Low-intensity transcranial focused ultrasound cerebral apoplexy rehabilitation animal experiment method and system

Abstract

The invention discloses a low-intensity transcranial focused ultrasound cerebral apoplexy rehabilitation animal experiment method and system. The method sequentially comprises the following steps of ultrasonic parameter safety calibration, animal screening and baseline establishment, cerebral apoplexy animal model preparation and establishment, ultrasonic intervention treatment, neurobehavioral dynamic evaluation, tissue sample acquisition and processing and multi-modal tissue inspection. The system comprises a stimulation unit, a positioning unit and a control unit, and is used for realizing accurate and parameter-controllable ultrasonic intervention. By constructing a complete research closed loop from parameter safety verification and standardized model intervention to multi-dimensional effect evaluation, the problems of blind parameter selection, single evaluation system and nonstandard experimental process in the prior art are solved, and a set of standardized and repeatable research normal form is provided. The rehabilitation effect of the LIFU on the cerebral apoplexy can be comprehensively and objectively evaluated, and parameter optimization can be guided.

Description

Technical Field

[0001] This invention relates to the fields of neuromodulation and stroke rehabilitation technology, and in particular to a systematic animal experimental research method based on low-intensity transcranial focused ultrasound (LIFU) to promote the recovery of neurological function after ischemic stroke. Background Technology

[0002] Ischemic stroke (cerebral infarction) is one of the leading causes of death and long-term disability in adults. Although revascularization therapy (such as intravenous thrombolysis) has been applied clinically, the treatment window is narrow, and many patients still suffer from varying degrees of neurological dysfunction. Therefore, developing novel rehabilitation techniques that can effectively promote neurological repair after stroke is of great significance.

[0003] Low-intensity transcranial focused ultrasound (LIFU), as an emerging non-invasive neuromodulation technique, shows great potential in the intervention of neuropsychiatric diseases because it can non-invasively focus sound energy on deep brain regions and modulate neuronal activity. However, translating LIFU into an effective stroke rehabilitation therapy faces three major challenges: First, ultrasound waves attenuate and distort when they propagate through the skull. Accurately assessing and ensuring their effective focusing and biosafety in the live target area is a prerequisite for clinical application. Second, the biological effects of LIFU are closely related to its acoustic parameters (such as sound intensity, pulse repetition frequency, duty cycle, etc.), but there is currently a lack of systematic research to clarify the differences in the effects of different parameter combinations on stroke rehabilitation, and parameter selection relies heavily on experience. Third, existing animal experiment evaluation systems are often too simplistic, failing to organically combine behavioral functional assessment, histological analysis, and molecular mechanism exploration, making it difficult to comprehensively and objectively evaluate efficacy and guide parameter optimization.

[0004] Therefore, it is urgent to establish a standardized and systematic set of animal experimental research methods to scientifically evaluate the rehabilitation effect of LIFU on stroke and to provide a basis for parameter optimization and mechanism exploration. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a low-intensity transcranial focused ultrasound method for animal rehabilitation of stroke. This method integrates ultrasound parameter safety calibration, standardized animal model construction, multi-time point neurobehavioral assessment, and multi-level histopathological and molecular biological analysis, forming a complete evaluation system.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a low-intensity transcranial focused ultrasound (LCI-QU) method for stroke rehabilitation. The method is a systematic animal experimental evaluation method for evaluating the effect of low-intensity transcranial focused ultrasound on neurological function rehabilitation after ischemic stroke and optimizing its stimulation parameters. The method performs the following steps in a preset logical order to form a complete research closed loop from parameter safety verification, model intervention to multi-dimensional effect evaluation. S1: Ultrasound parameter safety calibration steps: Determine the safe and effective parameter window for low-intensity transcranial focused ultrasound through sound field simulation, in vitro acoustic measurement, phantom thermal damage experiment, and in vivo blood-brain barrier opening experiment; S2: Animal screening and baseline establishment steps: Select healthy experimental animals, exclude those with spontaneous neurological defects or abnormal activity, and after adaptive training, randomly divide them into control group, stroke model group and ultrasound stimulation group; S3: Steps for preparing animal models of stroke: Constructing a cerebral artery occlusion model in experimental animals using the suture occlusion method; S4: Ultrasound intervention treatment steps: After the establishment of the stroke animal model, low-intensity transcranial focused ultrasound stimulation is applied to the target area of ​​the animal brain using the parameters determined by the safety calibration steps described above; S5: Dynamic neurobehavioral assessment steps: Perform neurobehavioral tests on experimental animals at multiple time points before and after ultrasound intervention treatment to dynamically assess their neurological function recovery. S6: Tissue sample acquisition and processing steps: At the end of the experiment, the animal was euthanized, brain tissue was obtained and processed for morphological and molecular biological analysis; S7: Multimodal tissue testing steps: Perform multiple tests on the processed brain tissue samples, including infarct volume staining, histopathological staining, apoptosis detection, and specific protein expression analysis. Specifically, step S1 provides a safe and effective ultrasound parameter window for step S4; the evaluation results of step S5 are correlated with the test results of step S7, together forming a complete chain of evidence for evaluating the mechanisms of nerve function recovery and tissue repair.

[0007] Furthermore, in step S1, step S1 specifically includes the following three-level progressive calibration process: (1) Sound field simulation prediction: A simulation model was established based on the generalized Westervelt equation to simulate transcranial ultrasound propagation and predict focal characteristics; (2) Determination of safety boundaries in vitro: The skull is covered with tissue phantoms and subjected to ultrasound irradiation. The safe sound pressure and duty cycle range that do not produce thermal damage are determined by judging whether protein coagulative necrosis occurs in the phantoms. (3) In vivo focusing effectiveness verification: Within the specified safety parameters, the target area of ​​a live animal was irradiated with ultrasound after intravenous injection of microbubbles and dye to verify its ability to reversibly and precisely open the blood-brain barrier of the target area.

[0008] Furthermore, in step S2, the experimental animals are SD rats weighing 220-250g; the adaptive training includes balance beam walking and adhesion removal training, with a training time of 3-5 days; after grouping, each group contains 8 animals.

[0009] Furthermore, in step S3, a middle cerebral artery occlusion / reperfusion model is established using the suture embolization method. Specifically, a nylon suture embolization is advanced to the origin of the middle cerebral artery to cause ischemia. After ischemia lasts for 90 minutes, the suture embolization is removed to restore perfusion.

[0010] Furthermore, in step S4, a concave focusing transducer with a frequency of 1 MHz is used as the sound source, and the acoustic focus of the transducer is precisely located in the target brain region using a stereotaxic instrument; the treatment parameters are set and adjusted through a graphical user interface, including pulse repetition frequency, duty cycle, duration and stimulation interval; starting from the first day after surgery, treatment is performed once a day, each treatment lasting 15-30 minutes, for 5-10 consecutive days.

[0011] Furthermore, the target brain region is the sensorimotor cortex on the same side as the lesion.

[0012] Furthermore, in step S5, the neurobehavioral test is a modified neurological deficit score, and the scoring time points include at least postoperative day 1, day 3, day 7 and day 14, and the scoring process is conducted using a double-blind method.

[0013] Furthermore, in step S6, the animal is euthanized by cardiac perfusion. It is first rinsed with physiological saline, then perfused and fixed with 4% paraformaldehyde, and then the brain is removed and subjected to paraffin embedding or frozen sectioning.

[0014] Furthermore, in step S7, the brain tissue sample is subjected to tests including at least quantitative analysis of infarct volume, detection of apoptosis, and analysis of specific protein expression; and the test results obtained in step S7 are correlated with the neurobehavioral scores at the corresponding time points obtained in step S5.

[0015] The present invention provides a low-intensity transcranial focused ultrasound stimulation system for performing the above-described method, comprising a stimulation unit, a positioning unit, and a control unit; The stimulation unit includes a signal generator, a power amplifier, and a concave focusing transducer with a frequency of 1 MHz. The signal generator, power amplifier, and transducer are electrically connected in sequence to generate and output low-intensity focused ultrasound. The positioning unit is a stereo positioning device for experimental animals, used to fix the head of the experimental animal and adjust the spatial position of the transducer in three dimensions so that the acoustic focus of the transducer is accurately positioned on the predetermined target area of ​​the animal's brain. The control unit includes a controller with a graphical user interface, which is signal-connected to the stimulation unit and used to precisely set and regulate the ultrasound parameters output by the stimulation unit. The ultrasound parameters include pulse repetition frequency, duty cycle, duration of a single stimulation, and stimulation interval.

[0016] Furthermore, the control unit limits the sound pressure and duty cycle parameters of the ultrasound to a safe calibrated window range through the graphical user interface. The upper limit of the window range is that the sound pressure is no greater than 1.2 MPa and the duty cycle is no greater than 40%.

[0017] The beneficial effects of this invention are as follows: This invention provides a low-intensity transcranial focused ultrasound (LCI) method and system for animal experiments in stroke rehabilitation. This method integrates ultrasound parameter safety calibration, standardized animal model construction, multi-timepoint neurobehavioral assessment, and multi-level histopathological and molecular biological analysis, forming a complete evaluation system. This method can be used to evaluate the promoting effect of low-intensity transcranial focused ultrasound on neurological rehabilitation after ischemic stroke and to explore its potential mechanisms. It is a systematic, rigorous, and reproducible animal experimental method with the following advantages: Systematic and standardized: This invention integrates ultrasound physical parameter calibration, standardized disease model, dynamic functional assessment and multi-level mechanism exploration into a rigorous and closed-loop research process, which greatly improves the standardization and reproducibility of the experiment and provides a standardized model for LIFU stroke rehabilitation research.

[0018] Safety and precision are equally important: By conducting preliminary three-level parameter safety and efficacy calibration of “simulation-ex vivo-in vivo”, the safety of subsequent treatment experiments was ensured and the precision of transcranial focusing was verified, providing key experimental evidence for dosimetric exploration in clinical translation.

[0019] Comprehensive and objective evaluation: The longitudinal, double-blind behavioral scoring combined with multimodal histological tests such as TTC, HE, TUNEL, and IHC can comprehensively and objectively evaluate the therapeutic effect of LIFU from the macroscopic functional level to the microscopic cellular and molecular level, and reveal its potential mechanism of action (such as reducing infarct size, inhibiting apoptosis, and regulating inflammation).

[0020] Guiding parameter optimization: This methodological framework is particularly suitable for systematically comparing the efficacy differences of different LIFU parameters (such as different PRFs and DCs), thereby enabling the scientific selection of better or optimal stimulation parameter combinations for stroke rehabilitation and overcoming the blindness in parameter selection.

[0021] The above and other objects, advantages, and features of the present invention will be more fully set forth and demonstrated through the following detailed description of specific embodiments in conjunction with the accompanying drawings. Those skilled in the art, upon referring to the following detailed description and the accompanying drawings, will be able to better understand and realize the above advantages of the present invention. Other objects, features, and advantages of the present invention will become clearer after being described in detail in the detailed description section in conjunction with the accompanying drawings. Attached Figure Description

[0022] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following drawings are provided for illustration.

[0023] Figure 1 This is the overall flowchart of the low-intensity transcranial focused ultrasound (TCI) method for animal experiments in stroke rehabilitation. Figure 2 This is a schematic diagram of the principle of low-intensity transcranial focused ultrasound (TCI) animal experimental method for stroke rehabilitation; Figure 3 This is a schematic diagram of the nonlinear sound field simulation of the low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments. Figure 4 This is a schematic diagram of the ultrasound stimulation process in a low-intensity transcranial focused ultrasound (TCI) method for stroke rehabilitation in animal experiments. Figure 5 This is a schematic diagram of the statistical results of the modified neurobehavioral testing of the low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments. Figure 6 This is a statistical diagram of TTC staining results from a low-intensity transcranial focused ultrasound (TCI) method for animal rehabilitation of stroke patients. Figure 7 This is a schematic diagram of TUNEL staining results from a low-intensity transcranial focused ultrasound (TCI) method for animal rehabilitation of stroke patients. Figure 8 This is a statistical diagram of TUNEL staining results from a low-intensity transcranial focused ultrasound (TCI) method for stroke rehabilitation in animal experiments. Figure 9 This is a schematic diagram of the IL-10 immunohistochemical white light results of a low-intensity transcranial focused ultrasound (TCI) method for stroke rehabilitation in animal experiments. Figure 10 This is a statistical diagram of the IL-10 immunohistochemical white light results of low-intensity transcranial focused ultrasound (LCI) for stroke rehabilitation in animal experiments. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0025] Example 1 like Figure 1 As shown, Figure 1 This is a schematic diagram of the overall process of low-intensity transcranial focused ultrasound (LCI) for animal experiments in stroke rehabilitation. Figure 2 This is a block diagram of the principle of the low-intensity transcranial focused ultrasound (LCI-QUS) animal experiment method for stroke rehabilitation. The low-intensity transcranial focused ultrasound (LCI-QUS) animal experiment method for stroke rehabilitation provided in this embodiment is a systematic animal experimental evaluation method for evaluating the effect of low-intensity transcranial focused ultrasound on the rehabilitation of neurological function after ischemic stroke and optimizing its stimulation parameters. It performs the following steps in a preset logical order to form a complete research closed loop from parameter safety verification, model intervention to multi-dimensional effect evaluation. S1: Ultrasonic parameter safety calibration steps: Determine the safe and effective parameter window for low-intensity transcranial focused ultrasound through sound field simulation, in vitro acoustic measurement, phantom thermal damage experiment, and in vivo blood-brain barrier opening experiment. In this embodiment, the sound field simulation was based on the generalized Westervelt equation to establish a sound field simulation model, including a skull simulation model, verifying the feasibility of focal focusing when inserting the skull into the target area and tissue. Furthermore, a self-developed GUI software was used to control the signal generator, setting the focal sound pressure level to 1.2 MPa, the pulse repetition frequency (PRF) to 100 Hz, the duty cycle to 40%, the duration of each pulse to 0.4 s, the pulse interval to 2.6 s, and the total irradiation time to 20 min. No discoloration of the bovine whey protein phantom was observed, indicating that the temperature rise was within an acceptable range. Only when the parameters were further changed to 2 MPa, the duty cycle to 60%, and the pulse interval to 0.1 s did the bovine whey protein phantom show discoloration. Therefore, by ensuring lower sound pressure levels and lower duty cycles while increasing the pulse interval, the temperature rise can be kept within an acceptable and safe range.

[0026] In this embodiment, the safety and effectiveness parameter window can be set to sound pressure ≤ 1.2 MPa and duty cycle ≤ 40%. This parameter can be calibrated through a phantom thermal damage experiment. If protein coagulation occurs during the experiment, this parameter can be used as a safety parameter. The conclusion of its safety calibration can be supported by direct correlation through experiments. When the total energy is much greater than the parameter used, thermal damage will be caused. However, if the duty cycle or sound pressure is lower than this parameter, thermal damage will not be caused. This leads to the conclusion that the current parameter used is safe.

[0027] S2: Animal screening and baseline establishment steps: Select healthy experimental animals, exclude those with spontaneous neurological defects or abnormal activity, and after adaptive training, randomly divide them into control group, stroke model group and ultrasound stimulation group. In this study, 24 SD rats weighing 220–250g were selected, excluding those with spontaneous neurological deficits or abnormal behavior. The rats were randomly divided into a sham-operated control group, a stroke group, and an ultrasound stimulation group using a random number table. The sham-operated control group consisted of 6 rats, the stroke group of 6 rats, and the ultrasound stimulation group of 12 rats (3 rats for each parameter D1-D4) to enhance the rigor and reproducibility of the experiment. All rats underwent 3–5 days of acclimatization training before the experiment, including balance beam exercises and adhesion removal, to reduce stress interference and ensure consistency in baseline behavioral scores. S3: Steps for preparing an animal model of stroke: The cerebral artery occlusion model of experimental animals is constructed using the suture occlusion method; specifically, a nylon suture occluder is advanced through the internal carotid artery to the origin of the middle cerebral artery to cause focal cerebral ischemia. After ischemia lasts for 90 minutes, the suture occluder is removed to achieve reperfusion, thus establishing the middle cerebral artery occlusion / reperfusion model. S4: Ultrasound Intervention Treatment Procedure: After establishing the animal model of stroke, low-intensity transcranial focused ultrasound stimulation is applied to the target area of ​​the animal brain using the parameters determined in the aforementioned safety calibration procedure. S1 (parameter calibration) is a safety prerequisite for S4 (ultrasound intervention). In the ultrasound stimulation groups, rats in group D1 received PRF = 1000Hz and DC = 10%; rats in group D2 received PRF = 1000Hz and DC = 20%; rats in group D3 received PRF = 1000Hz and DC = 30%; and rats in group D4 received PRF = 1000Hz and DC = 40%. S5: Neurobehavioral assessment steps: At multiple time points before and after ultrasound intervention treatment, multiple neurobehavioral tests were performed on experimental animals to assess and statistically analyze the recovery of neurological function in different groups of rats. Among them, S2 (baseline establishment) is the basis for comparison in S5 (behavioral assessment). S6: Tissue sample acquisition and processing steps: At the end of the experiment, the animal was euthanized, brain tissue was obtained and processed for morphological and molecular biological analysis. S7: Multimodal tissue testing steps: Perform multiple tests on the processed brain tissue samples, including infarct volume staining, histopathological staining, apoptosis detection, and specific protein expression analysis. Specifically, step S1 provides a safe and effective ultrasound parameter window for step S4; the results of step S5 and step S7 are interrelated and together constitute a complete evaluation system for functional recovery and tissue repair mechanisms.

[0028] In this embodiment, step S1 specifically includes the following three-level progressive calibration process: (1) Sound field simulation prediction: A simulation model is established based on the generalized Westervelt equation to simulate transcranial ultrasound propagation and predict focal characteristics; In this embodiment, a sound field simulation model is established based on the generalized Westervelt equation, the acoustic parameters of the target skull are input, the transcranial ultrasound propagation is simulated, and the focal characteristics are predicted. (2) Determination of safety boundaries in vitro: The skull is covered with tissue phantoms and subjected to ultrasound irradiation. The safe sound pressure and duty cycle range that do not produce thermal damage are determined by judging whether protein coagulative necrosis occurs in the phantoms. (3) In vivo focusing effectiveness verification: Within the specified safety parameters, the target area of ​​a live animal was irradiated with ultrasound after intravenous injection of microbubbles and dye to verify its ability to reversibly and precisely open the blood-brain barrier of the target area.

[0029] like Figure 3 As shown, Figure 3 This is a nonlinear acoustic field simulation diagram of a low-intensity transcranial focused ultrasound (TCI) method for stroke rehabilitation in animal experiments (including a simulated skull structure, two-dimensional focal point distribution, and radial and axial sound pressure distribution). The results show that when the skull is located between the transducer and the focal point, the sound pressure amplitude at the focal point reaches its maximum value of approximately 4.8 kPa, a decrease of about 17.24% compared to the result without the skull, suggesting that the skull causes a certain degree of energy attenuation and distortion in sound wave propagation. The figure also shows that a clear energy accumulation zone is still formed at the focal point, indicating that ultrasound still possesses good focusing performance even with the skull present.

[0030] In this embodiment, in step S2, the grouped rats are labeled and repeatedly interacted with during pre-training to reduce resistance to the experimenters. The balance beam and adhesion removal experiments are repeated multiple times to ensure consistency in basic behavioral scores. In this embodiment, a temporary MCAO / R model was established in step S3 using the suture embolization method. Rats were fixed to the operating table under isoflurane anesthesia, and the common carotid artery, internal carotid artery, and external carotid artery were exposed. A nylon suture embolization was advanced to the origin of the middle cerebral artery to induce ischemia. After 90 minutes of ischemia, the suture embolization was removed to restore perfusion.

[0031] In this embodiment, in step S4, a 1 MHz concave focusing transducer is selected as the sound source. A signal generator produces the required pulse waveform, which is then amplified by a power amplifier before being output to the transducer to ensure a clear focusing point is formed beneath the rat's skull. The transducer's position is precisely controlled in three dimensions using a rat-specific stereotaxic instrument (Anhui Zhenghua Bio-Instrument Equipment Co., Ltd. ZH-Lanxing B / S type), ensuring accurate ultrasound irradiation covering the predetermined brain region. The low-intensity transcranial focused ultrasound stimulation module also includes a graphical user interface for precisely setting and adjusting the pulse repetition frequency, duty cycle, duration, and stimulation interval parameters. Treatment was administered daily at specific times (different groups used corresponding parameters, with LIFU stimulation for 20 minutes daily), for a total of three treatments on days 1, 3, and 7 after modeling. The rats were sacrificed for sampling on day 14 after testing and treatment completion.

[0032] like Figure 4 As shown, Figure 4 This is a schematic diagram of the ultrasound stimulation process of the low-intensity transcranial focused ultrasound (TCI) method for animal rehabilitation of stroke. In this embodiment, in step S5, three different groups of rats were given modified neurological scores, with the scoring time points being day 1, day 3, day 7, and day 14 after surgery.

[0033] like Figure 5 As shown, Figure 5 This is a schematic diagram of the modified neurobehavioral testing results of the low-intensity focused transcranial ultrasound (LCI) method for stroke rehabilitation in animal experiments (* indicates P≤0.05; ** indicates P≤0.01; *** indicates P≤0.001). On day 1 post-modeling, the MCAO group and all ultrasound stimulation groups (D1–D4) showed significantly elevated mNSS scores, approaching 11 points, indicating successful establishment of the middle cerebral artery occlusion model and inducing significant neurological deficits. Over time, the scores in all groups showed a decreasing trend, reflecting partial spontaneous recovery. However, compared with the MCAO group alone, the low-intensity focused ultrasound intervention group showed a greater decrease in scores on days 7 and 14.

[0034] In this embodiment, in step S6, after the final behavioral test, the rats underwent cardiac perfusion under overdose anesthesia. Blood was first removed with physiological saline, followed by perfusion with 4% paraformaldehyde for fixation. The brain was then removed, and the ischemic hemispheres were separated for subsequent paraffin embedding, frozen sectioning, or other molecular assays.

[0035] like Figure 6 As shown, Figure 6This is a statistical diagram of TTC staining results from a low-intensity transcranial focused ultrasound (LCU) animal experiment on stroke rehabilitation (* indicates P≤0.05; ** indicates P≤0.01; *** indicates P≤0.001). A comprehensive comparison of the control group, MCAO group, and each LIFU intervention group reveals the overall effect of LIFU intervention more intuitively. No infarct lesions were observed in the control group, which remained in a normal state; the MCAO group showed a significantly increased infarct volume, further confirming the stability and reliability of the model. All LIFU intervention groups significantly reduced the infarct area to varying degrees.

[0036] In this embodiment, in step S7, the brain tissue sample is subjected to tests including at least quantitative analysis of infarct volume, detection of apoptosis, and analysis of specific protein expression; and the test results obtained in step S7 are correlated with the neurobehavioral scores at the corresponding time points obtained in step S5.

[0037] This embodiment uses TTC staining to quantify cerebral infarction volume. Fresh brain slices are immersed in 2% TTC solution and incubated at 37°C in the dark for 15-20 minutes. Normal tissue appears red, while the infarcted area appears pale white due to dehydrogenase inactivation. The infarct volume is calculated after photographing. TUNEL staining is used to assess cell apoptosis. Paraffin sections are dewaxed and antigen-repaired, then incubated with TdT enzyme and labeled dUTP to reveal DNA breaks, allowing apoptotic cells to be identified and counted under a microscope. IL-10 immunohistochemical staining analysis is then performed.

[0038] like Figure 7 As shown, Figure 7 This is a schematic diagram of TUNEL staining results from a low-intensity transcranial focused ultrasound (TCI) method for stroke rehabilitation in animal experiments. In the control group, almost no obvious brownish-red positive cells were observed in the sections, with only a very small number of scattered signals. The overall tissue background was uniform and clear, suggesting that under physiological conditions, the level of neuronal DNA breakage remained low, and the incidence of apoptosis was extremely low. In contrast, the stroke group showed a large number of positive signals, especially concentrated in the ischemic penumbra and infarct core area, manifested as a dense distribution of brownish-red positive cells. The ultrasound stimulation group showed relatively milder results, indicating that stimulation had an ameliorative effect.

[0039] The low-intensity transcranial focused ultrasound stimulation system for performing the above method provided in this embodiment is characterized by comprising a stimulation unit, a positioning unit, and a control unit; The stimulation unit includes a signal generator, a power amplifier, and a concave focusing transducer with a frequency of 1 MHz. The signal generator, power amplifier, and transducer are electrically connected in sequence to generate and output low-intensity focused ultrasound. The positioning unit is a stereo positioning device for experimental animals, used to fix the head of the experimental animal and adjust the spatial position of the transducer in three dimensions so that the acoustic focus of the transducer is accurately positioned on the predetermined target area of ​​the animal's brain. The control unit includes a controller with a graphical user interface, which is signal-connected to the stimulation unit and used to precisely set and regulate the ultrasound parameters output by the stimulation unit. The ultrasound parameters include pulse repetition frequency, duty cycle, duration of a single stimulation, and stimulation interval.

[0040] In this embodiment, the control unit limits the sound pressure and duty cycle parameters of the ultrasound to a safe calibrated window range through the graphical user interface. The upper limit of the window range is that the sound pressure is no greater than 1.2 MPa and the duty cycle is no greater than 40%.

[0041] Example 3 The low-intensity transcranial focused ultrasound method for stroke rehabilitation provided in this embodiment is characterized by comprising the following steps performed in sequence: S1: Ultrasonic parameter safety calibration procedure This step is a core pretreatment process designed to ensure the safety and validity of subsequent in vivo experiments. Specifically, it includes: Sound field simulation and characteristic prediction: Based on the generalized Westervelt equation, a sound field simulation model is established. The real acoustic parameters (sound velocity, sound attenuation coefficient) of the skull of the target animal (such as rat) are input to simulate the transcranial propagation process of ultrasound at a specific frequency (such as 1 MHz) and predict its focal position, sound pressure distribution and possible distortion in the brain tissue.

[0042] In vitro validation and safety boundary determination: Isolated skull samples were covered with tissue phantoms (e.g., bovine serum albumin-polyacrylamide composite gel) and irradiated with ultrasound at different sound pressure levels and duty cycles. The occurrence of protein coagulative necrosis (manifested as localized whitening and coagulation) in the phantoms was used as a direct criterion to determine the safe sound pressure and duty cycle ranges that would not cause thermal damage. This established a clear energy safety upper limit for in vivo experiments.

[0043] In vivo efficacy verification: Within the established safety parameters, the target brain region of a live animal was irradiated with ultrasound by intravenous injection of microbubble contrast agent and tracer dye (such as Evans Blue). After the experiment, the precise location and extent of dye extravasation in brain tissue sections were observed to verify whether LIFU combined with microbubbles could achieve reversible and precise opening of the blood-brain barrier in vivo, thereby indirectly confirming the effective focusing of ultrasound energy in the live brain.

[0044] S2: Animal Selection and Baseline Establishment Steps Healthy adult SD rats weighing 220–250g were selected as experimental animals; the number could be determined according to the study design (e.g., 24 rats). Individuals with spontaneous neurological deficits or significant behavioral abnormalities were first excluded. All selected rats underwent 3–5 days of acclimatization and pre-training before the formal experiment. Pre-training included, but was not limited to, behavioral tests such as balance beam walking and removal of adhesives (stickers). This process aimed to: Reduce stress: Familiarize rats with the experimental environment and operators to reduce their tension and resistance during subsequent tests.

[0045] Ensure baseline consistency: Through repeated training, all rats achieved a relatively stable and consistent level of basic motor and sensory abilities before the formal experiment began, providing reliable baseline data for subsequent grouping and comparison.

[0046] Standardized procedures: Researchers repeatedly interacted with rats and followed standard operating procedures during pre-training.

[0047] After pre-training, all rats were randomly divided into several groups, such as: sham surgery control group (only undergoing surgery to expose blood vessels, without inserting sutures), stroke model group (MCAO model, without ultrasound treatment), LIFU treatment group (MCAO model followed by ultrasound intervention), etc., with each group usually consisting of 8 rats.

[0048] S3: Steps for establishing an animal model of stroke A rat model of temporary middle cerebral artery occlusion / reperfusion (MCAO / R) was established using the classic suture occlusion method to simulate clinical ischemic stroke. The procedure was as follows: Rats were fixed to a temperature-controlled operating table under inhalation anesthesia (e.g., isoflurane). A midline incision was made in the neck, and the left common carotid artery, internal carotid artery, and external carotid artery were separated and exposed. The distal end of the external carotid artery was ligated to temporarily block blood flow to the common carotid artery. A small incision was made in the external carotid artery, and a nylon suture with a silicone-coated tip was inserted and advanced into the cranium along the internal carotid artery for approximately 18-22 mm (the exact length varied slightly depending on the rat strain and weight) until slight resistance was encountered. At this point, the tip of the suture had reached the origin of the middle cerebral artery, blocking its blood flow and causing cerebral ischemia. After 90 minutes of ischemia, the suture was slowly removed, restoring blood flow to the middle cerebral artery and achieving reperfusion. Rats in the sham-operated group underwent the same surgical procedure except that no suture was inserted.

[0049] S4: Ultrasound Intervention Treatment Steps After successful establishment of the MCAO model (usually starting from day 1 post-surgery), ultrasound intervention was applied to rats in the LIFU treatment group. An ultrasound system calibrated for safety in step S1 was used, for example, a concave focusing transducer with a center frequency of 1 MHz as the sound source. This system consisted of a signal generator, power amplifier, transducer, and stereotaxic apparatus. Treatment parameters, including pulse repetition frequency (PRF, e.g., 100 Hz, 500 Hz, 800 Hz), duty cycle (DC, e.g., 10%, 20%, 30%), duration of a single stimulation, and stimulation interval, could be precisely set and adjusted via a graphical user interface (GUI).

[0050] During treatment, the anesthetized rat's head is fixed to a stereotaxic instrument, the hair on the top of the head is shaved, and an ultrasound coupling agent is applied. By adjusting the three-dimensional coordinates of the stereotaxic instrument, the acoustic focus of the transducer is precisely located on the target brain region (usually the sensorimotor cortex on the same side as the lesion). According to preset parameters, the treatment is performed for a specific duration each day (e.g., 20 minutes), and the entire intervention is completed within a certain cycle (e.g., 7 consecutive days of treatment).

[0051] S5: Steps for Dynamic Neurobehavioral Assessment To objectively and dynamically evaluate neurological deficits and recovery, double-blind neurobehavioral scoring was performed on rats in all groups (control group, model group, and treatment group) at several key time points before ultrasound intervention (baseline) and after treatment (e.g., days 1, 3, 7, and 14 post-surgery). Researchers unaware of the animals' grouping performed the scoring. The core assessment tool was the Modified Neurological Deficit Score (mNSS), which comprehensively evaluates the animals' motor, sensory, balance, and reflex functions, with a total score of 0-14, where higher scores indicate more severe neurological deficits. By comparing changes in mNSS scores at different time points and across different groups, a dynamic trajectory of neurological recovery could be mapped, and the intervention effect of LIFU treatment could be analyzed.

[0052] S6: Procedures for Obtaining and Processing Tissue Samples At the pre-set experimental endpoint (usually day 14 after the last behavioral test), rats were over-anesthetized and euthanized. Immediately, transcardiac perfusion was performed: first, ice-cold saline was rapidly infused to flush out blood from the vessels, followed by infusion of 4% paraformaldehyde fixative to fix the brain tissue in situ. After complete removal of the brain, the ischemic hemisphere (left side) and the non-ischemic hemisphere could be separated as needed for the experiment. The brain tissue was then post-fixed and processed according to the requirements of subsequent detection methods: one portion was used for fresh sections for TTC staining (immediate assessment of infarction); another portion was dehydrated, cleared, and embedded in paraffin to prepare serial coronal sections for further analysis such as HE staining, TUNEL staining, and immunohistochemistry.

[0053] S7: Multimodal Tissue Examination Procedure Multi-level pathological and molecular biological analyses were performed on the processed brain tissue to reveal the damage and repair process from different perspectives: Infarct volume assessment (TTC staining): Fresh brain tissue was coronally sectioned into 2 mm thick slices and immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) for 15-20 minutes in the dark at 37°C. Dehydrogenases in active brain tissue reduce TTC to red formazan, while the infarcted area remains pale due to loss of enzyme activity. After photographing, image analysis software was used to calculate the area of ​​the pale region, and the infarct volume percentage of the entire brain was calculated by overlaying the slice thickness, thus quantitatively assessing the degree of brain injury.

[0054] Histopathological observation (HE staining): Hematoxylin-eosin (HE) staining was performed on paraffin sections. Neuronal morphology and arrangement were observed under an optical microscope, as well as the presence of pathological changes such as nuclear condensation, cytoplasmic vacuolation, inflammatory cell infiltration, and tissue edema. Semi-quantitative pathological scoring was also performed.

[0055] like Figure 8 As shown, Figure 8 This is a statistical diagram of TUNEL staining results from a low-intensity transcranial focused ultrasound (LIFU) animal study on stroke rehabilitation (*: P≤0.05; **: P≤0.01; ***: P≤0.001). The apoptosis rate in the MCAO group was significantly higher than that in the control group, indicating that middle cerebral artery occlusion successfully induced severe neuronal damage and apoptosis. Simultaneously, all experimental groups (D1–D4) receiving LIFU intervention showed a significantly lower apoptosis rate compared to the MCAO group.

[0056] Apoptosis detection (TUNEL staining): The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method was used. Paraffin sections were dewaxed, hydrated, and digested with proteinase K, then incubated with a mixture containing TdT enzyme and fluorescent or enzyme-labeled dUTP. TdT enzyme ligates the labeled dUTP to the 3'-OH end of the DNA break in apoptotic cells, resulting in nucleus-specific staining (e.g., brown) in apoptotic cells. TUNEL-positive cells in the penumbra region surrounding the infarct were observed and counted under a microscope, and the apoptosis index was calculated to assess the inhibitory effect of LIFU on neuronal apoptosis.

[0057] The method provided in this embodiment may also include the following steps: Analyze the behavioral effects of LIFU on ischemic stroke in rats: By integrating the longitudinal mNSS score data obtained in step S5, and combining the results of other possible fine behavioral tests (such as corner test and sticker removal test), we comprehensively analyzed the improving effects of LIFU on motor, sensory, and coordination functions.

[0058] Analysis of the effect of LIFU on cerebral infarction volume: By comparing and analyzing the infarction volume data of each group obtained by TTC staining in step S7, the effect of LIFU intervention on cerebral infarction range can be quantitatively evaluated.

[0059] Analysis of the effect of LIFU on brain neuronal apoptosis: By comparing and analyzing the apoptosis index of each group obtained by TUNEL staining in step S7, the neuroprotective mechanism of LIFU was explored from the perspective of inhibiting programmed cell death.

[0060] Example 4 This embodiment further illustrates the method with specific illustrations and implementation procedures. The low-intensity transcranial focused ultrasound method for stroke rehabilitation animal experiments provided in this embodiment includes the following steps: Step 1: Parameter calibration: The effectiveness and safety of ultrasound parameters were determined through simulation. The ultrasound parameters for rats used in this embodiment were determined. The safe ultrasound parameter window used in the experiment was: sound pressure ≤ 1.2 MPa, duty cycle ≤ 40%.

[0061] Step 2: Animals and Grouping: 24 SD rats were randomly divided into 3 groups (n=8): sham operation group, MCAO model group, and LIFU treatment group. All rats underwent 3 days of pre-training.

[0062] Step 3: Model establishment: Left-sided MCAO surgery (90 min ischemia / reperfusion) was performed on rats in the Model and LIFU groups. In the Sham group, only the blood vessels were dissected.

[0063] Step 4: Ultrasound Treatment: Starting from day 1 post-surgery, rats in the LIFU group underwent LIFU treatment once daily for 20 minutes each time, for 7 consecutive days. Treatment parameters were set as follows: 1 MHz, ISPTA 5 W / cm². 2 PRF 100-1600 Hz, DC 30%. The transducer focus is located on the left sensorimotor cortex.

[0064] Step 5: Behavioral assessment: All rats were assessed using a double-blind mNSS score on postoperative days 1, 3, 7, and 14.

[0065] Step 6: Sample Processing and Testing: On day 14, rats were sacrificed and their brains were harvested. One portion of the brain slices was stained with TTC to calculate the infarct volume; the other portion was prepared into paraffin sections, stained with TUNEL to calculate the number of apoptotic cells, and subjected to IL-10 immunohistochemical staining analysis. The results were analyzed using ImageJ software.

[0066] like Figure 9 As shown, Figure 9This is a schematic diagram of the IL-10 immunohistochemical white light results in the low-intensity transcranial focused ultrasound (TCI) method for animal rehabilitation of stroke. The control group rats showed the lowest positive expression level of IL-10 in their brain tissue, with only sporadic positive signals and a uniform background, suggesting that under normal physiological conditions, the brain tissue is in a state of low-level anti-inflammatory factor expression.

[0067] Experimental results: (1) Behavioral: The experimental results showed that the behavioral scores of both the stroke groups that received LIFU stimulation and those that did not were significantly increased. However, the Model group that did not receive LIFU stimulation had a higher score than the control group. The mNSS score of the LIFU group on days 7 and 14 was significantly lower than that of the Model group (p<0.05), indicating faster recovery. Therefore, LIFU stimulation can improve the behavioral scores of stroke rats.

[0068] (2) Infarct volume: The experimental results showed that both the stroke groups that underwent LIFU stimulation and those that did not have infarcted brain regions. The percentage of infarct volume in the LIFU group was significantly smaller than that in the Model group (p<0.01). LIFU stimulation can reduce the infarct area in stroke rats.

[0069] (3) Apoptosis: The experimental results showed that the number of positive cells in the stroke group that was stimulated by LIFU and the group that was not stimulated by LIFU was greater than that in the control group. However, the number of TUNEL positive cells in the peri-infarct area of ​​the LIFU group was significantly less than that in the Model group (p<0.05). LIFU stimulation can reduce apoptosis in the brain region of stroke rats.

[0070] (4) Molecular expression: The IL-10 expression level in the LIFU group was higher than that in the Model group.

[0071] Conclusion: This embodiment demonstrates that the method of the present invention confirms the optimal parameters (1 MHz, 5 W / cm²). 2 LIFU treatment (PRF 100-1600 Hz, DC 30%) can effectively promote the recovery of neurological function in MCAO rats. The mechanism may be related to reducing infarct volume, inhibiting apoptosis and upregulating the anti-inflammatory factor IL-10.

[0072] like Figure 10 As shown, Figure 10This is a statistical diagram illustrating the immunohistochemical white light results of IL-10 in a low-intensity transcranial focused ultrasound (LIFU) method for stroke rehabilitation in animals (*: P≤0.05; **: P≤0.01; ***: P≤0.001). Compared with the control group, the expression of IL-10 in the brain tissue of rats in the MCAO model group was slightly upregulated (p < 0.05), suggesting that cerebral ischemia-induced injury can induce a certain degree of spontaneous anti-inflammatory response. However, the expression level was still relatively low and insufficient to effectively antagonize the strong pro-inflammatory response, consistent with its significant increase in infarct volume and neurological deficits. In contrast, the LIFU stimulation significantly increased the expression level of IL-10 in all parameter groups.

[0073] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A low-intensity transcranial focused ultrasound method for animal rehabilitation of stroke, characterized in that, The method described is a systematic animal experimental evaluation method for assessing the effect of low-intensity transcranial focused ultrasound on neurological function rehabilitation after ischemic stroke and optimizing its stimulation parameters. It executes the following steps in a preset logical order to form a complete research closed loop from parameter safety verification, model intervention to multi-dimensional effect evaluation. S1: Ultrasound parameter safety calibration steps: Determine the safe and effective parameter window for low-intensity transcranial focused ultrasound through sound field simulation, in vitro acoustic measurement, phantom thermal damage experiment, and in vivo blood-brain barrier opening experiment; S2: Animal screening and baseline establishment steps: Select healthy experimental animals, exclude those with spontaneous neurological defects or abnormal activity, and after adaptive training, randomly divide them into control group, stroke model group and ultrasound stimulation group; S3: Steps for preparing animal models of stroke: Constructing a cerebral artery occlusion model in experimental animals using the suture occlusion method; S4: Ultrasound intervention treatment steps: After the establishment of the stroke animal model, low-intensity transcranial focused ultrasound stimulation is applied to the target area of ​​the animal brain using the parameters determined by the safety calibration steps described above; S5: Dynamic neurobehavioral assessment steps: Perform neurobehavioral tests on experimental animals at multiple time points before and after ultrasound intervention treatment to dynamically assess their neurological function recovery. S6: Tissue sample acquisition and processing steps: At the end of the experiment, the animal was euthanized, brain tissue was obtained and processed for morphological and molecular biological analysis; S7: Multimodal tissue testing steps: Perform multiple tests on the processed brain tissue samples, including infarct volume staining, histopathological staining, apoptosis detection, and specific protein expression analysis. Specifically, step S1 provides a safe and effective ultrasound parameter window for step S4; the evaluation results of step S5 are correlated with the test results of step S7, together forming a complete chain of evidence for evaluating the mechanisms of nerve function recovery and tissue repair.

2. The low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments according to claim 1, characterized in that, In step S1, the specific steps include the following three-level progressive calibration process: (1) Sound field simulation prediction: A simulation model was established based on the generalized Westervelt equation to simulate transcranial ultrasound propagation and predict focal characteristics; (2) Determination of safety boundaries in vitro: The skull is covered with tissue phantoms and subjected to ultrasound irradiation. The safe sound pressure and duty cycle range that do not produce thermal damage are determined by judging whether protein coagulative necrosis occurs in the phantoms. (3) In vivo focusing effectiveness verification: Within the specified safety parameters, the target area of ​​a live animal was irradiated with ultrasound after intravenous injection of microbubbles and dye to verify its ability to reversibly and precisely open the blood-brain barrier of the target area.

3. The low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments according to claim 1, characterized in that, In step S3, a middle cerebral artery occlusion / reperfusion model is established using the suture embolization method. Specifically, a nylon suture embolization is advanced to the origin of the middle cerebral artery to cause ischemia. After ischemia lasts for 90 minutes, the suture embolization is removed to restore perfusion.

4. The low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments according to claim 1, characterized in that, In step S4, a concave focusing transducer with a frequency of 1 MHz is used as the sound source, and the acoustic focus of the transducer is precisely located in the target brain region using a stereotaxic instrument. The treatment parameters are set and adjusted through a graphical user interface, including pulse repetition frequency, duty cycle, duration, and stimulation interval. Starting from the first day after surgery, treatment is performed once a day for 15-30 minutes each time, for 5-10 consecutive days.

5. The low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments according to claim 4, characterized in that, The target brain region is the sensorimotor cortex on the same side as the lesion.

6. The low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments according to claim 1, characterized in that, In step S5, the neurobehavioral test is a modified neurological deficit score, and the scoring time points include at least postoperative day 1, day 3, day 7 and day 14, and the scoring process is conducted using a double-blind method.

7. The low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments according to claim 1, characterized in that, In step S6, the animal is euthanized by cardiac perfusion. It is first rinsed with physiological saline, then perfused and fixed with 4% paraformaldehyde, and then the brain is removed and subjected to paraffin embedding or frozen sectioning.

8. The low-intensity transcranial focused ultrasound method for stroke rehabilitation in animal experiments according to claim 1, characterized in that, In step S7, the brain tissue sample is subjected to tests including quantitative analysis of infarct volume, detection of apoptosis, and analysis of specific protein expression; and the test results obtained in step S7 are correlated with the neurobehavioral scores at the corresponding time points obtained in step S5.

9. A low-intensity transcranial focused ultrasound (TCU) system for stroke rehabilitation, used to perform the method described in any one of claims 1-8, characterized in that, Includes a stimulation unit, a positioning unit, and a control unit; The stimulation unit includes a signal generator, a power amplifier, and a concave focusing transducer with a frequency of 1 MHz. The signal generator, power amplifier, and transducer are electrically connected in sequence to generate and output low-intensity focused ultrasound. The positioning unit is a stereo positioning device for experimental animals, used to fix the head of the experimental animal and adjust the spatial position of the transducer in three dimensions so that the acoustic focus of the transducer is accurately positioned on the predetermined target area of ​​the animal's brain. The control unit includes a controller with a graphical user interface, which is signal-connected to the stimulation unit and used to precisely set and regulate the ultrasound parameters output by the stimulation unit. The ultrasound parameters include pulse repetition frequency, duty cycle, duration of a single stimulation, and stimulation interval.

10. The low-intensity transcranial focused ultrasound stroke rehabilitation animal experimental system according to claim 9, characterized in that, The control unit limits the sound pressure and duty cycle parameters of the ultrasound to a safe calibrated window range through the graphical user interface. The upper limit of the window range is that the sound pressure is no greater than 1.2 MPa and the duty cycle is no greater than 40%.

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