Compound for treating cerebral hemorrhage chronic stage delayed nerve injury and identification method

Abstract

The invention provides a method for identifying a compound for treating cerebral hemorrhage chronic stage delayed nerve injury. The method comprises the following steps: establishing a cerebral hemorrhage mouse model, applying a medicine to be identified to a treatment group, and applying immunoglobulin IgG to a non-treatment group; determining the dyskinesia through nerve function evaluation and a rotation angle experiment; evaluating the excitability of thalamic neurons by using a whole cell patch clamp recording technology; comparing the synaptic delivery functions of the therapeutic group and the non-therapeutic group; the spatial learning and memory ability is evaluated through a Morris water maze test; the improvement effects of the neurological function, synaptic delivery and cognitive function are comprehensively evaluated to determine the therapeutic effect of the drug. The method further comprises the steps of collecting autopsy brain tissues of a cerebral hemorrhage patient and a healthy control group, performing spatial transcriptome sequencing to identify a specific differential expression gene, and exploring a pathogenic mechanism in the cerebral hemorrhage animal model. The method can be used for accurately screening and identifying potential cerebral hemorrhage chronic stage nerve injury treatment medicines.

Description

Technical Field

[0001] The invention relates to a drug identification method, in particular to a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage and an identification method. Background Art

[0002] In the current field of medical research, there is still a lack of perfect drug identification methods for the treatment of delayed neurological damage in the chronic phase of cerebral hemorrhage. Intracerebral hemorrhage (ICH) is a serious neurological disease characterized by the accumulation of blood in the brain parenchyma due to rupture of intracerebral blood vessels. Primary and secondary injuries will occur after ICH. Primary brain injury refers to the increase in overall intracranial pressure and mechanical compression of local structures due to initial bleeding or continuous bleeding and hematoma expansion after cerebral hemorrhage, while secondary brain injury is mainly caused by the pathophysiological reaction of hematoma, including edema and inflammation as well as toxic components produced by clots. At present, conventional medical and surgical treatments for ICH cannot meet the long-term prognosis of patients. Therefore, it is more necessary to actively explore the pathological mechanism of ICH, find potential molecular therapeutic targets for ICH, reduce the inflammatory cascade reaction after ICH and the degree of edema of brain cells, and thus improve the prognosis and quality of life of patients.

[0003] At present, the treatment strategies for ICH mainly include controlling blood pressure, stopping bleeding, and surgical treatment. However, the existing treatment methods are mainly focused on the management of the acute phase, and the treatment effect on the neurological dysfunction and delayed neurological damage in the chronic phase is limited. In addition to the severe neurological damage in the early stage of cerebral hemorrhage, the survivors will face a variety of irreversible delayed neurological sequelae in the chronic phase, such as dementia, depression, fatigue, etc., which seriously affect the quality of life of patients. In addition, because the pathological mechanism of ICH has not been fully elucidated and there is a lack of targeted treatment plans, the prognosis of patients is poor, and there are significant long-term neurological damage and cognitive defects. There is currently no treatment for the delayed sequelae of chronic cerebral hemorrhage.

[0004] In the chronic phase, the activation of microglia is closely related to chronic inflammation, neuropathology, and degenerative changes in the chronic phase of brain injury. The complement system, especially complement C1q, plays an important role in the inflammatory response and neurological injury after ICH. Studies have shown that the expression of C1q is chronically increased in the cortex around the injury site and in the functionally connected cortical hypothalamus, which is consistent with the area of ​​neuronal loss and chronic inflammation. Therefore, treatment targeting complement C1q may have potential efficacy in improving neurological dysfunction in the chronic phase of ICH.

[0005] More and more clinical and animal experimental evidence supports that immune responses can be stimulated in distant sites after stroke, resulting in widespread neuroinflammation throughout the brain. Neuroinflammation starts in the acute phase and worsens and persists in the chronic phase. It is generally believed that the primary bleeding focus of cerebral hemorrhage initiates a local inflammatory response, which then spreads to a wide area of ​​the brain, and may therefore aggravate distal nerve damage and long-term functional deficits. This phenomenon reveals the mechanism by which secondary lesions in the chronic phase of acute brain injury continue to affect brain structure and function. Based on this, studying the characteristics of neuroinflammation in the chronic phase of cerebral hemorrhage is crucial to inventing treatments for chronic brain injury.

[0006] Spatial transcriptomics technology provides a map arrangement of gene expression patterns mapped onto tissue sections, which can connect structure and activity. This feature enables researchers to elucidate biological interactions at the cellular level, thereby generating new insights into complex tissues. This technology has been used to generate tissue atlases, providing valuable resources as references. Currently, in the field of cerebral hemorrhage, most of the studies using this technology are on the lesions and surrounding tissues in the acute phase of cerebral hemorrhage, but so far, no studies have been conducted on the gene expression patterns of distant lesion areas or even the whole brain in the chronic phase of cerebral hemorrhage. The molecular profiles expressed by damaged neurons in distant sites may show completely different characteristics compared with local lesions. Even if they are partially the same, they may show completely different functions and phenotypes at different times. The specific molecular mechanisms need further exploration.

[0007] Currently, there is a lack of effective drug identification methods for delayed neurological damage in the chronic phase of cerebral hemorrhage, and the key role of the complement system in the inflammatory response after cerebral hemorrhage has not been fully therapeutically regulated. These challenges have prompted researchers to develop new drug identification methods in order to find therapeutic drugs that can improve the prognosis of patients with cerebral hemorrhage. In response to the above problems, the present invention uses brain tissue samples in the chronic phase of cerebral hemorrhage, combined with spatial multi-omics research, to deeply explore the effects and molecular mechanisms of neuroinflammation on the long-term prognosis of cerebral hemorrhage, which will help us further understand the role of delayed brain damage after stroke and the feasibility of drug intervention. In addition, through preclinical intervention and in situ immune spectrum analysis, we will find targets for neural repair in the recovery period of cerebral hemorrhage. Summary of the invention

[0008] In order to solve the above problems, the present invention firstly carried out spatial transcriptome sequencing on brain tissue in the chronic phase of cerebral hemorrhage, and found that brain tissue around the lesions left after cerebral hemorrhage showed persistent neuroinflammatory activation, which showed the performance of reactive microglial transcriptome. Through differential gene enrichment pathway analysis, it was found that complement C1q is a key target for the treatment of delayed neurological dysfunction in the chronic phase of cerebral hemorrhage, and then verified in animal models to support the above findings.

[0009] Based on the above research results, the present invention provides a method for identifying a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage, comprising the following steps:

[0010] (a) A mouse model of cerebral hemorrhage was established and the mice were divided into a treatment group and a non-treatment group. The treatment group was treated with the drug to be identified, and the non-treatment group was treated with immunoglobulin IgG. Neurological function of the mice was evaluated N days after the model was induced, and residual motor dysfunction was measured by corner turning test.

[0011] (b) Whole-cell patch clamp recording was used to evaluate the excitability of thalamic neurons in mice N days after ICH. The amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) were measured to determine the basic synaptic transmission properties of thalamic neurons. The frequency and amplitude of sEPSCs in mice N days after ICH were compared with those in the control group to evaluate the effect of ICH on synaptic transmission.

[0012] (c) measuring the frequency and amplitude of sEPSCs in the treated mice and comparing the results with those in the untreated mice to evaluate the improvement effect of the drug treatment on synaptic transmission function;

[0013] (d) The spatial learning and memory abilities of mice in the treatment and non-treatment groups were evaluated by Morris water maze test N days after ICH and compared with those in the control group without ICH to determine the presence of cognitive dysfunction;

[0014] (e) Analyze the effects of the drug treatment to be identified in the treatment group on the spatial learning and memory ability of mice N days after ICH, and compare the Morris water maze test results with those of the non-treatment group to evaluate the improvement effect of the treatment on cognitive dysfunction;

[0015] (f) Based on the results of the above steps, determine whether the treatment group improves neurological function, synaptic transmission and cognitive function, alleviates long-term neurological damage and cognitive deficits associated with the chronic stage of ICH, and determines whether the drug can be used as a potential drug for the treatment of delayed neurological damage in the chronic stage of ICH by determining whether the treatment group improves neurological function, synaptic transmission and cognitive function, and alleviates long-term neurological damage and cognitive deficits associated with the chronic stage of ICH.

[0016] In step (a), the degree of neurological deficit was assessed by the modified neurological deficit score (mNSS scores), and motor coordination and balance ability were assessed by the rotarod test;

[0017] Step (b) and step (c) evaluate the excitability of the neuron by recording spontaneous excitatory postsynaptic currents sEPSCs, and evaluate the effect on synaptic transmission by a histogram and a cumulative probability graph of the frequency of sEPSCs and the amplitude of sEPSCs;

[0018] Step (d) and step (e) evaluate motor function through the movement speed and distance of different groups within N days. The path tracking diagram shows the path of the mice when searching for the hidden platform. The platform residence time and the number of times the target platform is crossed show the learning and memory ability of the mice.

[0019] In some embodiments: N≥35, and the drug to be identified is a complement C1q inhibitor.

[0020] In some embodiments, the complement C1q inhibitor is specifically an anti-C1q antibody.

[0021] In some embodiments:

[0022] Prior to step (a), there is also a step of obtaining specific differentially expressed genes in the chronic phase of cerebral hemorrhage, which specifically includes:

[0023] (1) Collect autopsy brain tissue from patients with cerebral hemorrhage and healthy controls;

[0024] (2) perform spatial transcriptome sequencing to identify specific differentially expressed genes;

[0025] (3) Compare the gene expression data of patients with cerebral hemorrhage with those of the healthy control group to identify differentially expressed genes.

[0026] In some embodiments:

[0027] Step (1) specifically comprises: collecting autopsy brain tissue from patients with cerebral hemorrhage and a healthy control group; collecting brain tissue within 4 hours after the death of the collected individuals to ensure the freshness of the tissue;

[0028] Step (2) is as follows: Use NanoString Spatial transcriptomic analysis was performed using a digital spatial profiler. Brain tissues were sliced ​​to 6 μm thickness and mounted on Leica BOND Plus slides. Subsequent steps were performed using DEPC-treated water under RNase-free conditions, including three consecutive washes of the slides in xylene for 5 min, followed by two washes in 100% ethanol for 5 min, one wash in 95% ethanol, and one wash in 1x PBS. Target retrieval was performed in the BioGenex ezretriverSystem with target retrieval reagent diluted to 1x at 95°C for 10 min. After washing in 1x PBS, the slides were incubated with RNA hybridization probes diluted in BufferR at 37°C overnight. The next day, the slides were stained with antibodies to Iba1, GFAP, NeuN, and Syto13 for 1 h at room temperature and then loaded into the GeoMx The DSP instrument scanned and selected the region of interest (ROI); DAPI immunofluorescence was used to identify morphological markers, and Iba1, NeuN and GFAP staining was used for segmentation, and the ROI selected for subsequent gene expression analysis was marked with a circle; each ROI was segmented into three illumination areas (AOI): microglia iba1-positive, neurons neuN-positive, and astrocytes gmap-positive, and the probe identity of each segment was captured by ultraviolet irradiation and moved to a 96-well plate; the DSP standard process was used to build the PCR system, construct the library, and use GeoMx NGSPipeline software to convert the original fastq file into a digital count file;

[0029] Step 3 specifically includes: segmenting the data and performing probe quality control to remove poor AOI / ROI and abnormal probes, and determining the detection limit of each segment, ignoring gene expression below LOQ; using quantile 3 normalization to normalize the data, and using edgeR for differential detection to compare the differences between the AOIs groups, that is, the ICH and control groups, and genes with a false discovery rate FDR < 0.05 and log2 (Fold Change) > 1 were considered differentially expressed genes;

[0030] The identified genes were analyzed using the Toppgene program, mainly for gene ontology annotation, and enrichment analysis of up-regulated and down-regulated genes was performed using the GO and KEGG methods; gene signature analysis was performed using the gene collection Hallmark in the Molecular Signatures Database, MSigDB, and statistical tests were used in the selected groups to test the hypothesis; the Wilcoxon rank sum test was used to compare two groups of independent AOI / ROI, and the Kruskal-Wallis method was used to test the rank sum of multiple groups of independent AOI / ROI to determine the specific differentially expressed genes in the chronic stage of cerebral hemorrhage.

[0031] In some embodiments:

[0032] After step (f), there is also a step of exploring the pathogenic mechanism in the animal model of cerebral hemorrhage, which specifically includes:

[0033] (i) Observe the pathological changes in the ICH animal model N days after ICH, including reactive microgliosis and neuropathological abnormalities, neuronal loss, neuronal degeneration, and decreased dendritic spine density;

[0034] (ii) using transmission electron microscopy to observe the separation of myelin sheets and / or vesicles, as well as the condensation of postsynaptic structures in animal models of intracerebral hemorrhage;

[0035] (iii) Treating animal models of cerebral hemorrhage with the drug to be identified and evaluating the therapeutic effects, including alleviating neuropathological abnormalities and improving chronic inflammation, neuropathology and degenerative changes in the chronic phase of brain injury.

[0036] The present invention further provides a pharmaceutical composition for treating delayed nerve damage in the chronic phase of cerebral hemorrhage obtained by the identification method, characterized in that:

[0037] The drugs include complement C1q inhibitors.

[0038] It also includes at least one drug carrier, the complement C1q inhibitor is an anti-C1q antibody, and the drug carrier is specifically a liposome or nanoparticle. The drug carrier can effectively deliver the complement C1q inhibitor to the treatment site.

[0039] The present invention further provides an application of the pharmaceutical composition in preparing a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage.

[0040] In view of the above method, the present invention further proposes a device system for identifying drugs for treating delayed nerve damage in the chronic phase of cerebral hemorrhage. The system is used to implement the above method, and the system includes:

[0041] (a) The module for establishing the mouse model of intracerebral hemorrhage is used to divide the mice into treatment group and non-treatment group, and to apply the drug to be identified to the treatment group, and to apply immunoglobulin IgG to the non-treatment group; (b) The module for evaluating the neurological function of mice N days after the induction of the model, and to determine the residual motor dysfunction by corner turning experiment; (c) The module for evaluating the neuronal excitability is used to evaluate the excitability of thalamic neurons of mice N days after intracerebral hemorrhage using the whole-cell patch clamp recording technique, and to determine the basic synaptic transmission properties of thalamic neurons by measuring the amplitude and frequency of spontaneous excitatory postsynaptic currents sEPSCs; (d) The module for evaluating the improvement of synaptic transmission function is used to measure the sEPSCs frequency of the mice in the treatment group. and amplitude, and compare the results with those of the non-treated group mice to evaluate the improvement effect of the drug treatment to be identified on synaptic transmission function; (e) cognitive function evaluation module, which evaluates the spatial learning and memory ability of mice in the treatment group and non-treatment group N days after cerebral hemorrhage through Morris water maze test, and compares them with the control group mice without cerebral hemorrhage to determine the presence of cognitive dysfunction; (f) comprehensive evaluation module for treatment effect, which is used to integrate the results of the above modules, by determining whether the treatment group improves neural function, synaptic transmission and cognitive function, alleviates long-term neural damage and cognitive defects associated with the chronic stage of cerebral hemorrhage, and determines whether the drug can be used as a potential drug for the treatment of delayed neural damage in the chronic stage of cerebral hemorrhage.

[0042] In module (a), the degree of neurological deficit was assessed by the modified neurological deficit score (mNSS) scores, and motor coordination and balance ability were assessed by the rotarod test.

[0043] Modules (b) and (c) evaluate the excitability of neurons by recording spontaneous excitatory postsynaptic currents sEPSCs, and evaluate the effects on synaptic transmission by histograms and cumulative probability plots of the frequency and amplitude of sEPSCs;

[0044] Modules (d) and (e) evaluate motor function through the movement speed and distance of different groups within N days. The path tracking diagram shows the path of the mice when searching for the hidden platform, and the learning and memory ability of the mice is displayed by the platform residence time and the number of times the target platform is crossed.

[0045] In some embodiments: N≥35, and the drug to be identified is a complement C1q inhibitor.

[0046] In some embodiments, the complement C1q inhibitor is specifically an anti-C1q antibody.

[0047] In some embodiments:

[0048] There is also a module for obtaining differentially expressed genes specific to the chronic stage of cerebral hemorrhage. The module is used to:

[0049] (1) Collect autopsy brain tissue from patients with cerebral hemorrhage and healthy controls;

[0050] (2) perform spatial transcriptome sequencing to identify specific differentially expressed genes;

[0051] (3) Compare the gene expression data of patients with cerebral hemorrhage with those of the healthy control group to identify differentially expressed genes.

[0052] In some embodiments:

[0053] (1) Specifically: Collect autopsy brain tissue from patients with cerebral hemorrhage and healthy controls; collect brain tissue within 4 hours after the death of the individual to ensure tissue freshness;

[0054] (2) Specifically: Use NanoString Spatial transcriptomic analysis was performed using a digital spatial profiler. Brain tissues were sliced ​​to 6 μm thickness and mounted on Leica BOND Plus slides. Subsequent steps were performed using DEPC-treated water under RNase-free conditions, including three consecutive washes of the slides in xylene for 5 min, followed by two washes in 100% ethanol for 5 min, one wash in 95% ethanol, and one wash in 1x PBS. Target retrieval was performed in the BioGenex ezretriverSystem with target retrieval reagent diluted to 1x at 95°C for 10 min. After washing in 1x PBS, the slides were incubated with RNA hybridization probes diluted in Buffer R at 37°C overnight. The next day, the slides were stained with antibodies to Iba1, GFAP, NeuN, and Syto13 for 1 h at room temperature and then loaded into the GeoMx The DSP instrument scanned and selected the region of interest (ROI); DAPI immunofluorescence was used to identify morphological markers, and Iba1, NeuN and GFAP staining was used for segmentation, and the ROI selected for subsequent gene expression analysis was marked with a circle; each ROI was segmented into three illumination areas (AOI): microglia iba1-positive, neurons neuN-positive, and astrocytes gmap-positive, and the probe identity of each segment was captured by ultraviolet irradiation and moved to a 96-well plate; the DSP standard process was used to build the PCR system, construct the library, and use GeoMx NGSPipeline software to convert the original fastq file into a digital count file;

[0055] (3) Specifically: segment the data and perform probe quality control to remove poor AOI / ROI and abnormal probes, determine the detection limit of each segment, and ignore gene expression below LOQ; use quantile 3 normalization to normalize the data, and use edgeR for differential detection to compare the differences between AOIs groups, that is, ICH and control groups. Genes with false discovery rate FDR < 0.05 and log2 (Fold Change) > 1 are considered differentially expressed genes;

[0056] The identified genes were analyzed using the Toppgene program, mainly for gene ontology annotation, and enrichment analysis of up-regulated and down-regulated genes was performed using the GO and KEGG methods; gene signature analysis was performed using the gene collection Hallmark in the Molecular Signatures Database, MSigDB, and statistical tests were used in the selected groups to test the hypothesis; the Wilcoxon rank sum test was used to compare two groups of independent AOI / ROI, and the Kruskal-Wallis method was used to test the rank sum of multiple groups of independent AOI / ROI to determine the specific differentially expressed genes in the chronic stage of cerebral hemorrhage.

[0057] In some embodiments:

[0058] There are also modules to explore the pathogenic mechanisms in animal models of cerebral hemorrhage, including:

[0059] (i) Observe the pathological changes in the ICH animal model N days after ICH, including reactive microgliosis and neuropathological abnormalities, neuronal loss, neuronal degeneration, and decreased dendritic spine density;

[0060] (ii) using transmission electron microscopy to observe the separation of myelin sheets and / or vesicles, as well as the condensation of postsynaptic structures in animal models of intracerebral hemorrhage;

[0061] (iii) Treating animal models of cerebral hemorrhage with the drug to be identified and evaluating the therapeutic effects, including alleviating neuropathological abnormalities and improving chronic inflammation, neuropathology and degenerative changes in the chronic phase of brain injury.

[0062] It should be pointed out that all methods and systems of the present invention are laboratory research methods and non-disease diagnosis and treatment methods.

[0063] The beneficial effects of the present invention are:

[0064] The present invention uses a variety of scientifically validated technical means, including modified neurological deficit score (mNSSscores), rotarod test, spontaneous excitatory postsynaptic current (sEPSCs), etc. The combined use of these methods provides a multi-dimensional and comprehensive evaluation system to ensure the comprehensiveness and accuracy of the evaluation results.

[0065] The complement C1q inhibitor proposed in the present invention, especially the anti-C1q antibody, is proposed based on the current in-depth understanding of the pathological mechanism of cerebral hemorrhage. The activation of the complement system plays a key role in the inflammatory response after cerebral hemorrhage. Therefore, the drug identification method targeting this target has a clear scientific basis.

[0066] The present invention is the first to perform spatial transcriptome sequencing on brain tissue in the chronic phase of cerebral hemorrhage, and found that brain tissue around the lesions left after cerebral hemorrhage showed persistent neuroinflammatory activation, which showed the performance of reactive microglial transcriptome. Through differential gene enrichment pathway analysis, it was found that complement C1q is a key target for the treatment of delayed neurological dysfunction in the chronic phase of cerebral hemorrhage, and further verification from animal models supports the above findings. The present invention is currently the only one that uses human brain tissue and animal models to discover targets for the treatment of delayed neurological dysfunction in cerebral hemorrhage.

[0067] The present invention provides a new treatment strategy by systematically identifying and applying the role of complement C1q inhibitors in treating delayed neurological damage in the chronic phase of cerebral hemorrhage. The present invention establishes a mouse model of cerebral hemorrhage and comprehensively uses multi-dimensional research methods such as neurological function assessment, whole-cell patch clamp recording technology, Morris water maze test, and spatial transcriptome sequencing to comprehensively evaluate the improvement effect of the drugs to be identified on neurological function, synaptic transmission, and cognitive function. It can not only accurately identify and quantify the damage and recovery process of neurons after cerebral hemorrhage, but also deeply explore the mechanism of action of complement C1q in the pathological process of ICH.

[0068] The present invention further verifies the therapeutic effect of complement C1q inhibitor on neurological dysfunction after cerebral hemorrhage by improving the neurological deficit score, corner test, spontaneous excitatory postsynaptic current recording and other technical means. It provides a scientific basis for the treatment of chronic cerebral hemorrhage, is expected to reduce the long-term neurological damage and cognitive defects of patients, improve the quality of life, and has important clinical transformation potential and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0070] Attached Figure 1 Results of spatial transcriptomic analysis of brain tissues in patients with intracerebral hemorrhage (ICH) and healthy controls.

[0071] Attached Figure 2 To compare the neurological recovery of intracerebral hemorrhage (ICH) model animals after receiving different treatments (control group Sham, ICH plus IgG, ICH plus Anti-C1q);

[0072] Attached Figure 3 These are the histological and cytological changes in intracerebral hemorrhage (ICH) model animals after receiving different treatments (Sham, ICH+IgG, ICH+Anti-C1q). DETAILED DESCRIPTION

[0073] Embodiment 1:

[0074] The present invention provides a method for identifying a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage, comprising the following steps:

[0075] (a) A mouse model of cerebral hemorrhage was established and the mice were divided into a treatment group and a non-treatment group. The treatment group was treated with the drug to be identified, and the non-treatment group was treated with immunoglobulin IgG. Neurological function of the mice was evaluated N days after the model was induced, and residual motor dysfunction was measured by corner turning test.

[0076] (b) Whole-cell patch clamp recording was used to evaluate the excitability of thalamic neurons in mice N days after ICH. The amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) were measured to determine the basic synaptic transmission properties of thalamic neurons. The frequency and amplitude of sEPSCs in mice N days after ICH were compared with those in the control group to evaluate the effect of ICH on synaptic transmission.

[0077] (c) measuring the frequency and amplitude of sEPSCs in the treated mice and comparing the results with those in the untreated mice to evaluate the improvement effect of the drug treatment on synaptic transmission function;

[0078] (d) The spatial learning and memory abilities of mice in the treatment and non-treatment groups were evaluated by Morris water maze test N days after ICH and compared with those in the control group without ICH to determine the presence of cognitive dysfunction;

[0079] (e) Analyze the effects of the drug treatment to be identified in the treatment group on the spatial learning and memory ability of mice N days after ICH, and compare the Morris water maze test results with those of the non-treatment group to evaluate the improvement effect of the treatment on cognitive dysfunction;

[0080] (f) Based on the results of the above steps, determine whether the treatment group improves neurological function, synaptic transmission and cognitive function, alleviates long-term neurological damage and cognitive deficits associated with the chronic stage of ICH, and determines whether the drug can be used as a potential drug for the treatment of delayed neurological damage in the chronic stage of ICH by determining whether the treatment group improves neurological function, synaptic transmission and cognitive function, and alleviates long-term neurological damage and cognitive deficits associated with the chronic stage of ICH.

[0081] In step (a), the degree of neurological deficit was assessed by the modified neurological deficit score (mNSS scores), and motor coordination and balance ability were assessed by the rotarod test;

[0082] Step (b) and step (c) evaluate the excitability of the neuron by recording spontaneous excitatory postsynaptic currents sEPSCs, and evaluate the effect on synaptic transmission by a histogram and a cumulative probability graph of the frequency of sEPSCs and the amplitude of sEPSCs;

[0083] Step (d) and step (e) evaluate motor function through the movement speed and distance of different groups within N days. The path tracking diagram shows the path of the mice when searching for the hidden platform. The platform residence time and the number of times the target platform is crossed show the learning and memory ability of the mice.

[0084] In some embodiments: N≥35, and the drug to be identified is a complement C1q inhibitor.

[0085] In some embodiments, the complement C1q inhibitor is specifically an anti-C1q antibody.

[0086] In some embodiments:

[0087] Prior to step (a), there is also a step of obtaining specific differentially expressed genes in the chronic phase of cerebral hemorrhage, which specifically includes:

[0088] (1) Collect autopsy brain tissue from patients with cerebral hemorrhage and healthy controls;

[0089] (2) perform spatial transcriptome sequencing to identify specific differentially expressed genes;

[0090] (3) Compare the gene expression data of patients with cerebral hemorrhage with those of the healthy control group to identify differentially expressed genes.

[0091] In some embodiments:

[0092] Step (1) specifically comprises: collecting autopsy brain tissue from patients with cerebral hemorrhage and a healthy control group; collecting brain tissue within 4 hours after the death of the collected individuals to ensure the freshness of the tissue;

[0093] Step (2) is as follows: Use NanoString Spatial transcriptomic analysis was performed using a digital spatial profiler. Brain tissues were sliced ​​to 6 μm thickness and mounted on Leica BOND Plus slides. Subsequent steps were performed using DEPC-treated water under RNase-free conditions, including three consecutive washes of the slides in xylene for 5 min, followed by two washes in 100% ethanol for 5 min, one wash in 95% ethanol, and one wash in 1x PBS. Target retrieval was performed in the BioGenex ezretriverSystem with target retrieval reagent diluted to 1x at 95°C for 10 min. After washing in 1x PBS, the slides were incubated with RNA hybridization probes diluted in BufferR at 37°C overnight. The next day, the slides were stained with antibodies to Iba1, GFAP, NeuN, and Syto13 for 1 h at room temperature and then loaded into the GeoMx The DSP instrument scanned and selected the region of interest (ROI); DAPI immunofluorescence was used to identify morphological markers, and Iba1, NeuN and GFAP staining was used for segmentation, and the ROI selected for subsequent gene expression analysis was marked with a circle; each ROI was segmented into three illumination areas (AOI): microglia iba1-positive, neurons neuN-positive, and astrocytes gmap-positive, and the probe identity of each segment was captured by ultraviolet irradiation and moved to a 96-well plate; the DSP standard process was used to build the PCR system, construct the library, and use GeoMx NGSPipeline software to convert the original fastq file into a digital count file;

[0094] Step 3 specifically includes: segmenting the data and performing probe quality control to remove poor AOI / ROI and abnormal probes, and determining the detection limit of each segment, ignoring gene expression below LOQ; using quantile 3 normalization to normalize the data, and using edgeR for differential detection to compare the differences between the AOIs groups, that is, the ICH and control groups, and genes with a false discovery rate FDR < 0.05 and log2 (Fold Change) > 1 were considered differentially expressed genes;

[0095] The identified genes were analyzed using the Toppgene program, mainly for gene ontology annotation, and enrichment analysis of up-regulated and down-regulated genes was performed using the GO and KEGG methods; gene signature analysis was performed using the gene collection Hallmark in the Molecular Signatures Database, MSigDB, and statistical tests were used in the selected groups to test the hypothesis; the Wilcoxon rank sum test was used to compare two groups of independent AOI / ROI, and the Kruskal-Wallis method was used to test the rank sum of multiple groups of independent AOI / ROI to determine the specific differentially expressed genes in the chronic stage of cerebral hemorrhage.

[0096] In some embodiments:

[0097] After step (f), there is also a step of exploring the pathogenic mechanism in the animal model of cerebral hemorrhage, which specifically includes:

[0098] (i) Observe the pathological changes in the ICH animal model N days after ICH, including reactive microgliosis and neuropathological abnormalities, neuronal loss, neuronal degeneration, and decreased dendritic spine density;

[0099] (ii) using transmission electron microscopy to observe the separation of myelin sheets and / or vesicles, as well as the condensation of postsynaptic structures in animal models of intracerebral hemorrhage;

[0100] (iii) Treating animal models of cerebral hemorrhage with the drug to be identified and evaluating the therapeutic effects, including alleviating neuropathological abnormalities and improving chronic inflammation, neuropathology and degenerative changes in the chronic phase of brain injury.

[0101] The present invention further provides a pharmaceutical composition for treating delayed nerve damage in the chronic phase of cerebral hemorrhage obtained by the identification method, characterized in that:

[0102] The drugs include complement C1q inhibitors.

[0103] It also includes at least one drug carrier, the complement C1q inhibitor is an anti-C1q antibody, and the drug carrier is specifically a liposome or nanoparticle. The drug carrier can effectively deliver the complement C1q inhibitor to the treatment site.

[0104] The present invention further provides an application of the pharmaceutical composition in preparing a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage.

[0105] Embodiment 2:

[0106] The results of spatial transcriptome sequencing of autopsy brain tissues from patients with cerebral hemorrhage and healthy controls revealed differentially expressed genes specific to the chronic phase of cerebral hemorrhage.

[0107] Human brain slices were obtained from Beijing Tiantan Hospital affiliated to Capital Medical University. The Ethics Committee of Beijing Tiantan Hospital affiliated to Capital Medical University, China, approved the establishment of a human brain tissue bank for neurological diseases (KY 2018-031-02). Brain tissue was collected within 4 hours after death. Three patients and three controls were used in the GeoMx spatial transcriptome experiment. The three patients were patients with right basal ganglia hemorrhage who died within 6 to 7 years after ICH. Control cases were individuals with no history of neurological or neuropsychiatric diseases but died of non-neurological diseases. The selected thalamic tissue slices were matched with those of ICH patients. All included patients had no acute myocardial infarction, heart failure, autoimmune diseases, hematological diseases, or infections at the time of death. There was no significant difference in the age of death between stroke patients and controls (ICH: 63.4 ± 2.2 years; control: 59.8 ± 2.3 years; mean ± SEM; P > 0.05; unpaired t test).

[0108] Using NanoString Spatial transcriptomics was performed using a digital spatial analyzer. Sections were cut at 6 μm thickness and mounted on slides (LeicaBOND Plus slides). All subsequent steps were performed using RNase-free conditions and DEPC (diethylpyrocarbonate)-treated water. Slides were washed three times in succession in xylene for 5 min, followed by two washes in 100% ethanol for 5 min, one wash in 95% ethanol, and one wash in 1x PBS. Target retrieval was performed at 95°C for 10 min in a BioGenex ezretriverSystem with target retrieval reagent (10x Invitrogen 00-4956-58EDTA pH 9.0) diluted to 1x. After washing in 1x PBS, the samples were incubated with RNA hybridization probe (NanoString, GMX-RNA-NGS-HuWTA-4) diluted in Buffer R (provided by GeoMx RNA Slide Prep Formalin-fixed paraffin-embedded (FFPE)-PCLN Kit, GMX-PREP-RNA-FFPE-PCLN-12) at 37°C overnight. The next day, the samples were stained with antibodies Iba1 (CST, 48934), GFAP (Novus, NBP2-33184AF532), NeuN (Abcam, ab190565), and Syto13 (Nanostring, GMX-RNA-MORPH-HST-12) for 1 hour at room temperature and then loaded into the GeoMx DSP instrument for scanning and selection of regions of interest (ROI). Morphological markers were identified by DAPI (4′, 6-diamidino-2-phenylindole) immunofluorescence. Segmentation was performed using Iba1, NeuN, and GFAP staining. The ROIs selected for subsequent gene expression analysis are marked with circles. Each ROI was segmented into three areas of illumination (AOI): microglia Iba1-positive, neurons NeuN-positive, and astrocytes Gmap-positive. The probe identity of each segment was captured by UV illumination and moved to a 96-well plate. The DSP standard process was used to construct the PCR system and construct the library.

[0109] The raw fastq files were converted to digital count files using GeoMxNGS Pipeline software (v2.0). The data were segmented and probe quality control (QC) was performed to remove poor AOI / ROI and aberrant probes. The limit of detection (limit of quantification) was determined for each segment, and gene expression below LOQ was ignored. LOQ was set as the geometric number of negative probes multiplied by the geometric standard deviation of negative probes. After the quality control step, the data were normalized using quantile 3 normalization. Based on the data type of the DSP-WTA technology platform, edgeR was used for differential detection. We compared the differences between AOIs groups (ICH and control group). Genes with a false discovery rate (FDR) < 0.05 and log2 (Fold Change) > 1 were considered deg. The identified genes were analyzed using the Toppgene program, which was mainly used for gene ontology annotation. The up-regulated and down-regulated genes were enriched by GO and KEGG methods, respectively. Gene signature analysis was performed using the gene collection Hallmark in the Molecular Signatures Database (MSigDB). Statistical tests were used to test the hypotheses in the selected groups. The Wilcoxon rank sum test was used to compare two independent groups of AOI / ROI, and the Kruskal-Wallis method was used to test the rank sum of multiple independent groups of AOI / ROI.

[0110] Regional heterogeneity of transcriptome signatures of reactive microglia in the chronic phase of ICH: Differential gene expression (DGE) analysis between ICH and control groups revealed 633 upregulated genes. The most highly upregulated genes in Iba1+ microglia were associated with activated transcriptome signatures, including components of the classical complement pathway (C1QA, C1QB, C1QC, C3) and effector molecules involved in endophagic and lysosomal processes (CTSA, CTSB, CTSC, CTSD, CTSS, CTSZ, CYBA, CYBB, CLTA, CD63, PPT1, ABCA2, FUCA1, NPC2, LAPTM5, LGMN), antigen processing and presentation (RGS1, FCER1G, CD74, HLA-B, IFI30, B2M, HLA-A, HLA-DRB1, HLA-DPA1, HLA-DMA, HLA-DRA, HLA-DQB1, HLA-E). and neurodegenerative phenotypes (TREM2, APOE, APOC1, SPP1, ITGAX). KEGG pathway analysis showed that the differentially expressed genes (DEGs) in thalamic microglia of ICH patients were enriched in immune response pathways (classical complement cascade, inflammatory response, antigen processing and presentation) and cell death regulation pathways (phagosome, lysosome, apoptosis, autophagy, endocytosis and cellular senescence) compared with controls. In addition, GeneOntology (GO) database analysis showed that immune response functions related to glial cell activation, inflammatory response, cytokine production and immune effector function were enriched. Gene signatures and gene set variation analysis (GSVA) ​​scores showed that inflammation-related pathways (complement and coagulation cascades, and IL-6 / JAK / STAT3 signaling) were significantly upregulated. In situ immunostaining showed that complement factor C1q was mainly expressed by thalamic microglia 6 years after ICH.

[0111] See also Figure 1 , Figure 1 The results of spatial transcriptomic analysis of brain tissues from intracerebral hemorrhage (ICH) and healthy controls are presented.

[0112] The upper left image shows the source of samples from brain hemorrhage and healthy controls. The image shows a side view of the human brain, with the location of the brain hemorrhage (Lesion), thalamus (Thalamus), neurons and astrocytes marked.

[0113] Heat map in the lower left corner: Comparison of gene expression differences between patients with intracerebral hemorrhage (ICH) and healthy controls. Each column represents a sample, and each row represents a gene. The depth of the color indicates the level of gene expression, red indicates upregulation (high expression), and blue indicates downregulation (low expression).

[0114] The volcano chart in the upper right corner shows the significance of changes in gene expression. The horizontal axis is the log2 Fold Change of gene expression, and the vertical axis is the negative log 10 p value (-log10 Pvalue), indicating statistical significance. Red dots indicate genes that are significantly upregulated in cerebral hemorrhage, blue dots indicate genes that are significantly downregulated, and gray dots indicate genes that have no significant changes.

[0115] The figure in the lower right corner: The figure above is an enrichment analysis graph, which shows the enrichment of genes significantly upregulated in ICH in different biological pathways. Different colors represent different pathways, such as antigen processing and presentation, complement and coagulation cascades, etc. The figure below is a gene set variation analysis (GSVA) ​​graph, which shows the enrichment scores of different gene sets in ICH. Each point represents a gene set, the horizontal axis is the ranking of the gene set in the sorted data set, and the vertical axis is the enrichment score.

[0116] Embodiment 3:

[0117] Application of complement C1q inhibitors in the treatment of delayed neurological damage in the chronic phase of cerebral hemorrhage.

[0118] To verify whether targeting complement C1q can improve the impairment of neurological and cognitive functions in the chronic stage of ICH, we used an ICH mouse model and assessed neurological function 35 days after induction of the model. Corner turning test showed residual motor dysfunction. Treatment with anti-C1q antibody significantly promoted neurological recovery and reduced residual motor dysfunction. To determine whether C1q was associated with synaptic transmission and cognitive dysfunction 35 days after ICH, we examined spatial learning using Morris water maze and assessed the excitability of thalamic neurons using whole-cell patch clamp recording. Basic synaptic transmission of thalamic neurons was determined by measuring the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs). Compared with the control group, the sEPSC frequency was reduced 35 days after ICH. Anti-C1q antibody treatment significantly increased the sEPSC frequency and changed the cumulative probability distribution curve (sham vs. ICH: P = 0.0368; ICH vs. ICH + anti-C1q: P = 0.0310). In addition, the sEPSC amplitude of thalamic neurons increased after anti-C1q antibody treatment (sham vs. ICH: P = 0.0262; ICH vs. ICH + anti-C1q: P = 0.0054). These data suggest that thalamic neurons treated with C1q antibodies contain more synaptic inputs or greater neurotransmitter release, which contributes to the recovery of functional connectivity and attention in the chronic stage of ICH. Using the Morris water maze, we found that learning and memory were impaired in ICH mice compared with controls 35 days after ICH. However, anti-C1q antibody treatment improved the impairment of spatial learning and memory. Our data identify the classical complement pathway as a therapeutic target to alleviate long-term cognitive deficits associated with the chronic stage of ICH.

[0119] See also Figure 2 , Figure 2 The neurological function recovery of intracerebral hemorrhage (ICH) model animals after receiving different treatments (control group Sham, ICH plus IgG, ICH plus Anti-C1q) was compared.

[0120] A: The bar graph shows the results of the rotarod test, which is used to evaluate motor coordination and balance, for different groups over 35 days. The ICH group showed a significant decline in performance, while the Anti-C1q treatment group showed a smaller decline.

[0121] B: The bar graph shows the modified neurological deficit score (mNSS scores), which is used to assess the degree of neurological deficit. The scores in the ICH group were higher, indicating more severe neurological deficit, while the scores in the Anti-C1q treatment group were lower.

[0122] C: shows the recording of spontaneous excitatory postsynaptic currents (sEPSCs), which is used to assess the excitability of neurons. The waveform and frequency of sEPSCs were different in different groups.

[0123] D: The bar graph and cumulative probability plot show the frequency of sEPSCs. The frequency of sEPSCs decreased in the ICH group, while it was restored in the Anti-C1q treated group.

[0124] E: The bar graph and cumulative probability plot show the amplitude of sEPSCs. The amplitude of sEPSCs was reduced in the ICH group, while it was restored in the Anti-C1q treated group.

[0125] F: The bar graph shows the movement speed and distance of different groups within 5 days. The movement speed and distance of the ICH group were significantly reduced, while the Anti-C1q treatment group improved.

[0126] G: Shows the results of the Morris water maze test, which is used to assess spatial learning and memory. The path-tracing graphs of the different groups show the paths they took while searching for the hidden platform.

[0127] H: The bar graph shows the duration in target quadrant and the number of crossings of target platform in the Morris water maze test. The Anti-C1q treatment group performed better in these parameters, indicating that their learning and memory abilities were restored.

[0128] Embodiment 4:

[0129] The results of spatial transcriptome sequencing were used to explore the pathogenic mechanism of complement C1q in an animal model of intracerebral hemorrhage. To further determine the mechanism by which anti-C1q antibodies play a therapeutic role in neurological dysfunction in the chronic phase of intracerebral hemorrhage, we observed reactive microgliosis (CD68+Iba1+) and neuropathological abnormalities, such as neuronal loss, neuronal degeneration, and reduced dendritic spine density, 35 days after intracerebral hemorrhage. Corresponding transmission electron microscopy showed separation of myelin sheets and / or vesicles, and less condensation of postsynaptic structures. Treatment with C1q function-blocking antibodies partially alleviated these injuries. These results confirm that targeting C1q works by improving chronic inflammation, neuropathology, and degenerative changes in the chronic phase of brain injury.

[0130] See also Figure 3 , showing the histological and cytological changes in intracerebral hemorrhage (ICH) model animals after receiving different treatments (Sham, ICH+IgG, ICH+Anti-C1q). Among them:

[0131] A: Immunofluorescence staining of whole brain sections and cortical regions for CD68 (macrophage / microglia marker) and CD86 (activated microglia marker) is shown. The bar graph below shows the number of CD68 and CD86 double positive cells in different treatment groups.

[0132] B: Immunofluorescence staining of F4 / 80 (another macrophage / microglia marker) and Iba1 (microglia marker) in brain sections is shown. The bar graph below shows the number of F4 / 80 and Iba1 positive cells in different treatment groups.

[0133] C: Histological staining of myelin (Luxol Fast Blue / PAS staining) and immunofluorescence staining of axons (MBP labeling) are shown. The bar graph below shows the number of myelinated axons and unmyelinated axons.

[0134] D: shows immunofluorescence staining of synapses (Synapsin and PSD-95 markers). The lower bar graph shows the thickness of the postsynaptic density.

[0135] E: Immunofluorescence staining of neurons (NeuN marker) and Golgi staining of dendritic spines in brain sections are shown. The bar graph below shows the number of dendritic spines per 10 microns.

[0136] Together, these figures demonstrate the potential protective effects of Anti-C1q therapy on brain tissue structure and cellular changes after ICH, including reducing microglial activation, protecting myelin integrity, maintaining synaptic structure, and promoting the preservation of neuronal dendritic spines. These results further support the potential application of Anti-C1q therapy in neuroprotection and functional recovery after ICH.

[0137] A method for treating neurological function damage in the chronic stage of cerebral hemorrhage is derived from this, which is to analyze the spatial transcriptome sequencing results in the brain tissue of patients with cerebral hemorrhage, and then target complement C1q to slow down the long-term neurological and cognitive damage of cerebral hemorrhage; the spatial transcriptome sequencing results include:

[0138] Classical complement pathway components (C1QA, C1QB, C1QC, C3) and effector molecules involved in endophagy and lysosomal processes (CTSA, CTSB, CTSC, CTSD, CTSS, CTSZ, CYBA, CYBB, CLTA, CD63, PPT1, ABCA2, FUCA1, NPC2, LAPTM5, LGMN), antigen processing and presentation (RGS1, FCER1G, CD74, HLA-B, IFI30, B2M, HLA-A, HLA-DRB1, HLA-DPA1, HLA-DMA, HLA-DRA, HLA-DQB1, HLA-E), and neurodegenerative phenotypes (TREM2, APOE, APOC1, SPP1, ITGAX).

[0139] A method for treating neurological function damage in the chronic stage of cerebral hemorrhage, wherein the method targets complement C1q and can slow down the long-term neurological and cognitive function damage in cerebral hemorrhage, and its mechanism is to reduce the pathological damage of chronic neuroinflammation and neurodegeneration, such as neuronal loss, neuronal degeneration and synaptic dysfunction.

[0140] Thus far, the description of the above-described embodiments is provided for the purpose of illustration and description. It is not intended to be exhaustive or to limit the present disclosure. The individual elements or features of a specific embodiment are generally not subject to the limitations of the specific embodiment, but when applicable, they can be interchanged and used for selected embodiments even if not specifically shown or described. In many respects, the same element or feature also can be changed. This variation is not considered to be a departure from the present disclosure, and all such modifications are intended to be included in the scope of the present disclosure.

[0141] Example embodiments are provided so that the present disclosure will be thorough and the scope will be fully conveyed to those skilled in the art. In order to thoroughly understand the embodiments of the present disclosure, numerous details are set forth, such as examples of specific parts, devices, and methods. Obviously, for those skilled in the art, it is not necessary to use specific details, the example embodiments can be implemented in many different forms, and neither should be construed as limiting the scope of the present disclosure. In certain example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0142] Here, specialized vocabulary is used only for the purpose of describing specific example embodiments, and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms "a" and "the" used herein may be intended to include plural forms as well. The terms "including" and "having" are inclusive, and therefore specify the presence of the claimed features, wholes, steps, operations, elements and / or components, but do not exclude the presence or additional presence of one or more other features, wholes, steps, operations, elements, components and / or combinations thereof. Unless the order of execution is clearly indicated, the method steps, processing and operations described herein are not interpreted as necessarily needing to be performed in the specific order discussed and shown. It should also be understood that additional or optional steps may be adopted.

Claims

1. A method for identifying a drug for treating delayed neurological damage in the chronic phase of cerebral hemorrhage, characterized in that The steps include: (a) A mouse model of cerebral hemorrhage was established and the mice were divided into a treatment group and a non-treatment group. The treatment group was treated with the drug to be identified, and the non-treatment group was treated with immunoglobulin IgG. Neurological function of the mice was evaluated N days after the model was induced, and residual motor dysfunction was measured by corner turning test. (b) Whole-cell patch clamp recording was used to evaluate the excitability of thalamic neurons in mice N days after ICH. The amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) were measured to determine the basic synaptic transmission properties of thalamic neurons. The frequency and amplitude of sEPSCs in mice N days after ICH were compared with those in the control group to evaluate the effect of ICH on synaptic transmission. (c) measuring the frequency and amplitude of sEPSCs in the treated mice and comparing the results with those in the untreated mice to evaluate the improvement effect of the drug treatment on synaptic transmission function; (d) The spatial learning and memory abilities of mice in the treatment and non-treatment groups were evaluated by Morris water maze test N days after ICH and compared with those in the control group without ICH to determine the presence of cognitive dysfunction; (e) Analyze the effects of the drug treatment to be identified in the treatment group on the spatial learning and memory ability of mice N days after ICH, and compare the Morris water maze test results with those of the non-treatment group to evaluate the improvement effect of the treatment on cognitive dysfunction; (f) Based on the results of the above steps, determine whether the treatment group improves neurological function, synaptic transmission and cognitive function, alleviates long-term neurological damage and cognitive deficits associated with the chronic stage of ICH, and determines whether the drug can be used as a potential drug for the treatment of delayed neurological damage in the chronic stage of ICH by determining whether the treatment group improves neurological function, synaptic transmission and cognitive function, and alleviates long-term neurological damage and cognitive deficits associated with the chronic stage of ICH.

2. The method for identifying a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage according to claim 1, characterized in that: In step (a), the degree of neurological deficit was assessed by the modified neurological deficit score (mNSS scores), and motor coordination and balance ability were assessed by the rotarod test; Step (b) and step (c) evaluate the excitability of the neuron by recording spontaneous excitatory postsynaptic currents sEPSCs, and evaluate the effect on synaptic transmission by a histogram and a cumulative probability graph of the frequency of sEPSCs and the amplitude of sEPSCs; Step (d) and step (e) evaluate motor function through the movement speed and distance of different groups within N days. The path tracking diagram shows the path of the mice when searching for the hidden platform. The platform residence time and the number of times the target platform is crossed show the learning and memory ability of the mice.

3. The method for identifying a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage according to claim 1 or 2, characterized in that: The N is ≥ 35, and the drug to be identified includes a complement C1q inhibitor.

4. The method for identifying a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage according to claim 3, characterized in that: The complement C1q inhibitor is specifically an anti-C1q antibody.

5. The method for identifying a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage according to claim 1, characterized in that: Prior to step (a), there is also a step of obtaining specific differentially expressed genes in the chronic phase of cerebral hemorrhage, which specifically includes: (1) Collect autopsy brain tissue from patients with cerebral hemorrhage and healthy controls; (2) perform spatial transcriptome sequencing to identify specific differentially expressed genes; (3) Compare the gene expression data of patients with cerebral hemorrhage with those of the healthy control group to identify differentially expressed genes.

6. The method for identifying a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage according to claim 5, characterized in that: Step (1) specifically comprises: collecting autopsy brain tissue from patients with cerebral hemorrhage and a healthy control group; collecting brain tissue within 4 hours after the death of the collected individuals to ensure the freshness of the tissue; Step (2) is as follows: Use NanoString Spatial transcriptomic analysis was performed using a digital spatial profiler. Brain tissues were sliced ​​to 6 μm thickness and mounted on Leica BOND Plus slides. Subsequent steps were performed using DEPC-treated water under RNase-free conditions, including three consecutive washes of the slides in xylene for 5 min, followed by two washes in 100% ethanol for 5 min, one wash in 95% ethanol, and one wash in 1x PBS. Target retrieval was performed in the BioGenex ezretriverSystem with target retrieval reagent diluted to 1x at 95°C for 10 min. After washing in 1x PBS, the slides were incubated with RNA hybridization probes diluted in BufferR at 37°C overnight. The next day, the slides were stained with antibodies to Iba1, GFAP, NeuN, and Syto13 for 1 h at room temperature and then loaded into the GeoMx The DSP instrument scanned and selected the region of interest (ROI); DAPI immunofluorescence was used to identify morphological markers, and Iba1, NeuN and GFAP staining was used for segmentation, and the ROI selected for subsequent gene expression analysis was marked with a circle; each ROI was segmented into three illumination areas (AOI): microglia iba1-positive, neurons neuN-positive, and astrocytes gmap-positive, and the probe identity of each segment was captured by ultraviolet irradiation and moved to a 96-well plate; the DSP standard process was used to build the PCR system, construct the library, and use GeoMx NGSPipeline software to convert the original fastq file into a digital count file; Step 3 specifically includes: segmenting the data and performing probe quality control to remove poor AOI / ROI and abnormal probes, and determining the detection limit of each segment, ignoring gene expression below LOQ; using quantile 3 normalization to normalize the data, and using edgeR for differential detection to compare the differences between the AOIs groups, that is, the ICH and control groups, and genes with a false discovery rate FDR < 0.05 and log2 (Fold Change) > 1 were considered differentially expressed genes; The identified genes were analyzed using the Toppgene program for gene ontology annotation, and enrichment analysis of up-regulated and down-regulated genes was performed using GO and KEGG methods; gene signature analysis was performed using Hallmark, a gene collection in the Molecular Signatures Database, MSigDB, and statistical tests were used in the selected groups to test the hypothesis; The Wilcoxon rank sum test was used to compare two groups of independent AOI / ROI, and the Kruskal-Wallis method was used to test the rank sum of multiple groups of independent AOI / ROI to determine the specific differentially expressed genes in the chronic stage of ICH.

7. The method for identifying a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage according to claim 1, characterized in that: After step (f), there is also a step of exploring the pathogenic mechanism in the animal model of cerebral hemorrhage, which specifically includes: (i) Observe the pathological changes in the ICH animal model N days after ICH, including reactive microgliosis and neuropathological abnormalities, neuronal loss, neuronal degeneration, and decreased dendritic spine density; (ii) using transmission electron microscopy to observe the separation of myelin sheets and / or vesicles, as well as the condensation of postsynaptic structures in animal models of intracerebral hemorrhage; (iii) Treating animal models of cerebral hemorrhage with the drug to be identified and evaluating the therapeutic effects, including alleviating neuropathological abnormalities and improving chronic inflammation, neuropathology and degenerative changes in the chronic phase of brain injury.

8. A pharmaceutical composition for treating delayed nerve damage in chronic cerebral hemorrhage obtained by the identification method according to any one of claims 1 to 7, characterized in that: The drugs include complement C1q inhibitors.

9. The pharmaceutical composition according to claim 8, characterized in that: It also includes at least one drug carrier, the complement C1q inhibitor is an anti-C1q antibody, and the drug carrier is specifically a liposome or nanoparticle. The drug carrier can effectively deliver the complement C1q inhibitor to the treatment site.

10. Use of the pharmaceutical composition according to claim 8 or 9 in the preparation of a drug for treating delayed nerve damage in the chronic phase of cerebral hemorrhage.

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