Method for constructing an acute hypoperfusion animal model and applications thereof

Abstract

The application provides a construction method of an acute low-perfusion animal model and application thereof, and relates to the technical field of biomedicine. The construction method of the acute low-perfusion animal model of the application is aimed at brain capillary endothelial cells, and the animal model constructed is globally and durably low-perfused in brain microvessels, and the animal model has no infection or death risk and low cost. The animal model constructed by the application has important significance for researching the molecular mechanism of central nervous system injury caused by acute cerebral ischemia and developing a stroke treatment method.

Description

Technical Field

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

[0002] Cerebral stroke, also known as apoplexy, includes ischemic and hemorrhagic strokes. Stroke is a group of diseases caused by brain tissue damage due to blockage of blood vessels, preventing blood flow to the brain. Based on this, there are many methods and types for preparing acute cerebral ischemia models, mainly including vascular occlusion, open craniotomy with mechanical occlusion, mechanical compression cortical infarction, and suture occlusion. Furthermore, based on the severity and location of ischemia, it is classified as focal ischemia or global cerebral ischemia.

[0003] However, the above model construction method still has the following drawbacks:

[0004] Routine skin preparation is required for rats, and the entire surgical procedure must be kept warm; it is easy to puncture the subarachnoid vessels, causing intracranial hemorrhage; postoperative care of the animals is necessary to prevent serious infection; currently, hypoperfusion models using mice as experimental subjects are all focal and temporary.

[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 an acute hypoperfusion animal model to solve the aforementioned technical problems.

[0007] The second objective of this invention is to provide the application of the acute hypoperfusion animal model constructed by the above-described method in screening drugs for the treatment of stroke.

[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 an acute hypoperfusion animal model, which uses genetic engineering technology to prevent or suppress the expression of the Emc10 gene in microglia or monocytes / macrophages of experimental animals.

[0010] As a further technical solution, the construction method includes using genetic engineering technology to prevent or suppress the expression of the second exon sequence of the Emc10 gene in microglia or monocytes / macrophages of experimental animals.

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

[0012] As a further technical solution, the genetic engineering technology is any one or a combination of gene knockout technology and RNA interference technology.

[0013] As a further technical solution, the gene knockout technology is Cre-loxp gene knockout technology.

[0014] As a further technical solution, the construction method includes:

[0015] Animal A was constructed with the CreERT2 gene inserted at the 3' end of the Tmem119 gene; animal B was constructed with the Loxp sequence inserted on both sides of the second exon of the Emc10 gene; animal C was obtained by crossing animal A and animal B to obtain animal C carrying both the CreERT2 gene and the Loxp sequence.

[0016] Alternatively, construct experimental animal D with the CreERT2 gene inserted at the 3' end of the Cx3cr1 gene; construct experimental animal E with the Loxp sequence inserted on both sides of the second exon of the Emc10 gene; and cross experimental animal D and experimental animal E to obtain experimental animal F that carries both the Cx3cr1 gene and the Loxp sequence.

[0017] As a further technical solution, the construction method is to achieve conditional knockout of the Emc10 gene by activating the CreERT2 protein.

[0018] As a further technical solution, the CreERT2 protein is activated by tamoxifen.

[0019] As a further technical solution, the experimental animals include mice, rats, rabbits, or dogs.

[0020] Secondly, this invention provides the application of the acute hypoperfusion animal model constructed by the above-mentioned construction method in screening drugs for the treatment of stroke.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a method for constructing an acute hypoperfusion animal model targeting brain capillary endothelial cells. The resulting animal model exhibits global and persistent microvascular hypoperfusion, with no risk of infection or death, and is cost-effective. The animal model constructed using this invention is of great significance for studying the molecular mechanisms of central nervous system injury caused by acute cerebral ischemia and for developing treatments for stroke. Attached Figure Description

[0023] 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.

[0024] Figure 1 The study investigated how microglia-derived Emc10 knockout induced decreased cerebral blood flow in mice. A. Laser speckle imaging showed that 7 days after conditional Emc10 gene knockout in microglia, blood perfusion was measured using a laser speckle contrast analysis (LASCA)-based blood perfusion imaging system. The mouse population was 6-12, with an equal number of males and females. Unpaired t-tests were used for statistical analysis. **: p < 0.01; ***: p < 0.001. B. Transmission electron microscopy (TEM) showed that after Emc10 gene knockout in microglia, the cross-sectional area of ​​cortical vessels was significantly reduced, while the cross-sectional area of ​​endothelial cells showed no significant change. The mouse population was 6-7, with an equal number of males and females. 2-9 TEM images containing complete microvascular cross-sections were selected from each sample for statistical analysis. Unpaired t-tests were used for statistical analysis. ns: not statistically significant; *: p < 0.05; ***: p < 0.001; Scale bar = 5 μm.

[0025] Figure 2 The results showed hypoperfusion in the cerebral cortex of mice. A. Immunofluorescence of cerebral cortex sections from 7-week-old mice showed reduced microvascular perfusion in the brain after Emc10 gene conditional knockout. Blue: DAPI (labeled nucleus); Red: EB (specifically referring to cerebral blood flow perfusion). Statistical analysis was performed using the unpaired t-test. *: p<0.05; Scale bar = 100 μm. B. Immunofluorescence of cerebral cortex sections from 7-week-old mice showed decreased EBA expression in brain microvascular endothelial cells after Emc10 gene conditional knockout, while EBA expression in microglia showed no significant change. Blue: DAPI (labeled nucleus); Green: IBA1 (specifically labeled microglia); Red: EBA (specifically referring to brain vascular endothelial cells), EB (specifically referring to cerebral blood flow perfusion). Statistical analysis was performed using the unpaired t-test. *: p<0.05; Scale bar = 100 μm.

[0026] Figure 3 After conditional knockout of microglia (Emc10), iNOS and CD206 staining results showed that the microglia phenotype did not change 7 days after Emc10 knockout; green: IBA1 (specifically labeled microglia); red: iNOS (M1 macrophage marker); purple: CD206 (M2 macrophage marker); scale bar = 200 μm; unpaired t-test was used for statistical analysis; ns: no statistical significance; *: p < 0.05, **: p < 0.01, ***: p < 0.001; Bio-Plex analysis of chemokines and cytokines in the cerebral cortex of B.cWT and cKO mice, 8 mice in each group, cKO(T): cKO-Tmem119 mice, cKO(Cx): cKO-Cx3cr1 mice;

[0027] Figure 4 A. Western blot detection of MBP expression in the cortex of cWT and cKO mice, quantified as shown on the right, *P<0.05, **P<0.01, Student's t-test; N=4 mice; B. Immunofluorescence of MBP (red) and DAPI (blue) in the cingulate tract, scwm of the corpus callosum, and internal capsule, scale bar, 200 μm; C. Luxol Fast Blue (LFB) staining of white matter tracts in cWT and cKO mice, scale bar, 2500 μm; D. Quantification of myelin density (r) and LFB density (s) by ImageJ, ns, not significant, *P<0.05, Student's t-test; MBP staining n=3, LFB staining n=4. 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 an acute hypoperfusion animal model, which uses genetic engineering technology to prevent or suppress the expression of the Emc10 gene in microglia or monocytes / macrophages of experimental animals.

[0030] This invention provides a method for constructing an acute hypoperfusion animal model targeting brain capillary endothelial cells. The resulting animal model exhibits global and persistent microvascular hypoperfusion, with no risk of infection or death, and is cost-effective. The animal model constructed using this invention is of great significance for studying the molecular mechanisms of central nervous system injury caused by acute cerebral ischemia and for developing treatments for stroke.

[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 microglia or monocytes / macrophages of 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 is suppressed in microglia or monocytes / macrophages of experimental animals.

[0033] In some alternative implementations, the genetic engineering technology is a gene editing technology, such as CRISPR / Cas9 technology.

[0034] In some alternative implementations, the genetic engineering technology is any one or a combination of gene knockout technology and RNA interference technology.

[0035] In some alternative implementations, the gene knockout technology is the Cre-loxp gene knockout technology.

[0036] In some alternative implementations, the construction method includes:

[0037] Animal A was constructed with the CreERT2 gene inserted at the 3' end of the Tmem119 gene; animal B was constructed with the Loxp sequence inserted on both sides of the second exon of the Emc10 gene; animal C was obtained by crossing animal A and animal B to obtain animal C carrying both the CreERT2 gene and the Loxp sequence.

[0038] Alternatively, construct experimental animal D with the CreERT2 gene inserted at the 3' end of the Cx3cr1 gene; construct experimental animal E with the Loxp sequence inserted on both sides of the second exon of the Emc10 gene; and cross experimental animal D and experimental animal E to obtain experimental animal F that carries both the Cx3cr1 gene and the Loxp sequence.

[0039] In some alternative implementations, the construction method involves conditionally knocking out the Emc10 gene by activating the CreERT2 protein.

[0040] In some alternative implementations, the CreERT2 protein is activated via tamoxifen.

[0041] 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.

[0042] Secondly, this invention provides the application of the acute hypoperfusion animal model constructed by the above-mentioned construction method in screening drugs for the treatment of stroke.

[0043] 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.

[0044] Example

[0045] 1. Experimental Methods

[0046] 1.1 Construction of a mouse conditional knockout model of Emc10 gene

[0047] Two types of conditional knockout Emc10 C57BL / 6 mice (cKO) were constructed using the cre-loxp system: microglia-specific Emc10 knockout mice and monocyte-macrophage-specific Emc10 knockout mice.

[0048] Cre mouse construction: The C57BL / 6-Tmem119-2A-CreERT2 mouse (Tmem119: microglia marker gene) was obtained by adding the CreERT2 sequence to the 3' end of the Tmem119 gene; the C57BL / 6-Cx3cr1-2A-Creert2 mouse (Cx3cr1: monocyte / macrophage marker gene) was obtained by adding the CreERT2 sequence to the 3' end of the Cx3cr1 gene. CreERT2 mice can express the Cre enzyme and ERT2 fusion protein.

[0049] Loxp mouse construction: The Loxp sequence was added flanking the second exon of the Emc10 gene to obtain C57BL / 6-Emc10-floxed (exon 2) mice. When Cre mice were crossed with Loxp mice to produce mice carrying both the CreERT2 and loxp gene sequences (Flox... + / + Tmem119 + Mice and Flox + / + Cx3cr1 + In mice, double-positive mice express the CreERT2 protein. After activation of this protein via intraperitoneal injection of tamoxifen (20 μg / μL, injection dose 5 μL / g), the CRE enzyme removes the exon 2 gene sequence of the Emc10 gene sandwiched between two Loxp fragments by cleaving the Loxp fragment. Therefore, the Cre-Loxp system can be used to specifically knock out the Emc10 gene in microglia or monocytes / macrophages.

[0050] 1.2 Transmission Electron Microscopy

[0051] 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. 3Small tissue blocks were prepared 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.

[0052] 1.3 Laser speckle contrast imaging for monitoring cerebral blood flow

[0053] Eight mice of different genotypes, each 7 weeks old, half male and half female, were used. Changes in cerebral blood flow (CBF) were monitored using laser speckle contrast imaging (LSCI), a technique based on speckle contrast analysis for full-field imaging of blood flow. Prior to use, 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 placing the mouse in a stereotactic frame. A 4×4 mm incision was carefully made above the right parietal bone 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 cWT and cKO 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.

[0054] 1.4 Immunocytochemistry

[0055] 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. Cells were then reverse-stained with 40,6-diamino-2-phenylindophenol (DAPI, 0.5 mg / ml, Thermo Scientific, Cat#62248) for 10 minutes. Finally, the cells were washed three 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.

[0056] 2. Experimental Results

[0057] To investigate the effects of microglia-derived Emc10 deficiency on mammalian cerebral blood vessels, we conducted a series of comprehensive studies.

[0058] Two types of conditionally knocked-out Emc10 gene C57BL / 6 mice (cKO mice) were used for 7 days after Emc10 gene knockout. Cerebral blood flow in these mice was analyzed using laser speckle contrast analysis (LASCA), with wild-type C57BL / 6 mice serving as the control group (cWT mice). Each group consisted of 6-12 mice, 7 weeks old, with an equal number of males and females. The results showed that compared to cWT mice, cKO mice exhibited a significant 75% reduction in cerebral cortical blood flow. Figure 1 A in the middle.

[0059] Tissue vascular diameter analysis is commonly performed using electron microscopy. Further transmission electron microscopy (TEM) analysis was conducted on the experimental and control mice. The results showed that, compared to cWT mice, cKO mice exhibited a 75% reduction in the diameter of cortical microvessels, while the cross-sectional area of ​​endothelial cells showed no significant change. Figure 1 (B in the middle).

[0060] To assess whether there was leakage of the blood-brain barrier in cKO mice, two types of conditionally knocked-out Emc10 gene C57BL / 6 mice and wild-type C57BL / 6 mice (5 mice in each group, 7 weeks old) were subjected to EB perfusion and endothelial barrier antigen (EBA) staining. EBA is a protein specifically expressed by endothelial cells, known as a "barrier protein," and is used as a marker of an intact blood-brain barrier. The results showed that, compared with cWT mice, cKO mice had reduced EB perfusion in all brain regions. Figure 2 (A) Sagittal brain slice immunostaining further showed that capillary perfusion in the cortex of cKO mice was reduced by 30%, while blood-brain barrier permeability was not abnormal. Figure 2 (B in the text). Furthermore, immunofluorescence staining of cerebral cortex sections showed no significant difference in cortical barrier function between cKO and cWT mice, although endothelial signaling was reduced (…). Figure 2 (C in the middle).

[0061] Microglia are a type of brain macrophage that exhibit different M1 / M2 or resting-state immunophenotypes. Common markers of the M1 phenotype include iNOS, IL-1β, and TNF-α, while markers of the M2 phenotype include CD206, IL-6, and TGF-β. This study used iNOS and CD206 staining of cKO mouse brain sections to assess the effect of Emc10 deficiency on microglia immunophenotype. The results showed that the M1 or M2 phenotype of IBA1-positive cells was not altered. Figure 3 This provides new experimental evidence for the study of changes in the secretory phenotype of microglia under acute cerebral ischemia.

[0062] 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 acute hypoperfusion induced by Emc10 deficiency on brain injury, we systematically examined the coronal plane covering the temporal cortex of cKO and cWT mice 7 days after Emc10 knockout using histological methods, observing changes in the expression of axonal and myelin protein markers. Western blot analysis confirmed reduced expression of myelin basic protein (MBP) in the cKO cortex. Figure 4 (A) Immunofluorescence and Luxol FastBlue (LFB) staining showed that demyelination was limited to the cingulate tract and supracallous white matter (scwm), but not limited to the internal capsule (int). Figure 4 (BD in the middle).

[0063] 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; and these 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 an acute hypoperfusion animal model, characterized in that, Through Cre-loxp gene knockout technology Emc10 Gene knockout in microglia of laboratory animals; The experimental animal was a mouse; The construction method is achieved by activating the CreERT2 protein. Emc10 Conditional gene knockout.

2. The construction method according to claim 1, characterized in that, The construction method includes: Build Tmem119 Insertion at the 3' end of the gene CreERT2 Experimental animal A for gene generation; construction Emc10 Insertion between the second exon of the gene Loxp Experimental animal B carrying the sequence; hybridization of experimental animal A and experimental animal B yields animals that simultaneously carry the sequence. CreERT2 Genes and Loxp Experimental animal C with sequence; Or, build Cx3cr1 Insertion at the 3' end of the gene CreERT2 Experimental animals D for gene generation; construction Emc10 Insertion between the second exon of the gene Loxp Experimental animal E carrying the sequence; hybridization of experimental animal D and experimental animal E yields animals simultaneously carrying the sequence. Cx3cr1 Genes and Loxp Experimental animal F with sequence.

3. The construction method according to claim 1, characterized in that, Tamoxifen activates the CreERT2 protein.

4. The application of the acute hypoperfusion animal model constructed by the construction method according to any one of claims 1-3 in screening drugs for the treatment of stroke.

Images