Accelerated chemical decalcification of bone in vivo to improve ultrasound signal transmission

Abstract

Among other things, in general, methods, apparatus, systems, and compositions are provided for rendering bone tissue sufficiently acoustically transparent for ultrasound imaging or ultrasound therapy or both. The bone tissue is rendered acoustically transparent by use of a decalcifying treatment (with or without concomitant sonication) within a pre-thinned cavity of bone. Ultrasound imaging and therapy examining a region of interest within brain, spinal column, carpal tunnel, etc. is thereby enabled.

Description

Accelerated chemical decalcification of bone in vivo to improve ultrasound signal transmissionCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Patent Application Ser. No. 63 / 660,725, filed June 17, 2024, the content of which is hereby incorporated by reference in its entirety.BACKGROUN D

[0001] Ultrasound therapies, such as ultrasonic mechanical ablation ("histotripsy"), ultrasound thermal ablation, high-intensity focused ultrasound, and ultrasound neuromodulation, can provide real-time imaging guidance in concert with therapy and are therefore highly valuable and uniquely suited for delivery of ultrasound therapy in many body tissues. Interest in the use of ultrasound in brain applications is growing, not only for more traditional uses such as imaging and high intensity focused ultrasound (HIFU) therapy, but also for a wide range of recently developed applications, including histotripsy, blood brain barrier modulation, neuromodulation, immune modulation, and drug delivery. In particular, imaging and histotripsy of brain tumors is a promising approach against brain cancer.

[0002] However, ultrasound imaging of (and therapy on) brain tissue is nearly impossible to deploy in current practice. Non-invasive delivery of ultrasound requires transmission through and across the skull ("transcran ially"), yet transcranial ultrasound is significantly impacted by the dense bone environment, with high acoustic attenuation and reflection. For example, non-invasive transcranial histotripsy results in aberration errors that are difficult to correct. Indeed, these aberration errors are generally too difficult to correct in current practice without sacrificing the degree of precision required for ablative brain procedures. The same is true for other ultrasound therapies across the skull. For many species of imaging subject, and for many ultrasound frequencies, ultrasound device output capabilities, and desired ultrasound targeting accuracies, the skull poses too difficult a barrier for ultrasound imaging or HIFU therapy even when assisted by MRI guidance.

[0003] In order to bypass the difficulties of using ultrasound non-invasively in brain procedures, efforts have been made towards performing ultrasound imaging-guided histotripsy in "minimally invasive" neurosurgery. For example, a small hole can be made in the skull, and the procedure could be performed at least to some degree using miniaturizedimaging and histotripsy devices. In a modification of this approach, a section of skull can be removed and replaced by an acoustically transparent (sonolucent) implant, such as with glass or polymethyl methacrylate. Among the significant disadvantages of these approaches are that opening up the skull and exposing the brain is, per se, not "minimally invasive". Indeed, this is a significant surgery that results in tissue trauma, blood loss (potentially resulting in death), and long subject recovery times. Based on preclinical animal research and the nascent literature around neuroinflammation risk factors, opening the skull (even transiently to install the implant) also has additional risks, as doing so has been preclinically observed to result in changes to the astrocytic and microglial environment of the brain that may require administration of anti-inflammatory or antibiotic drugs in the subject. Even assuming that patient risk is low for prophylactic, short-term administration of such drugs, these neuroinflammatory changes might not be prevented in a human subject even with such drugs. The resulting neuroinflammatory changes are per se undesirable and might be semi-permanent or permanent, which adds to the risk profile of open-skull approaches for ultrasound therapy.

[0004] Clinical uptake for the above approaches has therefore been limited. There remains a need to find more clinically acceptable ways to supply ultrasound modalities though bone tissue, especially through the skull.SUMMARY

[0005] Herein are disclosed compositions, methods, apparatus, and systems for enhancing acoustic signal transmission into a subject by mechanical thinning of a bone or other calcified tissue, followed by chemical decalcifying of the thinned region to the extent required to achieve a window for ultrasound imaging of or therapy on a subject; particularly, as it applies to enhancing acoustic signal transmission into the brain without exposing it (i.e., the dura mater remains intact).

[0006] In general, in an aspect, a method of ultrasound imaging of a region of interest using an ultrasound imaging probe is disclosed. The region of interest is within a brain encased by a skull. The imaging is performed through a thinned region of the skull (an area of the skull either previously thinned, or thinned as the first step of the method, so as to form a cavity capable of receiving the ultrasound imaging probe; in each case thus "pre-thinned" with respect to the following steps). The pre-thinned cavity of the skull is charged with a decalcifying treatment for a period of time sufficient to render at least the base of the prethinned cavity acoustically transparent, then the region of interest within the brain is imaged using ultrasound through the pre-thinned and treated region of skull. Implementations mayinclude one or more of the following. The decalcifying treatment is removed prior to the imaging. The ultrasound imaging is accompanied by ultrasound therapy. The pre-thinned cavity is thinned to between 20 and 50 microns. The pre-thinned cavity is thinned to between 23 and 47 microns. The pre-thinned cavity is thinned to between 26 and 44 microns. The pre-thinned cavity is thinned to between 29 and 41 microns. The pre-thinned cavity is thinned to between 33 and 37 microns. The pre-thinned cavity is thinned to about 35 microns. The thinning is followed by verifying that the thinning is complete. The verifying includes performing ultrasound imaging using the probe to obtain a measure of acoustic transparency of the cavity and comparing the measure to a previously established reference. The probe is inserted into the cavity during the ultrasound imaging. The decalcifying treatment is a calcium chelator. The decalcifying treatment contains an aqueous solution of ethylenediamine tetraacetic acid (EDTA) or a pharmaceutically acceptable salt thereof. The decalcifying treatment is an aqueous solution containing about 20% EDTA. The decalcifying treatment includes a semisolid that leaches EDTA. The period of time is about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 150 minutes, about 180 minutes, or more than 180 minutes. The period of time is at least about 30 minutes. The period of time is between 15 minutes and 30 minutes. The method also includes sonicating within the prethinned cavity during the period of time. The decalcification is accompanied by sonication using a sonicating device. The sonication is low intensity sonication at a frequency between 20 kHz and 30 MHz, or at a frequency between 500 kHz and 20 MHz, or at a frequency between 1 MHz and 10 MHz. The sonication is continuous at a frequency of about 1 MHz. The period of time is at least about 15 minutes when low intensity sonication is also in operation. The ultrasound therapy is ultrasound thermal ablation. The ultrasound therapy is thermal high-frequency focused ultrasound. The ultrasound therapy is ultrasound mechanical ablation. The ultrasound therapy is histotripsy. The ultrasound therapy is neuromodulation. The ultrasound therapy is focused ultrasound.

[0007] In general, in an aspect, a method of ultrasound imaging of a region of interest within a brain encased by a skull is disclosed. The method includes imaging using ultrasound through a pre-thinned and pre-treated region of the skull where it has been previously thinned and been made acoustically transparent with a decalcifying treatment to the extent desired for ultrasound. Implementations may include one or more of the following. The ultrasound imaging is accompanied by ultrasound therapy. The ultrasound therapy is ultrasound thermal ablation. The ultrasound therapy is thermal high-frequency focusedultrasound. The ultrasound therapy is ultrasound mechanical ablation. The ultrasound therapy is histotripsy. The ultrasound therapy is neuromodulation. The ultrasound therapy is focused ultrasound.

[0008] In general, in an aspect, a system for ultrasound imaging of a region of interest within a brain encased by a skull is disclosed, the system including an ultrasound imaging device, a pump capable of pumping a decalcifying treatment, and input and output tubing connected to the pump. The imaging is performed through a previously thinned region of the skull once decalcifying treatment has been pumped through the previously thinned region via the pump and tubing for a period of time sufficient to make the region acoustically transparent for ultrasound. Implementations may include one or more of the following. The system also includes an ultrasound therapy device. The system also includes a sonicating device equipped to operate while the pump operates. The decalcifying treatment contains an aqueous solution of ethylenediamine tetraacetic acid or a pharmaceutically acceptable salt thereof. The period of time is about 15 minutes, about 30 minutes, between 15 minutes and 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 150 minutes, about 180 minutes, or more than 180 minutes.

[0009] In general, in an aspect, a method of ultrasound imaging of a region of interest using an ultrasound imaging probe is disclosed. The region of interest lies within a subject behind calcified tissue (i.e., bone or other calcium-containing tissue), and the calcified tissue is prethinned in an area to form a pre-thinned cavity capable of receiving the probe. The method includes charging the pre-thinned cavity with a decalcifying treatment for a period of time sufficient to render the pre-thinned cavity acoustically transparent, and performing ultrasound imaging of the region of interest in the subject through the pre-thinned cavity. Implementations may include one or more of the following. The calcified tissue is bone. The calcified tissue is not anatomical bone. The calcified tissue is skull, spine, foot, shoulder, hip, rib, wrist, or leg.

[0010] With the foregoing and other advantages and features of the invention that will become hereafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the invention, drawings, examples, and appended claims.BRIEF DESCRIPTION OF DRAWI NGS

[0011] FIG. 1 shows an embodiment of a chemical decalcification system and method described herein on surgically thinned bone.

[0012] FIG. 2 reports histotripsy cavitation threshold voltage across a decalcified skull window, relative to a threshold voltage at a contralateral control site with all skull tissue removed.

[0013] FIG. 3 reports the difference in B-mode contrast between the decalcified side of a skull relative to the control condition (no skull; defined as 0 V solid line) over 60 minutes of a chemical decalcification process.

[0014] FIG. 4 illustrates B-mode images on the decalcifying side of a skull during the decalcification process at 0 min, 15 min, 30 min, and 60 min left-to-right (at top) compared to an image taken with no skull at 30 min (at bottom).

[0015] FIG. 5. A) Photo of a rat skull after thinning and 15 min decalcification of the treatment site (left circle) and completion of the craniectomy control site (right circle), prior to histotripsy. B) Photo of a rat skull undergoing decalcification with EDTA solution (cycled with immersed in / out port needles) held within a fluid well bordered by a sealed O-ring. The aluminum lens of a histotripsy device can also be seen above the O-ring. C) H&E stained cross section of untreated full thickness rat skull. (D) High magnification photograph of a thinned and decalcified (30 min EDTA) rat skull sample, cut and viewed edge-on. A 33 pm thickness example measurement point is shown.

[0016] FIG. 6 shows a further embodiment of a chemical decalcification system and method described herein on surgically thinned bone.

[0017] FIG. 7 shows benchtop hydrophone measurements of acoustic attenuation by different rat skull samples in the Examples. A) Raw data traces for 5 and 30 MHz presenting (from bottom to top) normal skull, thinned skull, thinned and decalcified skull, and control recording with no skull. B) Attenuation across frequencies for each skull sample relative to no skull.

[0018] FIG. 8 illustrates B-mode images of a single rat from the Examples as imaged over a 60 minute decalcifying process. A) Cropped B-mode image showing treatment with decalcifying EDTA solution over time and the associated thinning of the skull (high contrast white arc diffusing in the images left-to-right). B) Full-size B-mode image of the treated site 31 minutes after the 60 minute decalcification process had concluded. C) Full-size B-mode image of the craniectomy (no skull) control site in the same rat.

[0019] FIG. 9 shows a bar graph with analysis from the Examples. A) mean post-treatment B-mode brightness relative to pre-treatment across groups; B) mean cavitation threshold MPa differences from the within-animal no skull control. Error bars + / - 1 standard deviation; * indicates p < 0.05DETAILED DESCRIPTION

[0020] Figure Legend

[0021] 101 Surface of skull / bone tissue within pre-thinned cavity

[0022] 103 Skull, bone tissue

[0023] 105 Dura

[0024] 107 Brain

[0025] 109 Pre-thinned cavity of the skull / bone tissue charged with decalcifying therapy solution

[0026] 111 Region of interest within the brain

[0027] 113 Pump

[0028] 115 Output tubing

[0029] 117 Input tubing

[0030] 121 Ultrasound imaging device + / - ultrasound therapy device

[0031] 131 Sonicating device

[0032] 509 Pre-thinned cavity of the skull / bone tissue charged with decalcifying EDTA solution

[0033] 515 Needle connected to output tubing

[0034] 516 Fluid containment structure (O-ring)

[0035] 517 Needle connected input tubing

[0036] 521 Ultrasound therapy device

[0037] 590 Circular treated region (pre-thinned and decalcified)

[0038] 591 Circular control region (skull removed)

[0039] 600 Subject of imaging

[0040] 609 Pre-thinned cavity of the skull / bone tissue charged with decalcifying therapy solution

[0041] 613 Pump

[0042] 615 Output tubing

[0043] 616 Fluid containment structure (O-ring sealed to the surface of skull / bone tissue surrounding the pre-thinned cavity)

[0044] 617 Input tubing

[0045] 631 Sonicating device

[0046] 651 Waste syringe

[0047] 653 Fluid syringe charged with decalcifying solution

[0048] 661 Needle connected to output tubing

[0049] 663 Needle connected to input tubing

[0050] 665 Filter

[0051] 801 Surface of skull / bone tissue within pre-thinned cavity

[0052] 803 Skull, bone tissue

[0053] 807 Brain

[0054] 811 Example 2 mm x 2.5 mm region of interest within the brain (used for brain gray value calculation)

[0055] 880 Cropped window described by polygon within B-mode imaging window (FIG. 8A)

[0056] 882 Full-size B-mode imaging window (FIG. 8B, 8C)

[0057] 884 Thinned skull

[0058] 886 Brain surface

[0059] 888 Hippocampus

[0060] 890 Cortex

[0061] To facilitate acoustic signal transmission into the brain, e.g., for brain imaging or histotripsy, we herein describe creating an acoustic window in the bone tissue by surgically thinning a region of bone, such as by drilling or other means of bone removal, without breaking through to the dura mater underneath, followed by decalcification of the thinned bone via a topical chemical treatment. Decalcification greatly reduces the bone density, while leaving the other structural elements of the bone such as collagen intact, resulting in an acoustically transparent window of bone while maintaining a barrier of native tissue over the brain. The speed of decalcification can be further enhanced by simultaneous application of low intensity (continuous or intermittent) sonication.

[0062] Ultrasound imaging and therapies can then be performed across this window (particularly at frequencies up to about 30 MHz) into the brain immediately following the procedure, or in the future across the scalp after closing the surgical incision for, e.g., non- invasive longitudinal imaging and / or therapy. Other advantages of having acoustic access to the brain through a decalcified-but-closed skull include allowing for better recovery from the procedure (i.e., the brain is allowed to swell to some degree in order to avoid traumatic brain injury secondary to excessive intracranial pressure) and avoiding the risk of the brain "mushrooming out" of the therapy site.

[0063] In some embodiments, the subject of imaging is a mammal. In some embodiments, the subject of imaging is a rodent. In some embodiments, the subject of imaging is a human. In some embodiments, the bone removal is to the minimum extent required for creating a recessed region within the bone. In some embodiments, the bone removal is to the extentpreviously calibrated for a given species, sex, size, skull length / width, or some other factor or combination thereof that, with experience using the methods or systems disclosed herein, has been found in preclinical or clinical practice (as applicable) to provide for prethinning that supports acceptable-to-optimal application of the decalcifying treatment. In some embodiments, anthropometric calibration is used to support the desired amount of bone removal. In some embodiments, radiographic information is used to support the desired amount of bone removal. In some embodiments, prior within-subject experience with the methods and systems described herein is used to support the desired amount of bone removal (e.g., to "true up" the amount of bone removed in a subsequent session so as to at least approximately match the amount of bone that had been removed in an earlier session).

[0064] In some embodiments, the methods and systems described herein can be used in combination with endoscopic form factor devices capable of high frequency ultrasound imaging and histotripsy. Examples of preferred devices known in the art include those described in T. G. Landry et al., 'Endoscopic co-registered ultrasound imaging and precision histotripsy: Initial in vivo evaluation', BME Front, vol. 2022, no. 9794321, pp. 1-14, 2022, doi: 10.34133 / 2022 / 9794321; C. A. Samson, A. Bezanson, and J. A. Brown, 'A Sub-Nyquist, Variable Sampling, High-Frequency Phased Array Beamformer', IEEE Trans Ultrason Ferroelectr Freq Control, vol. 64, no. 3, pp. 568-576, 2017, doi:10.1109 / TUFFC.2016.2646925; J. K. Woodacre, T. G. Landry, and J. A. Brown, 'A low-cost 10 mm diameter histotripsy transducer for tissue ablation guided by a co-registered high- frequency endoscopic phased array', in 2017 IEEE International Ultrasonics Symposium, IUS, 2017, pp. 1-4. doi: 10.1109 / ULTSYM.2017.8092405; or T. G. Landry and J. A. Brown, 'Ultrasound imaging guided precision histotripsy: Effects of pulse settings on ablation properties in rat brain', J Acoust Soc Am, vol. 155, no. 4, pp. 2860-2874, Apr. 2024, doi: 10.1121 / 10.0025832. The contents of these references are hereby incorporated by reference in their entireties.

[0065] By "acoustically transparent", we mean, in general, that when the material or tissue so described happens to lie within an acoustic path between an ultrasound imaging or therapy device and a region of interest to be imaged or treated, the material or tissue does not pose an impediment to imaging or treating that region of interest. In reference to clinical practice of ultrasound imaging or therapy, acoustically transparent tissues or materials do not attenuate ultrasound waves to any clinically meaningful degree. Preferably, tissues ormaterials rendered acoustically transparent by methods or systems described herein will have minimal, or at most manageable, reflection or other artifact.

[0066] By "charging", we mean applying to, on, or into a pre-thinned (and thus at least partially recessed) region of a skull so as to substantially cover the bottom and sides of the region but not to appreciably escape over the top of the region. In some embodiments, the charging is with a liquid. In some embodiments, the liquid is also being continuously replenished by being pumped into and out of the pre-thinned cavity through tubing, fabricated of biocompatible material in whole or at least as to the portion of tubing that contacts the pre-thinned cavity. In some embodiments, the charging is with a semisolid such as an ointment or cream.

[0067] By "decalcifying treatment", we mean a chemical means of decalcification delivered in a form appropriate for clinical use. In some embodiments, the decalcification is effected by means of a calcium chelator. In some embodiments, the calcium chelator is ethylenediaminetetraacetic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the decalcifying treatment comprises an aqueous solution of EDTA or a pharmaceutically acceptable salt thereof. In some embodiments, the solution is buffered to a biocompatible pH, such as to about physiological pH (7.4). In some embodiments, the decalcifying treatment comprises a calcium chelator incorporated within a pharmaceutical preparation suitable for topical use. In some embodiments, the solution is an aqueous solution comprising over 10% but not greater than 30% EDTA. In some embodiments, the solution is pH-adjusted 20% EDTA aqueous solution. In some embodiments, the solution is pumped continuously through the pre-thinned cavity and the solution has a concentration gradient tailored to perform the treatment to the desired extent and in the desired period of time.

[0068] By "pre-thinned", we mean reduction of the thickness of bone or other calcium- containing tissue prior to imaging or therapy, in which reduced thickness is desirable to prepare the area being thinned to be decalcified using method or systems disclosed herein. Conventional ultrasound imaging of, or therapy into, regions of interest that lie behind bone or other calcified tissue can be significantly hampered in that unmodified bone is too acoustically opaque to allow for ultrasound to penetrate to the region of interest; removal of bone or calcium-containing tissue across an area results in a pre-thinned cavity with residual bone that can be decalcified in a clinically desirable timeframe. In some embodiments, the pre-thinning is accomplished using mechanical means, such as with a drill. In some embodiments, the pre-thinning is monitored by visual appearance and deemed completebased on clinical judgment. In some embodiments, the pre-thinning is monitored by manual palpation and deemed complete based on clinical judgment. In some embodiments, the prethinning is verified using ultrasound based on a predetermined output indicative of completion. In some embodiments, the pre-thinning is verified using optical coherence tomography based on a predetermined output indicative of completion. In some embodiments, the predetermined output indicative of completion is the degree of acoustic transparency inferred from the output, based on previously established references for a given calcified tissue / bone, species of subject, size of subject, age of subject, or the degree of transparency desired for the subject's clinical goal (e.g., diagnostic imaging, longitudinal imaging, therapy). In some embodiments, the thinning results in the base of the pre-thinned cavity having a total thickness of between 20 and 50 microns. In some embodiments, the thinning results in the base of the cavity having a total thickness of between 23 and 47 microns. In some embodiments, the thinning results in the base of the cavity having a total thickness of between 26 and 44 microns. In some embodiments, the thinning results in the base of the cavity having a total thickness of between 29 and 41 microns. In some embodiments, the thinning results in the base of the cavity having a total thickness of between 33 and 37 microns, such as 35 microns. In some embodiments, the thinning results in the base of the cavity having a total thickness of 20, 21, 22, 23, 24, 25, 26, 1 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 microns.

[0069] In some embodiments, the period of time is about 15 minutes. In some embodiments, the period of time is about 30 minutes. In some embodiments, the period of time is between 15 minutes and 30 minutes. In some embodiments, the period of time is about 45 minutes. In some embodiments, the period of time is about 60 minutes. In some embodiments, the period of time is about 75 minutes. In some embodiments, the period of time is about 90 minutes. In some embodiments, the period of time is about 105 minutes. In some embodiments, the period of time is about 120 minutes. In some embodiments, the period of time is about 150 minutes. In some embodiments, the period of time is about 180 minutes. In some embodiments, the period of time is more than 180 minutes. Whether a period of time is sufficient or optimal for producing the desired effect on ultrasound imaging or therapy depends on a variety of factors including the type of therapy (if any), species, sex, size, skull length / width, or some other factor or combination thereof that, with experience using the methods or systems disclosed herein, in preclinical or clinical practice (as applicable) has been found to provide for acceptable-to-optimal application of the desired ultrasound modality.

[0070] In some embodiments, the speed of decalcification can be further enhanced by simultaneous application of low intensity sonication. In some embodiments, the acoustic field of the sonication device is tuned mechanically or electronically to produce uniform acoustic exposure over the area to be decalcified. In some embodiments, the low intensity sonication is at a frequency between 20 kHz and 30 MHz, or at a frequency between 500 kHz and 20 MHz, or at a frequency between 1 MHz and 10 MHz. In some embodiments, the sonication is continuous. In some embodiments, the low intensity sonication is continuous at a frequency of about 1MHz. In some embodiments, a sonication on / off duty cycle is established to balance intensity of sonication (energy input) with the desired frequency. In some embodiments, the duty cycle is 10 milliseconds on / 10 milliseconds off. In some embodiments, the duty cycle is 20 milliseconds on / 20 milliseconds off. In some embodiments, the enhanced speed is about twice as fast when compared to decalcifying according to the methods described herein when not applying sonication at the same time. In some embodiments, the frequency and power of the sonication are optimized to enhance the speed of decalcification while maintaining thermal safety and avoiding cavitation.

[0071] In some embodiments, the ultrasound modality is ultrasound imaging. In some embodiments, the ultrasound modality is ultrasound thermal ablation. In some embodiments, the ultrasound modality is thermal high-intensity focused ultrasound (HIFU). In some embodiments, the ultrasound modality is ultrasound mechanical ablation. In some embodiments, the ultrasound modality is histotripsy. In some embodiments, the ultrasound modality is neuromodulation. In some embodiments, the ultrasound modality is focused ultrasound. In some embodiments, the ultrasound modality is diagnostic. In some embodiments, the ultrasound modality is therapeutic. In some embodiments, the ultrasound modality is intended to open the blood-brain barrier at least in part so as to enable adjunctive therapy. In some embodiments, the ultrasound modality is applied clinically for human clinical or research use. In some embodiments, the ultrasound modality is applied for veterinary use. In some embodiments, the ultrasound modality is applied for preclinical or research use.

[0072] In some embodiments, an ultrasound imaging device is used. In some embodiments, both an ultrasound imaging and an ultrasound therapy device is used. In some embodiments, a device capable of simultaneous or sequential ultrasound imaging and ultrasound therapy is used.

[0073] In general, under conventional clinical practice, when a subject has a lesion or other region of interest that is the intended object of imaging, therapy, or both, it cannot beaccessed with ultrasound if it lies behind bone or other calcified tissue. In some embodiments, the methods or systems disclosed herein are used to decalcify skull bone in order to ultrasonically access a region of interest within the brain. In some embodiments, the methods or systems disclosed herein are used to decalcify spine. In some embodiments, the methods or systems disclosed herein are used to decalcify foot, shoulder, hip, rib, wrist, or leg bone. In some embodiments, the methods or systems disclosed herein are used to decalcify a calcified tissue that is not anatomical bone.

[0074] Chemical means of decalcification are in some embodiments used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. Pharmaceutically acceptable salt(s) are well-known in the art. For clarity, the term "pharmaceutically acceptable salts" as used herein generally refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable non-toxic acids include inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids. Examples of specific salts thus may include hydrochloride and mesylate salts. The preparation and use of acid addition salts, carboxylate salts, amino acid addition salts, and zwitterion salts of compounds of the present invention may also be considered pharmaceutically acceptable if they are, within the scope of sound medical judgment, suitable for use in contact with the soft tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, are commensurate with a reasonable benefit / risk ratio, and do not appreciably counterbalance the intended decalcifying effect. Such salts may also include various solvates and hydrates.

[0075] Dosage forms for topical administration may include creams, ointments, poultices, and other semisolid dosage forms. A decalcifying therapy described herein can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which may be required.

[0076] In some embodiments, during treatment a sonicating device is used to apply an acoustic or other vibrational or mechanical signal to the site, in order to agitate the agenttissue interface or to enhance the molecular interaction of the agent with tissue and clearance of agent-bound calcium from the tissue.

[0077] A schematic of a method and system of decalcification is shown in an embodiment in FIG. 1. The process may be accelerated by flowing decalcifying solution via pump 113, input tubing 117, and output tubing 115; and / or sonication via sonicating device 131, though these features are optional elements as in some embodiments, the treatment requires only to charge the pre-thinned cavity 109 of the bone tissue 103 with decalcifying therapy such that the surface 101 of the bone tissue 103 within the pre-thinned cavity is suitably acoustically transparent for imaging and / or therapy through the dura 105 into the region of interest 111 within the brain 107 using an ultrasound device 121. Similarly, an embodiment is shown in FIG. 6 with a subject of imaging (e.g., a rat) 600.

[0078] In some embodiments, a sealant or fluid containment structure (e.g., an O ring 616 as FIG. 6) may be applied or attached around the edge of the treatment site in order to contain the decalcifying agent and prevent it contacting non-target areas. In some embodiments, the O ring is adhered upon its bottom via wax or glue to the bone tissue immediately surrounding the edge of the treatment site. For ultrasound imaging or therapy regimes that require treatment across the window at future time points, additional decalcification may be desirable if the bone becomes re-calcified over time. This additional treatment could be achieved by re-opening the scalp, or by the installation of a system for infusion via catheter or implanted minipump for example.

[0079] In some embodiments, a pump recirculates decalcifying agent through the region for decalcification. In some embodiments, a pump moves the decalcifying agent through the region for decalcification. In some embodiments, a system for ultrasound imaging is so equipped as to allow for pumping out of the decalcifying agent once the decalcifying period of time is elapsed. See for example FIG. 6 in which a pump 613 moves previously unused decalcifying fluid from an input syringe 653 through input tubing 617, passing though a filter 665 to trap particulate, through a needle 663 into the region for decalcification 609. This used fluid is siphoned via output syringe 661 through the output tubing 615 and into the output / waste syringe 651 for disposal, e.g., into a waste reservoir. In some embodiments, the input syringe 653 is connected to a reservoir of unused decalcifying fluid.

[0080] In a preferred embodiment described in the Examples below, thinning the skull to approximately 35 microns thickness and decalcifying with 20% EDTA for at least 30 minuteseffectively rendered the treated bone (i.e., skull) acoustically transparent with respect to 5.4MHz ultrasound therapy (e.g., for histotripsy) and with respect to 30 MHz ultrasound imaging signals.

[0081] In another preferred embodiment described in the Examples below, thinning the skull to approximately 35 microns thickness, and decalcifying with 20% EDTA for at least 15 minutes while simultaneously sonicating at about 1 MHz effectively rendered the treated bone (i.e., skull) acoustically transparent with respect to 5.4 MHz ultrasound therapy (e.g., for histotripsy) and with respect to 30 MHz ultrasound imaging signals.

[0082] Data presented below includes one or more of the following: decalcifying with 20% EDTA up to 60 mins in rats; EDTA clearance and replenishment using a syringe pump; and continuous sonication at 1 MHz.

[0083] Example 1 - Details of ultrasound equipment and general experimental procedures

[0084] The equipment used for ultrasound and histotripsy was that described in the preferred device references incorporated herein. Briefly, the imaging probe was a 64 element 30 MHz phased array design with a 3.6x3.8 mm tip size. The histotripsy device was 10 mm in diameter with an aluminum lens giving a fixed 7 mm focus depth, and operated at 5.4 MHz. The histotripsy device had a 3.9 mm square hole in the center through which the imaging device was positioned and epoxied in place, situating the histotripsy focal spot at a fixed 6 mm depth in the imaging window.

[0085] The imaging system used FPGA-based beamforming hardware with display software showing B-mode data in a ±32° sector window up to 15 mm depth. A full frame power Doppler display could be activated which was useful for displaying active bubble clouds during histotripsy. The histotripsy drive hardware consisted of a 0-650 V variable high voltage supply (TDK Lambda, Tokyo, Japan), a function generator to control the pulse shape (Tektronix, Beaverton, USA), and a custom interposer PCB to drive the device. Drive voltages during in vivo experiments were typically in the 200-300 V range. All histotripsy pulses were 5.4 MHz 12 cycle bursts presented at a rate of ~938 Hz (triggers synchronized to imaging system). To estimate the MPa output by the histotripsy device during ablation, benchtop pressure measurements were recorded using a fiber optic hydrophone (FOHS v2, Precision Acoustics, Dorset, UK) located at the focal spot while driving the device at 1-10 V. Using the output intensity at these low voltages, the non-linear output at high voltages was estimated using the HITU simulator v2.0 script developed by Soneson (https: / / github.com / jsoneson / HITU_Simulator). (All MPa values given refer to the peak negative pressure estimated from the HITU model for the drive voltage and device used.)

[0086] For treatment groups undergoing continuous 1MHz skull sonication during decalcification, this sonication device consisted of a 9.5 mm diameter Pz39 piezo disc (1 MHz resonance) fixed in a 3D-printed case with an empty space for air backing. 10 pm of parylene film was deposited over the surface for biocompatibility and device protection. The plastic case was attached to a support rod for mounting in a manipulator. A function generator sent a 1 MHz 10 V peak-peak continuous sine signal during sonication. Because the working distance is in the ultrasound near field, the pressure pattern was very nonuniform, but had a maximum peak-to-peak pressure of about 180 kPa. The total power reaching the skull within a 6 mm O-ring well (described later for the in vivo preparation) is estimated to be 24 mW. In order to offset the near field nonuniformity and present the skull target region with a more even in sonification over time, the device was translated under motorized control in a 3 mm diameter circular path parallel to the skull surface at a rate of 1 rotation per minute over the course of treatment. A benchtop hydrophone recording of the acoustic field of this device at 3 mm distance (the approximate distance it was used in vivo) showed that the cumulative signal exposure at 3 mm depth over time in this translated configuration produce an acoustic exposure over time that was more spatially even, especially within the 6 mm target circle of the intended treatment zone.

[0087] Prior to each in vivo experiment, acoustic cavitation emission recording was performed in freshly poured tap water to measure the histotripsy device cavitation threshold, utilizing a setup previously constructed for assessing cavitation probability (T. G. Landry, M. G. Mallay, and J. A. Brown, 'A Simple and Cost-Effective Benchtop Setup for Assessing Ultrasound-Induced Inertial Cavitation Probability', IEEE Sens Lett, vol. 7, no. 6, pp. 1-4, 2023, doi: 10.1109 / LSENS.2023.3282076; hereby incorporated by reference in its entirety). The cavitation threshold was defined as the voltage yielding cavitation in 50% of trials. The threshold value measured in fresh tap water was usually within 20 V (~1.7 MPa) of the value that later achieved cavitation in the brain, making this test a simple indicator of the approximate expected cavitation threshold voltage in brain and an efficient check procedure for changes in device performance from experiment to experiment.

[0088] All procedures were approved by the Dalhousie University Committee for Laboratory Animals (protocol 23-077).

[0089] Example 2 - Decalcification procedure (preliminary study)

[0090] In a preliminary study, the decalcification procedure was performed in 20 normal rats (n=5 each of 15 min, 30 min, 60 min treatments, and 60 min sham treatment with saline). The decalcification procedure used 20% EDTA (pH adjusted to 7.4) on a circular 6mm diameter region of skull that was thinned by drilling (mean post-drilling thickness 35 pm, s.d. 12 pm). The fluid was contained by a silicone O-ring that was first glued around the site. Fresh EDTA was continuously infused and waste removed at a rate of 0.15 ml / min via syringe pump. The simultaneous sonication / agitation concept described herein was not used for these 20 rats; but see Example 5 ff for further study in additional animals.

[0091] Example 3 - Measurement of histotripsy cavitation threshold through decalcified window

[0092] Analyzing the 20 animals treated in Example 2, FIG. 2 shows the effect of the treatment on histotripsy cavitation (5.4 MHz) threshold through the decalcified window relative to the threshold measured at a control site with no skull present. For the 0-30 minute treatment data, there was a significant correlation between threshold change and treatment time (Pearson correlation, r = -0.881, p < 0.001) with increasing EDTA treatment times resulting in cavitation thresholds that approached the threshold seen with no skull. There was no significant difference between the 30 minute treatment values and zero (one sample t-test, t(4) = 0.885, p > 0.05), indicating that the EDTA treatment effectively resulted in a completely acoustically transparent window by 30 minutes for the 5.4 MHz histotripsy signal. There was no significant difference in threshold change between the 30 and 60 minute treatment groups (independent samples t-test, t(8) = 0.614, p > 0.05). Data points shown by "x" indicate points where cavitation was not observed on the treated skull up to the maximum drive voltage used (~300 V, in order to minimize risk of damaging the histotripsy device). The values assigned to these points are just above the maximum voltage tested and therefore represent the smallest possible threshold differences for those experiments but true values may in fact be higher. If true values were indeed higher it would appear to further strengthen the negative correlation.

[0093] Average skull thickness (after any thinning or decalcification) in this study ranged from about 20 microns to about 80 microns. There was no statistically significant effect on threshold change with respect to skull thickness, which suggests that the decalcification treatment is the primary driver of producing an acoustically transparent window for ultrasound.

[0094] Example 4 - B-mode imaging through decalcified window

[0095] Analyzing the animals treated in Example 2, regarding 30 MHz ultrasound imaging through the decalcified skull window, B-mode contrast was measured between the brain cortex and the empty space above the skull. As with the cavitation threshold analysis, the contrast values were compared to the same measure from the control side (no skull) withinanimals, and the differences were compared statistically. Data points and images at 0 min (treatment not yet begun) were treated as sham treatment. A one-way ANOVA found a significant effect of treatment time (F(3) = 4.057, p = 0.025), with post hoc contrast analysis showing that 30 and 60 minute EDTA treatments were significantly higher contrast than the sham treatment (30 min p = 0.014, 60 min p = 0.027), whereas 15 minute treatment was not significantly different from the sham group (p > 0.05). The contrast difference values are shown in FIG. 3 along with representative examples of B-mode images from the various conditions in FIG. 4.

[0096] Example 5 - Decalcification procedure and additional experimental details (preliminary plus further study)

[0097] In further work, 11 additional adult Wistar rats (228-412 g weight) were added to the experiment (preliminary plus further study totals n = 31, 16 male, 15 female) were used. These additional rats were assigned to perform decalcification in conjunction with General anesthesia was induced with isoflurane in oxygen (4% induction, 1.5-2.5% maintenance) and body temperature was maintained at 37°C with a homeothermic heat pad. The head was secured in a stereotaxic frame and the scalp was incised. The periosteum was cleared away with a curette and alcohol swab to expose and clean most of the skull surface covering the cerebrum. A tissue marker was used to mark the boundaries of the skull target sites relative to the skull reference point bregma (P) : targets were two 6 mm diameter circles, each centered at p-3.5 mm and 3.2 mm to the left and right of midline. See FIG. 5.

[0098] Skull treatment: The left or right side was pseudo-randomly chosen as the treatment side and the other side was the craniectomy control (i.e. no skull, or "NS" condition). Bone thinning was achieved by careful manual drilling with a rotary dental drill, mostly using a 1.6 mm ball cutting bit followed by a 1 mm ball cutting bit to further even out the bone, especially around the edges of the site. During drilling the skull was frequently irrigated with saline to minimize heating due to drilling friction. Bone thinness was judged visually by the appearance of dura and brain blood vessels through the translucent thinned bone, and by the easy deformation of the thinned bone with gentle prodding. As described later, the bone was found to be an average across animals of about 35 pm thick after this procedure. For the NS side, the bone was thinned in a ring shape but otherwise left unopened at this stage. A fluid well was then created at the site by using bone wax and cyanoacrylate glue to fix a silicone O-ring (inner diameter 6.1 mm; McMaster-Carr, Aurora, USA; Cat. 1182N01) to the skull, being careful not to allow glue onto the thinned patch, as any glue may impedeultrasound signals. Prior to any further treatment the well was first filled with saline and the untreated skull was imaged with ultrasound for pre-treatment reference.

[0099] To decalcify the thinned bone patch, 20% EDTA (Fisher Scientific, Ottawa, Canada; Cat. E478) dissolved in deionized water was used. It was found that EDTA concentrations >20% led to precipitate formation too soon after preparation to be practically useful. The stock 20% EDTA solution was stored at a pH of 9-10 for shelf stability and pH was adjusted to 7.4 with HCI several hours before use. To achieve pH of 9-10 to dissolve the EDTA powder, 10-11 g of NaOH pellets (Fisher Cat. S318) per 100 ml were required, and to adjust pH back down to 7.4 before in vivo use, about 0.6 ml of 37% HCI was used (Fisher Cat. A144). The resulting concentrations of Na+and CT for the in vivo solution are estimated to be 2.65 M and 0.42 M, respectively. A syringe pump (model BS-8000; Braintree Scientific, Braintree, USA) was used to continuously eject EDTA into the O-ring well at a rate of 0.15 ml / min via a 25 gauge needle positioned inside the well above the skull. A second syringe and needle simultaneously withdrew EDTA at the same rate as ejection, resulting in a constant fluid volume in the well with a continuously refreshed EDTA supply and removal of calcium-bound EDTA waste. Any EDTA applied to the skull was passed through a 0.22 pm pore size PVDF membrane filter just before the ejection needle to capture potential contaminants and solution precipitates. A schematic of the setup used in this and following Examples is given in FIG. 6.

[0100] EDTA treatment lasted for one of four durations: 15 min, 20 min, 30 min, or 60 min (n = 5 per duration group, except n = 6 for 20 min). The 20 min group was added after earlier results from the 15 and 30 min group suggested that an intermediate time might be informative. A sham treatment group (n = 5) was also included that received identical procedures but 60 min of saline was used rather than EDTA. This 60 min saline sham treatment group is sometimes referred to as the 0 min EDTA treatment group. Finally, a 15 min EDTA treatment group that also included 1 MHz continuous insonification was also included to determine if this could increase decalcification rate. For this group the sonication device was positioned over the O-ring ~3 mm from the skull. The device face was in contact with the EDTA and the pump needles remained in the small gap between the sonicator and O-ring. The sonicator was activated with a 1 MHz 10 V signal for the 15 min treatment while translating the device in a 3 mm diameter circle.

[0101] Ultrasound imaging and histotripsy: For ultrasound imaging, the histotripsy lens bowl was filled with ultrasound gel and any bubbles in the gel were removed under a microscope. The O-ring well was filled with saline and the ultrasound device, held by a rigidmotorized micropositioner attached to the stereotaxic frame, was positioned over the thinned skull and ultrasound imaging data was collected. The saline was then wicked out with a tightly rolled Kimwipe (Kimberly-Clark, Roswell, USA), the well was manually filled with EDTA, and the syringe pump activated. For 30 and 60 min treatment groups, every 15 min the pump was stopped, the EDTA was wicked out, the well flushed several times with saline, and ultrasound imaging performed at the site. This intermediate imaging step was done after 10 min in the 20 min treatment group. Replacing the EDTA with saline was found to be necessary for image collection as the EDTA significantly attenuated the image, making images collected when coupled with EDTA harder to analyze and not comparable to other images collected before and after treatment through saline / gel. A photograph of the tissue was also usually taken through the surgical microscope during the ultrasound imaging break. After any intermediate ultrasound imaging in the 20-60 min groups, the saline was then wicked out and manually replaced by EDTA as above, and the treatment resumed with the syringe pump until the cumulative target treatment time was reached. After the full treatment period the well was again filled with saline and imaging immediately performed. The O-ring and glue were then removed and the site cleaned well. The craniectomy on the control NS side was then completed to expose the dura.

[0102] Both left and right sites were covered in ultrasound gel and the probe was positioned over the center of the NS site with the fixed histotripsy focal spot location at 0.8 mm below the brain surface. While recording imaging data, the histotripsy signal was turned on with the voltage set to 20 V below the cavitation threshold measured in tap water prior to the experiment. The histotripsy signal was left on for at least 3 sec, and if no bubble cloud appeared in the Doppler imaging, the signal was switched off, the voltage was increased by 5 V (~0.45 MPa), and the process repeated until cavitation occurred or the maximum allowed voltage of 300 V was reached, beyond which there was an increasing risk of damaging the histotripsy device. If cavitation occurred, as determined by a strong Doppler signal spot appearing, the histotripsy signal was maintained for an additional 3 sec then stopped. The probe was then moved to the center of the thinned / decalcified treatment site and the same process was performed.

[0103] Tissue Processing: Within minutes of the second histotripsy treatment, rats were sacrificed by overdose injection of sodium pentobarbital (150 mg / kg, intraperitoneally). After breathing cessation, they were transcardially perfused with warm 0.01 M phosphate buffered saline (PBS) followed by cold neutral buffered formalin (NBF). The brain was then harvested and placed into NBF for overnight fixation at 4°C. The section of skull containingthe thinned patch was also retained and placed into NBF overnight. The following day the brain and skull samples were rinsed in PBS and the brain was placed in 15% sucrose. The fixed skull samples remained in PBS for storage. A sample of full thickness untreated skull was also saved from one rat in order to gain a better understanding of the untreated skull tissue structure and measure its thickness. This sample was fixed in NBF overnight then decalcified in 10% EDTA at room temperature on a shaker for several days with daily 10% EDTA changes followed by one day in 15% sucrose. (The decalcification of this full thickness bone sample for tissue processing is unrelated to the in vivo skull decalcification method described herein: decalcifying bone samples as described in this paragraph is a standard bone histology processing step to allow for thin sectioning.)

[0104] Once saturated in 15% sucrose, brains and the single untreated thick skull sample were placed into 30% sucrose until saturation. Samples were then cryoembedded in O.C.T. and stored at -80°C. Coronal plane brain sections with 15 pm thickness were then collected every 90 pm using a cryostat and sections stained with hematoxylin and eosin (H&E). The thick untreated skull sample was similarly sectioned and stained to view the skull edge-on (Fig.2D). H&E slides were scanned at 200x with an Aperio slide scanner and viewed with ImageScope software (Leica, Wetzlar, Germany).

[0105] The thinned skull samples were examined under a high magnification dissection microscope with the thinned region cut with a razor blade and viewed edge-on (e.g., Fig. 2D). Photographs were taken with 225x magnification at multiple locations per sample and the thickness was measured in several locations per image using ImageJ image analysis software (NIH, Bethesda, USA). The within-animal average of all the measurements was assigned as the skull thickness for that animal.

[0106] Ultrasound analysis: To quantify changes in ultrasound brain imaging signal with EDTA treatment, ultrasound B-mode video snapshots were imported into ImageJ. B-mode brightness of the brain was measured in a 2x2.5 mm region of interest placed below the skull, covering most of the cortical and hippocampus thickness, and the mean gray value was then calculated. When analyzing brightness differences between different conditions within animals, the differences were converted to dB by scaling to the known dynamic range setting in the imaging software (e.g. with the 0-255 grayscale range at a 60 dB dynamic range setting, a mean grayscale difference of 50 is converted by (50 / 255)x60 dB = 11.8 dB).

[0107] To quantify the ability of treated skull to constrain brain bulging, a segmented arc was traced on the bulged surface in both treated and NS before histotripsy, and the ratio was calculated for the arc length to the distance between the arc endpoints (e.g. a fullsemicircle bulge would give a value of w / 2, or 1.571). This normalized approach aimed to reduce variability based on small differences in drilled site diameter or errors in choosing the site edges in B-mode images, rather than using an absolute metric such as arc height. For reference, B-mode images of a thinned decalcified skull and no skull were made using the same rat; measurement of the brain bulge using the ratio of surface arc length to arc endpoint distance for each condition were 1.053 and 1.119 respectively.

[0108] Statistical testing was performed using SPSS software (IBM, Armonk, NY) and figures were generated using SPSS, Aperio ImageScope, ImageJ, Matlab 2024b (Mathworks, Natick, MA), and Inkscape software (GNU General Public License, www.inkscape.or ).

[0109] Benchtop measurements: To more directly determine the degree of ultrasound attenuation in different bone conditions, some benchtop measurements were performed. Acoustic signals were directed at the FOHS hydrophone in a tank of distilled water with the ultrasound device position under microstepper motor control. The signals were 10 V, 12 cycle sine wave pulses from a function generator, ranging from 1-9 MHz using an unfocused fragment of the same piezo material used for the histotripsy device, and from 10-40 MHz using a single element of an imaging probe. The two different drive devices were used because of differences in ability to output a clean sinusoid signal over different frequency ranges. This test was performed with the devices at a fixed position of about 6.5 mm without any bone sample as a baseline, and then while several different bone samples were held <0.5 mm in front of the hydrophone tip, including a full thickness normal nondecalcified rat skull sample. The thinned skull samples were from the in vivo experiments and the specific samples were chosen to be close to the average of 37 pm that was found across all rats. Hydrophone signals were averaged 256 times by an oscilloscope and the waveform saved. Using Matlab, the signals were bandpass filtered from 0.7-1.3x the stimulus frequency, then the waveform envelope was derived, and the average envelope amplitude was extracted over 4 cycles after signal onset (i.e. the mid-section of the pulse). The dB amplitude difference for each bone sample relative to no bone was then calculated for each frequency to generate attenuation curves.

[0110] Although it had little effect on interpreting in vivo results, the density of 20% EDTA (after adjusting pH to 7.4) was calculated by weighing a 10 ml sample. EDTA speed of sound at 30 MHz was also measured by exciting a single imaging array element and measuring the time-of-flight changes between pulse-echo responses from a flat quartz reflector at varying distances. The density of pH-adjusted 20% EDTA was measured as 1.12 g / ml, and speed of sound was 1695 m / s compared to 1484 m / s measured for distilled water.

[0111] For the thickness measurements of thinned skull samples, the mean across all rats was 37.4 pm (±11.2). A univariate ANOVA found no significant treatment group effect on skull thickness (p > 0.05), indicating no systematic bias in skull thickness between groups.

[0112] Example 6 - Analysis of further study

[0113] Acoustic attenuation values for the three skull samples measured are shown in FIG. 7. The thinned / decalcified skull sample showed essentially no attenuation in the histotripsy and imaging frequency ranges (i.e. around 5.4 and 30 MHz) although there was a dip of several dB from 10-15 MHz. Full thickness normal rat skull samples gave much greater attenuation at most frequencies, mostly on the order of 5-15 dB, with a general trend of more attenuation with increasing frequency. The thinned but not decalcified rat skull attenuated signals >7 MHz by about 5 dB. These one-way attenuation measurements would be doubled for return echo imaging signals.

[0114] The B-mode imaging data was available throughout the decalcification treatment period at regular intervals within each animal (see FIG. 8 for an example within a single rat; sonication was not performed during this treatment for this animal). The most instructive analysis was the difference in mean brightness of imaged bone between pre-treatment and post-treatment. The post-treatment images were collected after a period of time elapsed (average 26.2 min), during which the O-ring was removed, the site cleaned, and the craniectomy was completed for the NS side. During this post-treatment interval an increase in B-mode brightness (3.1 dB on average) and improved anatomy visibility was usually seen on the EDTA-treated side compared to immediately after treatment. In non-sonicated animals, the 3.1 dB increase was confirmed to be significant (one-sample two-sided t-test against a difference of 0; t(20) = 3.539, p = 0.002) and the sonicated group 0.8 dB decrease not significant (p > 0.05).

[0115] B-mode brightness: Following the post-treatment delay, the dB difference in B-mode brightness of the brain tissue between post-treatment and pre-treatment was calculated for each animal. There was a trend towards a ~10 dB increase in brightness following treatment (FIG. 9A), in agreement with the ~5 dB attenuation seen in hydrophone measurements across thinned skull (FIG. 7B), as two-way imaging signals are doubly attenuated. A univariate ANOVA was performed on the difference values across the treatment groups. The average treated skull thickness for each animal was also included as a covariate to test whether the degree of skull thinning had a significant influence on outcomes. The treatment group effect was significant (F(5) = 3.846, p = 0.011) whereas the skull thickness was not a significant factor (p > 0.05). A post hoc simple contrast analysis of each EDTA treatmentgroup versus the sham group showed significantly higher values at 15 min sonicated (p = 0.005), 20 min (p = 0.021), 30 min (p = 0.005), and 60 min treatments (p < 0.001), but not for non-sonicated 15 min versus sham (p > 0.05).

[0116] To examine how the brightnesses compared to NS control sides within animals, a two-sided one-sample t-test was performed within each treatment group, testing against a difference of 0. For sham and 20 min EDTA duration, brightness was significantly lower than NS (sham t(4) = -7.290, p = 0.002; 20 min t(5) = -5.853, p = 0.002), whereas brightnesses for the other groups were not different from NS (p > 0.05). Given that the 20 min treatment values were significantly below controls and all but one non-sonicated 15 min value were below control, it is likely that that a larger sample size would have found a significant difference in the 15 min group as well. Therefore, the B-mode results altogether suggest that by 30 min of EDTA treatment alone there was little attenuation of the 30 MHz signal with minimal change beyond 30 min, and that sonication accelerated decalcification, making the bone about as transmissive by 15 min as non-sonicated 30 min.

[0117] Cavitation threshold: For NS sites the mean cavitation threshold was 29.9 MPa (±2.9). The change in cavitation threshold MPa was calculated as the dB difference between the treatment side and NS control. Note that the actual MPa at the brain cavitation focus site itself would be expected to be unchanged in any condition, but rather the MPa values here reflect changes in the device output intensity that were required to overcome the additional attenuation caused by the bone. In two of the 20 min animals, glue leaked onto the thinned bone, raising a concern that cavitation threshold may have been increased in these animals due to attenuation by the glue. Therefore, these two animals were excluded from cavitation threshold analysis a priori, reducing the 20 min group size from 6 to 4. The leaked glue was far enough from the central area of the thinned patch that there was no concern that it may have affected imaging.

[0118] The same general ANOVA design was run for cavitation threshold data as with the B- mode change above: treatment group factor with skull thickness covariate, and post hoc contrasts of EDTA groups against the sham group (FIG. 9B). There was a significant treatment group effect (F(5) = 10.175, p < 0.001) but again no skull thickness effect (p > 0.05). Contrasts showed a significant difference between sham and each of the EDTA groups (non-sonicated 15 min p = 0.002; sonicated 15 min p < 0.001 ; 20 min p < 0.001; 30 min p < 0.001; 60 min p < 0.001). The difference values approached 0 dB with increasing duration, appearing to plateau by 30 min. Sonication appeared to provide a slight improvement or acceleration of the process.

[0119] As with B-mode brightness analysis, two-sided one-sample t-tests were run for each treatment group for cavitation threshold dB differences against a value of 0 to determine whether each group was significantly different from their NS controls. At 0 and 15 min treatment the threshold was significantly greater than NS (sham t(4) = 6.981, p = 0.002; 15 min t(4) = 4.785, p = 0.009), whereas the other four groups were not significantly different from NS (p > 0.05). These results together with the ANOVA suggest that the 5.4 MHz focused histotripsy signal was not significantly attenuated by the thinned skull by 20 min of EDTA treatment, with minimal change beyond 20 min, while 15 min treatment without sonication had a mitigating effect compared to sham but some attenuation was still occurring. As with the B-mode results, sonication accelerated the decalcification process.

[0120] In conclusion: this study examined the acute effects of decalcifying thinned rat skull with EDTA in vivo on ultrasound signal transmission and on the brain tissue appearance. Overall, the EDTA treatment was highly effective in rendering the bone acoustically transparent, with brain visibility and histotripsy threshold both being comparable to NS controls after at least 30 min. Furthermore, continuous low power 1 MHz sonication appeared to accelerate the decalcification process, with a 15 min combined treatment resulting in a similar effect as 30 min EDTA without sonication.

Claims

Claims1. A method of ultrasound imaging of a region of interest using an ultrasound imaging probe, wherein the region of interest is within a brain encased by a skull and the skull is pre-thinned in an area of the skull to form a pre-thinned cavity capable of receiving the probe, the method comprising: charging the pre-thinned cavity with a decalcifying treatment for a period of time sufficient to render the pre-thinned cavity acoustically transparent, and performing ultrasound imaging of the region of interest through the pre-thinned cavity.

2. The method of claim 1 further comprising applying sonication upon the pre-thinned cavity of the skull using a sonicating device during the period of time.

3. The method of claim 2 in which the sonication is at a frequency between 20 kHz and 30 MHz.

4. The method of claim 3 in which the sonication is at a frequency between 500 kHz and 20 MHz.

5. The method of claim 4 in which the sonication is continuous during the period of time and at a frequency of about 1 MHz.

6. The method of claim 1 in which the decalcifying treatment is removed prior to the ultrasound imaging.

7. The method of claim 1 in which the probe is inserted into the cavity during the ultrasound imaging.

8. The method of claim 1 further comprising performing an ultrasound therapy simultaneous with or after the ultrasound imaging.

9. The method of claim 8 in which the ultrasound therapy is selected from ultrasound thermal ablation, thermal high-intensity focused ultrasound, ultrasound mechanical ablation, histotripsy, neuromodulation, and focused ultrasound.

10. The method of claim 1 in which the decalcifying treatment comprises a calcium chelator.

11. The method of claim 1 in which the decalcifying treatment comprises an aqueous solution having ethylenediamine tetraacetic acid (EDTA) or a pharmaceutically acceptable salt thereof.

12. The method of claim 1 in which the decalcifying treatment comprises a semisolid that leaches a calcium chelator.

13. The method of claim 1 in which the period of time is selected from about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 150 minutes, and about 180 minutes.

14. The method of claim 13 in which the period of time is between 15 minutes and 30 minutes.

15. The method of claim 1 in which the period of time is more than 180 minutes.

16. The method of claim 1 further comprising, first, thinning a region of the skull to become the pre-thinned cavity.

17. The method of claim 16 in which the thinning is followed by verifying that the thinning is complete.

18. The method of claim 17 in which the verifying comprises performing ultrasound imaging using the probe to obtain a measure of acoustic transparency of the cavity and comparing the measure to a previously established reference.

19. The method of claim 1 in which the pre-thinned cavity of the skull is between 20 and 50 microns.

20. The method of claim 19 in which the pre-thinned cavity of the skull is between 23 and 47 microns.

21. The method of claim 20 in which the pre-thinned cavity of the skull is between 29 and 41 microns.

22. The method of claim 21 in which the pre-thinned cavity of the skull is between 33 and 37 microns.

23. The method of claim 1 in which the pre-thinned cavity of the skull is about 35 microns, the decalcifying treatment is an aqueous solution comprising about 20% ethylenediamine tetraacetic acid (EDTA) aqueous solution, and the period of time is at least about 30 minutes.

24. The method of claim 2 in which the pre-thinned cavity of the skull is about 35 microns, the decalcifying treatment is an aqueous solution comprising about 20% ethylenediamine tetraacetic acid (EDTA) aqueous solution, and the period of time is at least about 15 minutes.

25. A method of ultrasound imaging of a region of interest within a brain encased by a skull, the method comprising performing ultrasound imaging of the region of interest through a acoustically transparent pre-thinned cavity of the skull, the pre-thinned cavity having previously been made acoustically transparent with a decalcifying treatment.

26. The method of claim 25 further comprising performing an ultrasound therapy simultaneous with or after the ultrasound imaging.

27. The method of claim 26 in which the ultrasound therapy is selected from ultrasound thermal ablation, thermal high-intensity focused ultrasound, ultrasound mechanical ablation, histotripsy, neuromodulation, and focused ultrasound.

28. A system for ultrasound imaging of a region of interest within a brain encased by a skull, the imaging to occur through a pre-thinned cavity of the skull, the system comprising an ultrasound imaging device, a pump capable of pumping a decalcifying treatment, and input and output tubing connected to the pump, whereby ultrasound imaging can be performed once the input and output tubing is placed within the pre-thinned cavity and decalcifying treatment is pumped through the tubing for a period of time sufficient to render the prethinned cavity acoustically transparent.

29. The system of claim 28 equipped to also perform ultrasound therapy, further comprising an ultrasound therapy device.

30. The system of claim 29 further comprising a sonicating device equipped to operate while the pump operates.

31. The system of claim 29 in which the decalcifying treatment is an aqueous EDTA (or pharmaceutically acceptable salt thereof) solution and the period of time is selected from about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 150 minutes, and about 180 minutes.

32. The system of claim 29 in which the decalcifying treatment is an aqueous EDTA (or pharmaceutically acceptable salt thereof) solution and the period of time is more than 180 minutes.

33. The system of claim 29 in which the ultrasound therapy is selected from ultrasound thermal ablation, thermal high-intensity focused ultrasound, ultrasound mechanical ablation, histotripsy, neuromodulation, and focused ultrasound.

34. A method of ultrasound imaging of a region of interest using an ultrasound imaging probe, wherein the region of interest lies within a subject behind calcified tissue, wherein the calcified tissue is pre-thinned in an area to form a pre-thinned cavity capable of receiving the probe, the method comprising: charging the pre-thinned cavity with a decalcifying treatment for a period of time sufficient to render the pre-thinned cavity acoustically transparent, and performing ultrasound imaging of the region of interest in the subject through the prethinned cavity.

35. The method of claim 34 in which the calcified tissue is selected from skull, spine, foot, shoulder, hip, rib, wrist, and leg.

36. The method of claim 34 in which the calcified tissue is a calcified region of the subject that is not anatomical bone.

Images