Construction method and application of mouse brain small vessel disease model

Abstract

The invention discloses a construction method and application of a mouse brain small vascular disease model, and belongs to the technical field of mouse brain small vascular disease models. According to the construction method, a Gucy1a3 gene knockout mouse is constructed. The Gucy1a3 gene knockout mouse has high pathological relevance: the Gucy1a3 gene knockout mouse systematically reproduces the core pathological characteristics of the cerebral small vascular disease, including blood brain barrier injury, white matter demyelination and pericyte and endothelial cell dysfunction, meanwhile, the Gucy1a3 gene knockout mouse has no expression of great vasculopathy, and has better repeatability and stability; the mouse cerebral small vascular disease model is suitable for research of cerebral small vascular disease pathogenesis, screening and evaluation of blood brain barrier protection drugs, development of microvascular protective agents or myelin sheath regeneration drugs and verification of related treatment strategies, and has important scientific research value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of mouse cerebral small vessel disease models, and particularly relates to a construction method of a mouse cerebral small vessel disease model and application thereof. BACKGROUND

[0002] Cerebral small vessel disease (CSVD) is a clinical, imaging, and pathological syndrome caused by various causes affecting the small arteries and their distal branches, arterioles, capillaries, venules, and small veins in the brain. As an important type of cerebrovascular disease, CSVD accounts for 30% of all ischemic strokes and is the primary cause of vascular cognitive impairment. Its characteristic pathological changes include blood-brain barrier damage, white matter damage, pericyte / endothelial cell dysfunction, etc. With the acceleration of population aging and the high incidence of metabolic-related diseases, the incidence of CSVD continues to rise and has become an important factor in the global burden of neurological diseases in the elderly. Therefore, establishing a reliable CSVD animal model to in-depth analyze the pathogenesis of CSVD has important practical significance for promoting the prevention and treatment of the disease.

[0003] Current CSVD animal models mainly include low perfusion animal models and chronic hypertension animal models. These models can reflect the characteristics of CSVD to some extent, but still have the following defects: 1. Difficulty in inducing multiple CSVD pathological changes simultaneously: For example, the low perfusion model (bilateral carotid artery stenosis) only simulates ischemic white matter damage and cannot effectively simulate pericyte / endothelial cell coupling disorders; the chronic hypertension animal model only simulates blood-brain barrier damage.

[0004] 2. Non-specific interference: For example, the spontaneously hypertensive rat model can simulate hypertension-related CSVD lesions, but may also be accompanied by other hypertension-related systemic changes (such as large vessel disease), which interferes with the independent study of the core mechanism of CSVD; 3. Complexity of operation: Exogenous injury models (such as stereotactic drug injection) require invasive surgery, have large individual differences, and the success rate of the model and the consistency of pathological performance are unstable. SUMMARY

[0005] The application provides a construction method of a mouse CSVD model and application thereof. By constructing a Gucy1a3 KO (knockout) mouse model, the key pathological characteristics of CSVD such as blood-brain barrier damage, white matter demyelination, and pericyte / endothelial cell dysfunction are systematically reproduced, and large vessel disease is avoided, which exhibits significant advantages in the accuracy of CSVD pathological simulation, the stability of the phenotype, and the applicability of mechanism research.

[0006] To solve the above technical problems, the present application adopts the following technical solutions: A construction method of a mouse CSVD model, the construction method comprising knocking out a Gucy1a3 gene of the mouse.

[0007] Further, the Gucy1a3 KO mouse is constructed by CRISPR / Cas9 gene editing technology.

[0008] Further, the target point of the Gucy1a3 KO is the 5th exon.

[0009] Further, the construction method comprises the following steps: S1, designing and synthesizing a target site sequence targeting the Gucy1a3 gene of the mouse; S2, using CRISPR / Cas9 gene editing technology, respectively preparing sgRNA and Cas9 mRNA by in vitro transcription, and microinjecting into mouse zygote pronuclei; S3, culturing the embryo of step S2 to the blastocyst stage, and then pseudopregnant transplantation to a pseudopregnant female mouse, and the born mouse is F0 generation; S4, expanding and identifying the F0 generation mouse; S5, mating the F0 generation mouse with a wild type C57BL / 6J mouse to obtain F1 generation heterozygote mice, and then obtaining F2 generation homozygote mouse strains by self-crossing the F1 generation mice.

[0010] Further, when identifying the constructed mouse CSVD model, at least one of the following is identified: blood-brain barrier integrity, white matter lesions, pericyte function, and endothelial cell reduction.

[0011] Further, when identifying the constructed mouse CSVD model, intracranial large vessel lesions should also be identified.

[0012] The present application also provides an application of a mouse CSVD model, and the mouse CSVD model constructed by the present application is applied to the research of the pathogenesis of CSVD.

[0013] The present application also provides an application of a mouse CSVD model, and the mouse CSVD model constructed by the present application is applied to the screening or evaluation of blood-brain barrier protection drugs.

[0014] The present application also provides an application of a mouse CSVD model, characterized in that the mouse CSVD model constructed by the present application is applied to the development of microvascular protective agents or myelin regeneration drugs.

[0015] Compared with the prior art, the present application has the following beneficial effects: 1. High physiological relevance: Gucy1a3 KO mice systematically recapitulate the core pathological features of CSVD, including blood brain barrier impairment (increased Evans blue extravasation, tight junction opening), white matter demyelination (thinning of corpus callosum, abnormal LFB staining) and pericyte dysfunction (decreased expression of markers such as PDGFRβ), endothelial cell dysfunction (decreased cell proliferation and migration ability), without large vessel pathology.

[0016] 2. Reproducibility and stability: Gucy1a3 KO mouse model achieves genotype consistency through stable passage, with consistent and stable pathological phenotypes. No traumatic operation is required for the mice, avoiding surgical stress and other factors, reducing the experimental period and ensuring the reproducibility of the experiment.

[0017] 3. Wide application potential: suitable for the study of CSVD pathogenesis, drug target verification and treatment strategy development, with important scientific research value. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0019] Figure 1 Construction and verification of Gucy1a3 KO mice.

[0020] Figure 2 Blood brain barrier impairment (increased Evans blue extravasation, abnormal expression of ZO1 and MMP-9 protein, electron microscopy results suggesting tight junction damage). Figure 3 Corpus callosum LFB staining shows white matter demyelination.

[0021] Figure 4 Decreased expression of pericyte markers PDGFRβ, Desmin and CD146 protein.

[0022] Figure 5 CD31 immunohistochemistry shows decreased cortical vascular density. Figure 6 Gucy1a3 gene knockdown inhibits the proliferation and migration ability of mouse brain microvascular endothelial cells.

[0023] Figure 7 No stenosis or occlusion of intracranial large vessels (magnetic resonance angiography, blue latex perfusion and ink perfusion angiography).

[0024] Figure 8Intracranial large artery histopathology detection: hematoxylin and eosin (HE) staining, elastic van Gieson (EVG) staining and alpha-smooth muscle actin (α-SMA) immunohistochemical staining. DETAILED DESCRIPTION

[0025] In order to better understand the present application, the following examples are used in conjunction with the accompanying drawings, which are within the scope of the present application, but do not limit the scope of the present application.

[0026] EMBODIMENT A method for constructing a mouse CSVD model, the construction method being knocking out a Gucy1a3 gene of the mouse.

[0027] The Gucy1a3 KO mouse is constructed by CRISPR / Cas9 gene editing technology.

[0028] The target of the Gucy1a3 KO is the 5th exon.

[0029] The construction method comprises the following steps: S1, designing and synthesizing a target site sequence targeting a Gucy1a3 gene of a mouse; S2, using CRISPR / Cas9 gene editing technology, respectively preparing sgRNA and Cas9 mRNA by in vitro transcription, and microinjecting into a mouse pronucleus; S3, culturing the embryo of step S2 to the blastocyst stage, and then pseudopregnant transplantation to a pseudopregnant female mouse, and the born mouse is F0 generation; S4, expanding and identifying the F0 generation mouse.

[0030] S5, mating the F0 generation mouse with a wild type C57BL / 6J mouse to obtain F1 generation heterozygote mice, and then obtaining F2 generation homozygote mouse strains by self-crossing the F1 generation mice.

[0031] When identifying the constructed mouse CSVD model, at least one of the following is identified: blood-brain barrier integrity, white matter lesions, pericyte function, and endothelial cell function.

[0032] When identifying the constructed mouse CSVD model, intracranial large vessel lesions should also be identified.

[0033] Application of a mouse CSVD model, the mouse CSVD model constructed by the present application is applied to research the pathogenesis of CSVD.

[0034] The application of the mouse CSVD model, the mouse CSVD model constructed by the application is applied to screening or evaluation of blood-brain barrier protection drugs.

[0035] The application of the mouse CSVD model, characterized in that the mouse CSVD model constructed by the application is applied to development of microvascular protection agents or myelin regeneration drugs.

[0036] Test I. Construction of mouse model The mouse strain used is C57BL / 6J.

[0037] S1, design and synthesis of target site sequence targeting mouse Gucy1a3 gene (target site sequence and identification primer see Table 1); Table 1. Mouse Gucy1a3 gRNA target site sequence and identification primer sequence

[0038] S2, using CRISPR / Cas9 gene editing technology, respectively preparing sgRNA and Cas9 mRNA by in vitro transcription, and injecting Cas9 mRNA / sgRNA into mouse zygote pronucleus in a 1:1 ratio by microinjection; S3, the embryos of step S2 are cultured to the blastocyst stage, and then are transferred to a pseudopregnant mother mouse for gestation, and the born mice are F0 generation; S4, F0 generation mice are expanded and identified; S5, F0 generation mice are mated with wild type C57BL / 6J mice to obtain F1 generation heterozygous mice, and then F2 generation homozygous mouse strain is obtained by self-crossing of F1 generation mice.

[0039] The above steps involve microinjection technology, embryo culture, and F0 generation mouse breeding, expansion, F1 generation breeding, F2 generation breeding, etc., which are prior art and are not the innovation points of the present application, and will not be described here.

[0040] II. Identification of mouse model 1. Detection instruction: each item of model identification uses existing equipment, such as enzyme label instrument, electrophoresis instrument, transmission electron microscope, optical microscope, dissecting microscope, small animal magnetic resonance imaging instrument, etc., the model, installation, use, etc. of the equipment are not the innovation points of the present application, and will not be described here; the reagents, auxiliary instruments, mouse disposal methods, cell culture methods, etc. used in each detection item are existing products / technologies, which meet the relevant experimental use requirements.

[0041] Statistical analysis of each detection item was completed using SPSS 25.0 software. Each item was independently performed at least 3 biological repeats (n≥3), and the data were expressed as mean ± standard deviation (SD); two-tailed independent sample t test was used for comparison between two groups; the statistical significance threshold was set as a = 0.05, and the statistical significance was marked with an asterisk in the description related to the drawings: *P<0.05, **P<0.01, ***P<0.001.

[0042] 2. Construct a Gucy1a3 KO mouse model using CRISPR / Cas9 technology, breed the mice to the F2 generation, and confirm the successful model construction by gene sequencing and WB. Genotype identification of mouse tail: cut the mouse tail tissue to extract DNA, and then perform Sanger sequencing on the PCR amplification product. Select the genotype peak chart results for listing.

[0043] The obtained peak chart results are shown in Figure 1 (A-B): A single peak appears in the Gucy1a3 KO mouse gene sequence, indicating a -13 bp homozygous mutant. The sGCα1 protein expression of Gucy1a3 KO mice and wild type (Wild Type, WT) mice was detected by Western Blot after extracting brain tissue protein, and the results are shown in Figure 1 (C-D).

[0044] 3. Evans blue exudation experiment: Gucy1a3 KO mice and WT mice were intravenously injected with 4% Evans blue (1 mL / kg), 2 h later, 40 ml PBS was used to clear the intravascular dye by heart perfusion, the brain tissue was weighed and placed in 3 mL formamide, 60°C constant temperature water bath for 24 h, 4000 r / min centrifugation for 15 min, then the absorbance of supernatant at 620 / 630 nm was measured, and the dye content (μg / g) was calculated.

[0045] As can be seen from Figure 2 , the amount of Evans blue exudation can reflect the integrity of the blood-brain barrier in mice, and the more exudation, the more severe the barrier damage. As can be seen from Figure 2 (A-B), the exudation amount of the Gucy1a3 KO group was significantly higher than that of the WT group, indicating that Gucy1a3 KO can cause blood-brain barrier damage. 4. Transmission electron microscopy: After perfusion with 4% paraformaldehyde + 4% glutaraldehyde mixed solution, the brain tissues of Gucy1a3 KO mice and WT mice were separated, and samples were taken within 1-3 min. During sampling, mechanical damage such as tissue traction, bruising and extrusion should be minimized. The size of the sampling tissue is 1 mm 3; Brain tissue blocks were immediately immersed in 2.5% glutaraldehyde for 2h (4°C overnight fixation); the tissue blocks were removed, washed with 1x PBS, fixed with 1% osmium acid for 2h; washed with 1x PBS; dehydrated with gradient ethanol-acetone, permeated and polymerized with acetone-embedding agent, and after ultrathin section, double stained with uranium-lead, and observed under electron microscope for tight junction and myelin ultrastructure.

[0046] Figure 2 C The results showed that there was no obvious abnormality in the ultrastructure of the blood-brain barrier in the WT group, while the tight junction structure was damaged in the Gucy1a3 KO group, and some were in an open state.

[0047] 5. Western blot detection of related proteins: brain tissue proteins of Gucy1a3 KO mice and WT mice were extracted, transferred after SDS-PAGE electrophoresis, incubated with ZO1, MMP9, PDGFRβ, Desmin, CD146 primary antibodies, and developed with secondary antibodies, and the gray value of the band was quantified by ImageJ.

[0048] From Figure 2 As can be seen from (D-E), Z01, Occludin, Claudin5 are key proteins for maintaining the tight junction of the blood-brain barrier, and MMP-9 is a matrix metalloproteinase, and its abnormal expression suggests barrier damage. The results showed that compared with the WT group, the expression of Z01 protein in the Gucy1a3 KO group was significantly reduced, and the expression of MMP-9 protein was significantly increased, indicating that Gucy1a3 KO can cause damage to the blood-brain barrier. From Figure 4 (A-B) shows the expression of pericyte markers (PDGFR-β, CD146 and Desmin) in mouse brain, and the results show that compared with the WT group, the expression of pericyte markers in the Gucy1a3 KO group is significantly decreased. It is indicated that Gucy1a3 may play a role in maintaining the expression of pericyte-related proteins and pericyte function, and its deletion may affect the level of pericyte markers, which may further affect the function of pericytes and related physiological processes, such as blood-brain barrier-related functions. 6. LFB staining and analysis: brain tissue sections of Gucy1a3 KO mice and WT mice were deparaffined with xylene, gradient ethanol rehydrated, and then immersed in LFB staining solution at 60°C for 4h, then washed; 70% ethanol differentiation to gray / white matter, dehydrated and transparent, and then mounted with neutral resin, and the myelin sheath thickness of the corpus callosum was measured under a microscope, and the staining intensity was analyzed by Image software.

[0049] Figure 3 (A) shows that the myelin sheath in the corpus callosum is blue-purple under the microscope, and the background is light purple, and the outline of the corpus callosum is clear. Figure 3(B-C) showed that compared with WT group, Gucy1a3 KO group corpus callosum thinned and could be seen lighter color, suggesting that Gucy1a3 KO group appeared myelin integrity damage, white matter demyelination changes. 7. CD31 immunohistochemistry: after paraffin section antigen repair, anti-CD31 primary antibody (1:200) was incubated overnight at 4°C, and DAB was developed. Five cortical fields were randomly selected for each sample, and the number and total length of CD31 positive blood vessels were counted to calculate the vascular density (vessel length / field area).

[0050] Figure 5 The results of CD31 immunohistochemical staining (CD31 labeled vascular endothelial cells, reflecting the distribution of blood vessels). Figure 5 (A) is a mouse brain section and local enlargement (box area), the results show that the vascular staining density in the Gucy1a3 KO group is lower than that in the WT group, suggesting that the number or distribution of blood vessels is reduced.

[0051] Compared with the WT group, the vascular density in the Gucy1a3 KO group in the cerebral cortex of the mouse was reduced, and the vascular diameter was also quantitatively analyzed, and the results showed that the brain microvessel vascular diameter in the Gucy1a3 KO group was smaller than that in the WT group. The results showed that the loss of Gucy1a3 gene function could inhibit the generation and remodeling of small blood vessels in the cerebral cortex.

[0052] 8. Evaluation of the proliferation and migration ability of mouse brain microvascular endothelial cells after Gucy1a3 gene knockdown: mouse brain microvascular endothelial cells (Punox bEnd.3, item number: CL-0598) were cultured, and after the cell state was stable, siRNA technology was used to transfect cells for Gucy1a3 gene knockdown, and a negative control group (Negative control, NC) transfected with non-targeting siRNA was set up.

[0053] Figure 6 (A) shows that Western blot was used to detect the protein expression level of Gucy1a3 to verify the gene knockdown efficiency. Subsequently, EdU and flow cytometry experiments were used to evaluate the cell proliferation ability, Figure 6 (B-C) showed that Gucy1a3 knockdown significantly inhibited the proliferation of brain microvascular endothelial cells; in addition, Transwell migration experiment and scratch healing experiment were further used to analyze the cell migration ability, Figure 6 (D-E) showed that the migration ability of Gucy1a3 knockdown group was significantly weaker than that of the control group. The above results showed that Gucy1a3 gene knockdown could effectively inhibit the proliferation and migration ability of mouse brain microvascular endothelial cells.

[0054] 9. Magnetic resonance imaging and perfusion staining evaluation of intracranial large vessels: Magnetic resonance angiography: Gucy1a3 KO mice and WT mice were fasted and anesthetized with isoflurane, fixed in supine position and monitored physiological parameters. After positioning scan, parameters were set (repetition time 100-500 ms, echo time 10-30 ms, flip angle 30°-90°, slice thickness 0.5-1.5 mm, matrix ≥ 256 x 256) for high resolution scan. After scan, three-dimensional vascular images were reconstructed, and the diameters of internal carotid artery, middle cerebral artery and basilar artery were measured to evaluate the vascular condition.

[0055] Blue latex / carbon black gelatin solution perfusion: After anesthesia, the heart was exposed by opening the chest, and the aorta was cannulated. The blood was first flushed out with heparin saline (liver turned white as a sign). According to the ratio of 60% latex: 20% ammonia water: 1 blue pigment / India ink, 10% gelatin solution was mixed and slowly perfused at 0.5-1 mL (pressure <20 kPa). The blood vessel tree was shaped by freezing on ice for 30 min, and the brain tissue was taken for fixation and observed under a microscope.

[0056] Figure 7 (A) Magnetic resonance angiography. No stenosis or occlusive changes of intracranial large vessels were found in the magnetic resonance angiography results of the two groups of mice; Figure 7 (B-C) Blue latex solution and ink gelatin solution perfusion, respectively, directly showing the three-dimensional structure of blood vessels. The results showed that there was no significant difference in the anatomical structure of the basilar artery of the two groups of mice, and no stenosis or occlusion of intracranial large vessels was observed. Therefore, magnetic resonance angiography, latex perfusion, and ink perfusion all verified that Gucy1a3 KO did not cause stenosis or occlusion of intracranial large vessels.

[0057] 10. Intracranial large artery histopathology examination: After the Gucy1a3 KO mice and WT mice were sacrificed, the brain tissues were taken and fixed with 4% paraformaldehyde for 48 h. The bifurcation of the middle cerebral artery, the end of the internal carotid artery and the end of the basilar artery were located under the body microscope, and three parts of brain tissue containing blood vessels were cut along the coronal plane. After dehydration and paraffin embedding, 4 μm thick sections were cut every 30 μm for subsequent staining. HE staining: deparaffinization and hydration, hematoxylin nuclear staining (5-15 min), eosin cytoplasmic staining (2-5 min), gradient alcohol dehydration and xylene transparency, and finally neutral resin mounting. After staining, the cell nucleus is blue and the cytoplasm is pink, and the basic pathological changes of the tissue can be clearly observed; EVG elastic fiber staining: used to show the distribution of elastic fibers in the blood vessel wall. Key technical links include potassium permanganate oxidation (5-10 min), oxalic acid bleaching, EVG staining solution (15-30 min), and Van Gieson restaining (5-10 min). After staining, the elastic fibers are dark blue, the collagen fibers are red, and the muscle tissue is yellow, which can accurately evaluate the integrity of the elastic layer of the blood vessel; α-SMA immunohistochemical staining by antigen repair, anti-SMA primary antibody (4°C overnight), secondary antibody (30-60 min) incubation, 3,3'-diaminobenzidine (DAB) color development (3-10 min), etc. specifically label vascular smooth muscle cells. After staining, the cytoplasm of α-SMA positive cells is brownish yellow, and the nucleus is located by hematoxylin restaining, which can be used for quantitative analysis of the distribution and thickness of the vascular media smooth muscle.

[0058] Figure 8 (A) HE staining results show that the intima of the blood vessels in the Gucy1a3 KO group mice is composed of 1-2 layers of endothelial cells, and there is no fibrous thickening, which has no significant difference with the WT group. Figure 8 (B) EVG staining shows that compared with the WT group, the elastic layer of the blood vessels in the Gucy1a3 KO group mice is complete, without abnormal changes such as rupture, redundancy or stratification. Figure 8 (C) The results of α-SMA immunohistochemical staining show that the media smooth muscle structure of the blood vessels in the Gucy1a3 KO group mice is complete, and the thickness has no significant difference compared with the WT group, and there is no media thinning phenomenon. In summary, the Gucy1a3 KO group mice do not show pathological changes of intracranial large arteries.

[0059] The above tests and the annotations in the figure part all adopt the conventional way in the art, and the abbreviations of experimental groups / control groups, indicators, etc. are all in the conventional way, which will not be repeated here.

[0060] It is to be noted that, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" can include a combination of two or more components, and the term "an element" can include comparable reference to a plurality of elements. Also, as used in this specification and the appended claims, the term "or" as used in the context of "A / B or C" means any of the following: A; B; or C. Also, the term "comprising" as used in the claims should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Rather, the term "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof.

[0061] The foregoing is considered as illustrative only of the principles of the application. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and practice described. Accordingly, all such variations are intended to be included within the scope of present application as defined in the following claims.

Claims

1. A method for constructing a mouse model of cerebral small vessel disease, characterized by, The construction method comprises knocking out a Gucy1a3 gene of a mouse.

2. The method according to claim 1, wherein the method is characterized by, The Gucy1a3 gene knockout mouse is constructed by a CRISPR / Cas9 gene editing technology.

3. The method according to claim 2, wherein the mouse model of cerebral small vessel disease is constructed by, The target of the Gucy1a3 gene knockout is the 5th exon.

4. The method according to claim 3, wherein the mouse model of cerebral small vessel disease is constructed by, The method comprises the following steps: S1. Designing and synthesizing a target site sequence targeting a Gucy1a3 gene of a mouse; S2. Using a CRISPR / Cas9 gene editing technology, preparing sgRNA and Cas9 mRNA by in vitro transcription, and microinjecting into a mouse pronucleus; S3. Culturing the embryo of step S2 to a blastocyst stage, and then performing pseudopregnant transplantation to a pseudopregnant female mouse, and the born mouse is an F0 generation; S4. Propagating the F0 generation mouse and identifying; S5. Crossing the F0 generation mouse with a wild-type C57BL / 6J mouse to obtain an F1 generation heterozygote mouse, and then obtaining an F2 generation homozygote mouse strain by self-crossing the F1 generation mouse.

5. The method according to claim 4, wherein the mouse model of cerebral small vessel disease is constructed by, When the constructed mouse cerebral small vessel disease model is identified, at least one of the following is identified: Blood-brain barrier integrity, white matter lesion, pericyte function, endothelial cell function.

6. The method according to claim 5, wherein the mouse model of cerebral small vessel disease is constructed by, When the constructed mouse cerebral small vessel disease model is identified, intracranial large vessel lesion should also be identified.

7. Use of a mouse model of cerebral small vessel disease, characterized in that, The mouse cerebral small vessel disease model constructed by the construction method of any one of claims 1-6 is applied to research the pathogenesis of cerebral small vessel disease.

8. Use of a mouse model of cerebral small vessel disease, characterized in that, The mouse cerebral small vessel disease model constructed by the construction method of any one of claims 1-6 is applied to screening or evaluation of blood-brain barrier protection drugs.

9. Use of a mouse model of cerebral small vessel disease, characterized in that, The mouse cerebral small vessel disease model constructed by the construction method of any one of claims 1-6 is applied to the development of microvascular protection agents or myelin regeneration drugs.

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