Construction method for microglial cell imaging animal model and application thereof

Abstract

The invention particularly discloses a construction method and application of an animal model for microglial cell imaging, and relates to the technical field of diagnostic imaging. The invention provides a construction method of an animal model for microglial cell imaging. The animal model overexpresses Oatp1a1 protein. According to the present invention, after the clinical contrast agent Gd-EOB-DTPA is injected, the Gd-EOB-DTPA can be specifically ingested by the microglial cells through the Oatp1a1 transport protein, and the microglial cells absorbing the Gd-EOB-DTPA can show the high signal under the T1 image at the specific time point so as to achieve the microglial cell tracking, and the good biological safety is provided; the invention provides a theoretical basis and an experimental basis for developing a new treatment strategy, and has good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of diagnostic imaging technology, and particularly relates to a construction method and application of an animal model for microglia imaging. Background Art

[0002] Microglia are the main immune cells in the central nervous system (CNS) and have key functions. They can continuously monitor the CNS environment and respond quickly to injury or infection; regulate synaptic connections and affect neural circuits during development and disease; regulate neuroinflammation by releasing cytokines and chemokines; clear cell debris, promote tissue repair and regeneration; and may have dual protective or toxic effects in diseases. Studying microglia helps to understand their complex behaviors in health and disease.

[0003] Imaging technology is an important tool for studying microglia. Different imaging technologies can reveal the location, state, morphology and other characteristics of microglia in different diseases and physiological states, which plays an important role in studying the functions of microglia. For example, in vivo imaging technology (such as two-photon microscopy) can be used to observe the dynamic behaviors of microglia in physiological and pathological states in real time. High-resolution imaging (such as confocal microscopy) can reveal the morphological changes and subcellular structures of microglia. Imaging technology can combine fluorescence labeling and functional probes to study the calcium signals, metabolic activities and molecular mechanisms of microglia. In vivo imaging technology can long-term monitor the changes of microglia during disease progression or treatment. The development of imaging technology has promoted the in-depth study of microglia and provided intuitive evidence for understanding their functions.

[0004] However, although the current optical microscopy imaging technology has developed rapidly and its high-resolution imaging of microglia has been very mature, these methods are all ex vivo imaging technologies based on slices and cannot show the morphology and location and other information of microglia in the living state. And now the relatively advanced multi-photon in vivo imaging technology can observe the behaviors of microglia in living tissues in real time, provide subcellular resolution, can clearly observe the morphology and dynamic processes of microglia, and at the same time use a variety of fluorescent probes to study the interactions of multiple cells or molecules. And this kind of imaging technology (taking two-photon microscopy imaging as an example) often has a high system cost and complex maintenance, which limits its popularization. Compared with other technologies (such as light sheet microscopy), two-photon imaging is slower and not suitable for fast dynamic processes. Its sample preparation requirements are also relatively high. In vivo imaging requires surgical exposure of the target area or the use of clearing techniques, which increases the experimental complexity. The most important point is that its penetration depth is limited and it cannot completely observe the entire brain parenchyma. Although it is superior to confocal microscopy, the penetration depth is still limited in very dense or highly scattering tissues.

[0005] Therefore, it is particularly important to develop a new MRI-based imaging method to trace microglia in living mice. Summary of the Invention

[0006] (1) Technical problems to be solved

[0007] In view of this, one of the main objectives of the present invention is to provide a method for constructing an animal model for microglia imaging, in which the animal model overexpresses the Oatp1a1 protein. After injecting the clinical contrast agent Gd-EOB-DTPA, microglia can specifically uptake Gd-EOB-DTPA through the Oatp1a1 transporter. At a specific time point, microglia that have absorbed Gd-EOB-DTPA show high signal intensity in T1 images, thereby realizing the tracing of microglia and having good biosafety.

[0008] (2) Technical solutions

[0009] To achieve the above objective, the present invention provides a method for constructing an animal model for microglia imaging, in which the animal model overexpresses the Oatp1a1 protein.

[0010] In one embodiment, the construction method includes:

[0011] (1) Transfecting an animal with a targeting vector containing a promoter, loxp-stop-loxp, the Oatp1a1 gene, and polyA to obtain a chimeric animal;

[0012] (2) Mating the chimeric animal with the opposite sex expressing Cre to obtain the animal model.

[0013] In one embodiment, the targeting vector includes a viral vector or a non-viral vector.

[0014] In one embodiment, the viral vector includes an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a radovirus vector, a retrovirus vector, a herpesvirus vector, a poxvirus vector, or a lentivirus vector.

[0015] In one embodiment, the viral vector is an adeno-associated virus (AAV) vector.

[0016] In one embodiment, the targeting site of the targeting vector includes the Rosa26 gene locus.

[0017] In one embodiment, the construction method further includes screening for offspring expressing Oatp1a1 through genotype identification.

[0018] In one embodiment, the offspring expresses Oatp1a1.

[0019] In one embodiment, the offspring expresses Cre.

[0020] In one embodiment, the offspring expresses both Oatp1a1 and Cre.

[0021] In one embodiment, the imaging implementation method includes one or a combination of MRI, CT, optical imaging, X-ray, PET, PET-CT, SPECT, SPECT / CT, and PEM.

[0022] In one embodiment, the imaging implementation method is MRI.

[0023] The present invention also provides, in another aspect, an application of an animal model obtained by the above construction method, and the application is selected from: 1) application in constructing a microglia-related disease model; 2) application in studying the pathogenesis of microglia-related diseases; 3) application in screening candidate drugs for microglia-related diseases.

[0024] In one embodiment, the microglia-related diseases include one or a combination of pinhole brain injury, middle cerebral artery occlusion, and neuronal ablation.

[0025] In one embodiment, the animals include mammals and non-mammals.

[0026] In one embodiment, the animal is a mammal.

[0027] In one embodiment, the mammals include one or a combination of apes, monkeys, rodents, artiodactyls, and perissodactyls.

[0028] In one embodiment, the mammal is a rodent.

[0029] In one embodiment, the rodents include one or a combination of rats, mice, and guinea pigs.

[0030] In one embodiment, the rodent is a mouse.

[0031] (III) Beneficial effects

[0032] The present invention provides a construction method for an animal model for microglia imaging, and the animal model overexpresses the Oatp1a1 protein. Compared with the prior art, it has the following beneficial effects:

[0033] 1. Traditional microglial imaging techniques (such as immunofluorescence and multiphoton microscopy) rely on ex vivo samples or invasive operations, making it difficult to achieve long-term in vivo dynamic monitoring. In the present invention, the Oatp1a1 protein is specifically expressed in microglia through gene editing technology, and combined with the MRI signal enhancement effect of Gd-EOB-DTPA, non-invasive tracing of microglia is achieved in living animals for the first time. This method breaks through the spatial and temporal limitations of the existing technology and provides a new visualization tool for neuroscience research.

[0034] 2. The effectiveness of the construction method provided by the present invention was verified in models of pinhole injury, MCAO, and neuron ablation. For example, in the MCAO model, MRI signals clearly showed the aggregation of microglia in the ischemic penumbra, which was highly consistent with the results of immunofluorescence. This multi-model verification not only proves the reliability of the technology but also reveals the spatio-temporal response rules of microglia in different pathological scenarios, providing important clues for the study of disease mechanisms.

[0035] 3. As a clinically commonly used contrast agent, the safety of Gd-EOB-DTPA has been widely verified in liver imaging. The present invention further confirmed its safety in nervous system applications through cytotoxicity experiments (CCK-8 survival rate > 90%), blood biochemical analysis (no abnormalities in indicators such as ALT and AST), and histopathological evaluation (no damage seen in HE staining), laying the foundation for subsequent clinical translation.

[0036] (IV) Terms and Definitions

[0037] As used herein, the term "microglia" is a common innate immune cell in the central nervous system (CNS). It accounts for 10 - 15% of all glial cells and is commonly referred to as the tissue-resident macrophage of the CNS. However, different from meningeal, choroid plexus, and perivascular macrophages, microglia originate from the yolk sac and populate the CNS before its vasculogenesis. Resting microglia in the adult brain have small cell bodies and are highly branched - this is the morphological basis for distinguishing them from macrophages and dendritic cells. These cells are involved in CNS development and homeostasis. As part of their homeostatic function, microglial cell bodies remain stationary, but their tentacles constantly scan the surrounding extracellular space and communicate directly with neurons, astrocytes, and blood vessels.

[0038] As used herein, the terms "Magnetic Resonance Imaging" and "(Magnetic Resonance Imaging, MRI)" are important non-invasive diagnostic techniques in modern medical imaging, with advantages such as high soft tissue contrast, multi-parameter imaging, and no radiation. Its basic principle is based on the nuclear magnetic resonance phenomenon of atomic nuclei in a strong magnetic field, and images of the internal structure of the human body are obtained by detecting the weak electromagnetic waves emitted by the human body. MRI technology is widely used not only in clinical diagnosis but also plays an important role in basic research, such as tumor molecular imaging, brain functional imaging, etc.

[0039] T1-weighted imaging mainly reflects the T1 characteristics of tissues. In T1-weighted images, tissues with short T1 relaxation times (such as fat) have high signal intensities, while tissues with long T1 relaxation times (such as cerebrospinal fluid) have low signal intensities. T1-weighted imaging is often used to display anatomical structures, such as the brain, spinal cord, liver, kidneys, etc.

[0040] T2-weighted imaging mainly reflects the T2 characteristics of tissues. In T2-weighted images, tissues with long T2 relaxation times (such as cerebrospinal fluid) have high signal intensities, while tissues with short T2 relaxation times (such as bone) have low signal intensities. T2-weighted imaging is often used to display lesions, such as cerebral infarction, encephalitis, spinal cord lesions, etc. Contrast-enhanced imaging enhances the signal of specific tissues by injecting contrast agents containing iron or gadolinium, improving the detection rate of lesions. Contrast-enhanced imaging is often used to display tumors, inflammation, vascular lesions, etc.

[0041] As used in the text, the term "hepatic organic anion transporting polypeptide" or "OATP" is a new class of MRI reporter proteins. OATP is a membrane protein of approximately 700 amino acids with 12 transmembrane helices, and its expression is conserved in vertebrates. Hepatic OATP transports commercially available, FDA-approved, clinically used MRI contrast agents into cells. In clinical situations, after intravenous injection of the two liver-specific MRI contrast agents Gd-EOB-DTPA or Gd-BOPTA, hepatocytes become hyperintense on T1-weighted MRI due to the intracellular accumulation of Gd-based contrast agents. This is clinically used to detect tumors in the liver because tumors (usually) do not express OATP and remain hypointense relative to the bright liver. OATPs that effectively transport Gd-EOB-DTPA and are related to the use of OATP as an MRI reporter protein include human / primate OATP1B1 and OATP1B3, rat Oatp1a1, and rat OATP1B2. Liver MRI has demonstrated that many other species, including mice, rabbits, dogs, and pigs, exhibit hepatic accumulation of Gd-EOB-DTPA and Gd-BOPTA, so other members of the hepatic OATP1B family (such as dog OATP1B4) may also transport these drugs.

[0042] In C57BL / 6 mice, Oatp1a1 (organic anion transporting polypeptide 1A1) is mainly expressed in the liver, responsible for transporting a variety of endogenous and exogenous substances, while its expression in the kidney is relatively low or absent, and it is basically not expressed in other tissues, mainly playing a role in hepatocytes.

[0043] As used herein, the term "Gd-EOB-DTPA" is a clinically widely used MRI contrast agent, which is formed by adding lipophilic ethoxybenzyl (EOB) to the molecular structure of Gd-EOB-DTPA. Brief Description of the Drawings

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0045] Figure 1 It is a construction diagram of the Oatp1a1 plasmid. (A) Construction strategy diagram of the Oatp1a1 plasmid vector. (B) Schematic diagram of the pcDNA3.1(+)-mCherry-Oatp1a1 plasmid. (C) Schematic diagram of the pcDNA3.1(+)-mCherry empty plasmid.

[0046] Figure 2 It is a schematic diagram of the Oatp1a1 gene mouse strategy.

[0047] Figure 3 It is a construction and identification diagram of the MRI and optical dual-modal imaging gene mouse. (A) Construction strategy of the MRI and optical dual-modal imaging gene mouse. The constructed dual-modal imaging gene mouse can specifically express Cre enzyme in microglia, which can cut off the loxp site containing the STOP sequence, so that such a gene mouse can specifically express Oatp1a1 protein and EGFP protein in microglia. (B) Genotype identification results of the MRI and optical dual-modal imaging gene mouse. The band near the lower part of 500bp in the upper left indicates Cx3cr1 P1P2, and the band near the upper part of 500bp in the lower left indicates Cx3cr1 P3P4; the band between 750 - 1000bp in the upper right indicates Oatp1a1 P1P2, and the band near 500bp in the lower right indicates Oatp1a1P3P4; the presence or absence of the P3P4 band indicates the presence or absence of this gene, and the presence or absence of the P1P2 band indicates the heterozygosity and homozygosity of this gene.

[0048] Figure 4It is the localization of Oatp1a1 in BV2 cells. The Oatp1a1 protein is mainly distributed on the cell membrane of BV2 cells (green), and the merged image further confirms this. Neuron-dio is used as a cell membrane green fluorescent dye for cell membrane staining. mCherry in the control group is mainly distributed in the cytoplasm. The scale bar in the image represents 20 μm.

[0049] Figure 5 It is the uptake of Gd-EOB-DTPA by BV2 cells. O-M (red) represents the Gd concentration after co-incubating the transfected mCherry-Oatp1a1 fusion protein plasmid (experimental group) with 50 μg / ml Gd-EOB-DTPA for 30 min, 1 h, 2 h, 4 h, and 6 h. M (black) represents the Gd concentration after co-incubating the transfected mCherry plasmid (control group) with Gd-EOB-DTPA for 30 min, 1 h, 2 h, 4 h, and 6 h. There is a significant difference (****, p < 0.0001; n = 3).

[0050] Figure 6 It is the result diagram of in vitro MRI experiment. (A) T1-weighted axial scan of BV2 cells (B) Signal intensities of OM+Gd, M+Gd, and PBS. PBS represents the T1 signal under only PBS; M+Gd represents the T1 signal after co-culturing BV2 cells transfected with the empty mCherry plasmid (control group) with Gd; OM+Gd represents the T1 signal after co-culturing BV2 cells transfected with the mCherry-Oatp1a1 fusion protein plasmid (experimental group) with Gd. The cells were continuously cultured for 2 hours with a Gd ion concentration of 50 μg / mL, and there was a significant difference in the signal intensity (*, p < 0.05; n = 3).

[0051] Figure 7 It is the specificity of Cx3cr1-labeled microglia. (A) & (B) Autofluorescence and IBA-1 staining of the dual-modal imaging gene mouse. The green fluorescence represents Cx3cr1-eGFP, the red fluorescence represents the IBA-1 antibody, the blue fluorescence represents DAPI, and the Merge channel represents the fluorescence image after merging the above three colors. The experiment was repeated three times. The scale bar in Figure A is 50 μm, and the scale bar in Figure B is 10 μm. (C) Co-localization analysis of the two channels within the field of view of Figure (B). The horizontal axis represents each position point in the image, and the vertical axis represents the change in the gray value of each channel.

[0052] Figure 8It is the bimodal imaging of gene mice under normal conditions. (A) T1W imaging results. The baseline represents the T1W image before intrathecal injection of Gd-EOB-DTPA, which is used as the baseline. After injection of the contrast agent, it represents the image scanned 24 h after intrathecal injection of Gd-EOB-DTPA. (B) Comparison of T1W imaging results and fluorescence images. The left side represents the T1W image of microglia in gene mice with bimodal imaging under normal physiological conditions, and the right side represents the immunofluorescence optical image. The green fluorescence represents the autofluorescence of Cx3cr1-EGFP in gene mice with bimodal imaging, which is used to label microglia. There are three replicates in each group, the thickness of the frozen section is 35 μm, and the scale bar of the fluorescence image is 1 mm.

[0053] Figure 9 It is the time point and method for establishing the pinhole brain injury model. (A) Time points for establishing the pinhole brain injury model and experiments. Gene mice were selected as the experimental group, and wildtype C57BL / 6 mice were used as the control group, with three in each group. The mice were subjected to pinhole injury modeling. After 14 days, their T1W images were collected at 9.4 T, which was used as the baseline. Immediately after collection, 10 μl of Gd-EOB-DTPA was injected by intrathecal injection. One day later, their 9.4 T T1W images were collected again. After collection, the mice were sacrificed, the brains were taken, and optical images were collected. (B) Schematic diagram of the method and location for establishing the pinhole brain injury model. Under the stereotaxic apparatus, using a glass electrode as a tool, the PO brain region of the mice was selected and punctured to cause pinhole injury.

[0054] Figure 10 It is the 9.4 T T1W image of the pinhole brain injury. (A) T1W imaging results of the control group with pinhole brain injury. (B) T1W imaging results of the experimental group with pinhole brain injury. The baseline represents the T1W image before intrathecal injection of Gd-EOB-DTPA. After injection of the contrast agent, it represents the image collected 24 h after intrathecal injection of Gd-EOB-DTPA.

[0055] Figure 11 It is the comparison between magnetic resonance imaging and optical imaging. The upper row represents the T1W image (left) and immunofluorescence optical image (middle) of microglia in gene mice with bimodal imaging after pinhole brain injury, and the right side is the enlarged view of the optical image. The green fluorescence represents the autofluorescence of Cx3cr1-EGFP in gene mice with bimodal imaging, which is used to mark the location of microglia. The lower row represents the T1W image (left) and immunofluorescence optical image (middle) of wildtype mice after pinhole brain injury. The red color represents the staining result of IBA-1, and the right side is the enlarged fluorescence image. There are three replicates in each group, the thickness of the frozen section is 35 μm, the scale bar of the fluorescence image is 500 μm, and the scale bar after enlargement is 200 μm.

[0056] Figure 12It is about the modeling time points and methods of the MCAO injury model. (A) MCAO injury modeling and experimental time points. (B) Schematic diagram of the modeling method and location of the pinhole brain injury model. Gene mice were selected as the experimental group, and Wildtype C57BL / 6 mice were used as the control group, with three mice in each group. MCAO modeling was performed on the mice, and the common carotid artery was blocked with a thread embolism. The thread embolism was removed 60 minutes later.

[0057] Figure 13 It is about the MCAO model causing cerebral ischemia and infarction. (A) TTC staining results of MCAO mice modeling and sham operation group. The mice were sacrificed 24 h after MCAO modeling to obtain the brain for TTC staining. The section thickness was 2 mm, and 2% TTC was used for staining. The right scale bar is 1 cm. (B) T2W images of MCAO mice modeling and sham operation. The MRI T2W images were collected 24 h after MCAO modeling. Black represents normal tissue, and white high signal indicates the infarct area.

[0058] Figure 14 It is about the 9.4t T1W images of the MCAO injury model. (A) T1W imaging results of the control group of the MCAO model. (B) T1W imaging results of the experimental group of the MCAO model. Baseline represents the T1W image before intrathecal injection of Gd-EOB-DTPA, and this was used as the baseline. After injection of the contrast agent, it represents the image scanned 24 h after intrathecal injection of Gd-EOB-DTPA.

[0059] Figure 15 It is about the comparison between magnetic resonance imaging and optical imaging. The upper row shows the T1W image (left) and immunofluorescence optical image (middle) of microglia in gene mice with dual-modal imaging after MCAO modeling. The right side is the enlarged view of the optical image. Green fluorescence represents the spontaneous fluorescence of Cx3cr1-EGFP in dual-modal imaging gene mice, which is used to label the location of microglia. The lower row shows the T1W image (left) and immunofluorescence optical image (middle) of Wildtype mice after MCAO modeling. Red represents the staining result of IBA-1, and the right side is the enlarged fluorescence image. Each group was repeated three times. The cryostat section thickness was 35 um. The scale bar of the fluorescence image is 1 mm, and the scale bar after enlargement is 200 um.

[0060] Figure 16 It is about the schematic diagram of the modeling and data acquisition of the neuron ablation model. (A) Modeling method of the neuron ablation model and data acquisition time points. (B) Schematic diagram of virus injection. On day 0, 300 nl of rAAV-hsyn-DTR-tdTomato-WPREs virus was injected into the PO brain region of the mice. After the virus expression (usually 21 days), diphtheria toxin was injected twice on days 22 and 24, with a dose of 50 ug / kg each time. MRI data was collected on the fourth day after the last injection of the drug.

[0061] Figure 17 The DTR virus is used in combination with DT to achieve neuronal ablation. The upper row shows the fluorescence images of mouse slices after injecting the virus but not injecting the DT drug; the lower row shows the fluorescence images of mouse slices after injecting the virus and injecting the DT drug after the virus is fully expressed. Blue represents DAPI (405 nm), red represents tdTomato (554 nm), and the last column shows the merged image of the two. 300 nl of the DTR virus was injected into each mouse, and diphtheria toxin was injected in two doses, with a two-day interval between each dose, and the dose was 50 μg / kg. The slice thickness was 35 μm, the scale of the whole-brain fluorescence image was 1 mm, and the scale of the enlarged image was 100 μm.

[0062] Figure 18 It is a 9.4T T1W image of the neuronal ablation model. (A) T1W imaging results of the control group of the neuronal ablation model. (B) T1W imaging results of the experimental group of the neuronal ablation model. The baseline represents the T1W image before intrathecal injection of Gd-EOB-DTPA, and this is used as the baseline. The image after injection of the contrast agent represents the image scanned 24 h after intrathecal injection of Gd-EOB-DTPA.

[0063] Figure 19 It is a comparison between magnetic resonance imaging and optical imaging. The upper row shows the T1W image (left) and immunofluorescence optical image (middle) of microglia after neuronal ablation in the dual-modal imaging transgenic mouse, and the right side is the enlarged view of the optical image. Green fluorescence represents the autofluorescence of Cx3cr1-EGFP in the dual-modal imaging transgenic mouse, which is used to label the location of microglia. The lower row shows the T1W image (left) and immunofluorescence optical image (middle) of microglia after neuronal ablation in Wildtype mice. Green represents the staining result of IBA-1 (since the injected virus carried a red fluorescent protein, the secondary antibody of IBA-1 was changed to green fluorescence labeling), and the right side is the enlarged fluorescence image. Each group had three replicates. The thickness of the frozen section was 35 μm, the scale of the fluorescence image was 1 mm, and the scale after enlargement was 200 μm. Specific implementation manners

[0064] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0065] As used herein, "comprising", "having", or "including" includes "containing", "consisting essentially of", "substantially consisting of", and "consisting of"; "consisting essentially of", "substantially consisting of", and "consisting of" are subordinate concepts of "comprising", "having", or "including".

[0066] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The reagents, methods, and equipment used are all conventional reagents, methods, and equipment in this technical field unless otherwise specified.

[0067] Example 1

[0068] Construction of Oatp1a1 plasmid vector:

[0069] 1. Plasmid construction: The gene sequence of Oatp1a1 protein was searched in the nucleic acid database of NCBI (NM_013797.5). Recombinant rAAV-EF1a-Oatp1a1-P2A-EGFP-WPRE-pA virus was constructed and recombinant plasmid was constructed on pcDNA3.1. (1) Extract pcDNA3.1(+) vector DNA: Use a plasmid extraction kit to extract pcDNA3.1(+) plasmid DNA from transformed bacteria. (2) Verify the integrity of the plasmid by agarose gel electrophoresis. (3) PCR amplify the Oatp1a1 gene sequence: Design specific primers, add XhoI restriction enzyme site to the upstream primer and NotI restriction enzyme site to the downstream primer. (4) Use high-fidelity DNA polymerase for PCR amplification. Reaction conditions: Denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles. (5) Verify the size of the PCR product by agarose gel electrophoresis. (6) Digest the pcDNA3.1(+) vector: Reaction system (20ul): Plasmid DNA: 1ug; 10×Buffer: 2ul; XhoI: 1ul; NotI: 1ul; ddH2O: Make up to 20ul. Reaction conditions: Incubate at 37°C for 1 hour. (7) Verify the digestion effect by agarose gel electrophoresis. (8) Use a DNA purification kit to purify the digested vector and target gene fragment respectively. (9) Transform the ligation reaction product into competent cells (DH5α).

[0070] (10) Pick monoclonal colonies and inoculate them into LB liquid medium containing antibiotics, and culture them overnight on a shaker at 37°C. (11) Extract plasmid DNA and verify whether the construction of the recombinant plasmid is successful by restriction enzyme analysis. Select the clones with correct restriction enzyme digestion results and send them for sequencing verification. The schematic diagram of the constructed plasmid vector is as Figure 1 shown.

[0071] 2. Plasmid Amplification and Extraction: (1) Inoculate 2 - 3 μl of the bacterial solution containing the target plasmid into 5 ml of LB medium (containing appropriate antibiotics). (2) Incubate at 37 °C with shaking at 200 rpm for 12 - 16 hours (until OD600 ≈ 2.0). (3) Take 1.5 mL of the bacterial solution, centrifuge at 12,000 g at 4 °C for 1 minute to collect the bacterial cells. (4) Add 100 μL of pre - cooled Solution I (50 mM glucose, 25 mM Tris - HCl, 10 mM EDTA) and resuspend the bacterial cells thoroughly. (5) Add 200 μL of freshly prepared Solution II (0.2 M NaOH, 1% SDS), gently invert and mix 5 - 6 times, and incubate on ice for 5 minutes. Note: Avoid vigorous shaking to prevent genomic DNA breakage. (6) Add 150 μl of pre - cooled Solution III (3 M potassium acetate), gently invert and mix 5 - 6 times, and incubate on ice for 10 minutes. (7) Centrifuge at 12,000 g at 4 °C for 10 minutes. (8) Transfer the supernatant to a new EP tube, add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 minutes. (9) Centrifuge at 12,000 g at 4 °C for 10 minutes and discard the supernatant. (10) Add 1 mL of pre - cooled 70% ethanol to wash the pellet.

[0072] (11) Centrifuge at 12,000 g at 4 °C for 5 minutes and discard the supernatant. (12) Open the lid and air - dry for 5 - 10 minutes, then add 50 μl of TE (10 mM Tris - HCl, 1 mM EDTA) buffer to dissolve the DNA. (13) Concentration determination: Use OneDrop to measure the A260 / A280 ratio, ideal range: 1.8 - 2.0.

[0073] Example 2

[0074] Cx3cr1 - icre and Oatp1a1 Gene Mice:

[0075] By gene - editing method, the gene of Oatp1a1 protein is inserted into the Rosa26 gene locus of mice. The sequence is as Figure 2 shown. At the same time, a STOP sequence is inserted in front of the Oatp1a1 gene. The STOP sequence contains loxp sites on both sides. Under normal conditions, Oatp1a1 protein is not expressed. When Cre enzyme is expressed in cells, the STOP sequence is cleaved and Oatp1a1 protein is normally expressed.

[0076] 1. Animal Rearing: All animals in this experiment are reared in the SPF experimental animal center of the First Affiliated Hospital of the University of Science and Technology of China (Anhui Provincial Hospital). Mice enjoy good animal welfare in the barrier, can freely access water and food, and are reared under light - dark (12 hours / 12 hours) conditions. All experiments use 6 - to 8 - week - old C57BL / 6 female mice.

[0077] 2. Gene mouse breeding: The Cx3cr1-iCre mice used were purchased from Nanjing Model Organisms Company. The Oatp1a1 transgenic mice were constructed by our research group. The homozygous male Cx3cr1-iCre mice were mated with the homozygous female Oatp1a1 mice (specifically, two male mice were mated with three female mice) to obtain the F1 generation, and the genotypes were identified to ensure their correctness. The F1 generation was crossed to obtain mice with various genotypes, and the mice we needed were screened by genotype identification. During the breeding process, the supply of feed and water should be maintained: Check the supply of feed and water every day to ensure it is sufficient and fresh. The cages can be changed once a week, but the water should be changed at least twice a week. Observe and record the birth of mice: Observe the behavior and health status of mice every day. Record the mating behavior of the breeding pairs and the pregnancy status of female mice, record the birth dates of the mice, and separate the cages in time after the mice reach adulthood.

[0078] 3. Genotype identification: The three-week-old mice were separated into cages, and the tips of their tails were cut for genotype identification. The identification steps are as follows: (1) Tail lysis: Add 80 μl of 50 mM NaOH to a 0.1 ml EP tube containing the mouse tail, and heat at 100 °C for 90 minutes. (2) Add 40 μl of 0.1 mM Tris Hcl (Ph = 1.46) for neutralization. (3) Remove impurities: Centrifuge at 12,000 rpm for 5 minutes, take 100 μl of the supernatant and transfer it to a new EP tube, and make a good mark. (4) Perform PCR according to the systems and cycling temperatures shown in Tables 1 - 5. (5) Prepare 1% agarose gel: Weigh 1.2 g of agarose powder, add 120 ml of TAE solution, mix well, and heat to boiling in a microwave oven. Add gelred nucleic acid dye at a ratio of 1:10,000, pour it into the agarose gel mold, insert the comb, and let it solidify at room temperature for 30 minutes. (6) Load the samples onto the agarose gel and perform electrophoresis at a constant voltage of 150 V for 30 minutes in a conventional electrophoresis tank. (7) Use a UV302 to detect the band distribution to determine the genotype.

[0079] The results are as Figure 3As shown, the constructed Cx3cr1-Cre-Oatp1a1 double-positive mice can specifically express Cre enzyme in microglia, which can cleave the loxp sites containing the STOP sequence, so that such genetically engineered mice can specifically express Oatp1a1 protein and EGFP protein in microglia. It can achieve efficient and specific labeling of microglia, providing a reliable technical platform for subsequent cell tracing and functional research. (The band near the lower part of 500bp in the upper left indicates Cx3cr1 P1P2, and the band near the upper part of 500bp in the lower left indicates Cx3cr1 P3P4; the band between 750-1000bp in the upper right indicates Oatp1a1 P1P2, and the band near 500bp in the lower right indicates Oatp1a1 P3P4; the presence or absence of the P3P4 band indicates the presence or absence of the gene, and the presence or absence of the P1P2 band indicates the heterozygosity and homozygosity of the gene).

[0080] Table 1 PCR Loading System

[0081]

[0082] Table 2 Primer Sequences of Cx3cr1 Gene

[0083]

[0084] Table 3 PCR Program of Cx3cr1 Gene

[0085]

[0086] Table 4 Primer Sequences of Oatp1a1 Gene

[0087]

[0088]

[0089] Table 5 PCR Program of Oatp1a1 Gene

[0090]

[0091] Example 3

[0092] Verification of the Localization and Function of Oatp1a1 Protein in Vitro:

[0093] The BV2 microglial cell line is derived from microglia of neonatal C57 / BL6 mice (newborns). This cell line is immortalized by infecting with the J2 retrovirus carrying the v-raf / v-myc oncogenes. BV2 cells have some characteristics of primary microglia and are an alternative model for studying microglial function and responses. Therefore, in this study, the BV2 microglial cell line was used instead of primary microglia for in vitro experiments.

[0094] For the maintenance of the above cell lines, high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was uniformly used. These cells were cultured under standard conditions in a constant temperature incubator at 37°C with 5% CO2.

[0095] 1. Culture of BV2 cells:

[0096] Cell resuscitation: (1) Instrument preparation: Preheat the water bath to 37°C in advance, prepare pipette tips, pipettes, culture dishes, complete medium, etc., place them in the laminar flow hood, and turn on the ultraviolet sterilization 30 minutes in advance. (2) Take out the cryopreservation tube: Take out the cell cryopreservation tube from the liquid nitrogen tank or -80°C refrigerator. If it is far from the water bath, it can be placed on dry ice first and then sent to the water bath. (3) Rapid thawing: Quickly put the cryopreservation tube into the 37°C water bath, hold it with forceps and shake it constantly to rapidly thaw it within 1 - 2 minutes. Note that the mouth of the cryopreservation tube should not be immersed in the water bath to avoid contamination. Stop resuscitation when there is still a small piece of ice (3 - 4 mm in diameter) left in the cryopreservation tube. (4) Transfer: Wipe the outside of the cryopreservation tube with an alcohol cotton ball for disinfection, and then transfer it to the laminar flow hood. Unscrew the cap, add 1 ml of preheated medium to the cryopreservation tube, mix well and transfer it to a centrifuge tube. Add another 2 mL of fresh medium to the centrifuge tube. (5) Centrifugation: Centrifuge at 900 rpm for 5 minutes to remove the cell cryopreservation solution. (6) Resuspend the cells: Discard the supernatant, resuspend the cells with fresh medium, and inoculate them into a new sterile 10 cm culture vessel.

[0097] Cell passage: Observe the cell density under the microscope. When the cell density reaches about 80%, cell passage can be carried out (this cell line is semi-adherent, and some cells will be in suspension). (1) Prepare reagents: Preheat reagents such as complete medium, PBS buffer, trypsin containing EDTA, etc. to 37°C, and place them in the ultra-clean bench for 30 minutes of ultraviolet disinfection in advance. (2) Remove the old medium: Pour out the old culture solution in the culture dish, then add 2 ml of PBS, shake gently for a moment, and pour out the solution to remove the residual serum and senescent and detached cells. (3) Digestion: Add 1 ml of trypsin digestion solution (0.25% trypsin solution + 0.02% EDTA solution) to the culture flask to cover the cell surface, and place it at room temperature or in the incubator for 1 minute. At the same time, observe under the inverted microscope. When the cells retract to nearly spherical and the cell gaps increase, the digestion is completed. (4) Collect cells: Add 1 ml of fresh medium containing serum, and pipette the digested cells repeatedly to make them detach from the wall until a dispersed cell suspension is formed. Transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 900 rpm for 5 minutes. (5) Resuspend: Discard the supernatant, and add 1 ml of fresh complete medium to resuspend the cells. (6) Inoculation: Transfer to a new 10 cm sterile culture dish at a ratio of 1:3 or 1:5, and place it in the 37°C incubator for continued culture.

[0098] Cell cryopreservation: When the cells are in the logarithmic growth phase, that is, when the cell density reaches 80%-90%, cell cryopreservation can be carried out. (1) Prepare reagents: Preheat reagents such as complete medium, PBS buffer, trypsin containing EDTA, cell rapid cryopreservation solution, etc. to 37°C, and place them in the ultra-clean bench for 30 minutes of ultraviolet disinfection in advance. (2) Remove the old medium: Pour out the old culture solution in the culture dish, then add 2 ml of PBS, shake gently for a moment, and pour out the solution to remove the residual serum and senescent and detached cells. (3) Digestion: Add 1 ml of trypsin digestion solution (0.25% trypsin solution + 0.02% EDTA solution) to the culture flask to cover the cell surface, and place it at room temperature or in the incubator for 1 minute. At the same time, observe under the inverted microscope. When the cells retract to nearly spherical and the cell gaps increase, the digestion is completed. (4) Collect cells: Add 1 ml of fresh medium containing serum, and pipette the digested cells repeatedly to make them detach from the wall until a dispersed cell suspension is formed. Transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 900 rpm for 5 minutes. (5) Discard the supernatant, add 1 ml of cell rapid cryopreservation solution to resuspend the cells, make a good mark, and place it in the -80°C refrigerator for storage.

[0099] 2. CCK-8 cytotoxicity assay: (1) Preparation of cell suspension: Select cells in the logarithmic growth phase with good growth status to prepare a cell suspension and count the cells. (2) Seeding cells: Seed the cell suspension (100 μl / well) in a 96-well plate, divided into a blank group and five concentration gradient groups, with three replicates for each sample. The seeding density of cells is 1000 cells / well, and the blank control group only adds 100 μl of culture medium. (3) Cell adhesion: Place the culture plate in a cell culture incubator at 37 °C and 5% CO2 to allow the cells to adhere for 24 h. (4) Drug treatment: Add 10 μl of Gd-EOB-DTPA at different concentrations to each well. The five concentrations are 10 mM, 5 mM, 2.5 mM, 1.25 mM, and 0 mM (control), and continue to culture for the required time. The time gradients are 6 h, 12 h, and 24 h. (5) Adding CCK-8 solution: After culturing for the corresponding time, add 10 μl of CCK-8 solution to each well, and try to avoid generating bubbles during the addition process. (6) Incubation: Incubate the 96-well plate in a cell culture incubator at 37 °C with 5% CO2 for 1 - 4 h. It is better when the absorbance is around 1.0, and the liquid turns orange visibly to the naked eye. (7) Measuring absorbance: As soon as possible, measure the absorbance (OD value) of each well at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The level of absorbance reflects the cell proliferation. (8) Evaluating cell proliferation and viability: According to the change in absorbance, the cell proliferation and viability can be evaluated. Cell proliferation viability (%) = (ODtreatment group - ODblank group) / (ODcontrol group - ODblank group) × 100%.

[0100] 3. Transfection of Oatp1a1 plasmid into BV2 cell line: Transfect the Oatp1a1-mCherry plasmid and the control m-Cherry plasmid into the BV2 cell line to express them in BV2 cells for subsequent research.

[0101] (1) Seeding plates: One day before transfection, seed the cells in a suitable vessel (taking a 24-well plate as an example) so that the density is 70% - 80% at the time of transfection. (2) Before transfection, replace with fresh complete culture medium. (3) Preparation of transfection reagent / DNA mixture: Add 25 μl of Opti-MEM to a 1.5 ml sterile centrifuge tube, add 0.5 μg of DNA, and gently mix with a pipette. Add 25 μl of Opti-MEM to another 1.5 ml sterile centrifuge tube, add 1.5 μl of transfection reagent, and gently mix with a pipette. Drop the transfection reagent - Opti-MEM into the DNA - Opti-MEM, gently mix with a pipette, and let it stand at room temperature for 10 - 15 min before use for transfection. (4) Transfection: Drop 50 μl of the transfection reagent / DNA mixture into the culture medium, and gently shake the culture vessel to evenly disperse the transfection reagent / DNA. (5) Continue to culture for 24 - 72 h, harvest the cells, and perform subsequent experiments.

[0102] 4. Oatp1a1 Protein Localization Experiment: (1) Transfect the Oatp1a1-mCherry plasmid (experimental group) and the m-Cherry plasmid (control group) into the BV2 cell line. (2) Seed the cells in a 24-well plate with coverslips and place them in an incubator at 37 °C for 24 - 48 h. (3) Fixation: Remove the well plate, aspirate the old medium, rinse 3 times with 1 ml of PBS, add 1 ml of pre-cooled methanol to each well, and fix in a -20 °C refrigerator for five minutes. (4) Cell membrane staining: Aspirate the methanol, rinse 3 times with PBS, incubate with 5 μM NeuroDiO cell membrane dye at 37 °C for 30 min, and then wash 3 times with PBS. (5) Mounting: Take out the cell coverslips and mount them with a fluorescence quenching mounting agent. (6) Imaging: Collect images at mCherry (594 nm) and NeuroDio (488 nm) using a laser confocal microscope.

[0103] The results of fluorescence and differential interference imaging are as Figure 4 shown. The mCherry-Oatp1a1 fusion protein is located on the cell membrane, while mCherry is mainly distributed in the cytoplasm of the control group cells. (The fluorescence images show the localization of Oatp1a1 in BV2 cells after transfection. The Oatp1a1 protein is mainly distributed on the cell membrane of BV2 cells (green), and the merged images further confirm this. Neuron-dio is used as a cell membrane green fluorescence dye for cell membrane staining. mCherry in the control group is mainly distributed in the cytoplasm. The scale bar in the image represents 20 μm.)

[0104] This in vitro experiment preliminarily demonstrates that, like the Oatp1a1 transporter expressed in the liver and kidneys, the Oatp1a1 protein expressed in microglia is also localized on the membrane and has the same membrane localization.

[0105] Example 4

[0106] Determination of Gd-EOB-DTPA Uptake:

[0107] The Oatp1a1 protein expressed in liver and kidney cells can specifically uptake Gd-EOB-DTPA, the generic name of which is gadoxetic acid, a highly water-soluble contrast agent, where a lipophilic EOB group is attached to gadolinium (Gd)-DTPA. Due to the presence of the lipophilic EOB group, Gd-EOB-DTPA is actively transported into hepatocytes through Oatp1a1. Therefore, it is possible to determine whether it has normal function by measuring the uptake of Gd-EOB-DTPA by BV2 cells (expressing the Oatp1a1 protein).

[0108] 1. Inductively Coupled Plasma-Mass Spectrometry (ICP-MS): (1) Transfect the Oatp1a1-mCherry plasmid (experimental group) and the m-Cherry plasmid (control group) into the BV2 cell line. (2) Seed the cells in a 6-well plate and culture them in an incubator at 37 °C for 24 - 48 h. The success of transfection can be verified by fluorescence microscopy. (3) Incubate with Gd-EOB-DTPA at a concentration of 50 μg / ml for 30 min, 1 h, 2 h, 4 h, and 6 h respectively. (4) Collect the cells: Aspirate the original culture medium, wash the cells thoroughly with PBS three times. Digest the cells with trypsin digestion solution, count the cells. Centrifuge at 900 rpm for 3 minutes. (5) Transfer the cells to a flask, dry the cells at 60 °C, add 500 μl of nitric acid, boil for 30 minutes until there is no precipitate, and make up the volume to 4 ml with deionized water. (6) Send the samples to the Physical and Chemical Experiment Center of the University of Science and Technology of China, and determine the content of Gd in the cells by ICP-MS (Inductively Coupled Plasma-Mass Spectrometry).

[0109] The experimental results are as Figure 5 shown. The Oatp1a1 protein expressed on BV2 cells can normally uptake Gd-EOB-DTPA, that is, it has normal function. (O-M (red) represents the Gd concentration after co-incubation of the transfected mCherry-Oatp1a1 fusion protein plasmid (experimental group) with 50 μg / ml Gd-EOB-DTPA for 30 min, 1 h, 2 h, 4 h, and 6 h. M (black) represents the Gd concentration after co-incubation of the transfected mCherry plasmid (control group) with Gd-EOB-DTPA for 30 min, 1 h, 2 h, 4 h, and 6 h. Significant difference (****, p < 0.0001; n = 3)).

[0110] Example 5

[0111] In vitro MRI:

[0112] In vitro MRI: (1) Transfect the Oatp1a1-mCherry plasmid (experimental group) and the m-Cherry plasmid (control group) into the BV2 cell line. (2) Seed the cells in a 6-well plate and incubate them in an incubator at 37 °C for 24 - 48 h. The success of transfection can be verified by a fluorescence microscope. (3) Add Gd-EOB-DTPA to the experimental group and the control group at a concentration of 50 μg / ml and co-incubate for 2 h. Use only PBS as the blank control group. (4) Aspirate the old culture medium, rinse with PBS, digest the cells with trypsin digestion solution, collect the cells, and centrifuge at 900 rpm for 3 minutes. (5) Wash: Discard the supernatant, resuspend the cells with PBS, centrifuge at 900 rpm for 3 minutes, and repeat three times. (6) Add 1.5 ml of PBS to the cells in each group for volume determination and transfer them to 1.5 ml EP tubes. (7) Acquire the T1 sequence image data under a 3T NMR machine.

[0113] The results are as Figure 6 shown. The MRI signal intensity of BV2 cells transfected with the mCherry-Oatp1a1 fusion protein plasmid (OM+Gd group) under the T1W sequence is significantly higher than that of the control group transfected only with the empty vector (M+Gd group) and the baseline group without adding Gd-EOB-DTPA (PBS group). Specifically, the signal intensity of the OM+Gd group is significantly increased compared with the PBS group, while there is no significant difference in the signal intensity between the M+Gd group and the PBS group. (PBS represents the T1 signal under only PBS; M+Gd represents the T1 signal after co-culturing BV2 cells transfected with the empty mCherry plasmid (control group) with Gd; OM+Gd represents the T1 signal after co-culturing BV2 cells transfected with the mCherry-Oatp1a1 fusion protein plasmid (experimental group) with Gd. The cells were continuously cultured for 2 hours with a Gd ion concentration of 50 μg / mL, and the signal intensity showed a significant difference numerically (*, p < 0.05; n = 3)).

[0114] This result indicates that the distribution of Gd-EOB-DTPA outside the cell is non-specific and can hardly enter the cell spontaneously. However, when the Oatp1a1 protein is expressed on the cell membrane, this transporter can function normally to uptake Gd-EOB-DTPA into the cell, thus generating a high signal under the T1W sequence.

[0115] The results of this in vitro experiment further confirmed the functional expression of Oatp1a1 on microglia. As a member of the organic anion transporting polypeptide family, Oatp1a1 can mediate the uptake of various substrates. Its successful expression and functional verification on the cell membrane are of great significance for understanding the metabolic and transport mechanisms of Oatp1a1 on microglia. In addition, this result is also consistent with the conclusion drawn from the cell uptake experiment, that is, Oatp1a1 has a normal transport function on microglia and can effectively uptake Gd-EOB-DTPA.

[0116] Example 6

[0117] Specificity of Cx3cr1-labeled microglia:

[0118] IBA-1 is an evolutionarily conserved calcium-binding protein. As an F-actin binding protein, IBA-1 can reshape the actin cytoskeleton of microglia. IBA-1 is specifically expressed in microglia. Therefore, antibodies targeting this protein are widely used to label microglia in the brain and other tissues. If the fluorescence signal labeled by IBA-1 has a good co-localization effect with the autofluorescence of the imaging gene mouse, it can prove that the microglia labeled by this gene mouse have high specificity.

[0119] 1. Establishment of the unilateral common carotid artery occlusion model (MCAO): (1) Preparation of experimental animals: Select male mice aged 6 - 8 weeks with a body weight of 23 - 25 g. (2) Anesthesia and fixation: Use 5% chloral hydrate (300 μL / 100 g) for general anesthesia. Fix the animal in the supine position on the operating table and secure the four limbs with tape to ensure the stability of the head. (3) Disinfection and incision of the neck skin: Disinfect the neck skin with iodophor and 75% ethanol. The disinfection range is centered on the incision with a radius of about 3 - 5 cm. Make a longitudinal incision about 2 - 3 cm long in the midline of the neck, bluntly separate the subcutaneous tissue and muscle, and expose the carotid sheath. (4) Separation of blood vessels: Separate the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). First, separate the CCA and thread it for standby, then separate the ECA and ICA. Temporarily clamp the distal end of the CCA with an arterial clip and ligate the proximal end of the ECA. (5) Insertion of the embolization wire: Insert the pre-prepared embolization wire from the stump of the ECA and slowly push it into the ICA. Continue to push the embolization wire until a slight resistance is felt. At this time, the tip of the embolization wire should reach the origin of the middle cerebral artery (MCA) to block the blood flow. (6) Reperfusion: After 60 minutes, remove the wire embolization and suture. Place the animal on a heating pad to recover from anesthesia and provide soft feed such as jelly after the operation.

[0120] 2. Immunofluorescence of mouse brain sections: (1) Brain extraction: After anesthetizing the mouse, expose the heart, insert the prepared PBS syringe into the left ventricle, direct it towards the aorta, and perfuse the mouse. Subsequently, switch the PBS to 4% paraformaldehyde for fixation. Decapitate the mouse to remove the brain, place the mouse brain tissue in 4% paraformaldehyde fixative for overnight fixation, and then dehydrate it with 30% sucrose solution. (2) Freezing: Embed the dehydrated brain tissue with embedding agent and place it on a -20°C freezing table for freezing. (3) Sectioning: Trim the frozen brain tissue block to be flat and coat it with OCT. Use a cryostat to section the tissue, with a thickness of generally 35 μm. Keep the low temperature during the sectioning process to avoid tissue damage. (4) Collecting sections: Use a disposable pipette to add 1–1.5 mL of antifreeze solution (50% PBS, 30% ethylene glycol, 20% glycerol) to each well of a 24-well plate, and collect the brain sections into the 24-well plate in order with a fine brush. (5) Rewarming: After restoring the collected brain sections to room temperature, wash them three times with 1×PBS for 5 minutes each time. (6) Blocking: Add 1 ml of immunofluorescence rapid blocking solution to each well of the 24-well plate, transfer the brain sections into it, and incubate at room temperature for 60 minutes. (7) Primary antibody incubation: Remove the blocking solution, add 300-500 μl of the prepared primary antibody working solution to the well plate, and incubate at 4°C overnight. (8) Primary antibody elution: Take out the well plate the next day and rewarm it for 30 minutes, then elute it three times with PBST for 5 minutes each time. (9) Fluorescent secondary antibody: Dilute the fluorescent secondary antibody with antibody diluent and incubate at room temperature for 1 hour, paying attention to avoiding light. (10) Secondary antibody elution: Elute it three times with PBST for 5 minutes each time. (11) Nuclear staining: Add 1 μg / mL DAPI working solution to the well plate, avoid light, incubate at room temperature for 30 minutes. Remove the DAPI working solution and wash it 3 times with PBS for 5 minutes each time.

[0121] (12) Mounting: Mount the sections with an anti-fluorescence quenching mounting medium. (13) Imaging: After air-drying the mounted sections, observe them under a fluorescence microscope or slide scanner to obtain fluorescence images.

[0122] After establishing a model by performing unilateral common carotid artery ligation (MCAO) surgery on the imaging gene mice, sacrifice the mice and select appropriate brain slices for immunofluorescence. The results of the co-localization experiment are as Figure 7As shown, it can be found that in the larger field of view of Figure A (cell population), most of the red fluorescence signals (IBA-1) and the cells labeled with green fluorescence signals (Cx3cr1) are almost the same, indicating that both can almost label microglia. In the smaller field of view of Figure B (cell soma visible), it can be found that the positions labeled by the red fluorescence signal (IBA-1) and the green fluorescence signal (Cx3cr1) are almost the same. According to the cell morphology, it can be judged that both label microglia in the brain parenchyma. Figure C is a quantitative analysis of the co-localization results in the area of Figure B, representing the change trend of the gray values of the two channels at each point in the area. It can be found that the IBA-1 signal and the Cx3cr1 signal have extremely similar change trends, indicating that they have a good co-localization effect. Therefore, the experimental results prove that the microglia labeled by Cx3cr1 have high specificity.

[0123] Example 7

[0124] Imaging of microglia under normal physiological conditions:

[0125] MRI T1W images were collected from gene mice at 6 - 8 weeks in the normal state. Taking the results of the initially collected images as the baseline, after collection, Gd-EOB-DTPA was injected by intrathecal injection, and the final T1W images were collected 24 hours later. After the collection was completed, the mice were sacrificed, and the brains were taken to collect fluorescence images (the experimental steps were the same as in Example 6).

[0126] 1. MRI data acquisition: In vivo MRI imaging was performed using a 9.4T (uMR 9.4T, United Imaging Life Science Instrument Co., Ltd., Wuhan, China) scanner. During imaging, mice were anesthetized with isoflurane using an anesthetic machine. During the induction period, mice were anesthetized with 1%-1.5% isoflurane and an air flow of 0.3-0.5 L / min, and during imaging, maintenance anesthesia was performed with 1%-1.5% isoflurane and 0.3-0.4 L / min air. For 9.4T, a mouse head surface coil developed by United Imaging was used for mouse brain imaging. For Gd-EOB-DTPA experimental T1W image acquisition, a mouse abdominal VTC42 coil was used, and for r1 determination experiments, a VTC24 coil was used. For the 9.4T imaging sequence, 2D T2 fast spin echo (T2-FSE) sequence was used to acquire T2W images (TR = 3000 ms; TE = 43.68 ms; pixel size = 78.13×78.13 um2; slice thickness = 0.5 mm; scan time = 4 min), 2D T1-FLAIR sequence (TI = 770 ms; TE = 5.9 ms; TR = 2000 ms; pixel size = 84.9×84.9 um2; slice thickness = 0.5 mm; scan time = 18 min); 3D T1-Quick sequence was used to obtain T1W images (TE = 1.39 ms; TR = 3.98 ms; pixel size = 104×104 um2; slice thickness = 0.1 mm; scan time = 28 min, flip angle = 10°); 3D T1-FSP sequence was used to obtain T1W images (TI = 770 ms; TE = 1.4 ms; TR = 3.9 ms; pixel size = 100×100 um2; slice thickness = 0.1 mm; scan time = 32 min, flip angle = 10°) to obtain T1W images. T1-mapping data acquisition parameters were as follows (TR = 35 ms; TE = 1.97 ms; pixel size = 172×172 um2; slice thickness = 0.2 mm; scan time for each flip angle = 14 min; flip angles = 5°, 10°, 20°, 30°, 40°, 50°) to obtain T1 maps at different Gd-EOB-DTPA concentrations at 6 flip angles.

[0127] 2. Data analysis: MATLAB (R2021a) was used to analyze the MRI images obtained from mouse brains. T1-weighted (T1W) and T2-weighted (T2W) images were registered using FSL (Oxford, UK). To illustrate the resolution differences between human and mouse MRI images, the resolution of all images acquired from mice was increased by 10 times before registration.

[0128] The region of interest (ROI) was drawn on the MRI image and the statistically significant signal differences were calculated. Additionally, the MRI image was matched to the mouse brain atlas (TMBTA) using affine registration, and PO, RT, SSp, SSs, MOs, MOp, VIS, and EPv were selected as the target ROIs. The resolution of the fluorescence image was reduced to match the MRI and it was converted to grayscale. Then, the MRI image was registered into the fluorescence image using the deformable image registration toolbox. The z-score and p-value of the signal intensity of the T1W image were calculated using ANOVA in MATLAB. The fluorescence image and paraffin section image were processed using imageJ, the 3D image was reconstructed into a 3D stereoscopic image using amira software and U_VIEWER software, and the molecular experiment was performed for sequence alignment and sequence design using SnapGene software.

[0129] The results are as Figure 8 shown. No obvious hyperintensity was observed in the image 24 h after injection of Gd-EOB-DTPA, which was similar to the green signal in the optical image, and this was consistent with the distribution and quantity of microglia in the basal state.

[0130] Example 8

[0131] Verification of dual-modal imaging of mouse microglia in the pinhole model:

[0132] Microglia have a reparative and therapeutic effect on the damaged area. Therefore, when a physical injury occurs in the brain parenchyma, microglia will gather at the injury site within a certain period of time to exert their injury repair function. Based on this property of microglia, physical injury can be used to aggregate microglia. A special injury model was used to make microglia gather regionally to better verify the accuracy of the dual-modal imaging of our transgenic mice.

[0133] 1. Pinhole-shaped brain injury model establishment: (1) Refer to the atlas to determine the injection site coordinates: According to the experimental requirements, refer to the relevant brain atlas to determine the three-dimensional coordinates (X, Y, Z axes) of the target PO brain region. The coordinates are X = 0.5 mm, Y = -1.8 mm, Z = -2.0 mm. (2) Instrument preparation: Seal the connection between the micro syringe and the glass electrode with hot melt adhesive, and fix it on the stereotaxic apparatus after standing and curing. (3) Anesthesia and fixation: Inject anesthetic into the animal with 5% chloral hydrate until it enters a deep anesthesia state. Shave the hair on the head, fix the animal on the stereotaxic apparatus adapter, insert the bilateral ear rods into the external auditory canals, adjust the scales to be consistent and tighten them to ensure that the head is centered. Gently clamp the incisors with the fixator to avoid being too tight. (4) Expose the skull: Disinfect the scalp with 70% alcohol, cut a wound about 2 cm long, wipe off the soft tissue on the skull surface with a dry cotton ball to completely expose the skull. Locate the anterior fontanelle (Bregma point) and use it as the origin of the three-dimensional coordinate system. Touch the Bregma point with the syringe tip and zero the readings of the X, Y, and Z axes of the stereotaxic apparatus. (5) Left-right leveling: Using the Bregma point as the midpoint, move the same distance left and right (such as 3.0 mm), lower the needle to touch the skull surface, read the Z-axis coordinate, and adjust the height of the ear rod to make it level left and right. Front-back leveling: Level with the anterior fontanelle point and the posterior fontanelle point (Lambda point), and the method is the same as left-right leveling. (6) Injury operation: According to the coordinates of the target brain region, move the syringe to the target position, mark it after touching the skull, and then drill a hole at the marked position with a cranial drill. Slowly insert the glass electrode needle to the target depth, stay for 2 - 10 minutes, and then slowly pull out the needle. (7) Suture the scalp and disinfect the wound with iodophor. Put the animal back into the cage and keep it warm on a heating pad if necessary.

[0134] The modeling time and method of this model are as Figure 9 shown. The interval between the modeling time and the MRI data collection is two weeks to enable better aggregation of microglia. After collecting the baseline, inject Gd-EOB-DTPA into each mouse, and sacrifice the mouse to collect optical images 24 hours after collecting the final results.

[0135] Collect the MRI T1W images of the control group and the experimental group 14 days after modeling as the baseline control after injecting Gd-EOB-DTPA. After the first collection, inject the Gd-EOB-DTPA contrast agent into each mouse, collect its T1W image again 24 hours later, and then sacrifice the mouse and obtain the data of its optical imaging. Gd-EOB-DTPA shows high signal in the T1W sequence and can be taken up by the Oatp1a1 protein on microglia and enter the cells. If the high signal in the T1W sequence in this experiment can correspond to the microglia signal in the optical results, it can indicate the correctness of using MRI to trace microglia.

[0136] The results are as Figure 10As shown, in the control group (modeling was performed on wildtype C57BL / 6 mice), no obvious hyperintense regions appeared after injection of Gd-EOB-DTPA. In the experimental group (modeling was performed on genetically modified mice), linear needle-like hyperintense regions appeared near the brain regions with pinhole injuries (indicated by the white arrows in Figure B).

[0137] After collecting the images after injection of the contrast agent, the mice were immediately sacrificed to obtain the results of optical imaging. The magnetic resonance images were compared with the optical images as Figure 11 shown. For the hyperintense regions observed on magnetic resonance, after obtaining mouse brain slices, fluorescence images were collected at the same level (the experimental steps were the same as in Example 6). The results showed that in the hyperintense regions of T1W in the experimental group, the aggregation of microglia was also observed at the same positions in the fluorescence results. And in the further magnified images, the aggregated green signals could clearly show the cell bodies and branches of microglia, which preliminarily proved the accuracy of the dual-modal imaging in this genetically modified mouse for dual-modal imaging, and also indicated that the above method could trace microglia by magnetic resonance in living small animals.

[0138] Example 9

[0139] Verification of dual-modal imaging of microglia in mice with middle cerebral artery occlusion model:

[0140] The MCAO (middle cerebral artery occlusion) model can temporarily or permanently block the blood flow of the middle cerebral artery through surgical methods, and can accurately simulate the pathophysiological processes of clinical cerebral ischemia and reperfusion injury. It is one of the most widely used experimental models for studying ischemic stroke.

[0141] The modeling method was the same as in Example 6, and the time points and methods for modeling and data collection Figure 12 as shown. MRI data were collected on the fifth day after modeling. As Figure 12 shown, the time point for reperfusion after cerebral ischemia used was 60 minutes, which was a time point with less damage to the mice and good phenotypes. And baseline images were collected and intrathecal injection was performed on the fifth day after mouse modeling, and the final nuclear magnetic resonance images were collected and the mice were sacrificed to collect optical images on the sixth day.

[0142] To ensure the reliability and reproducibility of experimental data, establishing a standardized model quality assessment system is an important prerequisite for carrying out subsequent research. Histological staining and imaging detection were used to systematically verify the construction quality of the MCAO model. Through 2,3,5-triphenyltetrazolium chloride (TTC) staining and MRI technology (the experimental steps were the same as in Example 5), the cerebral infarction regions were characterized from the aspects of histopathology and in vivo imaging, respectively.

[0143] TTC Staining: Prepare 1% TTC staining solution. Weigh 0.15 g of TTC powder and place it in a covered petri dish wrapped with tin foil. Pipette 15 ml of PBS stored at room temperature to dissolve it, and store it at 4°C while avoiding light as much as possible during the whole process. The staining solution needs to be prepared and used immediately. (1) Specimen collection: After anesthetizing the mice, decapitate them and take out the brains. Place the brains in a brain trough and put them in an -80°C refrigerator for 3 minutes to slightly freeze them to facilitate sectioning. (2) Sectioning: Cut the brains coronally into four 2-mm sections, taking care not to damage the integrity of the brain slices (cut immediately after taking them out of the -80°C refrigerator). The first cut is made at the midpoint of the line connecting the anterior pole of the brain and the optic chiasm; the second cut is at the optic chiasm; the third cut is at the infundibular stalk; the fourth cut is between the infundibular stalk and the caudal pole of the posterior lobe. The cut brain slices are rewarmed in PBS at room temperature. (3) Staining: Place the brain slices in 1% TTC solution and incubate them in the dark in a 37°C incubator for 15 - 30 minutes, turning the brain slices midway to ensure uniform staining. (4) Observation and preservation: After staining, photos can be taken for observation. If subsequent quantitative photo detection of the infarct area is required, the brain slices can be fixed and preserved in 4% formalin solution.

[0144] The results are as Figure 13 shown. The TTC staining results show (Figure A) that the model group presented significant pathological changes compared with the sham operation control group. Normal brain tissue showed characteristic rose red after TTC incubation (reflecting mitochondrial dehydrogenase activity), while the ischemic infarct area showed a clearly demarcated pale lesion due to the loss of enzyme activity. This result conforms to the internationally recognized successful criteria for the MCAO model (infarct volume > 20% of the ipsilateral hemisphere volume). In corroboration, MRI examination (Figure B) observed clear ischemic lesions in the T2-weighted imaging (T2WI) sequence: the infarct area showed high signal changes on T2WI, reflecting the increased local water content caused by vasogenic edema; the pathological changes in the early stage of cytotoxic edema were accurately captured.

[0145] The above two verification systems not only confirmed the stability of the model construction, but also laid a reliable foundation for subsequent molecular imaging research. Subsequently, experimental animals meeting the criteria (infarct volume 20 - 35% of the ipsilateral hemisphere volume) will be selected based on this verification system to enter the imaging research cohort, so as to minimize the impact of model variation on the experimental results.

[0146] After confirming the stability of the MCAO model, the dual-modal imaging effect of microglia in this disease model was verified. The imaging protocol adopted a dual-time-point dynamic contrast-enhanced MRI strategy: First, on the fifth day after modeling, a baseline T1-weighted imaging (T1WI) was obtained before the injection of the contrast agent, and then gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) was injected intrathecally. This hepatobiliary-specific contrast agent can enter microglia through the active transport mechanism mediated by Oatp1a1, and its T1 shortening effect can make the cells expressing this transporter show characteristic hyperintensity on T1W. A delayed-phase MRI scan was performed 24 hours after injection, and the optical imaging data of the brain tissue was collected synchronously to verify the correspondence between the MRI signal and the spatial distribution of microglia.

[0147] The results were as Figure 14 shown. Imaging analysis showed ( Figure 14 A), no obvious signal enhancement area was seen in the wild-type control group after the injection of the contrast agent. In sharp contrast, regional hyperintensity appeared in the ischemic penumbra area of the experimental group gene mice ( Figure 14 B white arrow), and the signal intensity increased compared with the baseline.

[0148] After collecting the images after injecting the contrast agent, the mice were immediately sacrificed to obtain the results of optical imaging. The comparison of magnetic resonance images and optical images was as Figure 15 shown. The hyperintense area observed on magnetic resonance. After obtaining the mouse brain slices, fluorescence images were collected at the same level (the experimental steps were the same as in Example 6). The results showed that in the hyperintense area of T1W in the experimental group, the aggregation of microglia was also observed at the same position in the fluorescence results. And in the further magnified images, it can be seen that the aggregated green signal can clearly show the cell bodies and branches of microglia, which preliminarily proves the accuracy of the imaging gene mice in dual-modal imaging and also indicates that the above method can trace microglia by magnetic resonance in living small animals.

[0149] Example 10

[0150] Verification of dual-modal imaging of mouse microglia in the neuron ablation model:

[0151] The diphtheria toxin (DT) and diphtheria toxin receptor (DTR) system is a commonly used genetic tool that is widely applied in cell-specific ablation studies in mouse experiments. First, the rAAV-hsyn-DTR-tdTomato-WPREs virus was constructed. This virus specifically infects neurons at the injection site through the hysn promoter, causing the expression of diphtheria toxin receptor (DTR) on the infected neurons. Then, by injecting diphtheria toxin (DT), these cells are specifically ablated.

[0152] The model establishment and data collection time of this model are as Figure 16 shown. First, the constructed virus was injected into the PO nucleus of transgenic mice (experimental group) and wild type C57BL / 6 mice (control group) to infect neurons in this area. After the virus was fully expressed, the drug diphtheria toxin was injected to kill these infected neurons. We found that on the sixth day after the drug injection, that is, the sixth day after the neurons were killed, more activated microglia could be observed.

[0153] To verify the function of the viral vector and the neuron ablation efficiency, a bifunctional recombinant virus carrying the diphtheria toxin receptor (Diazepam Toxin Receptor, DTR) and the red fluorescent protein tdTomato was injected intracranially. After the virus was expressed, the infected neurons could be visualized through the tdTomato fluorescence signal (excitation / emission wavelength: 554 nm / 581 nm). After injecting diphtheria toxin (DT), specific apoptosis occurred in DTR-positive neurons, and the tdTomato fluorescence signal mostly disappeared as the neurons were killed. After injecting the virus, coronal cryosections of the mouse brain tissue in the experimental group were prepared and subjected to immunofluorescence staining.

[0154] The results are as Figure 17 shown. Confocal microscopy analysis showed that neurons at the virus injection site were specifically infected, and the number of tdTomato+ neurons was significantly lower in the DT-treated group than in the control group, confirming the spatio-temporal specific ablation ability of this virus system and providing an important basis for subsequent imaging verification.

[0155] After confirming that the combination of DTR virus and diphtheria toxin can specifically ablate neurons in specific nuclei, the imaging effect of microglia was then continuously studied. The imaging protocol adopted a dual-time-point dynamic contrast-enhanced MRI strategy: First, on the 4th day after modeling (the 4th day after the last injection of diphtheria toxin), a baseline T1-weighted imaging (T1WI) was obtained before the injection of the contrast agent. Subsequently, gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) was injected intrathecally. This hepatobiliary-specific contrast agent can enter microglia through the active transport mechanism mediated by Oatp1a1, and its T1 shortening effect can make the cells expressing this transporter show characteristic hyperintensity on T1W. Delayed-phase MRI scanning was performed 24 hours after injection, and optical imaging data of the brain tissue was collected synchronously to verify the correspondence between the MRI signal and the spatial distribution of microglia.

[0156] The results are as Figure 18 shown. Imaging analysis showed ( Figure 18 A), no obvious signal enhancement area was seen on T1W in the wild-type control group after the injection of the contrast agent. In sharp contrast, regional hyperintensity appeared in the ischemic penumbra area of the experimental group gene mice ( Figure 18 B white arrow), and its signal intensity increased compared with the baseline.

[0157] After collecting the images after injecting the contrast agent, the mice were immediately sacrificed to obtain the results of optical imaging. The comparison of magnetic resonance images and optical images is as Figure 19 shown. The hyperintense area observed on magnetic resonance. After obtaining the mouse brain slices, fluorescence images were collected at the same level. The results showed that in the hyperintense area of T1W in the experimental group, the aggregation of microglia was also observed at the same position in the fluorescence results. And in the further magnified images, it can be seen that the aggregated green signal can clearly show the cell bodies and branches of microglia, which preliminarily proves the accuracy of the gene mice in dual-modal imaging and further indicates that the above method can trace microglia by magnetic resonance in live small animals.

[0158] It should be noted that, in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the existence of additional identical elements in the process, method, article or device comprising the said element.

[0159] 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 foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an animal model for microglia imaging, characterized in that, The animal model overexpresses the Oatp1a1 protein.

2. The construction method according to claim 1, characterized in that, The construction method includes: (1) Transfecting an animal with a targeting vector containing a promoter, loxp-stop-loxp, the Oatp1a1 gene, and polyA to obtain a chimeric animal; (2) Mating the chimeric animal with the opposite sex expressing Cre to obtain the animal model.

3. The construction method according to claim 2, wherein The targeting site of the targeting vector includes the Rosa26 gene locus.

4. The construction method according to claim 3, wherein, It also includes screening for offspring expressing Oatp1a1 through genotype identification.

5. The construction method according to claim 1, characterized in that The imaging implementation method includes one or a combination of MRI, CT, optical imaging, X-ray, PET, PET-CT, SPECT, SPECT / CT, and PEM.

6. The construction method according to claim 5, characterized in that The imaging implementation method is MRI.

7. Use of the animal model obtained by the construction method according to any one of claims 1-6, characterized in that, The applications include: 1) Application in constructing a microglia-related disease model; 2) Application in studying the pathogenesis of microglia-related diseases; 3) Application in screening candidate drugs for microglia-related diseases.

8. The application according to claim 7, characterized in that, The microglia-related diseases include one or a combination of pinhole brain injury, middle cerebral artery occlusion, and neuronal ablation.

9. The construction method according to any one of claims 1-6 or the application according to claim 8, characterized in that, The animal is a mammal.

10. The construction method or application according to claim 9, characterized in that, The mammals include one or a combination of apes, monkeys, rodents, artiodactyls, and perissodactyls.

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