Method for improving cognitive functionality by regulating gamma rhythm oscillation with microwave

Abstract

The present disclosure relates to the field of biological technology, and in particular, to a method for improving intracerebral gamma rhythm oscillation and reducing intracerebral Aβ deposition and neurofibrillary tangles, a method for preparing an ex-vivo brain slice of an animal and use thereof, and a method for improving learning ability, spatial memory, and episodic memory. The method for improving the intracerebral gamma rhythm oscillation is for non-therapeutic purposes, and comprises: exposing an individual of interest to microwave radiation, wherein the repetition frequency of the microwave radiation is 20-100 Hz, and the frequency of the microwave radiation is 800-2500 MHz.

Description

Microwave-mediated gamma rhythm oscillations to improve cognitive function Technical Field

[0001] The present disclosure relates to the field of biotechnology, and specifically to methods for increasing gamma rhythm oscillations in the brain, reducing Aβ deposition and neurofibrillary tangles in the brain, methods for preparing animal brain slices in vitro and their applications, and methods for improving learning ability, spatial memory, and event memory ability. Background Art

[0002] Neural rhythmic oscillations, arising from the synchronized interaction of information exchange between neural populations, underlie many functional changes in both normal physiological and pathological states and are involved in a variety of cognitive functions. Neuronal network oscillations of varying frequencies are crucial for brain information processing. Gamma rhythmic oscillations (30-100 Hz), in particular, are believed to underlie cognitive processes such as attention, sensory perception, and memory. These oscillations, also known as "gamma rhythms" or "gamma neural oscillations," are thought to underlie cognitive processes such as attention, sensory perception, and memory. Gamma rhythmic oscillations participate in diverse cognitive processes, such as associative learning, language processing, perception and memory formation, and selective attention, by modulating the activity of local neuronal populations, particularly those separated by functional or spatial associations. Gamma rhythmic oscillations are commonly found in numerous brain regions, including the hippocampus, visual cortex, and entorhinal cortex. Gamma rhythmic oscillations generated in the hippocampus and entorhinal cortex are the most active brain regions involved in learning and memory, regulating higher-level cognitive behaviors such as perception, attention, the generation of learning and memory, and spatial localization and integration.

[0003] Studies have shown that impaired gamma oscillations in neuropsychiatric disorders such as Alzheimer's disease, epilepsy, and schizophrenia are closely associated with the cognitive impairments associated with these brain diseases. As cognitive impairment worsens, gamma rhythm oscillations in AD patients gradually decrease. Depending on the patient's disease course, cognitive state, and related functional brain regions, gamma rhythm oscillations can weaken to varying degrees; the degree of weakening of gamma rhythm oscillations is directly correlated with the patient's degree of cognitive decline. Recent studies have found that gamma rhythm oscillations are impaired in the hippocampus of AD mouse models, and this damage can even occur before the appearance of Aβ plaques. Studies have found that sensory stimulation such as sound and light, or techniques such as transcranial magnetic and transcranial electrical stimulation, can enhance gamma rhythm oscillations in the brain and improve cognitive function, but the therapeutic effects vary. The effects of microwaves on gamma rhythm oscillations have not been reported.

[0004] Therefore, it is urgent to explore the effects of microwaves on gamma rhythm oscillations in the brain and reveal the conditions and methods of microwave exposure that affect neuronal activity. Summary of the Invention

[0005] The present disclosure aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present disclosure provides a method for increasing gamma rhythm oscillations in the brain, reducing Aβ deposition and neurofibrillary tangles in the brain, a method for preparing animal in vitro brain slices and their application, and a method for improving learning ability, spatial memory, and event memory ability. The method disclosed in the present disclosure can enhance the gamma rhythm oscillations in the brain of an individual or inhibit the weakening of gamma rhythm oscillations. The microwave action time is short, and the gamma rhythm oscillation synergy is significantly enhanced, thereby improving the learning ability, spatial memory, event memory and other related cognitive levels of the subject. The method disclosed in the present disclosure can obtain in vitro brain slices of non-human mammals with enhanced electrical processes related to gamma rhythm oscillations.

[0006] The present disclosure is based on the inventors' findings and understanding of the following facts and problems:

[0007] While studying the effects of microwave radiation on gamma rhythm oscillations, the inventors unexpectedly discovered that microwave radiation with a repetition rate of 20 to 100 Hz can enhance gamma rhythm oscillations in the hippocampus of experimental animals. Further experimental results showed that microwave radiation with a repetition rate of 40 Hz can significantly enhance gamma rhythm oscillations or promote their early onset.

[0008] Therefore, in the first aspect of the present disclosure, the present disclosure proposes a method for increasing gamma rhythm oscillations in the brain. The method is used for non-therapeutic purposes, comprising: subjecting the individual to be improved to microwave radiation, the repetition frequency of the microwave radiation is 20 to 100 Hz, and the frequency of the microwave radiation is 800 to 2500 MHz. According to the method of the embodiment of the present disclosure, the gamma rhythm oscillations in the individual's brain can be enhanced or the weakening of the gamma rhythm oscillations can be suppressed, the microwave action time is short, and the gamma rhythm oscillations are significantly enhanced, thereby improving the learning ability, spatial memory, event memory and other related cognitive levels of the subject. Therefore, the method of the present disclosure can also be further used in scientific research and drug development.

[0009] It should be noted that the method according to the embodiment of the present disclosure is used for non-therapeutic purposes. For example, one purpose of the method of the present disclosure is to be used for related scientific research or drug development.

[0010] As used herein, the term "pulse repetition frequency" refers to the pulse repetition frequency, which is the number of microwave pulses emitted per second (usually expressed in Hertz (Hz)).

[0011] According to an embodiment of the present disclosure, the above method may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present disclosure, the repetition frequency of the microwave radiation is 30-50 Hz, preferably 40 Hz, thereby further enhancing the gamma rhythm oscillation of the individual to be improved or promoting the early occurrence of gamma rhythm oscillation.

[0013] According to an embodiment of the present disclosure, the frequency of the microwave radiation is 900 MHz. The inventors have determined the optimal microwave radiation frequency through extensive experiments, whereby gamma rhythm oscillations achieve optimal enhancement.

[0014] According to an embodiment of the present disclosure, the average power density is 0.8-4 mW / cm 2 . The inventors have determined the optimal average power density of microwave radiation through a large number of experiments, thereby achieving optimal enhancement of gamma rhythm oscillation.

[0015] According to an embodiment of the present disclosure, the microwave radiation time is not less than 8 days. The inventors have determined the optimal microwave radiation time through a large number of experiments, thereby achieving the best enhancement of gamma rhythm oscillation.

[0016] Illustratively, in some embodiments of the present disclosure, the time of microwave radiation is 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 42 days.

[0017] In the second aspect of the present disclosure, the present disclosure proposes a method for reducing Aβ deposition and / or neurofibrillary tangles in the brain. The method is for non-therapeutic purposes and comprises: subjecting the brain of the individual to be reduced or the sample to be processed to microwave radiation, wherein the repetition frequency of the microwave radiation is 20 to 100 Hz, the frequency of the microwave radiation is 800 to 2500 MHz, and the sample to be processed comprises at least one of the hippocampus, cerebral cortex, or thalamus. According to the method of the embodiment of the present disclosure, Aβ deposition and neurofibrillary tangles in the brain of the subject or in the sample to be processed can be reduced.

[0018] For example, the sample to be processed can be an animal in vitro brain slice comprising the hippocampus, cerebral cortex or thalamus. In this article, the term "neurofibrillary tangles" refers to the aggregation of hyperphosphorylated tau protein.

[0019] As used herein, the term "β-amyloid protein" is equivalent to "Aβ protein".

[0020] It should be noted that the method according to the embodiment of the present disclosure is used for non-therapeutic purposes. For example, one purpose of the method of the present disclosure is to be used for related scientific research or drug development.

[0021] According to an embodiment of the present disclosure, the above method may further include at least one of the following additional technical features:

[0022] According to an embodiment of the present disclosure, the repetition frequency of the microwave radiation is 30-50 Hz, preferably 40 Hz, thereby further enhancing the electrical processes related to gamma rhythm oscillations in the brain and reducing Aβ protein deposition or neurofibrillary tangles in the brain.

[0023] According to an embodiment of the present disclosure, the frequency of the microwave radiation is 900 MHz.

[0024] According to an embodiment of the present disclosure, the average power density of the microwave radiation is 0.8-4 mW / cm2.

[0025] According to an embodiment of the present disclosure, the microwave radiation time is not less than 30 days.

[0026] In some specific embodiments of the present disclosure, the microwave radiation has a frequency of 900 MHz, an average power density of 0.8-4 mW / cm2, and a duration of no less than 30 days, thereby significantly reducing Aβ deposition in the brain of the subject.

[0027] According to an embodiment of the present disclosure, the microwave radiation time is not less than 42 days.

[0028] In some specific embodiments of the present disclosure, the microwave radiation has a frequency of 900 MHz, an average power density of 4 mW / cm2, and a duration of no less than 42 days. Thus, neurofibrillary tangles in the brain of the subject can be significantly reduced.

[0029] It should be noted that, as used herein, the term "scientific research" may, for example, include electrophysiological research, pathophysiological research, biochemical research, pharmacological research, molecular biology research, and clinical medicine research. As used herein, the term "drug development" may, for example, include drug discovery, drug screening, pharmacology and toxicology research, drug formulation research, drug quality research, and drug safety evaluation.

[0030] In a third aspect, the present disclosure provides a method for preparing isolated brain slices from animals. The method comprises: treating isolated brain slices from non-human mammals with microwave radiation, wherein the microwave radiation has a repetition frequency of 20 to 100 Hz and a frequency of 800 to 2500 MHz.

[0031] Thus, an isolated brain slice of a non-human mammal with enhanced electrical processes associated with gamma rhythm oscillations is obtained. This isolated brain slice can be further used in scientific research or drug development, for example, for gamma rhythm oscillation-related research or for screening or identifying factors that can enhance gamma rhythm oscillations in the brain.

[0032] According to an embodiment of the present disclosure, the above method may further include at least one of the following additional technical features:

[0033] According to an embodiment of the present disclosure, the frequency of the microwave radiation is 900 MHz. After extensive experiments, the inventors determined that this optimal microwave radiation frequency further enhanced the electrical processes related to gamma rhythm oscillations in isolated brain slices.

[0034] According to an embodiment of the present disclosure, the repetition frequency of the microwave radiation is 40 Hz. The inventors have determined the optimal microwave repetition frequency through extensive experiments, thereby further enhancing the electrical processes related to gamma rhythm oscillations in isolated brain slices.

[0035] According to the embodiments of the present disclosure, the average power density of the microwave radiation is 0.1 to 4 mW / cm², preferably 0.4 mW / cm². The inventors, through extensive experiments, determined this optimal average microwave power density, which enables enhanced gamma rhythm oscillation-related electrical processes in isolated non-human mammalian brain slices.

[0036] According to an embodiment of the present disclosure, the microwave irradiation time is not less than 20 minutes. Under this microwave irradiation time, an in vitro brain slice of a non-human mammal with enhanced electrical processes related to gamma rhythm oscillations can be obtained.

[0037] In some embodiments of the present disclosure, the average power density of the microwave radiation is 0.1 mW / cm2, and the time of the microwave radiation is not less than 80 minutes.

[0038] In some embodiments of the present disclosure, the average power density of the microwave radiation is 0.4 mW / cm2, and the time of the microwave radiation is not less than 20 minutes.

[0039] In the fourth aspect of the present disclosure, the present disclosure proposes a use of an isolated brain slice of a non-human mammal prepared according to the aforementioned method, wherein the isolated brain slice is used for research related to gamma rhythm oscillations or for screening or identifying factors that can enhance gamma rhythm oscillations in the brain.

[0040] Exemplarily, the factors that can enhance gamma rhythm oscillations in the brain can be physical or chemical. Herein, the term "physical factors" includes, but is not limited to, microwave exposure, sensory stimulation such as sound and light, transcranial magnetic exposure, transcranial electrical stimulation, and hyperoxia. The term "chemical factors" includes, but is not limited to, pharmaceutical compounds or their precursor compounds, macromolecular drugs, traditional Chinese medicine, and drug delivery vehicles.

[0041] Those skilled in the art will understand that the features and advantages described above for the method of enhancing gamma rhythm oscillations, the method of reducing neuronal Aβ deposition or neurofibrillary tangles, and the method of preparing animal isolated brain slices are also applicable to this use and will not be repeated here.

[0042] In a fifth aspect, the present disclosure proposes a method for improving learning ability, spatial memory, and event memory. According to an embodiment of the present disclosure, the method is used for non-therapeutic purposes and comprises: exposing the individual to be improved to microwave radiation, wherein the microwave radiation has a repetition rate of 20 to 100 Hz and a frequency of 800 to 2500 MHz. This improves the individual's learning ability, spatial memory, event memory, and other related cognitive abilities, which can be further used in scientific research and drug development.

[0043] According to an embodiment of the present disclosure, the above method may further include at least one of the following additional technical features:

[0044] According to an embodiment of the present disclosure, the repetition frequency of the microwave radiation is 30 to 50 Hz; preferably, 40 Hz.

[0045] According to an embodiment of the present disclosure, the frequency of the microwave radiation is 900 MHz.

[0046] According to an embodiment of the present disclosure, the average power density of the microwave radiation is 0.8 to 4 mW / cm2. According to an embodiment of the present disclosure, the duration of the microwave radiation is not less than 30 days.

[0047] Those skilled in the art will understand that the features and advantages described above for the method of increasing gamma rhythm oscillations in the brain and reducing neuronal Aβ deposition or neurofibrillary tangles are also applicable to this method and will not be repeated here.

[0048] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0050] FIG1 is a graph showing the results of a study on gamma rhythm oscillations in brain slices under microwave irradiation at 600 MHz, 900 MHz, 1800 MHz, and 2400 MHz in Example 1 of the present disclosure, wherein A is a graph showing the results of a study on the changes in the peak frequency of gamma rhythm oscillations in brain slices; B is a graph showing the results of a study on the changes in the power of gamma rhythm oscillations in brain slices;

[0051] FIG2 is a graph showing the statistical analysis results of gamma rhythm oscillation power density in isolated brain slices after continuous microwave irradiation for 80 minutes in Example 1 of the present disclosure;

[0052] FIG3 is a graph showing the results of investigating the peak frequency of gamma rhythm oscillations in isolated brain slices under 900 MHz microwave irradiation at different repetition frequencies in Example 1 of the present disclosure;

[0053] FIG4 is a diagram showing the analysis results of gamma rhythm oscillations in in vitro brain slices under 900 MHz microwave irradiation at different repetition frequencies in Example 1 of the present disclosure, wherein A is the FFT power density spectrum; B is the power density value;

[0054] FIG5 is a graph showing the results of an investigation of changes in the peak frequency of gamma rhythm oscillations in brain slices under 900 MHz (repetition frequency 40 Hz) microwave irradiation at average power densities of 0.1 mW / cm2 and 0.4 mW / cm2 in Example 1 of the present disclosure;

[0055] FIG6 is a graph showing analysis results of changes in gamma rhythm oscillations in brain slices under 900 MHz (repetition frequency 40 Hz) microwave irradiation at average power densities of 0.1 mW / cm2 and 0.4 mW / cm2 in Example 1 of the present disclosure;

[0056] FIG7 is a graph showing the statistical analysis results of the power density of gamma rhythm oscillations in in vitro brain slices under 900 MHz (repetition frequency 40 Hz) microwave irradiation with average power densities of 0.1 mW / cm2 and 0.4 mW / cm2 in Example 1 of the present disclosure;

[0057] FIG8 is a graph showing the results of an investigation of changes in peak frequency of gamma rhythm oscillations in brain slices during microwave irradiation at 900 MHz (repetition frequency 40 Hz; average power density 0.4 mW / cm2) in Example 1 of the present disclosure;

[0058] FIG9 is a graph showing the analysis results of gamma rhythm oscillation changes in brain slices during 900 MHz (repetition frequency 40 Hz; average power density 0.4 mW / cm2) microwave irradiation in Example 1 of the present disclosure;

[0059] FIG10 is a graph showing statistical analysis of gamma rhythm oscillation power in 5xFAD mouse brain slices during 20 min of microwave irradiation at 900 MHz (repetition frequency 40 Hz; average power density 0.4 mW / cm2) in Example 1 of the present disclosure;

[0060] FIG11 is a graph showing the results of an investigation into the effect of microwave irradiation on the LG rhythmic oscillation power in the hippocampal CA1 region of 5xFAD mice in Example 2 of the present disclosure;

[0061] FIG12 is a statistical diagram of the peak node power of LG oscillations in the hippocampal CA1 region of 5xFAD mice irradiated with microwaves in Example 2 of the present disclosure;

[0062] FIG13 is a graph showing the results of an investigation into the effect of microwave irradiation on the HG rhythmic oscillation power in the hippocampal CA1 region of 5xFAD mice in Example 2 of the present disclosure;

[0063] FIG14 is a statistical diagram of the peak node power of HG oscillations in the hippocampal CA1 region of 5xFAD mice irradiated with microwaves in Example 2 of the present disclosure;

[0064] FIG15 is a graph showing the change in discrimination index in a novel object recognition experiment of 5xFAD mice after microwave irradiation in Example 3 of the present disclosure, wherein A is a sample diagram of the experimental scheme; B is a statistical diagram of the discrimination index;

[0065] FIG16 is a graph showing changes in the discrimination index of 5xFAD mice in a novel arm exploration test in a Y-maze after microwave irradiation in Example 3 of the present disclosure, wherein A is a sample diagram of the experimental scheme; B is a statistical graph of the discrimination index;

[0066] FIG17 is a diagram showing the results of Congo red staining of the hippocampus of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0067] FIG18 is a diagram showing the results of Congo red staining of the cerebral cortex of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0068] FIG19 is a diagram showing the Congo red staining results of the thalamus of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0069] FIG20-A is a graph showing the statistical analysis results of Congo red staining of the hippocampus of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0070] FIG20-B is a graph showing the statistical analysis results of Congo red staining of the cerebral cortex of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0071] FIG20-C is a diagram showing the statistical analysis results of Congo red staining of the thalamus region of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0072] FIG21 is a diagram showing the immunohistochemical staining results of APP in the hippocampus of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0073] FIG22 is a diagram showing the results of immunohistochemical staining of APP in the cerebral cortex of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0074] FIG23 is a diagram showing the immunohistochemical staining results of APP in the thalamus of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0075] FIG24-A is a diagram showing the results of immunohistochemical analysis of APP in the hippocampus of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0076] FIG24-B is a diagram showing the results of immunohistochemical analysis of APP in the cerebral cortex of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0077] FIG24-C is a diagram showing the results of immunohistochemical analysis of APP in the thalamus of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure;

[0078] FIG25-A shows the results of p-Tau immunofluorescence and quantitative analysis of the cerebral cortex of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure, wherein (top) shows the results of p-Tau immunofluorescence staining, and (bottom) shows the results of quantitative analysis; control (C57) indicates a blank control group of C57BL / 6 mice placed in an irradiation box without microwave irradiation, sham-4w indicates a blank control group of 5xFAD mice placed in an irradiation box for 4 weeks without microwave irradiation, sham-6w indicates a blank control group of 5xFAD mice placed in an irradiation box for 6 weeks without microwave irradiation, MW-4w indicates an experimental group irradiated with 4 mW / cm2 microwaves for 4 weeks, and MW-6w indicates an experimental group irradiated with 4 mW / cm2 microwaves for 6 weeks; the white horizontal line in the figure indicates a scale of 200 μm;

[0079] FIG25-B shows the results of p-Tau immunofluorescence and quantitative analysis of the hippocampus of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure, wherein (top) shows the results of p-Tau immunofluorescence staining, and (bottom) shows the results of quantitative analysis; control (C57) indicates a blank control group of C57BL / 6 mice placed in an irradiation box without microwave irradiation, sham-4w indicates a blank control group of 5xFAD mice placed in an irradiation box for 4 weeks without microwave irradiation, sham-6w indicates a blank control group of 5xFAD mice placed in an irradiation box for 6 weeks without microwave irradiation, MW-4w indicates an experimental group irradiated with 4 mW / cm2 microwaves for 4 weeks, and MW-6w indicates an experimental group irradiated with 4 mW / cm2 microwaves for 6 weeks; the white horizontal line in the figure indicates a scale bar of 200 μm;

[0080] FIG25-C shows the results of p-Tau immunofluorescence and quantitative analysis of the thalamus region of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure, wherein (top) shows the results of p-Tau immunofluorescence staining, and (bottom) shows the results of quantitative analysis; control (C57) indicates a blank control group of C57BL / 6 mice placed in an irradiation box without microwave irradiation, sham-4w indicates a blank control group of 5xFAD mice placed in an irradiation box for 4 weeks without microwave irradiation, sham-6w indicates a blank control group of 5xFAD mice placed in an irradiation box for 6 weeks without microwave irradiation, MW-4w indicates an experimental group irradiated with 4 mW / cm2 microwaves for 4 weeks, and MW-6w indicates an experimental group irradiated with 4 mW / cm2 microwaves for 6 weeks; the white horizontal line in the figure indicates a scale of 200 μm;

[0081] Figure 25-D shows the results of p-Tau immunofluorescence and quantitative analysis of the internal capsule region of 5xFAD mice after microwave irradiation in Example 4 of the present disclosure, wherein (top) is the result of p-Tau immunofluorescence staining, and (bottom) is the result of quantitative analysis; control (C57) represents the blank control group of C57BL / 6 mice placed in an irradiation box without microwave irradiation, sham-4w represents the blank control group of 5xFAD mice placed in an irradiation box for 4 weeks without microwave irradiation, sham-6w represents the blank control group of 5xFAD mice placed in an irradiation box for 6 weeks without microwave irradiation, MW-4w represents the experimental group irradiated with 4 mW / cm2 microwaves for 4 weeks, and MW-6w represents the experimental group irradiated with 4 mW / cm2 microwaves for 6 weeks; the white horizontal line in the figure represents the scale: 200 μm. DETAILED DESCRIPTION

[0082] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0083] The key materials and methods used in the embodiments of the present disclosure are as follows:

[0084] In vitro brain slice preparation method

[0085] 1) Brain perfusion: Mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital solution (50 mg / kg). The limbs of the mice were secured to a foam board using a 1 mL syringe needle. The thoracic cavity was exposed by transverse incision along the abdomen and chest using ophthalmic scissors, fully exposing the heart and liver. Using a 20 mL syringe and replacing the needle with a 1 mL syringe needle, 20 mL of slice solution (ice-water mixture) was withdrawn and the excess air in the syringe was expelled. The needle was then inserted into the apex of the mouse's heart. A small incision was made in the right atrial appendage using ophthalmic scissors. Approximately 15 mL of slice solution (ice-water mixture) was injected from the apex of the heart until the liver turned white. The head was quickly decapitated using surgical scissors, and the scalp was removed. Next, the skull was carefully and quickly removed using ophthalmic scissors, and the pia mater was separated to fully expose the brain tissue. During this time, the brain tissue was continuously rinsed with artificial slice solution (ice-water mixture) to maintain a cool and moist state. The removed brain tissue was placed in a flat dish pre-filled with artificial slicing fluid (ice-water mixture), and the brain stem and cerebellum were removed with a blade.

[0086] 2) Sectioning: Before sectioning, prepare agar blocks with deionized water and agar powder and allow to dry. Use a blade to cut small squares and adhere them to the slicing table of an oscillating microtome with 404 glue. The agar blocks provide support and cushioning for the brain tissue during sectioning. Use a spatula to scoop the brain tissue from the dish and remove excess water from the bottom of the tissue with filter paper. Use ophthalmic forceps to place the brain tissue (frontal lobe facing up) after the brainstem and cerebellum have been removed, in front of the agar block on the oscillating microtome coated with 404 glue. Next, place the slicing jar in the slicing chamber of the oscillating microtome, which has been pre-filled with an ice-water mixture. A mixed gas (95% oxygen, 5% carbon dioxide) is continuously aerated. Adjust the oscillating microtome to appropriate sectioning parameters. Slices approximately 300 μm thick are cut coronally and quickly transferred to a brain slice incubator containing aCSF using a homemade slicing net.

[0087] 3) Brain slice incubation: Place the brain slice incubation tank in a constant temperature water bath. Continuously monitor the cerebrospinal fluid temperature in the incubation tank. Incubate at 37°C for at least 30 minutes. Then remove the brain slice incubation tank and incubate at room temperature for 30 minutes before starting the electrophysiological experiment.

[0088] In vitro electrophysiological signal recording and analysis of brain slices

[0089] 1) Real-time recording of electrophysiological signals in isolated brain slices during microwave irradiation

[0090] To avoid noise and baseline drift during recording, as well as cytotoxicity caused by exposed silver wire, the reference and recording silver wires were immersed in 84 disinfectant for 20 minutes to chlorinate before the experiment. A homemade slice scoop was used to transfer the brain slice from the incubation tank to the recording tank. The position of the isolated brain slice was adjusted with a fine brush. The fully immersed recording tank cover was then placed to secure the slice in the recording tank, ensuring that the recorded hippocampus was centered in the microwave irradiation field. Before recording, the slices were inspected under a stereomicroscope for complete hippocampal shape, clear structure, and good refractive index. Slices with incomplete hippocampal shape, unclear structure, and poor refractive index were not selected for isolated brain slice electrophysiology experiments. A glass electrode (resistance approximately 1 MΩ) was drawn and filled with aCSF. Excess bubbles were ejected from the glass electrode, and the glass electrode was then secured to the electrode micromanipulator. The recording silver wire is placed in the glass electrode, and the reference electrode is placed in the immersion recording tank. At the same time, it is ensured that the reference electrode is outside the irradiation recording field to avoid electromagnetic interference to the recording signal after turning on the microwave device.

[0091] Under stereomicroscope observation, the glass electrode tip was first placed in contact with the liquid surface to form a reference-recording loop. Complex software then performed real-time bandpass filtering (15–500 Hz), and the built-in Hum Silencer noise cancellation function was used to remove excess power-frequency noise. All instruments were further grounded to minimize noise during the experiment. The coarse and fine spirals on the glass electrode micromanipulator were adjusted to slowly insert the glass electrode into the pyramidal cell layer (left side) of the hippocampal CA1 region at a moderate depth, ensuring that the glass electrode tip was intact and accurately positioned. The recorded electrophysiological signals were amplified 500-fold by a microelectrode amplifier and transmitted to an analog-to-digital converter for acquisition and recording at a sampling rate of 5000 Hz. After the glass electrode is placed in the target brain area, a 1-minute baseline signal is recorded. Then, aCSF containing 20 μmol / L carbachol (CCH) is introduced. After about 2 minutes, gamma rhythm oscillations induced by the isolated brain slice can be seen. After 30 minutes, when the induced stable state is reached, a 10-minute electrophysiological signal baseline is recorded. After that, the broadband electromagnetic exposure device is turned on, and the electrophysiological signals of the hippocampal CA1 region are synchronously recorded during the irradiation period.

[0092] 2) In vitro brain slice electrophysiological signal processing and analysis

[0093] After importing the recorded data into Matlab 2016b, the raw LFP data were processed using built-in and modified specific codes. A notch filter (bandwidth 8, bandwidth attenuation approximately 0.0004) was used to remove 50 Hz power frequency noise and machine noise. A second-order infinite impulse response digital filter was used for bandpass filtering from 15 to 100 Hz. Baseline drift was removed using polynomial fitting. All acquired data were segmented into 1-minute time windows. FFT analysis was performed on each 1-minute window. The power density spectrum was obtained using the Welch-FFT method (Hamming window, 2-second window width, 50% overlap). Gamma rhythm oscillation power was calculated by integrating the 25-45 Hz range. All normalized data were calculated by averaging the gamma rhythm oscillation power over a 10-minute period and dividing this average by the gamma rhythm oscillation power across all recorded time periods. Data between groups were analyzed using a 1-minute window.

[0094] Unless otherwise specified, the two-sample data of the in vitro brain slice electrophysiology in the embodiments of the present disclosure were statistically analyzed using the Wilcoxon test; the multi-sample data were statistically analyzed using the Mann-Whitney U test. The differences in gamma rhythm oscillation power were compared, and P < 0.05 indicated that the difference was statistically significant.

[0095] Experimental animals and groups of microwave irradiation method

[0096] Healthy 3-month-old C57BL / 6 and 5xFAD mice, weighing 24-28 g each, were purchased and randomly divided into group C, sham group, 0.8 mW / cm² irradiation group, and 4 mW / cm² irradiation group, with 30 mice in each group. Group C served as a blank control group for C57BL / 6 mice, and the sham group served as a blank control group for 5xFAD mice. The mice were placed in the irradiation box for the same period of time but were not exposed to microwaves.

[0097] Electrode implantation and placement

[0098] 1) Preoperative Preparation: To reduce stress responses to handling during the experiment, mice underwent daily habituation training. Mice were deprived of food and water for 12 hours before surgery. The stereotaxic apparatus was wiped with 75% alcohol. All surgical instruments were disinfected by soaking in 75% alcohol the night before the experiment and rinsed with saline before use.

[0099] 2) Anesthesia and Fixation: Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (50 mg / kg). After the mice showed no obvious response to painful stimuli such as foot pinching or tail clamping, they were placed on a digital stereotaxic instrument with their incisors clamped on the nose clip of the stereotaxic instrument. The sides of the mouse's head were fixed with bilateral ear bars. The head was firmly fixed by shaking the mouse's body. Erythromycin ointment was applied to both eyes to prevent cold light stimulation and dry eyes. A heater was used to maintain the operating room temperature throughout the operation. The anesthesia status of the mouse was closely observed during the operation.

[0100] 3) Skull Exposure: Disinfect the surgical area with iodine. Shave the mouse's head hair with ophthalmic curved scissors and a razor. Disinfect the head skin again with iodine. Make an approximately 1 cm sagittal incision in the shape of an inverted T using an ophthalmic needle. Apply pressure to any bleeding from the incision with a cotton swab. Separate the connective fascia with a cotton swab dipped in saline to ensure adequate exposure of the bregma and lambdoid sutures.

[0101] 4) Positioning the Window: Using the Bregma point as the coordinate origin, use the balance needle on the digital stereotaxic instrument to balance the mouse's head front-to-back and left-to-right, ensuring a 0.02mm error. Using a standard mouse brain atlas, locate the coordinates of the hippocampal CA1 region (A / P: -2.0mm; M / L: 1.5mm; D / V: -1.5mm). Mark the electrode implantation location with a marker. Using a 0.8mm diameter skull drill, create a 4×3mm rectangular window centered at this point. Simultaneously, drill four small holes in the thicker areas of the skull surrounding the window. Four stainless steel skull screws are inserted to secure the dental cement to the mouse skull surface. Under a microscope, rinse the head bone scraps with saline. Carefully peel and remove the dura mater using ophthalmic forceps. During this process, flush the window with saline to keep the brain tissue moist.

[0102] 5) Electrode implantation: Secure the electrode to the electrode micro-advancer of the digital stereotaxic instrument and slowly push until the electrode is implanted in the marked area. Wrap the two ground wires at one end of the electrode around the skull screw, ensuring that the ground wires are tightly wrapped around the skull screw. Dissolve the PEG glue on the electrode array with saline to ensure that the microwire electrode is fully exposed. Slowly implant the electrode vertically into the target brain area at a speed of 100 μm / min.

[0103] 6) Electrode Fixation: After the electrodes are lowered into the target brain area, they are sealed with liquid paraffin to reduce irritation caused by dental cement. Pour an appropriate amount of denture base resin powder and aqueous solution into a 35mm culture dish and mix them in a certain proportion (first thin and then thick). Apply a layer of dental cement evenly to cover the electrodes and skull screws. After it solidifies, apply a second layer of dental cement until the screws, ground wire, and microwire electrodes are completely covered with dental cement. After the dental cement completely solidifies, disinfect the surgical wound on the mouse scalp with iodine tincture and trim the sharp parts of the dental cement to prevent subsequent damage to the mouse scalp. After the surgery, remove the mouse from the digital positioning instrument.

[0104] 7) Postoperative care: Observe the condition of the mice during this period. Once the mice resume normal activities, place them in individual cages for maintenance.

[0105] Microwave irradiation method

[0106] Using a custom-built microwave irradiation antenna, signal generator, and microwave power amplifier, whole-body irradiation was performed on 5xFAD mice in each experimental group. Irradiation conditions were: 900 MHz, repetition rates of 20 Hz, 40 Hz, 60 Hz, and 80 Hz, with average power densities of 0.8 mW / cm2 and 4 mW / cm2, respectively. Irradiation was performed for 1 hour daily for 28 consecutive days. Starting three days before irradiation, mice in the irradiated groups were placed in an open 30 cm × 30 cm × 19 cm acrylic box (divided into 16 small compartments approximately 7 cm × 7 cm by baffles) for 30 minutes daily to prevent stress reactions in the mice. During irradiation, the acrylic box containing the mice was placed below the center of the microwave antenna to ensure uniform irradiation intensity for all irradiated mice. Unless otherwise noted, all statistical analyses in the disclosed examples were performed using SPSS 25.0 software, and plotted using GraphPad Prism 9.0 software. Data are presented as mean ± standard error (SEM). Repeated measures analysis of variance was used to evaluate the differences in LG and HG rhythmic oscillation power measured in the sham group, 0.8 mW / cm2 group, and 4 mW / cm2 group before and after irradiation at the two irradiation doses; one-way analysis of variance was used to evaluate the differences in cognitive ability, Aβ deposition, APP content, and p-Tau content of mice. P < 0.05 indicated that the difference was statistically significant.

[0107] Example 1: Effects of microwave irradiation on gamma rhythm oscillations in isolated brain slices

[0108] First, the hippocampus of isolated brain slices from C57BL / 6 mice was irradiated with microwaves at 600 MHz, 900 MHz, 1800 MHz, and 2400 MHz, all with a repetition rate of 40 Hz and an average power density of 0.1 mW / cm². Electrophysiological signals from the CA1 region of the hippocampus were recorded during irradiation to identify the optimal microwave frequency for enhancing gamma rhythmic oscillations in the CA1 region. Subsequently, microwave irradiation was performed at optimal repetition rates of 10 Hz, 20 Hz, 30 Hz, and 40 Hz to identify the optimal repetition rate for enhancing gamma rhythmic oscillations in the CA1 region. The output of the microwave irradiation device was connected to a microwave power amplifier, which transmitted the emitted microwaves to a microwave transmitting board. By calculating and adjusting the microwave transmitter and microwave power amplifier parameters, the optimal microwave frequency and repetition rate were selected using an average power density of 0.4 mW / cm² for exposure to brain slices. The effects of different microwave parameters with different average power densities on gamma rhythmic oscillations in the CA1 region were determined. All sham group brain slices were placed in the center of the brain slice recording tank, but the microwave irradiation device was not turned on after the baseline electrophysiological signal recording (10 minutes). The specific experimental methods and results are as follows:

[0109] 1. Effects of microwave irradiation at different frequencies on gamma rhythm oscillations in the hippocampus of C57BL / 6 mouse brain slices

[0110] To investigate the effects of microwaves of different frequencies (600MHz, 900MHz, 1800MHz, and 2400MHz) on gamma rhythm oscillations in the CA1 region of the hippocampus of C57BL / 6 mouse brain slices in vitro, the same repetition frequency of 40Hz and average power density of 0.1mW / cm2 were used to record the changes in gamma rhythm oscillation power of brain slices in real time within 120min during irradiation.

[0111] The results are shown in Figure 1: (1) No changes were found in the peak frequency of gamma rhythm oscillations during microwave irradiation at 600MHz, 900MHz, 1800MHz, and 2400MHz, indicating that the induction of gamma rhythm was stable (Figure 1: A). (2) By standardizing the power density of gamma rhythm oscillations in isolated brain slices within 120 minutes and comparing the changes in power density of the hippocampal CA1 region of isolated brain slices from C57BL / 6 mice after microwave irradiation at 600MHz, 900MHz, 1800MHz, and 2400MHz, it was found that gamma rhythm oscillations in isolated brain slices from C57BL / 6 mice gradually increased when exposed to microwaves at 900MHz, 1800MHz, and 2400MHz, and the power of gamma rhythm oscillations in isolated brain slices from C57BL / 6 mice tended to stabilize after 80 minutes of irradiation, while no significant changes were observed under the same conditions of 600MHz microwave irradiation (Figure 1: B).

[0112] The power density spectra were recorded at the first minute and at 600MHz, 900MHz, 1800MHz and 2400MHz for 80 minutes, and the standardized power density of the gamma rhythm oscillation frequency band was further quantified and compared.

[0113] The results are shown in Figure 2: When the isolated brain slices were irradiated with 900 MHz (repetition frequency 40 Hz; average power density 0.1 mW / cm2) microwaves for 80 minutes, the power density of gamma rhythm oscillations was significantly enhanced (P<0.05).

[0114] 2. Effects of microwave irradiation at different repetition frequencies on gamma rhythm oscillations in the hippocampus of C57BL / 6 mouse brain slices

[0115] After screening out the optimal microwave frequency of 900 MHz for enhancing gamma rhythm oscillations in the CA1 region of the hippocampus of C57BL / 6 mice in vitro, in order to further explore the effects of microwave irradiation with different repetition frequencies on gamma rhythm oscillations in brain slices, 900 MHz microwave irradiation with an average power density of 0.1 mW / cm2 and repetition frequencies of 10 Hz, 20 Hz, 30 Hz, and 40 Hz was used, and the changes in gamma rhythm oscillations in the CA1 region of the hippocampus of C57BL / 6 mice in vitro were synchronously recorded during 5 minutes of microwave irradiation.

[0116] The results are shown in Figures 3 and 4: (1) 900 MHz microwave irradiation with repetition frequencies of 10 Hz, 20 Hz, 30 Hz, and 40 Hz did not cause significant changes in the peak frequency of gamma rhythm oscillations (P>0.05), indicating that the induction of gamma rhythm oscillations in isolated brain slices was stable (Figure 3). (2) The power density spectrum of gamma rhythm oscillations in brain slices under 900 MHz microwave irradiation with different repetition frequencies is shown in Figure 4 (A); the average power density of the 25-45 Hz gamma rhythm oscillation frequency range in the power density spectrum was calculated by integration, and the power density of gamma rhythm oscillations in brain slices under irradiation with different repetition frequencies was compared. The results are shown in Figure 4 (B). The results in Figure 4 show that 900 MHz microwave irradiation with a repetition frequency of 40 Hz can significantly enhance the gamma rhythm oscillations in the CA1 region of the hippocampus of isolated brain slices of C57BL / 6 mice (P<0.05).

[0117] 3. Effects of 900 MHz (40 Hz repetition rate) microwave irradiation at different average power densities on gamma rhythm oscillations in the hippocampus of C57BL / 6 mouse brain slices

[0118] Based on the clarification of the influence of 900MHz (repetition frequency 40Hz; average power density 0.1mW / cm2) microwaves on the gamma rhythm oscillations of C57BL / 6 mouse brain slices in vitro, a microwave power amplifier was used to amplify its average power density to a SAR value of 0.4W / kg, that is, an average power density of 0.4mW / cm2, and continuously irradiate the mouse brain slices in vitro to explore the effects of different average power densities on the gamma rhythm oscillations of brain slices.

[0119] The results are shown in Figures 5 and 6: (1) Based on the peak frequency of gamma rhythm oscillations in brain slices irradiated with microwaves at an average power density of 0.1 mW / cm2 and 0.4 mW / cm2 at 900 MHz (repetition frequency 40 Hz), it can be seen that the induction of gamma rhythm oscillations is stable (Figure 5). (2) By analyzing and standardizing the power density of gamma rhythm oscillations in isolated brain slices within 170 minutes, and comparing the power changes in the hippocampal CA1 region of isolated brain slices from C57BL / 6 mice irradiated with microwaves at an average power density of 0.1 mW / cm2 and 0.4 mW / cm2 at 900 MHz (repetition frequency 40 Hz), it was found that irradiation with microwaves at an average power density of 0.4 mW / cm2 for 20 minutes can cause the power density of gamma rhythm oscillations in isolated brain slices to reach the peak level earlier, which is significantly earlier than the peak time of irradiation with microwaves at an average power density of 0.1 mW / cm2 (Figure 6).

[0120] According to the power density spectra at the peak moments of gamma rhythm oscillation power density during 900MHz (repetition frequency 40Hz) microwave irradiation with average power density of 0.1mW / cm2 and 0.4mW / cm2, the standardized power densities at the peak moments of gamma rhythm oscillation of these two microwave parameters were further compared.

[0121] The results are shown in Figure 7: Irradiation of isolated brain slices with 900 MHz microwaves at an average power density of 0.4 mW / cm2 for 20 minutes can significantly enhance the gamma rhythm oscillation of isolated brain slices (P<0.05), and the enhancement effect is similar to that of irradiation with 900 MHz at an average power density of 0.1 mW / cm2 for 80 minutes (P>0.05).

[0122] 4. Effects of 900 MHz (40 Hz repetition rate) microwave irradiation with an average power density of 0.4 mW / cm2 on gamma rhythm oscillations in the hippocampus of isolated brain slices from 5xFAD mice

[0123] Comparison of gamma rhythm oscillations in LFPs during resting state in APP / PS1 and 5xFAD mice revealed a more pronounced decrease in 5xFAD mice. Therefore, this study selected 5xFAD mice, which exhibit a more pronounced neurophysiological phenotype, to prepare isolated brain slices. These slices were irradiated with 900 MHz (40 Hz repetition rate) at an average power density of 0.4 mW / cm² for 120 minutes.

[0124] The results are shown in Figures 8 to 10: (1) 900 MHz (40 Hz repetition rate) with an average power density of 0.4 mW / cm2 was used for continuous irradiation for 120 min, and the induction of gamma rhythm oscillation was stable during the irradiation period (Figure 8). (2) By standardizing the gamma rhythm oscillation power of isolated brain slices within 120 min, it was found that 20 min of 900 MHz (40 Hz repetition rate; average power density 0.4 mW / cm2) microwave irradiation of the hippocampal CA1 region of 5xFAD mouse isolated brain slices could cause the gamma rhythm oscillation to reach a peak level (Figure 9). (3) Based on the power density spectrum of the peak time point of irradiation of 900 MHz (40 Hz repetition rate; average power density 0.4 mW / cm2) microwave irradiation of 5xFAD mouse isolated brain slices, the normalized gamma rhythm oscillation power was further compared. The results showed that 20 min of 0.4 mW / cm2 irradiation could significantly enhance the gamma rhythm oscillation of isolated brain slices (P < 0.01) (Figure 10).

[0125] The above results show that: (1) 900MHz microwave irradiation with a repetition frequency of 40Hz and an average power density of 0.1mW / cm2 and 0.4mW / cm2 can enhance the gamma rhythm oscillation in C57BL / 6 mouse brain slices in vitro, and 0.4mW / cm2 can advance the time when the peak of gamma rhythm oscillation appears.

[0126] (2) Microwave irradiation at 900 MHz (repetition frequency 40 Hz, average power density 0.4 mW / cm2) for 20 min can significantly enhance the gamma rhythm oscillations in the isolated brain slices of 5xFAD mice.

[0127] Example 2: Effects of microwave irradiation on gamma rhythm oscillations in the CA1 region of the mouse hippocampus

[0128] In this example, mice were irradiated with microwaves, and LFPs from the CA1 region of the hippocampus of the mice were collected and analyzed. The specific method is as follows:

[0129] Three to four days after electrode implantation, LFPs from the CA1 region of 5xFAD mice were recorded using a Plexon multi-channel electrophysiological acquisition system for 5 minutes before irradiation. Following irradiation, the head electrodes were connected to the OmniPlex multi-channel in vivo recording system amplifier. All electrical equipment was turned off and grounded to minimize interference from power frequency noise. After the mice adjusted to the weight of the head amplifier and were awake and quiet, electrophysiological signals were recorded at a 40 kHz sampling rate for 10 minutes. The system automatically downsampled the acquired LFPs to 1000 Hz. A control group was similarly placed under the microwave antenna but not exposed to microwave irradiation, and electrophysiological signals were recorded in the same manner for 10 minutes.

[0130] The acquired electrophysiological data were imported into Matlab 2016b. Raw LFPs were processed using built-in and modified code. A notch filter (bandwidth 8, bandwidth attenuation approximately 0.0004) was used to remove 50 Hz power frequency noise and machine noise. A second-order infinite impulse response digital filter was used for 1-90 Hz bandpass filtering. Baseline drift was removed using polynomial fitting. All acquired data were segmented into 1-minute time windows. Welch-FFT analysis (using a Hamming window with a 2-second window width and 50% overlap) was performed on each segmented 1-minute window to obtain a minute-by-minute power density spectrum. The power of the LG and HG rhythmic oscillations was calculated by averaging the 30-50 Hz and 50-90 Hz frequency bands, respectively. All normalized data were obtained by averaging the gamma rhythmic oscillation power within the 5-minute pre-irradiation period and the 10-minute gamma rhythmic oscillation power recorded daily after irradiation. The values ​​were then averaged and normalized to obtain power density.

[0131] Preliminary observation results showed that when the repetition frequency was 20Hz, 40Hz, 60Hz, 80Hz, and 100Hz, the gamma rhythm oscillations in the hippocampus of the experimental animals in the 0.8mW / cm2 group and the 4mW / cm2 group were enhanced; when the repetition frequency was 40Hz, the above changes were more significant (Figures 11 to 14).

[0132] The results show:

[0133] (1) By standardizing the changes in gamma rhythm oscillation power in the CA1 region of the hippocampus of 5xFAD mice after 15 days of irradiation (Figures 11 and 13), it was found that the power density of LG and HG rhythm oscillations in the CA1 region of the hippocampus of 5xFAD mice reached peak levels after 12 days of continuous irradiation with 900 MHz microwaves at an average power density of 0.8 mW / cm2 (repetition frequency 40 Hz). In contrast, continuous irradiation with 900 MHz microwaves at an average power density of 4 mW / cm2 (repetition frequency 40 Hz) for 8 days advanced the peak of the power density of LG and HG rhythm oscillations. Moreover, the enhancement effect under both irradiation intensities still existed at day 30.

[0134] (2) Further quantitative comparison of the LG and HG oscillation power levels in the CA1 region of the hippocampus of 5xFAD mice in the 8-day, 12-day, and 30-day irradiation groups before and after irradiation (Figures 12 and 14) revealed that compared with the sham group, continuous irradiation with 900 MHz (repetition frequency 40 Hz) microwaves with an average power density of 0.8 mW / cm2 for 12 days and 30 days significantly increased the LG and HG oscillation power densities in the CA1 region of the hippocampus of 5xFAD mice (P<0.05 or P<0.01), while no significant changes were observed after 8 days of irradiation (P>0.05). Continuous irradiation with microwaves at an average power density of 4 mW / cm2 for 8, 12, and 30 days showed a significant increase in the power density (P<0.01).

[0135] (3) Further comparison of the average power density of the LG and HG in the hippocampal CA1 region of 5xFAD mice in the sham group and the two microwave irradiation intensities (Figures 12 and 14) revealed that after 8 days of irradiation, the enhancement effect of the average power density of 4 mW / cm2 was more significant than that of 0.8 mW / cm2 (P<0.05); the effects of 8 days of irradiation with an average power density of 4 mW / cm2 and 12 days of irradiation with an average power density of 0.8 mW / cm2 were similar (P>0.05). These results indicate that 12 days of 900 MHz (repetition frequency 40 Hz; average power density 0.8 mW / cm2) microwave irradiation can modulate and enhance the abnormally attenuated gamma rhythm oscillation of AD mice to the peak level, and 4 mW / cm2 can advance the peak.

[0136] Example 3: Effects of microwave irradiation on cognitive ability of mice

[0137] In this example, mice were irradiated with microwaves, and the effects of microwave irradiation on their cognitive abilities were investigated using novel object recognition and Y-maze tests. The specific methods are as follows:

[0138] 1. New object recognition experiment

[0139] The experimental equipment consists of a 40cm×40cm×40cm white acrylic box. The camera is located directly above the box and is covered with curtains on all sides to ensure that the ambient light is uniform and soft.

[0140] On days 28 and 29 of irradiation, a novel object recognition acclimation experiment was conducted. Experimental mice were placed in a corner of the chamber, facing the chamber wall, to acclimate for 10 minutes. The environment was kept quiet. After the experiment, the mice were removed, their excrement was cleaned with toilet paper, and the chamber was sprayed and wiped with 75% alcohol to remove any remaining odors and prevent interference with the next experiment.

[0141] On the 30th day after irradiation, a familiarization and testing phase for novel object recognition was conducted. During the familiarization phase, two identical 4cm×4cm×4cm square blue blocks were placed in the box at 1 / 3 of the box's diagonal, with all other blocks remaining unchanged. After 5 minutes, the mice were removed. One hour after the familiarization phase, each mouse entered the testing phase. At this time, one of the blue blocks (the old object) was replaced with a block composed of a 4cm×4cm×1cm green flat block and a 3cm×3cm×3cm pink hexagonal block (the new object). All other blocks remained unchanged. The time the mouse spent exploring the new and old objects was recorded over a 5-minute period. The discrimination index was used to evaluate the mice's recognition memory ability, as shown in the following formula:

[0142] Discrimination index = (new object exploration time - old object exploration time) / total exploration time

[0143] 2. Y-maze experiment

[0144] The experimental equipment consists of a 72cm×72cm×72cm black acrylic Y-shaped maze (the bottom of the maze is white acrylic). The maze is divided into a starting arm, other arms, and a novel arm. The ends of the maze arms are affixed with 2cm×2cm triangular, square, and hexagonal clues. The camera is located above the center of the maze and is covered with curtains on all sides to ensure uniform and soft ambient light.

[0145] The novel arm exploration experiment was carried out 30 days after irradiation, and the experiment was divided into a familiarization period and a test period. Before the experiment, the mice were placed in the experimental environment to adapt for 30 minutes. During the familiarization period, the entrance to the novel arm was closed with a baffle, and the mouse was placed facing the center of the maze. After 5 minutes, the mouse was taken out, and the mouse excrement in the box was cleaned with toilet paper and sprayed and wiped with 75% alcohol to remove the remaining odor in the box to avoid interfering with the next mouse experiment. Each mouse entered the test period 1.5 hours after the end of the familiarization period. At this time, the novel arm baffle was removed, and the others remained unchanged. The time the mouse explored each maze arm within 5 minutes was recorded. The discrimination index was used to evaluate the spatial memory ability of mice. The formula is as follows:

[0146] Discrimination index = (exploration time of novel arm - exploration time of other arms) / (exploration time of novel arm + exploration time of other arms)

[0147] 3. Results Analysis

[0148] The results are shown in Figures 15 and 16.

[0149] The results show:

[0150] (1) Compared with the sham group, 30 days of microwave irradiation at 900 MHz (repetition frequency 40 Hz) with an average power density of 4 mW / cm2 significantly increased the discrimination index of 5xFAD mice in the novel object recognition test and the novel arm exploration test of the Y maze (P < 0.05 or P < 0.01).

[0151] (2) In the Y-maze novel arm exploration experiment, the discrimination index of the 4 mW / cm2 irradiation group was significantly higher than that of the 0.8 mW / cm2 irradiation group (P<0.01).

[0152] Example 4: Effects of microwave irradiation on mouse brain structure

[0153] In this example, mice were irradiated with microwaves, and the effects of microwave irradiation on the brain structure of mice were investigated using methods such as pathological sections, Congo red staining, immunohistochemical staining of amyloid procursor-like protein (APP), and immunofluorescent staining of phosphorylated Tau (p-Tau). The specific methods are as follows:

[0154] 1. Preparation of pathological sections

[0155] (1) Sampling: 30 days after irradiation, the mice were decapitated and the brains were taken out. Routinely, 3 mm thick slices were taken and the tissues were placed in an embedding mold.

[0156] (2) The order of dehydration, transparency and wax immersion is: 75% ethanol for 1 hour, 85% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol (I) for 1 hour, 95% ethanol (II) for 1 hour, 100% ethanol (I) for 1 hour, 100% ethanol (II) for 1 hour, ethanol and xylene for 40 minutes, xylene (I) for 25 minutes, xylene (II) for 15 minutes, wax immersion at 65°C for 1 hour, wax immersion at 65°C for 1 hour, wax immersion at 65°C for 1 hour.

[0157] (3) Embedding: Embed the tissue with the cross section facing downward. After the wax block cools down, remove it from the mold and trim the block.

[0158] (4) Sectioning: Pre-cool the wax block on a freezing table for 20 minutes, then slice it into 4 μm sections on a microtome using a coronal section. Then, spread the cut tissue wax slices in a constant temperature water area in a slide spreader. After the tissue wax slices are flattened, adhere them to the slide.

[0159] (5) Slide baking: Place the glass slide with the tissue wax sheet on a slide baking machine to dry the residual moisture, then place it in a 65°C oven and bake for 1 hour. Then, perform Congo red staining and immunohistochemical staining respectively.

[0160] 2. Congo red staining

[0161] The sections were incubated with xylene (I) for 20 min, xylene (II) for 20 min, anhydrous ethanol (I) for 5 min, anhydrous ethanol (II) for 5 min, 75% alcohol for 5 min, washed with tap water, stained with Congo red A solution overnight, washed with tap water for 2 min, differentiated with Congo red B solution for 1 s until positive plaques were obvious and the background was basically colorless, washed with tap water, stained with Congo red C solution for 1 min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with bluing solution, rinsed with running water, incubated with anhydrous ethanol (I) for 5 min, anhydrous ethanol (II) for 5 min, anhydrous ethanol (III) for 5 min, xylene (I) for 5 min, and xylene (II) for 5 min until transparent, and then sealed with neutral gum. The sections were observed and photographed under a light microscope. The positive areas of different brain regions were quantitatively analyzed using Image Pro Plus software.

[0162] 3. APP immunohistochemical staining

[0163] Xylene (I) 10 min, xylene (II) 10 min, xylene, 100% ethanol (1:1) 2 min, 100% ethanol (I) 5 min, 100% ethanol (II) 5 min, 80% ethanol 5 min, water washing 5 min, PBS washing 3 times for 5 min each, antigen repair solution repair 15 min (microwave repair method), cooled to room temperature, 3% hydrogen peroxide blocking and incubation at room temperature for 30 min, PBS washing 2 times for 5 min each, drying and circle the tissue with an immunohistochemistry pen, 3% BSA incubation at room temperature for 30 min, add primary antibody (APP Goat Anti-Rabbit IgG H&L), incubate overnight at 4°C, rinse three times with PBS for 5 minutes each, add the secondary antibody working solution corresponding to the primary antibody, incubate at 37°C for 30 minutes, rinse three times with PBS for 5 minutes each, develop with DAB (protect from light, observe under a microscope until brown) for about 3 minutes, wash with water for 5 minutes, stain with hematoxylin solution for 5 minutes, wash with water for 5 minutes, differentiate with hydrochloric acid ethanol differentiation solution for 1 second, blue with ammonia solution for 10 seconds, wash with water for 30 seconds, 95% ethanol (Ⅰ) for 1 minute, 95% ethanol (Ⅱ) for 1 minute, 100% ethanol (Ⅰ) for 3 minutes, 100% ethanol (Ⅱ) for 3 minutes, xylene (Ⅰ) for 3 minutes, xylene (Ⅱ) for 3 minutes, and seal with neutral gum.

[0164] The cells were observed and photographed under a light microscope, and the integrated optical density (IOD) of the positive areas in different brain regions was quantitatively analyzed using Image Pro Plus software.

[0165] 4. Frozen Section Preparation and p-Tau Immunofluorescence Staining

[0166] At 4 and 6 weeks of irradiation, brains were perfused from 5xFAD mice, dehydrated with a sucrose gradient, and snap-frozen at -80°C. After embedding in OCT, 25-μm-thick frozen sections were cut using a cryostat. The frozen sections were circled with an immunohistochemistry pen and permeabilized with 0.3% Triton X-100 solution for 30 minutes at room temperature. The primary antibody (p-Tau Ser404, Thermofisher) was diluted 1:300 in the permeabilization buffer to serve as the antibody diluent and applied dropwise to the sections. The sections were incubated in a humidified chamber at 4°C overnight. Unbound antibody was removed by washing the sections with PBS four times for 5 minutes. Secondary antibody (goat anti-rabbit FTIC, Zhongshan Jinqiao) was diluted 1:100 and applied dropwise to the sections. The sections were incubated in a humidified chamber at room temperature for 1.5 hours. Unbound antibody was removed by washing the sections four times for 5 minutes. Nuclei were stained with DAPI (mounting medium, Zhongshan Jinqiao) and then mounted with coverslips. Scan the slides using a digital scanner.

[0167] 5. Results Analysis

[0168] 1) The results of Congo red staining are shown in Figures 17 to 20.

[0169] Congo red staining results showed that 5xFAD mice had scattered pink plaque deposits mainly in the hippocampus, cerebral cortex and thalamus (Figures 17 to 19). After 30 days of microwave irradiation at 900 MHz (repetition frequency 40 Hz) with an average power density of 0.8 mW / cm2 and 4 mW / cm2, the pink plaque deposits in these brain regions were reduced.

[0170] Further quantitative comparative analysis showed (Figure 20) that compared with the sham group, the 0.8 mW / cm2 group and the 4 mW / cm2 group were irradiated with 900 MHz (repetition frequency 40 Hz) microwaves for 30 days, and the pink plaque areas in the hippocampus (Figure 20-A), cerebral cortex (Figure 20-B) and thalamus (Figure 20-C) of 5xFAD mice were significantly reduced (P<0.01), indicating that Aβ deposition in these brain regions was reduced after irradiation.

[0171] 2) The results of APP immunohistochemical staining are shown in Figures 21 to 23.

[0172] APP immunohistochemical staining revealed abundant APP-positive neurons in the hippocampus, cerebral cortex, and thalamus of 5xFAD mice, with positive expression concentrated in the cytoplasm and appearing tan or brown (Figures 21-23). ​​APP-positive expression in these brain regions was reduced after 30 days of microwave irradiation at an average power density of 0.8 mW / cm² and 4 mW / cm² at 900 MHz (40 Hz repetition rate).

[0173] Further quantitative comparison results showed (Figure 24) that compared with the sham group, after 30 days of 900 MHz (repetition frequency 40 Hz) microwave irradiation at 0.8 mW / cm2 and 4 mW / cm2, the IOD values ​​of APP positive expression in the hippocampus (Figure 24-A), cortex (Figure 24-B) and thalamus (Figure 24-C) of 5xFAD mice were significantly weakened (P<0.01), indicating that the APP content in these brain regions was reduced after microwave irradiation.

[0174] 3) The results of p-Tau immunofluorescence and quantitative analysis are shown in Figure 25.

[0175] p-Tau immunofluorescence results showed that compared with the control (C57), the sham blank control group showed a significant increase in p-Tau (Ser404) expression in the cerebral cortex (Figure 25-A), hippocampus (Figure 25-B), thalamus (Figure 25-C), and internal capsule (Figure 25-D) of 5xFAD mice. Four weeks of 4 mW / cm2 microwave radiation significantly reduced p-Tau (Ser404) expression in the cerebral cortex of 5xFAD mice, and six weeks of radiation significantly reduced p-Tau expression in the cerebral cortex, thalamus, internal capsule, and hippocampus (P>0.05). This suggests that six weeks of 4 mW / cm2 microwave radiation can significantly reduce neurofibrillary tangles in the brains of 5xFAD mice.

[0176] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0177] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A method for enhancing gamma rhythm oscillation in the brain, characterized in that, The method is for non-therapeutic purposes and includes: exposing an individual to be enhanced to microwave radiation, where the repetition frequency of the microwave radiation is 20 to 100 Hz and the frequency of the microwave radiation is 800 to 2500 MHz.

2. The method according to claim 1, wherein The repetition frequency of the microwave radiation is 30 to 50 Hz.

3. The method according to claim 1, wherein The repetition frequency of the microwave radiation is 40 Hz.

4. The method according to claim 1, wherein The frequency of the microwave radiation is 900 MHz.

5. The method according to claim 1, characterized in that The average power density of the microwave radiation is 0.8 to 4 mW / cm2.

6. The method according to claim 1, characterized in that, The time of the microwave radiation is not less than 8 days.

7. A method for reducing Aβ deposition and / or neurofibrillary tangles in the brain, characterized in that, The method is for non-therapeutic purposes and includes: exposing the brain of an individual to be reduced or a sample to be processed to microwave radiation, where the repetition frequency of the microwave radiation is 20 to 100 Hz, the frequency of the microwave radiation is 800 to 2500 MHz, and the sample to be processed contains at least one of the hippocampus, cerebral cortex, or thalamus.

8. The method according to claim 7, characterized in that The repetition frequency of the microwave radiation is 30 to 50 Hz.

9. The method according to claim 7, wherein The repetition frequency of the microwave radiation is 40 Hz.

10. The method according to claim 7, characterized in that, The frequency of the microwave radiation is 900 MHz.

11. The method according to claim 7, wherein The average power density of the microwave radiation is 0.8 to 4 mW / cm2.

12. The method according to claim 7, wherein The time of the microwave radiation is not less than 30 days.

13. The method according to claim 7, wherein The time of the microwave radiation is not less than 42 days.

14. A method for preparing an isolated animal brain slice, characterized in that, Including: Processing an ex vivo brain slice of a non-human mammal with microwave radiation, where the repetition frequency of the microwave radiation is 20 to 100 Hz and the frequency of the microwave radiation is 800 to 2500 MHz.

15. The method according to claim 14, wherein The frequency of the microwave radiation is 900 MHz.

16. The method according to claim 14, characterized in that, The repetition frequency of the microwave radiation is 40 Hz.

17. The method according to claim 14, wherein The average power density of the microwave radiation is 0.1 to 4 mW / cm2.

18. The method according to claim 14, wherein The average power density of the microwave radiation is 0.4 mW / cm2.

19. The method according to claim 14, wherein The time of the microwave radiation is not less than 20 minutes.

20. Use of an in vitro brain slice of a non-human mammal prepared by the method according to claim 14, characterized in that, The ex vivo brain slice is used for gamma rhythm oscillation-related research or screening or identifying factors that can enhance gamma rhythm oscillation in the brain.

21. A method for improving learning ability, spatial memory, and episodic memory ability, characterized in that, The method is for non-therapeutic purposes and includes: exposing an individual to be enhanced to microwave radiation, where the repetition frequency of the microwave radiation is 20 to 100 Hz and the frequency of the microwave radiation is 800 to 2500 MHz.

22. The method according to claim 21, characterized in that, The repetition frequency of the microwave radiation is 30 to 50 Hz.

23. The method according to claim 21, wherein The repetition frequency of the microwave radiation is 40 Hz.

24. The method according to claim 21, wherein The frequency of the microwave radiation is 900 MHz.

25. The method according to claim 21, wherein The average power density of the microwave radiation is 0.8 to 4 mW / cm2.

26. The method according to claim 21, wherein The time of the microwave radiation is not less than 30 days.

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