Experimental method for whole brain microglial cell transplantation

Abstract

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

Description

Technical Field

[0001] The invention relates to an experimental method for whole-brain microglial cell transplantation, and belongs to the technical field of neuroscience. Background Art

[0002] Despite tremendous advances in medical technology, the treatment of neurological diseases remains a great challenge. Cell therapy has emerged as a promising approach due to its potential for lasting effects. Increasing genetic evidence has established a strong link between microglial gene mutations and various neurological diseases. For example, CSF1R mutations are associated with adult leukoencephalopathy with axonal bulbs and pigmented glial cells, homozygous TREM2 mutations cause Nasu-Hakola disease, and heterozygous TREM2 mutations, especially the R47H variant, significantly increase the risk of Alzheimer's disease. Correcting microglial mutant genes has the potential to treat these microglia-based diseases. However, gene editing within microglia faces considerable challenges, such as difficulties in gene delivery and potential immune responses, which limits its therapeutic application. Therefore, microglia replacement therapy has become a more feasible and effective approach to treat these diseases.

[0003] Currently, the existing microglia replacement therapies mainly include bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), which is a cell therapy widely used for hematological malignancies. This process requires the use of radiotherapy or chemotherapy to pre-treat the body to remove the recipient's bone marrow, thereby establishing a receptive niche for donor stem cell implantation. Since bone marrow-derived cells can partially replace microglia in the brain, this traditional transplantation method shows potential therapeutic effects in the treatment of neuropathological diseases, such as leukodystrophy and lysosomal storage diseases. However, the cell replacement efficiency of this method is still low, and it has little effect in alleviating neurological symptoms. Preclinical attempts to apply this method often fail to achieve the expected results.

[0004] Recent studies have shown that the removal of microglia using CSF1R inhibitors significantly increases the infiltration of peripheral cells into the brain during bone marrow transplantation. However, the preconditioning required for transplantation triggers a brain injury response that may affect the expected treatment outcome. For example, radiotherapy or chemotherapy can disrupt the integrity of the blood-brain barrier (BBB) ​​and trigger a neuroinflammatory response. In addition, busulfan-mediated bone marrow ablation can severely impair the brain regenerative population, leading to permanent loss of neurogenesis in adulthood and potentially exacerbating cognitive deficits and disease progression. Although genetic models lacking microglia promote the engraftment of peripheral cells without the need for harsh conditions, translating this approach into clinical practice is not feasible. Peripheral bone marrow-derived cells can spatially replace microglia, but they are often different from the initial microglia in characteristics and functions.

[0005] In addition, when microglia are transplanted into the brain by intracranial injection (traditional microglia transplantation, tMT), only a very small number of microglia can be integrated into the brain, accounting for only 0.03% of the total number of microglia in the brain. In recent years, many research groups internationally have achieved microglial transplantation through similar ideas: transplanting exogenous microglia into immunodeficient mice in the early stages of development (P0-P1). However, this type of method relies on immunodeficient transplant recipient mice, and the transplantation must be completed in the early stages of development (when microglia have not completed colonization in the brain), and cannot be used for disease treatment. Therefore, how to efficiently replace microglia in the brain of adult mice has become a key issue of concern in the field.

[0006] Given the limitations of current microglia replacement transplantation methods, it is imperative to develop an alternative approach that improves transplantation efficiency, reduces preconditioning-related side effects, and preserves the properties and functions of naive microglia. Summary of the invention

[0007] The present invention provides an experimental method for whole-brain microglial cell transplantation, which can effectively solve the above problems.

[0008] The present invention is achieved in that:

[0009] An experimental method for whole-brain microglial transplantation, using a CSF1R inhibitor to perform three cycles of microglial depletion, and then injecting exogenous microglial cells into the brain of an experimental animal; the CSF1R inhibitor is PLX3397 or PLX5622.

[0010] In some embodiments, the three cycles of microglial depletion using a CSF1R inhibitor are as follows: feeding the experimental animals with a CSF1R inhibitor feed for 7 days to induce microglial depletion; then switching to a normal feed for 7 days for microglial regeneration; and repeating the process twice.

[0011] In some embodiments, the concentration of the CSF1R inhibitor in the CSF1R inhibitor feed is 200-620 mg / kg.

[0012] In some embodiments, the experimental animal is a mammal.

[0013] In some embodiments, the exogenous microglia are one or more of primary microglia, microglia cell lines, or stem cell-induced microglia.

[0014] In some embodiments, the site where exogenous microglia are injected into the brain of an experimental animal is the brain parenchyma.

[0015] An application of the above whole-brain microglia transplantation experimental method in experimental research on the treatment of neurodegenerative lysosomal storage diseases.

[0016] An application of the above-mentioned whole-brain microglial cell transplantation experimental method in experimental research on the treatment of Alzheimer's disease.

[0017] An application of the above whole-brain microglia transplantation experimental method in experimental research on the treatment of microglial dysfunction.

[0018] An application of the above whole-brain microglial cell transplantation experimental method in experimental research on the treatment of nervous system diseases.

[0019] The beneficial effects of the present invention are:

[0020] The present invention creates a continuous microglia depletion environment by cyclically using CSF1R inhibitors, which is conducive to the implantation of exogenous microglia. The method achieves efficient replacement of resident microglia, with an implantation rate of up to 90%, which is significantly higher than traditional methods. At the same time, primary cultured microglia can restore their initial characteristics and functions after implantation into the brain. This experimental method provides an innovative, practical and potentially transformable method for scientific research on neurological diseases related to microglial dysfunction.

[0021] The method of the present invention does not require transplantation of bone marrow cells or blood cells first, therefore, chemotherapy or radiotherapy is not required, thus avoiding damage to the blood-brain barrier caused by chemotherapy or radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is a diagram verifying the effect of PLX3397 in clearing microglia.

[0024] Figure 2 Schematic diagram of tricyclic microglia depletion and transplantation (TCMDT) (a) and flow cytometry analysis of the percentage of Cx3cr1-GFP positive microglia (b).

[0025] Figure 3 This is the result of immunofluorescence staining analysis of the high replacement ratio of TCMDT microglia.

[0026] Figure 4 This is the result of flow cytometry analysis of the high replacement ratio of TCMDT microglia.

[0027] Figure 5 This is the result of TCMDT enabling cultured microglia to restore their microglial characteristics after being transplanted into the brain.

[0028] Figure 6 This is a diagram showing the results of TCMDT transplanted microglia restoring their functions.

[0029] Figure 7 This is a graph showing that TCMDT significantly improved the movement disorder in LSD mice.

[0030] Figure 8 This figure shows the results of TCMDT significantly reducing neurodegeneration in LSD mice.

[0031] Fig. 9 This is a graph showing that TCMDT significantly reduced neuroinflammation in LSD mice.

[0032] Fig.10 This is a diagram showing that TCMDT significantly reduced Aβ pathology in AD mice carrying the Trem2 R47H mutation.

[0033] Fig.11 This is a graph showing that TCMDT significantly alleviates the related neurotoxicity in AD mice carrying the Trem2 R47H mutation. DETAILED DESCRIPTION

[0034] In order to make the purpose, 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 in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The embodiment of the present invention provides an experimental method for whole-brain microglial transplantation, wherein a CSF1R inhibitor is used to perform three cycles of microglial depletion, and then exogenous microglial cells are injected into the brain of an experimental animal; the CSF1R inhibitor is PLX3397 or PLX5622.

[0036] According to the results of previous experiments, the elimination effect of endogenous microglia is poor after 1 or 2 cycles of microglia depletion, and a large number of endogenous microglia still exist, which affects the transplantation of exogenous microglia; after 3 cycles of microglia depletion, endogenous microglia have been completely eliminated and will not regenerate in a short time, and the replacement efficiency of exogenous microglia transplantation is high; if 4 or more cycles of microglia depletion are used, the time required is too long, which increases the pretreatment cost and related side effects. Therefore, 3 cycles of microglia depletion were selected.

[0037] The method provided by the present invention is called "Tri-Cyclic Microglial Depletion for Transplantation" (TCMDT), which is a simple and efficient microglial transplantation method. The method uses CSF1R inhibitors to perform three cycles of microglial depletion, creating an optimal window period for the effective implantation of exogenous microglia. The primary cultured exogenous microglia successfully restored the characteristics and functions of the original microglia through TCMDT, providing a substantial method for scientific research on the treatment of diseases related to microglial dysfunction.

[0038] In some embodiments, the three cycles of microglial depletion using a CSF1R inhibitor are: feeding the experimental animals with a CSF1R inhibitor feed for 7 days to induce microglial depletion; then switching to a normal feed for 7 days for microglial regeneration; and repeating the process twice. The method can remove microglial cells in the brain, significantly reduce the regeneration rate of microglial cells in the brain, and has a very high efficiency of replacing microglial cells in the brain.

[0039] In some embodiments, the concentration of the CSF1R inhibitor in the CSF1R inhibitor feed is 200-620 mg / kg. This concentration can ensure its effectiveness and consistency in experimental or therapeutic applications. Specifically in some embodiments, the concentration of the CSF1R inhibitor in the CSF1R inhibitor feed is 580-620 mg / kg.

[0040] 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 the physiological and pathological processes of humans, thereby providing important data support for medical research and drug development.

[0041] 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 amplified and used under in vitro culture conditions. Microglia cell lines are derived from primary microglia through a series of screening and culture processes, and eventually form cell lines that can proliferate indefinitely under laboratory conditions. Stem cells have the characteristics of self-renewal and high differentiation. The mass production of stem cell-induced microglia can be achieved through stem cell in vitro induction technology. Stem cell-induced microglia have a gene expression profile that is highly similar to that of microglia in the adult central nervous system. They can all be used in various biological studies and experiments to simulate and study the functions and behaviors of microglia under physiological and pathological conditions.

[0042] In some embodiments, the site where exogenous microglia are injected into the brain of the experimental animal is the brain parenchyma, such as the hippocampus and / or the cerebral cortex. The hippocampus is an important part of the brain that is closely related to learning and memory functions, while the cerebral cortex is the gray matter layer on the surface of the brain, which is responsible for processing various complex cognitive functions. By precisely injecting exogenous microglia into these specific areas, researchers hope to better study and understand the role of microglia in these brain regions and their impact on related neural functions.

[0043] An experimental method of whole-brain microglia transplantation is used in experimental research on the treatment of neurodegenerative lysosomal storage diseases. This method aims to study the effect of healthy whole-brain microglia on the disease process and potential therapeutic effect by transplanting healthy whole-brain microglia into experimental animals with the disease.

[0044] An experimental method of whole-brain microglia transplantation described above is used in experimental studies of Alzheimer's disease treatment. By transplanting whole-brain microglia in experimental models, researchers hope to better understand the role of these cells in Alzheimer's disease and explore new treatment strategies.

[0045] An experimental method of whole-brain microglia transplantation is used in experimental studies of the treatment of microglial dysfunction. Microglial dysfunction is considered to be a key factor in a variety of neurological diseases, so by transplanting healthy whole-brain microglia, researchers hope to restore the normal function of these cells, thereby alleviating or treating related diseases.

[0046] The application of the above-mentioned whole-brain microglia transplantation experimental method in experimental research on the treatment of neurological diseases. Because microglia play an important role in maintaining the health of the nervous system and responding to injuries, by transplanting these cells, researchers hope to provide new treatment ideas and methods for a variety of neurological diseases.

[0047] Example 1 Verification of the effect of PLX3397 in removing microglia

[0048] To eliminate microglia, PLX3397 (Selleck, S7818) was formulated into a standard diet with a concentration of 600 mg / kg. The mice were 3-8 months old, and each mouse was fed about 4 g of PLX3397 feed per day. The feeding environment was a 12-hour light environment, with unlimited drinking water and free movement. After 7 days of feeding, the mice were anesthetized with isoflurane, perfused with 50 mL of pre-cooled 1× PBS through the heart, and then the brain tissue was removed from the skull and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148) overnight. The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until the brain tissue sank to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding agent for frozen sectioning with a section thickness of 15 μm, and then immunofluorescence staining was performed. Immunofluorescence staining of microglia Iba1 (Wako, 019-19741, 1:200) and nucleus DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000) was performed, and panoramic immunofluorescence staining images of brain tissue were taken to count the number of microglia.

[0049] The experimental results are as follows Figure 1 The results showed that compared with the control group, microglia were basically eliminated in mice fed with PLX3397 feed.

[0050] Example 2 Tricyclic Microglia Depletion and Transplantation (TCMDT)

[0051] like Figure 2As shown in a, adult mice (3-8 months old) were fed with PLX3397 feed for 7 days to induce microglial depletion, and then switched to standard feed for 7 days for microglial regeneration. This process was repeated twice, for a total of 21 days of feeding mice with PLX3397 feed, for 3 cycles. The feeding amount and feeding environment were the same as in Example 1. Then, Cx3cr1-GFP microglia were transplanted into mice (Cx3cr1-GFP newborn mouse brain tissue, Cx3cr1-GFP microglia were obtained by primary culture), and the mice were anesthetized by injecting sodium pentobarbital according to their weight, and then fixed on a stereotaxic injection instrument. After disinfection with iodine, the skin on the mouse head was carefully cut to expose the skull, and the anterior fontanelle was found. The injection position was located with the anterior fontanelle as the origin (hippocampus area: x = ± 1.75mm, y = -2mm; cortical area: x = ± 1.75mm, y = -2mm), and a hole was made at the injection position. Microglia were injected using a microinjector (Hamilton). A total of 4 sites were injected in the left and right hemispheres, with a hippocampal injection depth of 2mm and a cortical injection depth of 0.85mm. The concentration of the microglial suspension was 2-3×10 4 cells / μL, 2μL was injected at each site, the injection speed was 0.5μL / min, after the injection, the needle was kept for 5min to allow the microglia to fully diffuse, then the syringe was slowly withdrawn, the wound was sutured with medical needle and thread, and the mice were returned to the breeding cage.

[0052] One month after transplantation, mice were anesthetized with isoflurane, and 50 mL of pre-cooled 1×PBS was perfused through the heart to obtain brain tissue. The hippocampus and cortex tissues on both sides 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, DNaseI, RNase Inhibitor) was added to the grinder in advance. The tissue was first ground 10 times with a looser grinding rod A, and then ground 10 times with a tighter grinding rod B to grind the tissue into a homogenous state. Filter the cell homogenate with a 100 μm cell sieve into a 50 mL RNase-Free EP tube, then add 1 mL 1×HBSS to rinse the grinder, transfer the cell homogenate obtained twice into a new 15 mL RNase-Free EP tube, add SIP solution prepared by Percoll (Sigma, P1644) and 10×HBSS (Sigma, H4641) to make the final solution 30% Percoll solution, gently invert and mix, centrifuge at 800g for 40 min in a 4°C centrifuge, quickly rise and slowly fall (9:3), and discard the supernatant containing myelin fragments. Add 6 mL 1×PBS to resuspend the cell pellet, gently centrifuge at 800g for 5 min, and discard the supernatant. Use 250 μL blocking solution (anti-mouse CD16 / CD32, BD Bioscience, 553141, 1:400, 1×PBS dilution) to block nonspecific binding antigens, resuspend the cell pellet, and block on ice for 10 min. Take 20 μL of the blocked cell suspension and dilute it to 400 μL with FACS buffer (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-related 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), and added directly to the blocked cell suspension. The whole process was kept on ice and away from light, and the staining was carried out for 20 minutes. After the staining was completed, 6 mL FACS Buffer was added to resuspend the cells, inverted to mix, and the cells were washed. Centrifuged at 800 g for 5 minutes, the supernatant was discarded, and 6 mL FACS Buffer was added to repeat the washing once, and the supernatant was discarded. Finally, add 500 μL FACS Buffer (containing DNaseI, RNase inhibitor) to resuspend the cells, and analyze the proportion of Cx3cr1-GFP positive microglia by instrument. Cx3cr1-GFP positive microglia can also be sorted by instrument.

[0053] The experimental results are as follows Figure 2 As shown in b. Figure 2 As can be seen from b, the proportion of GFP-positive microglia is 75.8%, indicating that the replacement efficiency of microglia is 75.8%.

[0054] Example 3 Immunofluorescence staining analysis of high replacement ratio of TCMDT microglia

[0055] Adult mice (3-8 months old) were divided into 3 groups: no PLX3397 feed group (0×PLX), single PLX3397 feed group (1×PLX), and TCMDT PLX3397 feed group (3×PLX). The feeding amount and feeding environment were the same as in Example 1. The method of transplanting microglia in mice was the same as in Example 2. One month after transplantation, the mice were anesthetized with isoflurane, and 50 mL of pre-cooled 1×PBS was perfused through the heart. Then the brain tissue was removed from the skull and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148) overnight. The brain tissue was then transferred to 25% and 30% sucrose solution for dehydration until the brain tissue sank to the bottom of the container. Finally, the brain tissue was embedded in OCT embedding agent for frozen sectioning. The section thickness was 15 μm, and then immunofluorescence staining was performed. Immunofluorescence staining of microglia Iba1 (Wako, 019-19741, 1:200) and nucleus DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000) was performed, and panoramic immunofluorescence staining images of brain tissue were taken. The proportion of Cx3cr1-GFP positive microglia to all microglia was counted, and the microglia replacement efficiency was calculated.

[0056] The experimental results are as follows Figure 3 As shown. From the results, it can be seen that there was no microglia engraftment in the brain of the untreated group (0×PLX). In mice treated with PLX3397 for 7 days (1×PLX), a small number of microglia (about 10% of the brain area) around the injection site were replaced. After 3 cycles of microglia depletion (3×PLX, called TCMDT), the transplanted microglia occupied about 70% of the brain area, significantly improving the engraftment efficiency.

[0057] In addition, after three cycles of microglial depletion, the microglial engraftment efficiency was further improved to approximately 90% three months after transplantation.

[0058] Example 4 Flow cytometry analysis of high replacement ratio of TCMDT microglia

[0059] Adult mice were divided into 3 groups: no PLX3397 feed group (0×PLX), single PLX3397 feed group (1×PLX), and TCMDT PLX3397 feed group (3×PLX). The feeding amount and feeding environment were the same as in Example 1. The method of transplanting microglia in mice was the same as in Example 2. One month after transplantation, the mice were anesthetized with isoflurane, and 50 mL of pre-cooled 1×PBS was perfused through the heart to obtain brain tissue. The hippocampus and cortical tissues on both sides were separated and placed in pre-cooled 1×HBSS (gibco, C14175500BT) for standby use. Flow cytometry analysis of the proportion of Cx3cr1-GFP positive microglia to all microglia was the same as in Example 2.

[0060] The experimental results are as follows Figure 4 The results showed that no microglial implantation was detected in the brain of the untreated group (0×PLX), the replacement efficiency of microglial cells in the 1×PLX group was only about 3%, and the replacement efficiency of microglial cells in the 3×PLX group was significantly increased to about 70%.

[0061] Similarly, 3 months after transplantation, the replacement efficiency of microglia in the 3×PLX group was as high as 90%.

[0062] Example 5 TCMDT can restore the microglial characteristics of cultured microglia after transplantation into the brain

[0063] TCMDT eliminates microglia in the mouse brain, exogenously transplants Cx3cr1-GFP microglia, and the transplantation method is the same as in Example 2. After 3 months of transplantation, mice are anesthetized with isoflurane, and 50 mL of pre-cooled 1 × PBS is perfused through the heart to take brain tissue, and the hippocampus and cortical tissue on both sides are separated and placed in pre-cooled 1 × HBSS (gibco, C14175500BT) for standby use. Flow cytometry sorting of Cx3cr1-GFP positive microglia is the same as in Example 2. 500 μL Trizol was added to the sorted Cx3cr1-GFP positive microglia, placed at room temperature for 5 min, and vortexed for 15 s to completely dissociate the cell precipitation complex. Subsequently, 100 μL chloroform was added, vortexed for 20 s, placed at room temperature for 3 min, and centrifuged at 12000 g for 15 min. Take about 350 μL of supernatant, add an equal volume of isopropanol, add 1 μL of glycogen at the same time, gently invert to mix, precipitate at -20℃ for 1h, and centrifuge at 12000rpm for 15min. Discard the supernatant with a pipette, add 500 μL of pre-cooled 75% ethanol, and centrifuge at 7500g for 5min. Discard the supernatant with a pipette (there may be a little residue), and centrifuge again at 7500g for 2min. Discard the residual liquid, open the lid, air dry for about 2min, add 15 μL of DEPC water to dissolve the RNA, and use the obtained RNA for sequencing.

[0064] The sequencing results are as follows Figure 5 The sequencing results were analyzed and principal component analysis (PCA) revealed significant gene expression differences between TM (transplanted microglia) and PM (primary microglia), but both TM and NM showed a high Pearson correlation coefficient and significant overlap, indicating that TM was very similar to NM (naive microglia) in transcriptional profile, indicating that microglia cultured in vitro restored their microglial characteristics after transplantation into the brain and were very close to microglia in the brain.

[0065] Example 6 TCMDT can restore the function of transplanted microglia

[0066] TCMDT eliminated microglia in the mouse brain, exogenously transplanted Cx3cr1-GFP microglia, and the transplantation method was the same as in Example 2. Three months after transplantation, the mice were anesthetized with isoflurane, and 50 mL of pre-cooled 1×PBS was perfused through the heart, and then the brain tissue was removed from the skull and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148) overnight. The brain tissue was then transferred to a 25% and 30% sucrose solution for dehydration until the brain tissue sank to the bottom of the container, and finally the brain tissue was embedded in an OCT embedding agent for frozen sectioning, with a section thickness of 15 μm, and then immunofluorescence staining was performed. Immunofluorescence staining of microglia for Iba1 (Wako, 019-19741, 1:200) and nuclear DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000) was performed, and images were taken at 60× using an inverted fluorescence microscope to analyze microglial morphology, which was evaluated by intersections, endpoints, and neurite length.

[0067] The experimental results are shown in 6. The results show that in the absence of LPS treatment, transplanted microglia exhibited the same cell morphology as the initial microglia in the brain, indicating that primary cultured microglia can reconstruct their microglial phenotype after brain transplantation, which is consistent with our transcriptional analysis. After LPS treatment, both initial and transplanted microglia showed shorter neurite lengths and fewer intersections and termini, showing similar morphological changes in response to stimulation.

[0068] Example 7 TCMDT significantly improves movement disorders in LSD mice

[0069] TCMDT eliminates Hexb-KO mice (a mouse model of neurodegenerative lysosomal storage disease Sandhoff disease, from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., C57BL / 6JGpt-Hexb em5cd3129 / Gpt) brain microglia, exogenously transplanted Cx3cr1-GFP microglia, the transplantation method was the same as in Example 2, and 2 months after the transplantation, the mice were subjected to behavioral experiments related to the rotarod and balance beam and exercise at 4 months of age. The rotarod experiment recorded the time when the mice lost balance and fell on the rotarod, and the balance beam experiment recorded the time when the mice passed the balance beam, so as to evaluate the movement balance and coordination ability of the mice.

[0070] The experimental results are as follows Figure 7 As shown. The rotarod test showed that there was no difference in the latency to fall between untreated (Ctrl), sham-operated (Sham), and microglia-transplanted (MT) WT mice, indicating that TCMDT and microglia transplantation did not affect the motor function of WT mice. In contrast, the latency to fall of KO mice was significantly shortened, highlighting the obvious motor dysfunction, while microglia transplantation significantly improved the motor function of KO mice. Similarly, the results of the balance beam test were consistent with the rotarod test. There was no difference in the crossing time and fall scores among the three groups of WT mice, while in KO mice, MT significantly shortened the crossing time and reduced the fall score.

[0071] Example 8 TCMDT significantly reduces neurodegeneration in LSD mice

[0072] After the behavioral experiment in Example 7, the mice were anesthetized with isoflurane and perfused with 50 mL of pre-cooled 1×PBS through the heart. The brain tissue was then removed from the skull and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148) overnight. The brain tissue was then transferred to a 25% and 30% sucrose solution for dehydration until the brain tissue sank to the bottom of the container. Finally, the brain tissue was embedded in an OCT embedding agent 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 for 10 minutes at room temperature. After the sections were stained, H 2 The brain was washed with 1% ethanol and 1% anhydrous ethanol for 3 times, and then quickly washed with 95% ethanol and anhydrous ethanol for 3 times, and then soaked in xylene twice, each time for 5 minutes. Finally, the slides were sealed with neutral resin. The whole brain images were scanned using a panoramic digital slice scanner to analyze the number of neurons.

[0073] The experimental results are as follows Figure 8 The results showed that the number of neurons in KO mice was significantly reduced compared with WT mice, especially in the primary motor cortex and midbrain, areas related to motor function. After microglia transplantation, the number of neurons in KO mice was significantly increased.

[0074] Example 9 TCMDT significantly reduces neuroinflammation in LSD mice

[0075] After the behavioral experiment in Example 7, the mice were anesthetized with isoflurane, 50 mL of pre-cooled 1×PBS was perfused through the heart, and then the brain tissue was removed from the skull and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148) overnight. The brain tissue was then transferred to 25% and 30% sucrose solutions for dehydration until the brain tissue sank to the bottom of the container, and finally the brain tissue was embedded in OCT embedding agent for frozen sectioning, with a section thickness of 15 μm, and then immunohistochemical staining was performed. Immunofluorescence staining of microglia Iba1 (Wako, 019-19741, 1: 200), microglia phagocytic marker CD68 (BioLegend, 137001, 1: 200) and nucleus DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1: 1000), an inverted fluorescence microscope was used to take 60× images, and the microglia CD68 area percentage was counted.

[0076] The experimental results are shown in Figure 9. Compared with the control group, the number of CD68-positive microglia in KO mice increased significantly, and the CD68 area increased significantly, which completely returned to normal levels after microglia transplantation, indicating that microglia transplantation has a positive effect on reducing neuroinflammation in LSD mice.

[0077] Example 10 TCMDT significantly reduces Aβ pathology in AD mice carrying Trem2 R47H mutation TCMDT eliminates Aβ pathology in AD mice carrying Trem2 R47H mutation (Shanghai Model Organisms, C57BL / 6Smoc-Trem 2em3(p.R47H)smoc ) Brain microglia, exogenously transplanted Cx3cr1-GFP microglia, the transplantation method is the same as Example 2, 4 months after transplantation, mice were anesthetized with isoflurane, 50 mL of pre-cooled 1×PBS was perfused through the heart, and then the brain tissue was removed from the skull and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148) overnight. The brain tissue was then transferred to 25% and 30% sucrose solution for dehydration until the brain tissue sank to the bottom of the container, and finally embedded in OCT embedding agent for frozen sectioning, the section thickness was 15 μm, and then immunohistochemical staining was performed. Immunofluorescence staining of microglia Iba1 (Wako, 019-19741, 1:200), amyloid protein MOAB2 (Abcam, ab26649, 1:400) and nuclear DAPI (Sigma-Aldrich, D9542, 1 mg / mL, 1:1000) was performed, and 60x images were taken under an inverted fluorescence microscope to count the area of ​​Aβ plaques and the number of microglia.

[0078] The experimental results are as follows Fig.10The results showed that microglial transplantation significantly reduced Aβ plaque deposition in R47H mice, and the number of microglial cells was also restored after transplantation.

[0079] Example 11 TCMDT significantly reduces the associated neurotoxicity in AD mice carrying Trem2 R47H mutation

[0080] TCMDT eliminates microglia in the brain of AD mice carrying Trem2 R47H mutation, exogenously transplants Cx3cr1-GFP microglia, and the transplantation method is the same as in Example 2. Four months after transplantation, mice are anesthetized with isoflurane, and 50 mL of pre-cooled 1×PBS is perfused through the heart, and then the brain tissue is removed from the skull and fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, P6148) overnight. The brain tissue is then transferred to 25% and 30% sucrose solutions for dehydration until the brain tissue sinks to the bottom of the container, and finally the brain tissue is embedded in OCT embedding agent for frozen sectioning, the section thickness is 15 μm, and then immunohistochemical staining is performed. Immunofluorescence staining of microglia Iba1 (Wako, 019-19741, 1:200), lysosomal-associated membrane protein Lamp1 (DB Biosciences, 553792, 1:200) marking dystrophic neurites, and amyloid core dense plaque ThioS (Sigma-Aldrich, T1892, 0.1% in 50% ethanol) was performed, and images were taken at 60x under an inverted fluorescence microscope to analyze the Lamp1-positive area.

[0081] The experimental results are as follows Fig.11 The results showed that the Lamp1-positive area, a marker of neurite dystrophy, decreased around each plaque, indicating that microglia transplantation alleviated the neurotoxicity caused by plaques.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An experimental method for whole-brain microglial transplantation, characterized in that: The microglia were depleted for three cycles using a CSF1R inhibitor, and then exogenous microglia were injected into the brain of the experimental animals; the CSF1R inhibitor was PLX3397 or PLX5622.

2. The whole-brain microglial transplantation experimental method according to claim 1, characterized in that: The three cycles of microglial depletion using CSF1R inhibitors are as follows: feeding the experimental animals with CSF1R inhibitor feed for 7 days to induce microglial depletion; then switching to normal feed for 7 days for microglial regeneration; and repeating the process twice.

3. The experimental method for whole-brain microglia transplantation according to claim 2, characterized in that: The concentration of the CSF1R inhibitor in the CSF1R inhibitor feed is 200-620 mg / kg.

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

5. 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.

6. The experimental method for whole-brain microglia transplantation according to claim 1, characterized in that: The site where exogenous microglia are injected into the brain of experimental animals is the brain parenchyma.

7. Use of the whole-brain microglial cell transplantation experimental method according to any one of claims 1 to 6 in experimental research on the treatment of neurodegenerative lysosomal storage diseases.

8. Use of the whole-brain microglial cell transplantation experimental method according to any one of claims 1 to 6 in experimental research on the treatment of Alzheimer's disease.

9. Use of the whole-brain microglial cell transplantation experimental method according to any one of claims 1 to 6 in experimental research on the treatment of microglial cell dysfunction.

10. Use of the experimental method of whole-brain microglial transplantation according to any one of claims 1 to 6 in experimental research on the treatment of nervous system diseases.

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