A compound for treating delayed neurological injury in the chronic phase of cerebral hemorrhage and its identification method.

Abstract

This invention provides a method for identifying compounds for treating delayed neurological injury in the chronic phase of cerebral hemorrhage. The method includes establishing a mouse model of cerebral hemorrhage, administering the identified drug to a treatment group and immunoglobulin IgG to a non-treatment group; assessing motor dysfunction through neurological function evaluation and a turning angle test; evaluating the excitability of thalamic neurons using whole-cell patch-clamp recording technology; comparing synaptic transmission function in the treatment and non-treatment groups; assessing spatial learning and memory abilities using the Morris water maze test; and comprehensively evaluating the improvement in neurological function, synaptic transmission, and cognitive function to determine the therapeutic effect of the drug. The method also includes collecting autopsy brain tissue from cerebral hemorrhage patients and healthy controls, performing spatial transcriptome sequencing to identify differentially expressed genes, and exploring the pathogenic mechanism in the animal model of cerebral hemorrhage. This method can accurately screen and identify potential therapeutic drugs for chronic neurological injury in cerebral hemorrhage.

Description

Technical Field

[0001] This invention relates to drug identification methods, specifically to drugs and identification methods for treating chronic delayed neurological damage in the cerebral hemorrhage phase. Background Technology

[0002] In current medical research, there is a lack of comprehensive drug identification methods for the treatment of delayed neurological damage in the chronic phase of intracerebral hemorrhage (ICH). ICH is a serious neurological disease characterized by the rupture of blood vessels in the brain, leading to the accumulation of blood in the brain parenchyma. ICH results in both primary and secondary brain injury. Primary brain injury refers to the increase in overall intracranial pressure and mechanical compression of local structures caused by the initial or continued bleeding and hematoma expansion. Secondary brain injury is mainly caused by the pathophysiological response of the hematoma, including edema, inflammation, and toxic components produced by the clot. Currently, conventional medical and surgical treatments for ICH are insufficient for long-term patient outcomes. Therefore, it is crucial to actively explore the pathological mechanisms of ICH, identify potential molecular therapeutic targets, reduce the inflammatory cascade and the degree of brain cell edema after ICH, and ultimately improve the patient's prognosis and quality of life.

[0003] Currently, treatment strategies for ICH mainly include blood pressure control, hemostasis, and surgical intervention. However, existing treatments primarily focus on acute-phase management, with limited effectiveness in addressing chronic neurological dysfunction and delayed neurological damage. In addition to severe early neurological damage, survivors of cerebral hemorrhage face various irreversible delayed neurological sequelae in the chronic phase, such as dementia, depression, and fatigue, severely impacting their quality of life. Furthermore, because the pathological mechanisms of ICH are not fully understood, the lack of targeted treatment options leads to poor patient prognosis, significant long-term neurological damage, and cognitive deficits. Currently, there are no treatments available for delayed sequelae in the chronic phase of cerebral hemorrhage.

[0004] In the chronic phase, microglial activation is closely associated with chronic inflammation, neuropathology, and degenerative changes characteristic of chronic brain injury. The complement system, particularly complement C1q, plays a crucial role in the inflammatory response and neurological damage following ICH. Studies have shown that C1q expression is chronically increased in the cortex surrounding the injury site and in the cortical hypothalamus, corresponding to areas of neuronal loss and chronic inflammation. Therefore, treatment targeting complement C1q may have potential therapeutic benefits in improving neurological dysfunction during the chronic phase of ICH.

[0005] Increasing clinical and animal evidence supports the ability of an immune response to be triggered even at sites distant from the lesion after stroke, leading to widespread neuroinflammation throughout the brain. This neuroinflammation begins in the acute phase and intensifies and persists in the chronic phase. It is generally believed that the primary hemorrhage site initiates a local inflammatory response, which then spreads to extensive areas of the brain, potentially exacerbating distal neurological damage and long-term functional deficits. This phenomenon reveals the mechanism by which secondary lesions from acute brain injury persistently affect brain structure and function in the chronic phase. Therefore, studying the characteristics of neuroinflammation in the chronic phase of stroke is crucial for developing treatments for chronic brain injury.

[0006] Spatial transcriptomics provides a map of gene expression patterns mapped onto tissue slices, linking structure and activity. This capability allows researchers to elucidate biological interactions at the cellular level, leading to new insights into complex tissues. This technology has been used to generate tissue atlases, providing a valuable resource for reference. Currently, in research on intracerebral hemorrhage, this technology is mostly used to study acute lesions and surrounding tissues, but to date, gene expression patterns in distant lesion regions or even the entire brain during the chronic phase of intracerebral hemorrhage have not been studied. The molecular profile of neurons damaged in distant sites may exhibit completely different characteristics compared to those in localized lesions; even if some aspects are similar, they may present entirely different functions and phenotypes at different times. The specific molecular mechanisms require further exploration.

[0007] Currently, there is a lack of effective drug identification methods for delayed neurological injury in the chronic phase of cerebral hemorrhage, and the crucial role of the complement system in the inflammatory response after cerebral hemorrhage has not been adequately therapeutically modulated. These challenges have prompted researchers to develop new drug identification methods in order to find therapeutic agents that can improve the prognosis of patients with cerebral hemorrhage. To address these issues, this invention utilizes brain tissue samples from the chronic phase of cerebral hemorrhage, combined with spatial multi-omics research, to deeply explore the impact and molecular mechanisms of neuroinflammation on the long-term prognosis of cerebral hemorrhage. This will help us further understand the role of delayed brain injury after stroke and the feasibility of drug intervention. Furthermore, through preclinical intervention and in situ immunoprofiling analysis, targets for neurological repair during the rehabilitation phase of cerebral hemorrhage are identified. Summary of the Invention

[0008] To address the aforementioned issues, this invention, for the first time, performed spatial transcriptome sequencing on brain tissue during the chronic phase of cerebral hemorrhage. The study revealed persistent neuroinflammatory activation in the brain tissue surrounding the residual lesions after cerebral hemorrhage, exhibiting characteristics of reactive microglia transcriptomes. Through differential gene enrichment pathway analysis, complement C1q was identified as a key target for treating delayed neurological dysfunction in the chronic phase of cerebral hemorrhage. This finding was further validated in animal models.

[0009] Based on the above research findings, this invention provides a method for identifying drugs used to treat chronic delayed neurological injury in cerebral hemorrhage, comprising the following steps:

[0010] (a) A mouse model of cerebral hemorrhage was established and 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. N days after model induction, the mice were assessed for neurological function and residual motor dysfunction was determined by the cornering test.

[0011] (b) The excitability of thalamic neurons in mice N days after cerebral hemorrhage was assessed using whole-cell patch-clamp recording technology. The basic synaptic transmission characteristics of thalamic neurons were determined by measuring the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs). The frequency and amplitude of sEPSCs in mice N days after cerebral hemorrhage were compared with those in control mice to assess the effect of cerebral hemorrhage on synaptic transmission.

[0012] (c) The frequency and amplitude of sEPSCs were measured in the mice in the treatment group and the results were compared with those in the non-treatment group to evaluate the effect of the drug to be identified on the improvement of synaptic transmission function.

[0013] (d) Spatial learning and memory abilities of mice in the treatment and non-treatment groups were assessed by the Morris water maze test N days after cerebral hemorrhage and compared with control mice without cerebral hemorrhage to determine the presence of cognitive impairment.

[0014] (e) The effects of the drug treatment to be identified in the treatment group on the spatial learning and memory abilities of mice N days after cerebral hemorrhage were analyzed, and the Morris water maze test results were compared with those in the non-treatment group to assess the effect of treatment on improving cognitive impairment.

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

[0016] In step (a), the degree of neurological deficit is assessed by modified neurological deficit scores (mNSS scores), and motor coordination and balance are assessed by the rotarod test.

[0017] Steps (b) and (c) assess neuronal excitability by recording spontaneous excitatory postsynaptic currents (sEPSCs) and evaluate the impact on synaptic transmission by histograms and cumulative probability plots of sEPSC frequency and amplitude.

[0018] Steps (d) and (e) assessed motor function by measuring movement speed and distance over N days in different groups. Path tracking maps showed the paths mice took when searching for hidden platforms, and the time spent on the platforms and the number of times the mice crossed the target platforms demonstrated their learning and memory abilities.

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

[0020] In some implementations: complement C1q inhibitors are specifically anti-C1q antibodies.

[0021] In some implementation methods:

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

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

[0024] (2) Perform spatial transcriptome sequencing to identify specifically differentially expressed genes;

[0025] (3) Compare gene expression data between patients with cerebral hemorrhage and healthy controls to identify differentially expressed genes.

[0026] In some implementation methods:

[0027] Step (1) specifically involves: collecting autopsy brain tissue from patients with cerebral hemorrhage and healthy control groups; brain tissue collection should be performed within 4 hours after the death of the individual to ensure tissue freshness;

[0028] Step (2) specifically involves using NanoString. Spatial transcriptomics analysis was performed using a digital spatial analyzer. Brain tissue sections were sliced ​​to a thickness of 6 μm and mounted on Leica BOND Plus slides. Subsequent steps were performed using DEPC-treated water under RNase-free conditions, including three consecutive 5-minute washes in xylene, followed by two 5-minute washes in 100% ethanol, one wash in 95% ethanol, and one wash in 1x PBS. Target searching was performed at 95°C for 10 minutes using a BioGenex ezretriver System diluted 1x with a target search reagent. After washing in 1x PBS, the tissue was diluted with RNA hybridization probes in Buffer R and incubated overnight at 37°C. The next day, the tissue was stained with antibodies Iba1, GFAP, NeuN, and Syto13 at room temperature for 1 hour, and then loaded onto GeoMx. Regions of interest (ROIs) were scanned and selected in the DSP instrument. Morphological markers were identified using DAPI immunofluorescence assay, and the ROIs were segmented using Iba1, NeuN, and GFAP staining. ROIs selected for subsequent gene expression analysis were marked with circles. Each ROI was segmented into three illuminated areas (AOIs): microglia iba1-positive, neurons neuN-positive, and astrocyte gmap-positive. The probe identity of each segment was captured by ultraviolet irradiation and transferred to a 96-well plate. A PCR system was constructed using the standard DSP procedure, a library was built, and the original fastq file was converted into a digital count file using GeoMx NGSPipeline software.

[0029] Step 3 specifically involves: segmenting the data and performing probe quality control to remove defective AOIs / ROIs and abnormal probes, determining the detection limit for each fragment, and ignoring gene expression below the LOQ; normalizing the data using quantile 3 normalization, and performing differential detection using edgeR to compare the differences between the AOIs group (i.e., ICH and the control group); genes with a false detection rate (FDR) < 0.05 and log2(Fold Change) > 1 are considered differentially expressed genes (deg).

[0030] The Toppgene program was used to analyze the identified genes, primarily for gene ontology annotation. GO and KEGG methods were used for enrichment analysis of upregulated and downregulated genes. Gene feature analysis was performed using Hallmarks from the Molecular Signatures Database (MSigDB), and statistical tests were used to test hypotheses in selected groups. The Wilcoxon rank-sum test was used to compare the independent AOI / ROIs between two groups, and the Kruskal-Wallis method was used to test the rank sums of multiple independent AOI / ROIs to identify differentially expressed genes specific to the chronic phase of cerebral hemorrhage.

[0031] In some implementation methods:

[0032] Following step (f) are steps to explore the pathogenic mechanisms in animal models of cerebral hemorrhage, specifically including:

[0033] (i) Observe pathological changes in an animal model of cerebral hemorrhage N days after the hemorrhage, including reactive microglial proliferation and neuropathological abnormalities, neuronal loss, neuronal degeneration and decreased dendritic spine density.

[0034] (ii) Use transmission electron microscopy to observe the separation of myelin sheaths and / or vesicles, as well as the aggregation of postsynaptic structures in animal models of cerebral hemorrhage;

[0035] (iii) Treat animal models of cerebral hemorrhage with the drug to be identified and evaluate the treatment effect, including reducing neuropathological abnormalities and improving chronic inflammation, neuropathological and degenerative changes in the chronic phase of brain injury.

[0036] The present invention further proposes a pharmaceutical composition for treating chronic delayed neurological injury in the cerebral hemorrhage phase, obtained using an identification method, characterized in that:

[0037] The drug includes a complement C1q inhibitor.

[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, which can effectively deliver the complement C1q inhibitor to the treatment site.

[0039] The present invention further proposes the use of the pharmaceutical composition described herein in the preparation of a medicament for treating chronic delayed neurological injury in cerebral hemorrhage.

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

[0041] (a) A mouse model establishment module for cerebral hemorrhage, used to divide mice into treatment and non-treatment groups, administering the identified drug to the treatment group and immunoglobulin IgG to the non-treatment group; (b) A neurological function assessment module, used to assess the neurological function of mice N days after model induction and to determine residual motor dysfunction through a cornering test; (c) A neuronal excitability assessment module, using whole-cell patch-clamp recording technology to assess the excitability of thalamic neurons in mice N days after cerebral hemorrhage, and determining the basic synaptic transmission characteristics of thalamic neurons by measuring the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs); (d) A synaptic transmission function improvement assessment module, used to measure the frequency of sEPSCs in the treatment group mice. (e) A cognitive function assessment module, which assesses the spatial learning and memory abilities of mice in the treatment and non-treatment groups N days after cerebral hemorrhage using the Morris water maze test, and compares them with those of mice in the control group without cerebral hemorrhage, to determine the presence of cognitive impairment; and (f) A comprehensive treatment effect assessment module, which integrates the results of the above modules to determine whether the treatment group improves neurological function, synaptic transmission and cognitive function, reduces long-term neurological damage and cognitive deficits associated with the chronic phase of cerebral hemorrhage, and determines whether the drug can be used as a potential treatment for delayed neurological damage in the chronic phase of cerebral hemorrhage.

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

[0043] Modules (b) and (c) assess neuronal excitability by recording spontaneous excitatory postsynaptic currents (sEPSCs) and evaluate the impact on synaptic transmission by histograms and cumulative probability plots of sEPSC frequency and amplitude.

[0044] Modules (d) and (e) assessed motor function by measuring movement speed and distance over N days in different groups. Path tracking maps showed the paths mice took when searching for hidden platforms, and the time spent on the platform and the number of times the target platform was crossed indicated the mice's learning and memory abilities.

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

[0046] In some implementations: complement C1q inhibitors are specifically anti-C1q antibodies.

[0047] In some implementation methods:

[0048] The module also includes a section for identifying differentially expressed genes specific to the chronic phase of cerebral hemorrhage. This module is used for:

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

[0050] (2) Perform spatial transcriptome sequencing to identify specifically differentially expressed genes;

[0051] (3) Compare gene expression data between patients with cerebral hemorrhage and healthy controls to identify differentially expressed genes.

[0052] In some implementation methods:

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

[0054] (2) Specifically: using NanoString Spatial transcriptomics analysis was performed using a digital spatial analyzer. Brain tissue sections were sliced ​​to a thickness of 6 μm and mounted on Leica BOND Plus slides. Subsequent steps were performed using DEPC-treated water under RNase-free conditions, including three consecutive 5-minute washes in xylene, followed by two 5-minute washes in 100% ethanol, one wash in 95% ethanol, and one wash in 1x PBS. Target searching was performed at 95°C for 10 minutes using a target search reagent diluted 1x in the BioGenex ezretriver System. After washing in 1x PBS, the tissue was diluted with RNA hybridization probes in Buffer R and incubated overnight at 37°C. The next day, the tissue was stained with antibodies Iba1, GFAP, NeuN, and Syto13 at room temperature for 1 hour, and then loaded onto GeoMx. Regions of interest (ROIs) were scanned and selected in the DSP instrument. Morphological markers were identified using DAPI immunofluorescence assay, and the ROIs were segmented using Iba1, NeuN, and GFAP staining. ROIs selected for subsequent gene expression analysis were marked with circles. Each ROI was segmented into three illuminated areas (AOIs): microglia iba1-positive, neurons neuN-positive, and astrocyte gmap-positive. The probe identity of each segment was captured by ultraviolet irradiation and transferred to a 96-well plate. A PCR system was constructed using the standard DSP procedure, a library was built, and the original fastq file was converted into a digital count file using GeoMx NGSPipeline software.

[0055] (3) Specifically, the data was segmented and probe quality was controlled to remove bad AOI / ROI and abnormal probes, and the detection limit of each fragment was determined, ignoring gene expression below LOQ; the data were normalized using quantile 3 normalization, and edgeR was used for differential detection to compare the differences between the AOIs group, i.e., ICH and the control group. Genes with false detection rate FDR < 0.05 and log2 (Fold Change) > 1 were considered differentially expressed genes deg;

[0056] The Toppgene program was used to analyze the identified genes, primarily for gene ontology annotation. GO and KEGG methods were used for enrichment analysis of upregulated and downregulated genes. Gene feature analysis was performed using Hallmarks from the Molecular Signatures Database (MSigDB), and statistical tests were used to test hypotheses in selected groups. The Wilcoxon rank-sum test was used to compare the independent AOI / ROIs between two groups, and the Kruskal-Wallis method was used to test the rank sums of multiple independent AOI / ROIs to identify differentially expressed genes specific to the chronic phase of cerebral hemorrhage.

[0057] In some implementation methods:

[0058] The module also includes sections for exploring the pathogenic mechanisms in animal models of cerebral hemorrhage, specifically:

[0059] (i) Observe pathological changes in an animal model of cerebral hemorrhage N days after the hemorrhage, including reactive microglial proliferation and neuropathological abnormalities, neuronal loss, neuronal degeneration and decreased dendritic spine density.

[0060] (ii) Use transmission electron microscopy to observe the separation of myelin sheaths and / or vesicles, as well as the aggregation of postsynaptic structures in animal models of cerebral hemorrhage;

[0061] (iii) Treat animal models of cerebral hemorrhage with the drug to be identified and evaluate the treatment effect, including reducing neuropathological abnormalities and improving chronic inflammation, neuropathological and degenerative changes in the chronic phase of brain injury.

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

[0063] The beneficial effects of this invention are as follows:

[0064] This invention comprehensively utilizes various scientifically validated techniques, including modified neurological deficit scores (mNSS scores), the rotarod test, and spontaneous excitatory postsynaptic currents (sEPSCs). The combined use of these methods provides a multi-dimensional and comprehensive assessment system, ensuring the comprehensiveness and accuracy of the assessment results.

[0065] The complement C1q inhibitors, particularly anti-C1q antibodies, proposed in this invention are based on a current in-depth understanding of the pathological mechanisms of cerebral hemorrhage. Activation of the complement system plays a crucial role in the inflammatory response following cerebral hemorrhage; therefore, drug identification methods targeting this specific target have a clear scientific basis.

[0066] This invention is the first to perform spatial transcriptome sequencing on brain tissue in the chronic phase of intracerebral hemorrhage, revealing persistent neuroinflammatory activation in the brain tissue surrounding the residual lesions after hemorrhage, exhibiting reactive microglial transcriptome characteristics. Differential gene enrichment pathway analysis identified complement C1q as a key target for treating delayed neurological dysfunction in the chronic phase of intracerebral hemorrhage, and this finding was further validated in animal models. This invention is currently the only one to utilize human brain tissue and animal models to identify a target for treating delayed neurological dysfunction caused by intracerebral hemorrhage.

[0067] This invention provides a novel treatment strategy by systematically identifying and applying the role of complement C1q inhibitors in treating delayed neurological injury in the chronic phase of intracerebral hemorrhage (ICH). By establishing a mouse model of ICH, this invention comprehensively evaluates the effects of the identified drugs on neurological function, synaptic transmission, and cognitive function using a multi-dimensional research approach, including neurological function assessment, whole-cell patch-clamp recording, the Morris water maze test, and spatial transcriptome sequencing. This not only allows for precise identification and quantification of neuronal damage and recovery processes after ICH but also enables in-depth exploration of the mechanism of complement C1q in the pathological process of ICH.

[0068] This invention further validates the therapeutic effect of complement C1q inhibitors on neurological dysfunction after cerebral hemorrhage by using improved neurological deficit scoring, angle-turning tests, and spontaneous excitatory postsynaptic current recording techniques. This provides a scientific basis for the treatment of chronic cerebral hemorrhage, and is expected to reduce long-term neurological damage and cognitive deficits, improve patients' quality of life, and has significant clinical translational potential and broad application prospects. Attached Figure Description

[0069] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0070] Appendix Figure 1 Spatial transcriptomic analysis results of brain tissue from intracerebral hemorrhage (ICH) and healthy controls.

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

[0072] Appendix Figure 3 Histological and cytological changes in animal models of intracerebral hemorrhage (ICH) after receiving different treatments (Sham, ICH+IgG, ICH+Anti-C1q). Detailed Implementation

[0073] Example 1:

[0074] This invention provides a method for identifying a drug for treating chronic delayed neurological injury in the cerebral hemorrhage phase, comprising the following steps:

[0075] (a) A mouse model of cerebral hemorrhage was established and 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. N days after model induction, the mice were assessed for neurological function and residual motor dysfunction was determined by the cornering test.

[0076] (b) The excitability of thalamic neurons in mice N days after cerebral hemorrhage was assessed using whole-cell patch-clamp recording technology. The basic synaptic transmission characteristics of thalamic neurons were determined by measuring the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs). The frequency and amplitude of sEPSCs in mice N days after cerebral hemorrhage were compared with those in control mice to assess the effect of cerebral hemorrhage on synaptic transmission.

[0077] (c) The frequency and amplitude of sEPSCs were measured in the mice in the treatment group and the results were compared with those in the non-treatment group to evaluate the effect of the drug to be identified on the improvement of synaptic transmission function.

[0078] (d) Spatial learning and memory abilities of mice in the treatment and non-treatment groups were assessed by the Morris water maze test N days after cerebral hemorrhage and compared with control mice without cerebral hemorrhage to determine the presence of cognitive impairment.

[0079] (e) The effects of the drug treatment to be identified in the treatment group on the spatial learning and memory abilities of mice N days after cerebral hemorrhage were analyzed, and the Morris water maze test results were compared with those in the non-treatment group to assess the effect of treatment on improving cognitive impairment.

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

[0081] In step (a), the degree of neurological deficit is assessed by modified neurological deficit scores (mNSS scores), and motor coordination and balance are assessed by the rotarod test.

[0082] Steps (b) and (c) assess neuronal excitability by recording spontaneous excitatory postsynaptic currents (sEPSCs) and evaluate the impact on synaptic transmission by histograms and cumulative probability plots of sEPSC frequency and amplitude.

[0083] Steps (d) and (e) assessed motor function by measuring movement speed and distance over N days in different groups. Path tracking maps showed the paths mice took when searching for hidden platforms, and the time spent on the platforms and the number of times the mice crossed the target platforms demonstrated their learning and memory abilities.

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

[0085] In some implementations: complement C1q inhibitors are specifically anti-C1q antibodies.

[0086] In some implementation methods:

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

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

[0089] (2) Perform spatial transcriptome sequencing to identify specifically differentially expressed genes;

[0090] (3) Compare gene expression data between patients with cerebral hemorrhage and healthy controls to identify differentially expressed genes.

[0091] In some implementation methods:

[0092] Step (1) specifically involves: collecting autopsy brain tissue from patients with cerebral hemorrhage and healthy control groups; brain tissue collection should be performed within 4 hours after the death of the individual to ensure tissue freshness;

[0093] Step (2) specifically involves using NanoString. Spatial transcriptomics analysis was performed using a digital spatial analyzer. Brain tissue sections were sliced ​​to a thickness of 6 μm and mounted on Leica BOND Plus slides. Subsequent steps were performed using DEPC-treated water under RNase-free conditions, including three consecutive 5-minute washes in xylene, followed by two 5-minute washes in 100% ethanol, one wash in 95% ethanol, and one wash in 1x PBS. Target searching was performed at 95°C for 10 minutes using a BioGenex ezretriver System diluted 1x with a target search reagent. After washing in 1x PBS, the tissue was diluted with RNA hybridization probes in Buffer R and incubated overnight at 37°C. The next day, the tissue was stained with antibodies Iba1, GFAP, NeuN, and Syto13 at room temperature for 1 hour, and then loaded onto GeoMx. Regions of interest (ROIs) were scanned and selected in the DSP instrument. Morphological markers were identified using DAPI immunofluorescence assay, and the ROIs were segmented using Iba1, NeuN, and GFAP staining. ROIs selected for subsequent gene expression analysis were marked with circles. Each ROI was segmented into three illuminated areas (AOIs): microglia iba1-positive, neurons neuN-positive, and astrocyte gmap-positive. The probe identity of each segment was captured by ultraviolet irradiation and transferred to a 96-well plate. A PCR system was constructed using the standard DSP procedure, a library was built, and the original fastq file was converted into a digital count file using GeoMx NGSPipeline software.

[0094] Step 3 specifically involves: segmenting the data and performing probe quality control to remove defective AOIs / ROIs and abnormal probes, determining the detection limit for each fragment, and ignoring gene expression below the LOQ; normalizing the data using quantile 3 normalization, and performing differential detection using edgeR to compare the differences between the AOIs group (i.e., ICH and the control group); genes with a false detection rate (FDR) < 0.05 and log2(Fold Change) > 1 are considered differentially expressed genes (deg).

[0095] The Toppgene program was used to analyze the identified genes, primarily for gene ontology annotation. GO and KEGG methods were used for enrichment analysis of upregulated and downregulated genes. Gene feature analysis was performed using Hallmarks from the Molecular Signatures Database (MSigDB), and statistical tests were used to test hypotheses in selected groups. The Wilcoxon rank-sum test was used to compare the independent AOI / ROIs between two groups, and the Kruskal-Wallis method was used to test the rank sums of multiple independent AOI / ROIs to identify differentially expressed genes specific to the chronic phase of cerebral hemorrhage.

[0096] In some implementation methods:

[0097] Following step (f) are steps to explore the pathogenic mechanisms in animal models of cerebral hemorrhage, specifically including:

[0098] (i) Observe pathological changes in an animal model of cerebral hemorrhage N days after the hemorrhage, including reactive microglial proliferation and neuropathological abnormalities, neuronal loss, neuronal degeneration and decreased dendritic spine density.

[0099] (ii) Use transmission electron microscopy to observe the separation of myelin sheaths and / or vesicles, as well as the aggregation of postsynaptic structures in animal models of cerebral hemorrhage;

[0100] (iii) Treat animal models of cerebral hemorrhage with the drug to be identified and evaluate the treatment effect, including reducing neuropathological abnormalities and improving chronic inflammation, neuropathological and degenerative changes in the chronic phase of brain injury.

[0101] The present invention further proposes a pharmaceutical composition for treating chronic delayed neurological injury in the cerebral hemorrhage phase, obtained using an identification method, characterized in that:

[0102] The drug includes a complement C1q inhibitor.

[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, which can effectively deliver the complement C1q inhibitor to the treatment site.

[0104] The present invention further proposes the use of the pharmaceutical composition described herein in the preparation of a medicament for treating chronic delayed neurological injury in cerebral hemorrhage.

[0105] Example 2:

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

[0107] Human brain tissue sections were obtained from Beijing Tiantan Hospital, affiliated with Capital Medical University. The Ethics Committee of Beijing Tiantan Hospital, affiliated with Capital Medical University, approved the establishment of a human brain tissue bank for neurological diseases (KY 2018-031-02). Brain tissue was collected within 4 hours post-mortem. The GeoMx spatial transcriptome experiment included 3 patients and 3 controls. The 3 patients were those with right basal ganglia hemorrhage who died within 6–7 years after cerebral hemorrhage. The control cases were individuals without a history of neurological or neuropsychiatric diseases but who died from non-neurological causes. Thalamic tissue sections were matched with those from patients with cerebral hemorrhage. All included patients had no acute myocardial infarction, heart failure, autoimmune diseases, hematologic disorders, or infections at death. There was no significant difference in age at 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. Slides were cut to a thickness of 6 μm and mounted on LeicaBOND Plus slides. All subsequent steps were performed using RNase-free and DEPC (diethyl pyrocarbonate)-treated water. Slides were washed three times consecutively in xylene for 5 min, then twice in 100% ethanol for 5 min, once in 95% ethanol, and once in 1x PBS. Target searching was performed for 10 min at 95°C using a BioGenex ezretriver System diluted 1x with target search reagent (10x Invitrogen 00-4956-58 EDTA pH 9.0). After washing in 1x PBS, the RNA hybridization probe (NanoString, GMX-RNA-NGS-HuWTA-4) was diluted in Buffer R (provided by the GeoMx RNA Slide Prep Formalin-Fixed Paraffin-Embedded (FFPE)-PCLN Kit, GMX-PREP-RNA-FFPE-PCLN-12) and incubated overnight at 37°C. The next day, the sample was stained for 1 hour at room temperature with antibodies Iba1 (CST, 48934), GFAP (Novus, NBP2-33184AF532), NeuN (Abcam, ab190565), and Syto13 (Nanostring, GMX-RNA-MORPH-HST-12), and then loaded into the GeoMx DSP instrument for scanning and selection of regions of interest (ROIs). Morphological markers were identified using DAPI (4′, 6-diamino-2-phenylindole) immunofluorescence. Segmentation was performed using Iba1, NeuN, and GFAP staining. ROIs selected for subsequent gene expression analysis were marked with circles. Each ROI was divided into three illuminated areas (AOIs): microglia iba1-positive, neuronal NEUN-positive, and astrocyte gmap-positive. Probe identification for each segment was captured by UV irradiation and transferred to 96-well plates. PCR systems were constructed using standard DSP procedures to build libraries.

[0109] The raw fastq files were converted to numeric count files using GeoMxNGS Pipeline software (v2.0). Data segmentation and probe quality control (QC) were performed to remove defective AOIs / ROIs and anomalous probes. The limit of detection (LOQ) was determined for each fragment, ignoring gene expression below the LOQ. The LOQ was set as the geometric number of negative probes multiplied by the geometric standard deviation of the negative probes. After completing the QC steps, the data was 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 the AOI groups (ICH and control groups). Genes with a false detection rate (FDR) < 0.05 and log2 (Fold Change) > 1 were considered deg. The identified genes were analyzed using the Toppgene program, primarily for gene ontology annotation. Upregulated and downregulated genes were enriched using GO and KEGG methods, respectively. Gene signature analysis was performed using the gene acquisition hallmark from 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 the independent AOI / ROIs of two groups, and the Kruskal-Wallis method was used to test the rank sum of multiple independent AOI / ROIs.

[0110] Regional heterogeneity of transcriptome features in reactive microglia during the chronic phase of intracerebral hemorrhage: Differential gene expression (DGE) analysis between the ICH group and the control group revealed 633 upregulated genes. The genes most highly upregulated in Iba1+ microglia were associated with activated transcriptome features, including components of the classical complement pathway (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). KEGG pathway analysis showed that, compared with the control group, 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 (phagosomes, lysosomes, apoptosis, autophagy, endocytosis and cellular senescence). In addition, GeneOntology (GO) database analysis showed enrichment of immune response functions related to glial cell activation, inflammatory response, cytokine production and immune effector function. Gene marker and gene set variation analysis (GSVA) ​​scores showed significant upregulation of inflammation-related pathways (complement and coagulation cascade, and IL-6 / JAK / STAT3 signaling). In situ immunostaining showed that complement factor C1q was mainly expressed by thalamic microglia 6 years after intracerebral hemorrhage.

[0111] See Figure 1 , Figure 1 Spatial transcriptomic analysis results of brain tissue from intracerebral hemorrhage (ICH) and healthy controls are presented. Among them:

[0112] The top-left image shows the sample sources for the brain hemorrhage and healthy control group. The image displays a side view of the human brain, labeling the location of the hemorrhage (Lesion), the thalamus, and neurons and astrocytes.

[0113] The heatmap in the bottom left corner compares gene expression differences between patients with intracerebral hemorrhage (ICH) and healthy controls. Each column represents a sample, and each row represents a gene. The intensity of the color indicates the level of gene expression, with red indicating upregulation (high expression) and blue indicating downregulation (low expression).

[0114] The volcano plot in the upper right corner illustrates the significance of gene expression changes. The horizontal axis represents the log-2 change in gene expression (log2 Fold Change), and the vertical axis represents the negative log-10 p-value (-log10 P-value), indicating statistical significance. Red dots represent genes that are significantly upregulated in cerebral hemorrhage, blue dots represent genes that are significantly downregulated, and gray dots represent genes that do not show significant changes.

[0115] The bottom right image shows an enrichment analysis plot, illustrating the enrichment of significantly upregulated genes in different biological pathways during intracerebral hemorrhage. Different colors represent different pathways, such as antigen processing and presentation, complement and coagulation cascades, etc. The bottom image shows a Gene Set Variation Analysis (GSVA) ​​plot, displaying the enrichment scores of different gene sets in intracerebral hemorrhage. Each point represents a gene set, the horizontal axis is the rank of the gene set in the sorted dataset, and the vertical axis is the enrichment score.

[0116] Example 3:

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

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

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

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

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

[0122] C: This shows the recordings of spontaneous excitatory postsynaptic currents (sEPSCs) used to assess neuronal excitability. The waveforms and frequencies of sEPSCs differ between groups.

[0123] D: The bar chart and cumulative probability plot show the frequency of sEPSCs. The frequency of sEPSCs decreased in the ICH group, while it recovered in the Anti-C1q treatment group.

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

[0125] F: The bar chart shows the movement speed and distance of different groups over 5 days. The movement speed and distance were significantly reduced in the ICH group, while the Anti-C1q treatment group showed improvement.

[0126] G: Shows the results of the Morris water maze test, used to assess spatial learning and memory abilities. Path-tracking maps for different groups show their paths while searching for hidden platforms.

[0127] H: The bar chart shows the duration in the target quadrant and the number of crosses of the target platform in the Morris water maze test. The Anti-C1q treatment group performed better on these parameters, indicating a recovery in their learning and memory abilities.

[0128] Example 4:

[0129] We investigated the pathogenic mechanism of complement C1q in an animal model of intracerebral hemorrhage based on spatial transcriptome sequencing results. To further determine the mechanism by which anti-C1q antibodies exert therapeutic effects in chronic neurological dysfunction following intracerebral hemorrhage, we observed reactive microglial proliferation (CD68+Iba1+) and neuropathological abnormalities, such as neuronal loss, neuronal degeneration, and decreased dendritic spine density, 35 days after hemorrhage. Corresponding transmission electron microscopy revealed myelin sheath separation and / or vesicles, and less postsynaptic aggregation. Treatment with C1q function-blocking antibodies partially alleviated these impairments. These results confirm that targeting C1q exerts its effects by improving chronic inflammation, neuropathological changes, and degenerative alterations during the chronic phase of brain injury.

[0130] See Figure 3 This study demonstrates the histological and cytological changes in an animal model of intracerebral hemorrhage (ICH) after receiving different treatments (Sham, ICH+IgG, ICH+Anti-C1q). Among them:

[0131] A: Immunofluorescence staining of whole brain sections and cortical regions is shown, marking CD68 (macrophage / microglia marker) and CD86 (activated microglia marker). The bar chart 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 (a microglia marker) in brain slices is shown. The bar chart below shows the number of F4 / 80 and Iba1 positive cells in different treatment groups.

[0133] C: Shows histological staining of the myelin sheath (Luxol Fast Blue / PAS staining) and immunofluorescence staining of the axon (MBP labeling). The bar chart below shows the number of myelinated and unmyelinated axons.

[0134] D: Shows immunofluorescence staining of the synapse (Synapsin and PSD-95 labeling). The bar chart below shows the thickness of the postsynaptic density.

[0135] E: Shows immunofluorescence staining (NeuN labeling) of neurons and Golgi staining of dendritic spines in the brain slice. The bar chart below shows the number of dendritic spines per 10 micrometers in length.

[0136] These charts collectively demonstrate the potential protective effects of Anti-C1q therapy against structural and cellular changes in brain tissue following intracerebral hemorrhage, including reducing microglia 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 intracerebral hemorrhage.

[0137] This leads to a method for treating chronic neurological impairment caused by cerebral hemorrhage. The method involves analyzing spatial transcriptome sequencing results from the brain tissue of cerebral hemorrhage patients, and then targeting complement C1q to alleviate long-term neurological and cognitive function impairment. The spatial transcriptome sequencing results include...

[0138] Components of the classical complement pathway (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, FCERT1G, 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 chronic neurological function impairment caused by cerebral hemorrhage, wherein the method targets complement C1q to slow down long-term neurological and cognitive function impairment caused by cerebral hemorrhage. The mechanism is to reduce pathological damage such as chronic neuroinflammation and neurodegeneration, such as neuronal loss, neuronal degeneration and synaptic dysfunction.

[0140] Thus far, the description of the above embodiments has been provided for illustrative and descriptive purposes. This is not intended to be exhaustive or limiting of the present disclosure. Individual elements or features of particular embodiments are generally not limited to those particular embodiments, but may be interchanged and used in selected embodiments where applicable, even if not specifically shown or described. In many respects, the same elements or features may also be varied. Such variations are not considered a departure from this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

[0141] Example embodiments are provided so that this disclosure will become thorough and will fully convey the scope to those skilled in the art. Numerous details, such as examples of specific parts, apparatus, and methods, are set forth to provide a thorough understanding of embodiments of this disclosure. It will be apparent to those skilled in the art that the specific details are not required, and the example embodiments may be implemented in many different forms, neither of which should be construed as limiting the scope of this disclosure. In some example embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0142] Technical terms are used herein for the purpose of describing specific exemplary embodiments only and are not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein may also refer to the plural forms. The terms “comprising” and “having” are inclusive and therefore specify the presence of the stated features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or additional having of one or more other features, integrals, steps, operations, elements, components, and / or combinations thereof. Unless expressly indicated in order of execution, the method steps, processes, and operations described herein are not to be construed as necessarily requiring performance in the specific order discussed and shown. It should also be understood that additional or optional steps may be employed.

Claims

1. A method for identifying drugs used for delayed neurological injury in the chronic phase of cerebral hemorrhage, said method being a non-diagnostic and non-therapeutic method, characterized in that... Includes the following steps: (a) A mouse model of cerebral hemorrhage was established and 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. N days after model induction, the mice were assessed for neurological function and residual motor dysfunction was determined by the cornering test. (b) The excitability of thalamic neurons in mice N days after intracerebral hemorrhage was assessed using whole-cell patch-clamp recording technology. The basic synaptic transmission characteristics of thalamic neurons were determined by measuring the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs). The frequency and amplitude of sEPSCs in mice N days after intracerebral hemorrhage were compared with those in control mice to assess the effect of intracerebral hemorrhage on synaptic transmission. (c) The frequency and amplitude of sEPSCs were measured in the mice in the treatment group and the results were compared with those in the non-treatment group to evaluate the effect of the drug to be identified on the improvement of synaptic transmission function. (d) Spatial learning and memory abilities of mice in the treatment and non-treatment groups were assessed using the Morris water maze test N days after cerebral hemorrhage and compared with control mice without cerebral hemorrhage to determine the presence of cognitive impairment. (e) The effects of the drug treatment to be identified in the treatment group on spatial learning and memory abilities in mice N days after cerebral hemorrhage were analyzed, and the Morris water maze test results were compared with those in the non-treatment group to assess the effect of treatment on improving cognitive impairment. (f) Based on the results of the above steps, determine whether the treatment group improves neurological function, synaptic transmission and cognitive function, and reduces long-term neurological damage and cognitive deficits associated with the chronic phase of cerebral hemorrhage, and determine whether the drug can be used as a potential drug for treating delayed neurological damage in the chronic phase of cerebral hemorrhage. in: In step (a), the degree of neurological deficit is assessed by modified neurological deficit scores (mNSS scores), and motor coordination and balance are assessed by the rotarod test. Steps (b) and (c) assess neuronal excitability by recording spontaneous excitatory postsynaptic currents (sEPSCs) and evaluate the impact on synaptic transmission by histograms and cumulative probability plots of sEPSC frequency and amplitude. Steps (d) and (e) assess motor function by measuring movement speed and distance in different groups over N days. The path tracking map shows the path the mice took when searching for the hidden platform. The learning and memory abilities of the mice are shown by the time spent on the platform and the number of times they crossed the target platform. The N≥35; Prior to step (a), there is a step of identifying differentially expressed genes specific to the chronic phase of cerebral hemorrhage, which specifically includes: (1) Collect autopsy brain tissue from patients with cerebral hemorrhage and healthy control groups; (2) Perform spatial transcriptome sequencing to identify differentially expressed genes; (3) Compare gene expression data between patients with cerebral hemorrhage and healthy controls to identify differentially expressed genes.

2. The method according to claim 1, Its features are: Specifically, step (2) involves: performing spatial transcriptomics analysis using the NanoString GeoMx® digital spatial analyzer; slicing brain tissue to a thickness of 6 μm and mounting it on a Leica BOND Plus slide; and performing subsequent steps using DEPC-treated water under RNase-free conditions, including washing the slide three times for 5 minutes in xylene, then twice for 5 minutes in 100% ethanol, once in 95% ethanol, and once in 1x PBS; in the BioGenex ezretriver System, diluting the target search reagent to 1x and performing target search at 95°C for 10 minutes; after washing in 1x PBS, diluting the RNA hybridization probe in Buffer R and incubating overnight at 37°C; the next day, staining with antibodies Iba1, GFAP, NeuN, and Syto13 at room temperature for 1 hour, and then loading it into the GeoMx system. Regions of interest (ROIs) were scanned and selected in the DSP instrument. Morphological markers were identified using DAPI immunofluorescence assay, and the ROIs were segmented using Iba1, NeuN, and GFAP staining. ROIs selected for subsequent gene expression analysis were marked with circles. Each ROI was segmented into three illuminated areas (AOIs): microglia iba1-positive, neurons neuN-positive, and astrocyte gmap-positive. The probe identity of each segment was captured by ultraviolet irradiation and transferred to a 96-well plate. A PCR system was constructed using the standard DSP procedure, a library was built, and the original fastq file was converted into a digital count file using GeoMx NGSPipeline software. Step (3) specifically involves: segmenting the data and performing probe quality control to remove undesirable AOI / ROIs and abnormal probes, and determining the detection limit for each fragment, ignoring gene expression below LOQ; normalizing the data using quantile 3 normalization, and performing differential detection using edgeR, comparing the differences between the AOIs group, i.e., ICH and the control group, with genes having a false detection rate FDR <0.05 and log2 (Fold Change) >1 considered as differentially expressed genes deg; The identified genes were analyzed using the Toppgene program for gene ontology annotation, and enrichment analysis of upregulated and downregulated genes was performed using GO and KEGG methods. Gene feature analysis was performed using Hallmarks collected from the Molecular Signatures Database (MSigDB), and statistical tests were used to test hypotheses in selected groups. The Wilcoxon rank-sum test was used to compare the independent AOI / ROI of two groups, and the Kruskal-Wallis method was used to test the rank sum of multiple independent AOI / ROIs to identify differentially expressed genes specific to the chronic phase of cerebral hemorrhage.

3. The method according to claim 1, characterized in that: The drugs to be identified include complement C1q inhibitors.

4. The method according to claim 3, characterized in that: Complement C1q inhibitors are specifically anti-C1q antibodies.

5. The method according to claim 1, characterized in that: Step (1) specifically involves collecting autopsy brain tissue from patients with cerebral hemorrhage and healthy control groups; brain tissue collection is performed within 4 hours after the death of the individual to ensure tissue freshness.

6. The method according to claim 1, characterized in that: Following step (f) are steps to explore the pathogenic mechanisms in animal models of cerebral hemorrhage, specifically including: (i) Observe pathological changes in an animal model of cerebral hemorrhage N days after the hemorrhage, including reactive microglial proliferation and neuropathological abnormalities, neuronal loss, neuronal degeneration and decreased dendritic spine density. (ii) Use transmission electron microscopy to observe the separation of myelin sheaths and / or vesicles, as well as the aggregation of postsynaptic structures in animal models of cerebral hemorrhage; (iii) Treat animal models of cerebral hemorrhage with the drug to be identified and evaluate the treatment effect, including reducing neuropathological abnormalities and improving chronic inflammation, neuropathological and degenerative changes in the chronic phase of brain injury.

7. The method according to claim 1, characterized in that: The drug includes a complement C1q inhibitor.

Images