Chronic hypoperfusion animal model construction method and application thereof

Abstract

The invention provides a chronic hypoperfusion animal model construction method and application thereof, and relates to the technical field of biomedicine. The construction method of the chronic hypoperfusion animal model, provided by the invention, aims at brain capillary endothelial cells, and the constructed chronic hypoperfusion animal model is global and lasting brain hypoperfusion, has no infection or death risk and is low in cost. The animal model constructed by using the method is more convincing in the aspects of reperfusion research and drug curative effect evaluation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for constructing a chronic hypoperfusion animal model and application thereof. Background Art

[0002] Chronic cerebral hypoperfusion (CCH) is a major cause of cerebral small vessel disease and a major pathogenic factor for cognitive decline and neurodegeneration. Currently, methods for establishing CCH models in rats and mice include bilateral common carotid artery occlusion (BCCAO), bilateral common carotid artery stenosis (BCAS), bilateral vertebral artery + bilateral common / internal carotid artery ligation (4VO), gradual common carotid artery stenosis (GCAS), and asymmetric common carotid artery stenosis (ACAS). Among these, the microspring BCAS method is the most widely used in clinical practice.

[0003] However, the above technical solution has the following disadvantages:

[0004] Microsprings are expensive and cannot be scanned with MRI. Using ex vivo brain tissue for MRI examinations prevents further experiments and can result in discrepancies between test results and actual results. Existing methods require neck surgery on mice, which carries a risk of mortality, and the inflammatory response during recovery can affect other experimental results. Current hypoperfusion models used in mice are generally focal and temporary.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The first purpose of the present invention is to provide a method for constructing a chronic hypoperfusion animal model to solve the above technical problems.

[0007] The second purpose of the present invention is to provide an application of the chronic hypoperfusion animal model constructed by the above construction method in drug efficacy evaluation.

[0008] In order to achieve the above objectives, the following technical solutions are adopted:

[0009] In a first aspect, the present invention provides a method for constructing a chronic hypoperfusion animal model, wherein genetic engineering technology is used to prevent the expression of the Emc10 gene in experimental animals or to inhibit its expression.

[0010] As a further technical solution, the construction method includes using genetic engineering technology to prevent the second exon sequence of the Emc10 gene from being expressed or suppressing its expression in experimental animals.

[0011] As a further technical solution, the genetic engineering technology is gene editing technology.

[0012] As a further technical solution, the gene editing technology is CRISPR / Cas technology.

[0013] As a further technical solution, the construction method includes:

[0014] Emc10 gene knockout fertilized eggs were constructed using the CRISPR / Cas system, and the fertilized eggs were cultured to obtain F0 generation heterozygous experimental animals; the F0 generation heterozygous experimental animals were then mated with wild-type experimental animals to obtain F1 generation heterozygous experimental animals; the F1 generation heterozygous experimental animals were then mated and screened to obtain homozygous experimental animals with Emc10 gene knockout. The homozygous experimental animals with Emc10 gene knockout are the chronic hypoperfusion animal model.

[0015] As a further technical solution, the screening method includes PCR detection.

[0016] As a further technical solution, the experimental animals include mice, rats, rabbits or dogs.

[0017] In a second aspect, the present invention provides the use of the chronic hypoperfusion animal model constructed by the above construction method in the evaluation of drug efficacy.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The method for constructing a chronic hypoperfusion animal model provided by this invention targets brain capillary endothelial cells. The resulting chronic hypoperfusion animal model exhibits global and persistent cerebral hypoperfusion without the risk of infection or death, and is low-cost. The animal model constructed using this invention is more convincing in reperfusion studies and drug efficacy evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Schematic diagram for constructing the mouse Emc10 knockout model;

[0022] Figure 2 Emc10 deficiency causes reduced cerebral blood flow in mice. A. Three-dimensional reconstruction of the entire mouse brain vasculature shows that Emc10 knockout results in reduced vessel diameters across all brain regions. Iso: neocortex; OLF: olfactory region; HPF: hippocampal formation; CTXsp: cortical floor plate; STR: striatum; PAL: globus pallidus; TH: thalamus; HY: hypothalamus; MB: midbrain; HB: hindbrain; CB: cerebellum. Scale bar = 1000 μm. B. Laser speckle imaging (LSI) shows that cerebral blood flow in the KO mouse cerebral cortex is reduced to 75% of that in the wild-type. Blood perfusion was measured using a blood perfusion imager based on laser speckle contrast analysis (LASCA). Seven mice were used, half male and half female. Statistics were performed using an unpaired t-test. **: p < 0.01; ***: p < 0.001. C. Transmission electron microscopy (TEM) shows a significant decrease in the cross-sectional area of ​​cerebral vessels in the KO mouse cerebral cortex, while the cross-sectional area of ​​endothelial cells remains unchanged. The number of mice was 5, half male and half female; 2-9 electron microscopy images containing complete microvascular cross-sections were selected for each sample for statistical analysis; unpaired t-test was used for statistical analysis; ns: statistically not significant; *: p < 0.05. Scale bar = 5 μm;

[0023] Figure 3The mouse cerebral cortex showed hypoperfusion, but no obvious leakage was found. A. Immunofluorescence analysis of cerebral cortical sections from 8-week-old mice showed significantly reduced microvascular perfusion and weakened endothelial cell signals in Emc10 homozygous knockout mice. Red: EBA (specifically labeled cerebral endothelial cells); Purple: EB (plasma protein marker, indicating blood perfusion); Blue: DAPI (labeled nuclei). Six WT mice and six KO mice (one died). Unpaired t-test was used for statistical analysis. **: p < 0.01, scale bar = 100 μm; B. Immunofluorescence analysis of cerebral cortical sections from 8-week-old mice showed significantly reduced cerebral microvascular perfusion and weakened endothelial cell signals in homozygous Emc10 knockout mice; Green: FITC-dextran (indicating cerebral blood perfusion, 10 kDa); Yellow: CD31 (endothelial cells); Blue: DAPI (labeling nuclei); WT and KO mice were analyzed in 4 mice each; unpaired t-test was used for statistical analysis; **: p < 0.01, scale bar = 50 μm;

[0024] Figure 4 KO mice showed decreased physical coordination and depression. The rotarod test (A) and tail suspension test (B) showed that the performance of Emc10 knockout homozygous mice was significantly worse than that of wild-type mice. Unpaired t-test was used for statistics. ns: statistically not significant; #: p < 0.05; ##: p < 0.01.

[0025] Figure 5 A. Cerebral blood flow (CBF) in the mouse cerebral cortex was measured using a blood perfusion imager based on laser speckle contrast analysis (LASCA) technology. CBF in the mouse cerebral cortex was quantitatively analyzed, and the results are shown in the right figure. Values ​​are expressed as mean ± standard error. ns: no significant difference, *p < 0.05, ***p < 0.001, by Student's t-test. B. Representative immunofluorescence images of CD31 staining in the cerebral cortex of WT, KO, and scEMC10-supplemented KO mice. Scale bar = 30 μm. Aging markers in the right figure were quantitatively analyzed using Imaris software. Values ​​are expressed as mean ± standard error. ns: no significant difference, *p < 0.05, **p < 0.01, by Student's t-test.

[0026] Figure 6: A. Representative western blotting images of MBP protein expression in the cortex of WT and KO mice. The images on the right are the results of ImageJ quantitative analysis. The values ​​are plotted as mean ± SEM. The unpaired t-test was used for statistics. **p < 0.01. n = 6 mice. B. Representative immunofluorescence images of MBP staining in WT and KO mice. Red: MBP, blue: DAPI. Black and white images are the results of ImageJ analysis. Scale bar = 200 μm. A' and A": central and central brain regions, B' and B": cerebral cortex and ccb region, cing: cingulate gyrus, ccb: corpus callosum, alv: hippocampal nerve fiber, scwm: supracallosal white matter. The images on the right are the results of ImageJ quantitative analysis. The values ​​are plotted as mean ± SEM and normalized to the WT group. The results were analyzed by Student's t-test, p<0.05, n=4 mice; C. Representative images of LFB staining in WT and KO mice, blue: LFB, purple: H&E staining, scale bar = 2500 μm, A' and A": central and central brain regions; B' and B": cerebral cortex and ccb region, cing: cingulate gyrus, ccb: corpus callosum, alv: hippocampal nerve fiber, scwm: supracallosal white matter, the right image is the result of ImageJ quantitative analysis, the values ​​are plotted as mean ± SEM and normalized to the WT group, Student's t-test, p<0.05, n=4 mice;

[0027] Figure 7A. Representative western blotting images of MBP protein expression in WT, KO, and KO-supplemented scEMC10 mice. The right image shows quantification of the results using ImageJ. Values ​​are plotted as mean ± SEM and were compared with Student's t-test. ns: not statistically significant. *p < 0.05, **p < 0.01. n = 6 mice. B. Representative immunofluorescence images of MBP staining in WT, KO, and KO-supplemented scEMC10 mice. Red: MBP; blue: DAPI. Black-and-white images analyzed using ImageJ. Scale bar = 1000 μm. A', A", A'': cingulate gyrus of the corpus callosum; B', B", B'': cerebral cortex and ccb. The right image shows quantification of myelin density using ImageJ. Values ​​are plotted as mean ± SEM and normalized to those in the WT + PBS group. Student's t-test. ns: not statistically significant. *p < 0.05. n = 4 mice; C. Representative images of LFB staining in WT, KO, and KO-supplemented scEMC10 mice. Blue: local feedback; purple: HE staining. Scale bar = 2500 μm. A', A", A'': cingulate gyrus of the corpus callosum; B', B", B'': cerebral cortex and CCB. Right: Quantification of LFB density using ImageJ. Values ​​are plotted as mean ± SEM and normalized to those in the WT + PBS group. Student's t-test, ns: not statistically significant, *p < 0.05. n = 4 mice. DETAILED DESCRIPTION

[0028] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0029] In a first aspect, the present invention provides a method for constructing a chronic hypoperfusion animal model, wherein genetic engineering technology is used to prevent the expression of the Emc10 gene in experimental animals or to inhibit its expression.

[0030] The method for constructing a chronic hypoperfusion animal model provided by this invention targets brain capillary endothelial cells. The resulting chronic hypoperfusion animal model exhibits global and persistent cerebral hypoperfusion without the risk of infection or death, and is low-cost. The animal model constructed using this invention is more convincing in reperfusion studies and drug efficacy evaluation.

[0031] In some optional embodiments, the construction method includes using genetic engineering technology to prevent the second exon sequence of the Emc10 gene from being expressed or suppressing its expression in experimental animals.

[0032] Those skilled in the art may also choose to knock out or silence other exons of the Emc10 gene so that the Emc10 gene is not expressed or its expression is suppressed in experimental animals.

[0033] In some optional embodiments, the genetic engineering technology is gene editing technology.

[0034] In some optional embodiments, the gene editing technology is CRISPR / Cas technology.

[0035] In some optional embodiments, the construction method includes:

[0036] Emc10 gene knockout fertilized eggs were constructed using the CRISPR / Cas system, and the fertilized eggs were cultured to obtain F0 generation heterozygous experimental animals; the F0 generation heterozygous experimental animals were then mated with wild-type experimental animals to obtain F1 generation heterozygous experimental animals; the F1 generation heterozygous experimental animals were then mated and screened to obtain homozygous experimental animals with Emc10 gene knockout. The homozygous experimental animals with Emc10 gene knockout are the chronic hypoperfusion animal model.

[0037] In some optional embodiments, the screening method includes but is not limited to PCR detection.

[0038] In some optional embodiments, the experimental animals include but are not limited to mice, rats, rabbits or dogs, or other experimental animals well known to those skilled in the art.

[0039] In a second aspect, the present invention provides the use of the chronic hypoperfusion animal model constructed by the above construction method in the evaluation of drug efficacy.

[0040] The drugs include drugs for treating cerebral small vessel diseases.

[0041] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0042] Example 1

[0043] 1. Experimental Methods

[0044] 1.1 Construction of Emc10 knockout mouse model

[0045] All mouse experiments in this study were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University (registration numbers 2019-436, 2019-694). In this study, wild-type or Emc10 knockout mice were from the C57BL / 6J strain and provided by Saiye Biotechnology (C57BL / 6JCya-Emc10em2 / Cya). All mice were raised in a specific pathogen-free (SPF) facility. The CRISPR / Cas system was used to construct fertilized eggs of C57BL / 6 mice with the Emc10 gene knockout. By embryo transplantation, F0 generation heterozygous mice were constructed, and after mating with wild-type mice, F1 generation heterozygous mice were generated. After mating, the F1 generation heterozygous mice were screened for homozygous mice, i.e., Emc10 - / - mice (KO mice), heterozygous mice were designated as Emc10 + / - Mouse genotypes were determined by PCR using the following thermal cycling conditions: 94°C for 30 seconds (denaturation), 60°C for 30 seconds (annealing), and 72°C for 90 seconds (extension), for 35 cycles. In subsequent experiments, mice were 7 weeks old, with half male and half female, unless otherwise specified.

[0046] 1.2 Transmission electron microscopy

[0047] Mice were anesthetized with ketamine (80 mg / kg)-thiazide (5 mg / kg), and the heart was perfused with pre-cooled PBS to obtain blood. The heart was then perfused with pre-cooled electron microscopy fixative. After perfusion, the skin was cut open, the skull was peeled off with forceps, and the brain was removed and immersed in electron microscopy fixative. The anterior cortex of the mouse brain was separated and cut into 1 mm 3 Small pieces of tissue were collected. The tissue blocks were immersed in electron microscopy fixative at 4°C and stored in the dark. The tissue was fixed with 2.5% glutaraldehyde. After dehydration with ethanol, the tissue was embedded in LX112 resin (LADD Research Industries) and sliced ​​into ultrathin sections using an ultramicrotome (EMUC7 (Leica, Germany)), and then stained with uranyl acetate and lead citrate. All grids were observed under a 200 kV transmission electron microscope (Tecnal G2 20TWIN, FEI, USA). After thresholding, morphometric analysis was performed using ImageJ on 15-20 different micrographs for each case.

[0048] 1.3 Laser Speckle Contrast Imaging for Monitoring Cerebral Blood Flow

[0049] Eight mice of different genotypes, 7 weeks old, half male and half female, were enrolled in each group. Laser speckle contrast imaging (LSCI) was used to monitor cerebral blood flow (CBF). LSCI is a technique based on speckle contrast analysis for full-field imaging of blood flow. [5-7]Before, each mouse was anesthetized with ketamine (80 mg / kg)-thiazide (5 mg / kg), the skin and tissue around the skull were removed, and then placed in a stereotaxic frame. A 4 × 4 mm cranial burr was carefully opened along the sagittal suture above the right parietal bone. 2 A cranial window was created for the cerebral cortex, and the surface was frequently rinsed with cold saline to avoid brain damage. In this study, changes in CBF were measured in WT and KO mice using a perfusion speckle imager (Perimed, Stockholm, Sweden). A laser non-contact probe was positioned approximately 20 cm above the parietal cortex region of the brain. The dynamics and spatial distribution of CBF were then recorded for 5 minutes.

[0050] 1.4 Immunocytochemistry

[0051] All solutions were prepared in PBS (GIBCO, Cat#10-010-023). At the indicated time points, samples were fixed with 4% paraformaldehyde (Thermo Scientific, Cat#28906) for 15 minutes. Subsequently, the aldehyde-free group was treated with 0.1 M glycine, and the cells were washed three times with PBS. After permeabilization with 0.3% Triton X-100 for 20 minutes, the cells were washed three times with PBS. The cells were then incubated with blocking solution (10% goat serum (Beyotime, Cat#C0265)) for 30 minutes. The primary antibody (EBA 1:100) was added in 10% blocking solution for 1 hour. After four washes in PBS, the cells were incubated with the appropriate Alexa Fluor secondary antibody (anti-mouse 488; anti-rabbit 594, 1:500) for 30 minutes. The cells were then counterstained with 4,0,6-diamidino-2-phenylindol (DAPI, 0.5 mg / ml, Thermo Scientific, Cat#62248) for 10 minutes. Finally, the cells were washed three more times in PBS and then mounted on microscope slides using Fluoromount (Sigma, Cat# F4680-25). Images were analyzed using ImageJ analysis software (NIH, USA). To quantify fluorescence intensity, the mean grayscale value of the region of interest was measured and background was subtracted to obtain fluorescence intensity. High-resolution imaging was performed using an Airyscan super-resolution confocal microscope and a Leica SP8 confocal microscope, and analyzed using ImageJ analysis software.

[0052] 1.5 Mouse fatigue rod (rotarod) test

[0053] Training: The mice were placed on a fatigue rod with a specific speed and exercised for 300 seconds for training. The training was conducted twice, with an interval of more than 15 minutes between each training.

[0054] Formal experiment: Place mice on a rotarod with rotation speeds of 30, 35, and 40 rpm, respectively. Record the duration of the mice's movement on the rotarod at different speeds. If the duration exceeds 300 s, it will be recorded as 300 s. Measure each speed three times, with an interval of more than 15 minutes between each measurement. An interval of more than 30 minutes should be maintained before changing the speed.

[0055] 1.6 Mouse tail suspension test

[0056] Gently remove the animal from its cage and secure its tail as quickly as possible, avoiding any unnecessary stress or strain. Suspend the animal's tail from the apparatus's suspension rod using medical tape 1 cm from the tip of the tail. The height between the tip of the tail and the ground should be approximately 30 cm, placing the mouse in a head-down position. The total recording duration for the experiment is 200 seconds.

[0057] 2. Experimental Results

[0058] The diameters of blood vessels in various brain regions of KO mice and wild-type mice were detected. The results showed that after knocking out the Emc10 gene, the diameters of blood vessels in various brain regions of mice decreased, and the areas with reduced blood vessel diameters in KO mice were mainly concentrated in the motor area, taste area, visceral area, lateral visual area, posterior parietal association area, temporal association area of ​​the neocortex, the anterior hypothalamus and hypothalamus of the hippocampus, and the cerebellum ( Figure 2 A).

[0059] To investigate the effects of microglial-derived Emc10 deficiency on mammalian cerebral vasculature, we conducted a comprehensive series of studies. First, laser speckle contrast analysis (LASCA) was used to study cerebral blood flow in these mice. The results showed that compared with wild-type mice, the KO mice had a significant decrease in cerebral cortical blood flow by 75% ( Figure 2 Transmission electron microscopy (TEM) analysis of the inner diameter of tissue blood vessels showed that the diameter of microvessels in the cerebral cortex of KO mice was reduced by 75% compared with that of WT mice, while the area of ​​endothelial cells did not change significantly ( Figure 2 C).

[0060] Endothelial barrier antigen (EBA) is a protein specifically expressed by endothelial cells, known as "barrier protein", and is used as a marker for the integrity of the blood-brain barrier. After Evans Blue (EB) perfusion and EBA labeling treatment of 8-week-old KO mice (6, half male and half female) and wild-type mice (6, half male and half female), immunofluorescence analysis of mouse cerebral cortical sections showed that brain perfusion volume and endothelial cell signals were significantly reduced in Emc10 KO mice. However, no significant changes in blood-brain barrier permeability were observed ( Figure 3To determine whether small molecule dextran can penetrate microvessels, we perfused the hearts of 8-week-old KO mice (4 mice, half male and half female) and wild-type mice (4 mice, half male and half female) with FITC-labeled 10kDa dextran and stained endothelial cells with the endothelial cell marker CD31. The results showed that there was no vascular leakage in the KO mouse cerebral cortex and endothelial cell signaling was reduced ( Figure 3 B) in.

[0061] 8-week-old homozygous mice Emc10 - / - Mice (8, half male and half female), heterozygous mice Emc10 + / - The rotarod test and tail suspension test were performed on KO mice (6, half male and half female) and wild-type mice (6, half male and half female). The results showed that the neurobehavioral performance of KO mice also declined. Among them, the rotarod test showed that the motor coordination and endurance of KO mice were significantly lower than those of wild-type mice ( Figure 4 A in the figure); the tail suspension experiment showed that the KO mice showed no signs of struggling for a long time, indicating that the mice showed a tendency to depression ( Figure 4 These are all direct evidences obtained by applying the present invention that chronic cerebral hypoperfusion causes damage to the mammalian nervous system.

[0062] KO mice were given scEMC10 (0.5 μg / kg) twice a day, morning and evening, for 14 consecutive days (KO mice were supplemented with scEMC10), and a control group of KO mice supplemented with PBS and a control group of wild-type mice supplemented with PBS were set up (8 mice in each group, half male and half female). The blood flow in the cerebral cortex of the mice was then tested. The results showed that the reduction in cerebral cortical blood flow in KO mice was improved two weeks after the tail vein injection of secretory EMC10 recombinant protein. Figure 5 A).

[0063] Three KO mice were supplemented with scEMC0, three wild-type mice were supplemented with PBS, and four KO mice were supplemented with PBS. CD31 immunofluorescence staining analysis of the cerebral cortex of these mice showed that the cross-sectional area of ​​blood vessels increased significantly after two weeks of supplementation with secretory EMC10 recombinant protein ( Figure 5 B) in.

[0064] One of the negative effects of reduced cerebral blood flow is damage to the white matter. Previous studies have shown that the structural integrity of white matter is particularly susceptible to vascular damage caused by chronic hypoperfusion. To examine the effects of chronic hypoperfusion caused by EMC10 deficiency on brain damage, we systematically examined coronal sections covering the temporal cortex of WT and KO mice using histological methods to observe changes in the expression of axonal and myelin protein markers. Western blotting analysis showed that EMC10 deficiency led to a loss of MBP protein in the KO mouse brain ( Figure 6 Myelin basic protein (MBP) is a myelin fiber marker. Immunofluorescence also showed that EMC10 deficiency induced demyelination in the cingulate bundle (cing) and supracallosal white matter (scwm) regions. LFB staining also confirmed that the white matter in the cing and scwm regions of KO mice was damaged ( Figure 6 B, C).

[0065] Next, we injected scEMC10 into KO mice aged 12-18 weeks via the tail vein for 14 days. After supplementation with scEMC10, MBP protein levels in the cortex were also increased ( Figure 7 In addition, MBP and LFB staining of mouse brain sections showed that demyelination in the cing and scwm regions, which are supplied by long perforating artery branches, was inhibited after replenishment of scEMC10 ( Figure 7 B, C), in summary, the supplementation of scEMC10 can improve the demyelination caused by brain white matter lesions.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a chronic hypoperfusion animal model, characterized in that: Through genetic engineering technology, the Emc10 gene is not expressed or its expression is inhibited in experimental animals.

2. The construction method according to claim 1, characterized in that The construction method includes using genetic engineering technology to make the second exon sequence of the Emc10 gene not expressed or inhibited in experimental animals.

3. The construction method according to claim 1 or 2, characterized in that The genetic engineering technology is gene editing technology.

4. The construction method according to claim 3, characterized in that The gene editing technology is CRISPR / Cas technology.

5. The construction method according to claim 4, characterized in that The construction method comprises: Emc10 gene knockout fertilized eggs were constructed using the CRISPR / Cas system, and the fertilized eggs were cultured to obtain F0 generation heterozygous experimental animals; the F0 generation heterozygous experimental animals were then mated with wild-type experimental animals to obtain F1 generation heterozygous experimental animals; the F1 generation heterozygous experimental animals were then mated and screened to obtain homozygous experimental animals with Emc10 gene knockout. The homozygous experimental animals with Emc10 gene knockout are the chronic hypoperfusion animal model.

6. The construction method according to claim 5, characterized in that: The screening method includes PCR detection.

7. The construction method according to claim 1, characterized in that The experimental animals include mice, rats, rabbits or dogs.

8. Use of the chronic hypoperfusion animal model constructed by the construction method according to any one of claims 1 to 7 in drug efficacy evaluation.

Images