Method for establishing cerebral ischemia reperfusion injury model

Abstract

The invention discloses a method for establishing a cerebral ischemia reperfusion injury model, and relates to the technical field of biomedicine. Comprising the following steps: S1, preparing experimental materials, and selecting healthy adult male SD rats; s2, anesthesia and fixation: placing the to-be-treated SD rat in a closed container containing 7% isoflurane until the to-be-treated SD rat is unconscious, then transferring the to-be-treated SD rat to an operating table, continuing to give isoflurane with the concentration of 2%-3% to maintain a deep anesthesia state, and keeping the body temperature by adopting a constant-temperature blanket; s3, surgical operation: cutting a 1cm long mouth of the skin along the median line of the neck, and carefully separating common carotid artery and vagus nerve on two sides to avoid damage to surrounding tissues; the left side external jugular vein telecentric end is ligated with a silk thread to reduce the amount of bleeding. According to the scheme, the establishment process of the cerebral ischemia reperfusion injury model is described in detail, the establishment process comprises the steps of experimental material preparation, anesthesia and fixation, surgical operation, ischemia induction, reperfusion, nursing, data analysis and the like, and complete reference is provided for related research.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to a method for establishing a cerebral ischemia-reperfusion injury model. Background Art

[0002] Cerebral ischemia-reperfusion injury is an important pathophysiological process of cerebrovascular disease, and its occurrence and development are related to multiple factors, including oxidative stress, inflammatory response, and cell apoptosis. Establishing an effective model of cerebral ischemia-reperfusion injury is of great significance for studying these mechanisms. Currently, commonly used methods include suture method and photochemical induction method, but these methods have problems such as complex operation and unstable results.

[0003] After searching, the application scheme of Chinese patent application number CN202311625123.X discloses the use of gastrodin in the preparation of JAK2-STAT3 signaling pathway inhibitors and anti-cerebral ischemia reperfusion injury drugs. The middle cerebral artery ischemia (MCAO) model of SD rats was constructed by the thread embolism method, and the glucose-oxygen deprivation / reoxygenation (OGD / R) model of BMEC cells was constructed in vitro. The gastrodin was intervened with different concentrations, and the expression of JAK2, STAT3, phosphorylated JAK2, phosphorylated STAT3, oxidative stress and apoptosis levels before and after the intervention of gastrodin was detected. In the above patent, the middle cerebral artery ischemia (MCAO) model of SD rats was constructed by the thread embolism method, which has the above-mentioned problems such as complex operation and unstable results. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for establishing a cerebral ischemia-reperfusion injury model.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for establishing a cerebral ischemia-reperfusion injury model, comprising:

[0007] S1: Preparation of experimental materials, selection of healthy adult male SD rats;

[0008] S2: Anesthesia and fixation: The SD rats to be treated were placed in a sealed container containing 7% isoflurane until they lost consciousness, and then transferred to the operating table and continued to be given 2%-3% isoflurane to maintain a deep anesthesia state, and a constant temperature blanket was used to maintain body temperature;

[0009] S3: Surgical operation: make a 1 cm long skin incision along the midline of the neck, carefully separate the bilateral common carotid arteries and vagus nerves, and avoid damaging the surrounding tissues; use silk thread to ligate the distal end of the left external jugular vein to reduce bleeding; then, temporarily block the right internal carotid artery with a micro artery clamp 5 mm above the bifurcation point for no more than 30 minutes to prevent irreversible damage;

[0010] S4: Ischemia induction: According to the predetermined plan, the pre-prepared thrombosis accelerator solution was injected into the left internal carotid artery at a ratio of 0.1 mL / kg body weight. The specific composition ratio was a mixture of FeCl3·6H2O 0.05 mol / L and disodium ethylenediaminetetraacetate 0.01 mol / L, in which Fe 3+ The ion concentration must be precisely controlled to ensure effectiveness without overstimulating the blood vessel wall and causing additional damage. Immediately after the injection, the clamp on the right internal carotid artery is released to allow natural reflow.

[0011] S5: Reperfusion: the time interval from the release of the micro-arterial clamp to the restoration of normal blood circulation was recorded as one of the indicators of reperfusion duration. At the same time, the laser Doppler technique was used to continuously monitor the changes in blood flow velocity in the middle cerebral artery supply area.

[0012] S6: Nursing: move the animal to a warm and quiet environment to wake it up, regularly check the wound healing status and give antibiotics to prevent infection;

[0013] S7: Data analysis.

[0014] Preferably: the anesthesia and fixation of S2 includes:

[0015] S21: Anesthesia preparation, ensure the sterility of the surgical area, control the temperature between 22-24°C to prevent the animal from getting cold or overheated during the operation; calculate the anesthesia dose based on the weight of the experimental animal;

[0016] S22: Induce anesthesia, use 7% isoflurane as the induction anesthetic, and perform inhalation general anesthesia through a closed container; closely observe the animal's reaction, and quickly transfer it to the operating table after it loses consciousness;

[0017] S23: Maintain anesthesia by reducing the isoflurane concentration to 2%-3% to maintain a deep anesthesia state; use a physiological monitor to continuously monitor heart rate and respiratory rate;

[0018] S24: Fix the animal, place the rat supinely on the operating table, stretch the limbs naturally and fix them gently with tape; use a head frame to fix the head and ensure that the neck is fully exposed for subsequent operations.

[0019] Preferably: the surgical operation of S3 includes:

[0020] S31: Skin incision, wipe the surgical area with 75% alcohol cotton balls, and then disinfect with iodine twice; make a 1cm long longitudinal incision along the midline of the neck;

[0021] S32: Separate the tissues and use forceps to separate the subcutaneous tissue to expose the bilateral common carotid arteries and their branches;

[0022] S33: Ligate the blood vessels. Use silk thread to ligate the distal end of the left external jugular vein 5 mm above the bifurcation point to reduce bleeding. Also use silk thread to temporarily block the blood flow of the right internal carotid artery. The duration should not exceed 30 minutes to prevent irreversible damage.

[0023] Preferably, the ischemic induction of S4 comprises:

[0024] S41: Preparation of thrombosis promoter: weigh the required chemical substances using a precision balance and dissolve them in normal saline according to the proportion to prepare a mixed solution of FeCl3·6H2O 0.05mol / L + disodium ethylenediaminetetraacetate 0.01mol / L;

[0025] S42: Prepare before injection, use a microinjector to draw a dose of 0.1 mL / kg body weight from the prepared solution;

[0026] S43: Thrombosis accelerator injection: find the specific location of the left internal carotid artery, and after confirming that it is correct, inject the drug into the target area at a constant speed. Immediately after the injection, release the micro-arterial clamp on the right internal carotid artery.

[0027] Preferably, the reperfusion of S5 includes:

[0028] S51: Blood flow recovery monitoring, using laser Doppler blood flow meter to monitor the changes in blood flow velocity in the middle cerebral artery supply area in real time; recording the blood flow velocity value at regular intervals until it returns to the baseline level;

[0029] S52: Observe behavioral performance and check animals regularly to see if they show any abnormal behavior.

[0030] Preferably: the S6 care includes:

[0031] S61: Management during the recovery period: place the animal in an incubator after surgery and keep the room temperature between 30-32°C; give glucose saline solution orally or intraperitoneally;

[0032] S62: Wound care: gently wipe the skin around the wound with an iodine cotton swab to prevent infection; give broad-spectrum antibiotics to prevent secondary infection;

[0033] S63: Long-term follow-up observation, record the animals' food intake, water intake and excrement color indicators for more than one week, weigh them once a week to understand whether their growth and development are normal.

[0034] Preferably, the data analysis of S7 includes:

[0035] Neurological function score: Garcia test was used to evaluate the degree of neurological deficit at different time points after surgery;

[0036] Determination of brain edema volume: Calculate the edema space ratio after obtaining image information through magnetic resonance imaging scan;

[0037] Detection of inflammatory factor levels: Peripheral blood samples were drawn and centrifuged to separate serum, and ELISA kits were used to quantitatively measure the content of TNF-α and IL-1β mediators;

[0038] Histological evaluation: Fresh brain tissue sections were taken for HE staining to observe the morphological and structural changes;

[0039] Molecular biology analysis: After RNA was extracted and reverse transcribed into cDNA, the target gene fragment was amplified by qPCR technology to explore the activation status of related signaling pathways.

[0040] Preferably, in S7, the brain edema volume determination comprises:

[0041] MRI scanning preparation: ensure that the magnetic resonance imaging machine is in the best working condition and all parameter settings meet the requirements. Fix the rat on the special scanning bed and fix the head with ear bars;

[0042] Image acquisition: T2-weighted image sequence was used for cross-sectional scanning of the brain, with the slice thickness set to 1 mm, and the image acquisition work within the whole brain was completed according to the preset plan;

[0043] Data analysis: use medical imaging analysis software to process the original images, remove background noise, manually or automatically identify the edema boundary, and calculate its space occupancy ratio;

[0044] In S7, the detection of inflammatory factor levels includes:

[0045] For sample collection, the first, third, and seventh days after surgery were selected as sampling days. 0.5 mL of peripheral blood was drawn by tail vein puncture and placed in EDTA anticoagulant tubes;

[0046] Serum separation: fresh whole blood samples were placed in a low-temperature centrifuge and spun at 3000 rpm for 10 minutes. The upper clear liquid was taken out as the serum. The obtained serum was divided into several small portions and frozen in a -80℃ refrigerator for later use.

[0047] For ELISA experiment operation, prepare various necessary reagents, including standard substances and enzyme-labeled antibodies, according to the instructions of the manual, add the samples to be tested and the control substances to the microplate pre-coated with specific antigens, let it stand at room temperature for 1 hour, discard the liquid in the wells and wash several times with PBST buffer, then add the substrate solution and incubate in the dark for an appropriate period of time until the color appears. After terminating the reaction, immediately read the OD value of each well with an enzyme reader, and convert the corresponding concentration value according to the standard curve.

[0048] Preferably, in S7, the histological evaluation comprises:

[0049] After fixation, the whole brain was taken out and placed on an operating table under ice bath condition, and then immediately immersed in 4% paraformaldehyde solution for at least 24 hours to allow it to be fully cross-linked and solidified;

[0050] HE staining sections were prepared by dehydrating with graded ethanol concentrations and then immersed in xylene until completely transparent. The processed sample blocks were embedded in a melted paraffin mold and cooled to form, cut into 5 μm slices, and then subjected to hematoxylin-eosin staining procedures;

[0051] Observe and take pictures under a microscope. First use a low-magnification lens to view the overall morphological features, then switch to a high-magnification objective to carefully observe changes in the cell structure and take pictures for archiving.

[0052] Preferably: in said S7, the molecular biological analysis comprises:

[0053] RNA extraction and purification: fresh or frozen tissues are added with TRIZOL reagent and shaken vigorously, then left to stand for a few minutes to release nucleic acids. An equal volume of chloroform is added and shaken vigorously for tens of seconds, then centrifuged and separated, and the upper aqueous phase is aspirated to continue the subsequent steps. 0.5 times the volume of isopropanol is added to the supernatant, mixed by inversion, and left for a while to precipitate RNA. The precipitate is repeatedly rinsed with 75% ethanol to remove residual impurities, and finally naturally air-dried to obtain pure RNA powder;

[0054] cDNA synthesis reverse transcription, using DNase I to digest the total RNA sample to eliminate potential genetic material interference factors, mix the oligo primers, dNTPs, and M-MLV reverse transcriptase components in proportion in an EP tube, and then heat and denature briefly, control the temperature cycle conditions, and promote the synthesis of complementary DNA chains, i.e. cDNA products, under the guidance of the mRNA template;

[0055] qPCR amplification detection, a pair of specific oligonucleotide probes are specially customized for the target gene sequence for subsequent quantitative analysis. The template cDNA is diluted and mixed with SYBR Green dye and buffer and evenly distributed into each PCR tube and sealed. The real-time quantitative PCR instrument is used to dynamically monitor the changes in fluorescence intensity generated during the entire amplification process, thereby indirectly reflecting the number of copies of the target fragment.

[0056] The beneficial effects of the present invention are:

[0057] 1. The scheme of the present invention describes in detail the process of establishing a cerebral ischemia-reperfusion injury model, including the steps of experimental material preparation, anesthesia and fixation, surgical operation, ischemia induction, reperfusion, nursing and data analysis, providing a complete reference for related research.

[0058] 2. The solution of the present invention ensures the effect of the thrombosis promoter by precisely controlling the concentrations of FeCl3·6H2O and disodium ethylenediaminetetraacetate and the injection volume of 0.1 mL / kg body weight, while avoiding excessive stimulation of the blood vessel wall to cause additional damage.

[0059] 3. The present invention uses laser Doppler technology to continuously monitor changes in blood flow velocity in the middle cerebral artery supply area to ensure data accuracy and completeness.

[0060] 4. The data analysis of the present invention is comprehensive, including neurological function scores, brain edema volume determination, inflammatory factor level detection, histological evaluation and molecular biological analysis, which comprehensively evaluates the effect of cerebral ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The present invention provides a flow chart of a method for establishing a cerebral ischemia-reperfusion injury model. DETAILED DESCRIPTION

[0062] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0063] Embodiment 1:

[0064] A method for establishing a cerebral ischemia-reperfusion injury model, comprising:

[0065] S1: Preparation of experimental materials: healthy adult male SD rats (weight 200-300 g) were selected;

[0066] S2: Anesthesia and fixation: The SD rats to be treated were placed in a sealed container containing 7% isoflurane until they lost consciousness, and then transferred to the operating table and continued to be given 2%-3% isoflurane to maintain a deep anesthesia state, and a constant temperature blanket was used to maintain the body temperature at around 37°C;

[0067] S3: Surgical operation: make a 1-cm long skin incision along the midline of the neck, carefully separate the bilateral common carotid arteries and vagus nerves, and avoid damaging surrounding tissues; use silk thread to ligate the distal end of the left external jugular vein to reduce bleeding; then, temporarily block the right internal carotid artery with a micro-arterial clamp about 5 mm above the bifurcation point for no more than 30 minutes to prevent irreversible damage;

[0068] S4: Ischemia induction: According to the predetermined plan, the pre-prepared thrombosis accelerator solution was injected into the left internal carotid artery at a ratio of 0.1 mL / kg body weight. The specific composition ratio was a mixture of FeCl3·6H2O 0.05 mol / L and disodium ethylenediaminetetraacetate 0.01 mol / L, in which Fe 3+ The ion concentration must be precisely controlled to ensure effectiveness without overstimulating the blood vessel wall and causing additional damage. Immediately after the injection, the clamp on the right internal carotid artery is released to allow natural reflow.

[0069] S5: Reperfusion: observe and record the time interval from the release of the micro-arterial clamp to the restoration of normal blood circulation as one of the indicators of reperfusion duration. At the same time, laser Doppler technology is used to continuously monitor the changes in blood flow velocity in the middle cerebral artery supply area to ensure data accuracy and completeness.

[0070] S6: Nursing: move the animal to a warm and quiet environment to wake it up, regularly check the wound healing status and give antibiotics to prevent infection; in addition, closely monitor and observe changes in general vital signs such as its behavior, food and water intake for at least one week;

[0071] S7: Data analysis.

[0072] Wherein, the anesthesia and fixation of S2 includes:

[0073] S21: Anesthesia preparation, ensure the sterility of the surgical area, control the temperature between 22-24°C to prevent the animal from getting cold or overheated during the operation; calculate the anesthesia dose based on the weight of the experimental animal;

[0074] S22: Induce anesthesia, use 7% isoflurane as the induction anesthetic, and perform inhalation general anesthesia through a closed container; closely observe the animal's reaction, and quickly transfer it to the operating table after it loses consciousness;

[0075] S23: Maintain anesthesia by reducing the isoflurane concentration to 2%-3% to maintain a deep anesthesia state; use a physiological monitor to continuously monitor basic vital signs such as heart rate and respiratory rate;

[0076] S24: Fix the animal, place the rat supinely on the operating table, stretch the limbs naturally and fix them gently with tape; use a head frame to fix the head and ensure that the neck is fully exposed for subsequent operations.

[0077] The surgical operation of S3 includes:

[0078] S31: Skin incision, wipe the surgical area with 75% alcohol cotton balls, and then disinfect with iodine twice; make a 1 cm long longitudinal incision along the midline of the neck, paying attention to avoid important blood vessels and nerves;

[0079] S32: Separate the tissues and use forceps to separate the subcutaneous tissue to expose the bilateral common carotid arteries and their branches;

[0080] S33: Ligate the blood vessels. Use silk thread to ligate the distal end of the left external jugular vein about 5 mm above the bifurcation point to reduce bleeding. Also use silk thread to temporarily block the blood flow of the right internal carotid artery. The duration should not exceed 30 minutes to prevent irreversible damage.

[0081] Wherein, the ischemic induction of S4 comprises:

[0082] S41: Preparation of thrombosis promoter: weigh the required chemical substances using a precision balance and dissolve them in normal saline according to the proportion to prepare a mixed solution of FeCl3·6H2O 0.05mol / L + disodium ethylenediaminetetraacetate 0.01mol / L;

[0083] S42: Prepare before injection, use a microinjector to draw a dose of 0.1 mL / kg body weight from the prepared solution;

[0084] S43: Thrombosis accelerator injection: find the specific location of the left internal carotid artery, and after confirming that it is correct, inject the drug into the target area at a constant speed. Immediately after the injection, release the micro-arterial clamp on the right internal carotid artery to allow natural reflow.

[0085] Wherein, the reperfusion of S5 includes:

[0086] S51: Blood flow recovery monitoring, using laser Doppler blood flow meter to monitor the changes in blood flow velocity in the middle cerebral artery supply area in real time; recording the blood flow velocity value at regular intervals until it returns to the baseline level;

[0087] S52: Observe behavioral manifestations and regularly check whether the animals show abnormal behaviors, such as convulsions, coma, etc.

[0088] The S6 care includes:

[0089] S61: Management during the recovery period: place the animal in an incubator after surgery and keep the room temperature between 30-32°C; give glucose saline solution orally or intraperitoneally;

[0090] S62: Wound care: gently wipe the skin around the wound with an iodine cotton swab to prevent infection; give broad-spectrum antibiotics to prevent secondary infection;

[0091] S63: Long-term follow-up observation, record the animal's food intake, water intake, excrement color and other indicators for more than one week, weigh it once a week to understand whether its growth and development are normal.

[0092] The data analysis of S7 includes:

[0093] Neurological function score: Garcia test was used to evaluate the degree of neurological deficit at different time points after surgery;

[0094] Brain edema volume measurement: Calculate the edema space ratio after obtaining image information through magnetic resonance imaging (MRI) scanning;

[0095] Detection of inflammatory factor levels: After peripheral blood samples were drawn and centrifuged to separate serum, ELISA kits were used to quantitatively measure the content of key mediators such as TNF-α and IL-1β;

[0096] Histological evaluation: Fresh brain tissue sections were taken for HE staining to observe the morphological and structural changes;

[0097] Molecular biology analysis: After RNA was extracted and reverse transcribed into cDNA, the target gene fragment was amplified by qPCR technology to explore the activation status of related signaling pathways.

[0098] Among them, in S7, based on the Garcia test of the degree of neurological deficit in rats at different time points after surgery, including spontaneous activity, limb symmetry, climbing ability and other aspects, each with a full score of 6 points and a total score of 36 points, as follows:

[0099] Spontaneous activity: Observe the rats' free movement in an open field;

[0100] Limb symmetry: Check the strength balance of the front and back limbs;

[0101] Climbing ability: let the rats try to climb up an inclined plane;

[0102] Whisker stimulation response: lightly touch the whiskers and observe the head turning speed;

[0103] Visual placement response: placing the rat on a high surface to see how it lands;

[0104] Auditory startle response: Make a sound and see if the rat shows a startled gesture.

[0105] Wherein, in said S7, the measurement of brain edema volume comprises:

[0106] MRI scanning preparation: ensure that the magnetic resonance imaging machine is in the best working condition and all parameter settings meet the requirements. Fix the rat on the special scanning bed and fix the head with ear bars;

[0107] Image acquisition: T2-weighted image sequence was used for cross-sectional scanning of the brain, with the slice thickness set to 1 mm, and the image acquisition work within the whole brain was completed according to the preset plan;

[0108] Data analysis: Medical imaging analysis software was used to process the original images, remove background noise, manually or automatically identify the edema boundary, and calculate its space occupancy ratio.

[0109] Wherein, in said S7, the detection of inflammatory factor levels includes:

[0110] For sample collection, the first, third, and seventh days after surgery were selected as sampling days. About 0.5 mL of peripheral blood was drawn by tail vein puncture and placed in EDTA anticoagulant tubes;

[0111] Serum separation: fresh whole blood samples were placed in a low-temperature centrifuge and spun at 3000 rpm for 10 minutes. The upper clear liquid was taken out as the serum. The obtained serum was divided into several small portions and frozen in a -80℃ refrigerator for later use.

[0112] For ELISA experiment operation, prepare various necessary reagents according to the instructions, including standard substances, enzyme-labeled antibodies, etc., add the samples to be tested and the control substances to the microplate pre-coated with specific antigens, let it stand at room temperature for 1 hour, discard the liquid in the wells and wash several times with PBST buffer, then add the substrate solution and incubate in the dark for an appropriate period of time until the color appears. After terminating the reaction, immediately read the OD value of each well with an enzyme reader, and convert the corresponding concentration value according to the standard curve.

[0113] Wherein, in said S7, the histological evaluation comprises:

[0114] After fixation, the whole brain was taken out and placed on an operating table under ice bath condition, and then immediately immersed in 4% paraformaldehyde solution for at least 24 hours to allow it to be fully cross-linked and solidified;

[0115] HE staining sections were prepared by dehydrating with graded ethanol concentrations and then immersed in xylene until completely transparent. The processed sample blocks were embedded in a melted paraffin mold and cooled to form, cut into slices with a thickness of about 5 μm, and then subjected to hematoxylin-eosin staining procedures;

[0116] For observation and photography under a microscope, first use a low-magnification lens to roughly browse the overall morphological features, then switch to a high-magnification objective lens to carefully observe changes in the cell structure, and select a typical field of view to take high-quality color photos for archiving.

[0117] Wherein, in said S7, the molecular biology analysis comprises:

[0118] RNA extraction and purification: fresh or frozen tissues are added with TRIZOL reagent and shaken vigorously, then left to stand for a few minutes to release nucleic acids. An equal volume of chloroform is added and shaken vigorously for tens of seconds, then centrifuged and separated, and the upper aqueous phase is aspirated to continue the subsequent steps. 0.5 times the volume of isopropanol is added to the supernatant, mixed by inversion, and left for a while to precipitate RNA. The precipitate is repeatedly rinsed with 75% ethanol to remove residual impurities, and finally naturally air-dried to obtain pure RNA powder;

[0119] cDNA synthesis reverse transcription, using DNase I to digest the total RNA sample to eliminate potential genetic material interference factors, mixing Oligo (dT) primers, dNTPs, M-MLV reverse transcriptase and other key components in an EP tube in proportion, and then briefly heating and denaturing, controlling the temperature cycle conditions, to promote the synthesis of complementary DNA chains, i.e. cDNA products, under the guidance of mRNA templates;

[0120] qPCR amplification detection, a pair of specific oligonucleotide probes are specially customized for the target gene sequence for subsequent quantitative analysis. The template cDNA is diluted to an appropriate concentration and mixed with SYBR Green dye and other auxiliary components (such as buffer, deoxynucleotide triphosphates, enhancers and additives) and evenly distributed into each PCR tube and sealed. The annealing temperature and other key parameters are flexibly adjusted according to different experimental requirements to optimize the amplification efficiency and specificity. The real-time quantitative PCR instrument is used to dynamically monitor the changes in fluorescence intensity generated during the entire amplification process, thereby indirectly reflecting the number of copies of the target fragment.

[0121] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for establishing a cerebral ischemia-reperfusion injury model, characterized in that: include: S1: Preparation of experimental materials, selection of healthy adult male SD rats; S2: Anesthesia and fixation: The SD rats to be treated were placed in a sealed container containing 7% isoflurane until they lost consciousness, and then transferred to the operating table and continued to be given 2%-3% isoflurane to maintain a deep anesthesia state, and a constant temperature blanket was used to maintain body temperature; S3: Surgical operation: make a 1 cm long skin incision along the midline of the neck, carefully separate the bilateral common carotid arteries and vagus nerves, and avoid damaging the surrounding tissues; use silk thread to ligate the distal end of the left external jugular vein to reduce bleeding; then, temporarily block the right internal carotid artery with a micro artery clamp 5 mm above the bifurcation point for no more than 30 minutes to prevent irreversible damage; S4: Ischemia induction: According to the predetermined plan, the pre-prepared thrombosis accelerator solution was injected into the left internal carotid artery at a ratio of 0.1 mL / kg body weight. The specific composition ratio was a mixture of FeCl3·6H2O 0.05 mol / L and disodium ethylenediaminetetraacetate 0.01 mol / L, in which Fe 3+ The ion concentration must be precisely controlled to ensure effectiveness without overstimulating the blood vessel wall and causing additional damage. Immediately after the injection, the clamp on the right internal carotid artery is released to allow natural reflow. S5: Reperfusion: the time interval from the release of the micro-arterial clamp to the restoration of normal blood circulation was recorded as one of the indicators of reperfusion duration. At the same time, the laser Doppler technique was used to continuously monitor the changes in blood flow velocity in the middle cerebral artery supply area. S6: Nursing: move the animal to a warm and quiet environment to wake it up, regularly check the wound healing status and give antibiotics to prevent infection; S7: Data analysis.

2. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 1, characterized in that: The anesthesia and fixation of S2 includes: S21: Anesthesia preparation, ensure the sterility of the surgical area, control the temperature between 22-24°C to prevent the animal from getting cold or overheated during the operation; calculate the anesthesia dose based on the weight of the experimental animal; S22: Induce anesthesia, use 7% isoflurane as the induction anesthetic, and perform inhalation general anesthesia through a closed container; closely observe the animal's reaction, and quickly transfer it to the operating table after it loses consciousness; S23: Maintain anesthesia by reducing the isoflurane concentration to 2%-3% to maintain a deep anesthesia state; use a physiological monitor to continuously monitor heart rate and respiratory rate; S24: Fix the animal, place the rat supinely on the operating table, stretch the limbs naturally and fix them gently with tape; use a head frame to fix the head and ensure that the neck is fully exposed for subsequent operations.

3. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 1, characterized in that: The surgical procedures of S3 include: S31: Skin incision, wipe the surgical area with 75% alcohol cotton balls, and then disinfect with iodine twice; make a 1cm long longitudinal incision along the midline of the neck; S32: Separate the tissues and use forceps to separate the subcutaneous tissue to expose the bilateral common carotid arteries and their branches; S33: Ligate the blood vessels. Use silk thread to ligate the distal end of the left external jugular vein 5 mm above the bifurcation point to reduce bleeding. Also use silk thread to temporarily block the blood flow of the right internal carotid artery. The duration should not exceed 30 minutes to prevent irreversible damage.

4. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 1, characterized in that: The ischemic induction of S4 includes: S41: Preparation of thrombosis promoter: weigh the required chemical substances using a precision balance and dissolve them in normal saline according to the proportion to prepare a mixed solution of FeCl3·6H2O 0.05mol / L + disodium ethylenediaminetetraacetate 0.01mol / L; S42: Prepare before injection, use a microinjector to draw a dose of 0.1 mL / kg body weight from the prepared solution; S43: Thrombosis accelerator injection: find the specific location of the left internal carotid artery, and after confirming that it is correct, inject the drug into the target area at a constant speed. Immediately after the injection, release the micro-arterial clamp on the right internal carotid artery.

5. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 1, characterized in that: The reperfusion of S5 includes: S51: Blood flow recovery monitoring, using laser Doppler blood flow meter to monitor the changes in blood flow velocity in the middle cerebral artery supply area in real time; recording the blood flow velocity value at regular intervals until it returns to the baseline level; S52: Observe behavioral performance and check animals regularly to see if they show any abnormal behavior.

6. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 1, characterized in that: The S6 care includes: S61: Management during the recovery period: place the animal in an incubator after surgery and keep the room temperature between 30-32°C; give glucose saline solution orally or intraperitoneally; S62: Wound care: gently wipe the skin around the wound with an iodine cotton swab to prevent infection; give broad-spectrum antibiotics to prevent secondary infection; S63: Long-term follow-up observation, record the animals' food intake, water intake and excrement color indicators for more than one week, weigh them once a week to understand whether their growth and development are normal.

7. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 1, characterized in that: The data analysis of S7 includes: Neurological function score: Garcia test was used to evaluate the degree of neurological deficit at different time points after surgery; Determination of brain edema volume: Calculate the edema space ratio after obtaining image information through magnetic resonance imaging scan; Detection of inflammatory factor levels: Peripheral blood samples were drawn and centrifuged to separate serum, and ELISA kits were used to quantitatively measure the content of TNF-α and IL-1β mediators; Histological evaluation: Fresh brain tissue sections were taken for HE staining to observe the morphological and structural changes; Molecular biology analysis: After RNA was extracted and reverse transcribed into cDNA, the target gene fragment was amplified by qPCR technology to explore the activation status of related signaling pathways.

8. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 7, characterized in that: In S7, the measurement of brain edema volume includes: MRI scanning preparation: ensure that the magnetic resonance imaging machine is in the best working condition and all parameter settings meet the requirements. Fix the rat on the special scanning bed and fix the head with ear bars; Image acquisition: T2-weighted image sequence was used for cross-sectional scanning of the brain, with the slice thickness set to 1 mm, and the image acquisition work within the whole brain was completed according to the preset plan; Data analysis: use medical imaging analysis software to process the original images, remove background noise, manually or automatically identify the edema boundary, and calculate its space occupancy ratio; In S7, the detection of inflammatory factor levels includes: For sample collection, the first, third, and seventh days after surgery were selected as sampling days. 0.5 mL of peripheral blood was drawn by tail vein puncture and placed in EDTA anticoagulant tubes; Serum separation: fresh whole blood samples were placed in a low-temperature centrifuge and spun at 3000 rpm for 10 minutes. The upper clear liquid was taken out as the serum. The obtained serum was divided into several small portions and frozen in a -80℃ refrigerator for later use. For ELISA experiment operation, prepare various necessary reagents, including standard substances and enzyme-labeled antibodies, according to the instructions of the manual, add the samples to be tested and the control substances to the microplate pre-coated with specific antigens, let it stand at room temperature for 1 hour, discard the liquid in the wells and wash several times with PBST buffer, then add the substrate solution and incubate in the dark for an appropriate period of time until the color appears. After terminating the reaction, immediately read the OD value of each well with an enzyme reader, and convert the corresponding concentration value according to the standard curve.

9. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 7, characterized in that: In S7, the histological evaluation includes: After fixation, the whole brain was taken out and placed on an operating table under ice bath condition, and then immediately immersed in 4% paraformaldehyde solution for at least 24 hours to allow it to be fully cross-linked and solidified; HE staining sections were prepared by dehydrating with graded ethanol concentrations and then immersed in xylene until completely transparent. The processed sample blocks were embedded in a melted paraffin mold and cooled to form, cut into 5 μm slices, and then subjected to hematoxylin-eosin staining procedures; Observe and take pictures under a microscope. First use a low-magnification lens to view the overall morphological features, then switch to a high-magnification objective to carefully observe changes in the cell structure and take pictures for archiving.

10. The method for establishing a cerebral ischemia-reperfusion injury model according to claim 7, characterized in that: In S7, the molecular biological analysis comprises: RNA extraction and purification: take fresh or frozen tissue, add TRIZOL reagent, shake vigorously, and let it stand for a few minutes to release the nucleic acid. Add an equal volume of chloroform, shake vigorously for tens of seconds, centrifuge and separate, and then aspirate the upper aqueous phase to continue the subsequent steps. Add 0.5 times the volume of isopropanol to the supernatant, mix it upside down and let it stand for a while to precipitate RNA. Rinse the precipitate repeatedly with 75% ethanol to remove residual impurities, and finally air-dry it naturally to obtain pure RNA powder; cDNA synthesis reverse transcription, using DNase I to digest the total RNA sample to eliminate potential genetic material interference factors, mix the oligo primers, dNTPs, and M-MLV reverse transcriptase components in proportion in an EP tube, and then heat and denature briefly, control the temperature cycle conditions, and promote the synthesis of complementary DNA chains, i.e. cDNA products, under the guidance of the mRNA template; qPCR amplification detection, a pair of specific oligonucleotide probes are specially customized for the target gene sequence for subsequent quantitative analysis. The template cDNA is diluted and mixed with SYBR Green dye and buffer and evenly distributed into each PCR tube and sealed. The real-time quantitative PCR instrument is used to dynamically monitor the changes in fluorescence intensity generated during the entire amplification process, thereby indirectly reflecting the number of copies of the target fragment.

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