Plasma sample processing method

Abstract

The invention relates to the technical field of plasma treatment, in particular to a plasma sample treatment method. The method comprises the following steps that S1, an injector is pretreated, the injector is washed with 0.5 M ethylenediamine tetraacetic acid, and the pH of the ethylenediamine tetraacetic acid is 8.0; s2, the syringe obtained in the step S1 is used for collecting plasma samples from mice through heart puncture, and the mice with the plasma samples collected are male mice of EKO, E4 and E3 genotypes; s3, treating the blood plasma sample obtained in the step S2: S31, centrifuging the blood sample under 5500xg, separating out blood plasma, and collecting the blood plasma according to genotypes; and S32, dialyzing the collected plasma by using a Slide-A-LyzerTM dialysis bag and a phosphate buffer solution, and then immediately storing the dialyzed plasma at the temperature of 80 DEG C below zero. According to the plasma sample treatment method provided by the invention, the plasma sample taken out from the mouse heart is treated, and then the treated plasma sample can be applied to a medical mouse model.

Description

Technical Field

[0001] The present invention relates to the technical field of plasma processing, and in particular to a method for processing plasma samples. Background Art

[0002] Tauopathies are a class of neurodegenerative diseases characterized by abnormal aggregation of Tau protein in the central nervous system (CNS). Apolipoprotein E (APOE) has three main subtypes - APOEε2, APOEε3, and APOEε4 - which can directly affect Tauopathies and their related neurodegenerative processes. Some common methods known include parabiosis, cerebrospinal fluid (CSF) replacement, and plasma exchange, and these methods have been reported to alleviate pathology and clinical manifestations. In recent years, various methods have been proposed to alleviate pathology and clinical manifestations, such as parabiosis, cerebrospinal fluid (CSF) replacement, and plasma exchange.

[0003] When using the above technologies, it is found that the following technical problems exist in the prior art: Plasma needs to be processed to a certain extent before application, and the existing processing methods are not complete enough. Therefore, a method for processing plasma samples is designed to provide another technical solution to the above technical problems. Summary of the Invention

[0004] Based on this, it is necessary to provide a method for processing plasma samples to solve the technical problems raised in the above background art.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for processing plasma samples, the steps are as follows:

[0007] S1: Pretreat the syringe;

[0008] S2: Use the syringe obtained in step S1 to collect plasma samples from mice by cardiac puncture;

[0009] S3: Process the plasma samples obtained in step S2.

[0010] As a preferred embodiment of the method for processing plasma samples provided by the present invention, in step S1, when pretreating the syringe, the steps are as follows:

[0011] Rinse the syringe with 0.5M ethylenediaminetetraacetic acid, and the pH of the ethylenediaminetetraacetic acid is 8.0.

[0012] As a preferred embodiment of the method for processing plasma samples provided by the present invention, in step S2, the male mice used for collecting plasma samples are of EKO, E4, and E3 genotypes.

[0013] As a preferred embodiment of the method for processing a plasma sample provided by the present invention, in step S3, the plasma sample obtained in step S2 is processed as follows:

[0014] S31: The blood sample is centrifuged at 5500 xg to separate the plasma, and the plasma is pooled according to the genotype;

[0015] S32: The pooled plasma is dialyzed using a Slide-A-Lyzer TM dialysis bag and phosphate buffer, and then immediately stored at -80 °C.

[0016] Application of the plasma sample, the steps are as follows: After thawing the processed plasma sample, it is injected into mice through retro-orbital injection.

[0017] As a preferred embodiment of the application of the plasma sample provided by the present invention, the steps are as follows:

[0018] The mice are anesthetized with isoflurane, and the plasma is injected within 10 s;

[0019] When the mice reach 9.5 months of age, they are perfused with heparin-containing PBS on ice to remove the blood.

[0020] It can be seen without doubt that through the above technical solutions of the present application, the technical problems to be solved by the present application can surely be solved.

[0021] Meanwhile, through the above technical solutions, the present invention has at least the following beneficial effects:

[0022] A method for processing a plasma sample provided by the present invention processes the plasma sample taken from the heart of a mouse, and then the processed plasma sample can be applied to a mouse model in medicine, thereby proving that young plasma has no alleviating effect on tau proteinopathy and is independent of the APOE subtype. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is a flowchart of plasma processing according to the present invention;

[0025] Figure 2 It is a schematic diagram of plasma characteristics and experimental design of different APOE subtype mice according to the present invention;

[0026] Figure 3 Schematic diagram showing that injection of young plasma of the present invention fails to alleviate Tau protein pathology in PS19 mice;

[0027] Figure 4 Schematic diagram showing no significant changes in microglia treated with plasma of different APOE subtypes of the present invention;

[0028] Figure 5 Schematic diagram showing up - regulation of GFAP - positive astrocytes and no change in AQP4 polarity in mice treated with APOE3 plasma of the present invention. Detailed implementation manners

[0029] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0031] It should be noted that, without conflict, the embodiments in the present invention and the features and technical solutions in the embodiments can be combined with each other.

[0032] It should be noted that similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0033] Example 1

[0034] Reference Figure 1 , a method for processing a plasma sample.

[0035] Animal

[0036] P301S Tau transgenic mice (B6;C3-Tg(Prnp-MAPT*P301S)PS19Vle / J, stock number #008169) were obtained from the Jackson Laboratory and backcrossed to C57BL / 6J mice for more than 10 generations and then maintained on a C57BL / 6J background. Human E3, E4, and APOE KO (EKO) mice were purchased from Taconic. All mice were maintained on a C57BL / 6J background. The care and experiments of all experimental animals were performed in accordance with the requirements of the Animal Ethics Committee of Chongqing Medical University. Mice were housed at a constant temperature with a 12-hour light / dark cycle and provided with free access to food and water. Only male mice were used because the Tau protein pathology and neurodegeneration in male PS19 mice were significantly higher than those in female mice, which might be more suitable for evaluating anti-phosphorylated Tau treatment strategies.

[0037] Animal Plasma Collection and Processing

[0038] Plasma was collected from male mice (5-6 weeks old) of EKO, E4, and E3 genotypes by cardiac puncture. Before collection, the syringe was rinsed with 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 8.0). Blood samples were centrifuged at 5500 x g to separate the plasma, and the plasma was pooled by genotype. To remove residual EDTA, the pooled plasma was dialyzed using a Slide-A-Lyzer TM dialysis cassette (3.5K MWCO, Thermo Fisher Cat#66130) against phosphate-buffered saline (PBS) and then immediately stored at -80 °C. Plasma samples underwent only one freeze-thaw cycle before use. During the experiment, plasma was injected into mice via retro-orbital injection. The experiment was divided into four groups, with each group containing 9-15 male PS19 mice at 5.5 months of age. Each group was injected with 150 μl of plasma of different APOE subtypes or PBS as a control once a week. Mice were injected under isoflurane anesthesia, and the injection process was controlled within 10 seconds. At 9.5 months of age, mice were perfused with heparinized PBS on ice to clear the blood.

[0039] Glial-Neuronal Co-Culture

[0040] Microglia were isolated from the meninges of male C57BL / 6 mice at postnatal day 2 (P2). The meninges were removed using pre-cooled calcium- and magnesium-free balanced salt solution (BSS), followed by dissection of the cerebral cortex. The cortical tissue was enzymatically digested with Hanks' balanced salt solution (HBSS) containing 0.25% trypsin (GIBCO Cat#15090-046) and 0.2 mg / mL deoxyribonuclease I (Sigma Cat#DN-25) at 37 °C for 10 minutes. The digested tissue was filtered through a 70-μm nylon cell strainer (Beyotime Cat#FSTR070). The resulting cell suspension was centrifuged at 1000 x g for 5 minutes, and the cell pellet was resuspended and washed with microglia medium containing 10% fetal bovine serum (FBS), 1x penicillin-streptomycin (Pen / Strep), and 1x GlutaMAX. The cells were seeded into a T75 culture flask (Thermo Fisher Scientific Cat#156499) for culture. After the monolayer cells grew to confluence, they were centrifuged to form a pellet, washed, and seeded onto 24-well tissue culture plates coated with Geltrex (Gibco Cat#A1413201) glass coverslips at a density of 75,000 cells per well and cultured using Neurobasal medium for 2 days.

[0041] Neurons were obtained from PS19 mouse embryos at embryonic day 17. The method of dissection and isolation of the cerebral cortex was the same as that for extracting microglia, except that Neurobasal medium (Neurobasal + 1x B27 + 1x penicillin / streptomycin + 1x L-glutamine) was used for washing. Then, the microglia were washed with 1 ml of Neurobasal medium (Neurobasal + 2% B27 + 1x penicillin / streptomycin + 1x L-glutamine) and placed in 400 μl of Neurobasal medium for use.

[0042] Prepared neurons were seeded onto glial cells at a density of 20,000 cells per well. In all cell culture experiments, only the central wells of the culture plates were used, and the surrounding wells were filled with 1 ml of sterile double-distilled water. After co-culturing for 2 weeks, 200 μl of fresh neuron medium containing 10 μl of different APOE subtypes (dialyzed through a 3.5 kDa dialysis membrane) or PBS as a control was added for 24 hours of co-culture. Then, neuron staining analysis was performed using a microtubule-associated protein 2 (MAP2) antibody (Thermo Fisher Scientific Cat#OSM00030W), and the cell nuclei were confirmed using 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific Cat#62248).

[0043] Immunocytochemical staining (IHC)

[0044] Cells were fixed in Dulbecco's phosphate-buffered saline (DPBS) containing 4% paraformaldehyde (PFA) for 10 minutes at room temperature and permeabilized with DPBS containing 0.25% Triton X-100 for 10 minutes. Then, they were blocked with DPBS containing 0.25% Triton X-100 and 3% bovine serum albumin (BSA) for 30 minutes at room temperature. Images were taken using an Olympus SpinSR10 confocal microscope at 10× magnification and quantitatively analyzed using ImageJ software.

[0045] Protein immunoblotting (WB)

[0046] Plasma protein detection:

[0047] Plasma samples were collected from 16-month-old and 6-week-old male mice of E4 / E3 / EKO genotypes. After diluting the plasma 50-fold, 5x SDS loading buffer was added, and Western blot analysis was performed.

[0048] Protein separation was performed using MOPS buffer and a 4-12% SurePAGE gel. The gel was transferred to a polyvinylidene difluoride (PVDF) membrane, which was then blocked with Tris-buffered saline (TBST) containing 5% non-fat dry milk and 0.05% Tween-20 for 60 minutes at room temperature. The membrane was incubated with anti-interleukin-6 (IL-6) antibody (Affinity rabbit polyclonal antibody 1:1000) and anti-tumor necrosis factor α (TNF-α) antibody (Affinity rabbit polyclonal antibody 1:1000) overnight at 4°C.

[0049] On the second day, the membrane was incubated with the corresponding horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. After color development, the antibody was eluted using Western Stripping Buffer (Beyotime Cat#P0025). The membrane was re-blocked and incubated overnight at 4°C with the anti-transferrin antibody (Proteintech rabbit polyclonal antibody 1:5000). On the second day, the membrane was incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1 hour and color development was performed.

[0050] Histone detection:

[0051] For each mouse, 5 mg of posterior cerebral cortex tissue was taken and lysed using radioimmunoprecipitation buffer (RIPA, stored at 4°C) supplemented with protease inhibitor (Thermo Fisher Scientific Cat#36978) and phosphatase inhibitor (Sigma Cat#P5726). Protein concentration was measured using the Micro Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific Cat#23235). 20 μg of total protein was loaded in MOPS buffer and separated using 4-12% polyacrylamide gel electrophoresis (PAGE). The gel was transferred to a PVDF membrane, and the membrane was blocked with 5% non-fat milk in TBST at room temperature for 60 minutes. The membrane was incubated overnight at 4°C with anti-AT8 antibody (Thermo Fisher rabbit monoclonal antibody MN1020, 1:1000), anti-Thr181 antibody (Thermo Fisher rabbit monoclonal antibody 701530, 1:1000), anti-total Tau antibody (Proteintech rabbit polyclonal antibody 10274-1-AP, 1:1000), and anti-2A7 antibody (Xiamen University). On the second day, the membrane was incubated with the corresponding HRP-labeled secondary antibody (1:5000) at room temperature for 1 hour and color development was performed. After color development, the antibody was eluted using Western Stripping Buffer (Beyotime P0025). The membrane was re-blocked and incubated overnight at 4°C with the anti-transferrin antibody (Proteintech rabbit polyclonal antibody 1:5000). On the second day, the membrane was incubated with the corresponding HRP-labeled secondary antibody (1:5000) at room temperature for 1 hour and color development was performed. Images were taken using an imaging system (Fusion FX SPECTRA) and analyzed using ImageJ.

[0052] Example 2

[0053] Based on the above Example 1, referring to Figures 2 - 5 , the processed plasma was applied to prove whether young plasma injection could alleviate Tau protein pathology in PS19 mice.

[0054] Quantitative polymerase chain reaction (qPCR)

[0055] The posterior cortex tissue (5 mg) of each mouse was used to extract RNA using the RNeasy Mini Kit (QIAGEN Cat#74104). The purity and concentration of RNA were evaluated by measuring the A260 / A280 ratio and A260 value using a NanoDrop One micro-spectrophotometer (Thermo Fisher). Complementary DNA (cDNA) was synthesized using the RT Master Mix for qPCR II-gDNA Digester Plus (MedChemExpress Cat#HY-K0510A). qPCR analysis was performed using a 480 instrument. The relative gene expression levels were quantitatively measured using TaqMan primers and normalized to the housekeeping gene GAPDH.

[0056] Volume analysis and measurement of neuronal layer thickness

[0057] The left half of the brain of each mouse was fixed in 4% paraformaldehyde overnight and then immersed in 30% sucrose solution for at least 24 hours. Coronal sections with a thickness of 50 μm were cut using a Leica SM2010 sliding microtome and stored as floating sections. The volumes of the hippocampus, piriform cortex, and ventricles were evaluated using stereological analysis. Six to seven brain sections of each mouse were selected according to the degree of brain atrophy and sampled at 300-μm intervals from bregma -1.3 mm to bregma -3.1 mm. All sections were mounted on glass slides, air-dried at room temperature for 2 hours, and then dried in an oven for 3 hours until completely dehydrated. The sections were rinsed with PBS for 2 minutes, stained with Nissl staining solution for 5 minutes, then briefly incubated in PBS for 3 seconds, and transferred to a new PBS bottle to remove excess staining solution. Subsequently, the sections were dehydrated through an ethanol series (50%-100%) and washed twice in xylene for 4 minutes each, then mounted with neutral resin and dried overnight. The sections were scanned using an Olympus VS-200 scanner at a magnification of 20 times. The areas of the hippocampus, piriform cortex, and ventricles were calibrated using VS-200 software. The volume calculation formula was: volume = (sum of areas) × 0.3 mm. The thickness of the granule cell layer of the dentate gyrus was measured by drawing scale lines in two different regions of the section, and the average value was calculated.

[0058] Immunofluorescence staining (IF)

[0059] Slices were washed three times with Tris-buffered saline (TBS) for 5 minutes each at room temperature. Subsequently, the samples were incubated in TBS containing 0.25% Triton X-100 (TBSX) for 30 minutes to enhance tissue permeability. After permeabilization, the slices were washed again with TBS three times for 5 minutes each. To block non-specific antibody binding, the slices were incubated in blocking buffer containing 10% donkey serum in TBSX for 1 hour and then incubated overnight at 4°C with the following primary antibodies: goat polyclonal ionized calcium-binding adapter molecule 1 (Iba1) (Abcam Cat#ab5076, 1:500), goat polyclonal glial fibrillary acidic protein (GFAP) (Abcam Cat#ab53554, 1:500), mouse monoclonal cluster of differentiation antigen 68 (CD68) (Bio-Rad Cat#MCA1957, 1:300), mouse monoclonal AT8 biotinylated antibody (Abcam Cat#ab54450, 1:500), rabbit polyclonal aquaporin-4 (AQP4) (Abcam Cat#ab46182, 1:500), rabbit polyclonal microtubule-associated protein 2 (MAP2) (Abcam Cat#ab32454, 1:500).

[0060] On the next day, the slices were washed again with TBS three times for 5 minutes each and then incubated with the corresponding fluorescently labeled secondary antibodies for 1 hour at room temperature. All washing and incubation steps were performed in the dark. The slices were further incubated with 2 μg / ml Hoechst (DAPI channel) for 10 minutes in the dark and then washed with TBS three times for 5 minutes each at room temperature. Finally, the slices were mounted with ProLong TM Gold antifade mounting medium (Invitrogen TM Cat#P36930). It should be noted that the value of "AQP4 polarity" was derived from the ratio of the low-stringency region to the high-stringency region. A higher AQP4 polarity value indicates a higher level of AQP4 expression in perivascular endfeet than in the surrounding tissue, while a lower AQP4 polarity value indicates that the AQP4 expression in perivascular endfeet is more similar to the AQP4 expression in the non-perivascular region. Images were acquired using an Olympus SpinSR10 confocal microscope and analyzed using ImageJ and Imaris software.

[0061] Statistical analysis

[0062] All results are presented as mean ± standard error of the mean (SEM), and all statistical analyses were performed using Prism 8 (GraphPad Software). Significance between two groups was determined by Student’s t-test. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) with multiple comparisons was used. A p-value less than 0.05 (p < 0.05) was considered statistically significant. The significance levels are shown as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0063] APOE4 plasma exacerbates neuronal injury in co-culture systems

[0064] Differences in the levels of certain inflammation-related proteins were confirmed between young and old mice, as well as between mice with different APOE subtypes. APOE was undetectable in EKO mice, and was statistically significant compared with E4 and E3 mice ( Figure 2 A). The APOE expression levels were nearly the same between young and old mice ( Figure 2 A). Representative inflammatory factors, especially TNF-α, showed lower protein levels in the plasma of young mice than in old mice, and were statistically significant in E4 plasma ( Figure 2 B). TNF-α showed the highest levels in E4 plasma, gradually decreasing in E3 and EKO plasma. To investigate the effect of lipoprotein-containing plasma on neuronal synapses, primary neurons from mice expressing P301S Tau were co-cultured with mixed primary glial cells from wild-type (WT) mice.

[0065] In neurons from mice treated with E4 plasma, significant synapse loss was observed, while neurons from mice treated with E3 and EKO plasma were less damaged ( Figure 2 C and 2D). This indicates that plasma containing APOE4 exacerbates synapse loss in vitro, possibly due to lipid component dysregulation. In addition to the lipid effect, the lower levels of pro-inflammatory factors in young plasma may also provide some protective effects, similar to neurons treated with EKO plasma.

[0066] Young plasma injection fails to alleviate Tau protein pathology or Tau-mediated neurodegeneration in PS19 mice

[0067] To further investigate the effect of young plasma with different APOE subtypes on Tau protein pathology in vivo, the P301S Tau mouse model was used. Young plasma was collected from 5-week-old EKO, E3, and E4 mice. The plasma was injected into P301S Tau mice via retro-orbital venous injection once a week starting at 6 months of age, and samples were collected at 9.5 months of age. Figure 3A). Whether the injection of young plasma with different APOE subtypes has different effects on brain atrophy was evaluated. Unfortunately, compared with the PBS-treated control group mice, none of the young plasma treatments could alleviate brain atrophy ( Figure 3 B). Statistical analysis showed that on specific atrophy indicators, such as the hippocampal volume ( Figure 3 C), piriform cortex volume ( Figure 3 D), ventricular volume ( Figure 3 E), and dentate gyrus (DG) layer thickness ( Figure 3 F), no significant differences were shown regardless of the APOE subtype contained in the treated plasma. In addition, it was also found that in P301S mice treated with young plasma, the total Tau protein level was significantly higher than that in the PBS group ( Figure 3 G). Previous studies have shown that the dysregulation of lipid components can disrupt the microenvironment and promote the spread of Tau protein pathology, which is consistent with the results. However, the phosphorylation sites of Tau protein, such as 2A7 ( Figure 3 H) and Thr181 ( Figure 3 I), did not show significant changes in WB, and the AT8 staining ( Figure 3 J and 3K) representing hyperphosphorylated Tau also did not show significant changes.

[0068] There were no significant changes in the phagocytosis of microglia and neuroinflammatory factors under young plasma treatment

[0069] Neuronal death and synaptic loss are mainly due to the overactivation of microglia. To evaluate the effects of young plasma with different APOE subtypes on the functional changes of microglia under Tau protein pathology, immunofluorescence staining was performed on the brain tissues of 9.5-month-old P301S mice. Microglia were stained with Iba1, and a slight increase was observed in the E4 young plasma treatment group, although statistical analysis showed no significant differences among the four groups ( Figure 4 A and 4B). Similar results were also shown in the analysis of the percentage of the area covered by CD68-labeled lysosomal granules ( Figure 4 C). The phagocytosis of microglia was evaluated by the co-localization and area ratio of CD68 and Iba1 ( Figure 4 D). The results showed that there were no significant changes in the phagocytic activity of microglia under E3, E4, and EKO young plasma treatments ( Figure 4 E).

[0070] In addition to staining analysis, a set of genes related to or activated by inflammation in the cortex, including Iba1, triggering receptor expressed on myeloid cells 2 (Trem2), CD68, glycoprotein non-metastatic melanoma protein b (Gpnmb), chemokine (C-X3-C motif) receptor 1 (Cx3cr1), and dendritic cell-associated C-type lectin-1 (Dectin-1, also known as Clec7a), were quantified by qPCR. These genes were relatively stable after injection of young plasma in the presence of Tau pathology ( Figure 4 F). This indicates that microglia in P301S Tau mice did not respond significantly to young plasma treatment.

[0071] The characteristics of astrocytes under Tau pathology were not affected by young plasma injection

[0072] Astrocytes are the main producers of APOE in the CNS. It is of great significance to study whether young plasma of different APOE subtypes can affect astrocytes under Tau pathology. AQP4 is known to be upregulated in the rTg4510 Tauopathy model, but the response to Tau pathology in reactive astrocytes shows a decrease in the polarity of AQP4 towards astrocytic endfeet. However, the results showed no significant change in AQP4 polarity among the four groups ( Figure 5 A and 5B). Interestingly, the number of astrocytes in the hippocampal tissue of PS19 mice gradually increased from PBS treatment to E3 young plasma. In particular, the coverage area of GFAP in Tau mice treated with E3 young plasma was significantly higher than that in the control group ( Figure 5 C). In addition, several inflammation-related genes related to astrocytes were analyzed by qPCR. Except for the expression of GFAP, other genes, including major histocompatibility complex class II region gene 23 (H2.T23), vimentin (Vim), complement component 3 (C3), and transforming growth factor β (TGF-β), did not show significant changes among the four groups ( Figure 5 D). In summary, although the number of astrocytes increased slightly, the activation state of astrocytes did not change significantly, suggesting that the activation of astrocytes may not have made a significant contribution to Tau-mediated neurodegeneration after injection of young plasma.

[0073] The in vitro experimental results are consistent with existing studies, indicating that different APOE subtypes have different effects on neuronal damage, suggesting that targeting APOE may be a feasible strategy. However, due to the complexity of the internal environment, our in vivo experiments did not show significant changes in key indicators of Tau pathology. This indicates that young plasma injection may not be able to effectively improve Tau pathology. Figure 2Among them, (A) and (B) analyzed the protein levels of APOE, IL-6, TNF-α, and transferrin (TRF) in the plasma of human APOE knock-in (KI) E3, E4, and EKO mice by Western blot. (C) Representative images showing primary PS19 neurons co-cultured with glial cells (80–90% astrocytes, 10–20% microglia) from C57BL / 6 mice for 2 weeks. (D) Quantitative analysis of the neuronal area covered by MAP2 in plasma-treated neurons (4 wells per genotype, 9 random images per well). Scale bar = 500 μm; n = 9; *p < 0.05, **p < 0.01; Student's t-test (A)(B). One-way ANOVA; data are presented as mean ± standard error of the mean (SEM).

[0074] Figure 3 Among them, (A) Experimental design: Plasma was collected from male mice of different APOE genotypes at 5–6 weeks of age. Starting at 5.5 months of age, PS19 male mice were injected with 150 μl of plasma weekly until tissues were collected at 9.5 months of age. (B) Representative images of Nissl staining of 9.5-month-old PS19 mice in different treatment groups. Scale bar = 500 μm. (C-F) Quantitative analysis of brain structures: hippocampal volume (C), piriform cortex volume (D), ventricular volume (E), and dentate gyrus (DG) layer thickness (F). (G-I) Protein levels in the cortex (5 mg) of different groups: total Tau phosphorylation (G), 2A7 (H), and Thr181 (I), normalized with GAPDH as a loading control. (J) Co-staining of DAPI (blue) and AT8 (red) in hippocampal sections. (K) Percentage of AT8 coverage in the hippocampal region. Scale bar = 25 μm; n = 9–12; *p < 0.05; One-way ANOVA; data are presented as mean ± standard error of the mean (SEM).

[0075] Figure 4In (A), co-staining of DAPI (blue), CD68 (green), and IBA1 (magenta) in hippocampal slices. Scale bar = 25 μm; n = 9 - 12. (B) Percentage area covered by IBA1 in the hippocampal region. (C) Percentage area covered by CD68 in the hippocampal region. (D) Representative confocal images (upper panel) and corresponding 3D surface reconstruction images (lower panel) showing volume reconstruction of IBA1 (white) and CD68 (green) in the hippocampus. Scale bar = 3 μm; n = 9 - 12. (E) CD68 volume in the hippocampal region normalized to IBA1 volume. (F) Quantitative analysis of mRNA expression of microglia-related genes using qPCR. n = 6; *p < 0.05; one-way ANOVA; data are presented as mean ± standard error of the mean (SEM).

[0076] Figure 5 In (A), co-staining of DAPI (blue), AQP4 (green), and GFAP (red) in hippocampal slices. Scale bar = 25 μm; n = 9 - 12. (B) Percentage area covered by GFAP in the hippocampal region. (C) Low / high threshold measurement of AQP4 polarity. (D) Quantitative analysis of mRNA expression of astrocyte-related genes using qPCR. n = 6; *p < 0.05; one-way ANOVA; data are presented as mean ± standard error of the mean (SEM).

[0077] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A method for processing a plasma sample, characterized in that: Here are the steps: S1: pre-treat the syringe; S2: Use the syringe obtained in step S1 to collect plasma samples from mice through cardiac puncture; S3: Process the plasma sample obtained in step S2.

2. A method for processing a plasma sample according to claim 1, characterized in that: In step S1, the syringe is pretreated as follows: The syringe was flushed with 0.5 M EDTA, pH 8.

0.

3. The method for processing a plasma sample according to claim 1, characterized in that: In step S2, the mice from which plasma samples were collected were male mice of the EKO, E4, and E3 genotypes.

4. The method for processing a plasma sample according to claim 1, characterized in that: In step S3, the plasma sample obtained in step S2 is processed as follows: S31: Blood samples were centrifuged at 5500xg to separate plasma, which was then pooled by genotype; S32: Using Slide-A-Lyzer on Pooled Plasma TM The dialysis bags were dialyzed against phosphate buffered saline and then immediately stored at -80°C.

5. The use of a plasma sample according to any one of claims 1 to 4, characterized in that: The steps are as follows: After thawing, the processed plasma samples were injected into mice via retro-orbital injection.

6. The use of the plasma sample according to claim 5, characterized in that: Here are the steps: Mice were anesthetized with isoflurane and injected with plasma within 10S; Mice were perfused on ice at 9.5 months of age with heparin-containing PBS to remove blood.

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