Method for constructing animal traumatic brain injury model based on low-intensity pulsed ultrasound regulation and control and application thereof

Abstract

The invention belongs to the technical field of medical treatment, and relates to a method for constructing an animal traumatic brain injury model based on low-intensity pulsed ultrasound regulation and application thereof. The method comprises the following steps: (1) establishing a TBI mouse by adopting controlled cortical injury so as to simulate neurological impairment caused by open brain trauma; (2) setting low-intensity pulse ultrasonic conditions, and performing low-intensity pulse ultrasonic treatment on the TBI mouse; and (3) comparing the influence of the space learning and long-term memory ability of the TBI mouse before and after the low-intensity pulsed ultrasound treatment and the change of the intracerebral clearance ability, and detecting the level of phosphorylated tau protein in the damaged cerebral hemisphere, if the influence on the TBI mouse has statistical significance, judging that the TBI mouse is damaged by the phosphorylated tau protein. And if not, constructing a low-intensity pulsed ultrasound regulation animal traumatic brain injury model by using the low-intensity pulsed ultrasound condition. By regulating and controlling the key parameters of the low-intensity pulsed ultrasound, the brain lymphatic drainage and nerve repair of traumatic brain injury of animals can be regulated and controlled.

Description

Technical Field

[0001] This invention belongs to the field of medical technology and relates to a method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation and its application. Specifically, it relates to a method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation and the application of low-intensity pulsed ultrasound in regulating cerebral lymphatic drainage and nerve repair in animals with traumatic brain injury. Background Technology

[0002] Traumatic brain injury (TBI) is one of the leading causes of death from traumatic injuries, with a persistently high incidence rate. In my country, the annual incidence of TBI is 55–64 per 100,000, resulting in nearly 100,000 deaths and hundreds of thousands of disabilities each year. Studies have shown that TBI can lead to blood-brain barrier disruption, intracranial metabolic disorders, and endocrine abnormalities, subsequently inducing secondary cerebral edema and increased intracranial pressure. Increased intracranial pressure can cause brain tissue displacement, compressing important brain regions and blood vessels, severely affecting cerebral blood and oxygen supply, further exacerbating metabolic disorders and endocrine imbalances, forming a vicious cycle of "high intracranial pressure—cerebral edema—perfusion impairment," ultimately causing irreversible brain tissue damage. Currently, the mainstream international consensus on TBI treatment still focuses on reducing intracranial pressure and maintaining adequate cerebral perfusion pressure. Common interventions include adjusting patient position (e.g., midline position, head elevation), administering analgesia and sedation, inducing hypothermia, hypertonic dehydration therapy, and barbiturate sedation to reduce brain tissue volume. When the condition progresses to severe intracranial hypertension, decompressive craniectomy or combined surgical treatment to remove traumatic hematoma is often used. The common goal of these treatment strategies is to reduce intracranial pressure and maintain cerebral perfusion by regulating intracranial volume, thereby meeting the metabolic needs of brain tissue. Despite decades of interventions, the global improvement in the diagnosis and treatment efficiency of TBI remains limited. The mortality rate of severe TBI is still close to 30%. Therefore, exploring new strategies for effectively regulating or intervening in secondary brain injury after TBI and promoting brain tissue repair and regeneration has become a challenging, hot, and key research direction in the field of TBI.

[0003] Low-intensity pulsed ultrasound (LIPUS), as a novel non-invasive neuromodulation technique, has gradually gained widespread attention in the fields of nerve and tissue repair due to its advantages such as high safety, non-invasiveness, good reversibility, and precise spatial positioning. Unlike high-intensity focused ultrasound (HIFU), which is based on thermal effects, LIPUS mainly relies on its mechanical action and cavitation effect to apply minute and continuous mechanical stimulation to target tissues without damaging tissue structure. This stimulation can induce reversible stretching of cell membranes, thereby regulating the permeability of ion channels and changes in membrane protein conformation, affecting cell function.

[0004] Existing studies have confirmed that LIPUS can regulate the expression of integrin on the cell membrane and further activate the focal adhesion kinase (FAK) signaling pathway, enhancing its phosphorylation level. This, in turn, activates downstream signaling molecules such as ERK, p38, and JNK in the MAPK pathway, playing an important role in regulating cell proliferation, apoptosis, and survival. Furthermore, LIPUS has shown broad application potential in tissue regeneration and anti-inflammation. Numerous studies have demonstrated its ability to effectively promote fresh fracture healing and reduce inflammatory responses. The U.S. Food and Drug Administration (FDA) approved LIPUS for the treatment of fresh fractures, delayed healing, and nonunion in 1994 and 2000, respectively, marking the gradual recognition of its clinical value. Preliminary studies have shown that LIPUS can promote intracranial fluid exchange and lymphatic drainage, suggesting a potential role in the clearance of brain metabolic waste and neuroprotection in TBI mice. Related literature documents that this effect can be applied to AD mice. However, this mechanism currently lacks systematic validation, and there are no systematic studies applying LIPUS to improve cerebral lymphatic drainage function in TBI mice. Its mechanism of action, optimal parameters, and safety require further investigation. In summary, research on the dysfunction of the brain's metabolic waste clearance system in TBI mice is rapidly developing, and the brain-lymphatic and lymphoid systems have become key pathological mechanisms and intervention targets. Developing novel, controllable, and non-invasive methods for constructing animal models of traumatic brain injury based on low-intensity pulsed ultrasound (LPI) modulation, and applying LPI in regulating cerebral lymphatic drainage and neural repair, will provide new breakthroughs for improving the prognosis of TBI. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation and its application. Specifically, it is a method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation and the application of low-intensity pulsed ultrasound in regulating cerebral lymphatic drainage and nerve repair in animals with traumatic brain injury.

[0006] The specific implementation process of this invention is as follows: A method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation includes: (1) TBI mice were established by controlled cortical injury to simulate the neurological deficits caused by open brain injury; (2) Set low-intensity pulsed ultrasound conditions and perform low-intensity pulsed ultrasound treatment on TBI mice; (3) Compare the effects of low-intensity pulsed ultrasound treatment on spatial learning and long-term memory abilities and changes in brain clearance ability in TBI mice before and after treatment, and detect the level of phosphorylated tau protein in the damaged brain hemisphere. If the effect on TBI mice is statistically significant, then a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury can be constructed using the low-intensity pulsed ultrasound conditions.

[0007] Furthermore, the low-intensity pulsed ultrasound conditions in step (2) include at least the effective sound intensity, frequency, pulse repetition frequency, duty cycle, and ultrasound processing time and frequency.

[0008] Furthermore, the effective sound intensity is 0.3 W / cm. 2 The frequency was 3MHz, the pulse repetition frequency was 100Hz, the pulse duty cycle was 50%, the ultrasonic treatment time was 10min, once a day for 3 consecutive days.

[0009] Furthermore, the spatial learning and long-term memory abilities of TBI mice before and after low-intensity pulsed ultrasound treatment were assessed using a water maze. The platform latency, crossing time, and swimming trajectory were recorded. If the experimental results were statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

[0010] Furthermore, the changes in brain clearance capacity of TBI mice before and after low-intensity pulsed ultrasound treatment were observed using a fluorescent tracer. If the experimental results were statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

[0011] Furthermore, the level of phosphorylated tau protein in the damaged brain hemisphere of TBI mice before and after low-intensity pulsed ultrasound treatment was quantitatively detected using Western blotting. If the experimental results are statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

[0012] The aforementioned low-intensity pulsed ultrasound was used to regulate cerebral lymphatic drainage and nerve repair in animals with traumatic brain injury.

[0013] The positive effects of this invention: Low-intensity pulsed ultrasound (HIFU) offers advantages such as high safety, non-invasiveness, good reversibility, and precise spatial positioning. It primarily relies on its mechanical action and cavitation effect to apply minute, continuous mechanical stimulation to target tissues without damaging their structure. This stimulation can induce reversible stretching of the cell membrane, thereby regulating ion channel permeability and membrane protein conformational changes, thus influencing cell function.

[0014] In TBI animal models, there are currently no high-quality experimental studies clarifying its effects on cerebral lymphatic drainage and its potential for neural repair. This invention utilizes low-intensity pulsed ultrasound (LIPUS) to activate or inhibit neural activity by controlling key parameters (including sound intensity, frequency, pulse repetition frequency, duty cycle, and application time), thereby regulating cerebral lymphatic drainage and neural repair in animals with traumatic brain injury. Attached Figure Description

[0015] Figure 1 It is a low-intensity pulsed ultrasound stimulation device; Figure 2 The performance of Sham (n=10), TBI (n=10), and TBI + LIPUS (n=10) in the Morris Water Maze (MWM) behavioral experiment is shown in the top row as a trajectory map reflecting the movement path and the bottom row as a heat map reflecting the activity density distribution. Figure 3 The left figure shows the number of times the platform area was traversed, reflecting spatial memory; the middle figure shows the time spent in the target quadrant; and the right figure shows the time required to reach the platform (Days 15–19). Figure 4 Comparison of tau protein and phosphorylated tau protein content in brain tissue of TBI group mice; Figure 5 The effects of LIPUS on the distribution of fluorescent tracers in the rat brain under different sound intensities (0.1W, 0.3W, 0.5W) and treatment times (20 min, 30 min); Figure 6 The distribution of fluorescent tracers in brain tissue during the acute phase after TBI (TBI-3D) and the changes before and after LIPUS regulation; Figure 7 Immunofluorescence imaging of meningeal lymphatic vessels in the transverse sinus region (TVS) of rats in different treatment groups; green fluorescence indicates meningeal lymphatic vessel structure. Figure 8The quantitative analysis of the morphology of meningeal lymphatic vessels in three groups of rats is shown in the left figure, which shows the area of ​​LYVE-1 positive lymphatic vessels, the middle figure shows the number of lymphatic vessel buds, and the right figure shows the average diameter of lymphatic vessels. Figure 9 The distribution and quantitative analysis of fluorescent tracers in dCLN of the Sham, TBI and TBI+LIPUS groups are shown. The left side is a representative immunofluorescence image, and purple-red is the tracer signal. Figure 10 This is a schematic diagram of the CCI instrument structure; Figure 11 This is a schematic diagram of the water maze; Figure 12 These are images taken using an inverted fluorescence microscope. Figure 13 Photo of a cryostat slicer. Detailed Implementation

[0016] The present invention will be further described below with reference to the embodiments.

[0017] This invention utilizes low-intensity pulsed ultrasound (LIPUS) to activate or inhibit neural activity by controlling key parameters (including sound intensity, frequency, pulse repetition frequency, duty cycle, and application time), thereby regulating cerebral lymphatic drainage and neural repair in animals with traumatic brain injury. This invention discloses a method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound, comprising: (1) TBI mice were established by controlled cortical injury to simulate the neurological deficits caused by open brain injury; (2) Set low-intensity pulsed ultrasound conditions and perform low-intensity pulsed ultrasound treatment on TBI mice; (3) Compare the effects of low-intensity pulsed ultrasound treatment on spatial learning and long-term memory abilities and changes in brain clearance ability in TBI mice before and after treatment, and detect the level of phosphorylated tau protein in the damaged brain hemisphere. If the effect on TBI mice is statistically significant, then a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury can be constructed using the low-intensity pulsed ultrasound conditions.

[0018] Furthermore, the low-intensity pulsed ultrasound conditions in step (2) include at least the effective sound intensity, frequency, pulse repetition frequency, duty cycle, and ultrasound processing time and frequency.

[0019] Furthermore, the effective sound intensity is 0.3 W / cm. 2 The frequency was 3MHz, the pulse repetition frequency was 100Hz, the pulse duty cycle was 50%, the ultrasonic treatment time was 10min, once a day for 3 consecutive days.

[0020] Furthermore, the spatial learning and long-term memory abilities of TBI mice before and after low-intensity pulsed ultrasound treatment were assessed using a water maze. The platform latency, crossing time, and swimming trajectory were recorded. If the experimental results were statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

[0021] Furthermore, the changes in brain clearance capacity of TBI mice before and after low-intensity pulsed ultrasound treatment were observed using a fluorescent tracer. If the experimental results were statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

[0022] Furthermore, the level of phosphorylated tau protein in the damaged brain hemisphere of TBI mice before and after low-intensity pulsed ultrasound treatment was quantitatively detected using Western blotting. If the experimental results are statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

[0023] The aforementioned low-intensity pulsed ultrasound was used to regulate cerebral lymphatic drainage and nerve repair in animals with traumatic brain injury.

[0024] Example 1 A method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation includes: (1) TBI mice were established by controlled cortical injury to simulate the neurological deficits caused by open brain injury; The specific procedure involves selecting adult male C57BL mice weighing 25-28g, and feeding and handling the experimental animals according to internationally accepted animal husbandry and protection principles. The C57BL mice were anesthetized by intraperitoneal injection of 2.5% tribromoethanol and fixed on a small animal stereotactic apparatus. A circular bone flap with a diameter of approximately 5mm was drilled 2mm to the left of the sagittal suture below the coronal suture of the mouse skull using a specially designed conical drill. The bone flap was gently lifted and removed with forceps, preserving the integrity of the hard mold. The CCI instrument parameters were then calibrated, and the mouse was fixed on a pre-calibrated impact stereotactic apparatus. The tip of the impact pin, with a diameter of 3.5mm, was placed in the center of the bone window, lightly against the dura mater. The impact parameters were set as follows: impact speed 5.00 m / sec, impact dwell time 0.15s, and impact depth 2mm. After the impact, the incision was sutured, aligned with the skin edge, and disinfected with iodine. The mouse was then placed on a 37±0.1℃ electric heating blanket to obtain the TBI mouse.

[0025] (2) Set low-intensity pulsed ultrasound conditions and perform low-intensity pulsed ultrasound treatment on TBI mice; The low-intensity pulsed ultrasound stimulation device used is described in [link to documentation]. Figure 1, for Chattanooga® Intelect Transport Ultrasound, DJO Global Inc., Mouguerre, France.

[0026] The specific procedure involves anesthetizing mice with 2.5% tribromoethanol, shaving the hair from the top of their heads, and fixing them to a stereotaxic apparatus. Medical ultrasound coupling gel is evenly applied to the mouse's scalp. An ultrasound probe is then fixed in place using a test tube rack, with the probe's plane aligned with the mouse's head, ensuring no air bubbles are present at the contact surface. Different ultrasound parameters (pulse duty cycle 20-50%, effective sound intensity 0.1-3 W / cm²) were tested. 2 After that, we selected parameters of 50% pulse duty cycle, 0.3 W / cm2 effective sound intensity (frequency 3MHz, pulse frequency 100Hz), and 10 min ultrasonic treatment time, once a day for 3 consecutive days. The specific parameter selection process is described in the following experiment.

[0027] (3) Compare the effects of low-intensity pulsed ultrasound treatment on spatial learning and long-term memory abilities and changes in brain clearance ability in TBI mice before and after treatment, and detect the level of phosphorylated tau protein in the damaged brain hemisphere. If the effect on TBI mice is statistically significant, then a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury can be constructed using the low-intensity pulsed ultrasound conditions.

[0028] (3.1) Specific experimental methods: (3.1.1) Morris Water Maze The water maze was used to assess the spatial learning and long-term memory abilities of TBI mice before and after low-intensity pulsed ultrasound treatment. The training phase was set from day 15 to 19 post-surgery, with four localization training sessions per day, and the latency to find the hidden platform was recorded. On day 19, a spatial exploration test was conducted, and after the platform was removed, the number of times the original platform location was crossed and the time spent in the target quadrant were recorded.

[0029] The experimental procedure is briefly described below: the training phase lasted five days, and the testing phase lasted only one day. During training from days 15 to 19, mice were required to find a hidden platform within 90 seconds and stay there for 15 seconds. If the target platform was not found within 90 seconds, researchers guided the mice to the target location and held it for 15 seconds; the delay time was recorded as 90 seconds. During training, mice received training in quadrants one through four four times a day. Testing was conducted on day 20, after which the platform was removed and the mice were placed on the other side of the platform. We used video tracking software (EthoVision XT 13, Noldus Information Technology, Wageningen, Netherlands) to record and analyze the platform latency, crossing time, and swimming trajectory.

[0030] (3.1.2) Dynamic tracking of cerebrospinal fluid clearance efficiency Fluorescent tracer (FITC-DEXTRAN, 40 kDa) was injected into the cisterna magna at different time points to simulate cerebrospinal fluid flow, and changes in brain clearance capacity before and after low-intensity pulsed ultrasound treatment were observed. (i) Anesthetize the mice and fix them in a stereotactic apparatus; (ii) Location coordinates: occipital cisternae, injection volume 10 μL (Gastight #1710, 1.46-mminnerdiameter; Hamilton). (iii) The animal was euthanized 30 minutes after the injection and perfused with PBS + 4% PFA; (iv) After freezing sections (CM1950, Leica Biosystems), whole-brain IVIS imaging analysis was performed to analyze fluorescence distribution and intensity (IX73, Olympus) to reflect the residual amount of tracer in brain tissue; (v) The lower the fluorescence intensity, the worse the drainage function. (3.1.3) Western Blotting Western blot was used to quantitatively detect the level of phosphorylated tau (p-tau) protein in the damaged brain hemisphere of TBI mice before and after low-intensity pulsed ultrasound treatment.

[0031] Tissue samples were collected after euthanasia and perfusion with cold PBS on day 3 following traumatic brain injury (TBI). Lysis buffer was prepared using radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (P1260, Solarbio, Beijing, China), and protein concentration was determined using a BCA (biuret acid) protein quantification kit (ThermoFisher Scientific, Waltham, MA). 10 μL of protein sample was separated by 10% SDS-PAGE at a constant voltage and then transferred to a polyvinylidene fluoride (PVDF) membrane using a Bio-Rad wet transfer apparatus. The membrane was then incubated overnight at 4°C and bound to the following primary antibodies: rabbit antiphosphorylated tau (Thr205) (1:2000, Abclonal, catalog number AP0168), rabbit antiphosphorylated tau (Ser404) (1:2000, Abclonal, catalog number AP0170), and rabbit antiphosphorylated tau. After incubation with the primary antibody, the membrane was thoroughly washed and then incubated with a horseradish peroxidase-conjugated secondary antibody. Protein density was quantitatively analyzed using FIJI (ImageJ) software, and relative expression levels were normalized using GAPDH as an internal control.

[0032] (3.1.4) Dissection and staining of the dura mater During the collection of whole meningeal specimens, we peeled away the skin and muscle layers of the skull from TBI mice before and after low-intensity pulsed ultrasound treatment, removed the top cranial bone using surgical scissors, and fixed it overnight with 4% paraformaldehyde at 4°C. The meningeal tissue (dura mater and arachnoid mater) was carefully separated from the skull. However, only the dorsal and occipital meninges remained intact. Intact meningeal samples were transferred to PBS buffer for further immunofluorescence staining. The meninges were washed with PBS and blocked for 1.5 hours at room temperature with PBS containing 3% bovine serum albumin, 0.2% Triton, and 0.05% Tween 20. Primary antibody was incubated overnight at 4°C, followed by secondary antibody incubation for 2 hours at room temperature. The specimens were observed under an inverted fluorescence microscope (IX73, Olympus).

[0033] (3.1.5) Extraction and staining of deep cervical lymph nodes (dCLN) After euthanizing the mice using the above method, the deep cervical lymph nodes of the TBI mice before and after low-intensity pulsed ultrasound treatment were carefully dissected under a microscope, fixed in 4% paraformaldehyde (PFA) for 24 hours, and dehydrated in gradients with 15% and 30% sucrose.

[0034] Subsequently, deep cervical lymph nodes (dCLNs) were immersed in the optimal cutting temperature compound (OCT) (Sakura Finetek USA, Torrance, CA, USA) and cut into 8-micron-thick coronal sections using a cryostat. The meninges were washed with PBS and blocked for 1.5 hours at room temperature with PBS containing 3% bovine serum albumin, 0.2% Triton, and 0.05% Tween 20. Primary antibodies were incubated overnight at 4°C, followed by incubation with secondary antibodies for 2 hours at room temperature. The stained sections were observed under an inverted fluorescence microscope (IX73, Olympus).

[0035] In the experiment, the Sham group represents the sham-operated group of mice, where only a cranial bone window was opened without any trauma; TBI refers to mice that have undergone controlled cortical injury to establish TBI. Experimental data are expressed as mean ± standard deviation. One-way ANOVA or paired t-test with one repeated measure was performed using GraphPad Prism9 statistical software. A p < 0.05 was considered statistically significant.

[0036] (3.2) Experimental results: Figure 2The image shows the performance of Sham (n=10), TBI (n=10), and TBI + LIPUS (n=10) in the Morris Water Maze (MWM) behavioral experiment. The top row shows the trajectory plot, reflecting the movement path; the bottom row shows the heatmap, reflecting the activity density distribution. Here, n represents the number of mice in each group. The TBI group showed significantly reduced activity and restricted exploration, while LIPUS modulation improved the range and frequency of movement, approaching the levels of the Sham group (the Sham group represents the sham-operated group, where only a cranial bone window was opened without impact). Figure 2 This study visually demonstrated the inhibitory effect of TBI on spontaneous behavior in mice and how LIPUS intervention reversed this behavioral disorder to some extent. The LIPUS group showed an improvement trend in both trajectory plots and heatmaps, suggesting its potential for neurobehavioral function repair.

[0037] Figure 3 The left figure shows the number of times the platform area was traversed, reflecting spatial memory; the middle figure shows the time spent in the target quadrant; and the right figure shows the time required to reach the platform (Days 15–19). Compared with the Sham group, the TBI group showed a significant decline in spatial memory and learning ability; while the LIPUS treatment group showed significant improvement in the above indicators. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001. Figure 3 The study revealed that Sham mice exhibited significantly higher platform crossing frequency (p<0.01) and target quadrant dwell time (p<0.001), indicating stronger spatial memory. The TBI group showed significant decreases in both indicators, while the TBI+LIPUS group performed significantly better than the TBI group (further illustrating that LIPUS regulation improves cognitive function). Sham mice showed a gradually shortening escape latency, suggesting normal learning ability. In contrast, the TBI group showed a significantly prolonged escape latency overall, with a slow decreasing trend, reflecting impaired spatial learning ability. Notably, the latency in the TBI+LIPUS group was significantly shorter than that in the TBI group, and decreased with the number of training days, suggesting that LIPUS regulation can improve learning impairment in TBI mice.

[0038] Figure 4The results showed that the expression level of p-tau-T205 in the brain tissue of mice in the TBI group was significantly increased (p<0.01 compared with the Sham group), while LIPUS regulation could reduce its expression (p<0.05 compared with the TBI group), but there was no significant difference between the TBI and LIPUS groups (ns). The expression level of p-tau-Ser404 was significantly increased in the TBI group (p<0.0001), while LIPUS treatment significantly downregulated the expression of this phosphorylation site (vs. TBI, p<0.0001; vs. Sham, p<0.01). Total tau protein (tau5) was also significantly upregulated in the TBI group (vs. Sham, p<0.0001), and its expression was significantly reduced after LIPUS regulation (vs. TBI, p<0.0001). GAPDH was used as an internal control protein for normalization. Figure 4 TBI leads to an increase in total tau protein and its phosphorylation levels, which can be partially reversed by LIPUS treatment.

[0039] Figure 5 This study investigated the effects of different sound intensities (0.1W, 0.3W, 0.5W) and treatment times (20 min, 30 min) on the distribution of fluorescent tracers in the rat brain. Under certain sound intensities and treatment times, LIPUS can enhance the inflow function of the lymphoid system, which is related to promoting brain-lymphatic drainage or altering the blood-brain barrier. Figure 5 As shown, the fluorescence signal in the brain tissue of the Sham-20min group was weaker, while the Sham-30min group showed a significantly wider distribution due to the prolonged tracer circulation time. This indicates that the influx of tracers into the brain can be increased by prolonging the circulation time. After 20 minutes of LIPUS regulation, both the 0.3W and 0.5W groups showed stronger fluorescence signals, suggesting that LIPUS enhances the permeability or mobility of substances in the brain. Quantitative results of fluorescence area showed no statistically significant differences between the 0.1W (20min) and Sham-20min groups, and between the 0.3W (20min) and 0.5W (20min) groups.

[0040] like Figure 6As shown, the distribution of fluorescent tracers in the brain tissue of TBI mice was significantly reduced, suggesting impaired brain-lymphatic drainage function after brain injury. In contrast, TBI mice treated with LIPUS showed significantly enhanced fluorescence signals in the cerebral cortex and deep regions, with a distribution similar to the Sham group. Quantitative analysis of fluorescence area showed that the fluorescence signal in the TBI-3D group was significantly lower than that in the Sham group (p<0.0001), while the fluorescence signal in the LIPUS-regulated group was significantly higher than that in the TBI group (p<0.0001). Although there is still a certain gap compared to the Sham group, LIPUS can reverse the lymphoid drainage impairment caused by TBI to some extent. These results suggest that LIPUS has the potential to promote the recovery of brain clearance function in TBI mice.

[0041] Figure 7 Immunofluorescence imaging of meningeal lymphatic vessels in the transverse sinus region (TVS) of rats in different treatment groups. Green fluorescence indicates the structure of meningeal lymphatic vessels. The Sham group shows intact, clearly distributed, and continuous lymphatic vessels. The TBI group shows significantly reduced fluorescence signal in the lymphatic vessels, exhibiting fragmentation and sparseness, indicating structural damage. After LIPUS intervention, the morphology of the lymphatic vessels was significantly improved, showing enhanced fluorescence, increased branching structures, and restored vessel continuity. The yellow dashed boxes mark the areas of structural repair, suggesting that LIPUS regulation promotes meningeal lymphatic vessel remodeling. Figure 7 As shown, immunofluorescence staining in the transverse sinus (TVS) region of the dura mater revealed that the lymphatic vessels in the Sham group were clear and continuous, representing a typical normal morphology. The TBI group exhibited significantly disordered lymphatic vessel structures with sparse and broken fluorescence signals, suggesting damage to the meningeal lymphatic vessel structure. In contrast, the LIPUS-regulated group displayed more complete and clear lymphatic vessel morphology, with continuous diameters and abundant branching structures, particularly around the transverse sinus region (shown by the yellow dashed box), indicating that it may have promoted lymphatic vessel regeneration or repair. These results suggest that LIPUS possesses certain functions in meningeal lymphatic repair and lymphogenesis promotion.

[0042] Figure 8To further quantitatively assess the effect of LIPUS on the morphology of meningeal lymphatic vessels in TBI mice, the results showed that the LYVE-1 positive area in the TBI group was significantly lower than that in the Sham group (p<0.0001), indicating significant damage to the meningeal lymphatic vessel structure caused by traumatic brain injury. LIPUS regulation significantly increased the LYVE-1 positive area (p<0.001 vs. TBI; p<0.0001 vs. Sham), suggesting that LIPUS may have a role in lymphatic vessel repair or promoting its regeneration. Regarding the number of lymphatic vessel buds, the TBI group showed a slight increase compared to the Sham group (p<0.05), while the LIPUS group had a significantly higher number of buds than both groups (p<0.001 vs. Sham; p<0.001 vs. TBI), suggesting that LIPUS can significantly promote the angiogenesis or remodeling of meningeal lymphatic vessels. In addition, the lymphatic vessel diameter was significantly increased in the LIPUS group (p<0.0001 vs. Sham; p<0.001 vs. TBI), further indicating its potential promoting effect on lymphatic vessel dilation and functional enhancement. Figure 8 Quantitative analysis of meningeal lymphatic vessel morphology in three groups of rats. The left figure shows the proportion of LYVE-1 positive lymphatic vessel area, which was significantly reduced in the TBI group and significantly restored by LIPUS intervention; the middle figure shows the number of lymphatic vessel buds, which was significantly increased by LIPUS; the right figure shows the average diameter of lymphatic vessels, which was significantly larger in the LIPUS group than in the TBI and Sham groups. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0043] Figure 9 The distribution of fluorescent tracers in deep cervical lymph nodes (dCLNs) was demonstrated. Results showed that fluorescence signals were widely distributed in the dCLNs of the Sham group, with a higher proportion of fluorescent area. Fluorescence signals were significantly reduced in the TBI group (p<0.0001), suggesting that brain injury significantly inhibits the transport of intracranial metabolic waste to the peripheral lymphatic system. LIPUS treatment significantly enhanced the fluorescence accumulation level in the dCLNs (p<0.05 vs. TBI; p<0.0001 vs. Sham), indicating that it can promote the recovery of brain-lymphatic drainage function to some extent. Figure 9 Distribution and quantitative analysis of fluorescent tracers in dCLN of the Sham, TBI, and TBI+LIPUS groups. The left side shows representative immunofluorescence images, with purple-red representing tracer signals. Fluorescence distribution was widespread in the Sham group, significantly reduced in the TBI group, and enhanced in the LIPUS group. The right side shows the quantitative analysis of the percentage of fluorescent area. Data are expressed as mean ± standard deviation; *p<0.05, ****p<0.0001.

[0044] This invention also involves a schematic diagram of a CCI instrument structure (see attached diagram). Figure 10 See the diagram of the water maze. Figure 11 See the inverted fluorescence microscope image. Figure 12 See photos of the cryostat slicer. Figure 13 .

[0045] Other embodiments of this application will readily conceive of by those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and embodiments are to be considered exemplary only, and this application is not limited to the precise structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation, characterized in that, include: (1) TBI mice were established by controlled cortical injury to simulate the neurological deficits caused by open brain injury; (2) Set low-intensity pulsed ultrasound conditions and perform low-intensity pulsed ultrasound treatment on TBI mice; (3) Compare the effects of low-intensity pulsed ultrasound treatment on spatial learning and long-term memory abilities and changes in brain clearance ability in TBI mice before and after treatment, and detect the level of phosphorylated tau protein in the damaged brain hemisphere. If the effect on TBI mice is statistically significant, then a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury can be constructed using the low-intensity pulsed ultrasound conditions.

2. The method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation according to claim 1, characterized in that: The low-intensity pulsed ultrasound conditions in step (2) include at least the effective sound intensity, frequency, pulse repetition frequency, duty cycle, and ultrasound processing time and frequency.

3. The method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation according to claim 2, characterized in that: The effective sound intensity is 0.3 W / cm². 2 The frequency was 3MHz, the pulse repetition frequency was 100Hz, the pulse duty cycle was 50%, the ultrasonic treatment time was 10min, once a day for 3 consecutive days.

4. The method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation according to claim 1, characterized in that: Spatial learning and long-term memory abilities of TBI mice before and after low-intensity pulsed ultrasound treatment were assessed using a water maze. Platform latency, crossing time, and swimming trajectory were recorded. If the experimental results were statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

5. The method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation according to claim 1, characterized in that: The changes in brain clearance capacity of TBI mice before and after low-intensity pulsed ultrasound treatment were observed using a fluorescent tracer. If the experimental results were statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

6. The method for constructing an animal model of traumatic brain injury based on low-intensity pulsed ultrasound modulation according to claim 1, characterized in that: The level of phosphorylated tau protein in the damaged brain hemisphere of TBI mice before and after low-intensity pulsed ultrasound treatment was quantitatively detected by Western blotting. If the experimental results were statistically significant, a low-intensity pulsed ultrasound-regulated animal model of traumatic brain injury was constructed using the aforementioned low-intensity pulsed ultrasound conditions.

7. Application of low-intensity pulsed ultrasound in regulating cerebral lymphatic drainage and nerve repair in animals with traumatic brain injury.

Images