Method for constructing chronic hypoperfusion animal model and application thereof

Abstract

The application provides a chronic hypoperfusion animal model construction method and application thereof, and relates to the field of biomedical technology. The chronic hypoperfusion animal model construction method provided by the application is aimed at brain capillary endothelial cells, and the chronic hypoperfusion animal model constructed by the method is global and persistent brain hypoperfusion, has no risk of infection or death, and has low cost. The animal model constructed by the application is more convincing in reperfusion research and drug efficacy evaluation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for constructing a chronic hypoperfusion animal model and its application. Background Technology

[0002] Chronic cerebral hypoperfusion (CCH) is a major cause of small vessel disease in the brain and a leading cause of cognitive decline and neurodegenerative changes. 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 micro-spring BCAS method is the most widely used clinically.

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

[0004] Microsprings are expensive and cannot be scanned using MRI; using ex vivo brain tissue for MRI examination makes it impossible to continue the experiment, and the detection results may differ from the actual results; existing methods require neck surgery on mice, which carries a risk of death, and the inflammatory response during the recovery process may affect other experimental results. Currently, hypoperfusion models using mice as experimental subjects are all focal and transient.

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

[0006] The primary objective of this invention is to provide a method for constructing a chronic hypoperfusion animal model to solve the aforementioned technical problems.

[0007] The second objective of this invention is to provide the application of the chronic hypoperfusion animal model constructed by the above-described method in the evaluation of drug efficacy.

[0008] To achieve the above objectives, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a method for constructing a chronic hypoperfusion animal model, which uses genetic engineering technology to make the Emc10 gene not expressed or its expression suppressed in experimental animals.

[0010] As a further technical solution, the construction method includes using genetic engineering techniques to prevent or suppress the expression of the second exon sequence of the Emc10 gene 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 zygotes were constructed using the CRISPR / Cas system, and F0 generation heterozygous experimental animals were obtained by culturing the zygotes. Then, the F0 generation heterozygous experimental animals were mated with wild-type experimental animals to obtain F1 generation heterozygous experimental animals. After mating the F1 generation heterozygous experimental animals, homozygous Emc10 gene knockout experimental animals were obtained. The homozygous Emc10 gene knockout experimental animals 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] Secondly, this invention provides the application of the chronic hypoperfusion animal model constructed by the above-mentioned 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 brain hypoperfusion without the risk of infection or death, and is low in cost. Animal models constructed using this invention are more convincing in reperfusion studies and drug efficacy evaluations. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 A schematic diagram illustrating the construction of a mouse model for knocking out the Emc10 gene.

[0022] Figure 2 Emc10 knockout caused reduced cerebral blood flow in mice. A. Three-dimensional reconstruction of the entire cerebral blood vessels in mice showed that after the Emc10 gene was knocked out, the diameter of blood vessels in various brain regions decreased. Iso: neocortex; OLF: olfactory region; HPF: hippocampal structure; CTXsp: cortical lamina; STR: striatum; PAL: globus pallidus; TH: thalamus; HY: hypothalamus; MB: midbrain; HB: hindbrain; CB: cerebellum; scale bar = 1000 μm; B. Laser speckle imaging showed that the cerebral cortex blood flow in KO mice was reduced to 75% of that in wild-type mice. Blood perfusion was measured using a blood perfusion imaging system based on laser speckle contrast analysis (LASCA). The number of mice was 7, half male and half female. Unpaired t-test was used for statistical analysis. **: p < 0.01; ***: p < 0.001. C. Transmission electron microscopy (TEM) showed that the cross-sectional area of ​​the cerebral cortex blood vessels in KO mice was significantly reduced, while the cross-sectional area of ​​endothelial cells did not change significantly. Five mice were used, half male and half female. Two to nine electron micrographs containing complete microvascular cross-sections were selected from each sample for statistical analysis. Unpaired t-tests were used. ns: not statistically significant; *: p < 0.05. Scale bar = 5 μm.

[0023] Figure 3Hypoperfusion was observed in the cerebral cortex of mice, with no obvious leakage. Immunofluorescence of cerebral cortical sections from 8-week-old mice showed significantly reduced microvascular perfusion and weakened endothelial cell signaling in homozygous Emc10 knockout mice. Red: EBA (specifically labeled cerebral vascular endothelial cells); Purple: EB (plasma protein marker, indicating blood perfusion); Blue: DAPI (labeled nucleus). Six WT mice and six KO mice (one died) were included. Statistical analysis was performed using an unpaired t-test. **: p<0.01, scale bar = 100 μm; Immunofluorescence of cerebral cortex sections from B. 8-week-old mice showed significantly reduced cerebral microvascular perfusion and weakened endothelial cell signaling in homozygous Emc10 knockout mice; green: FITC-glucan (indicating cerebral blood flow perfusion, 10 kDa); yellow: CD31 (endothelial cells); blue: DAPI (labeled nucleus); WT and KO mice were counted in groups of 4 each; unpaired t-tests were used for statistical analysis; **: p<0.01, scale bar = 50 μm;

[0024] Figure 4 KO mice exhibited decreased physical coordination and depressive tendencies; mouse rotarod test (A) and tail suspension test (B) showed that the performance of Emc10 gene knockout homozygous mice was significantly worse than that of wild-type mice; unpaired t-test was used for statistical analysis; ns: not statistically significant; #: p<0.05; ##: p<0.01;

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

[0026] Figure 6A. Representative Western blotting images of cortical MBP protein expression in WT and KO mice. The right image shows the results of ImageJ quantitative analysis. Values ​​are plotted as mean ± SEM. Statistical analysis was performed using an unpaired t-test. **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 fibers, SCWM: supra-corpus callosum white matter. The right image shows the results of ImageJ quantitative analysis. Values ​​are plotted as mean ± SEM and normalized to the WT group. t-test, p<0.05, n=4 mice; Representative images of LFB staining in C.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 fibers, SCWM: supra-corpus callosum white matter. The right image is the result of ImageJ quantitative analysis. The values ​​are plotted as mean ± SEM and normalized with reference 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 the quantitative analysis of the results using ImageJ. Values ​​are plotted as mean ± SEM. Student's t-test showed no statistical significance (ns: *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. ImageJ was used to analyze the black and white images. Scale bar = 1000 μm. A', A”, A”': cingulate gyrus of the corpus callosum; B', B”, and B”': cerebral cortex and ccb region. The right image shows the quantitative analysis of myelin density using ImageJ. Values ​​are plotted as mean ± SEM and normalized to the WT+PBS group. Student's t-test showed no statistical significance (ns: *p<0.05). n = 4 mice; C.WT, KO, and KO supplemented scEMC10 mice with representative images of LFB staining, blue: local feedback, purple: he staining, scale bar = 2500 μm, A', A”, A”': cingulate gyrus of the corpus callosum, B', B”, and B”': cerebral cortex and ccb region; the right figure shows the quantitative analysis of LFB density using ImageJ. Values ​​are plotted as mean ± SEM and normalized to the WT+PBS group. Student's t-test showed no statistical significance in ns, *p < 0.05. n = 4 mice. Detailed Implementation

[0028] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] In a first aspect, the present invention provides a method for constructing a chronic hypoperfusion animal model, which uses genetic engineering technology to make the Emc10 gene not expressed or its expression suppressed in experimental animals.

[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 brain hypoperfusion without the risk of infection or death, and is low in cost. Animal models constructed using this invention are more convincing in reperfusion studies and drug efficacy evaluations.

[0031] In some alternative embodiments, the construction method includes using genetic engineering techniques to prevent or suppress the expression of the second exon sequence of the Emc10 gene 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 alternative implementations, the genetic engineering technology is gene editing technology.

[0034] In some alternative implementations, the gene editing technology is CRISPR / Cas technology.

[0035] In some alternative implementations, the construction method includes:

[0036] Emc10 gene knockout zygotes were constructed using the CRISPR / Cas system, and F0 generation heterozygous experimental animals were obtained by culturing the zygotes. Then, the F0 generation heterozygous experimental animals were mated with wild-type experimental animals to obtain F1 generation heterozygous experimental animals. After mating the F1 generation heterozygous experimental animals, homozygous Emc10 gene knockout experimental animals were obtained. The homozygous Emc10 gene knockout experimental animals are the chronic hypoperfusion animal model.

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

[0038] In some alternative implementations, 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] Secondly, this invention provides the application of the chronic hypoperfusion animal model constructed by the above-mentioned construction method in the evaluation of drug efficacy.

[0040] The drugs include those used to treat small vessel diseases of the brain.

[0041] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0042] Example 1

[0043] 1. Experimental Methods

[0044] 1.1 Construction of a mouse model with Emc10 gene knockout

[0045] All mouse experiments in this study were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University (Registration Nos. 2019-436, 2019-694). In this study, wild-type or Emc10 knockout mice were derived from the C57BL / 6J strain and provided by Cyagen Biosciences (C57BL / 6JCya-Emc10em2 / Cya). All mice were housed in a pathogen-specific free (SPF) facility. Emc10 knockout C57BL / 6 mouse zygotes were constructed using the CRISPR / Cas system. F0 generation heterozygous mice were generated through embryo transfer and mated with wild-type mice to produce F1 generation heterozygous mice. The F1 generation heterozygous mice were then mated, and homozygous mice, i.e., Emc10 mice, were selected. - / - Mice (KO mice), heterozygous mice are designated Emc10 + / - The mouse genotype was determined by PCR under the following thermal cycling conditions: 94℃ for 30 seconds (denaturation), 60℃ for 30 seconds (annealing), 72℃ for 90 seconds (extension), for 35 cycles. Unless otherwise specified, all mice used in subsequent experiments were 7 weeks old, with an equal number of males and females.

[0046] 1.2 Transmission Electron Microscopy

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

[0048] 1.3 Laser speckle contrast imaging for monitoring cerebral blood flow

[0049] Eight mice of different genotypes, each group, were 7 weeks old and consisted of half males and half females. Changes in cerebral blood flow (CBF) were monitored using laser speckle contrast imaging (LSCI), a technique based on speckle contrast analysis used for full-field imaging of blood flow. [5-7]Previously, each mouse was anesthetized with ketamine (80 mg / kg) and thiazide (5 mg / kg), and the skin and tissue around the skull were removed before being placed in a stereotactic frame. Above the right parietal bone, a 4×4 mm opening was carefully made along the sagittal suture using a cranial drill. 2 A cranial window was created in the cerebral cortex, and the surface was frequently rinsed with cold saline to avoid damaging the brain. In this study, changes in brain fat flow (CBF) in WT and KO mice were measured using a perfusion speckle imaging system (Perimed, Stockholm, Sweden). A laser-guided non-contact probe was positioned approximately 20 cm above the parietal cortex. 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 specified time points, samples were fixed with 4% paraformaldehyde (Thermo Scientific, Cat#28906) for 15 minutes. Subsequently, the free aldehyde group was treated with 0.1M glycine and washed three times with PBS. After infiltration with 0.3% Triton X-100 for 20 minutes, cells were washed three more times with PBS. Cells were then incubated with blocking solution (10% goat serum (Beyotime, Cat#C0265)) for 30 minutes. Primary antibody (EBA 1:100) was added to 10% blocking solution for 1 hour. After rinsing four times in PBS, cells were incubated with appropriate Alexa Fluor secondary antibodies (anti-mouse 488; anti-rabbit 594, 1:500) for 30 minutes. Then, reverse staining with 40,6-diamino-2-phenylindophenol (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 onto microscope slides using Fluoromount (Sigma, Cat#F4680-25). Images were analyzed using ImageJ analysis software (NIH, USA). To quantify fluorescence intensity, the average gray value of the region of interest was measured and the background was subtracted to obtain the fluorescence intensity. High-resolution imaging was performed using an Airyscan super-resolution confocal microscope with a Leica SP8 confocal microscope, and the images were analyzed using ImageJ analysis software.

[0052] 1.5 Mouse fatigue bar (rotatable bar) test

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

[0054] Formal experiment: The mice were placed on rotators at speeds of 30, 35, and 40 rpm, and the duration of continuous movement of the mice on the rotators at different speeds was recorded. If the duration exceeded 300 seconds, it was recorded as 300 seconds. Each speed was measured 3 times, with an interval of more than 15 minutes between each measurement. There was an interval of more than 30 minutes before changing the speed.

[0055] 1.6 Mouse tail suspension test

[0056] Gently remove the animal from the cage and quickly secure its tail, avoiding any unnecessary stress or pressure on the mouse. Specifically, use medical tape to secure the tail 1 cm from the tip, suspending the animal's tail from the suspension rod. 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 experimental time is recorded for 200 seconds.

[0057] 2. Experimental Results

[0058] The diameter of blood vessels in various brain regions of KO mice and wild-type mice was measured. The results showed that knocking out the Emc10 gene reduced the diameter of blood vessels in various brain regions of mice. The areas of reduced blood vessel diameter in KO mice were mainly concentrated in the motor cortex, gustatory cortex, visceral cortex, lateral visual cortex, posterior parietal cortex, and cortical temporal cortex; the precolon and inferior colliculus of the hippocampus; and the cerebellum. Figure 2 (A) in the middle.

[0059] To investigate the effects of microglia-derived Emc10 deficiency on mammalian cerebral blood vessels, 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 to wild-type mice, KO mice exhibited a significant 75% reduction in cortical blood flow. Figure 2 (B in the text). Transmission electron microscopy (TEM) analysis of tissue vascular diameter showed that, compared with WT mice, KO mice had a 75% reduction in the diameter of cortical microvessels, while the endothelial cell area remained largely unchanged. Figure 2 (C in the middle).

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

[0061] 8-week-old homozygous mice Emc10 - / - Mice (8 in total, half male and half female), heterozygous mice Emc10 + / - Six KO mice (half male and half female) and six wild-type mice (half male and half female) were used in rotarod and tail suspension experiments. The results showed that the neurobehavioral performance of KO mice was also reduced. Specifically, the rotarod experiment revealed that KO mice had significantly lower motor coordination and endurance compared to wild-type mice. Figure 4 (A) The tail suspension test showed that KO mice showed signs of not struggling for a long time, indicating that the mice had a depressive tendency. Figure 4 (B) These are all direct evidence obtained by applying this invention of the damage caused by chronic cerebral hypoperfusion to the mammalian nervous system.

[0062] KO mice were administered scEMC10 (0.5 μg / kg) twice daily for 14 consecutive days (KO mice were re-supplemented with scEMC10). Control groups included KO mice re-supplemented with PBS and wild-type mice re-supplemented with PBS (n=8 per group, half male and half female). Cerebral cortical blood flow was then measured. Results showed that the reduced cerebral cortical blood flow in KO mice was improved two weeks after intravenous injection of re-supplementation with secretory EMC10 recombinant protein via the tail vein. Figure 5 (A) in the middle.

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

[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 vulnerable to vascular injury caused by chronic hypoperfusion. To examine the impact of chronic hypoperfusion induced by EMC10 deficiency on brain injury, we systematically examined the coronal plane covering the temporal cortex of WT and KO mice using histological methods, observing changes in the expression of axonal and myelin protein markers. Western blotting analysis showed that EMC10 deficiency led to the loss of MBP protein in the brains of KO mice (…). Figure 6 (A) Myelin basic protein (MBP) is a marker of myelin fibers. Immunofluorescence also shows that EMC10 deficiency in the cingulate tract and the white matter region of the corpus callosum induces demyelination. LFB staining results also confirm white matter damage in the cing and scwm regions of KO mice. Figure 6 (B and C in the text).

[0065] Next, we injected scEMC10 into KO mice aged 12-18 weeks via tail vein for 14 days. Following scEMC10 supplementation, the level of MBP protein in the cortex also increased. Figure 7 (A in the text). Furthermore, MBP and LFB staining of mouse brain slices showed that demyelination in the cing and scwm regions, supplied by branches of long perforating arteries, was inhibited after scEMC10 refill (…). Figure 7 In summary, scEMC10 supplementation can improve demyelination caused by white matter lesions in the brain.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions 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, The construction method includes: Built using the CRISPR / Cas system Emc10 Gene knockout zygotes were obtained and cultured to obtain F0 generation heterozygous experimental animals; then, the F0 generation heterozygous experimental animals were mated with wild-type experimental animals to obtain F1 generation heterozygous experimental animals; and finally, the F1 generation heterozygous experimental animals were mated and screened to obtain... Emc10 Homozygous gene knockout laboratory animals Emc10 Homozygous gene knockout experimental animals are the animal models of chronic hypoperfusion. The experimental animal was a mouse.

2. The construction method according to claim 1, characterized in that, The screening methods include PCR detection.

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