Method for constructing microwave radiation animal brain injury model based on medial septal nucleus-hippocampal CA3 cholinergic nerve loop regulation
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ACADEMY OF MILITARY MEDICAL SCIENCES
- Filing Date
- 2024-06-17
- Publication Date
- 2026-07-24
Smart Images
Figure CN118661686B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of medical technology. More specifically, this invention relates to a method for constructing an animal model of microwave radiation-induced brain injury based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region. Background Technology
[0002] Epidemiological studies have shown that long-term exposure to microwave radiation can lead to headaches, insomnia, abnormal brain waves, neurasthenia, memory loss / impairment, poor concentration, and sleep disorders. Rodent experiments have shown that exposure to electromagnetic radiation under certain conditions can cause a decline in memory abilities (including motor memory, short-term memory, and long-term memory), accompanied by neurobehavioral abnormalities such as excitement and anxiety. These studies suggest that the brain is a significant and sensitive target area for microwave radiation.
[0003] The hippocampus (HPC) is involved in learning, memory, cognition, and emotion, playing a crucial role in memory orientation, conversion, and storage. It is the primary neural center in the brain responsible for learning and memory tasks. The medial septum (MS), acting as the pacemaker for the HPC's theta rhythm, sends cholinergic nerve fibers through the hippocampal umbrella, dorsal fornix, and amygdala complex to target points in the hippocampal cortex, as well as the entorhinal area, cingulate cortex, post-splenic area, and hypothalamic cortex. The MS participates in regulating emotion and cognition through cholinergic input to the HPC. Therefore, the MS-HPC cholinergic neural circuit may play a significant regulatory role in emotion and cognitive behavior.
[0004] Since the molecular mechanism of electromagnetic radiation brain injury has not been elucidated, and the types of target cells sensitive to electromagnetic radiation and their molecular targets are not clearly defined, there is currently a lack of good methods for constructing animal models of electromagnetic radiation brain injury, resulting in a lack of relevant preventive and treatment drugs. Therefore, it is urgent to design a technical solution that can overcome the above-mentioned deficiencies to a certain extent. Summary of the Invention
[0005] One objective of this invention is to provide a method for constructing an animal model of microwave radiation-induced brain injury based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region. This method can accurately construct an animal model of electromagnetic radiation-induced brain injury, increasing the possibility of elucidating the molecular mechanism of electromagnetic radiation-induced brain injury and developing drugs for its prevention and treatment.
[0006] To achieve these objectives and other advantages of the present invention, the present invention provides a method for constructing a microwave radiation animal brain injury model based on the regulation of the cholinergic neural circuit between the medial septal nucleus and the CA3 region of the hippocampus, comprising: setting electromagnetic radiation conditions and subjecting experimental animals to electromagnetic radiation treatment; comparing whether the cholinergic neural circuit between the medial septal nucleus and the CA3 region of the hippocampus is activated before and after electromagnetic radiation treatment; if activated, constructing an electromagnetic radiation animal brain injury model using the electromagnetic radiation conditions.
[0007] Furthermore, an anterograde transsynaptic neurotropic virus carrying fluorescent protein was injected into the medial septal nucleus region of the experimental animals, and a retrograde non-transsynaptic neurotropic virus carrying fluorescent protein was injected into the CA3 region of the hippocampus of the experimental animals. Based on the fluorescence signals of the medial septal nucleus region and the CA3 region of the hippocampus, the cholinergic nerve fibers projecting from the medial septal nucleus region to the CA3 region of the hippocampus and the nerve fibers retrogradely projecting from the CA3 region of the hippocampus to the medial septal nucleus region were counted. If the amount of nerve fiber projection was statistically significant before and after electromagnetic radiation treatment, the cholinergic neural circuit was considered to be activated.
[0008] Furthermore, it also includes: comparing the action potential firing and energy oscillation of neurons in the CA3 region of the hippocampus before and after electromagnetic radiation treatment; if the reduction in action potential firing and energy oscillation is statistically significant, then an electromagnetic radiation animal brain injury model is constructed using the electromagnetic radiation conditions.
[0009] Furthermore, it also includes: using an optogenetic system to inhibit cholinergic neurons in the medial septal nucleus brain region, and comparing the action potential firing and energy oscillation of neurons in the CA3 region of the hippocampus before and after electromagnetic radiation treatment. If the increase in action potential firing and energy oscillation is statistically significant, then an electromagnetic radiation animal brain injury model is constructed using the electromagnetic radiation conditions.
[0010] Furthermore, it also includes: conducting conditioned fear memory tests and fear conditioned memory retrieval tests on experimental animals, comparing the percentage of lag time in experimental animals before and after electromagnetic radiation treatment, and if the decrease in the percentage of lag time is statistically significant, then an electromagnetic radiation animal brain injury model is constructed using the electromagnetic radiation conditions.
[0011] Furthermore, it also includes: activating cholinergic neurons in the medial septal nucleus brain region using an optogenetic system, and conducting conditioned fear memory behavioral experiments on experimental animals to compare the effects of optogenetic activation and non-activation of cholinergic neurons in the medial septal nucleus brain region on the percentage of lag time in experimental animals after electromagnetic radiation treatment. If the reduction in the percentage of lag time is statistically significant, then an electromagnetic radiation animal brain injury model is constructed using the electromagnetic radiation conditions.
[0012] Furthermore, it also includes: using chemical genetics to inhibit cholinergic neurons in the medial septal nucleus brain region, and conducting conditioned fear memory behavioral experiments on experimental animals. If the inhibition of cholinergic neurons in the medial septal nucleus brain region has a statistically significant effect on the increase in the percentage of lag time in experimental animals before and after electromagnetic radiation, then an electromagnetic radiation animal brain injury model is constructed using the electromagnetic radiation conditions.
[0013] Furthermore, the electromagnetic radiation conditions include at least the center frequency, power density, repetition frequency, pulse width, specific absorptivity, and radiation time.
[0014] The present invention has at least the following beneficial effects: This invention involves subjecting experimental animals to electromagnetic radiation and comparing the activation of the cholinergic neural circuit between the medial septal nucleus and the CA3 region of the hippocampus before and after electromagnetic radiation treatment. If activation is detected, an animal model of electromagnetic radiation-induced brain injury is constructed using electromagnetic radiation conditions. This invention can help elucidate the molecular mechanism of electromagnetic radiation-induced brain injury and provides new ideas for exploring biological targets of microwave radiation-induced brain injury and preventing and treating brain injury.
[0015] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description
[0016] Figure 1 This is an embodiment of the experimental protocol for testing conditioned fear memory in mice according to this application.
[0017] Figure 2 This is an embodiment of the present application of an experimental protocol for optogenetic activation of cholinergic neurons in the MS region of mice to induce conditioned fear behavior.
[0018] Figure 3 This is an embodiment of the experimental protocol for chemically genetically suppressing cholinergic neurons in the MS region of mice in this application, which is used to study conditioned fear behavior.
[0019] Figure 4 This application illustrates the effect of microwave radiation on conditioned fear memory in mice (N=10). AB. Changes in the percentage of freezing time during the conditioned fear learning and memory test, conditioned fear memory expiration, and conditioned fear memory recall phases in mice after microwave radiation; Freezing % = Freezing time during CS duration / CS duration × 100%; Con: blank control group, MR: microwave radiation group, N=10, independent samples t Test; compared with the blank control group, * indicates P <0.05, **indicates P <0.01.
[0020] Figure 5 This is a schematic diagram illustrating the confirmation of the AAV stereotactic injection target site in the MS region of a mouse according to an embodiment of this application.
[0021] Figure 6 This application describes the effect of microwave radiation on the projection of cholinergic nerve fibers in mouse MS-HPC (AAV anterograde tracing) in one embodiment of the present application. A. Schematic diagram of the viral injection target site; B. Schematic diagram of fluorescent protein expression verification at the injection target site; C. Schematic diagram of fluorescent protein expression in the HPC region downstream of MS cholinergic nerve fiber projection; D. Schematic diagram of fluorescent protein expression in the HPC region and its CA3 subregion in the blank control group (Con); E. Schematic diagram of fluorescent protein expression in the HPC region and its CA3 subregion in the microwave irradiation group (MR); FG. Quantitative analysis of fluorescent protein expression in cholinergic nerve fibers projected from MS to the HPC CA3 region 7 days after microwave irradiation; N=3, independent samples t Test; compared with the blank control group, * indicates P <0.05, **indicates P <0.01.
[0022] Figure 7 This application describes the effect of microwave radiation on fiber projection of mouse MS-HPC cholinergic neurons (AAV retrograde tracing) in one embodiment of the present application. A. Schematic diagram of the viral injection target site; B. Quantitative analysis of fluorescent protein expression in cholinergic nerve fibers retrogradely projected from HPC CA3 to the MS region 7 days after microwave irradiation; C. Schematic diagram of fluorescent protein expression verification at the injection target site; D. Schematic diagram of fluorescent protein expression in the MS region of the blank control group (Con); E. Schematic diagram of fluorescent protein expression in the MS region of the microwave irradiation group (MR); N=3, independent samples t Test; compared with the blank control group, * indicates P <0.05, **indicates P <0.01.
[0023] Figure 8 This application provides an embodiment of the effect of MS cholinergic neuron inhibition on neuronal firing activity in the CA3 region of mouse HPC after microwave irradiation; A. Schematic diagram of mouse head microfilament electrode and ceramic fiber ferrule implantation surgery, recovery, and testing; B. Changes in action potential firing rate of neurons in the HPC CA3 region of mice before and after microwave irradiation; C. Changes in the Theta oscillation power spectral density of the HPC CA3 region of mice at frequencies of 4–12 Hz before and after microwave irradiation; D. Changes in Theta energy of the HPC CA3 region of mice at frequencies of 4–12 Hz before and after microwave irradiation; E. Schematic diagram of the time spectrum (thermograph) of the HPC CA3 region of mice before and after microwave irradiation; F. Effect of optogenetic inhibition of MS cholinergic neurons on changes in action potential firing rate of neurons in the HPC CA3 region of mice before and after microwave irradiation; G. Effect of optogenetic inhibition of MS cholinergic neurons on changes in Theta energy of the HPC CA3 region of mice before and after microwave irradiation; H. Effect of optogenetic inhibition of MS cholinergic neurons on changes in Theta oscillation power spectral density of the HPC CA3 region of mice 7 days after microwave irradiation. Pre: Before microwave radiation; Post-6h: 6 hours after microwave radiation; Post-3d: 3 days after microwave radiation; Post-7d: 7 days after microwave radiation; OFF: Photoinactive; ON: Photoactivated; N=8, paired samples. t Test; compared with before microwave radiation, *shown P <0.05, **indicates P <0.01.
[0024] Figure 9 This application describes an embodiment of the regulatory effect of MS cholinergic neuron activation on microwave radiation-induced changes in conditioned fear memory in mice. AB. Changes in the percentage of lag time during the learning and memory phase of conditioned fear in mice; CD. Changes in the percentage of lag time during the expiration phase of conditioned fear memory in mice; EF. Changes in the percentage of lag time during the recall phase of conditioned fear memory in mice; Con-OFF: blank control - non-activation group, Con-ON: blank control - activation group, MR-OFF: microwave radiation - non-activation group, MR-ON: microwave radiation - activation group; N=10, two-way ANOVA; * indicates P <0.05, **indicates P <0.01.
[0025] Figure 10 This application describes an embodiment of the regulatory effect of MS cholinergic neuron inhibition on microwave radiation-induced changes in conditioned fear memory in mice. AB. Changes in the percentage of lag time during the learning and memory phase of conditioned fear in mice; CD. Changes in the percentage of lag time during the expiration phase of conditioned fear memory in mice; EF. Changes in the percentage of lag time during the recall phase of conditioned fear memory in mice; Con-NS: blank control-saline group, Con-CNO: blank control-CNO group, MR-NS: microwave radiation-saline group, MR-CNO: microwave radiation-CNO group; N=10, two-way ANOVA; * indicates P<0.05, ** indicates P<0.01. Detailed Implementation
[0026] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.
[0027] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.
[0028] The embodiments of this application provide a method for constructing a microwave radiation-induced animal brain injury model based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region, including: S1: Set electromagnetic radiation conditions and subject experimental animals to electromagnetic radiation treatment; For example, electromagnetic radiation conditions are set using factors including frequency, power, and time, and experimental animals are subjected to electromagnetic radiation. For example, the test animals include various types of rats and mice; S2: Compare whether the cholinergic neural circuit between the medial septal nucleus and the CA3 region of the hippocampus is activated before and after electromagnetic radiation treatment. If activated, construct an electromagnetic radiation animal brain injury model using the electromagnetic radiation conditions. For example, neurotropic virus tracing was used to determine whether the cholinergic neural circuit between the medial septal nucleus and the CA3 region of the hippocampus was activated. This embodiment was able to construct an animal brain injury model by characterizing and confirming the neural circuit, thereby helping to elucidate the molecular mechanism of electromagnetic radiation-induced brain injury and providing new ideas for exploring biological targets of microwave radiation-induced brain injury and preventing and treating brain injury.
[0029] In another embodiment, an anterograde transsynaptotropic virus carrying fluorescent protein is injected into the medial septal nucleus region of the experimental animal, and a retrograde non-transsynaptotropic virus carrying fluorescent protein is injected into the CA3 region of the hippocampus of the experimental animal. The cholinergic nerve fibers projecting from the medial septal nucleus region to the CA3 region of the hippocampus and the nerve fibers retrogradely projecting from the CA3 region of the hippocampus to the medial septal nucleus region are counted based on the fluorescence signals of the medial septal nucleus region and the CA3 region of the hippocampus. If the amount of nerve fiber projection is statistically significant before and after electromagnetic radiation treatment, the cholinergic neural circuit is considered to be activated.
[0030] For example, anterograde transsynaptic neurotropic virus (pAAV-EF1a-DIO-EGFP-WPRE) was injected into the MS brain region, and retrograde non-transsynaptic neurotropic virus (pAAV-EF1a-DIO-EGFP-WPRE) was injected into the HPC CA3 region.
[0031] For example, after the virus is stably expressed and the injection site is confirmed, the animal is subjected to microwave radiation, anesthesia, euthanasia and brain tissue is dissected. After the brain tissue is embedded by OCT, frozen sections with a thickness of 20 μm are prepared, and anti-fluorescence quenching mounting medium containing DAPI is added. The expression of green fluorescent protein EGFP in the MS and HPC regions is observed by confocal fluorescence microscopy to record cholinergic nerve fibers projected to the HPC CA3 region.
[0032] In another embodiment, the method further includes: comparing the action potential firing and energy oscillation of neurons in the CA3 region of the hippocampus before and after electromagnetic radiation treatment; if the reduction in action potential firing and energy oscillation is statistically significant, then an electromagnetic radiation animal brain injury model is constructed using the electromagnetic radiation conditions.
[0033] For example, using Chat + / + Using iCre gene-edited mice as the research subject, a yellow optogenetic system combined with an in vivo multichannel electrophysiological recording system was used to study the effect of MS cholinergic neuron activity inhibition on microwave-induced changes in the firing activity of the HPC CA3 region in mice.
[0034] In another embodiment, the method further includes: using an optogenetic system to inhibit cholinergic neurons in the medial septal nucleus brain region, and comparing the action potential firing and energy oscillation of neurons in the CA3 region of the hippocampus before and after electromagnetic radiation treatment. If the increase in action potential firing and energy oscillation is statistically significant, then an electromagnetic radiation animal brain injury model is constructed using the electromagnetic radiation conditions.
[0035] For example, 200 nL of optogenetic repressive adeno-associated virus (rAAV-hSyn-DIO-eNpHR3.0-P2A-EGFP) containing green fluorescent protein (EGFP) was injected into the MS region of experimental animals via stereotactic injection into the brain; simultaneously, channel microwire electrodes were implanted in the HPC CA3 region; the wires of the microwire electrodes were wound clockwise around the skull nail 4-6 times and sealed with dental cement; before and after microwave radiation, the activity of MS cholinergic neurons was inhibited using a yellow optogenetic system, and the firing activity of neurons in the mouse HPC CA3 region was recorded using an in vivo multichannel electrophysiological system.
[0036] In another embodiment, the method further includes: conducting conditioned fear memory tests and fear conditioned memory retrieval tests on experimental animals, comparing the percentage of lag time in experimental animals before and after electromagnetic radiation treatment, and if the decrease in the percentage of lag time is statistically significant, then constructing an electromagnetic radiation animal brain injury model using the electromagnetic radiation conditions.
[0037] Cholinergic neurons in the medial septal nucleus brain region were activated using an optogenetic system, and conditioned fear memory behavioral experiments were conducted on experimental animals. The effects of activation and inactivation of cholinergic neurons in the medial septal nucleus brain region on the percentage of lag time in experimental animals after electromagnetic radiation treatment were compared. If the reduction in the percentage of lag time was statistically significant, an electromagnetic radiation animal brain injury model was constructed using the electromagnetic radiation conditions.
[0038] Chemogenetic techniques were used to inhibit cholinergic neurons in the medial septal nucleus brain region, and conditioned fear memory behavioral experiments were conducted on experimental animals. If the inhibition of cholinergic neurons in the medial septal nucleus brain region had a statistically significant effect on the increase in the percentage of lag time in experimental animals before and after electromagnetic radiation, then an electromagnetic radiation animal brain injury model was constructed using the electromagnetic radiation conditions.
[0039] For example, an experiment was conducted using a conditioned fear testing system. After microwave radiation, a conditioned fear learning experiment, a conditioned fear memory test, a conditioned fear memory extinction experiment, a conditioned fear memory extinction test, and a conditioned fear memory recall test were performed sequentially. The percentage of the animals' lapse time in each experimental stage was recorded to assess their conditioned fear memory ability.
[0040] For example, optogenetic technology was used to suppress cholinergic neurons in the medial septal nucleus brain region via adeno-associated virus.
[0041] For example, chemogenetic techniques were used to suppress cholinergic neurons in the medial septal nucleus brain region via adeno-associated virus.
[0042] In another embodiment, the electromagnetic radiation conditions include at least the center frequency, power density, repetition frequency, pulse width, specific absorptivity, and radiation time.
[0043] The following explanation uses mice as experimental animals.
[0044] 1. Materials and Methods 1.1 Establishment of an animal model of microwave radiation Ten-week-old C57 BL / 6 N mice, weighing 24±2g, with an equal number of males and females, were randomly divided into a microwave radiation (MR) group and a blank control (Con) group. Conditioned fear behavior tests were performed using wild-type mice, with 10 mice in each group; in vivo multichannel electrophysiological recordings were performed using Chat. + / +-iCre gene-edited mice, 8 mice per group; optogenetic regulation combined with behavioral testing was performed using Chat. + / + -iCre:Rosa26 + / + ChR2 gene-edited mice, 10 mice per group; neurotropic virus tracing was performed using Chat. + / + -iCre gene-edited mice, 3 mice per group; chemical genetic regulation combined with behavioral testing was performed using Chat. + / + -iCre gene-edited mice, 10 mice per group.
[0045] Using a high-power microwave radiation simulation source built by the Academy of Military Medical Sciences, with a center frequency of 2.856 GHz and an average power density of 8 mW / cm², the experiment was conducted. 2 Peak power density is 200 W / cm³ 2 Mice were subjected to a single, uniform whole-body irradiation for 15 min using microwaves with a repetition frequency of 80 Hz, a pulse width of 500 ns, and a specific absorptivity (SAR) of 6.12 W / kg. The mice were placed in a transparent, perforated circular plexiglass box, which was placed on a rotatable radiation table in a microwave darkroom. The microwave source provided uniform irradiation from top to bottom. The blank control group received sham irradiation under the same conditions.
[0046] 1.2 Test of Conditioned Fear Memory Ability in Mice The experiment used a conditioned fear testing system (Med Associates, USA) and its accompanying Video Freeze 3.00.01 software. Conditioned fear learning was conducted 6 hours after microwave radiation, conditioned fear memory was tested 8 hours after radiation, conditioned fear memory extinction was conducted 30 hours after radiation, conditioned fear memory recall was tested 32 hours after radiation, and conditioned fear memory recall was tested 3 and 7 days after radiation. The percentage of time frozen in time at each experimental stage was recorded to assess the animals' conditioned fear memory ability. The experimental procedure is as follows: Figure 1 The specific experimental steps are as follows: (1) Fear learning experiment After placing the mice in the conditioned fear chamber for 2 minutes, they were given four sound (CS, 2 kHz, 80 dB) adaptations, 10 seconds each, with an interval (ITI) of 60 seconds. Then, they were given five sound stimuli (CS, 2 kHz, 80 dB) combined with plantar electric shocks, 10 seconds each, and plantar electric shocks (US, 0.5 mA, 1 s) were given at the 9th second, with an ITI of 60 seconds. Two minutes after the last stimulation, the mice were removed from the conditioned fear chamber, the chamber was wiped with an appropriate amount of 75% alcohol, and the next mouse was replaced.
[0047] (2) Conditioned fear memory test The conditioned fear memory test was performed 2 hours after conditioned fear learning. After acclimatizing in the conditioned fear chamber for 2 minutes, mice were given three auditory stimuli (CS, 2 kHz, 80 dB) at 10 s each, with an ITI of 60 s. The mice were removed 2 minutes after the CS stimulation ended.
[0048] (3) Fear extinction experiment The conditioned fear memory extinction experiment was conducted 24 h after the conditioned fear memory test. Mice were placed in a conditioned fear chamber for 2 min to adapt, and then given 10 sound stimuli (CS, 2 kHz, 80 dB) for 30 s each time, with an ITI of 60 s. The mice were removed 2 min after the sound stimuli ended.
[0049] (4) Fear extinction test The conditioned fear memory extinction test was conducted 2 hours after the conditioned fear memory extinction experiment. The testing method was the same as the conditioned fear memory test, that is, three auditory stimuli (CS, 2 kHz, 80 dB, 10 s / stimuli) were given, with an ITI of 60 s.
[0050] (5) Renewal test for conditioned fear memories The conditioned fear memory recall test was conducted 24 hours after the conditioned fear memory extinction test. The testing method was the same as the conditioned fear memory test, that is, three auditory stimuli (CS, 2 kHz, 80 dB, 10 s / test) were given, with an ITI of 60 s.
[0051] 1.3 Stereoscopic injection into the brain and neurotropic virus tracing Chat using specifically labeled cholinergic neurons expressing the red fluorescent protein tdTomato + / + -iCre mice were used as experimental subjects. Mice were anesthetized with isoflurane inhalation. Adeno-associated virus (AAV) was quantitatively injected into the HPC CA3 region (coordinates AP: -2.30 mm, ML: +2.60 mm, DV: -2.60 mm) and MS region (coordinates AP: +0.96 mm, ML: +0.00 mm, DV: -4.00 mm) using a stereotaxic system and a microinfusion pump. The injection volume was 80 nmol. The needle was left in place for 15 minutes after AAV injection, followed by implantation of a fiber optic ceramic ferrule and sealing with dental cement. Four weeks post-surgery, after the virus expression was stable, the mice underwent cardiac perfusion fixation and frozen sectioning of brain tissue. Fluorescence microscopy was used to confirm the accuracy of the injection site.
[0052] The effects of microwave radiation on cholinergic nerve fiber projection in MS-HPC were investigated using neurotropic viral tracing. Anterograde transsynaptic neurotropic virus (pAAV-EF1a-DIO-EGFP-WPRE, serotype AAV2 / 1, titer 1.60E+13 v.g. / mL) was injected into the MS brain region, while retrograde non-transsynaptic neurotropic virus (pAAV-EF1a-DIO-EGFP-WPRE, serotype AAV2 / Retro, titer 1.53E+13 v.g. / mL) was injected into the HPC CA3 region. After stable viral expression and confirmation of the injection sites, the animals were subjected to microwave radiation. Seven days post-radiation, mice were anesthetized with 1% sodium pentobarbital (50 mg / kg), euthanized, and their brain tissue was dissected. Brain tissue was embedded by OCT, and frozen sections with a thickness of 20 μm were prepared. A DAPI-containing anti-fluorescence quenching mounting medium was added, and the expression of green fluorescent protein (EGFP) in the MS and HPC regions was observed using a confocal fluorescence microscope (Dragonfly 200). Fluorescence images of the MS and HPC regions were acquired, and quantitative image analysis was performed using the Colocalization plugin in ImageJ.
[0053] 1.4 Optogenetic inhibition of MS cholinergic neuronal binding and in vivo electrophysiological recording of HPC CA3 neuronal firing activity Chat + / + Using iCre gene-edited mice as the research subject, a yellow (excitation wavelength 594nm) optogenetic system (Qianao Xingke Nanjing Biotechnology Co., Ltd., China) combined with an in vivo multichannel electrophysiological recording system (Blackrock Microsystems, USA) was used to study the effect of MS cholinergic neuron activity inhibition on microwave-induced changes in HPC CA3 region discharge activity in mice.
[0054] 200 nL of optogeneticly inhibited adeno-associated virus (rAAV-hSyn-DIO-eNpHR3.0-P2A-EGFP, serum type AAV / 9, titer 5.09 E+12 vg / mL) containing green fluorescent protein (EGFP) was injected into the MS region of mice via stereotactic injection. Simultaneously, an 8-channel microfilament electrode was implanted in the HPCCA3 region. The microfilament electrode wire was wound clockwise around a cranial screw 4–6 times and sealed with dental cement. Four weeks after mouse treatment, MS cholinergic neuronal activity was inhibited using a yellow optogenetic system (frequency 10 Hz, power 5 mW) at 2 days before microwave irradiation, 6 hours after irradiation, 3 days after irradiation, and 7 days after irradiation. The firing activity of neurons in the HPCCA3 region of mice was recorded using an in vivo multichannel electrophysiological system. Electrophysiological data were analyzed using NeuroExplorer 5 software.
[0055] 1.5 Optogenetic activation of MS cholinergic neurons and the detection of neurobehavioral changes in mice using a conditioned fear memory behavioral paradigm. Chat + / + -iCre:Rosa26 + / + Using ChR2 gene-edited mice as the research subject, these mice were specifically labeled with cholinergic neurons and expressed red fluorescent protein tdTomato, while simultaneously expressing the light-sensitive channel protein ChR2 labeled with green fluorescent protein EGFP. Using a blue (excitation wavelength 473nm) optogenetic system (Qianao Xingke Nanjing Biotechnology Co., Ltd., China), combined with the conditioned fear memory behavioral paradigm, the effects of MS cholinergic neuron activation on microwave radiation-induced changes in conditioned fear memory in mice were studied.
[0056] Ceramic inserts were implanted into the MS region of mice using stereotactic injection technique and sealed with dental cement. After 4 weeks of care, the mice underwent microwave irradiation, followed by placement in a conditioned fear chamber for sequential conditioned fear learning, conditioned fear memory testing, conditioned fear memory extinction, and conditioned fear memory recall testing. During the conditioned fear learning experiment, blue light was applied to activate MS cholinergic neurons, with the light and sound stimulation times being identical. The remaining experimental methods were the same as described in section 1.2. For detailed experimental procedures, please refer to [link to experimental procedure]. Figure 2 The percentage of lag time in mice at each experimental stage was recorded to assess the effect of MS cholinergic neuron activation on conditioned fear learning and memory ability in mice after microwave radiation. The optogenetic blue light activation parameters were as follows: stimulation frequency 10 Hz, power 5 mW, pulse duration 10 ms, duty cycle 10%, pulse train duration 1.5 s, interval 1 s, total duration 10 s.
[0057] 1.6 Chemical genetic inhibition of MS cholinergic neurons binding to conditioned fear memory behavioral paradigm to detect neurobehavioral changes in mice Chat + / +Using iCre gene-edited mice as the research subjects, 200 nL of adeno-associated virus (rAAV-EF1a-DIO-hM4D(Gi)-EGFP, serotype AAV / 9, titer 3.20 E+12 vg / mL) containing green fluorescent protein (EGFP) was injected into the MS region of the mice via stereotactic injection. Four weeks post-surgery, mice underwent microwave irradiation, and 30 minutes before conditioned fear learning, they received an intraperitoneal injection of clozapine nitric oxide (CNO) at a dose of 1 mg / kg, with an injection volume of 0.01 mL / g, selectively inhibiting cholinergic neurons in the MS region. Thirty minutes after CNO injection, the mice were placed in a conditioned fear chamber, and conditioned fear learning, conditioned fear memory testing, conditioned fear memory extinction, and conditioned fear memory recall tests were performed sequentially, following the same experimental methods as described in section 1.2. For detailed experimental procedures, please refer to [link to experimental procedure]. Figure 3 The percentage of lapse time in mice at each experimental stage was recorded to assess the effect of MS cholinergic neuron inhibition on conditioned fear memory in mice after microwave radiation.
[0058] 1.7 Statistical Analysis Experimental data are expressed as mean ± standard deviation and were analyzed using SPSS 25.0 statistical software. t Tests, independent samples t Tests or two-way ANOVA, to P <0.05 indicates statistical significance.
[0059] 2. Experimental Results 2.1 Effects of microwave radiation on conditioned fear memory in mice The results of the conditioned fear memory test showed that, compared with the control group, the percentage of lag time in the conditioned fear memory test was significantly reduced in the radiation group mice 6 hours after microwave radiation. P <0.01); 3 days after microwave radiation, the percentage of lag time in the conditioned fear memory retrieval test of the radiation group mice was significantly reduced ( P <0.05), results are shown in Figure 4 .
[0060] The above results indicate that microwave radiation can lead to a decrease in the ability of mice to learn and remember conditioned fear and to reproduce conditioned fear memories.
[0061] 2.2 Confirmation of AAV injection target site in mouse MS region Four weeks after stereotactic injection of AAV into the MS brain region of C57BL / 6N mice, the mice were fixed by cardiac perfusion, and frozen sections of brain tissue were harvested for observation of green fluorescent protein (EGFP) expression using fluorescence microscopy. The results showed that, compared with mouse brain atlases, the green fluorescent signal was specifically expressed in the MS brain region, and the fluorescence signal was strong, indicating accurate localization of the virus injection site to the MS brain region. (See attached figures). Figure 5 .
[0062] 2.3 Effects of microwave radiation on cholinergic nerve fiber projection in mouse MS-HPC The results of anterotropic neurotropic virus tracing showed that green fluorescent protein (EGFP) expression was observed in the MS region and all subregions of the HPC in mice, and the green fluorescence was similar to that of Chat. + / + -iCre mice exhibited colocalization of the spontaneous red fluorescent protein tdTomato, with a stronger green fluorescent signal in the HPCCA3 region; this indicates that after anterograde tracking virus labeling of cholinergic neurons injected into the MS brain region, cholinergic nerve fibers from the MS region projected to the bilateral HPC regions, with more projections from HPCCA3. Figure 6 AE). For cholinergic nerve fibers projecting to the HPC CA3 region, quantitative image analysis was performed based on their fluorescent protein expression, and their Pearson correlation coefficient was calculated. The results showed that ( Figure 6 FG), 7 days after microwave irradiation, the number of cholinergic nerve fibers projecting from the MS region to the HPC CA3 region in the irradiated group mice was significantly higher than that in the blank control group. P <0.01), and no statistically significant difference was observed in the projection of cholinergic nerve fibers from the MS region to the left and right sides of HPC CA3. P＞ 0.05).
[0063] The above results indicate that microwave radiation can increase the projection of cholinergic nerve fibers from the MS to the CA3 region of the HPC, suggesting that the MS-HPC cholinergic nerve circuit is activated after microwave radiation.
[0064] Retrograde neurotropic virus (AAV) tracing results showed that green fluorescent protein (EGFP) was specifically expressed in the CA3 region of the hippocampus, with a strong fluorescence signal, indicating the precision of the AAV injection site. Simultaneously, some cholinergic neurons in the MS region expressed EGFP, further confirming that some cholinergic nerve fibers in the MS region could project to the HPC CA3 region. Quantitative analysis of cholinergic neurons expressing EGFP in the MS region and calculation of their Pearson correlation coefficients revealed that 7 days after microwave irradiation, the number of cholinergic nerve fibers retrogradely projecting from the HPC CA3 region to the MS region in the irradiated group mice was significantly higher than that in the blank control group. P<0.01), which further indicates that microwave radiation leads to increased cholinergic nerve fiber projection in mouse MS-HPC, suggesting activation of this neural circuit after radiation.
[0065] 2.4 Inhibition of MS cholinergic neuronal activity and increased neuronal firing activity in the CA3 region of mouse HPC after microwave irradiation In vivo multichannel electrophysiological recording analysis results ( Figure 8 AE) showed that at 6 h, 3 d, and 7 d after microwave irradiation, the firing rate of action potentials in neurons in the HPCCA3 region of mice was significantly lower than before irradiation. P <0.01, Figure 8 B). The Theta band with frequencies of 4–12 Hz was selected for field potential filtering, and its energy change (Theta Power) was quantitatively analyzed. The results showed that the Theta Power of the mouse HPC CA3 region was significantly lower at 3 and 7 days after microwave radiation compared with that before radiation. P <0.01, Figure 8 (CE). The above results indicate that microwave radiation reduces the firing of action potentials and decreases Theta oscillations in neurons of the HPC CA3 region in mice.
[0066] Optogenetic inhibition of MS cholinergic neurons binding in vivo multichannel electrophysiological recording results ( Figure 8 FH showed that 6 h and 7 d after microwave radiation, selective inhibition of MS cholinergic neuron activity via the yellow optogenetic system led to a significant increase in the firing rate of action potentials in HPC CA3 neurons. P <0.01, Figure 8 F). Simultaneously, 7 days after microwave irradiation, optogenetic inhibition of MS cholinergic neuron activity significantly increased Theta Power in HPC CA3 region neurons (F). P <0.01, Figure 8 GH).
[0067] The above results indicate that microwave radiation reduced the firing of action potentials and Theta oscillations in the HPC CA3 region of mice. Optogenetic inhibition of MS cholinergic neurons significantly increased the firing activity of neurons in the HPC CA3 region of mice after microwave radiation, suggesting that MS cholinergic neurons have an important negative regulatory effect on the firing activity of neurons in the HPC CA3 region after microwave radiation.
[0068] 2.5 Optogenetic activation of MS cholinergic neurons exacerbates the decline in conditioned fear memory in mice after microwave radiation. Results of an experiment on optogenetic activation of MS cholinergic neurons combined with conditioned fear memory ( Figure 9(AF) showed that, during the conditioned fear learning phase in mice, selective activation of MS cholinergic neurons resulted in a significantly lower percentage of lapse time in the blank control-activated group (Con-ON) compared to the blank control-unactivated group (Con-OFF) during the conditioned fear memory test. P <0.01, Figure 9 (AB) indicates that activation of MS cholinergic neurons can reduce conditioned fear memory in mice. Simultaneously, the percentage of lapse time in the microwave radiation-activated group (MR-ON) was significantly lower than that in the microwave radiation-unactivated group (MR-OFF). P <0.05, Figure 9 B). Furthermore, the percentage of lag time in the MR-OFF group was significantly lower than that in the Con-OFF group ( P <0.05, Figure 9 B and E), and no statistically significant differences were observed in the others. P >0.05, Figure 9 CF).
[0069] The above results indicate that optogenetic activation of MS cholinergic neurons can further reduce the ability of mice to learn and remember conditioned fear after microwave radiation, suggesting that MS cholinergic neurons have a negative regulatory effect on the decline in conditioned fear memory caused by microwave radiation.
[0070] 2.6 Chemogenetic inhibition of MS cholinergic neurons improves conditioned fear memory in mice after microwave radiation exposure. Results of a chemogenetic study on inhibiting MS cholinergic neurons binding to conditioned fear memory ( Figure 10 AF) showed that intraperitoneal injection of CNO into mice prior to conditioned fear learning selectively inhibited MS cholinergic neurons, resulting in decreased conditioned fear memory performance during tests. Figure 10 In the microwave-CNO group (MR-CNO), the percentage of retardation time was significantly higher than that in the microwave-saline group (MR-NS). P <0.01, Figure 10 B), and the percentage of lag time in mice in the blank control-CNO group (Con-CNO) was significantly higher than that in the blank control-saline group (Con-NS). P <0.05, Figure 10 B); During the conditioned fear memory extinction test ( Figure 10 CD), the percentage of stasis time in the MR-CNO group was significantly higher than that in the Con-CNO group ( P <0.05, Figure 10 D); in the stage of conditioned fear memory re-enactment ( Figure 10 EF), the percentage of lag time in mice in the MR-CNO group was significantly higher than that in the MR-NS group ( P<0.05, Figure 10 E). Furthermore, the percentage of lag time was significantly lower in the MR-NS group compared to the Con-NS group ( P <0.01, Figure 10 B, E). No statistically significant differences were observed in the remaining cases. P >0.05).
[0071] The above results indicate that chemogenetic inhibition of MS cholinergic neuron activity can significantly improve microwave radiation-induced decline in conditioned fear learning and memory, as well as the ability to reproduce fear memories in mice. This further suggests that MS cholinergic neurons have a negative regulatory effect on the decline in microwave radiation-induced conditioned fear memory in mice.
[0072] 3. Conclusion This embodiment utilizes 2.856 GHz and 8 mW / cm². 2 Microwave radiation to mice with a SAR value of 6.12 W / kg for 15 min resulted in decreased conditioned fear learning and memory, and reduced fear memory retrieval ability; increased MS-HPC cholinergic neuron projection; decreased action potential firing rate and Theta oscillation in the HPC CA3 region; and significantly increased HPC CA3 neuron firing activity after microwave radiation by inhibiting MS cholinergic neuron activity. Activation of MS cholinergic neurons exacerbated the decline in conditioned fear memory after microwave radiation, while inhibition of MS cholinergic neurons improved the decline in conditioned fear memory after microwave radiation. This suggests that microwave radiation can activate the MS-HPC cholinergic neural circuit, leading to increased inhibition of hippocampal CA3 neurons by MS cholinergic neurons, which in turn weakens hippocampal CA3 neuron firing activity and Theta rhythm, ultimately resulting in decreased conditioned fear memory. The bidirectional regulatory effect of MS cholinergic neurons on changes in conditioned fear memory after microwave radiation suggests that the MS-HPC cholinergic neural circuit plays an important negative regulatory role in microwave radiation-induced changes in conditioned fear memory. Therefore, in this embodiment, after electromagnetic radiation was applied to mice, the electromagnetic radiation parameters were characterized and confirmed by the MS-HPC cholinergic neural circuit, combined with optogenetics, chemogenetic regulation, and neurobehavioral studies, ensuring the accurate construction of the animal brain injury model.
[0073] The number of devices and processing scale described herein are for simplification of the invention. Applications, modifications, and variations of the method for constructing a microwave radiation-induced animal brain injury model based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region according to this invention will be readily apparent to those skilled in the art.
[0074] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.
Claims
1. A method for constructing a microwave radiation-induced animal brain injury model based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region, characterized in that, include: Electromagnetic radiation conditions were set up to subject experimental animals to electromagnetic radiation treatment; the microwave radiation conditions were: a center frequency of 2.856 GHz and an average power density of 8 mW / cm². 2 Peak power density 200 W / cm³ 2 Mice were subjected to a single, uniform whole-body irradiation for 15 minutes using microwaves with a repetition frequency of 80 Hz, a pulse width of 500 ns, and a specific absorption rate of 6.12 W / kg. Compare whether the cholinergic neural circuit between the medial septal nucleus and the CA3 region of the hippocampus is activated before and after electromagnetic radiation treatment. If activated, construct an electromagnetic radiation animal brain injury model using the electromagnetic radiation conditions. Anterior transsynaptic neurotropic virus with fluorescent protein was injected into the medial septal nucleus brain region of experimental animals, and retrograde non-transsynaptic neurotropic virus with fluorescent protein was injected into the CA3 region of the hippocampus of experimental animals. Based on the fluorescence signals of the medial septal nucleus and the hippocampal CA3 region, the cholinergic nerve fibers projecting from the medial septal nucleus to the hippocampal CA3 region and the nerve fibers projecting retrogradely from the hippocampal CA3 region to the medial septal nucleus are statistically significant. If the amount of nerve fiber projection is statistically significant before and after electromagnetic radiation treatment, the cholinergic neural circuit is considered to be activated.
2. The method for constructing a microwave radiation-induced animal brain injury model based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region as described in claim 1, characterized in that, Also includes: Compare the action potential firing and energy oscillation of neurons in the CA3 region of the hippocampus before and after electromagnetic radiation treatment. If the reduction in action potential firing and energy oscillation is statistically significant, then an electromagnetic radiation animal brain injury model can be constructed using the electromagnetic radiation conditions.
3. The method for constructing a microwave radiation-induced animal brain injury model based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region as described in claim 1, characterized in that, Also includes: The optogenetic system was used to suppress cholinergic neurons in the medial septal nucleus brain region, and the action potential firing and energy oscillation of neurons in the CA3 region of the hippocampus were compared before and after electromagnetic radiation treatment. If the increase in action potential firing and energy oscillation was statistically significant, an electromagnetic radiation animal brain injury model was constructed using the electromagnetic radiation conditions.
4. The method for constructing a microwave radiation-induced animal brain injury model based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region as described in claim 1, characterized in that, Also includes: The experimental animals were subjected to conditioned fear memory tests and fear conditioned memory retrieval tests. The percentage of lag time in the experimental animals before and after electromagnetic radiation treatment was compared. If the decrease in the percentage of lag time was statistically significant, an electromagnetic radiation animal brain injury model was constructed using the electromagnetic radiation conditions.
5. The method for constructing a microwave radiation-induced animal brain injury model based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region as described in claim 4, characterized in that, Also includes: Cholinergic neurons in the medial septal nucleus brain region were activated using an optogenetic system, and conditioned fear memory behavioral experiments were conducted on experimental animals. The effects of activation and inactivation of cholinergic neurons in the medial septal nucleus brain region on the percentage of lag time in experimental animals after electromagnetic radiation treatment were compared. If the reduction in the percentage of lag time was statistically significant, an electromagnetic radiation animal brain injury model was constructed using the electromagnetic radiation conditions.
6. The method for constructing a microwave radiation-induced animal brain injury model based on the regulation of the cholinergic neural circuit in the medial septal nucleus-hippocampal CA3 region as described in claim 4, characterized in that, Also includes: Chemogenetic techniques were used to inhibit cholinergic neurons in the medial septal nucleus brain region, and conditioned fear memory behavioral experiments were conducted on experimental animals. If the inhibition of cholinergic neurons in the medial septal nucleus brain region had a statistically significant effect on the increase in the percentage of lag time in experimental animals before and after electromagnetic radiation, then an electromagnetic radiation animal brain injury model was constructed using the electromagnetic radiation conditions.