An experimental method for whole brain microglial cell transplantation

Abstract

The application discloses an experimental method for whole brain microglial cell transplantation, which comprises the following steps: performing microglial cell depletion for three cycles by using a CSF1R inhibitor PLX3397, and then injecting exogenous microglial cells into the brain of an experimental animal; the CSF1R inhibitor is PLX3397 or PLX5622. The method realizes efficient replacement of resident microglial cells, and the implantation rate is as high as 90%. Meanwhile, the primary cultured microglial cells can restore their initial characteristics and functions after being implanted into the brain.

Description

Technical Field

[0001] This invention relates to an experimental method for whole-brain microglia transplantation, belonging to the field of neuroscience technology. Background Technology

[0002] Despite significant advancements in medical technology, the treatment of neurological diseases remains a major challenge. Cell therapy has emerged as a promising approach due to its potential for durable effects. A growing body of genetic evidence has established a strong link between microglia gene mutations and various neurological disorders. For example, CSF1R mutations are associated with adult leukoencephalopathy with axons and pigment glial cells, homozygous TREM2 mutations lead to Nasu-Hakola disease, while heterozygous TREM2 mutations, particularly the R47H variant, significantly increase the risk of Alzheimer's disease. Correcting mutated genes in microglia holds the potential to treat these microglia-based diseases. However, gene editing within microglia faces considerable challenges, such as difficulties in gene delivery and the potential for triggering immune responses, which limit its therapeutic applications. Therefore, microglia replacement therapy represents a more feasible and effective approach for treating these diseases.

[0003] Currently, existing microglia replacement therapies mainly include bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), which are widely used cell therapies for hematologic malignancies. This process requires pretreatment with radiotherapy or chemotherapy to remove the recipient's bone marrow, thereby creating a receiving niche for donor stem cell implantation. Because bone marrow-derived cells can partially replace microglia in the brain, this traditional transplantation method has shown potential therapeutic efficacy in treating neuropathological diseases such as leukodystrophy and lysosomal storage diseases. However, the cell replacement efficiency of this method remains low, and its efficacy in relieving neurological symptoms is minimal. Preclinical trials using this method often fail to achieve the expected results.

[0004] Recent studies have shown that using CSF1R inhibitors to remove microglia can significantly increase peripheral cell infiltration into the brain during bone marrow transplantation. However, the pretreatment required for transplantation triggers brain injury responses that may affect expected treatment outcomes. For example, radiation or chemotherapy can disrupt the integrity of the blood-brain barrier (BBB) ​​and trigger neuroinflammatory responses. Furthermore, busulfan-mediated bone marrow ablation can severely impair the brain regeneration population, leading to permanent loss of neurogenesis in adulthood and potentially exacerbating cognitive deficits and disease progression. While genetic models lacking microglia have facilitated peripheral cell engraftment without demanding conditions, translating this approach into clinical practice is not feasible. Peripheral bone marrow-derived cells can spatially replace microglia, but they are often characteristically and functionally different from the original microglia.

[0005] Furthermore, intracranial microglia transplantation (tMT) results in only a very small percentage of microglia integrating into the brain, accounting for only 0.03% of the total number of microglia in the brain. In recent years, several research groups internationally have achieved microglia transplantation using a similar approach: transplanting exogenous microglia into immunodeficient mice during early development (P0-P1). However, this method relies on immunodeficient recipient mice, and the transplantation must be completed early in development (before the microglia have fully colonized in the brain), making it unsuitable for disease treatment. Therefore, how to efficiently replace microglia in the brains of adult mice has become a key focus in this field.

[0006] Given the limitations of current microglia replacement transplantation methods, it is necessary to develop an alternative method to improve transplantation efficiency, reduce pretreatment-related side effects, and preserve the characteristics and functions of the original microglia. Summary of the Invention

[0007] This invention provides an experimental method for whole-brain microglia transplantation, which can effectively solve the above-mentioned problems.

[0008] This invention is implemented as follows:

[0009] An experimental method for whole-brain microglia transplantation involves using a CSF1R inhibitor to deplete microglia for three cycles, followed by injecting exogenous microglia into the brain of an experimental animal; the CSF1R inhibitor is either PLX3397 or PLX5622.

[0010] In some embodiments, the process of using a CSF1R inhibitor to deplete microglia for three cycles, followed by injecting exogenous microglia into the brain of an experimental animal, includes the following steps:

[0011] (1) Experimental animals were fed with CSF1R inhibitors at a concentration of 200-620 mg / kg for 7 days to induce endogenous microglia depletion.

[0012] (2) The experimental animals were fed normal feed for 7 days for microglia regeneration;

[0013] (3) Repeat steps (1) and (2) twice, and then perform one exhaustion operation of step (1) again, for a total of 3 cycles of exhaustion processing.

[0014] (4) After the exhaustion phase of the third cycle, exogenous microglia were injected into the brain of the experimental animals.

[0015] In some embodiments, the experimental animal is a mammal.

[0016] In some embodiments, the exogenous microglia are one or more of primary microglia, microglia cell lines, or stem cell-induced microglia.

[0017] In some embodiments, the site in which exogenous microglia are injected into the brain of an experimental animal is the cerebral parenchyma.

[0018] The application of the above-mentioned experimental method for whole-brain microglia transplantation in an experimental study on the treatment of neurodegenerative lysosomal storage disease.

[0019] The application of the above-mentioned experimental method for whole-brain microglia transplantation in experimental research on the treatment of Alzheimer's disease.

[0020] The application of the above-mentioned experimental method for whole-brain microglia transplantation in experimental research on the treatment of microglia dysfunction.

[0021] The application of the above-mentioned experimental method for whole-brain microglia transplantation in experimental research on the treatment of nervous system diseases.

[0022] The beneficial effects of this invention are:

[0023] This invention creates a continuously depleted microglia environment by cyclically using CSF1R inhibitors, which is beneficial for the implantation of exogenous microglia. This method achieves highly efficient replacement of resident microglia, with an implantation rate as high as 90%, significantly higher than traditional methods. Simultaneously, primary cultured microglia can recover their initial characteristics and functions after implantation into the brain. This experimental method provides an innovative, practical, and potentially translatable approach for scientific research on neurological diseases related to microglia dysfunction.

[0024] The method of this invention does not require the prior transplantation of bone marrow cells or blood cells, thus eliminating the need for chemotherapy or radiotherapy and avoiding damage to the blood-brain barrier caused by chemotherapy or radiotherapy. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This image shows the effect of PLX3397 in clearing microglia.

[0027] Figure 2 The diagram shows a schematic of Tricyclic Microglia Removal and Transplantation (TCMDT) and a flow cytometry analysis of the percentage of Cx3cr1-GFP positive microglia (b).

[0028] Figure 3 This is a graph showing the results of immunofluorescence staining analysis of the high replacement rate of microglia in TCMDT.

[0029] Figure 4 This is a flow cytometry analysis of the high microglia replacement rate in TCMDT.

[0030] Figure 5 The image shows the results of TCMDT in restoring the microglia characteristics of cultured microglia after transplantation into the brain.

[0031] Figure 6 This is a diagram showing the functional recovery of microglia transplanted via TCMDT.

[0032] Figure 7 The image shows the results of TCMDT significantly improving motor dysfunction in LSD mice.

[0033] Figure 8 This is a diagram showing the results of TCMDT significantly reducing neurodegeneration in LSD mice.

[0034] Figure 9 This is a graph showing the results of TCMDT significantly reducing neuroinflammation in LSD mice.

[0035] Figure 10 This is a diagram showing the pathological results of Aβ in AD mice carrying the Trem2 R47H mutation, which were significantly reduced by TCMDT.

[0036] Figure 11The figure shows the results of TCMDT significantly reducing the neurotoxicity associated with AD mice carrying the Trem2 R47H mutation. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] This invention provides an experimental method for whole-brain microglia transplantation, which involves using a CSF1R inhibitor to deplete microglia for three cycles, and then injecting exogenous microglia into the brain of an experimental animal; the CSF1R inhibitor is PLX3397 or PLX5622.

[0039] Based on previous experimental results, after one or two cycles of microglia depletion, the elimination of endogenous microglia was ineffective, leaving a large number of endogenous microglia that affected exogenous microglia transplantation. After three cycles of microglia depletion, endogenous microglia were completely eliminated and would not regenerate in a short period, resulting in high efficiency of exogenous microglia transplantation. However, using four or more cycles of microglia depletion would require too much time, increasing pretreatment costs and related side effects. Therefore, three cycles of microglia depletion were chosen.

[0040] The method provided in this invention, termed "Tri-Cyclic Microglial Depletion for Transplantation" (TCMDT), is a simple and efficient microglial transplantation method. This method utilizes a CSF1R inhibitor to deplete microglia for three cycles, creating an optimal window for the effective engraftment of exogenous microglia. Primary cultured exogenous microglia, after TCMDT, successfully restored the characteristics and function of the original microglia, providing a substantial method for scientific research on the treatment of diseases related to microglial dysfunction.

[0041] In some embodiments, the process of using a CSF1R inhibitor to deplete microglia for three cycles, followed by injecting exogenous microglia into the brain of an experimental animal, includes the following steps:

[0042] (1) Depletion stage: Experimental animals were fed with CSF1R inhibitor at a concentration of 200-300 mg / kg for 7 days to induce endogenous microglia depletion;

[0043] (2) Regeneration stage: The experimental animals were fed normal feed for 7 days for microglia regeneration;

[0044] (3) Loop processing: After repeating steps (1) and (2) twice, perform one exhaustion operation of step (1) again, for a total of 3 cycles of exhaustion processing;

[0045] (4) Transplantation stage: During the window period after the exhaustion stage of the third cycle and before the endogenous microglia have regenerated in large quantities, exogenous microglia are injected into the brain of the experimental animal.

[0046] The method described above can clear microglia in the brain, significantly reduce the regeneration rate of microglia in the brain, and has extremely high microglia replacement efficiency.

[0047] In some embodiments, the concentration of CSF1R inhibitor in the CSF1R inhibitor diet is 200-620 mg / kg. This concentration ensures its effectiveness and consistency in experimental or therapeutic applications. Specifically, in some embodiments, the concentration of CSF1R inhibitor in the CSF1R inhibitor diet is 580-620 mg / kg.

[0048] In some embodiments, the experimental animals are mammals. These mammals may include, but are not limited to, mice, rabbits, dogs, cats, monkeys, and other similar animals. By selecting these mammals as experimental subjects, researchers can better simulate and understand human physiological and pathological processes, thereby providing important data support for medical research and drug development.

[0049] In some embodiments, the exogenous microglia are one or more of primary microglia, microglia cell lines, or stem cell-induced microglia. Primary microglia are directly isolated from animal or human tissue samples and expanded and used under in vitro culture conditions. Microglia cell lines are formed from primary microglia through a series of screening and culture processes, ultimately creating cell lines capable of unlimited proliferation under laboratory conditions. Stem cells possess characteristics such as self-renewal and high differentiation; stem cell-induced microglia can be mass-produced through in vitro stem cell induction technology. Stem cell-induced microglia have a gene expression profile highly similar to that of microglia in the adult central nervous system. Both can be used in various biological research and experiments to simulate and study the function and behavior of microglia under physiological and pathological conditions.

[0050] In some embodiments, exogenous microglia are injected into the brain parenchyma of experimental animals, such as the hippocampus and / or the cerebral cortex. The hippocampus is a crucial part of the brain closely related to learning and memory functions, while the cerebral cortex is the gray matter layer on the surface of the brain responsible for processing various complex cognitive functions. By precisely injecting exogenous microglia into these specific regions, researchers hope to better study and understand the role of microglia in these brain regions and their impact on related neural functions.

[0051] This paper describes the application of the aforementioned whole-brain microglia transplantation method in an experimental study of the treatment of neurodegenerative lysosomal storage disease. This method involves transplanting healthy whole-brain microglia into experimental animals suffering from the disease, aiming to investigate its impact on disease progression and potential therapeutic effects.

[0052] This study explores the application of the aforementioned experimental method for whole-brain microglia transplantation in experimental research on the treatment of Alzheimer's disease. By transplanting whole-brain microglia into an experimental model, researchers hope to better understand the role of these cells in Alzheimer's disease and explore new treatment strategies.

[0053] This paper describes the application of the aforementioned experimental method for whole-brain microglia transplantation in experimental research on the treatment of microglia dysfunction. Microglia dysfunction is considered a key factor in many neurological diseases; therefore, by transplanting healthy microglia from the whole brain, researchers hope to restore the normal function of these cells, thereby alleviating or treating related diseases.

[0054] This paper describes the application of the aforementioned experimental method for whole-brain microglia transplantation in experimental research on the treatment of neurological diseases. Since microglia play a crucial role in maintaining nervous system health and responding to damage, researchers hope that by transplanting these cells, they can provide new therapeutic ideas and methods for various neurological diseases.

[0055] Example 1: Verification of the effect of PLX3397 in clearing microglia

[0056] To eliminate microglia, PLX3397 (Selleck, S7818) was formulated into a standard diet at a concentration of 600 mg / kg. Mice aged 3-8 months were fed approximately 4 g of PLX3397 daily, kept in an environment with 12 hours of light, unrestricted water intake, and free movement. After 7 days of feeding, mice were anesthetized with isoflurane, and 50 mL of pre-cooled 1×PBS was perfused through the heart. Brain tissue was then removed from the skull and fixed overnight in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148). The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until it settled to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding medium for frozen sectioning at a thickness of 15 μm, followed by immunofluorescence staining. Immunofluorescence staining was performed on microglia Iba1 (Wako, 019-19741, 1:200) and cell nuclei DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000). Panoramic immunofluorescence staining images of brain tissue were captured, and the number of microglia was counted.

[0057] Experimental results are as follows Figure 1 As shown in the figure. The results showed that, compared with the control group, mice fed PLX3397 diet had almost completely cleared microglia.

[0058] Example 2: Tricyclic microglia removal and transplantation method (TCMDT)

[0059] like Figure 2As shown in Figure a, adult mice (3-8 months old) were fed PLX3397 diet for 7 days to induce microglia exhaustion, then switched to a standard diet for 7 days for microglia regeneration. Adult mice were fed PLX3397 diet for 7 days a second time to induce microglia exhaustion, then switched to a standard diet for 7 days for microglia regeneration. Adult mice were fed PLX3397 diet for 7 days a third time to induce microglia exhaustion. A total of 21 days of PLX3397 diet were administered to the mice, in 3 cycles. The feeding amount and rearing environment were the same as in Example 1. Cx3cr1-GFP microglia (Cx3cr1-GFP neonatal mouse brain tissue, obtained through primary culture) were then transplanted into mice. Mice were anesthetized with sodium pentobarbital based on their body weight, and then fixed on a stereotactic injection device. After disinfection with iodine, the mouse's head skin was carefully cut open to expose the skull. The anterior fontanelle was located, and the injection sites were positioned using the anterior fontanelle as the origin (hippocampal region: x = ± 1.75mm, y = -2mm; cortical region: x = ± 1.75mm, y = -2mm). Holes were punched at the injection sites, and microglia were injected using a Hamilton's syringe. A total of four injection sites were made in both hemispheres, with an injection depth of 2mm in the hippocampus and 0.85mm in the cortex. The concentration of the microglia suspension was 2-3 × 10⁻⁶. 4 Inject 2 μL of the solution at a rate of 0.5 μL / min, with the needle held for 5 minutes after injection to allow the microglia to spread fully. Then, slowly withdraw the syringe, suture the wound with medical needle and suture, and return the mouse to its rearing cage.

[0060] One month after transplantation, mice were anesthetized with isoflurane, and brain tissue was harvested by perfusion of 50 mL of pre-cooled 1×PBS. The bilateral hippocampus and cortical tissues were separated and placed in pre-cooled 1×HBSS (Gibco, C14175500BT) for later use. The tissue was cut into small pieces with scissors and transferred to a 2 mL small grinder. 1 mL of prepared Dounce Buffer (1×HBSS, DNase I, RNase Inhibitor) was added to the grinder beforehand. The tissue was first ground 10 times with a looser grinder (A), and then ground 10 times with a tighter grinder (B), until the tissue was homogenized. Cell homogenate was filtered through a 100 μm cell strainer and transferred to a 50 mL RNase-Free EP tube. 1 mL of 1×HBSS was added to wash the homogenizer. The resulting cell homogenates were then transferred to a new 15 mL RNase-Free EP tube. SIP solution prepared with Percoll (Sigma, P1644) and 10×HBSS (Sigma, H4641) was added to make a final solution of 30% Percoll. The mixture was gently inverted and centrifuged at 800 g for 40 min at 4 °C using a rapid incubation and slow descent ratio (9:3). The supernatant containing myelin fragments was discarded. The cell pellet was resuspended in 6 mL of 1×PBS, and centrifuged gently at 800 g for 5 min. The supernatant was discarded. Non-specific binding antigens were blocked with 250 μL of blocking buffer (anti-mouse CD16 / CD32, BD Bioscience, 553141, 1:400, diluted with 1×PBS). The cell pellet was resuspended and blocked on ice for 10 min. Take 20 μL of the blocked cell suspension and dilute it to 400 μL with FACSbuffer (50 mL 1×PBS, 200 μL 0.5M EDTA (Invitrogen, 15575-038), 500 μL FBS (NTC)) as a negative control. The remaining cell suspension was stained with microglia-associated antibodies CD11b-APC (eBioscience, 17-0112-83, 1:200, 1×PBS dilution) and CD45-PE-Cyanine7 (eBioscience, 25-0451-82, 1:200, 1×PBS dilution). These antibodies were added directly to the blocked cell suspension and stained on ice in the dark for 20 min. After staining, resuspend the cells in 6 mL of FACS Buffer, invert to mix, wash the cells, centrifuge at 800g for 5 min, discard the supernatant, add 6 mL of FACS Buffer and wash again, discard the supernatant.Finally, 500 μL of FACSBuffer (containing DNase I and RNase inhibitor) was added to resuspend the cells. The percentage of Cx3cr1-GFP positive microglia was analyzed by instrument, and Cx3cr1-GFP positive microglia could also be sorted by instrument.

[0061] Experimental results are as follows Figure 2 As shown in b. From Figure 2 b shows that GFP-positive microglia accounted for 75.8%, indicating that the microglia replacement efficiency was 75.8%.

[0062] Example 3: Immunofluorescence staining analysis of high microglia replacement ratio in TCMDT

[0063] Adult mice (3-8 months old) were divided into three groups: a group not fed PLX3397 (0×PLX), a group fed PLX3397 once (1×PLX), and a group fed PLX3397 via TCMDT (3×PLX). Feeding amounts and rearing environments were the same as in Example 1. Microglia transplantation in mice was performed using the same method as in Example 2. One month after transplantation, mice were anesthetized with isoflurane and perfused with 50 mL of pre-cooled 1×PBS via the heart. Brain tissue was then removed from the skull and fixed overnight in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148). The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until it settled to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding medium for frozen sectioning at a thickness of 15 μm, followed by immunofluorescence staining. Immunofluorescence staining was performed on Iba1 microglia (Wako, 019-19741, 1:200) and their nuclei (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000). Panoramic immunofluorescence staining images of brain tissue were captured. The proportion of Cx3cr1-GFP positive microglia among all microglia was counted, and the microglia replacement efficiency was calculated.

[0064] Experimental results are as follows Figure 3 As shown in the results, no microglia were implanted in the brain of the untreated group (0×PLX). In mice treated with PLX3397 for 7 days (1×PLX), a small number of microglia (approximately 10% of the brain region) were replaced around the injection site. After 3 cycles of microglia depletion (3×PLX, referred to as TCMDT), the transplanted microglia occupied approximately 70% of the brain region, significantly improving implantation efficiency.

[0065] In addition, after three cycles of microglia depletion, the engraftment efficiency of microglia was further improved to approximately 90% three months after transplantation.

[0066] Example 4: Flow cytometry analysis of high microglia replacement ratio in TCMDT

[0067] Adult mice were divided into three groups: a group not fed PLX3397 (0×PLX), a group fed a single dose of PLX3397 (1×PLX), and a group fed PLX3397 via TCMDT (3×PLX). Feeding amounts and rearing conditions were the same as in Example 1. Microglia transplantation in mice was performed using the same method as in Example 2. One month after transplantation, mice were anesthetized with isoflurane, and brain tissue was harvested by perfusing 50 mL of pre-cooled 1×PBS into the heart. The bilateral hippocampus and cortical tissues were separated and placed in pre-cooled 1×HBSS (gibco, C14175500BT) for later use. Flow cytometry analysis was performed to determine the proportion of Cx3cr1-GFP positive microglia among all microglia, as in Example 2.

[0068] Experimental results are as follows Figure 4 As shown in the figure. The results showed that no microglia were detected in the brain of the untreated group (0×PLX), the microglia replacement efficiency of the 1×PLX group was only about 3%, while the microglia replacement efficiency of the 3×PLX group was significantly improved to about 70%.

[0069] Similarly, three months after transplantation, the replacement efficiency of microglia in the 3×PLX group was as high as 90%.

[0070] Example 5: TCMDT can restore the microglia characteristics of cultured microglia after transplantation into the brain.

[0071] TCMDT eliminated microglia in the mouse brain, and exogenous Cx3cr1-GFP microglia were transplanted using the same method as in Example 2. Three months after transplantation, mice were anesthetized with isoflurane, and brain tissue was harvested by perfusion of 50 mL of pre-cooled 1×PBS. The bilateral hippocampus and cortical tissues were separated and placed in pre-cooled 1×HBSS (gibco, C14175500BT) for later use. Cx3cr1-GFP positive microglia were sorted by flow cytometry, as in Example 2. 500 μL of Trizol was added to the sorted Cx3cr1-GFP positive microglia, incubated at room temperature for 5 min, and vortexed for 15 s to completely dissociate the cell pellet complex. Then 100 μL of chloroform was added, vortexed for 20 s, incubated at room temperature for 3 min, and centrifuged at 12000g for 15 min. Take approximately 350 μL of the supernatant, add an equal volume of isopropanol, and simultaneously add 1 μL of glycogen. Gently invert to mix, incubate at -20°C for 1 hour, and centrifuge at 12000 rpm for 15 minutes. Discard the supernatant with a pipette, add 500 μL of pre-chilled 75% ethanol, and centrifuge at 7500 g for 5 minutes. Discard the supernatant with a pipette (a small amount may remain), and centrifuge again at 7500 g for 2 minutes. Discard the residual liquid, open the cap, air dry for approximately 2 minutes, add 15 μL of DEPC water to dissolve the RNA, and use the obtained RNA for sequencing.

[0072] Sequencing results as follows Figure 5 As shown. Analysis of the sequencing results revealed significant gene expression differences between TM (transplanted microglia) and PM (primary microglia) through principal component analysis (PCA). However, both TM and NM showed high Pearson correlation coefficients and significant overlap, indicating that TM is very similar to NM (naive microglia) in its transcriptional profile. This suggests that microglia cultured in vitro recover their microglia characteristics after transplantation into the brain, closely resembling microglia in the brain.

[0073] Example 6: TCMDT can restore the function of transplanted microglia.

[0074] TCMDT eliminated microglia in the mouse brain, and exogenous Cx3cr1-GFP microglia were transplanted using the same method as in Example 2. Three months after transplantation, mice were anesthetized with isoflurane and perfused with 50 mL of pre-cooled 1×PBS via the heart. Brain tissue was then removed from the skull and fixed overnight in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148). The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until it settled to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding medium for frozen sectioning at a thickness of 15 μm, followed by immunofluorescence staining. Immunofluorescence staining was performed on microglia Iba1 (Wako, 019-19741, 1:200) and their nuclei DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000). 60× images were captured using an inverted fluorescence microscope to analyze microglia morphology, which was evaluated by the length of crossover points, endpoints, and processes.

[0075] The experimental results are shown in Figure 6. The results indicate that, without LPS treatment, transplanted microglia exhibited the same cell morphology as the naïve microglia in the brain, suggesting that primary cultured microglia can reconstruct their microglia phenotype after brain transplantation, consistent with our transcriptional analysis. After LPS treatment, both naïve and transplanted microglia showed shorter process lengths and fewer crossovers and endpoints, demonstrating similar morphological changes in response to stimulation.

[0076] Example 7: TCMDT significantly improved kinetic disorders in LSD mice.

[0077] TCMDT-eliminated Hexb-KO mice (a mouse model of neurodegenerative lysosomal storage disease, Sandhoff's disease, derived from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., C57BL / 6JGpt-Hexb) em5cd3129 Cx3cr1-GFP microglia (Gpt) were exogenously transplanted into mice using the same transplantation method as in Example 2. Two months after transplantation, at 4 months of age, mice underwent rotarod and balance beam behavioral experiments related to movement. The rotarod experiment recorded the time it took for mice to lose balance and fall off the rotarod, while the balance beam experiment recorded the time it took for mice to cross the balance beam, assessing the mice's motor balance and coordination abilities.

[0078] Experimental results are as follows Figure 7As shown in the diagram. The rotundus test revealed no difference in fall latency among untreated (Ctrl), sham-operated (Sham), and microglia transplanted (MT) WT mice, indicating that TCMDT and microglia transplantation do not affect motor function in WT mice. In contrast, KO mice showed a significantly shortened fall latency, highlighting marked motor dysfunction, while microglia transplantation significantly improved motor function in KO mice. Similarly, the balance beam test results were consistent with the rotundus test; there were no differences in crossing time and fall scores among the three groups of WT mice, while in KO mice, MT significantly shortened crossing time and reduced fall scores.

[0079] Example 8: TCMDT significantly reduced neurodegeneration in LSD mice.

[0080] After the behavioral experiments in Example 7 above, mice were anesthetized with isoflurane and perfused with 50 mL of pre-cooled 1×PBS via cardiac perfusion. Brain tissue was then removed from the skull and fixed overnight in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148). The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until it settled to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding medium for frozen sectioning, with a section thickness of 15 μm, followed by immunohistochemical staining. The brain sections were washed three times in 1×PBS and then stained with Nissl solution at room temperature for 10 minutes. After staining, the sections were rinsed three times with H2O, then rapidly rinsed three times each with 95% ethanol and anhydrous ethanol, and then soaked twice in xylene for 5 minutes each time. Finally, the sections were mounted with neutral resin. Whole-brain images were scanned using a panoramic digital slide scanner to analyze the number of neurons.

[0081] Experimental results are as follows Figure 8 As shown in the figure. The results showed that, compared with WT mice, KO mice had a significantly reduced number of neurons, especially in the primary motor cortex and midbrain, areas related to motor function. Microglia transplantation significantly increased the number of neurons in KO mice.

[0082] Example 9: TCMDT significantly reduced neuroinflammation in LSD mice.

[0083] After the behavioral experiments in Example 7 above, mice were anesthetized with isoflurane and perfused with 50 mL of pre-cooled 1×PBS via the heart. Brain tissue was then removed from the skull and fixed overnight in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148). The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until it settled to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding medium for frozen sectioning, with a section thickness of 15 μm, followed by immunohistochemical staining. Immunofluorescence staining was performed on microglia Iba1 (Wako, 019-19741, 1:200), microglia phagocytic marker CD68 (BioLegend, 137001, 1:200), and cell nucleus DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000). 60× images were taken using an inverted fluorescence microscope, and the percentage of CD68 area in microglia was calculated.

[0084] The experimental results are shown in Figure 9. Compared with the control group, KO mice showed a significant increase in CD68-positive microglia and a marked increase in CD68 area. After microglia transplantation, the microglia completely recovered to normal levels, indicating that microglia transplantation has a positive effect on reducing neuroinflammation in LSD mice.

[0085] Example 10: TCMDT significantly reduced Aβ pathology in AD mice carrying the Trem2 R47H mutation.

[0086] TCMDT eliminated AD mice carrying the Trem2 R47H mutation (Nanjing Modeling Biotechnology, C57BL / 6Smoc-Trem). 2em3(p.R47H )smoc Microglia in the brain were exogenously transplanted using Cx3cr1-GFP microglia, following the same transplantation method as in Example 2. Four months after transplantation, mice were anesthetized with isoflurane and perfused with 50 mL of pre-cooled 1×PBS via the heart. Brain tissue was then extracted from the skull and fixed overnight in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148). The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until it settled to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding medium for frozen sectioning, with sections 15 μm thick, followed by immunohistochemical staining. Immunofluorescence staining was performed on microglia Iba1 (Wako, 019-19741, 1:200), amyloid MOAB2 (Abcam, ab26649, 1:400), and cell nuclei DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000). 60x images were captured using an inverted fluorescence microscope, and the area of ​​Aβ plaques and the number of microglia were counted.

[0087] Experimental results are as follows Figure 10 As shown in the figure. The results showed that microglia transplantation significantly reduced Aβ plaque deposition in R47H mice, and the number of microglia was restored after transplantation.

[0088] Example 11: TCMDT significantly reduced the associated neurotoxicity in AD mice carrying the Trem2 R47H mutation.

[0089] TCMDT eliminated microglia in the brains of AD mice carrying the Trem2 R47H mutation. Cx3cr1-GFP microglia were then exogenously transplanted using the same method as in Example 2. Four months after transplantation, mice were anesthetized with isoflurane and perfused with 50 mL of pre-cooled 1×PBS via the heart. Brain tissue was then extracted from the skull and fixed overnight in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148). The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until it settled to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding medium for frozen sectioning at a thickness of 15 μm, followed by immunohistochemical staining. Immunofluorescence staining was performed on microglia Iba1 (Wako, 019-19741, 1:200), lysosomal-associated membrane protein Lamp1 (DB Biosciences, 553792, 1:200) which marks dystrophic neurites, and dense plaques with amyloid cores ThioS (Sigma-Aldrich, T1892, 0.1% in 50% ethanol). 60x images were captured using an inverted fluorescence microscope to analyze Lamp1-positive areas.

[0090] Experimental results are as follows Figure 11 As shown in the figure. The results showed that the Lamp1 positive area, a marker of neurite dystrophy, was reduced around each plaque, indicating that microglia transplantation alleviated plaque-induced neurotoxicity.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. An experimental method for whole-brain microglia transplantation, characterized in that, Includes the following steps: (1) Experimental animals were fed with CSF1R inhibitors at a concentration of 200-620 mg / kg for 7 days to induce endogenous microglia depletion. (2) The experimental animals were fed normal feed for 7 days for microglia regeneration; (3) Repeat steps (1) and (2) twice, and then perform one exhaustion operation of step (1) again, for a total of 3 cycles of exhaustion processing. (4) After the exhaustion phase of the third cycle, exogenous microglia were injected into the brain of the experimental animals. The CSF1R inhibitor is either PLX3397 or PLX5622.

2. The experimental method for whole-brain microglia transplantation according to claim 1, characterized in that, The experimental animals were mammals.

3. The experimental method for whole-brain microglia transplantation according to claim 1, characterized in that, The exogenous microglia are one or more of primary microglia, microglia cell lines, or stem cell-induced microglia.

4. The experimental method for whole-brain microglia transplantation according to claim 1, characterized in that, The part of the brain into which exogenous microglia are injected is the cerebral parenchyma.

5. The application of an experimental method for whole-brain microglia transplantation according to any one of claims 1 to 4 in experimental research on the treatment of neurodegenerative lysosomal storage diseases.

6. The application of an experimental method for whole-brain microglia transplantation according to any one of claims 1 to 4 in experimental research on the treatment of Alzheimer's disease.

7. The application of an experimental method for whole-brain microglia transplantation according to any one of claims 1 to 4 in experimental research on the treatment of microglia dysfunction.

8. The application of an experimental method for whole-brain microglia transplantation according to any one of claims 1 to 4 in experimental research on the treatment of nervous system diseases.

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