Method for constructing animal model of spatial reward learning and memory function impairment caused by microwave radiation based on hippocampal astrocyte regulation

Abstract

The application discloses a method for constructing an animal model of microwave radiation-induced spatial reward learning and memory function damage based on hippocampal astrocyte regulation, which comprises the following steps: randomly dividing test animals into a sham radiation activation control group and a microwave radiation activation group; making the left or right hippocampal CA1 region astrocytes of the test animals specifically express excitatory light-sensitive channel proteins; setting microwave radiation conditions, performing microwave radiation on the microwave radiation activation group, and performing pseudo-radiation under the same conditions on the sham radiation activation control group; activating the unilateral hippocampal CA1 region astrocytes of the test animals, and comparing the influence of hippocampal CA1 region astrocyte activity regulation on the changes in the spatial reward learning ability and the memory ability of the microwave radiation test animals; and if the activation of the hippocampal CA1 region astrocytes has a regulatory effect on the changes in the spatial reward learning ability and / or the memory ability of the microwave radiation test animals, then the microwave radiation conditions are used to construct a microwave radiation animal brain damage model. The application first proposes to use hippocampal astrocytes as sensitive target cells of microwave radiation, to use a wireless optical genetic system, and to establish a new behavioral paradigm suitable for constructing a microwave radiation brain damage animal model based on spatial reward learning and memory evaluation, so as to open up a new way for clarifying the mechanism of microwave radiation brain damage effects and the research and development of related prevention and treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of microwave radiation injury technology, and relates to a method for constructing an animal model of microwave radiation-induced spatial reward learning and memory function impairment based on hippocampal astrocyte regulation. Background Technology

[0002] With the rapid development of electronic information technology and the widespread application of various electronic devices, microwave radiation in human living spaces is increasing daily, and the impact of microwave fields on life activities and human health is gradually attracting attention. Microwave radiation, commonly referred to as non-ionizing radiation, mainly includes radio frequency radiation (100kHz–300GHz) and power frequency radiation (50Hz–60Hz). Among these, microwaves (300MHz–300GHz) are widely used in wireless communication, induction heating, household appliances, radar, and medical fields, making microwave radiation closely related to human life. It is well known that the biological effects of microwave radiation are closely related to its radiation frequency; the biological effects and mechanisms of action caused by microwave radiation in different frequency bands may be completely different.

[0003] Existing research indicates that the brain is one of the sensitive target organs for microwave radiation injury. Under certain conditions, microwave radiation can cause pathological changes in hippocampal tissue, abnormalities in cell signal transduction and synaptic transmission, and related behavioral dysfunctions. Because the molecular mechanisms of the neurobiological effects of microwave radiation are not yet fully understood, the sensitive target sites and target cell types require further clarification. Consequently, there is a lack of effective methods for constructing and evaluating animal models of microwave radiation-induced brain injury, and related preventative and therapeutic drugs are also scarce. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0005] Another objective of this invention is to provide a method for constructing an animal model of microwave-induced spatial reward learning and memory impairment based on hippocampal astrocyte regulation.

[0006] Therefore, the technical solution provided by this invention is as follows:

[0007] Methods for constructing an animal model of microwave-induced spatial reward learning and memory impairment based on hippocampal astrocyte regulation include:

[0008] The experimental animals were randomly divided into a sham radiation activation control group and a microwave radiation activation group;

[0009] The experimental animals in the sham radiation activation control group and the microwave radiation activation group were made to specifically express excitatory light-sensitive channel proteins in the astrocytes of the CA1 region of the left or right hippocampus.

[0010] Microwave radiation conditions were set, and microwave radiation was applied to the microwave radiation activation group of astrocytes that specifically expressed excitatory light-sensitive channel proteins, while the sham radiation activation control group was subjected to sham radiation under the same conditions.

[0011] The astrocytes in the CA1 region of the hippocampus of the experimental animals in the sham radiation activation control group and the microwave radiation activation group were activated to compare the effects of the regulation of astrocyte activity in the CA1 region of the hippocampus on microwave radiation-induced changes in spatial reward learning ability and memory ability.

[0012] If activation of astrocytes in the CA1 region of the hippocampus has a regulatory effect on changes in spatial reward learning ability and / or memory ability in microwave radiation-treated animals, then a microwave radiation-induced brain injury model can be constructed using the microwave radiation conditions.

[0013] Preferably, in the method for constructing an animal model of microwave radiation-induced spatial reward learning and memory function impairment based on hippocampal astrocyte regulation, the experimental animals are randomly divided into a sham radiation group, a microwave radiation group, a sham radiation activation control group, and a microwave radiation activation group.

[0014] The microwave radiation group is subjected to microwave radiation under the aforementioned microwave radiation conditions, and the dummy radiation group is subjected to pseudo-radiation under the same conditions.

[0015] Preferably, in the method for constructing an animal model of spatial reward learning and memory function impairment induced by microwave radiation based on hippocampal astrocyte regulation, a wireless optogenetic system is used in conjunction with a behavioral paradigm to activate unilateral hippocampal CA1 region astrocytes in the experimental animals of the sham radiation activation control group and the microwave radiation activation group. The optogenetic modulation parameters include: blue optogenetic system excitation wavelength 470nm, stimulation frequency 20Hz, power 5mW, pulse duration 10ms, duty cycle 20%, pulse train duration 3s, and interval 2s.

[0016] Preferably, in the method for constructing an animal model of microwave radiation-induced spatial reward learning and memory impairment based on hippocampal astrocyte regulation, the method for comparing the effects of astrocyte activity regulation in the CA1 region of the hippocampus on microwave radiation-induced changes in spatial reward learning and memory abilities includes:

[0017] An open field box is used, with an acrylic plate with circular holes placed at the bottom. The process is divided into the following two stages:

[0018] Phase I: At 6 h, 1 d, and 2 d after microwave radiation, 0.2 ml of sugar water was added to a fixed hole in one corner of the open field box. The experimental animals were placed into the box facing any inner wall. The time it took for the experimental animals to find the sugar water and stay for 3 seconds to drink was recorded to analyze the spatial reward learning ability of the experimental animals. The experimental animals were placed into the box from each of the four inner walls once a day, and the average latency for the experimental animals to find the sugar water was calculated.

[0019] For the experimental animals in the sham radiation activation control group and the microwave radiation activation group, blue light stimulation was administered through the optogenetic system from the moment the experimental animals entered the open field box to selectively activate the astrocytes in the CA1 region of the hippocampus. The average latency of the experimental animals to find the sucrose solution was calculated to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the spatial reward learning ability of mice.

[0020] Phase II: 4, 5, and 6 days after microwave radiation, 0.2 ml of sugar water was added to the corner hole diagonally opposite the hole from Phase I. The experimental animals were placed in the open field box facing any inner wall, and the time it took for the experimental animals to find the sugar water and stay for 3 seconds to drink was recorded to analyze the animals' reverse spatial reward learning ability. Mice were placed in the box from each of the four inner walls once a day, and the average latency for the experimental animals to find the sugar water was calculated.

[0021] For the experimental animals in the sham radiation activation control group and the microwave radiation activation group, blue light stimulation was administered via an optogenetic system from the moment the experimental animals entered the open field box to selectively activate the astrocytes in the CA1 region of the hippocampus. The average latency for the experimental animals to find sucrose water was calculated to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the experimental animals' reverse spatial reward learning ability.

[0022] Preferably, in the method for constructing an animal model of microwave radiation-induced spatial reward learning and memory function impairment based on hippocampal astrocyte regulation, the regulatory effect is as follows: hippocampal CA1 region astrocytes have a positive regulatory effect on the decline in spatial reward learning ability of experimental animals caused by microwave radiation.

[0023] Preferably, in the method for constructing an animal model of microwave radiation-induced spatial reward learning and memory impairment based on hippocampal astrocyte regulation, the regulatory effect is as follows: hippocampal CA1 region astrocytes have a positive regulatory effect on the decline in spatial reward memory ability in mice induced by microwave radiation.

[0024] Preferably, in the method for constructing an animal model of spatial reward learning and memory function impairment induced by microwave radiation based on hippocampal astrocyte regulation, the method for specifically expressing excitatory light-sensitive channel proteins in the left or right hippocampal CA1 region astrocytes of the experimental animals in the sham radiation activation control group and the microwave radiation activation group includes:

[0025] Adeno-associated virus containing excitatory light-sensitive channel protein, astrocyte-specific promoter, and green fluorescent protein was injected into the CA1 region of the left or right hippocampus of the experimental animal. After the adeno-associated virus was stably expressed, the accuracy of the virus injection site was confirmed to obtain experimental animals in which the hippocampal astrocytes successfully expressed excitatory light-sensitive channel protein.

[0026] Preferably, in the method for constructing an animal model of microwave radiation-induced spatial reward learning and memory function impairment based on hippocampal astrocyte regulation, the experimental animal is a mouse.

[0027] The present invention has at least the following beneficial effects:

[0028] This invention is the first to propose using hippocampal astrocytes as sensitive target cells for microwave radiation. It utilizes a wireless optogenetic system and establishes a novel behavioral paradigm suitable for constructing animal models of microwave radiation-induced brain injury based on spatial reward learning and memory evaluation. This is expected to open up new avenues for elucidating the mechanism of microwave radiation-induced brain injury and developing related preventive and therapeutic drugs.

[0029] 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

[0030] Figure 1 The following diagrams and flowcharts illustrate the optogenetic combined spatial reward learning and memory experimental apparatus and its flowchart in an embodiment of the present invention: A: Schematic diagram of the optogenetic combined spatial reward learning and memory experimental apparatus; B: Flowchart of the optogenetic combined spatial reward learning and memory experiment.

[0031] Figure 2 This image shows the confirmation of the AAV stereotactic injection target site in the CA1 region of the mouse hippocampus in an embodiment of the present invention.

[0032] Figure 3This invention illustrates the effect of hippocampal CA1 astrocyte activation on microwave-induced changes in spatial reward learning ability in mice. A: Schematic diagram of mouse movement trajectories in each experimental group during stage I of spatial reward learning; B: Effect of hippocampal CA1 astrocyte activation on the average latency of mice to find sucrose after microwave radiation; C: Schematic diagram of mouse movement trajectories during stage II of spatial reward learning; D: Effect of hippocampal CA1 astrocyte activation on the average latency of mice to find sucrose after microwave radiation. Con: Sham radiation group; MW: Microwave radiation group; Con+ON: Sham radiation activation control group; MW+ON: Microwave radiation activation group. N=6, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001.

[0033] Figure 4 This invention illustrates the effect of hippocampal CA1 astrocyte activation on microwave-induced changes in spatial reward memory in mice. A: Schematic diagram of mouse movement trajectories in each experimental group during Phase I of spatial reward memory; B: Effect of hippocampal CA1 astrocyte activation on the number of times mice explored target holes after microwave radiation during Phase I of spatial reward memory; C: Effect of hippocampal CA1 astrocyte activation on the quadrant time of mice exploring target holes after microwave radiation during Phase I of spatial reward memory; D: Schematic diagram of mouse movement trajectories during Phase II of spatial reward memory; E: Effect of hippocampal CA1 astrocyte activation on the number of times mice explored target holes after microwave radiation during Phase II of spatial reward memory; F: Effect of hippocampal CA1 astrocyte activation on the quadrant time of mice exploring target holes after microwave radiation during Phase II of spatial reward memory; Con: Sham radiation group; MW: Microwave radiation group; Con+ON: Sham radiation activation control group; MW+ON: Microwave radiation activation group. N=6, * indicates P<0.05. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0037] This invention provides a method for constructing an animal model of microwave radiation-induced spatial reward learning and memory impairment based on hippocampal astrocyte regulation, comprising:

[0038] The experimental animals were randomly divided into a sham radiation activation control group and a microwave radiation activation group;

[0039] The experimental animals in the sham radiation activation control group and the microwave radiation activation group were made to specifically express excitatory light-sensitive channel proteins in the astrocytes of the CA1 region of the left or right hippocampus.

[0040] Microwave radiation conditions were set, and microwave radiation was applied to the microwave radiation activation group expressing excitatory light-sensitive channel proteins and astrocyte-specific promoters, while the sham radiation activation control group was subjected to sham radiation under the same conditions.

[0041] The astrocytes in the CA1 region of the hippocampus of the experimental animals in the sham radiation activation control group and the microwave radiation activation group were activated to compare the effects of the regulation of astrocyte activity in the CA1 region of the hippocampus on microwave radiation-induced changes in spatial reward learning ability and memory ability.

[0042] If activating astrocytes in the CA1 region of the hippocampus has a regulatory effect on changes in spatial reward learning ability and / or memory ability in microwave radiation-treated animals, then a microwave radiation-induced brain injury model can be constructed using the microwave radiation conditions.

[0043] Optionally, the experimental animals are randomly divided into a sham radiation group, a microwave radiation group, a sham radiation activation control group, and a microwave radiation activation group.

[0044] The microwave radiation group is subjected to microwave radiation under the aforementioned microwave radiation conditions, and the dummy radiation group is subjected to pseudo-radiation under the same conditions.

[0045] Optionally, a wireless optogenetic system, combined with a behavioral paradigm, is used to activate unilateral astrocytes in the CA1 region of the hippocampus of the experimental animals in the sham radiation activation control group and the microwave radiation activation group. The optogenetic modulation parameters include: blue optogenetic system excitation wavelength of 470 nm, stimulation frequency of 20 Hz, power of 5 mW, pulse duration of 10 ms, duty cycle of 20%, pulse train duration of 3 s, and interval of 2 s.

[0046] Optionally, methods for comparing the effects of regulation of astrocyte activity in the CA1 region of the hippocampus on microwave-induced changes in spatial reward learning and memory abilities include:

[0047] An open field box is used, with an acrylic plate with circular holes placed at the bottom. The process is divided into the following two stages:

[0048] Phase I: At 6 h, 1 d, and 2 d after microwave radiation, 0.2 ml of sugar water was added to a fixed hole in one corner of the open field box. The experimental animals were placed into the box facing any inner wall. The time it took for the experimental animals to find the sugar water and stay for 3 seconds to drink was recorded to analyze the spatial reward learning ability of the experimental animals. The experimental animals were placed into the box from each of the four inner walls once a day, and the average latency for the experimental animals to find the sugar water was calculated.

[0049] For the experimental animals in the sham radiation activation control group and the microwave radiation activation group, blue light stimulation was administered through the optogenetic system from the moment the experimental animals entered the open field box to selectively activate the astrocytes in the CA1 region of the hippocampus. The average latency of the experimental animals to find the sucrose solution was calculated to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the spatial reward learning ability of mice.

[0050] Phase II: 4, 5, and 6 days after microwave radiation, 0.2 ml of sugar water was added to the corner hole diagonally opposite the hole from Phase I. The experimental animals were placed in the open field box facing any inner wall, and the time it took for the experimental animals to find the sugar water and stay for 3 seconds to drink was recorded to analyze the animals' reverse spatial reward learning ability. Mice were placed in the box from each of the four inner walls once a day, and the average latency for the experimental animals to find the sugar water was calculated.

[0051] For the experimental animals in the sham radiation activation control group and the microwave radiation activation group, blue light stimulation was administered via an optogenetic system from the moment the experimental animals entered the open field box to selectively activate the astrocytes in the CA1 region of the hippocampus. The average latency for the experimental animals to find sucrose water was calculated to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the experimental animals' reverse spatial reward learning ability.

[0052] The regulatory effect is as follows: astrocytes in the CA1 region of the hippocampus have a positive regulatory effect on the decline in spatial reward learning ability of experimental animals induced by microwave radiation.

[0053] The regulatory effect is as follows: astrocytes in the CA1 region of the hippocampus have a positive regulatory effect on the decline in spatial reward memory ability in mice induced by microwave radiation.

[0054] Optionally, a method for specifically expressing excitatory light-sensitive channel proteins in astrocytes of the left or right hippocampus CA1 region of the experimental animals in the sham radiation activation control group and the microwave radiation activation group includes:

[0055] Adeno-associated virus containing excitatory light-sensitive channel protein, astrocyte-specific promoter, and green fluorescent protein was injected into the CA1 region of the left or right hippocampus of the experimental animal. After the adeno-associated virus was stably expressed, the accuracy of the virus injection site was confirmed to obtain experimental animals in which the hippocampal astrocytes successfully expressed excitatory light-sensitive channel protein.

[0056] Optionally, the test animal is a mouse.

[0057] To enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for illustration:

[0058] 1. Materials and Methods

[0059] Ten-week-old wild-type C57BL / N6 mice, weighing 22±2g, with half males and half females, were randomly divided into a sham radiation group (Con), a microwave radiation group (MW), a sham radiation activation control group (Con+ON), and a microwave radiation activation group (MW+ON). Three animals were confirmed to be the target site for viral injection, and six animals were included in each group for optogenetic regulation combined with behavioral testing.

[0060] A high-power microwave radiation simulation source built by the Academy of Military Medical Sciences was used, with a center frequency of 2.856 GHz and an average power density of 8 mW / cm². 2 Peak power density 200W / cm³ 2 The repetition frequency was 80 Hz, the pulse width was 500 ns, the SAR value was 6.12 W / kg, and the radiation time was 15 min. Mice were placed in transparent perforated plexiglass boxes, and the radiation boxes were placed in a rotatable radiation table in a microwave anechoic chamber. The microwave source radiated uniformly from top to bottom, and the sham radiation group was subjected to sham radiation under the same conditions.

[0061] 1.2 Stereotactic injection of AAV and probe implantation into the brain

[0062] Mice were anesthetized with isoflurane inhalation. Using a stereotaxic instrument and a microinfusion pump, adeno-associated virus (rAAV-GfaABC1D-hChR2-EGFP) containing excitatory light-sensitive channel protein (hChR2), astrocyte-specific promoter (GfaABC1D), and green fluorescent protein (EGFP) was injected into the CA1 region of the left or right hippocampus (2.06 mm posterior to the anterior fontanelle, 1.5 mm lateral to the fontanelle, and 1.3 mm below the skull surface). The injection volume was 80 nmol, and the needle was left in place for 10 min. A wireless implantable probe was then inserted, and the area was sealed with dental cement and the scalp was sutured with medical sutures. Three weeks post-surgery, after the virus expression had stabilized, the mice underwent cardiac perfusion fixation, and frozen sections of brain tissue were examined using a fluorescence microscope to confirm the precision of the injection site.

[0063] 1.3 Regulation of hippocampal astrocyte activity based on wireless optogenetics

[0064] Using a wireless optogenetic system (Neurolux, USA) combined with a behavioral paradigm, unilateral astrocytes in the CA1 region of the hippocampus were activated to investigate the effects of microwave radiation-induced changes in spatial reward learning and memory abilities. Figure 1 As shown. The optogenetic modulation parameters are as follows: blue optogenetic system excitation wavelength 470nm, stimulation frequency 20Hz, power 5mW, pulse duration 10ms, duty cycle 20%, pulse train duration 3s, interval 2s.

[0065] 1.4 Optogenetics combined with spatial reward learning experiment to detect changes in spatial reward learning ability in mice

[0066] A 30cm×30cm×30cm open field box was used, with an acrylic plate with 2cm diameter circular holes placed at the bottom. Mice were deprived of water for 24 hours before microwave radiation; after microwave radiation, they were deprived of water for another 7 days, and were given water for 10 minutes each day after the reward behavior test following radiation. The experimental setup diagram and procedure are shown below. Figure 1 Specifically, it is divided into the following two stages:

[0067] (1) Phase I: At 6 h, 1 d, and 2 d after microwave radiation, 0.2 ml of sugar water was added to a fixed hole in one corner of the open field box. Mice were placed into the box facing any inner wall. The time it took for the mice to find the sugar water and stay for 3 seconds to drink was recorded using automatic video tracking and Anymaze behavioral analysis software to analyze the mice's spatial reward learning ability. Mice were placed into the box from each of the four inner walls once a day, and the average latency for the mice to find the sugar water was calculated. For mice in the optogenetic activation group (Con+ON, MW+ON), blue light stimulation was administered via optogenetic system from the moment the mice entered the open field box to selectively activate astrocytes in the CA1 region of the hippocampus. The average latency for the mice to find the sugar water was calculated to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the spatial reward learning ability of mice.

[0068] (2) Phase II: 4, 5, and 6 days after microwave radiation, 0.2 ml of sugar water was added to the corner hole diagonally opposite the hole from Phase I. Mice were placed in the open field box facing any inner wall. Using automated video tracking and Anymaze behavioral analysis software, the time it took for the mice to find the sugar water and remain for 3 seconds to drink was recorded, analyzing the mice's reverse spatial reward learning ability. Mice were placed from each of the four inner walls once a day, continuously, and the average latency for finding the sugar water was calculated. For optogenetic activation group mice (Con+ON, MW+ON), blue light stimulation was administered via an optogenetic system from the moment the mice entered the open field box, selectively activating astrocytes in the CA1 region of the hippocampus. The average latency for finding the sugar water was calculated, evaluating the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the reverse spatial reward learning ability of mice.

[0069] 1.5 Optogenetics combined with spatial reward memory assay to detect changes in spatial reward memory ability in mice

[0070] A 30cm×30cm×30cm open field box was used, with an acrylic plate with a 2cm diameter circular hole placed at the bottom. The experiment consisted of the following two stages:

[0071] (1) Phase I: Three days after microwave radiation, mice were placed in an open field chamber from the diagonal side of the hole where sugar water was placed in Phase I of the spatial reward learning experiment, facing the inner wall. Automatic video tracking and Anymaze behavioral analysis software were used to record the mice's activity trajectory within 60 seconds, and the time the mice spent in the target quadrant where the sugar water hole was located was calculated to analyze the mice's spatial reward memory ability. For mice in the optogenetic activation group (Con+ON, MW+ON), blue light stimulation was administered via an optogenetic system from the moment the mice entered the open field chamber to selectively activate astrocytes in the CA1 region of the hippocampus. The activity trajectory of the mice in the open field chamber within 60 seconds after optogenetic activation was recorded to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the spatial reward memory ability of mice.

[0072] (2) Phase II: Seven days after microwave radiation, mice were placed in an open field chamber from the diagonal side of the hole where sugar water was placed in Phase II of the spatial reward learning experiment, facing the inner wall. Automatic video tracking and Anymaze behavioral analysis software were used to record the mice's activity trajectory over 60 seconds. The time the mice spent in the target quadrant where the sugar water hole was located was calculated to analyze the mice's reverse spatial reward memory ability. For mice in the optogenetic activation group (Con+ON, MW+ON), blue light stimulation was administered via an optogenetic system from the moment the mice entered the open field chamber to selectively activate astrocytes in the CA1 region of the hippocampus. The activity trajectory of the mice in the open field chamber over 60 seconds after optogenetic activation was recorded to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the reverse spatial reward memory ability of mice.

[0073] 1.6 Statistical Analysis

[0074] Experimental data are expressed as mean ± standard error. Repeated measures ANOVA was performed using SPSS 22.0 statistical software. P < 0.05 was considered statistically significant.

[0075] 2. Experimental Results

[0076] 2.1 Confirmation of AAV injection target site in the CA1 region of the mouse hippocampus

[0077] Three weeks after injection of adeno-associated virus (AAV) containing the excitatory light-sensitive channel protein hChR2 and labeled with green fluorescent protein (EGFP), mice underwent cardiac perfusion fixation. Frozen sections of brain tissue were harvested and observed under a laser scanning confocal microscope. Referring to a mouse brain stereotaxic atlas, EGFP was specifically expressed in the CA1 region of the hippocampus, with a strong fluorescence signal, indicating accurate stereotaxic targeting of the CA1 region. (See...) Figure 2 .

[0078] 2.2 Regulatory role of hippocampal CA1 astrocytes in microwave-induced changes in spatial reward learning ability in mice

[0079] Results of experiments combining optogenetics with spatial reward learning ( Figure 3 The results showed that, at 1, 2, 4, 5, and 6 days after microwave radiation, the average latency for mice in the radiation group (MW) to find sucrose water was significantly longer than that in the sham radiation group (Con) (P<0.05 or P<0.01). Figure 3 B and D) indicate that microwave radiation can cause a significant decrease in the spatial reward learning ability of mice.

[0080] In the reverse spatial reward learning phase (spatial reward learning II), specifically 4, 5, and 6 days after microwave radiation, the average latency for mice in the radiation-activated group (MW+ON) to find sucrose was significantly shorter than that in the microwave-radiated group (P<0.05). Figure 3 D), and no statistical differences were found in the others (P>0.05), which further indicates that activation of astrocytes in the CA1 region of the hippocampus can significantly improve the decline in spatial reward learning ability in mice induced by microwave radiation, but has no significant effect on the spatial reward learning ability of normal mice.

[0081] The above results suggest that astrocytes in the CA1 region of the hippocampus play an important positive regulatory role in the decline of spatial reward learning ability in mice induced by microwave radiation.

[0082] 2.3 Regulatory role of hippocampal CA1 astrocytes in microwave-induced changes in spatial reward memory in mice

[0083] Results of an experiment combining optogenetics and spatial reward memory ( Figure 4 AF) showed that 7 days after microwave radiation, in the reverse spatial reward memory test, the time taken by mice in the radiation group (MW) to explore the target hole quadrant in the sham radiation group (Con) was significantly shorter than that in the sham radiation group (Con) (P<0.05). Figure 4 F) indicates that microwave radiation can cause a significant decrease in spatial reward memory in mice.

[0084] Furthermore, 7 days after microwave radiation, the number of times mice in the radiation group explored the target hole in the sugar water was reduced compared to the sham radiation group, but no statistically significant difference was observed (P>0.05). Figure 4 E). Furthermore, 3 days after microwave radiation, the number of times the irradiated mice explored the target hole and the time spent in the target quadrant were both significantly reduced compared to the sham-irradiated group (P>0.05). Figure 4 B, C).

[0085] In the first stage of spatial reward memory, 3 days after microwave radiation, the time taken by mice in the radiation-activated group (MW+ON) to explore the target hole quadrant in the sucrose solution was significantly longer than that in the microwave-radiated group (P<0.05). Figure 4 C), and at the same time, the number of times the mice in the radiation-activated group explored the target hole was more than that in the microwave-irradiated group, but no statistical difference was observed (P>0.05). Figure 4 B). Furthermore, the number of times the sham-radiated activated control (Con+ON) mice explored the target hole and the time spent in the target quadrant were not significantly different from those in the sham-radiated group (P>0.05). Figure 4 (B, C, E, F). The above results indicate that activation of astrocytes in the CA1 region of the hippocampus can significantly improve the decline in spatial reward memory in mice induced by microwave radiation, but has no significant effect on the spatial reward memory in normal mice.

[0086] In the reverse spatial reward memory stage (spatial reward memory II), 7 days after microwave radiation, the number of times mice in the radiation-activated group explored the target hole in the sugar water and the time spent exploring the target quadrant were both significantly increased compared to the microwave radiation group, and there were no statistically significant differences between the radiation-activated group and the sham radiation group (P>0.05). Figure 4 E, F). The target quadrant time in the microwave radiation group was significantly shorter than that in the sham radiation group (P<0.05). Figure 4 F) further suggests that activating astrocytes in the CA1 region of the hippocampus can significantly improve the decline in reverse spatial reward memory in mice induced by microwave radiation.

[0087] In conclusion, astrocytes in the CA1 region of the hippocampus play an important positive regulatory role in the decline of spatial reward memory in mice induced by microwave radiation.

[0088] 3. Conclusion

[0089] In summary, our research found that microwave radiation under certain conditions can lead to impairment of spatial reward learning and memory functions. Astrocytes in the CA1 region of the hippocampus play a crucial positive regulatory role in the changes in spatial reward learning and memory abilities induced by microwave radiation; specifically, activation of astrocytes in the CA1 region significantly improves the decline in radiation-induced spatial reward learning and memory abilities. Therefore, this invention proposes using astrocytes in the CA1 region of the hippocampus as sensitive target cells for microwave radiation, combined with optogenetic regulation and behavioral evaluation of spatial reward learning and memory, for the construction of an animal model of microwave radiation-induced brain injury. This establishes a novel behavioral paradigm suitable for evaluating the neurobiological effects of microwave radiation and constructing animal models of brain injury, which is of great significance for research on the mechanisms of microwave radiation-induced brain injury and the development of preventive drugs.

[0090] The number of modules and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0091] 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 an animal model of spatial reward learning and memory function impairment caused by microwave radiation based on hippocampal astrocyte regulation, characterized in that, include: The experimental animals were randomly divided into a sham radiation group, a microwave radiation group, a sham radiation activation control group, and a microwave radiation activation group. The experimental animals were mice. The experimental animals in the sham radiation activation control group and the microwave radiation activation group were made to specifically express excitatory light-sensitive channel proteins in the astrocytes of the CA1 region of the left or right hippocampus. Microwave radiation conditions were set, and microwave radiation was applied to the microwave radiation activation group of astrocytes that specifically expressed excitatory light-sensitive channel proteins. The sham radiation activation control group was subjected to sham radiation under the same conditions. The microwave radiation group was then subjected to microwave radiation under the same conditions, and the sham radiation group was subjected to sham radiation under the same conditions. The astrocytes in the CA1 region of the hippocampus of the experimental animals in the sham radiation activation control group and the microwave radiation activation group were activated to compare the effects of the regulation of astrocyte activity in the CA1 region of the hippocampus on microwave radiation-induced changes in spatial reward learning ability and memory ability. If activation of astrocytes in the CA1 region of the hippocampus has a regulatory effect on changes in spatial reward learning and memory abilities in animals subjected to microwave radiation, then a microwave radiation animal brain injury model can be constructed using the microwave radiation conditions described above. Using a wireless optogenetic system and combined with a behavioral paradigm, astrocytes in the CA1 region of the unilateral hippocampus of experimental animals in the sham radiation activation control group and the microwave radiation activation group were activated. The modulation parameters of the wireless optogenetic system included: blue optogenetic system excitation wavelength of 470nm, stimulation frequency of 20Hz, power of 5mW, pulse duration of 10ms, duty cycle of 20%, pulse train duration of 3s, and interval of 2s. Methods for comparing the effects of regulation of astrocyte activity in the CA1 region of the hippocampus on microwave-induced changes in spatial reward learning and memory include: An open field box was used, with an acrylic plate with circular holes placed at the bottom. Specifically, the experiment included a spatial reward learning experiment to detect changes in the spatial reward learning ability of mice and a spatial reward memory experiment to detect changes in the spatial reward memory ability of mice. The spatial reward learning experiment consists of the following two phases: Phase I: At 6 h, 1 d, and 2 d after microwave radiation, 0.2 ml of sugar water was added to a fixed hole in one corner of the open field box. The experimental animals were placed into the box facing any inner wall. The time it took for the experimental animals to find the sugar water and stay for 3 seconds to drink was recorded to analyze the spatial reward learning ability of the experimental animals. The experimental animals were placed into the box from each of the four inner walls once a day, and the average latency for the experimental animals to find the sugar water was calculated. For the experimental animals in the sham radiation activation control group and the microwave radiation activation group, blue light stimulation was applied through the wireless optogenetic system from the moment the experimental animals entered the open field box to selectively activate the astrocytes in the CA1 region of the hippocampus. The average latency of the experimental animals to find the sucrose solution was calculated to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the spatial reward learning ability of mice. Phase II: 4, 5, and 6 days after microwave radiation, 0.2 ml of sugar water was added to the corner hole diagonally opposite the hole from Phase I. The experimental animals were placed in the open field box facing any inner wall, and the time it took for the experimental animals to find the sugar water and stay for 3 seconds to drink was recorded to analyze the animals' reverse spatial reward learning ability. Mice were placed in the box from each of the four inner walls once a day, and the average latency for the experimental animals to find the sugar water was calculated. For the experimental animals in the sham radiation activation control group and the microwave radiation activation group, blue light stimulation was applied through the wireless optogenetic system from the moment the experimental animals entered the open field box to selectively activate the astrocytes in the CA1 region of the hippocampus. The average latency of the experimental animals to find the sucrose solution was calculated to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the experimental animals' reverse spatial reward learning ability. The spatial reward memory experiment consists of the following two phases: Phase I: Three days after microwave radiation, mice were placed in an open field box from the diagonal side of the hole where sugar water was placed in Phase I of the spatial reward learning experiment, facing the inner wall. The movement trajectory of the mice was recorded within 60 seconds, and the time the mice spent in the target quadrant where the sugar water hole was located was calculated to analyze the spatial reward memory ability of the mice. For mice in the sham radiation activation control group and microwave radiation activation group, blue light stimulation was given through the wireless optogenetic system from the moment the mice entered the open field box to selectively activate the astrocytes in the CA1 region of the mouse hippocampus. The movement trajectory of the mice in the open field box within 60 seconds after optogenetic activation was recorded to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the spatial reward memory ability of mice. Phase II: Seven days after microwave radiation, mice were placed in an open field box from the diagonal side of the hole where sugar water was placed in Phase II of the spatial reward learning experiment, facing the inner wall. The movement trajectory of the mice was recorded within 60 seconds, and the time the mice spent in the target quadrant where the sugar water hole was located was calculated to analyze the reverse spatial reward memory ability of the mice. For mice in the sham radiation activation control group and microwave radiation activation group, blue light stimulation was applied through the wireless optogenetic system from the moment the mice entered the open field box to selectively activate the astrocytes in the CA1 region of the mouse hippocampus. The movement trajectory of the mice in the open field box within 60 seconds after optogenetic activation was recorded to evaluate the effect of optogenetic regulation of hippocampal astrocyte activity on microwave radiation-induced changes in the reverse spatial reward memory ability of mice.

2. The method for constructing an animal model of impairment of spatial reward learning and memory function induced by microwave radiation based on hippocampal astrocyte regulation according to claim 1, wherein, The regulatory effect is as follows: astrocytes in the CA1 region of the hippocampus have a positive regulatory effect on the decline in spatial reward learning ability of experimental animals caused by microwave radiation.

3. The method for constructing an animal model of impairment of spatial reward learning and memory function induced by microwave radiation based on hippocampal astrocyte regulation according to claim 1, wherein, The regulatory effect is as follows: astrocytes in the CA1 region of the hippocampus have a positive regulatory effect on the decline in spatial reward memory ability in mice induced by microwave radiation.

4. The method for constructing an animal model of impairment of spatial reward learning and memory function induced by microwave radiation based on hippocampal astrocyte regulation according to claim 1, wherein, Methods for inducing specific expression of excitatory light-sensitive channel proteins in astrocytes of the left or right hippocampus CA1 region of the experimental animals in the sham radiation activation control group and microwave radiation activation group include: Adeno-associated virus containing excitatory light-sensitive channel protein, astrocyte-specific promoter, and green fluorescent protein was injected into the CA1 region of the left or right hippocampus of the experimental animal. After the adeno-associated virus was stably expressed, the accuracy of the virus injection site was confirmed to obtain experimental animals in which the hippocampal astrocytes successfully expressed excitatory light-sensitive channel protein.

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