A mouse model of depression and a method for constructing the same and applications thereof

Abstract

The application relates to the technical field of biological medicine, and particularly discloses a depression mouse model and a construction method and application thereof. The construction method of the depression mouse model comprises hybrid knock-out of Csf1r specific to microglia cells on the mouse. The depression mouse model is obtained by inducing the mouse to have a depression-like behavior through hybrid knock-out of Csf1r specific to microglia cells. Compared with the existing method of causing an animal to have a depression-like behavior through chronic unpredictable mild stress stimulation, the depression phenotype is stable, the depression phenotype appears fast, repeated experiments for depression-related research can be facilitated, and a highly repeatable experimental result can be obtained.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a mouse model of depression, its construction method, and its application. Background Technology

[0002] Depression is a common mental disorder and a disabling illness. According to the World Health Organization (WHO), approximately 3.8% of the population, including 2.6% of children and adolescents and 5.0% of adults, are affected by depression. The prevalence of depression in China continues to rise, with a generally higher incidence in women than men. Social stress, psychological stress, and biological factors can all trigger depression. Current research on the causes of depression and the evaluation of treatment drugs generally uses chronic, unpredictable, mild stress to induce a depressive phenotype in mice; however, this animal model is unstable, with significant individual variability, introducing uncertainty into experiments.

[0003] Therefore, existing technologies still need improvement. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide a mouse model of depression, its construction method and application, in order to solve the problems of low stability, large individual differences and uncertainty brought to the experiment by the existing animal models of depression.

[0005] The technical solution of this application is as follows:

[0006] A first aspect of this application provides a method for constructing a mouse model of depression, the method comprising: performing microglia-specific Csf1r heterozygous knockout on mice.

[0007] Optionally, the mouse is a Cre mouse.

[0008] Optionally, the step of performing microglia-specific Csf1r heterozygous knockout in mice includes: mating wild-type Cre mice, first Cre mice, and second Cre mice to obtain a mouse model of depression; inserting LoxP sites on both sides of the exon of the Csf1r gene in the first Cre mouse; and inserting the creERT2-WPRE-polyA sequence at the start codon of the Cx3cr1 gene in the second Cre mouse.

[0009] Optionally, the method for constructing the mouse model of depression further includes genotyping the mouse model of depression.

[0010] Optionally, the genes identified in the genotyping identification include the Cx3cr1-Cre gene and the Csf1r-flox gene.

[0011] Optionally, the method for constructing the mouse model of depression further includes evaluating the depressive-like behavior of the mouse model of depression.

[0012] Optionally, the assessment method for the depressive-like behavior includes at least one of the elevated cross maze test, open field test, sucrose preference test, and forced swimming test.

[0013] A second aspect of this application provides a mouse model of depression constructed using the method described in this application.

[0014] A third aspect of this application provides an application of the mouse model of depression provided in this application in theoretical research on depression.

[0015] A fourth aspect of this application provides an application of the mouse model of depression provided in this application in the screening of drugs for depression.

[0016] Compared with the prior art, this application has the following advantages:

[0017] This application induces depressive-like behavior in mice through microglia-specific Csf1r heterozygous knockout, thus obtaining a mouse model of depression. Compared with existing methods that induce depressive-like behavior in animals through chronic, unpredictable, mild stress CUMS stimulation, this method produces a stable and rapidly developing depressive phenotype, facilitating reproducible experiments in depression-related research and yielding highly reproducible results. Furthermore, the mouse model of depression prepared using this method can mimic microglia-related depression and neuroinflammation-related depression, providing a foundation for mechanistic studies and drug screening for microglia-related or neuroinflammation-related depression. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0019] Figure 1 The following are images of depressive-like behavior tests in various groups of mice (WT, Cre-, Cre+) provided in the embodiments of this application:

[0020] A shows the drug administration and behavioral testing schedule for a mouse depression model; B shows the behavioral results of the mouse elevated cruciate maze test; C shows the behavioral results of the mouse open field test; D shows the behavioral results of the mouse sucrose preference test; E shows the behavioral results of the mouse forced swimming test.

[0021] Figure 2 Immunofluorescence staining images of the mPFC brain regions of mice (WT, Cre-, Cre+) provided in the embodiments of this application:

[0022] A shows the immunofluorescence staining results of Iba1 in each group of mice; B shows the statistical data on the coverage area of ​​microglia in the mPFC brain region of each group of mice; C shows the statistical data on the total length of microglia branches in the mPFC brain region of each group of mice; D shows the statistical data on the total number of microglia branches in the mPFC brain region of each group of mice; E shows representative images of Golgi staining in the mPFC brain region of each group of mice; F shows the statistical data on the number of dendritic spines in the mPFC brain region of each group of mice; G shows representative images of the co-localization of Iba1 and PSD95 in the mPFC brain region of each group of mice; H shows the statistical data on the volume of PSD95 in Iba1 in the mPFC brain region of each group of mice.

[0023] Figure 3 Images of depressive-like behaviors in mice (Cre-, Cre+, Cre+_PLX) provided in the embodiments of this application:

[0024] A shows the drug administration and behavioral testing schedule for a mouse depression model; B shows the behavioral results of the mouse elevated cruciate maze test; C shows the behavioral results of the mouse open field test; D shows the behavioral results of the mouse sucrose preference test; E shows the behavioral results of the mouse forced swimming test.

[0025] Figure 4 Immunofluorescence staining images of the mPFC brain regions of mice (Cre-, Cre+, Cre+_PLX) in each group provided for the embodiments of this application:

[0026] A shows the immunofluorescence staining results of Iba1 in each group of mice; B shows the statistical data on the coverage area of ​​microglia in the mPFC brain region of each group of mice; C shows the statistical data on the total length of microglia branches in the mPFC brain region of each group of mice; D shows the statistical data on the total number of microglia branches in the mPFC brain region of each group of mice; E shows representative images of Golgi staining in the mPFC brain region of each group of mice; F shows the statistical data on the number of dendritic spines in the mPFC brain region of each group of mice; G shows representative images of the co-localization of Iba1 and PSD95 in the mPFC brain region of each group of mice; H shows the statistical data on the volume of PSD95 in Iba1 in the mPFC brain region of each group of mice.

[0027] Figure 5 Signaling pathway analysis diagram of the mPFC brain region in a mouse depression model provided in this application embodiment:

[0028] A shows the differential gene KEGG enrichment analysis between Cre- and Cre+ mice; B shows the NOD family gene enrichment heatmap; C shows the difference in the expression levels of various molecular proteins in the NLRP6 / caspase-1 / IL-1β / IL-18 pathway in the mPFC brain region of Cre- and Cre+ mice; D shows the immunofluorescence colocalization of Iba1 and Caspase-1 in the mPFC brain region of Cre- and Cre+ mice; E shows the linear statistics of Iba1 and Caspase-1 colocalization; F shows the immunofluorescence colocalization of NeuN and Caspase-1 in the mPFC brain region of Cre- and Cre+ mice; G shows the linear statistics of NeuN and Caspase-1 colocalization; H shows the immunofluorescence colocalization of GFAP and Caspase-1 in the mPFC brain region of Cre- and Cre+ mice; I shows the linear statistics of GFAP and Caspase-1 colocalization.

[0029] Figure 6 Images of depressive-like behavior in mice (Cre-, Cre+, Cre+_VX765) provided for embodiments of this application:

[0030] A shows the drug administration and behavioral testing schedule for a mouse depression model; B shows the behavioral results of the mouse elevated cruciate maze test; C shows the behavioral results of the mouse open field test; D shows the behavioral results of the mouse sucrose preference test; E shows the behavioral results of the mouse forced swimming test.

[0031] Figure 7 Immunofluorescence staining images of the mPFC brain regions of mice (Cre-, Cre+, Cre+_VX765) in each group provided in the embodiments of this application:

[0032] A shows the immunofluorescence staining results of Iba1 in each group of mice; B shows the statistical data on the coverage area of ​​microglia in the mPFC brain region of each group of mice; C shows the statistical data on the total length of microglia branches in the mPFC brain region of each group of mice; D shows the statistical data on the total number of microglia branches in the mPFC brain region of each group of mice; E shows representative images of Golgi staining in the mPFC brain region of each group of mice; F shows the statistical data on the number of dendritic spines in the mPFC brain region of each group of mice; G shows representative images of co-localization of Iba1 and PSD95 in the mPFC brain region of each group of mice; H shows the statistical data on the volume of PSD95 in Iba1 in the mPFC brain region of each group of mice. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this application. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0034] It should be noted that if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on enabling those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0035] Microglia play a crucial role in the pathogenesis of depression. One of the main functions of microglia in the brain is responding to immune signals and participating in inflammatory responses. When the brain is injured or infected, microglia are activated, releasing various inflammatory factors and cytokines (such as TNF-α, IL-1β, and IL-6), which may lead to neuroinflammation. Depression is often accompanied by neuroinflammation, and activated microglia promote inflammation, thus exacerbating depressive symptoms. Microglia also play a key role in neuroplasticity. They are involved in synapse formation and pruning, and depression is closely related to neuroplasticity disorders. Microglia dysfunction may lead to abnormal neural connections, thereby affecting mood. The pathological mechanisms of depression may include excessive activation of microglia and persistent neuroinflammation, which may lead to neuronal damage and death, and exacerbate depressive symptoms.

[0036] Csf1r (colony-stimulating factor 1 receptor) plays a crucial role in maintaining the function and survival of microglia. Microglia are precursors of bone marrow mononuclear cells derived from embryonic cells, and Csf1r signaling is essential for their proliferation and differentiation during development. Csf1r signaling not only promotes microglia survival but also participates in regulating their activation state. In response to nerve injury or disease, microglia regulate their function through Csf1r-mediated signaling, including the production and release of various inflammatory mediators, thereby influencing the course of neuroinflammation. In Alzheimer's disease and other neurodegenerative diseases, changes in Csf1r expression may directly affect microglia function, thus influencing disease progression; however, the function of Csf1r in depression remains to be explored.

[0037] Based on this, this application provides a method for preparing a mouse model of depression, including the steps of: obtaining a mouse model by microglia-specific Csf1r heterozygous knockout. The mouse is a Cre mouse.

[0038] In some embodiments, the step of performing microglia-specific Csf1r heterozygous knockout in mice can be as follows: wild-type Cre mice, first Cre mice, and second Cre mice are mated to obtain a mouse model of depression. Specifically, LoxP sites are inserted flanking the exons of the Csf1r gene in the first Cre mouse, and the creERT2-WPRE-polyA sequence is inserted at the start codon of the Cx3cr1 gene in the second Cre mouse.

[0039] Heterozygous knockout of the Csf1r gene in mouse microglia was achieved using the Cre-Lox gene recombination system. LoxP sites were inserted flanking the exons of the Csf1r gene in first-Cre mice, and the creERT2-WPRE-polyA sequence was inserted at the start codon of the Cx3cr1 gene in second-Cre mice. During mating of wild-type Cre mice, first-Cre mice, and second-Cre mice, Cre recombinase was expressed and could cleave the DNA sequence between the two LoxP sites and re-circularize the two ends, thus achieving specific knockout of the Csf1r gene.

[0040] In some embodiments, the method for constructing a mouse model of depression further includes: genotyping the mouse model of depression. Specifically, genotyping of the mouse model of depression is achieved by identifying the Cx3cr1-Cre and Csf1r-flox genes. The Cx3cr1-Cre and Csf1r-flox genes can be identified using primers targeting the Cx3cr1-Cre and Csf1r-flox genes, through PCR amplification combined with gel electrophoresis and sequencing.

[0041] In some embodiments, the method for constructing a mouse model of depression further includes evaluating the depressive-like behaviors of the mouse model. The evaluation methods for depressive-like behaviors include at least one of the elevated cruciate maze test, open field test, sucrose preference test, and forced swimming test.

[0042] This application also provides a mouse model of depression using the method described above for preparing a mouse model of depression.

[0043] This application also provides a method for alleviating depressive-like behavior in a mouse model of depression as described above: treating the mouse model of depression with a Csf1r inhibitor or a Caspase-1 inhibitor. Specifically, the method involves treating the mouse model of depression with a Csf1r inhibitor or a Caspase-1 inhibitor via gavage. PLX3397 can be used as the Csf1r inhibitor, and VX-765 can be used as the Caspase-1 inhibitor.

[0044] Experimental results showed that both Csf1r inhibitors and Caspase-1 inhibitors effectively alleviated depressive-like behaviors in a mouse model of depression. Csf1r inhibitors reduced microglial activation and synaptic phagocytosis in the mPFC brain region of the mouse model of depression, thereby alleviating depressive-like behaviors. Caspase-1 inhibitors, by inhibiting the activation of the NLRP6 / Caspase-1 / Il-1β / Il-18 signaling pathway, also reduced microglial activation and synaptic phagocytosis in the mPFC brain region of the mouse model of depression, thus alleviating depressive-like behaviors.

[0045] This application also provides an application of the mouse model of depression described above in theoretical research on depression.

[0046] This application also provides an application of the mouse model of depression described above in the screening of drugs for depression.

[0047] The following specific examples will provide further details.

[0048] Example 1

[0049] The following mouse strains were used in this embodiment: C57BL / 6 (GemPharmatech); C57BL / 6Smoc-Cx3cr1em1(creERT2-WPRE-polyA)Smoc (Shanghai Model Biotechnology, NM-KI-200157) and C57BL / 6Smoc-Csf1rem1(flox)Smoc (Shanghai Model Biotechnology, NM-CKO-200091). By mating these mouse strains, transgenic mice with conditionally knocked-down Csf1r microglia were obtained. Genotyping of these mice was performed using primers targeting Cx3cr1-Cre and Csf1r-flox, ultimately resulting in a mouse model of depression.

[0050] The correctly identified mice (Cx3cr1-CreERT2; Csf1r) f / + Mice were treated with either tamoxifen (dissolved in corn oil at a concentration of 75 mg / kg) or corn oil via intraperitoneal injection for 5 days. Age-matched C57BL / 6 mice that did not receive any special treatment were designated as the WT group and treated with corn oil-treated Cx3cr1-CreERT2 and Csf1r mice. f / + Mice were labeled as the Cre- group and treated with tamoxifen (Cx3cr1-CreERT2; Csf1r). f / + Mice were labeled the Cre+ group. Behavioral tests were performed on mice in each group at 3 months of age. Depressive-like behaviors were assessed using the elevated cruciate maze, open field test, sucrose preference test, and forced swimming test. The timeline for drug administration and behavioral testing is as follows: Figure 1 As shown in Figure A.

[0051] The results of the depression-like behavioral tests on mice in each group are as follows: Figure 1 As shown in B, C, D, and E. From Figure 1As shown in Figure B, compared with the control and Cre- mice, Cre+ mice exhibited reduced total distance traveled, distance walked in the open arm, time spent in the open arm, and number of times they entered the open arm in the Elevated Plus Maze (EPM) test. Similar results were obtained in the Open Field Test (OFT), where Cre+ mice showed reduced distance traveled in the open field center and reduced proportion of time spent there, while total distance traveled and speed showed no statistically significant difference. In the Sucrose Preference Test (SPT), Cre+ mice showed reduced sucrose intake compared with the control and Cre- mice. In the Forced Swimming Test (FST), Cre+ mice showed a significantly increased time spent pausing in the water compared with the control and Cre- mice. Combined with the above results of depressive-like behavioral tests, it can be seen that the transgenic mice with conditionally knocked-down Csf1r in microglia exhibited significant depressive-like behavior.

[0052] Example 2

[0053] Immunofluorescence staining was used to stain Iba1 and PSD95 cells in the mouse mPFC brain regions obtained in Example 1 to observe the morphological and numerical changes of microglia in this brain region, as well as their co-localization with PSD95. Golgi staining was used to stain neurons in the mouse mPFC brain region to observe the number and morphology of dendritic spines in this brain region. The results are as follows: Figure 2 As shown, values ​​are expressed as mean ± standard deviation (mean ± SEM), ***p < 0.001, ****p < 0.0001.

[0054] from Figure 2 As can be seen from A, the number of microglia branches in the mPFC brain region of Cre+ mice is significantly increased. From Figure 2 As shown in Figures B, C, and D, the microglia coverage area in the mPFC brain region of Cre+ mice is significantly larger than that of Cre- mice. The total length of microglia branches in the mPFC brain region of Cre+ mice is also greater than that of Cre- mice, as is the total number of microglia branches in the mPFC brain region of Cre+ mice. This indicates that, compared to Cre- mice, the microglia in the mPFC brain region of Cre+ mice exhibit a hyperbranched state. Through three-dimensional reconstruction, it can be seen that the number of cell branches is increased, longer, and the coverage area is increased, and the number of colocalizations with PSD95 is also increased. Figure 2 Golgi staining results of E in the middle and Figure 2 Statistical analysis of the number of dendritic spines in the mPFC brain region of Cre+ mice showed that, compared with Cre- mice, Cre+ mice had a significantly reduced number of dendritic spines on neurons in the mPFC brain region. Figure 2As can be seen from G and H, compared with Cre- mice, Cre+ mice showed increased phagocytosis of the postsynaptic protein PSD95 and dendritic spines.

[0055] Example 3

[0056] One week after injection of Tamoxifen or corn oil, Cre+ mice were treated with the Csf1r inhibitor PLX3397 at a dose of 40 mg / kg via gavage, using pure water as a control solvent. This treatment continued for 3 weeks. Subsequently, behavioral tests were performed on the mice in each group (Cre-, Cre+, Cre+_PLX). Depressive-like behaviors were assessed using the elevated cruciate maze, open field test, sucrose preference test, and forced swimming test. The timeline of drug administration and behavioral testing is as follows: Figure 3 As shown in Figure A.

[0057] The results of the depression-like behavioral tests on mice in each group are as follows: Figure 3 As shown in B, C, D, and E. From Figure 3 As shown in Figure B, Cre+ mice treated with PLX3397 exhibited significantly increased total distance traveled, distance walked in the open arm, time spent in the open arm, and number of times they entered the open arm in the elevated cruciate vortex test. Figure 3 As can be seen from C, PLX3397-treated Cre+ mice exhibited increased distance traveled and a higher proportion of dwell time in the open field center. From... Figure 3 As can be seen from the data in Figure D, PLX3397-treated Cre+ mice had increased sucrose intake compared to Cre+ mice. From... Figure 3 The results show that Cre+ mice treated with PLX3397 exhibited a significantly shorter time spent refusing to struggle in water compared to Cre+ mice. These results suggest that the Csf1r inhibitor PLX3397 can effectively alleviate depressive-like behavior induced by conditional knockdown of Csf1r in microglia.

[0058] Example 4

[0059] Immunofluorescence staining was used to stain Iba1 and PSD95 in the mPFC brain regions of mice (Cre-, Cre+, Cre+_PLX) obtained in Example 3 to observe the morphological and numerical changes of microglia in this brain region, as well as their co-localization with PSD95. Golgi staining was used to stain neurons in the mouse mPFC brain region to observe the number and morphology of dendritic spines in neurons in this brain region. The results are as follows: Figure 4 As shown, values ​​are expressed as mean ± standard deviation (mean ± SEM), ***p < 0.001, ****p < 0.0001.

[0060] from Figure 4As shown in Figures A to D, compared to Cre+ mice, the microglia in the mPFC brain region of Cre+ mice treated with PLX3397 exhibited a normal branching pattern and a significantly reduced number. Three-dimensional reconstruction revealed a reduction in cell branching, shortening, and a significantly reduced coverage area, along with a decrease in the number of microglia co-localized with PSD95. Figure 4 Middle E Golgi staining results and Figure 4 Statistical analysis of dendritic spines in the mPFC brain region of Cre+ mice showed that, compared with Cre+ mice, Cre+ mice treated with PLX3397 had a significantly increased number of dendritic spines on neurons in the mPFC brain region. This indicates that conditional knockdown of Csf1r in microglia activates microglia in the mouse mPFC brain region, resulting in a hyperbranched state. The Csf1r inhibitor PLX3397 can effectively alleviate the activation of microglia in the mouse mPFC brain region induced by conditional knockdown of Csf1r and reduce synaptic phagocytosis.

[0061] Example 5

[0062] RNA high-throughput sequencing was performed on primary microglia from the mPFC brain region of Cre- and Cre+ mice to identify differentially expressed genes between the two mouse species. These differentially expressed genes were then screened and enriched to identify related signaling pathways. The results were validated using immunofluorescence and Western blot methods. Figure 5 As shown.

[0063] from Figure 5 Figures A and B show that, compared to Cre- mice, Cre+ mice exhibited predominantly inflammatory activation in the microglia of the mPFC brain region, accompanied by abnormal expression of NOD-like signaling pathways and significant activation of genes within the NOD family, including NLRP6 and NLRP1. Figure C demonstrates, using Western blot analysis, that the NLRP6 / Caspase-1 / Il-1β / Il-18 signaling pathways were significantly activated in the microglia of the mPFC brain region of Cre+ mice. Figure 5 As shown in D, E, F, G, H, and I, immunofluorescence staining of Caspase-1 and co-localization analysis with Iba1, NeuN, and GFAP revealed that Caspase-1 co-localized with Iba1 in most cases in the mPFC brain region of Cre+ mice. This indicates that the NLRP6 / Caspase-1 / Il-1β / Il-18 signaling pathway may be an important mechanism by which conditional knockdown of Csf1r in microglia induces depressive-like behavior in mice, and that activation of this pathway mainly occurs in the microglia of the model mice.

[0064] Example 6

[0065] One week after injection of Tamoxifen or corn oil, Cre+ mice were treated with the Caspase-1 inhibitor VX-765 at a dose of 40 mg / kg via gavage, using purified water as a control solvent. This treatment continued for 3 weeks. Subsequently, behavioral tests were performed on the mice in each group (Cre-, Cre+, Cre+_VX-765). Depressive-like behaviors were assessed using the elevated cruciate maze, open field test, sucrose preference test, and forced swimming test. The timeline of drug administration and behavioral testing is as follows: Figure 6 As shown in Figure A.

[0066] The results of the depression-like behavioral tests on mice in each group are as follows: Figure 6 As shown in B, C, D, and E. From Figure 6 As can be seen from Figure B, VX-765-treated Cre+ mice exhibited significantly increased total distance traveled, distance walked in the open arm, time spent in the open arm, and number of times they entered the open arm in the elevated cruciate vortex test. Figure 6 As can be seen from C, VX-765-treated Cre+ mice exhibited increased distance traveled and a higher proportion of time spent in the open field center. From... Figure 6 As can be seen from D, VX-765-treated Cre+ mice had increased sucrose intake compared to Cre+ mice. From Figure 6 The results show that Cre+ mice treated with VX-765 exhibited significantly less time spent refusing to struggle in water compared to Cre+ mice. These results suggest that the Caspase-1 inhibitor VX-765 can effectively alleviate depressive-like behavior induced by conditional knockdown of Csf1r in microglia.

[0067] Example 7

[0068] Immunofluorescence staining was used to stain Iba1 and PSD95 in the mPFC brain region of mice in each group (Cre-, Cre+, Cre+VX-765) to observe the morphological and numerical changes of microglia in this brain region, as well as their co-localization with PSD95. Golgi staining was used to stain neurons in the mPFC brain region of each group of mice to observe the number and morphology of neuronal dendritic spines in this brain region. Western blot was used to detect molecular proteins related to the NLRP6 / Caspase-1 / Il-1β / Il-18 signaling pathway to observe the effect of VX-765 on this pathway. The results are as follows: Figure 7 As shown, values ​​are expressed as mean ± standard deviation (mean ± SEM), ***p < 0.001, ****p < 0.0001.

[0069] from Figure 7As shown in Figures A to D, compared to Cre+ mice, the microglia in the mPFC brain region of VX-765-treated Cre+ mice exhibited a normal branching pattern and a significantly reduced number. Three-dimensional reconstruction revealed a reduction in cell branching, shortening, and significantly reduced coverage area, along with a decrease in the number of microglia co-localized with PSD95. Figure 7 Middle E Golgi staining results and Figure 7 Statistical analysis of dendritic spines in the mPFC brain region of Cre+ mice showed that, compared with Cre+ mice, VX-765-treated Cre+ mice exhibited a significant increase in dendritic spines on neurons in the mPFC brain region. This indicates that conditional knockdown of Csf1r in microglia activates microglia in the mouse mPFC brain region, resulting in a superbranched state. The caspase-1 inhibitor VX-765 can effectively alleviate the activation of microglia in the mouse mPFC brain region induced by conditional knockdown of Csf1r in microglia and reduce synaptic phagocytosis, which may be related to its effective inhibition of the NLRP6 / Caspase-1 / Il-1β / Il-18 signaling pathway.

[0070] In summary, this application established a mouse model of depression through microglia-specific Csf1r heterozygous knockout and experimentally verified the depressive-like behavior of this mouse model. Compared with existing methods that induce depressive-like behavior in animals through chronic, unpredictable, mild stress CUMS stimulation, this method produces a stable and rapidly developing depressive phenotype, facilitating reproducible experiments in depression-related research and yielding highly reproducible results. Furthermore, experiments demonstrated that microglia-specific Csf1r heterozygous knockout induces microglia activation and increases synaptic phagocytosis in the mouse mPFC brain region, triggering activation of the NLRP6 / caspase-1 / IL-1β / IL-18 pathway in the mPFC brain region. Moreover, treatment of the mouse model with caspase-1 inhibitors and Csf1r inhibitors alleviated its depressive-like behavior, providing a foundation for mechanistic research and drug screening for microglia-related or neuroinflammation-related depression.

[0071] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for constructing a mouse model of depression, characterized in that, The method for constructing the mouse model of depression includes: performing microglia-specific Csf1r heterozygous knockout on mice; The mice were Cre mice; The step of performing microglia-specific Csf1r heterozygous knockout on mice includes: mating wild-type Cre mice, first Cre mice, and second Cre mice to obtain a mouse model of depression; The wild type is the C57BL / 6J mouse with the Wildtype genotype. LoxP sites were inserted on both sides of the exon of the Csf1r gene in the first Cre mouse; The second Cre mouse had a creERT2-WPRE-polyA sequence inserted at the start codon of the Cx3cr1 gene.

2. The method for constructing a mouse model of depression according to claim 1, characterized in that, The method for constructing the mouse model of depression also includes genotyping the mouse model of depression.

3. The method for constructing a mouse model of depression according to claim 2, characterized in that, The genes identified in the genotyping identification include the Cx3cr1-Cre gene and the Csf1r-flox gene.

4. The method for constructing a mouse model of depression according to claim 1, characterized in that, The method for constructing the mouse model of depression also includes evaluating the depression-like behavior of the mouse model of depression.

5. The method for constructing a mouse model of depression according to claim 4, characterized in that, The assessment methods for the depressive-like behavior include at least one of the elevated cross maze test, open field test, sucrose preference test, and forced swimming test.

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