one UCC treatment of the UCC treatment protocol output in step 450 and the UCC treatment model may be updated responsively to the feedback (step 460). Exemplary feedback includes, but is not limited to, a result of a diagnostic test, a result of a blood test for a biomarker of neurological waste, a chemical composition of lymphatic fluid extracted from a cervical lymph node, an indication of heat within the subject's scalp or brain while the subject is undergoing the at least one UCC treatment, an indication of cavitation within the subject’s scalp or brain while the subject is undergoing the at least one UCC treatment, an image of the subject’s brain, and a score on a medical questionnaire and / or patient reported outcome (PRO) instrument. In some embodiments, the updated UCC treatment model of step 460 may be used to execute, or re-execute, step 435 to generate one or more updated UCC treatment protocols.

[0087] FIG. 5 provides a flowchart illustrating an exemplary method 500 for determining operational and / or therapeutic parameters for a UCC device and providing UCC treatment and / or administering a UCC treatment protocol to a subject. Method 500 may be executed by, for example, system 200 and / or a component thereof.

[0088] Initially, in step 505, information about a subject (e.g., subject 100) may be received. Information about the subject received in step 505 may include, but is not limited to, identifying information, a diagnosis for the subject, comorbidities of the subject, a neurological state of the subject, images of the subject’s brain, head and / or brain size and / or volume measurements, medications the subject is taking, prior treatment treatments the subject has received, a medical condition for the subject, responsiveness of the subject to a therapy, information regarding the subject’s cognitive abilities, and information regarding previously received UCC treatments. Additionally, or alternatively, a size of use of different housings (e.g., housing 235, housing 235A, housing 235B, or housing 235C) for subjects of differentsizes. In some embodiments, information about the subject may be received from the subject’s, electronic medical record, which may be stored in EMR database 220 and accessed by computer and / or controller 210.

[0089] In step 510, one or more operational and / or therapeutic parameters for a protocol regarding the use of a UCC device, like UCC device 230, UCC device 230A, UCC device 230B, or UCC device 230C, for the subject and / or ways of testing efficacy of the protocol may be determined. Operational and / or therapeutic parameters determined in step 510 may include, but are not limited to, a frequency, pressure, pulse width, duration of the delivery, duty cycle, and / or a schedule for the delivery of ultrasound by one or more of the ultrasound devices (e.g., ultrasound devices 240A-240N) of the UCC device into the subject’s brain. For example, in some embodiments, an operational parameter for the ultrasound devices may be to project ultrasound into a subject’s brain with a frequency within a range of about 100 kHz to about 1000 kHz, about 200 kHz to about 500 kHz, about 250 kHz to about 450 kHz, at least about 100 kHz, at least about 200 kHz, at least about 250 kHz, at least about 350 kHz, at least about 450 kHz, up to about 1000 kHz, up to about 900 kHz, up to about 800 kHz, up to about 700 kHz, up to about 600 kHz, and / or up to about 500 kHz.

[0090] Additionally, or alternatively, execution of step 510 may include using subject information regarding, for example, head and / or brain size and / or volume measurements to select and / or adjust a size of a housing (e.g., housing 235, housing 235A, housing 235B, or housing 235C) and / or a geometrical feature (e.g., penetration depth, size, and / or shape of ultrasound column) for the ultrasound projected into the subject’s skull. In some embodiments, there may be a plurality of housing sizes (e.g., small, medium, and large) and / or configurations (e.g., number of ultrasound devices 240) that may be determined via execution of step 510. Additionally, or alternatively, one or more images and / or scans (e.g., CT and / or MRI images) of a subject’s brain may be received in step 505. These images may be analyzed to extract one or more features and / or characteristics (e.g., size, position of a pathological feature, etc.) thereof and, in step 510, the features and / or characteristics of the subject’s brain may be used to determine, query for, and / or select operational and / or therapeutic parameters for use of the UCC device and / or administration of a UCC treatment protocol. For example, the images and / or scans may be used to select, query for, generate, and / or determine operational parameters that may optimize intercranial pressure field(s) created by administration of a UCC treatment on the subject using.

[0091] Additionally, or alternatively, an operational parameter for the ultrasound devices may be to project ultrasound into a subject’s brain at an in situ negative pressure within a range of 0.05 MPa to 0.6 MPa, about 0.05 MPa to about 0.6 MPa, about 0.05 MPa to about 0.2 MPa, about 0.2 MPa to about 0.5 MPa, about 0.3 MPa to about 0.5 MPa, about 0.45 MPa, and / or an in situ peak negative pressure within a range of about 0.2-0.7MPa or of 0.45 MPa.Additionally, or alternatively, an operational parameter for the ultrasound devices may be to project ultrasound into a subject’s brain with a pulse width from about 5 ps to about 50 ms, from about 5 ps to about 100 ms, from about 5 ms, up to about 500 ms, from about 500 ps to about 500ms, from about 1 ms to about 100ms, about 100 ps, about 500 ps, about 1 ms, up to about 250 ms, up to about 100 ms, and / or about 50 ms.

[0092] Additionally, or alternatively, an operational parameter for the ultrasound devices may be a time period for the projection of ultrasound into a subject's brain. Exemplary durations include, but are not limited to, 1 -60 minutes, 10-30 minutes, about 20 minutes, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, up to about 60 minutes, up to about 45 minutes, and / or up to about 30 minutes. Additionally, or alternatively, an operational parameter for the ultrasound devices may be to project ultrasound into a subject’s brain with a duty cycle of from about 1 -100%, from about 5% to about 75%, from about 10% to about 50%, and from about 20% to about 40%.

[0093] Additionally, or alternatively, an operational parameter for the ultrasound devices may be a schedule of use of the UCC device. Exemplary schedules of use include, but are not limited to, a one-time use, once a day, a plurality of times over one day, one time per day for a plurality of days, a plurality of times over a plurality of days, once every two, three, four, five, six, or seven days for a period of from about 1 , about 2, about 3, about 4, about 5, about 6, about 7 or more weeks, up to a chronic maintenance level. Intervals between a single treatment can be weekly, monthly, or yearly. Intervals can also be irregular.

[0094] Additionally, or alternatively, an operational parameter for the ultrasound devices may be a schedule of use of the UCC device to administer treatment to a subject suspected of, or already suffering from, such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and / or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In therapeutic applications, a treatment at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, a prophylactic regime may be administered to the subject as needed. In prophylactic applications, an operational parameter for the ultrasound devices and / or a UCC device may be a schedule of relatively infrequent intervals over a long period of time (e.g., years or decades), wherein some subjects may continue to receive periodic and / or as-needed treatment for the rest of their lives. One exemplary prophylactic UCC treatment protocol may be designed for subjects who may be at a higher risk than the general population of encountering neuroinflammation and / or concussion as may be the case for certain types of athletes (e.g., American football players, boxers, soccer players, and / or martial artists) and / orprofessionals who may suffer frequent head injuries and / or encounter percussive force to the head as may be the case with soldiers and other defense personnel who work with artillery. In these instances, a prophylactic UCC treatment protocol may be designed for use on a regular basis (e.g., 30 minutes once a week) and / or on an as-needed basis such as after exposure to an event (e.g., game, match, and / or launching of artillery rounds) that may cause neuroinflammation and / or concussion.

[0095] In embodiments and / or use cases involving the treatment of an acute condition, including without limitation traumatic brain injury; hemorrhage, including subarachnoid and intraparenchymal hemorrhage; ischemic injury; and the like, operational parameters for the UCC device may include establishing a treatment schedule to be performed shortly (e.g., within about 3 days, within about 2 days, within about 1 day, and may be performed with 24 hours, within 18 hours, within 12 hours, within 6 hours, within 3 hours) after an injury occurs or is diagnosed.

[0096] In step 515, an indication that the UCC device is in position on the subject’s head and / or is ready to be used may be received and then emission of the ultrasound into the subject’s brain by one or more of the ultrasound devices of the UCC device may begin (step 520). In some embodiments, the indication received in step 515 may include an indication that the subject’s head has been properly prepared for receipt of a UCC treatment via the UCC device. This preparation may include, but is not limited to, application of gel or other substances to the head to facilitate communication of the ultrasound to the subject’s brain, evaluation of the subject’s head and / or hair to determine whether changes (e.g., shaving, cutting, and / or hairstyle adjustments (e.g., unbraiding and / or removing hair from behind the ears)) thereto are necessary and, if so, they may be performed. Optionally, in step 525, the subject and / or the subject’s brain may be monitored during and / or following execution of step 520 to, for example, determine treatment efficacy and / or whether any adverse side effects have occurred and / or are occurring and, if so, it may be determined (step 530) whether an adjustment to the UCC treatment protocol and / or a manner in which the ultrasound is delivered to the subject’s brain is needed. When an adjustment is not needed, execution of step 520 may continue until the conclusion of the UCC treatment protocol and / or method 500 may end. When an adjustment is needed, an operation and / or therapeutic parameter for the use of the UCC may be adjusted (step 535) and method 500 may proceed back to step 520 or process 500 may end. In some embodiments, results of step 525 may be used to modify an instant and / or ongoing UCC treatment protocol for the subject and / or provide feedback for updating a UCC treatment model and / or UCC treatment protocol generated by a UCC treatment model as part of execution of method 400 and, in particular, steps 455 and 460 of method 400.

[0097] In some embodiments, the monitoring of step 525 may include performance of brain imaging using, for example, functional magnetic resonance imaging (fMRI) and / or positronemission tomography (PET) to, for example, measure hematoma size and / or a severity of edema. Additionally, or alternatively, the monitoring of step 525 may include monitoring brain electrophysiology via, for example, EEG, monitoring tissue oximetry, monitoring cognition via, for example, providing a questionnaire to a subject and scoring his or her responses, measuring intercranial pressure, and / or performing a behavioral assessment. Additionally, or alternatively, the efficacy of the execution of step 520 may be monitored by sampling and testing CSF and / or lymph extracted from, for example cervical lymph nodes (e.g., cervical lymph nodes 120) for the presence of pathogenic products and / or waste cleared from the brain. Additionally, or alternatively, the efficacy of the execution of step 520 may be monitored by sampling and testing blood from intravenous samples for the presence of pathogenic products and / or waste cleared from the brain and / or markers in the bloodstream associated with neurologic conditions like concussion, traumatic brain injury, brain cancer, stroke, and / or neuroinflamation.

[0098] Additionally, or alternatively, one or more devices on a UCC device may provide information that may be used to execute step 525. For example, sensor 280 may be used to sense, or detect, backscattered and / or reflected ultrasound to, for example, monitor for efficient delivery and / or desired penetration of the ultrasound. When higher than expected and / or desired levels of reflected ultrasound are detected, this may indicate inefficient transmission of the ultrasound, which may be caused by, for example, cavitation (i.e., trapped air bubbles) that reflect the ultrasound / prevent the ultrasound from fully penetrating to the desired depth within the brain and / or an implant (e.g., surgical hardware) within the brain. Additionally, or alternatively, heat sensing device 270 may measure scalp temperature and / or brain temperature to determine if the subject’s scalp is becoming overheated, which may be a safety concern that would end execution of method 500 or require an adjustment to the UCC treatment protocol being used.

[0099] FIG. 6 provides a schematic diagram of an exemplary way in which step 420 may be executed wherein subject 100 is wearing second housing 235B of UCC device 230 as shown in FIG. 3B so that ultrasound may be delivered to subject’s 100 brain 1 10 as shown. In particular, FIG. 6 shows a first zone of ultrasound 620A emitted by first ultrasound device 240A that penetrates into the front right side of brain 110, a second zone of ultrasound 620B emitted by second ultrasound device 640B that penetrates into the front-right-lower side of brain 110, a fourth zone of ultrasound 620D emitted by fourth ultrasound device 240D that penetrates into the upper-right side of brain 1 10, a fifth zone of ultrasound 620E emitted by fifth ultrasound device 240E that penetrates into the front-left side of brain 1 10, a sixth zone of ultrasound 620F emitted by sixth ultrasound device 240F that penetrates into the front-left-lower side of brain 110, and a eighth zone of ultrasound 620H emitted by eighth ultrasound device 240H that penetrates into the upper-left side of brain 1 10 as shown.[000100] FIGs. 7A-7C provide beam plots of a simulation of ultrasound being projected into a subject’s brain using, for example, second UCC device 230B, wherein FIG. 7A provides a beam plot 701 of the subject's brain in the X-Z plane showing position in the Z-direction in mm as a function of position in the X-direction in mm, FIG. 7B provides a beam plot 702 of the subject’s brain in the X-Y plane showing position in the X-direction in mm as a function of position in the Y-direction in mm, and FIG. 7C provides a beam plot 703 of the subject’s brain in the Y-Z plane showing position in the Z-direction in mm as a function of position in the Y- direction in mm. In the simulation of FIGs. 7A-7C, most of the subject’s brain experiences ultrasound of some magnitude / pressure (e.g., approximately 0.1 -0.22MPa), which is not directly shown in the beam plots, and there are regions of the brain proximate to and / or underlying an ultrasound device exposed to ultrasound of a higher magnitude / pressure (e.g., 0.3-1 ,2MPa). Regions of the brain exposed to the ultrasound of the higher magnitude / pressure are shown in the beam plots of FIGs. 7A-7C as black marks and / or outlined in black (i.e., a region of the brain outlined in black is exposed to ultrasound above 0.3MPa).[000101] In particular, FIG. 7A shows a simulation of how fifth zone 620E, sixth zone 620F, eighth zone 620H, and a third zone 620A may propagate through tissue when fifth, sixth, eighth, and third ultrasound devices 240E, 240F, 240H, and 240C, respectively, project ultrasound into the subject's brain. FIG. 7B shows a simulation of how second zone 620B and sixth zone 620F may propagate through tissue when fifth, sixth, eighth, and third ultrasound devices 240E, 240F, 240H, and 240C, respectively, project ultrasound into the subject’s brain. FIG. 7C shows a simulation of how first zone 620B, third zone 620C, fifth zone 620E, and A seventh zone 620G may propagate through tissue when first, third, fifth, and seventh ultrasound devices 240A, 240C, 240E, and 240G, respectively, project ultrasound into the subject’s brain.[000102] As used herein, the terms “about” or “approximate” and the like are synonymous and are used to indicate that the value modified by the term has an understood range associated with it, where the range can be ± 20%, ± 15%, ± 10%, ± 5%, or ± 1 %. The terms “substantially” and the like are used to indicate that a value is close to a targeted value, where close can mean, for example, the value is within 80% of the targeted value, within 85% of the targeted value, within 90% of the targeted value, within 95% of the targeted value, or within 99% of the targeted value.Methods of Treatment[000103] Hemorrhagic Stroke. Cerebrovascular accident (CVA), otherwise called a stroke, is the third major cause of morbidity and mortality in many developed countries. Hemorrhagic stroke contributes to 10% to 20% of strokes annually. Stroke can be either ischemic orhemorrhagic. Ischemic stroke is due to the loss of blood supply to an area of the brain. It is a common type of stroke.[000104] Hemorrhagic stroke is due to bleeding into the brain by the rupture of a blood vessel. The common sites of the bleed are the basal ganglia (50%), cerebral lobes (10% to 20%), the thalamus (15%), pons and the brain stem (10% to 20%), and the cerebellum (10%). Hemorrhagic stroke may be further subdivided into intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). ICH is bleeding into the brain parenchyma, and SAH is bleeding into the subarachnoid space. Hemorrhagic stroke is associated with severe morbidity and high mortality. Early diagnosis and treatment are essential given the usual rapid expansion of hemorrhage, causing sudden deterioration of consciousness and neurological dysfunction.[000105] Hypertension is the most common cause of hemorrhagic stroke. Longstanding hypertension produces degeneration of media, breakage of the elastic lamina, and fragmentation of smooth muscles of arteries. Cerebral amyloid angiopathy (CAA) is another important cause of primary lobar intracerebral bleeding in older adults. Cigarette smoking and moderate or heavy alcohol consumption, and chronic alcoholism are significant risk factors. Chronic liver disease also increases the chance of ICH due to coagulopathy and thrombocytopenia. Decreased low-density lipoprotein cholesterol and low triglycerides are also risk factors. Dual antiplatelet therapy has an increased risk of ICH than monotherapy. Sympathomimetics such as cocaine, heroin, amphetamine, ephedrine, and phenylpropanolamine carry an increased risk of a cerebral hemorrhage. Cerebral microbleeds (CMBs) associated with hypertension, diabetes mellitus, and cigarette smoking increase the risk of ICH. The incidence of ICH increases after 55 years of age. The relative risk after 70 years is 7.[000106] The usual causes of spontaneous subarachnoid hemorrhage (SAH) are ruptured aneurysm, arteriovenous malformation, vasculitis, cerebral artery dissection, dural sinus thrombosis, and pituitary apoplexy. The risk factors are hypertension, oral contraceptive pills, substance abuse, and pregnancy. Intracranial hemorrhage of pregnancy (ICHOP- intracerebral or subarachnoid hemorrhage) occurs with eclampsia. It is due to the loss of cerebrovascular autoregulation.[000107] Hemorrhagic products. After a hemorrhagic stroke, the composition of blood can include a variety of products that reflect the underlying damage and physiological responses, which products may be deleterious to brain function and are desirably removed by the methods of the disclosure. These products include red blood cells (RBCs) released into the brain tissue and cerebrospinal fluid. Hemoglobin from lysed RBCs can break down into bilirubin, hemosiderin, and other iron-containing compounds. Elevated levels of inflammatorycytokines (such as interleukins, TNF-alpha) and C-reactive protein (CRP) can be found due to the inflammatory response triggered by the bleeding. Increased levels of WBCs can be present as the body mounts an immune response to the hemorrhage and tissue injury. Initially, platelets and clotting factors might be found in higher concentrations around the site of hemorrhage as the body attempts to form clots to stop the bleeding. Fibrin degradation products and D-dimers are byproducts of fibrinolysis. Glial fibrillary acidic protein (GFAP) is a marker of astrocyte injury and can be elevated in the blood following a hemorrhagic stroke due to brain tissue damage. Neurofilament light chain (NfL) is a marker of neuronal damage and can be elevated in the blood after a hemorrhagic stroke. Increased levels of LDH can occur due to tissue damage and the subsequent release of this enzyme from damaged cells. Myelin Basic Protein (MBP) can be elevated due to damage to the myelin sheath surrounding nerve fibers in the brain. These products and markers help in diagnosing the extent of the hemorrhagic stroke, understanding the degree of brain injury, and guiding treatment strategies.[000108] The terms "individual," "subject," "host," and "patient," to which administration is contemplated, are used interchangeably herein; these terms typically refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets, but can also include commercially relevant birds such as chickens, ducks, geese, quail, and / or turkeys. A mammalian subject may be human or other primate (e.g., cynomolgus monkey, rhesus monkey), or commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs. The subject can be a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult). In some embodiments, the subject may be murine, rodent, lagomorph, feline, canine, porcine, ovine, bovine, equine, or primate. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject may be female. In some embodiments, the subject may be male. In some embodiments, the subject may be an infant, child, adolescent or adult.[000109] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. , arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.[000110] As used herein, the term “modulating” means increasing, reducing or inhibiting the activity of a biological or physiological pathway. In some cases, "modulate" or "modulating" or “modulation” may be measured using an appropriate in vitro assay, cellular assay or in vivo assay. In some cases, the increase or decrease is 10% or more relative to a reference, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, up to 100% relative to a reference. For example, the increase or decrease may be 2 or more times, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 50 times or more, or 100 times or more relative to a reference.[000111] A "therapeutically effective dose" or "efficacious dose" means the level of therapy that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease, for example one or more UCC rounds of treatment. The "therapeutically effective dose" will vary depending on the treatment, the disease and its severity and the age, weight, etc., of the subject to be treated.[000112] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a first therapeutic modality and a second therapeutic modality. When administered in combination, each treatment can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.[000113] "Concomitant administration" of a first therapeutic modality and a second therapeutic modality at such time that both the known treatments will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of a therapy with respect to the administration of a therapy of the present disclosure. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.[000114] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.[000115] The treatment may be administered one or a plurality of days, and in some embodiments is administered daily, every two days, semi-weekly, weekly, etc. for a period of from about 1 , about 2, about 3, about 4, about 5, about 6, about 7 or more weeks, up to achronic maintenance level. Intervals between single treatment can be weekly, monthly or yearly. Intervals can also be irregular.[000116] In prophylactic applications, a treatment is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a treatment at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.[000117] In still yet some other embodiments, for therapeutic applications, therapeutic treatment of the present disclosure are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and / or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose.[000118] Provided herein are methods of increasing removal of pathogenic products from the brain, e.g. in the CSF or interstitial fluid. The methods modulate the interstitial fluid transport systems of the body including, e.g, the lymphatic system. The methods can increase removal of substances with the application of ultrasound, e.g., low intensity or low frequency ultrasound. As summarized above, the methods may include applying or administering transcranial ultrasound to a subject to modulate the lymphatic pathway to promote removal of byproducts of a disease or injury that are deleterious to the brain. The term “glymphatic pathway” is used in its conventional sense to refer to a brain-wide network of paravascular channels along which cerebrospinal fluid (CSF) moves into and through the brain parenchyma, facilitating the exchange of CSF and interstitial fluid (ISF) and the clearance of interstitial solutes from the brain. Increasing or promoting the glymphatic clearance system facilitate clearance of waste products from the brain, such as, e.g., amyloid-|3. In some cases, the method includes upregulating the glymphatic pathway with the application of ultrasound. In some cases, clearance is increased by a percentage ranging from 50% to 120%, from 50% to 1 10%, from 50% to 1 10%, from 60% to 110%, or from 70% to 110% compared to a control. The control may not be subjected to any ultrasound.[000119] The application of transcranial ultrasound may include the transmission of low- intensity and / or low frequency ultrasound through the skull of a subject as disclosed herein. The transcranial ultrasound is applied in a non-invasive manner. In some cases, the transcranial ultrasound does not produce any tissue damage, e.g., neuronal cell damage,when applied to the brain of a subject. In some cases, the transcranial ultrasound includes transcranial focused ultrasound.[000120] It is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.[000121] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.[000122] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.[000123] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, as known to those skilled in the art, and so forth.[000124] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication date, which may need to be independently confirmed.ExamplesUltrasonic cerebrospinal fluid clearance improves outcomes in hemorrhagic brain injury models[000125] Impaired clearance of the byproducts of neurologic injury exacerbates disease progression and severity. We have developed a noninvasive, low intensity transcranial focused ultrasound protocol that facilitates removal of pathogenic substances from the cerebrospinal fluid (CSF) and brain interstitium. This protocol clears blood products from the central nervous system in two mouse models of hemorrhagic brain injury. Cleared blood products accumulate in the deep cervical lymph nodes from both the CSF and interstitial compartments, indicating clearance through meningeal lymphatics. Treatment with this ultrasound protocol reduced neuroinflammation and neurocytotoxicity, improved behavioral outcomes, decreased morbidity and, importantly, increased survival. Clearance efficacy was independent of thermal effects, given the lack of significant heating noted by simulation and experimental verification. Instead, protocol application was mechanistically associated with a shift of microglia to the homeostatic state and an increase of astrocytic endfeet aquaporin-4 polarization. As these processes have been shown to be downstream of mechanosensitive channels, we confirmed that blockade of mechanosensitive channels blocks clearance and that the protocol was effective when applied in anesthetized subjects. This indicates that ultrasound acts as a non-pharmacologic agonist of mechanosensitive channels to induce these homeostatic immune and glymphatic effects, in a way that does not depend on sensory stimulation or a specific neural activity pattern. Notably, the low intensity of this protocol is within FDA guidelines for safe ultrasound application, making it readily clinically translatable. Overall, our results demonstrate that this low-intensity transcranial focused ultrasound protocol induces cellular processes that clear hemorrhagic products from the brain via the meningeal lymphatic system, offering a novel therapeutic tool for the clearance of other neurotoxic byproducts in varied neurologic disorders.Results[000126] Ultrasonic CSF Clearance Enhances Intracranial Hemorrhage Clearance. To assess the utility of low intensity transcranial focused ultrasound (FUS) to clear soluble factors from the CSF and interstitial brain compartments in a translational animal model, we first adapted our prior protocol that used continuous 650 kHz ultrasound in rats for use in mice given the multitude of translational models developed and validated in mice. We also noted that while our earlier protocol was effective, its ultrasound center frequency of 650 kHz may present challenges for translation to larger animals and humans given both the more prominent attenuation provided by the skull and the aberrating effect of the skull and human haircompared to lower frequencies like 200-250 kHz. We first confirmed that the prior effect showed no significant reduction when decreasing the center frequency to 250 kHz (Fig. Si alo). Next, since at 250 kHz the ultrasound focus would treat much of the mouse brain at once (Fig. 8a-b), to prevent heat accumulation during application, we used a pulsed protocol with a duty cycle (25%) estimated to not yield significant parenchymal heating per the bioheat equation. Finally, we completed a parameter variation of the applied ultrasound pressure (Fig. 15c) to yield a target ultrasound protocol for subsequent use.[000127] We next determined the impact of this ultrasonic CSF clearance (UCC) protocol in models of neurologic disease. We specifically turned our attention to hemorrhagic brain injury as impaired clearance of hemorrhagic products is thought to be pathogenic in both subarachnoid hemorrhage (SAH) and intracerebral (also known as intraparenchymal) hemorrhage (ICH). In a mouse SAH model, UCC treatment significantly reduced the amount of red blood cells detected in the CSF compared to sham treatment (2.42-fold reduction, p < 0.001 ; Fig 8c). This reduction was associated with a greater accumulation of red blood cells in the deep cervical lymph nodes, indicating efficient clearance through the meningeal lymphatic system (2.07-fold increase, p < 0.01 ; Fig. 8c). Similarly, in the ICH model, UCC treatment led to a significant reduction in interstitial red blood cells compared to sham (2.24- fold reduction, p < 0.01 ; Fig. 8e). This clearance was again accompanied by increased red blood cell accumulation in the deep cervical lymph nodes (1.89-fold increase, p < 0.05; Fig. 8e). These findings suggest that UCC facilitates the clearance of hemorrhagic debris from both the CSF and interstitial compartments, likely through the meningeal lymphatic pathway that is identified as a main egress path for CSF and interstitial fluid (ISF) dispersed solutes via the glymphatic system.[000128] UCC Reduces Post-Hemorrhagic Neuroinflammatory and Neurocytotoxic Profiles. Neuroinflammation and neurocytotoxicity are critical factors that contribute to poor outcomes following hemorrhagic brain injury. To evaluate the impact of UCC on these processes, we performed immunohistochemical analyses of brain tissue from UCC-treated SAH and ICH models (Fig. 9a, e). UCC treatment significantly reduced IBA-1 + cells, suggesting an attenuation of microglial responses in both models (SAH: 1.43-fold reduction, ICH: 1 .46-fold reduction, p < 0.05; Fig. 9b, f). Additionally, astrocytic activation, assessed by GFAP staining, was also significantly reduced in UCC-treated animals in both models (SAH: 1 .81 -fold reduction, ICH: 1 .39-fold reduction, p < 0.05; Fig. 9c, g). These results indicate that UCC effectively attenuates neuroinflammatory responses following hemorrhagic brain injury. Furthermore, histological assessment of neuronal degeneration using Fluoro-Jade C (FJ-C) staining revealed a significant reduction in degenerating neurons in UCC-treated animals compared to sham (SAH: 1.71 -fold reduction, ICH: 2.09-foldreduction, p < 0.01 ; Fig. 9d, h). This reduction in neurotoxicity highlights the protective effects of noninvasive early blood clearance post-hemorrhage in reducing neuronal damage.[000129] Improved Behavioral Outcomes and Survival with UCC Treatment. Next, we assessed functional recovery and survival as indicators of the efficacy of UCC as a potential therapeutic intervention for brain injury. Our pilot studies indicated that the SAH model did not produce a sustained behavioral deficit. Therefore, to assess the impact of UCC on behavioral outcomes, we conducted the behavioral, morbidity, and mortality outcomes analysis using the ICH model (Fig. 10a). UCC treatment significantly improved both the assayed behavioral and functional outcomes (corner turn test and grip strength) as early as six days posthemorrhage, with sustained improvements observed through 14 days (corner turn test: 1 .46- fold increase from post-ICH to day 14, grip strength: 1 .24-fold increase from post-ICH to day 14, p < 0.01 compared to sham; Fig. 10b).[000130] Morbidity outcomes, including body weight and brain water content (indicative of edema), were also assessed. UCC-treated mice exhibited significantly lower brain edema and recovered their body weight faster compared to sham-treated controls (brain edema: 1 .07-fold reduction, body weight: 1.18-fold increase, p < 0.05; Fig. 10c). Crucially, UCC treatment significantly increased survival rates compared to sham (83.3% vs. 50% at 14 days, p < 0.05; Fig. 10d). These disparate survival rates between the sham and UCC groups underscore the positive behavioral effects (Fig. 10a, b), which were observed despite a survivorship bias in these data that otherwise serves to lessen the observable behavioral effect sizes. These findings demonstrate that UCC not only enhances functional recovery but also reduces morbidity and mortality following hemorrhagic brain injury.[000131] UCC induces homeostatic neuroimmunologic changes and cellular glymphatic processes. To investigate the potential cellular and molecular bioeffects induced by UCC, we completed a spatial transcriptomic analysis of both healthy control and ICH model mice, treated with sham or UCC (Fig. 1 1 a). Consistent with prior literature and our assessment of activated microglia and astrocyte states (Fig. 9), the ICH model induced prominent upregulation of neuroinflammation and microglial activation (Fig. 11 b). In the ICH model, both inflammation and microglial scores showed reductions with UCC compared to sham, particularly in the perihematomal region with a similar degree of transcriptional change of key markers of both disease-associated and homeostatic microglial states (Fig. 1 1 b). Investigating specific factors of interest, there was substantial transcriptional upregulation of AQP4, GFAP, CD68, AIF1 (IBA1 ), LGALS3, P2RY6 and P2RY14 in the ICH model particularly in the perihematomal region. With UCC treatment of the ICH model, the neuroinflammatory genes CD68 and LGALS3 showed relative downregulation in the perihematomal region (Fig.1 1 c), agreeing with our prior analysis of reduced microglial activation with UCC (Fig. 9). In contrast, P2YR12, a homeostatic microglial marker showed potential increase with UCC treatment of ICH. At the transcriptional level, no definite change of AQP4 or GFAP was noted with UCC in the ICH model, in potential contrast to the protein expression level changes we observed of GFAP (Fig. 9c, g). No definite changes of these select markers were noted of UCC in healthy control subjects. The largest activation in perihematomal gene expression was observed between ICH vs. control (1777 genes upregulated and 19 genes downregulated in ICH). UCC restored ICH perihematomal regions to more baseline gene expression with substantial gene downregulation in IHC + UCC vs. IHC (486 genes downregulated in IHC+UCC) and only modest gene expression differences between ICH + UCC vs. healthy control subjects (466 genes upregulated and 14 genes downregulated in ICH + UCC). Additionally, UCC only drove modest gene expression differences in control + UCC vs. control (324 genes upregulated and 21 genes downregulated in UCC; Fig. 9d). Together, these data suggest that UCC reverses ICH-associated gene networks, without substantial off-target gene activation in control conditions.[000132] To confirm whether these transcriptional changes were reflected in changes at the protein level, a comprehensive immunohistochemistry panel was completed in ICH models with and without UCC treatment (Fig. 12a). First, following our finding of reduced astrocytic activation with UCC treatment of hemorrhagic brain injury (Fig. 9c, g) compared to the lack of transcriptional differences in GFAP with UCC treatment of ICH (Fig. 11 c), we assessed S1 OOP - an independent astrocytic marker - which showed no significant difference with UCC. This indicates that UCC does not reduce astrocyte numbers and that the earlier GFAP protein level decrease after UCC reflects a per astrocyte decrease of activation state (Fig. 12b). Next, following the microglial state changes suggested by spatial transcriptomics (Fig. 1 1 ), we assessed markers of different microglial states identified in the neuroinflammatory literature: disease-associated microglia and homeostatic microglia.[000133] Microglial ramification branch complexity level, with increasing branch level correlating to the homeostatic state, showed increasing microglial branch level with UCC (1.42-fold increase, p < 0.05; Fig. 12c). In agreement with this result and the spatial transcriptomics analysis, CD68 and GAL-3 each decreased with UCC, indicating reduced microglial activation (CD68: 8.8-fold reduction, GAL-3: 2.83-fold reduction, p < 0.05; Fig. 12d). Likewise, P2RY12, a cell surface marker of homeostatic microglia necessary for the clearing of cellular debris, increased with UCC (1 .76-fold increase, p < 0.05; Fig. 12e) . Additionally, we observed a notable increase in the colocalization of red blood cell markers with microglia in the peri-hematoma region, suggesting elevated microglial phagocytosis of erythrocytes with UCC treatment of ICH (6.5-fold increase, p < 0.05; Fig. 12f). Finally, in contrast to the overall transcriptomic upregulation of AQP4 in ICH (Fig. 1 1c), we observed a trend-wise decrease inastrocytic endfeet polarization of AQP4 after ICH (Fig. 12g). The decrease in AQP4 endfoot polarization was restored in ICH models with UCC treatment (2.42-fold increase, p < 0.05), a key requirement of glymphatic upregulation. Integrating the transcriptom ic and immunohistochemistry data, we observe that UCC yields its effects by inducing an overall decreased inflammatory, more homeostatic neuroimmunological state and by upregulating glymphatic function through increased AQP4 astrocytic endfeet polarization.[000134] UCC Acts Through Molecular Mechanotransduction Pathways to Induce CSF Clearance. To elucidate the underlying mechanisms of UCC-mediated clearance, we first investigated potential thermal effects (Fig. 16). At the intensity, duty cycle, and ultrasound frequency used here, simulations indicated that no significant parenchymal heating is induced in these experiments, with heat accumulation being minimal and preferentially in the skull, not the parenchyma. We confirmed this experimentally by measuring the temperature before and after protocol application with a thermocouple placed intracranially, immediately under the skull and noted a trend towards cooling over the experiment, presumably due to thermal conduction into the room temperature gel and water used for ultrasound coupling in contact with the dorsal scalp (peak intracranial temperature change: -0.5 ± 0.2°C; Fig. 16).[000135] Given the lack of significant intracranial temperature rise (Fig. 16) with this protocol, we hypothesized that ultrasound is acting mechanically to clear the CSF in UCC. Possible mechanisms for this mechanical effect include a direct induction of a convection or oscillation by ultrasound of the intracranial fluid medium to promote increased exchange of the CSF and interstitial fluid with or without an accompanying mechanical change of the matrix to then drive downstream CSF circulation. However, our prior results demonstrated that the response to this protocol extends for a substantial amount of time beyond the direct ultrasound exposure, implying induction of a prolonged cascade of events that leads to longer-term CSF circulation upregulation. Notably, pharmacological agonism of mechanosensitive channels has been shown to yield a similar shift of microglia from disease-associated to homeostatic states and to increase aquaporin polarization in other contexts as we observed with UCC (Figs. 1 1 , 12). Further, ultrasound of this intensity would be sufficient to activate these mechanosensitive channels. Since agonism of these channels also yields meningeal lymphatic vessel sprouting with resultant increased CSF egress, we hypothesized that UCC may yield its diverse cellular effects (Figs. 1 1 , 12) via activation of mechanosensitive channels expressed intracranially.[000136] To determine the relative contribution of these mechanosensitive channels to the effect of UCC, we cisternally administered a mechanosensitive ion channel blocker (GsMTx4, a selective blocker of Piezo and TRP channels) in the ICH model to determine its effect on UCC-mediated clearance (Fig. 13a). Blockade of mechanosensitive channels with GsMTx4significantly reduced the efficacy of blood product clearance by UCC, as evidenced by increased hematoma volumes in the UCC + GsMTx4 treatment group versus UCC treatment alone (2.03-fold increase) and no significant difference between the Sham + GsMTx4 and UCC + GsMTx4 groups (Fig. 13b). This finding indicates that mechanosensitive ion channels mediate the majority of the observed clearance effects of UCC, with a near elimination of resolvable effect with GsMTx4 blockade (Hedge’s g, corrected for N < 50 = 2.96 for Sham vs. UCC alone compared to 0.46 for Sham + GsMTx4 vs. UCC + GsMTx4).[000137] We observed no significant change in meningeal lymphatic vessel density with UCC (Fig. 13c), potentially reflecting the short, days long timeline of both this disease model and the intervention. In contrast, we did observe that UCC, when applied only once to healthy control brains, also yielded an acute increase of a marker of the homeostatic microglial state (1.10-fold increase in P2RY12, p < 0.05; Fig. 13d), supporting that a mechanosensitivechannel mediated shift of microglial state is a key primary mediator of the efficacy of UCC. Notably, AQP4 expression did not significantly change in healthy control brains following only one application of UCC (Fig. 13e), suggesting that UCC acts indirectly to drive the observed glymphatic enhancement in ICH. Instead, AQP4 polarization with UCC treatment of ICH may be secondarily induced through more direct UCC-driven reductions in neuroinflammation with secondary disinhibition of AQP4 polarization, highlighting a distinction between the direct immunomodulatory effects of UCC compared to its relatively indirect glymphatic effects via AQP4 polarization.[000138] Overall, our results demonstrate that UCC indeed facilitates the clearance of pathogenic substances from the brain, thereby reducing neuroinflammation and neurocytotoxicity, and improving functional outcomes and survival following hemorrhagic brain injury. We have experimentally confirmed that UCC acts via mechanosensitive channel transduction to mediate these effects, acting as a noninvasive and nonpharmacologic agonist of molecular mechanotransduction pathways to elicit increased aquaporin astrocytic endfeet polarization and a shift of microglia from the disease-associated to the homeostatic state (Figs. 1 1 , 12, 13d). These cellular effects combine with a potential ultrasound-induced increased mixing of CSF and interstitial fluid to yield increased clearance of hemorrhagic debris with increased CSF circulation and egress (Fig. 14).[000139] Notably, the design of the UCC protocol enables a ready path towards clinical translation. The 250 kHz ultrasound frequency used here is in the range of commercial clinical transcranial FUS systems currently on market worldwide and is further enabled by having relatively low attenuation and aberration when transmitting this ultrasound frequency through the human scalp and skull. Additionally, the target in situ peak negative pressure used in these experiments corresponds to a mechanical index (Ml) of 0.9, which is well within FDAguidelines for safe application and similar to current studies of ultrasound neuromodulation, indicating that a clinical trial of UCC should be able to receive a nonsignificant risk (NSR) designation from a local IRB, further lowering barriers for clinical translation. In addition, UCC does not require co-administration of exogenous agents like nanoparticles or microbubbles, further uncomplicating its eventual application. Finally, as its mechanism does not require a particular pattern of brain activity (i.e., it is effective in anesthetized animals), it is applicable in humans in a range of cognitive states including while awake, asleep, engaged in some other task, or obtunded in the immediate post-injury recovery period.[000140] Furthermore, UCC provides a method to drive CSF circulation and egress through a parallel mechanism to pharmacologic approaches and approaches that activate particular neural activity patterns that are correlated with this circulation. As a benchmark comparison, pharmacologic agonism of mechanosensitive channels in the ICH model proved to be counteractive, with decreased survival of treated mice and without similar efficacy for hematoma clearance, indicating that pharmacologic agonism of these pathways is less safe and efficacious than UCC (Fig. 17). Accordingly, UCC provides an alternate method for understanding the role of CSF circulation and egress in the variety of disorders in which it has been implicated including sleep, neurodegeneration, traumatic brain injury, and mental health that is non- pharmacologic and could be applied to subjects in varied states of cognition or wakefulness. Indeed, further validation of UCC for harnessing CSF clearance in the variety of disorders to which it has been implicated could and should be applied in appropriate clinical trials.Methods[000141] Animals. All experiments were approved by the Institutional Animal Care and Use Committees of Stanford University. Experiments were conducted with adult (3-4 month) and aged (18-20 month) male C57BL / 6NCrl mice with bodyweight 30-40 g (Charles River Laboratories, Wilmington, MA, USA). All mice were housed in a temperature-controlled (22 °C), humidity-controlled (33-39%) environment under a 12h / 12 h light-dark cycle and were provided with food and water ad libitum. Mice were randomly assigned to one of two treatment groups: (1 ) no treatment (sham), and (2) treatment with a focused ultrasound protocol, herein referred to as ultrasonic CSF clearance (UCC).[000142] Ultrasonic CSF Clearance Protocol. Focused ultrasound (250kHz center frequency, 0.45MPa peak in situ negative pressure, 50ms pulse-width, 25% duty cycle for 10 min via a 250 kHz center frequency, 70 mm or 100 mm aperture, f=1 .0) or sham (ultrasound power off) was applied transcranially throughout the brain (Fig. S2). Before application, the fur on the head was removed using chemical hair depilatory. The transducer was coupled withultrasound gel to the dorsal surface of the head, and ultrasound applied while the mice were anesthetized under ketamine / xylazine (90 mg / kg and 10 mg / kg, respectively). Body temperature, cardiac and respiratory rates, and 02 saturation were monitored throughout the experiment. Environmental heating was used to help maintain body temperature.[000143] Hemorrhagic Models. Two previously characterized models of hemorrhagic stroke, subarachnoid (SAH) and intracerebral (ICH), were implemented in this study. Briefly, for the SAH model, 25pl of autologous blood was withdrawn from the tail vasculature into heparinized capillary tubing and injected into the cisterna magna. For the ICH model, 25 pl of autologous blood was withdrawn from the tail vasculature and injected into the right striatum. Mice were anesthetized under ketamine / xylazine (90 mg / kg and 10 mg / kg, respectively). Body temperature, cardiac and respiratory rates, and O2saturation were monitored throughout the experiment. Environmental heating was used to help maintain body temperature.[000144] Cerebrospinal Fluid Collection. Cerebrospinal fluid was collected by inserting a pulled glass capillary, connected to an aspirator tube, into the cisterna magna. Collected CSF was centrifuged at 1 ,000 x g for 10 min at 4°C to remove any cellular debris. Neurofilament light polypeptide (NEFL) levels were quantified by a high-sensitive ELISA (Cat# EKU10307, Biomatik, Ontario, Canada). Tests were performed in duplicate using 10 uL CSF diluted in aCSF (Tocris Biosciences, Minneapolis, MN, USA). Absorbances were quantified using a Spark Multimode Microplate Reader (Tecan, Mannedorf Switzerland).[000145] Histology and Immunostaining. Mice were euthanized and fixed via transcardial perfusion with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA) at timepoints described in each experimental timeline (Fig 1 -4). Brains were fixed in 4% PFA for 24 h, cryoprotected by serial incubation in 15% and 30% sucrose solutions for 24 h each, and then frozen at -80 °C in optimal cutting medium (OCT) compound. Brains were sectioned at 30 pm thickness using a cryostat (LEICA CM 1950, Buffalo Grove, IL, USA). Every 5th section (150 pm apart) was collected for imaging. The specimen temperature was set at -21 °C. Tissue sections were stored in cryoprotectant solution prior to staining.[000146] For immunofluorescence, the free-floating tissue sections were blocked by 0.3% PBST with 10% normal goat serum for 1 h at room temperature, then incubated with primary antibodies overnight at 4 °C. After washing with PBS three times for 5 min each, sections were incubated in secondary antibodies for 2 h at room temperature. The primary antibodies used in immunofluorescence included rabbit anti-LYVE-1 antibody (1 :500; Abeam, Cat. No. ab14917), rat anti-TER-1 19 antibody (1 :500; Invitrogen, Cat No. 14-5921 -82), recombinant rabbit anti-l BA-1 (1 :500; Abeam, Cat No. ab178846), and chicken anti-GFAP antibody (1 :500; Abeam, Cat. No. ab4674). The corresponding secondary antibodies were used as follows: AlexaFluor 488-labeled goat anti-rabbit antibody (1 :500; ThermoFisher Scientific, Cat. No.A32731 ), AlexaFluor 555-labeled goat anti- rat antibody (1 :500; ThermoFisher Scientific, Cat. No. A48263), AlexaFluor 647-labeled goat anti- rabbit antibody (1 :500; ThermoFisher Scientific, Cat. No. A32733), and AlexaFluor 488-labeled goat anti-chicken antibody (1 :500; ThermoFisher Scientific, Cat. No. A32931 ).[0001 47] For neurodegeneration staining, tissue sections were incubated in 0.06 % potassium permanganate for 20 min and in 0.0001 % Fluoro-Jade C (Biosensis, Thebarton, SA, Australia). Tissue sections were mounted on microscope glass slides (Fisher, Pittsburgh, PA) and cover-slipped with Prolong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA). All images were collected with a fluorescence microscope (BZ-X800, Keyence Corp., Itasca, IL, USA) and processed with BZ-X Advanced Analysis Software (Keyence Corp., Itasca, IL, USA) or Imaged Software (Version 1 .53, National Institutes of Health, Bethesda, MD, USA).[000148] For hematoma volume, the mean area of blood was calculated in coronal segments using Imaged and reported as percent hematoma area over total hemispheric area. For immunofluorescence quantification involving fluorescence intensity, signals above manually thresholded background were used for ROI segmentation to calculate total mean fluorescence intensity (MFI) using BZ-X Advanced Analysis Software and or Bitplane IMARIS software (version 9.8.0). To quantify microglial branching complexity from I BA-1 immunoreactive cells in confocal z-stack images, we used Bitplane IMARIS software (version 9.8.0). The “Modeling” and “Filaments” modules were employed to reconstruct IBA-1 + microglia, focusing on cells with processes extending from the soma. IMARIS’s automated cell detection algorithm was applied to identify all microglia within each image. Detected structures were manually reviewed to ensure accuracy, and any misidentified cells were corrected to refine the detection parameters prior to quantitative analysis. For analyses of phagocytosis, 3D models of microglia and red blood cells were generated using IMARIS software based on lba-1 and TER119 immunostaining, as previously described. Internalization and colocalization events between microglia and RBCs were subsequently measured. All imaging and thresholding parameters were completed by personnel blinded to experimental conditions.[000149] Neurobehavioral Function Evaluation. Two behavioral tests were used to evaluate behavioral and functional outcomes as previously described: a corner turn test and a grip strength test. Briefly, for the corner turn test, quantification of turning preference upon approaching a 30° corner was used to assay sensorimotor deficits. The values were calculated as percent of left versus right limb use on turn for a total 3 min. duration per session. For the grip strength test, motor function was assessed via the peak force (G) required for mice to release their grip from a grid bar as quantified by a digital grip strength meter (Maze Engineers,Skokie, IL, USA). The average of 3 attempts was calculated per test session. All behavioral assessments were completed by personnel blinded to experimental conditions.[000150] Morbidity Evaluation. Total body weight (g) and brain water content (edema) percentage were used as indicators of post- hemorrhagic morbidity. Brain water content was assessed at day 14 post-hemorrhage. The wet weight of each brain was recorded following euthanasia and extraction. The brains were then dehydrated at 1 10°C for 72h. Dry weights were recorded, and brain edema was evaluated as the difference in percent brain water content.[000151] Reagents. For mechanosensitive ion channel inhibition, we mixed GsMTx4 (MedChem Express, Cat. No. HY-P1410) with artificial CSF and injected (5 pM in 5 pl) into the cisterna magna of mice 0.5 h prior to focused ultrasound application. For mechanosensitive ion channel activation, we mixed Yodal (Sigma-Aldrich) in DMSO at 710 pg / ml, then diluted in phosphate-buffered saline (PBS) at a 3:100 (vol / vol) ratio of DMSO to PBS. The final solution was administered at a dosage of 213 pg / kg body weight per day, based off prior studies.[000152] Tissue Collection and Processing for Stereo-seq. Animals were euthanized on day 6 (post-ICH, see experimental timeline in Figure 1c) and brain tissues were extracted and snap- frozen in isopentane prechilled in liquid nitrogen in Tissue-Tek OCT (Sakura, 4583) and transferred to a -80°C freezer for storage. Cryosections were cut sagittally at a thickness of 10 pm on a Leica CM1860 cryostat. Sections were matched to include equivalent regions of olfactory bulb, striatum, lateral and 4th ventricles, thalamus, midbrain, and cerebellum. Stereo- seq experiments were performed as previously described and followed STOmics stereo- seq chip-on-a-slide (1cm * 1 cm) v1 .3 kit with minor modifications. Cryosections were melted onto stereo-seq T-chips that had been rinsed with DI H2O and dried with dust off, by placing the section on the chip using tweezer at -20°C in a cryostat, and then removing the slide from the cryostat. Afterwards, slides were baked for 5 min. at 37°C, and then submerged in pre-chilled methanol at -20°C for 30 min. After drying slides, they were washed 1 X in 0.1 X SSC, then placed in a gasket to enable easy addition and removal of solution directly to the chip. To the gasket, 150 pL of pre- warmed 1 X permeabilization solution was added, and chips were incubated at 37°C in a thermocycler for 12 min. The reagent was then removed, washed 1 X with 0.1 X SSC, and then 200 pL of 1 X reverse transcriptase (R.T.) solution was added, and chips were incubated for 2 hrs at 45°C. After removing R.T. solution, chips were again washed 1 X with 0.1 X SSC, and then we added 180 pL of cDNA release solution and incubated for 10 minutes at 55°C. The solution was then mixed thoroughly to remove sample from the chip and collected into a new tube. We added 23 pL of neutralization buffer to the tube, split to sample to 3 PCR tubes, and incubated at 95°C for 5min. Afterwards we amplified cDNA with 34 pL ofPCR mix, and ran using the following settings: 95°C 5 min, 13 cycles of: 98°C - 20 sec, 58°C - 20 sec, 72°C - 3 min, followed by 72°C - 5 min. Afterwards, we performed 1 :0.8 bead cleanup on samples using AMPure beads following manufacturer’s protocol.[000153] Library construction and sequencing of Stereo-seq data. Libraries were constructed and barcoded using STOmics barcode library preparation kit v1 .0. We fragmented amplified 300ng cDNA with 10uL of KMB in a total reaction volume of 45 pL, and incubated in a thermocycler 95°C - 5 min, 40°C - 3 min. Reaction was stopped by adding 5 pL of KME, then incubated at 37C for 10min. Fragmented DNA was cleaned up using AMPure beads with a ratio of 1 :0.8 beads following manufacturer’s protocol. DNA was eluted in 25 uL of 1X TE, and combined with 25 pL of a unique barcode mix, and 50 pL of 2X PCR master mix, and amplified using the following program: 95°C 5 min, 13 cycles of : 98°C - 20 sec, 58°C - 20 sec, 72°C - 30 sec, followed by 72°C - 5 min. Final product was cleaned up using a double sided AMPure bead cleanup with a 1 :0.55 bead ratio following manufacturer’s protocol. Libraries were eluted in 20uL of 1 X TE. Prepared libraries were then sequenced using a DBSEQ-T7RS sequencer at the Stanford Genomics core, targeting 1 .5 billion 75bp PE reads per sample.[000154] Processing of Stereo-seq raw data. FASTQ corresponding to barcode indexes per sample along with the DNA nanoball (DNB) mask file were processed using the SAW v8.1 .3 count pipeline. The sequences from the FASTQ files were filtered to only include reads with 1 or fewer unmapped base pairs in the CID. Cl Ds were then mapped to the corresponding DNB mask file, and valid CIDs were associated with reads and retained for subsequent steps. Next, FASTQs were filtered to only include reads that contained >30 bp not including adapter, DNB, or poly-A sequences. Afterwards, sequences with low quality molecular identified (MID) sequences were removed (quality <= Q10 or unmapped base pairs >= 1). A reference file for GRCm39vM30 was assembled and reads were aligned using STAR. We used default parameters for SAW count pipeline which utilizes STAR and performs alignment, annotation, MID correction and deduplication, and binning. The outputted gene expression file (GEF) contained the spatial gene expression matrix at different bin sizes.[000155] Analysis of Stereo-seq data. The resulting GEF files for all 4 samples were loaded into a multi-sample data object (MSdata) at bin size 100 (50um x 50um) using Stereopy and integrated. Using Stereopy default parameters, the expression data was log-normalized per bin. Highly variable genes were calculated, PCA was performed on highly variable genes, and Harmony was run to minimize batch effects in the embedding space. Afterwards nearest neighbors were calculated using the first 50 principal components, UMAPs were constructed, and leiden was run at resolution = 2. Leiden clustering was further refined by running Stereopy’s spatial neighbors algorithm and performing leiden again at resolution = 2. Clusters containing fewer than 300 bins across all samples were left unannotated, and remainingclusters were annotated based on spatial location, known marker genes and previously annotated stereo-seq data. After annotation, the MSdata objected was converted into annotated data (anndata) objects and analyzed using Scanpy. Differentially expressed genes within the perihematomal region (striatum+thalamus) were found using Scanpy rank_gene_groups function using method=’wilcoxon’, and differential expressed genes were selected using |log2FC| > 1 and pvals- adj <0.05 as thresholds. Gene scores were calculated using Scanpy score_genes function based on gene lists for inflammation (GO: 0006954), microglial activity (GO: 1903978), or disease- associated microglial and homeostatic microglial scores from manually curated lists of genes (disease-associated: 'Ifng', 'Tnf, 'II6', '111 b', '1112a', '1112b' 'Ccl2', 'Cybb', 'Nos2', 'H2-Aa', 'H2-Ab1 ', 'Itgam', 'Itgax', 'Cd36', 'Ptprc', 'Cd47', 'FcgrT, 'Fcgr2b', 'Fcgr3', 'Fcgr4', 'Feeri g'. Homeostatic: '114', '1113', '1110', 'Tgfbl ', 'IgfT, 'Fgf2', 'CsfT, 'Ngf, 'Bdnf, Ntf3', 'Gdnf, 'Grn', 'Mrc1', 'Retnla', 'Chil3', 'Arg1 '). Spatial plots of annotations, gene scores, or individual genes were generated using Squidpy.[000156] Thermal Evaluation. A male C57BL / 6 mouse (weight: 35 g) was imaged on a Quantum GX micro-CT (Waltham, Massachusetts, USA) to simulate the pressure-field distributions during FUS treatment. The obtained micro-CT image was 474x504x921 with a cubic voxel size of 0.0499 mm. The images were resampled linearly to a cubic voxel size of 0.34 mm. The bone, soft tissue, and water were isolated based on their Hounsfield units (1200 HU for bone / soft tissue and 930 HU for soft tissue / water threshold). Density and sound speed were linearly interpolated in each region using hounsfield2density, a predefined function through the k-Wave MATLAB (Natick, Massachusetts, USA) toolbox. The region surrounding the animal was defined as water. The transducer was described as a bowl with a diameter and radius of curvature of 100 mm. At 250 kHz center frequency, the points per wavelength was 17.44 in water and Courant-Friedrichs-Lewy stability criterion of 0.1 leading to a time step of 22.8 ns. The simulation was run for 85 ps allowing the initial wave to travel to the length of the simulation grid and back past the top of the skull (135 mm). All simulations were conducted using the k-Wave toolbox. After the pressure field was complete, the time array was cropped to remove the expected travel time from the transducer surface to the top of the skull (60 to 85 ps). The field of view was reduced to double the focal region of the ultrasound (FWHM) and double the height of the skull. The resulting simulation space was 15x15x20 mm for 25 ps. The phase and pressure amplitude were extracted using the built-in k-Wave function extractAmpPhase. The heat disposition was calculated for each voxel using the original density, speed of sound, and attenuation coefficient determined previously. The starting temperature, thermal conductivity, and specific heat were set to 37 °C, 0.51 W / (m*K), and 3630 J / (kg*K), respectively. Tissue properties were obtained from the IT’IS database. Thethermal heating was determined for a single burst (50 ms on, 150 ms off) with a time step of 0.1 ms.[000157] Statistical Analysis. Sample size determination was guided by a power analysis performed using G*Power 3.1 software (Heinrich-Heine-Universitat Dusseldorf). Parameters used for the analysis included an effect size (Cohen’s d) ranging from 0.8 to 2.0, an a error probability of 0.05, power (1 -|3) of 0.8, and an allocation ratio (N2 / N1 ) of 1. Effect sizes were estimated based on preliminary pilot data. All experiments were randomized. Data collection and analysis were conducted by investigators blinded to treatment groupings. Data normality was assessed using the Kolmogorov-Smirnov test where appropriate. Based on the outcomes and given respective sample sizes, nonparametric statistical methods were applied. For comparison of two groups, a two-tailed Mann-Whitney U- test (Wilcoxon Rank Sum Test) was used. For comparisons among multiple groups, a Kruskal- Wallis H test followed by Dunn’s multiple comparisons test was used. 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Claims

CLAIMSWe claim:1 . A device comprising: a housing configured to fit over a portion of a subject’s head, house a plurality of ultrasound devices, and orient an active side of each of the plurality of ultrasound devices toward the subject’s head when worn; and the plurality of ultrasound devices being arranged in the housing and configured to project ultrasound into the subject’s brain.

2. The device of claim 1 , wherein the device is configured as an ultrasonic cerebrospinal fluid clearance (UCC) device.

3. The device of claim 1 , wherein each ultrasound device of the plurality of ultrasound devices is configured to emit ultrasound of the same frequency.

4. The device of claim 1 or 2, wherein the plurality of ultrasound devices includes a first set of ultrasound devices configured to emit ultrasound of a first frequency and a second set of ultrasound devices configured to emit ultrasound of a second frequency.

5. The device of any of the above claims, further comprising: a heat sensing device configured to sense heat within the subject’s brain.

6. The device of any of the above claims, further comprising: a sensor device configured to measure ultrasound reflected from the subject's head.

7. The device of any of the above claims, wherein the housing is adjustable to fit a size of the subject’s head when worn.

8. The device of any of the above claims, wherein the ultrasound emitted by at least one ultrasound device of the plurality of ultrasound devices is low-intensity focused ultrasound.

9. The device of any of the above claims, wherein the plurality of ultrasound devices includes a first ultrasound device arranged within the housing to project ultrasound into a front of the subject’s brain, a second ultrasound device arranged within the housing to project ultrasound into a back of the subject’s brain, a third ultrasound device arranged within the housing to project ultrasound into a first side of the subject’s brain, and a fourth ultrasounddevice arranged within the housing to project ultrasound into a second side of the subject's brain.

10. The device of any of the above claims, wherein the plurality of ultrasound devices are configured to project ultrasound into to a plurality of regions of a subject’s brain to increase a circulation rate of cerebrospinal fluid (CSF) within the subject’s brain and egress from the subject’s brain.1 1 . The device of any of the above claims, wherein the plurality of ultrasound devices are configured to project ultrasound into to a plurality of regions of a subject’s brain to activate mechanosensitive channels within the subject’s brain to increase the circulation rate flow of the CSF within the subject’s brain and egress from the subject’s brain.

12. The device of claim 10, wherein activation of the mechanosensitive channels shifts microglia from a disease-associated state to a homeostatic state.

13. The device of any of the above claims, wherein the plurality of ultrasound devices are configured to project ultrasound into to a plurality of regions of a subject’s brain to induce removal of waste from the subject’s brain via the increase of the circulation rate flow of the CSF within the subject’s brain and egress from the brain.

14. The device of any of the above claims, wherein the plurality of ultrasound devices are configured to project ultrasound into to a plurality of regions of a subject’s brain to mechanically induce mixing of CSF within different compartments of the subject’s brain.

15. The device of any of the above claims, wherein the plurality of ultrasound devices is a first plurality of ultrasound devices configured to project ultrasound of a first frequency, the device further comprising: a second plurality of ultrasound devices arranged in the housing and configured to project ultrasound of a second frequency into the subject’s brain.

16. The device of any of the above claims, wherein the plurality of ultrasound devices includes: a first ultrasound device arranged within the housing to project ultrasound into a region proximate to a front left side of the subject’s brain; a second ultrasound device arranged within the housing to project ultrasound into a region proximate to left side of the subject’s brain;a third ultrasound device arranged within the housing to project ultrasound into a region proximate to a rear-left side of the subject’s brain; a fourth ultrasound device arranged within the housing to project ultrasound into a region proximate to an upper-left side of the subject’s brain; a fifth ultrasound device arranged within the housing to project ultrasound into a region proximate to a front right side of the subject's brain; a sixth ultrasound device arranged within the housing to project ultrasound into a region proximate to right side of the subject’s brain; a seventh ultrasound device arranged within the housing to project ultrasound into a region proximate to a rear-right side of the subject’s brain; and an eighth ultrasound device arranged within the housing to project ultrasound into a region proximate to an upper-right side of the subject’s brain.

17. The device of any of the above claims, wherein an ultrasound device of the plurality of ultrasound devices is configured to emit ultrasound that generates pressure within the subject’s brain within a range of 0.1 -1 ,1 MPa, 0.1 -1 MPa, 0.1 -0.95MPa, 0.15-3MPa, 0.1 -9MPa, 0.1 -5MPa, 0.15-0.3MPa, or 0.15-0.95MPa.

18. A method for using the device of any of claims 1-17 for treating brain injury.

19. A method for using the device of any of claims 1 -17 for treating neurologic disease.

20. A method for using the device of any of claims 1 -17 for enhancing waste clearance from the brain.21 . A computer-executed method comprising: receiving a neurological diagnosis for a subject; using the neurological diagnosis to query a ultrasonic CSF clearance (UCC) treatment protocol database for a UCC treatment protocol that matches the neurological diagnosis; receiving a UCC treatment protocol that matches the neurological diagnosis responsively to the query; receiving an indication that any one of the devices of claims 1 -17 is positioned on the subject’s head; and initiating projection of ultrasound into the subject’s brain via one or more of the ultrasound devices of the plurality of ultrasound devices included in the device positioned on the subject’s head in accordance with the received UCC treatment protocol.

22. The computer-executed method of claim 21 , wherein the UCC treatment protocol database stores a plurality of UCC treatment protocols, each UCC treatment protocol of the plurality of UCC treatment protocols being indexed to one or more neurological diagnoses.

23. The computer-executed method of claim 21 or 22, wherein the UCC treatment protocol database stores a plurality of UCC treatment protocols generated by a UCC treatment model, each UCC treatment protocol of the plurality of UCC treatment protocols being indexed to one or more neurological diagnoses.

24. The computer-executed method of any of claims 21 -23, wherein one or more of the plurality of UCC treatment protocols is further indexed to one or more subject characteristics.

25. The computer-executed method of claim 24, further comprising: receiving a characteristic of the subject, wherein the characteristic of the subject is also used to query the UCC treatment protocol database for a UCC treatment protocol that matches the neurological diagnosis and the characteristic of the subject; and receiving a UCC treatment protocol that matches the neurological diagnosis and the characteristic of the subject responsively to the query.

26. The computer-executed method of any of claims 21 -25, wherein the UCC treatment protocol includes at least one of a schedule for administration of UCC treatments to the subject, a duty cycle for one or more of the ultrasound devices of the device over the duration of the UCC treatment, and an intensity of the ultrasound projected into the subject’s brain by one or more of the ultrasound devices of the device over the duration of the UCC treatment.

27. The method of any of claims 21 -26, wherein the neurological diagnosis is an acute diagnosis and the UCC treatment protocol provides instructions for administering approximately 30-60 minutes of UCC to the subject’s brain per session using the device in multiple sessions over a series of days.

28. The method of any of claims 21 -27, wherein the neurological diagnosis is a chronic diagnosis and the UCC treatment protocol provides instructions for administering approximately 15-30 minutes of UCC to the subject’s brain per session using the device in multiple sessions over a series of months.

29. The method of any of claims 21 -28, wherein the UCC treatment protocol defines at least one of a duration of a UCC treatment session, a set of instructions for a serial projection of ultrasound by the plurality of ultrasound devices, a set of instructions for an alternating projection of ultrasound by the plurality of ultrasound devices, a set of instructions for a simultaneous projection of ultrasound by the plurality of ultrasound devices, and a set of instructions for projection of ultrasound by a subset of the plurality of ultrasound devices.

30. A computer-executed method comprising: receiving a set of neurological information for a subject; inputting the set neurological information into a UCC treatment model; and receiving an output from the UCC treatment model that is responsive to the set of neurological information, the output including a UCC treatment protocol that includes a set of instructions for use of the device of any of claims 1-17 to provide a UCC treatment to the subject.31 . The computer-executed method of claim 30, wherein the set of neurological information includes at least one of an image of the subject’s brain, a dimension of the subject’s head, a diagnosis for the subject, an indication when an adverse neurological event occurred for the subject, an indication of the subject’s responsiveness to UCC treatment, an indication of a treatment the subject is received other than the UCC treatment.

32. The computer-executed method of claim 30 or 31 , wherein the UCC treatment protocol is configured to optimize the UCC treatment for the subject using the set neurological information.

33. The computer-executed method of any of claims 30-32, wherein the output further includes a predicted outcome of the UCC treatment protocol for the subject, the method further comprising: providing the predicted outcome to a display device.

34. The computer-executed method of any of claims 30-33, wherein the output further includes a plurality of UCC treatment protocols and a predicted outcome for each the plurality of UCC treatment protocols for the subject, the method further comprising: providing an indication of each UCC treatment protocol of the plurality of UCC treatment protocols and the predicted outcome of each UCC treatment protocol to a display device.

35. The computer-executed method of any of claims 30-34, wherein the UCC treatment protocol includes at least one of a schedule for administration of UCC treatments to the subject, a duty cycle for one or more of the ultrasound devices of the device over the duration of the UCC treatment, and an intensity of the ultrasound projected into the subject’s brain by one or more of the ultrasound devices of the device over the duration of the UCC treatment.

36. The computer-executed method of any of claims 30-35, further comprising: receiving feedback regarding the subject following administration of at least one UCC treatment of the UCC treatment protocol; and updating the UCC treatment model responsively to the feedback.

37. The computer-executed method of any of claims 30-36, wherein the feedback is at least one of a result of a diagnostic test, a result of a blood test for a biomarker of neurological waste, a chemical composition of lymphatic fluid extracted from a cervical lymph node, an indication of heat within the subject’s brain while the subject is undergoing the at least one UCC treatment, an indication of cavitation within the subject’s brain while the subject is undergoing the at least one UCC treatment, and an image of the subject’s brain.

38. The method of any of claims 30-37, wherein the set of instructions for use of the device defines at least one of a duration of a UCC treatment session, a set of instructions for a serial projection of ultrasound by the plurality of ultrasound devices, a set of instructions for an alternating projection of ultrasound by the plurality of ultrasound devices, a set of instructions for a simultaneous projection of ultrasound by the plurality of ultrasound devices, and a set of instructions for projection of ultrasound by a subset of the plurality of ultrasound devices.

39. A method comprising: receiving an image of a subject’s brain; determining an operational parameter for the device of any of the above claims using the image of the subject’s brain; receiving an indication that the device is positioned on the subject’s head; and initiating projection of ultrasound into the subject’s brain via one or more of the ultrasound devices of the plurality of ultrasound devices in accordance with the operational parameter.

40. The method of claim 39, wherein the operational parameter is at least one of a duration of a UCC treatment session, a set of instructions for a serial projection of ultrasoundby the plurality of ultrasound devices, a set of instructions for an alternating projection of ultrasound by the plurality of ultrasound devices, a set of instructions for a simultaneous projection of ultrasound by the plurality of ultrasound devices, and a set of instructions for projection of ultrasound by a subset of the plurality of ultrasound devices.41 . The method of claim 40, further comprising: receiving an indication of heat within the subject’s brain from a heat sensing device; and adjusting the operational parameter responsively to the indication of heat within the subject’s brain.

42. The method of any of claims 39-41 , further comprising: receiving a second image of the subject’s brain while the subject is being treated with the device; and analyzing the image; adjusting the operational parameter responsively to a result of the analysis.

43. The method of claim 42, wherein a result of the analysis indicates that cavitation is present within the subject’s brain.

44. The method of any of claims 39-43, wherein the operational parameter for the device defines at least one of a duration of a UCC treatment session, a set of instructions for a serial projection of ultrasound by the plurality of ultrasound devices, a set of instructions for an alternating projection of ultrasound by the plurality of ultrasound devices, a set of instructions for a simultaneous projection of ultrasound by the plurality of ultrasound devices, and a set of instructions for projection of ultrasound by a subset of the plurality of ultrasound devices.

45. A system configured to execute the method of any of claims 21-44.

46. A system comprising: the device of any of claims 1-17; a power source configured to provide electrical power to the device; a processor configured to control an operation of at least one of the plurality of ultrasound devices of the device in accordance with a UCC treatment protocol that defines a set of instructions for the operation of the device; and a memory storing the set of instructions defining the UCC treatment protocol.

47. A method of increasing removal of pathogenic substances from the cerebrospinal fluid (CSF) and the brain interstitium in a mammalian subject, the method comprising: applying noninvasive low intensity transcranial focused ultrasound to the subject in the absence of administration of exogenous agents, to promote removal of the pathogenic substances from the brain.

48. The method of Claim 47, wherein the method comprises applying focused transcranial ultrasound across the whole brain of the subject.

49. The method of Claim 47 or 48, wherein the transcranial ultrasound has (i) a frequency ranging from 100 kHz to 1000 kHz; (ii) a pressure ranging from 0.05 MPa to 0.6 MPa, (iii) a pulse width from about 5 (is to 50 ms; and / or (iv) a duty cycle of from 1 to 100%.

50. The method of any of claims 47-49, wherein the transcranial ultrasound is performed from 1 to 60 minutes, 10 to 30 minutes.51 . The method of any of claims 47-50, wherein the exogenous agents comprise nanoparticles or microbubbles.

52. The method of any of claims 47-51 , wherein the subject has suffered an acute injury or condition to the brain, including a hemorrhagic stroke.

53. The method of any of claims 47-52, wherein the subject is treated within 3 days of occurrence or diagnosis of an injury or disease of the brain, or is treated within 12 hours of occurrence or diagnosis of an injury or disease of the brain.

54. The method of any of claims 47-53, wherein the subject suffers from a condition selected from traumatic brain injury; hemorrhage, including subarachnoid and intraparenchymal hemorrhage; ischemic injury; autoimmune or infectious encephalitis; tumor- induced brain injury, edema, and intracranial pressure elevation; concussion, headache, migraine, hydrocephalus including normal pressure hydrocephalus, arachnoid cyst (ruptured or unruptured), intracranial hypertension; epilepsy; sleep and circadian rhythm disorders; neurodegenerative disease, and affective mental health disorders.

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