Microwave exposure method for reducing excitability of glutamate neurons

Abstract

The invention relates to the technical field of biology, in particular to a microwave exposure method for reducing excitability of glutamate neurons. The special microwave exposure condition provided by the invention can significantly reduce the co-expression proportion of medial prefrontal lobe glutamate neurons and c-Fos, the calcium activity of glutamate neurons and the activity of glutamate neural circuits, so that the expression and calcium signal level of immediate early protein c-Fos of glutamate neurons are significantly reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a microwave exposure method for reducing the excitability of glutamate neurons. Background Art

[0002] Microwaves (MW) are electromagnetic waves with frequencies ranging from 300 MHz to 300 GHz and wavelengths from 1 m to 1 mm. They possess penetrating, reflective, and absorptive properties and are widely used in communications, medicine, the military, and other fields. Their biological effects have garnered considerable attention. As non-ionizing radiation, microwaves can convert electromagnetic energy into heat within the body, leading to a heating effect. They can also alter the structure and function of related macromolecules through ion resonance. While the heat generated by microwaves is a key factor affecting biological function, their non-thermal effects can also lead to alterations in DNA synthesis, protein conformation, and energy metabolism. Studies have shown that ion channel-related molecules are particularly sensitive to microwave radiation, and the brain is a sensitive target organ for microwave radiation.

[0003] Studies have found that due to the interaction between the electromagnetic field oscillation frequency and the ion resonance frequency, there is no strict linear correlation between the intensity of microwave radiation and its induced biological effects. This suggests that the effects of microwaves on neurons may be influenced by parameters such as radiation frequency, exposure duration, and exposure intensity. Currently, little research has been conducted on the effects of microwave radiation on the excitability of glutamatergic neurons. Glutamate neuronal excitotoxicity can cause a variety of neurological diseases, such as Parkinson's disease, Alzheimer's disease, epilepsy, and Huntington's disease. Therefore, there is a need to find methods to reduce the excitability of glutamatergic neurons. Microwaves, as a physical manipulation method, have been applied to ablation, anti-inflammatory effects, and improving local blood circulation through their thermal effects. However, their non-thermal effects have been less widely used in neuromodulation and the treatment of neurological diseases. Therefore, there is an urgent need to develop new microwave manipulation technologies and explore their potential applications in the treatment of various neurological diseases. Summary of the Invention

[0004] The present invention aims to address, at least to some extent, one of the technical problems in the related art. To this end, one object of the present invention is to provide microwave exposure conditions and methods for reducing the excitability of glutamate neurons. The inventors have found that this exposure method can significantly reduce the co-expression ratio of glutamate neurons and c-Fos in the medial prefrontal cortex, glutamate neuron calcium activity, and glutamate neural circuit activity.

[0005] To this end, the first aspect of the present invention provides a microwave exposure condition for reducing the excitability of glutamate neurons. According to an embodiment of the present invention, the microwave exposure condition includes:

[0006] microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, total exposure 3 times, interval 5 minutes each time.

[0007] The inventors have found, through a large number of experiments, that a microwave exposure condition can be used to reduce the excitability of glutamate neurons, i.e. microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, total exposure 3 times, interval 5 minutes each time (preferably 9.375 GHz, 30 mW / cm2, exposure 15 minutes / time, total exposure 3 times, interval 5 minutes each time), which can significantly reduce the co-expression ratio of c-Fos and glutamate neurons in the medial prefrontal cortex, glutamate neuron calcium activity, and glutamate neuron circuit activity. The present application provides a physical intervention method suitable for reducing the excitability of glutamate neurons.

[0008] According to a preferred embodiment of the present application, the microwave exposure condition is 9.375 GHz, average power density 30 mW / cm2, exposure 15 minutes / time, total exposure 3 times, interval 5 minutes each time.

[0009] The second aspect of the present application provides the use of a microwave exposure condition in reducing the excitability of glutamate neurons. According to an embodiment of the present application, the microwave exposure condition comprises:

[0010] microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, total exposure 3 times, interval 5 minutes each time.

[0011] According to a preferred embodiment of the present application, the microwave exposure condition is 9.375 GHz, average power density 30 mW / cm2, exposure 15 minutes / time, total exposure 3 times, interval 5 minutes each time.

[0012] The third aspect of the present application provides the use of a microwave exposure condition in preparing a physical treatment condition for treating or alleviating a nervous system disease. According to an embodiment of the present application, the microwave exposure condition comprises:

[0013] microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, total exposure 3 times, interval 5 minutes each time,

[0014] The nervous system disease is related to excitotoxicity of glutamate neurons.

[0015] The special microwave exposure condition provided by the present application can be used as a physical treatment condition (magnetic therapy method) for treating or alleviating nervous system diseases related to glutamate neuron excitotoxicity, such as Parkinson's disease, Alzheimer's disease, epilepsy, Huntington's disease, etc.

[0016] According to a preferred embodiment of the present application, the microwave exposure condition is 9.375 gigahertz (GHz), an average power density of 30 milliwatts per square centimeter, exposure for 15 minutes each time, a total of 3 times, and each time interval is 5 minutes.

[0017] According to an embodiment of the present application, the glutamate neurons are derived from the vHPC region mPFC neural circuit and / or the PRH region mPFC neural circuit.

[0018] The fourth aspect of the present application provides a method for reducing the excitability of glutamate neurons. According to an embodiment of the present application, the method comprises:

[0019] subjecting the glutamate neurons to microwave exposure treatment,

[0020] The microwave exposure condition comprises:

[0021] a microwave carrier frequency of 9-10 GHz, an average power density of 20-40 milliwatts per square centimeter, exposure for 15 minutes each time, a total of 3 times, and each time interval is 5 minutes.

[0022] The inventors have established a microwave exposure method for reducing the excitability of glutamate neurons. The inventors have found that under the condition of a microwave carrier frequency of 9-10 GHz, an average power density of 20-40 milliwatts per square centimeter, exposure for 15 minutes each time, a total of 3 times, and each time interval is 5 minutes, the co-expression ratio of the medial prefrontal glutamate neurons and c-Fos, the calcium activity of the glutamate neurons, and the glutamate neural circuit activity can be significantly reduced, and the expression of immediate early protein c-Fos and the calcium signal level of the glutamate neurons are significantly reduced.

[0023] According to a preferred embodiment of the present application, the microwave exposure condition is 9.375 gigahertz (GHz), an average power density of 30 milliwatts per square centimeter, exposure for 15 minutes each time, a total of 3 times, and each time interval is 5 minutes.

[0024] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description. BRIEF DESCRIPTION OF DRAWINGS

[0025] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0026] Figure 1 Dose measurement and temperature detection of 9.375 GHz microwave radiation, wherein A is a schematic diagram of the platform and the radiation source; B is the position of the mouse in the irradiation box; C is a schematic diagram of the mouse model and the microwave radiation dose distribution; D is the change of the anal temperature of the mouse in the microwave exposure;

[0027] Figure 2 Fluorescence co-expression imaging and statistical analysis of VGLUT2, VGAT and c-Fos in the mPFC brain region of mice under microwave radiation, wherein A: fluorescence images of VGLUT2, VGAT and c-Fos in the mPFC brain region, scale bar = 20 microns or 1000 microns; B: statistical analysis of the co-expression ratio of VGAT and c-Fos positive cells in the mPFC brain region after microwave radiation; C: statistical analysis of the co-expression ratio of VGLUT2 and c-Fos positive cells in the mPFC brain region after microwave radiation; * indicates p < 0.05;

[0028] Figure 3 Calcium activity changes of glutamatergic neurons in the mPFC brain region of mice after microwave exposure, wherein A: a schematic diagram of stereotactic injection of calcium imaging virus; B: imaging of the expression position of the calcium imaging virus, wherein IL and PL are mPFC subregions, scale bar = 100 microns; C: calcium activity event curve graph of glutamatergic neurons in the mPFC brain region of mice before and after 10s of cognitive task after microwave exposure; D: statistical analysis results of the average calcium activity of glutamatergic neurons in the mPFC brain region of mice before and after 10s of exploring new objects after microwave exposure, * indicates p < 0.05;

[0029] Figure 4 Effect of microwave on the activity of HPC-mPFC glutamate loop, wherein A: EGFP and c-Fos expression imaging in the HPC brain region, scale bar = 100 microns; B: statistical analysis of the co-expression of EGFP and c-Fos in the vHPC brain region after microwave radiation, ** indicates P < 0.01;

[0030] Figure 5 Effect of microwave on the activity of PRH-mPFC glutamate loop, wherein A: EGFP and c-Fos expression imaging in the PRH brain region, scale bar = 100 microns; B: statistical analysis of the co-expression of EGFP and c-Fos in the PRH brain region after microwave radiation, * indicates P < 0.05. DETAILED DESCRIPTION

[0031] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only, and are not to be understood as limiting the present application.

[0032] It should be noted that the terms "first", "second" are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or a specific order of limiting the technical features indicated. Thus, features limited by "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality" is two or more, unless otherwise specified.

[0033] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. Within each range, other values are contemplated and should be considered as if specifically written out.

[0034] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless clearly indicated otherwise, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the present application belongs.

[0035] In the present document, the terms "comprising" or "including" are open-ended, that is, they mean including, but not limited to, what is specifically recited.

[0036] In the present document, the terms "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0037] According to one specific embodiment of the present application, the present application provides a microwave exposure condition for reducing excitability of glutamate neurons, the microwave exposure condition comprising:

[0038] a microwave carrier frequency of 9-10 GHz, an average power density of 20-40 milliwatts per square centimeter, exposure for 15 minutes per time, a total of 3 times, with an interval of 5 minutes each time.

[0039] The inventors have found that the special microwave exposure condition as described above, under which the excitability of glutamate neurons can be reduced, significantly reduces the co-expression ratio of medial prefrontal glutamate neurons and c-Fos, the calcium activity of glutamate neurons, and the glutamate neuronal circuit activity.

[0040] According to one preferred embodiment of the present application, the microwave exposure condition is 9.375 gigahertz (GHz), an average power density of 30 milliwatts per square centimeter, exposure for 15 minutes per time, a total of 3 times, with an interval of 5 minutes each time.

[0041] According to a specific embodiment of the present application, the present application provides the use of microwave exposure conditions in reducing the excitability of glutamate neurons. According to an embodiment of the present application, the microwave exposure conditions comprise:

[0042] a microwave carrier frequency of 9-10 GHz, an average power density of 20-40 mW / cm2, exposure for 15 minutes each time, a total of 3 times, with 5 minutes interval between each time.

[0043] The specific microwave exposure conditions provided by the present application can be used to reduce the excitability of glutamate neurons, and in turn, can be used to treat or alleviate neurological diseases associated with glutamate neuron excitotoxicity.

[0044] According to a preferred embodiment of the present application, the microwave exposure conditions are 9.375 GHz, an average power density of 30 mW / cm2, exposure for 15 minutes each time, a total of 3 times, with 5 minutes interval between each time.

[0045] According to a specific embodiment of the present application, the present application provides the use of microwave exposure conditions in preparing a physical treatment condition for treating or alleviating neurological diseases, the microwave exposure conditions comprising:

[0046] a microwave carrier frequency of 9-10 GHz, an average power density of 20-40 mW / cm2, exposure for 15 minutes each time, a total of 3 times, with 5 minutes interval between each time,

[0047] the neurological diseases are associated with glutamate neuron excitotoxicity.

[0048] The specific microwave exposure conditions provided by the present application can be used as a physical treatment condition (magnetic therapy method) for treating or alleviating neurological diseases associated with glutamate neuron excitotoxicity, such as Parkinson's disease, Alzheimer's disease, epilepsy, Huntington's disease, etc.

[0049] "Physical treatment" includes the use of sound, light, cold, heat, electricity, force, etc. physical factors for treatment, using non-invasive, non-drug treatment to restore the original physiological function of the body. The specific microwave exposure conditions provided by the present application can be used as a physical treatment condition to reduce the excitability of glutamate neurons, and in turn, to treat or alleviate neurological diseases associated with glutamate neuron excitotoxicity.

[0050] According to a preferred embodiment of the present application, the microwave exposure conditions are 9.375 GHz, an average power density of 30 mW / cm2, exposure for 15 minutes each time, a total of 3 times, with 5 minutes interval between each time.

[0051] According to an embodiment of the present application, the glutamate neurons are derived from the vHPC region mPFC neural circuit and / or the PRH region mPFC neural circuit.

[0052] According to a specific embodiment of the present application, the present application provides a method for reducing the excitability of glutamate neurons, the method comprising:

[0053] subjecting the glutamate neurons to microwave exposure treatment,

[0054] wherein the microwave exposure conditions comprise:

[0055] a microwave carrier frequency of 9-10 GHz, an average power density of 20-40 mW / cm2, exposure for 15 minutes each time, a total of 3 times, with an interval of 5 minutes each time.

[0056] The inventors have established a microwave exposure method for reducing the excitability of glutamate neurons, and have found that under the conditions of a microwave carrier frequency of 9-10 GHz, an average power density of 20-40 mW / cm2, exposure for 15 minutes each time, a total of 3 times, with an interval of 5 minutes each time, the co-expression ratio of medial prefrontal glutamate neurons and c-Fos, the calcium activity of glutamate neurons, and the glutamate neural circuit activity can be significantly reduced, and the expression of immediate early protein c-Fos and the calcium signal level of glutamate neurons are significantly reduced.

[0057] According to a preferred embodiment of the present application, the microwave exposure conditions are 9.375 GHz, an average power density of 30 mW / cm2, exposure for 15 minutes each time, a total of 3 times, with an interval of 5 minutes each time.

[0058] The schemes of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present disclosure and should not be considered as limiting the scope of the present disclosure. If 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 the art or according to the product instructions. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0059] Example 1: Microwave exposure of mice

[0060] 8-9 week old C57BL / 6N male mice were raised and randomly divided into C and R groups. After the X-band high-power emission source was turned on and preheated for 30 minutes, the microwave carrier frequency was set to 9.375 GHz and the average power density was set to 30 mW / cm2. The R group was irradiated for 15 minutes each time, a total of 3 times, with an interval of 5 minutes each time, and the temperature rise was 0.9 degrees Celsius. The C group of mice was treated the same way, but without microwave exposure. The dose detection and temperature measurement results are shown in Figure 1 . Figure 1The results show that the temperature of the mice does not exceed 1 degree Celsius after microwave exposure, indicating that the effect of microwave exposure on mice is mainly non-thermal effect, and the thermal effect on mice can be ignored.

[0061] Example 2: 9.375 GHz / 30 mW / cm2exposure for 15 minutes 3 times, with 5 minutes interval each time, can cause reduction of glutamate neuron activity

[0062] 6 hours after microwave exposure, the mice were perfused with heart and brain, and then in situ hybridization staining of c-Fos, VGLUT2 and VGAT in the medial prefrontal cortex was performed. The specific process is as follows: fixed with 4% paraformaldehyde solution for 24 hours, then dehydrated with 10%, 20%, 30% sucrose solution gradient. Prepare 7-15 micron sections, and bake at 60°C for 30 minutes. Fixed with 4% paraformaldehyde for 15 minutes, then treated with 50%, 70%, 100%, 100% ethanol solution for 5 minutes respectively. After 10 minutes of hydrogen peroxide treatment, target repair reagent was used for 5 minutes, 100% ethanol for 3 minutes, then proteinase III for 30 minutes. Put into the probe mixture for 2 hours, Amp1 for 30 minutes, Amp2 for 30 minutes, Amp3 for 15 minutes, and HRP-C1 for 15 minutes, TSA 520 fluorescent dye diluent for 30 minutes, then HRP blocking agent for 15 minutes, HRP-C2 for 15 minutes, TSA 570 fluorescent dye diluent for 30 minutes, and HRP blocking agent for 15 minutes, then HRP-C3 for 15 minutes. After 30 minutes of TSA 650 fluorescent dye diluent, HRP blocking agent was used for 15 minutes, DAPI was used for sealing, and confocal microscope was used for observation. The brain atlas was matched with the fluorescence picture, and the c-Fos positive cells, VGLUT2 positive cells and VGAT positive cells in the medial prefrontal cortex were counted by Image J software.

[0063] As shown in Figure 2 , the results of microwave radiation on the co-expression of VGLUT2, VGAT and c-Fos in the mPFC brain region of mice are shown, wherein Fig. A is the fluorescence image of VGLUT2, VGAT and c-Fos in the mPFC brain region, and the white arrow indicates the fluorescent region; Fig. B is the statistical result of the co-expression ratio of VGAT and c-Fos positive cells in the mPFC brain region after microwave radiation; Fig. C is the statistical result of the co-expression ratio of VGLUT2 and c-Fos positive cells in the mPFC brain region after microwave radiation. The above results show that the co-expression of c-Fos and VGLUT2 positive cells in the medial prefrontal cortex after microwave exposure is significantly reduced (P<0.05), indicating that the activity of glutamate neurons is reduced.

[0064] Example 3: 9.375 GHz / 30 mW / cm2exposure for 15 minutes 3 times with 5 minutes interval can cause decrease of glutamatergic neuron calcium activity

[0065] After the mice were weighed, sodium pentobarbital was injected intraperitoneally, and the mice were fixed on the stereotaxic instrument to expose the skull. The skull level was adjusted, and the mPFC brain region coordinates were AP: 1.7 mm, ML: ±0.3 mm, and DV: -2.5 mm. After marking the target brain region position, craniotomy was performed at the target mark point using a skull drill. The microsyringe pump parameters were set as follows: volume 200 nL, and speed 40 nL / min. The injection of AAV-CaMKIIa-GCaMP6f-EGFP virus (provided by Bionice (Wuhan) Biotechnology Co., Ltd.) was started. After the virus injection was completed, the injection pump was lifted after standing for 10 minutes. The optical fiber needle holder was fixed to the brain stereotaxic injection instrument, the origin was positioned, and the optical fiber needle was lowered to about 200-300 microns above the virus injection position. The optical fiber was fixed with dental cement, and the optical fiber recording was performed after 2 weeks of virus expression.

[0066] Two weeks after the virus injection of the mice, the calcium signals of the mice from 2 seconds before the behavior experiment to 10 seconds after the behavior experiment were recorded. The 470 nm laser was focused through the objective lens to activate the GCaMP6f channel. The photomultiplier tube was used to collect the calcium signals. The calcium signals were converted into electrical signals using Matlab 2017b. The calcium signal changes were described using ΔF / F.

[0067] As shown in Figure 3 , the changes in the calcium activity of glutamatergic neurons in the mPFC brain region of mice after microwave exposure are shown. A: schematic diagram of brain stereotaxic injection of calcium imaging virus; B: imaging of the expression position of the calcium imaging virus (IL and PL are mPFC subregions); C: curve graph of the calcium activity event of glutamatergic neurons in the mPFC brain region of mice before and after 2s to 10s of cognitive tasks after microwave exposure; D: statistical analysis results of the average calcium activity of glutamatergic neurons in the mPFC brain region of mice before and after 2s to 10s of exploring new objects after microwave exposure. Figure 3 The results show that the average ΔF / F of the mPFC brain region of mice after microwave radiation significantly decreases when the mice perform cognitive activities (P<0.05).

[0068] Example 4: 9.375 GHz / 30 mW / cm2exposure for 15 minutes 3 times with 5 minutes interval can cause decrease of glutamatergic neuron calcium activity

[0069] After the mice were weighed, they were injected intraperitoneally with sodium pentobarbital, fixed on a stereotaxic instrument to expose the skull, adjusted the skull level, the coordinates of the mPFC brain region were AP: 1.7 mm, ML: ± 0.3 mm, DV: -2.5 mm, after marking the target brain region position, craniotomy was performed at the target marking point using a skull drill, the parameters of the microsyringe pump were set as volume 200 nanoliters, speed 40 nanoliters per minute, and the injection of AAV-Retro-CaMKIIa-EGFP virus (provided by Bionice (Wuhan) Biotechnology Co., Ltd.) was started; after waiting for 2 weeks for virus expression, PRH and HPCc-Fos immunofluorescence staining was performed on the upstream brain region of the mPFC glutamate nerve circuit.

[0070] The results of vHPC-mPFC and c-Fos immunofluorescence co-staining after microwave irradiation of mice are shown in Figure 4 The results of independent sample t test showed that the proportion of EGFP and c-Fos positive cell co-expressed neurons in the vHPC brain region decreased after microwave irradiation (P<0.05). The results of PRH-mPFC and c-Fos immunofluorescence co-staining after microwave irradiation of mice are shown in Figure 5 The proportion of EGFP and c-Fos positive cell co-expressed neurons in the PRH brain region decreased after microwave irradiation (P<0.05), indicating that the microwave exposure condition can cause the activity of glutamate neurons in the mPFC nerve circuit of vHPC and PRH regions to decrease.

[0071] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms is not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0072] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A microwave exposure condition for reducing excitability of glutamate neurons, characterized by, Comprising: microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, a total of 3 times, each interval 5 minutes.

2. Use of microwave exposure conditions in reducing the excitability of glutamate neurons, characterized in that, The microwave exposure conditions comprise: microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, a total of 3 times, each interval 5 minutes.

3. Use of microwave exposure conditions in the preparation of physical treatment conditions for the treatment or alleviation of nervous system disorders, characterized in that, The microwave exposure conditions comprise: microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, a total of 3 times, each interval 5 minutes, The nervous system disease is related to glutamate neuron excitotoxicity.

4. Use according to claim 3, characterized in that, The glutamate neurons are derived from the vHPC region mPFC neural circuit and / or the PRH region mPFC neural circuit.

5. A method for reducing excitability of glutamate neurons, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of claim 1. Comprising: microwave exposure treatment is performed on the glutamate neurons, wherein the microwave exposure conditions comprise: microwave carrier frequency 9-10 GHz, average power density 20-40 mW / cm2, exposure 15 minutes / time, a total of 3 times, each interval 5 minutes.

Images