Method for establishing a mouse model of new-onset atrial fibrillation after ischemic stroke

Abstract

The application discloses a method for establishing an ischemic stroke new-onset atrial fibrillation mouse model, and belongs to the medical field.The method comprises the following processing steps on the mouse: (1) brain ischemia treatment; (2) behavior evaluation; (3) normally feeding the mouse, which is shown to be successfully modeled by the behavior evaluation, for 3 days; (4) measuring the threshold stimulation of the mouse after step (3); and (5) programmed electrical stimulation to induce atrial fibrillation.The ischemic stroke new-onset atrial fibrillation mouse model established by the method is stable, reliable, high in success rate, low in cost, short in modeling time, and more restores the human pathological model (brain ischemia) induced atrial fibrillation compared with other models, and normal electrocardiogram detection can be used without wearing a device.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a method for establishing a mouse model of new-onset atrial fibrillation after ischemic stroke. BACKGROUND

[0002] Atrial fibrillation (AF) is one of the most common arrhythmias. If not treated in time, AF can cause a series of serious complications, such as heart failure, myocardial infarction, etc., which greatly increases the mortality and morbidity of patients. At present, the pathogenesis of AF is not fully understood. Studies have shown that ischemic stroke is closely related to the occurrence of AF. However, the existing animal models have many limitations: large animal models (such as sheep, dogs) are complex to operate, take a long time to model, and are costly; drug-induced methods are commonly used, but they cannot accurately simulate the pathological process of clinical AF.

[0003] Therefore, it is of great scientific value to develop a mouse model that is easy to operate, low in cost, and can accurately simulate AF after stroke. SUMMARY

[0004] To solve the above technical problems, the present application provides the following technical solutions.

[0005] In a first aspect, the present application provides a method for establishing a mouse model of new-onset atrial fibrillation after ischemic stroke.

[0006] A method for establishing a mouse model of new-onset atrial fibrillation after ischemic stroke, comprising the following processing steps for the mouse:

[0007] (1) cerebral ischemia treatment;

[0008] (2) behavioral evaluation;

[0009] (3) normal feeding of the mouse showing successful modeling in the behavioral evaluation for 3 days;

[0010] (4) measurement of threshold stimulation of the mouse after step (3): the mouse normally fed for 3 days after step (3) is stimulated with a pulse current, the current frequency of the pulse current is 1.1 times the basal heart rate of the mouse, each stimulation lasts for 5 seconds, the stimulation intensity of the pulse current starts from 0.3 mA and gradually increases by 0.1 mA each time, until each P wave is replaced by a stimulation wave (stimulation wave: fluctuation appearing on the electrocardiogram of the mouse caused by the pulse current), and the threshold stimulation current intensity is obtained;

[0011] (5) programmed electrical stimulation to induce atrial fibrillation: the mouse is stimulated 5 times with a pulse current with a threshold stimulation current intensity of 2 times, a current frequency of 40 Hz, each stimulation lasts for 15-30 seconds, and the interval between stimulations is 3 minutes, keeping the interval consistent each time.

[0012] In some embodiments, the brain ischemia treatment of step (1) comprises a 45 minutes to 1 hour occlusion of the internal carotid artery of the mouse with a nylon thread.

[0013] In some embodiments, the brain ischemia treatment of step (1) comprises: anesthesia, shaving the neck, making an incision in the neck, blunt dissection to expose the common carotid artery and vagus nerve, treating the internal carotid artery of the mouse with a nylon thread for 45 minutes to 1 hour, withdrawing the nylon thread, restoring blood circulation, and suturing.

[0014] In some embodiments, the behavioral evaluation comprises: 3 hours after the treatment of step (1), performing a behavioral evaluation on the mouse, the behavioral evaluation using the Zea Longa 5-point scoring standard,

[0015] wherein,

[0016] normal activity is 0 points, indicating no signs of nerve damage;

[0017] inability to fully extend the contralateral forelimb during the tail-flick test is 1 point, indicating mild focal injury;

[0018] contralateral rotation during walking is 2 points, indicating moderate focal injury;

[0019] leaning to the contralateral side at rest is 3 points, indicating severe focal injury;

[0020] no voluntary activity with disturbance of consciousness is 4 points, indicating extremely severe focal injury;

[0021] death is 5 points;

[0022] a score of 1-4 points indicates successful modeling.

[0023] In some embodiments, the step (4) and step (5) use an esophageal electrode catheter for stimulation.

[0024] In some embodiments, the stimulation of step (4) and step (5) uses a square wave with a pulse width of 1 ms.

[0025] In some embodiments, the stimulation of step (4) and step (5) uses an S1S1 stimulation mode.

[0026] In some embodiments, the step (5) comprises: using a 2-fold threshold stimulation intensity, stimulating at a frequency of 40 Hz for 5 times, each lasting 15 seconds, with an interval of 3 minutes, and keeping each interval consistent.

[0027] In some embodiments, the mouse is a C57 mouse.

[0028] In a second aspect, the present application provides an application of the aforementioned establishment method.

[0029] The first aspect of the establishment method constructs the ischemic stroke newly developed atrial fibrillation mouse model in the mechanism research of cerebrovascular disease and heart disease and / or in the preparation of a drug for preventing or treating diseases related to ischemic stroke newly developed atrial fibrillation.

[0030] Advantages

[0031] Compared with the prior art, the embodiment of the present application has at least one of the following advantages:

[0032] (1) The establishment method provided by the present application is stable, reliable, has high success rate, low cost, and short modeling time.

[0033] (2) The model provided by the present application is more close to the pathological model (brain ischemia) induced by atrial fibrillation of human beings, and does not need to wear a device, and ordinary electrocardiogram detection can be used.

[0034] (3) The innovation of the present application is to combine the pathological model of brain ischemia, to induce the pathological model of atrial fibrillation by reducing the disease of human beings, and this model is more close to the disease state that human beings will face compared with other vagus nerve stimulation and lipopolysaccharide injection.

[0035] (4) When the programmatic electrical stimulation is used to induce atrial fibrillation, compared with other current frequencies (such as 10Hz, 20Hz, 30Hz, 50Hz), the pulse current of 40Hz is more conducive to improving the success rate of atrial fibrillation, the number of atrial fibrillation and the total duration of atrial fibrillation, and has unexpected technical effects.

[0036] (5) For the behavior evaluation of the mice successfully modeled, compared with other normal feeding times, the normal feeding time of 3 days is more conducive to improving the success rate of atrial fibrillation, the number of atrial fibrillation and the total duration of atrial fibrillation, and has unexpected technical effects.

[0037] (6) Compared with the other carotid line plug processing time, the carotid line plug processing time of 45 minutes to 1 hour in the present application is more conducive to improving the success rate of atrial fibrillation, the number of atrial fibrillation and the total duration of atrial fibrillation, and has unexpected technical effects.

[0038] Term explanation

[0039] In the description of the present application, the term "room temperature" means ambient temperature, which refers to a temperature of about 10℃ to about 30℃, or about 20℃ to 30℃, or about 25℃.

[0040] The term "wt%" means mass percentage.

[0041] The term "W / W" means mass ratio.

[0042] The term "S1S1 stimulation mode" is a periodic stimulation mode, i.e. the heart is continuously acted on by stimulation pulses with a fixed frequency and consistent intervals. Usually the frequency is close to or slightly higher than the natural heart rate, to simulate normal heart rhythm. Among them, "S1 stimulation" means normal stimulation pulse, and the interval (S1-S1) between each pulse in a group of pulses is constant. By controlling the interval time of S1-S1, the response of the heart at different rates can be explored.

[0043] The term "P wave" means atrial depolarization wave.

[0044] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0045] In the following, all the numbers disclosed herein are approximate, regardless of whether the words "approximately" or "about" are used. The value of each number can vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20% or the like. Whenever a number with a value of N is disclosed, any number with a value of N + / - 1%, N + / - 2%, N + / - 3%, N + / - 5%, N + / - 7%, N + / - 8%, N + / - 10%, N + / - 15% or N + / - 20% is explicitly disclosed, where "+" or "-" means plus or minus. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Statistical analysis graph of the number of atrial fibrillation in ischemic stroke group mice and control group.

[0047] Figure 2 Statistical analysis graph of the total duration of atrial fibrillation in ischemic stroke group mice and control group.

[0048] Figure 3 Comparison chart of brain laser speckle blood flow imaging before and after operation in ischemic stroke group mice.

[0049] Figure 4Statistical analysis chart of relative cerebral blood flow of the mice in the ischemic stroke group (referred to as the cerebral ischemia group) and the mice in the control group.

[0050] Figure 5 Comparison chart of cerebral infarction area of the mice in the ischemic stroke group and the mice in the control group.

[0051] Figure 6 Statistical analysis chart of infarction volume of the mice in the ischemic stroke group and the mice in the control group.

[0052] Figure 7 Comparison chart of Masson staining results of the left atrial part of the atrium of the mice in the ischemic stroke group and the mice in the control group.

[0053] Figure 8 Comparison chart of HE staining results of the left atrial part of the heart of the mice in the ischemic stroke group and the mice in the control group.

[0054] Figure 9 Comparison chart of the proportion of left atrial fibrous tissue of the left atrial part of the heart of the mice in the ischemic stroke group and the mice in the control group.

[0055] Figure 10 Comparison chart of CD68 immunohistochemical results of the left atrial part of the heart of the mice in the ischemic stroke group and the mice in the control group.

[0056] Figure 11 Comparison chart of the number of CD68 expression under 40 times of the left atrial part of the heart of the mice in the ischemic stroke group and the mice in the control group.

[0057] In the figure, “ns” represents no statistically significant difference, and “*” represents that there is a statistically significant difference.

[0058] In the figure, the cerebral ischemia group represents the ischemic stroke group. DETAILED DESCRIPTION

[0059] In order to enable those skilled in the art to better understand the technical solutions of the present application, some non-limiting embodiments are further disclosed below, and the present application is further described in detail.

[0060] The reagents used in the present application can be purchased from the market or can be prepared by the method described in the present application.

[0061] The experimental instruments used in the present application are as follows:

[0062] C57 mice: Murine (Wuhan) Biotechnology Co., Ltd.

[0063] Small animal anesthesia machine: Shanghai Yurui Scientific Instrument Co., Ltd.

[0064] Laser speckle blood flow imaging instrument: Xunwei Optoelectronics Technology Co., Ltd.

[0065] Desktop computer: China Hewlett-Packard Co., Ltd.

[0066] ALC-V8S small animal respirator: Shanghai Aurkort Biological Technology Co., Ltd.

[0067] 4.5F6 polar esophageal electrode catheter and adapter box: Chengdu Taimeng Software Co., Ltd.

[0068] BL-420N biological signal acquisition and analysis system: Chengdu Taimeng Software Co., Ltd.

[0069] YC-3 type program-controlled stimulator: Chengdu Instrument Factory

[0070] Threaded bolt: Changsha Mayue Bioscience Co., Ltd.

[0071] Others: hair removal machine, fine tweezers 2, ophthalmic scissors (small) 1, hemostatic forceps, 1 ml syringe, rewarming plate, mouse cage, mouse table, ophthalmic curved forceps 1, ophthalmic straight forceps 1, curved hemostatic forceps (small) 1, curved hemostatic forceps 1, operating table (foam board), and bottom line.

[0072] Experimental consumables: 22G core tracheal cannula, 3M medical adhesive, cotton ball, coarse silk thread, 1 ml syringe, acupuncture needle (conductive), plasticine, thick gloves, erythromycin ointment, erythromycin eye ointment, depilatory paste, iodophor, disinfectant alcohol, hydrate chloral, surgical suture and suture needle, surgical cotton thread, isoflurane, normal mouse feed and drinking water.

[0073] Example 1: Model construction

[0074] 1. Experimental method

[0075] (1) Experimental animal preparation: 10 C57 mice aged 8-10 weeks were selected for the experiment, and they were adaptively fed for 3 days to ensure that the mice fully adapted to the experimental environment and avoid environmental factors affecting the experimental results. The mice were randomly divided into two groups: 5 in the control group and 5 in the ischemic stroke group (model group). All mice were numbered for experimental recording and management.

[0076] (2) Anesthesia treatment: C57 mice were anesthetized with isoflurane / oxygen mixed gas. The isoflurane concentration was 3% during anesthesia induction and 1.5% during anesthesia maintenance. The mice were placed in the gas anesthesia device for 2 minutes, and after observing that their activity was significantly reduced, they were fixed on the experimental table and the anesthesia state was continued.

[0077] (3) Blood flow imaging: The mouse brain was shaved using a mouse-specific hair clipper, followed by application of depilatory cream and scraping of the remaining hair. The exposed skin was disinfected with alcohol. To protect the eyes, erythromycin ophthalmic ointment was applied to the mouse eyes. The mouse scalp was incised with sterile scissors to expose the skull, and the laser speckle blood flow imager was started for brain blood flow imaging observation and recording. After recording was completed, erythromycin ointment was applied to the surface of the skull.

[0078] (4) Neck surgical preparation: The mouse was fixed supine on the surgical board, and the heating pad temperature was set to 37°C ± 0.5°C to maintain its body temperature. The mouse limbs were fixed with medical tape, and its anesthetic state was continuously maintained. The neck hair was shaved using a hair clipper, and the remaining hair was scraped after applying depilatory cream, and the exposed skin was disinfected with alcohol. A 1.5 cm incision was made in the middle of the neck with sterile scissors, and the anterior cervical muscle group was exposed and the connective tissue between the sternohyoid muscles was separated to gradually expose the common carotid artery and vagus nerve.

[0079] (5) Brain ischemia treatment

[0080] Arterial line plug implantation in the ischemic stroke group (model group): Two surgical cotton threads were placed above the common carotid artery, one at the bifurcation of the common carotid artery to tie a knot for fixing the line plug, and the other at the proximal end of the common carotid artery and tied. The external carotid artery and internal carotid artery were then separated and tied with surgical cotton threads. A "V" shaped small opening was cut on the common carotid artery using ophthalmic scissors, the line plug was inserted into the artery, and the line plug was fixed at the knot at the bifurcation of the common carotid artery to ensure that no blood flows out when the line plug moves. Loosen the knot of the internal carotid artery, quickly insert the line plug into the internal carotid artery, and avoid bleeding and insertion resistance during operation. If resistance is encountered, the position of the line plug should be adjusted in time to avoid damage to the sphenopalatine artery. The line plug was inserted to a depth of about 1 cm to block the middle cerebral artery. After insertion was completed, the knot of the common carotid artery was tightened again to fix the line plug, and the occlusion state was maintained for 1 hour.

[0081] Control group: bluntly dissect the salivary gland, then place two surgical cotton threads above the common carotid artery, one at the bifurcation of the common carotid artery, tie a knot, and the other at the proximal end of the common carotid artery and tie, cut off the left carotid artery.

[0082] (6) Suture

[0083] Ischemic stroke group (model group) line plug removal and suture: while maintaining the state of anesthesia of the mouse, slowly withdraw the inserted line plug, and tie the slipknot again at the bifurcation of the common carotid artery to ensure normal recovery of blood flow. Then, using a surgical needle and suture, the neck incision is carefully sutured layer by layer. After the suture is completed, the surgical area is disinfected with iodophor and alcohol. The mouse is placed on the experimental table, and the laser speckle blood flow imager is used again to monitor the change of brain blood flow, to ensure the success of the construction of the cerebral ischemia model. After confirming the success of the modeling, the small animal anesthesia equipment is turned off, and the mouse is placed on the heating pad until it fully awakens.

[0084] Control group: while maintaining the state of anesthesia of the mouse, using a surgical needle and suture, the neck incision is carefully sutured layer by layer. After the suture is completed, the surgical area is disinfected with iodophor and alcohol. The mouse is placed on the experimental table, and the laser speckle blood flow imager is used again to monitor the change of brain blood flow, to ensure the success of the construction of the cerebral ischemia model. After confirming the success of the modeling, the small animal anesthesia equipment is turned off, and the mouse is placed on the heating pad until it fully awakens.

[0085] (7) Behavioral evaluation: after 3h of modeling, the C57 mice after modeling were scored for behavior, and the behavior score was scored according to the Zea Longa 5-point scoring standard: see Table 1. The results are shown in Table 2.

[0086] Table 1: Degree of neurological impairment score

[0087]

[0088] Among them, the score of 1-4 is considered to be a successful modeling (5 points are theoretically considered to be a successful modeling, but since the 5-point mouse died, it could not continue the experiment, so the present application takes 1-4 as the standard for successful modeling).

[0089] Table 2: Group ischemic stroke group and control group score

[0090]

[0091] (8) The C57 mice after modeling were returned to the mouse cage and normally fed.

[0092] (9) Induction of pre-arrhythmia preparation: On the third day after modeling, the mice were taken out and weighed. Then, 10% chloral hydrate (350 mg / kg) was given by intraperitoneal injection with a 1 ml syringe for anesthesia. When the mouse limbs were clamped with hemostatic forceps without obvious reaction, it was confirmed that the anesthesia was successful. Then the mouse was placed on the experimental table, and erythromycin eye ointment was applied to the eyes to protect the eyes. The cerebral blood flow of the mouse was monitored and recorded using a laser speckle blood flow imager to ensure that the brain was still in an ischemic state. The mouse was fixed on the operating table in a supine position, and the limbs were fixed in turn using medical tape, starting with the hind limbs, then straightening the mouse's lower jaw by pulling the body with a thread, straightening the body and fixing it with a hemostatic forceps, and then fixing the front limbs. After completing the fixation, the operating table was raised, and the mouse's head and neck were illuminated with a table lamp. The tongue was gently pulled to one side with tweezers, and the tongue was gently pushed towards the abdomen with a hemostatic forceps or straight forceps to ensure a sufficient operating field of view. The white bright spot that jumps is the tracheal marker, and the tracheal tube is inserted in parallel along the tracheal direction, and the metal core is quickly pulled out after insertion. Then the operating table was leveled and connected to the respirator, and the chest was confirmed to be synchronous with the respirator, and the mouse's limb circulation was good, to confirm the success of the tracheal tube.

[0093] (10) ECG recording and esophageal electrode adjustment: Sterilized acupuncture needles were inserted subcutaneously into the mouse's limbs, with an insertion depth of about 0.5 cm, and connected with electrocardiogram electrodes, which were fixed firmly with adhesive tape. According to the supine position of the mouse, the ECG lead wires and two leads were connected to the acupuncture needles. Start the BL-420N biological signal acquisition and analysis system software, select channels 1 to 3, lead selection aVR, sampling frequency set to 1 kHz or 2 kHz, ensure that the parameters of each channel are consistent, and the rate and time can use the default settings. At the same time, the position of the grounding wire needs to be adjusted to minimize external noise interference. Connect the 4.5F6 polar esophageal electrode catheter to the adapter box, and connect the two leads to the adapter box to record the ECG signal of the esophageal electrode catheter. Before inserting the esophageal electrode, it is first soaked in physiological saline and inserted into the mouse along the esophageal direction. The specific insertion depth should be adjusted according to the changes in the electrocardiogram signal, and the correct placement is confirmed by atrial capture. At this time, the esophageal electrode is fixed to prevent displacement.

[0094] (11) Stimulation program setting: Start the stimulator software and perform electrical stimulation according to the following steps. All stimulation programs use square waves with a pulse width of 1 ms, unless otherwise specified, and use the S1S1 stimulation mode by default.

[0095] (i) Threshold stimulation measurement: After the aforementioned normal feeding for 3 days, the mouse was stimulated with a pulse current, the current frequency of which was 1.1 times the basal heart rate of the mouse (calculated as the number of heartbeats per minute / 60). For example, if the measured basal heart rate of the mouse was 600 beats per minute, the current frequency of the pulse current was 600÷60×1.1 Hz = 11 Hz, and so on. Each stimulation lasted 5 seconds, and the pulse current stimulation intensity was gradually increased from 0.3 mA, with an increase of 0.1 mA each time, until each P wave was replaced by a stimulation wave, to obtain the threshold stimulation current intensity.

[0096] (ii) Atrial fibrillation induction: After the threshold stimulation was measured, the mouse was stimulated with a pulse current with a threshold stimulation current intensity of 2 times and a current frequency of 40 Hz, for 5 times, each lasting 15 seconds, with an interval of 3 minutes, and the interval was kept consistent each time.

[0097] (12) Experimental data collection: The number of times of atrial fibrillation induction and the duration of atrial fibrillation of the C57 mouse were recorded. The total duration of atrial fibrillation was the cumulative value of the time of each occurrence of atrial fibrillation.

[0098] 2. Experimental results:

[0099] Table 3 lists whether atrial fibrillation was successfully induced in each C57 mouse in the middle cerebral artery ischemia 1 hour model group and the control group, as well as the induction rate of atrial fibrillation and the total duration of atrial fibrillation.

[0100] Figure 1 The graph is a statistical analysis of the number of times of atrial fibrillation in the ischemic stroke group and the control group.

[0101] Figure 2 The graph is a statistical analysis of the total duration of atrial fibrillation in the ischemic stroke group and the control group.

[0102] In the graph, "ns" indicates no statistically significant difference, and "*" indicates a statistically significant difference.

[0103] According to the statistical analysis, the atrial fibrillation induction rate of the experimental group (brain ischemia model group) mice was significantly higher than that of the control group (P<0.05). At the same time, the total duration of atrial fibrillation of the experimental group mice was also significantly higher than that of the control group (P<0.05). These results show that the C57 mouse induced by a linear plug to induce brain ischemia can be used as a new type of atrial fibrillation induction model, which meets the requirements of the construction of an experimental animal model.

[0104] Table 3: Atrial fibrillation results of the model group and the control group

[0105]

[0106] Example 2: Changes in brain blood flow laser speckle imaging

[0107] 1) The cerebral blood flow of mice was measured using a laser speckle blood flow imaging system (LSCI). The system consists of a wheeled workstation, a laser speckle imaging head, and a flexible connecting arm that can move in both horizontal and vertical directions. The imaging head emits a laser beam with a wavelength of 785 nm, equipped with a 12-bit charge-coupled device (CCD) camera. The system captures laser speckle images through the CCD camera, and a filter is installed in front of the camera to eliminate background light interference. The exposure time of the CCD camera is set to 13 milliseconds.

[0108] 2) Each mouse was anesthetized with isoflurane / oxygen gas mixture, and its body temperature was maintained at 37°C ± 0.5°C by a heating pad. The mouse's skull was exposed and cleaned by cutting the median skin. During image acquisition, a sanitary cotton swab was used to keep the exposed area clean and dry, and to ensure that the mouse's brain was perpendicular to the incident light. The focal length of the CCD camera was adjusted as needed to achieve clear imaging.

[0109] 3) The distance between the CCD camera and the target area was maintained at 10-15 cm. The blood flow signal of the mouse's brain was collected at a wavelength of 785 nm, and the signal was converted into a blood perfusion image using LSCI software. One frame of image was collected per second, for a total of 20 frames. The collected brain blood flow images were analyzed by LSCI software, and a circular area with a diameter of 100 mm 2 was selected for observation at fixed positions symmetrically on both sides of the mouse's brain. The LSCI software automatically calculated and generated the average blood flow index (BFI) of the observed area. This study analyzed the dynamic changes of cerebral blood flow by comparing the changes in cerebral blood flow between the injured side and the contralateral side.

[0110] 4) The relative change in cerebral blood flow was calculated using the following formula:

[0111] Relative change in cerebral blood flow = (average blood flow index of injured side - average blood flow index of contralateral side) / average blood flow index of contralateral side

[0112] 5) Results

[0113] Figure 3 The contrast image of the laser speckle blood flow imaging of the brain of the ischemic stroke group of mice before and after surgery.

[0114] Figure 4 Statistical analysis of the relative cerebral blood flow changes of the ischemic stroke group of mice (referred to as the brain ischemia group) and the control group of mice.

[0115] In the figure, "ns" indicates no statistically significant difference, and "*" indicates a statistically significant difference.

[0116] 6) Results analysis

[0117] The experiment recorded the changes of cerebral blood flow of the mice in the middle cerebral artery ischemia 1 hour model group before and after the operation. The experimental results showed that compared with the control group, the cerebral blood flow of the mice in the cerebral ischemia model group was significantly reduced, indicating that the ischemia model was successfully constructed.

[0118] Example 3: TTC staining method to evaluate cerebral infarction area

[0119] 1) After deep anesthesia with 4% chloral hydrate in C57 mice, the head was cut off with scissors, the skull was opened through the foramen magnum, and the brain tissue was taken out.

[0120] 2) The removed brain tissue was quickly frozen in a refrigerator at-20℃ for 20 minutes. Then, the brain tissue was placed on a pre-cooled brain section mold and cut into 2mm-thick sections.

[0121] 3) The sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution and incubated at 37℃ for 30 minutes. Shake gently every 5 minutes to ensure uniform staining. After incubation, wash the sections in PBS buffer solution once, and then take pictures for record.

[0122] 4) Use Image J software to measure the infarction area and total brain area of each section, and calculate the proportion of infarction volume. The formula for calculating the proportion of infarction volume is:

[0123] Infarction volume percentage = (sum of infarction area / sum of total brain area) x 100%.

[0124] 5) Results

[0125] Figure 5 The figure is a comparison of the infarction area of the ischemic stroke group mice and the control group mice.

[0126] Figure 6 The figure is a statistical analysis result of the infarction volume of the ischemic stroke group mice and the control group mice.

[0127] In the figure, "ns" means no statistically significant difference, and "*" means statistically significant difference.

[0128] 6) Results analysis

[0129] Compared with the control group, the brain tissue sections of the middle cerebral artery ischemia 1 hour model group mice showed obvious cerebral infarction area under TTC staining, indicating that the model group mice had significant cerebral ischemic injury.

[0130] Example 4: Masson staining to detect myocardial fibrosis

[0131] Masson staining experimental method

[0132] 1) The embedded C57 mouse myocardial tissue was cut into continuous sections with a thickness of 4 μm. After sectioning, the specimen was baked in a 60°C oven for 2 hours. Subsequently, the section was sequentially immersed in environmentally friendly deparaffinizing solution I for 20 minutes, environmentally friendly deparaffinizing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 75% alcohol for 5 minutes, and tap water for cleaning.

[0133] 2) Rewarming and fixing of the frozen section: after the frozen section was taken out from the -20°C refrigerator and restored to room temperature, the section was fixed with a tissue fixing solution for 15 minutes, and then washed with running water.

[0134] 3) The section was immersed in a staining solution mixed with Masson B solution and Masson C solution in equal proportions for 1 minute, and then washed with tap water. Subsequently, the section was differentiated for a few seconds using a differentiation solution, and then washed with tap water.

[0135] 4) The section was immersed in Masson D solution for 6 minutes, and then washed with tap water.

[0136] 5) The section was immersed in Masson E solution for 1 minute.

[0137] 6) Without washing with water, after slightly draining, the section was directly immersed in Masson F solution for 2 to 30 seconds.

[0138] 7) The section was washed and differentiated with 1% acetic acid, and then dehydrated with two cylinders of anhydrous ethanol.

[0139] 8) Transparency and mounting: the section was immersed in the third cylinder of anhydrous ethanol for 5 minutes, and then immersed in xylene for 5 minutes for transparency treatment, and finally mounted with neutral balsam.

[0140] 9) The section was observed under a microscope, and the images were collected and analyzed.

[0141] 10) In the Masson staining result, the collagen fibers were blue, and the muscle fibers, cellulose and red blood cells were red.

[0142] 11) Results

[0143] Figure 7 Comparison chart of Masson staining results of the left atrial part of the atrium of the ischemic stroke group mice and the control group mice.

[0144] 12) Analysis of results

[0145] The results show that, after 1 hour of middle cerebral artery ischemia, the mouse model is normally fed for three days, and the atrial myocardial cells of the model group mice show blue after Masson staining, indicating that the atrial part has obvious myocardial fibrosis compared with the control group.

[0146] Example 5: HE Staining

[0147] HE Staining Experimental Method

[0148] 1) Paraffin section deparaffinization to water: sequentially immerse the section in environmental protection type deparaffinization liquid I for 10 minutes, and then immerse it in environmental protection type deparaffinization liquid II and environmental protection type deparaffinization liquid III respectively for 10 minutes. Subsequently, immerse the section in anhydrous ethanol I, II and III respectively for 5 minutes, and finally wash it with distilled water.

[0149] 2) Rewarming and fixing of frozen section: take the frozen section out of the refrigerator at-20℃, restore it to room temperature, and then fix it with tissue fixing liquid for 15 minutes, and then rinse it with running water.

[0150] 3) Pretreatment: immerse the section in high-definition constant dyeing pretreatment liquid for 1 minute.

[0151] 4) Hematoxylin staining: immerse the section in hematoxylin dyeing liquid for 3-5 minutes, and then rinse it with tap water. After differentiation treatment with differentiation liquid, rinse it with tap water again, and then return it to blue with blue returning liquid, and finally rinse it with running water.

[0152] 5) Eosin staining: dehydrate the section in 95% alcohol for 1 minute, and then immerse it in eosin dyeing liquid for 15 seconds.

[0153] 6) Dehydration and mounting: sequentially immerse the section in anhydrous ethanol I, II and III for 2 minutes respectively, and then immerse it in n-butanol I and II for 2 minutes respectively. Subsequently, immerse the section in xylene I and II for 2 minutes respectively. After drying the slide rack in the fume hood, mount it with neutral gum. Observe the section tissue morphology under an optical microscope, and the cell nucleus appears blue and the cytoplasm appears red.

[0154] 7) Microscope examination and image acquisition: observe the tissue morphology under a microscope, and acquire images for analysis.

[0155] 8) Results

[0156] In the HE staining results, the cell nucleus appears blue and the cytoplasm appears red.

[0157] Figure 8 Comparison chart of HE staining results of the left atrial part of the heart of the ischemic stroke group mice and the control group mice.

[0158] Figure 9Figure 4 shows the comparison of the proportion of left atrial fibrous tissue in the left atrial part of the heart of the ischemic stroke group and the control group.

[0159] 9) Result analysis

[0160] The HE staining result shows that the nucleus of the ischemic stroke group is larger than that of the control group, indicating that the left atrial part of the heart of the ischemic stroke group has a higher degree of inflammatory cell infiltration and more serious inflammation than the control group. It can be seen from Figure 9 that there is a statistically significant difference between the ischemic stroke group and the control group.

[0161] Example 6: CD68 immunohistochemical experiment

[0162] CD68 immunohistochemical experiment method

[0163] 1) Paraffin section deparaffinization to water: Put the section into environmental protection type deparaffinization liquid I, II and III for 10 minutes respectively, then put it into anhydrous ethanol I, II and III for 5 minutes respectively, and finally wash it with distilled water.

[0164] 2) Antigen repair: Put the section into EDTA pH 8.0 hot repair solution for 30 minutes, and prevent the repair solution from evaporating too much and the section from drying during the process. After natural cooling, put the section into PBS (pH 7.4) and wash it on the decolorization shaker for 3 times, 5 minutes each time. (The repair solution and repair conditions are determined according to the type of tissue)

[0165] 3) Block endogenous peroxidase: Put the section into 3% hydrogen peroxide solution, incubate at room temperature for 25 minutes in the dark, and then put the section into PBS (pH 7.4) and wash it on the decolorization shaker for 3 times, 5 minutes each time.

[0166] 4) Serum blocking: Add 3% BSA evenly in the histological circle to cover the tissue, and block at room temperature for 30 minutes. (If the primary antibody is goat-derived, use rabbit serum for blocking, and use BSA for blocking for other antibodies)

[0167] 5) Primary antibody reaction: After removing the excess blocking solution, add the CD68 antibody prepared in proportion on the section, and put the section flat in the wet box and incubate at 4°C overnight.

[0168] 6) Secondary antibody reaction: Put the section into PBS (pH 7.4) and wash it on the decolorization shaker for 3 times, 5 minutes each time. After shaking off the section, add goat anti-rabbit IgG secondary antibody labeled with HRP (horseradish peroxidase) in the histological circle, and incubate at room temperature for 50 minutes.

[0169] 7) DAB color development: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time. After spinning dry, drop fresh DAB color development solution on the section, control the color development time under microscope, the positive signal appears brownish yellow. Rinse the section with tap water to stop the color development reaction.

[0170] 8) Nucleus restaining: Place the section in hematoxylin staining solution for about 3 minutes, then rinse with tap water. After differentiating for a few seconds using differentiation solution, rinse with tap water again, then return to blue using blue return solution and rinse with running water.

[0171] 9) Dehydration and mounting: Place the section in 75% alcohol and 85% alcohol for 5 minutes each, then in anhydrous ethanol I and II for 5 minutes each, then in n-butanol and xylene for 5 minutes each for transparency treatment. After taking out the section and slightly drying, mount using mounting medium.

[0172] 10) Microscopy: Observe and interpret the results of the section under a white light microscope.

[0173] 11) Results

[0174] Figure 10 Comparison chart of CD68 immunohistochemical results of left atrial part of heart of ischemic stroke group mice and control group mice.

[0175] Figure 11 Comparison chart of CD68 expression quantity results under 40 times magnification of left atrial part of heart of ischemic stroke group mice and control group mice.

[0176] Result description: CD68 is a transmembrane glycoprotein that can be highly expressed in macrophages. The more CD68 is expressed, the more macrophages there are, and the cells may have an inflammatory response.

[0177] 12) Results analysis:

[0178] The CD68 immunohistochemical analysis results show that the CD68 protein in the left atrial part of the heart of the ischemic stroke group is significantly increased compared to the control group, indicating that the number of macrophages is significantly increased, and the inflammation of the left atrium of the heart of the ischemic stroke group is more serious.

[0179] Comparative example 1-comparative example 4: investigation of pulse current frequency

[0180] According to the method of reference example 1, adjust the 40 Hz pulse current frequency in step (ii) to the pulse current frequencies in Table 4, and the rest of the operations are the same as example 1. The success rate of model construction and the induction rate of atrial fibrillation of different pulse current frequencies are detected. The results are shown in Table 4.

[0181] Table 4: success rate of model construction of different pulse current frequencies

[0182]

[0183] Conclusion: Compared with other pulse current frequencies, the use of 40Hz is more conducive to improving the success rate of model construction, the number of atrial fibrillation and the total duration of atrial fibrillation, and has unexpected technical effects.

[0184] The method of the present application has been described by preferred embodiments, and the related personnel can obviously make changes or appropriate changes and combinations to the method and application described herein within the content, spirit and scope of the present application to realize and apply the present application technology. Those skilled in the art can refer to the content herein to realize by appropriately improving the process parameters. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all considered to be included in the present application.

Claims

1. A method for establishing a mouse model of ischemic stroke-induced new-onset atrial fibrillation, characterized in that, The method comprises the following steps for the mouse: (1) brain ischemia treatment; (2) behavior evaluation; (3) normal feeding of the mouse with successful modeling shown by the behavior evaluation for 3 days; (4) threshold stimulation measurement of the mouse after normal feeding for 3 days in step (3): the mouse after normal feeding for 3 days in step (3) is stimulated by pulse current, the pulse current has a current frequency of 1.1 times of the basic heart rate of the mouse, each stimulation lasts for 5 seconds, the stimulation intensity of the pulse current is gradually increased from 0.3 mA, each time by 0.1 mA, until each P wave of the electrocardiogram of the mouse is replaced by the stimulation wave, and the threshold stimulation current intensity is obtained; (5) programmed electrical stimulation to induce atrial fibrillation: the mouse is stimulated by pulse current with a threshold stimulation current intensity of 2 times, a current frequency of 40 Hz, 5 times, each time lasts for 15 seconds, and the interval is 3 minutes, and the interval is kept consistent each time; The brain ischemia treatment in step (1) comprises carotid linear plug for 45 minutes to 1 hour for the mouse; The behavior evaluation in step (2) comprises behavior evaluation for the mouse 3 hours after the treatment in step (1), and the behavior evaluation adopts Zea Longa 5-point scoring standard, wherein, normal activity is 0 point, indicating no neurological injury signs; inability to fully stretch the contralateral forelimb in the tail lifting experiment is 1 point, indicating mild focal injury; contralateral rotation during walking is 2 points, indicating moderate focal injury; leaning to the contralateral side during rest is 3 points, indicating severe focal injury; no spontaneous activity with consciousness disorder is 4 points, indicating extremely severe focal injury; death is 5 points; a score of 1-4 points indicates successful modeling.

2. The establishment method according to claim 1, wherein the stimulation in steps (4) and (5) is performed by using an esophageal electrode catheter.

3. The establishment method according to claim 1, wherein the current of the stimulation in steps (4) and (5) is a square wave with a pulse width of 1 ms.

4. The establishment method according to any one of claims 1-3, wherein the stimulation in steps (4) and (5) adopts an S1S1 stimulation mode.

5. The establishment method according to any one of claims 1-3, wherein the mouse is a C57 mouse.

6. Application of the ischemic stroke and new-onset atrial fibrillation mouse model established by the method according to any one of claims 1-5 in mechanism research of cerebrovascular diseases and heart diseases and / or in preparation of a drug for preventing or treating diseases related to ischemic stroke and new-onset atrial fibrillation.

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