Establishment method of cerebral arterial thrombosis new atrial fibrillation mouse model

Abstract

The invention discloses a method for establishing a new atrial fibrillation mouse model with cerebral arterial thrombosis, and belongs to the field of medicine. The method comprises the following treatment steps for mice: (1) cerebral ischemia treatment; (2) behavioral evaluation; (3) normally feeding the mice of which the behavioral evaluation shows that the modeling is successful for 3 days; (4) measuring the threshold stimulation of the mouse after the step (3); and (5) performing programmed electrical stimulation to induce atrial fibrillation. The cerebral arterial thrombosis new atrial fibrillation mouse model established by the method is stable, reliable, high in success rate, low in cost and short in modeling time; compared with other models, the model provided by the invention can restore atrial fibrillation induced by a pathological model (cerebral ischemia) of human beings, does not need wearable equipment, and only needs common electrocardiogram detection.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to a method for establishing a mouse model of newly-onset atrial fibrillation due to ischemic stroke. Background Art

[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 and myocardial infarction, which greatly increases the mortality and disability rates of patients. At present, the pathogenesis of AF has not been fully clarified. Studies have shown that ischemic stroke is closely related to the occurrence of AF. However, existing animal models have many limitations: large animal models (such as sheep and dogs) are complex to operate, take a long time to model, and are expensive; although drug induction methods are more commonly used, they cannot accurately simulate the pathological process of clinical AF.

[0003] Therefore, developing a mouse model that is easy to operate, low-cost, and can accurately simulate post-stroke atrial fibrillation has important scientific research value. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions.

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

[0006] A method for establishing a mouse model of newly-onset atrial fibrillation due to ischemic stroke, comprising the following steps of treating the mouse:

[0007] (1) Treatment of cerebral ischemia;

[0008] (2) behavioral assessment;

[0009] (3) Mice that showed successful modeling based on behavioral evaluation were kept normally for 3 days;

[0010] (4) measuring the threshold stimulation of mice after step (3): stimulating the mice after step (3) after normal feeding for 3 days with pulse current, wherein the current frequency of the pulse current is 1.1 times the basal heart rate of the mice, each stimulation lasts for 5 seconds, and the pulse current stimulation intensity gradually increases from 0.3 mA, each time increasing by 0.1 mA, until each P wave is replaced by a stimulation wave (stimulation wave: a fluctuation on the electrocardiogram of the mouse caused by the pulse current), and obtaining the threshold stimulation current intensity;

[0011] (5) Programmed electrical stimulation to induce atrial fibrillation: The mice were stimulated five times with a pulse current of 2 times the threshold stimulation current intensity and a current frequency of 40 Hz, each lasting 15 to 30 seconds, with a stimulation interval of 3 minutes, and the interval was kept consistent each time.

[0012] In some embodiments, the cerebral ischemia treatment of step (1) comprises applying carotid artery occlusion to mice for 45 minutes to 1 hour. In some embodiments, the cerebral ischemia treatment of step (1) comprises applying carotid artery occlusion to mice for 45 minutes, 50 minutes, 55 minutes or 1 hour.

[0013] In some embodiments, the brain ischemia treatment in step (1) includes: anesthesia, neck hair removal, neck incision, blunt separation to expose the common carotid artery and vagus nerve, treating the internal carotid artery of the mouse with a carotid artery suture for 45 minutes to 1 hour, removing the carotid artery suture, restoring blood circulation, and suturing.

[0014] In some embodiments, the behavioral assessment includes: 3 hours after the treatment in step (1), performing a behavioral assessment on the mice, wherein the behavioral assessment adopts the Zea Longa 5-point scoring standard.

[0015] in,

[0016] Normal activity was scored as 0, indicating no signs of neurological damage;

[0017] The inability to fully extend the contralateral forelimb during the tail-lift test was scored as 1 point, indicating a mild focal injury;

[0018] Rotation to the contralateral side during walking was scored as 2 points, indicating a moderate focal injury;

[0019] Falling to the contralateral side at rest was scored as 3 points, indicating severe focal injury;

[0020] No spontaneous activity with impaired consciousness is 4 points, indicating extremely severe focal injury;

[0021] Death is 5 points;

[0022] A score of 1 to 4 was considered a successful model.

[0023] In some embodiments, steps (4) and (5) are performed using an esophageal electrode catheter for stimulation.

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

[0025] In some embodiments, the stimulation in step (4) and step (5) adopts S1S1 stimulation mode.

[0026] In some embodiments, step (5) includes: using 2 times the threshold stimulation intensity, 5 stimulations at a frequency of 40 Hz, each lasting 15 seconds, with a stimulation 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 invention provides an application of the aforementioned establishment method.

[0029] A mouse model of newly-onset atrial fibrillation after ischemic stroke constructed by the method of the first aspect is used in the study of the mechanisms of cerebrovascular disease and heart disease and / or in the preparation of drugs for preventing or treating diseases related to newly-onset atrial fibrillation after ischemic stroke.

[0030] Beneficial Effects

[0031] Compared with the prior art, a certain embodiment of the present invention has at least one of the following beneficial effects:

[0032] (1) The mouse model of newly-onset atrial fibrillation after ischemic stroke established by the establishment method provided by the present invention is stable, reliable, has a high success rate, is low in cost, and takes a short time to establish the model.

[0033] (2) Compared with other models, the model provided by the present invention is more accurate in restoring atrial fibrillation induced by human pathological models (cerebral ischemia) and does not require wearable devices, and ordinary electrocardiogram testing is sufficient.

[0034] (3) The innovation of the present invention lies in combining the pathological model of cerebral ischemia with the human disease to induce a pathological model of atrial fibrillation. Compared with other models such as vagus nerve stimulation and lipopolysaccharide injection, this model is closer to the disease state that humans will face.

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

[0036] (5) For mice that were successfully modeled according to behavioral evaluation, the normal feeding time of 3 days was more conducive to improving the success rate of atrial fibrillation, the number of atrial fibrillation times and the total duration of atrial fibrillation compared with other normal feeding times, which had unexpected technical effects.

[0037] (6) Compared with other carotid artery embolization treatment times, the present invention uses a carotid artery embolization treatment time of 45 minutes to 1 hour, which is more conducive to improving the success rate of atrial fibrillation, the number of atrial fibrillation times and the total duration of atrial fibrillation, and has unexpected technical effects.

[0038] Terminology

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

[0040] The term "wt%" means percent by mass.

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

[0042] The term "S1S1 stimulation mode" refers to a periodic stimulation method, which is to continuously act on the heart with stimulation pulses with a fixed frequency and consistent intervals. Usually this frequency is close to or slightly higher than the natural heart rate to mimic the normal heart rhythm. Among them, "S1 stimulation" means a normal stimulation pulse. In a group of pulses, the interval between each pulse (S1-S1) 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" refers to the atrial depolarization wave.

[0044] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0045] In the following, all numbers disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. 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% will be explicitly disclosed, where "+ / -" means plus or minus. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a statistical analysis chart of the number of atrial fibrillation events in the ischemic stroke group mice and the control group.

[0047] Figure 2 This is a statistical analysis chart of the total duration of atrial fibrillation in mice with ischemic stroke and the control group.

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

[0049] Figure 4This is a statistical analysis chart of the relative cerebral blood flow changes between mice in the ischemic stroke group (referred to as the cerebral ischemia group) and mice in the control group.

[0050] Figure 5 This is a comparison of the cerebral infarction area between the ischemic stroke group mice and the control group mice.

[0051] Figure 6 This is the statistical analysis result of the infarct volume between the ischemic stroke group mice and the control group mice.

[0052] Figure 7 This is a comparison of the Masson staining results of the left atrium of the atria of the ischemic stroke group mice and the control group mice.

[0053] Figure 8 This is a comparison of the HE staining results of the left atrium of the hearts of mice in the ischemic stroke group and the control group.

[0054] Fig. 9 This is a comparison chart of the proportion of left atrial fibrous tissue in the left atrium of the heart of mice in the ischemic stroke group and mice in the control group.

[0055] Fig.10 This is a comparison of the CD68 immunohistochemistry results in the left atrium of the heart of mice in the ischemic stroke group and the control group.

[0056] Fig.11 This is a comparison of the expression results of CD68 in the left atrium of the heart of mice in the ischemic stroke group and the control group under a 40x microscope.

[0057] In the figure, "ns" indicates no statistically significant difference, and * indicates 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 solution of the present invention, some non-limiting embodiments are further disclosed below to further describe the present invention in detail.

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

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

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

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

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

[0065] Desktop computer: HP China Ltd.

[0066] ALC-V8S small animal ventilator: Shanghai Alcott Biotechnology Co., Ltd.

[0067] 4.5F6-pole 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 programmable stimulator: Chengdu Instrument Factory.

[0070] Thread plug: Changsha Maiyue Biotechnology Co., Ltd.

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

[0072] Experimental consumables: 22G endotracheal tube with core, 3M medical tape, cotton balls, thick silk thread, 1ml syringe, acupuncture needle (conductive), plasticine, thick gloves, erythromycin ointment, erythromycin eye ointment, depilatory cream, iodine tincture, disinfectant alcohol, chloral hydrate, surgical sutures and suture needles, surgical cotton thread, isoflurane, normal mouse feed and drinking water.

[0073] Example 1: Model construction

[0074] 1. Experimental methods

[0075] (1) Experimental animal preparation: Ten C57 mice aged 8-10 weeks were selected for the experiment and were subjected to adaptive feeding for 3 days to ensure that the mice fully adapted to the experimental environment and to avoid the influence of environmental factors on the experimental results. The mice were randomly divided into two groups: a control group of 5 mice and an ischemic stroke group (model group) of 5 mice. All mice were numbered for experimental records and management.

[0076] (2) Anesthesia: C57 mice were anesthetized using a mixture of isoflurane and oxygen. The isoflurane concentration was 3% for anesthesia induction and 1.5% for anesthesia maintenance. The mice were placed in a gas anesthesia device for 2 minutes. After their activity was significantly reduced, they were fixed on the experimental table and the anesthesia state was maintained.

[0077] (3) Blood flow imaging observation: Use a mouse-specific hair remover to shave the brain hair, then apply depilatory cream and shave off the remaining hair, and disinfect the exposed skin with alcohol. To protect the eyes, apply erythromycin eye ointment to the mouse eyes. Use sterilized scissors to cut the mouse scalp, expose the skull, and start the laser speckle blood flow imager to observe and record brain blood flow imaging. After the recording is completed, apply erythromycin ointment on the skull surface.

[0078] (4) Preparation for neck surgery: The mouse is fixed on the operating board in a supine position, and the heating pad is set to a constant temperature of 37°C ± 0.5°C to maintain its body temperature. Use medical tape to fix the mouse's limbs and continue to maintain its anesthesia. Use an epilator to shave the neck hair, apply depilatory cream and shave off the remaining hair, and disinfect the exposed skin with alcohol. Use sterilized scissors to make an incision of about 1.5 cm in the middle of the neck, use sterilized fine tweezers for blunt separation, expose the anterior cervical muscles, and separate the connective tissue between the sternohyoid muscles, gradually revealing the common carotid artery and vagus nerve.

[0079] (5) Treatment of cerebral ischemia

[0080] Arterial suture implantation in the ischemic stroke group (model group): Two surgical cotton threads were placed above the common carotid artery, one was placed at the bifurcation of the common carotid artery and a slipknot was tied to fix the suture, and the other was placed at the proximal end of the common carotid artery and ligated. The external carotid artery and the internal carotid artery were then separated and each was tied with a slipknot using surgical cotton thread. A "V"-shaped incision was cut on the common carotid artery using ophthalmic scissors, and the suture was inserted into the artery, and the suture was fixed at the slipknot at the bifurcation of the common carotid artery to ensure that no blood flowed out when the suture was moved. The slipknot of the internal carotid artery was loosened, and the suture was quickly inserted into the internal carotid artery. Bleeding and insertion resistance should be avoided during the operation. If resistance is encountered, the position of the suture should be adjusted in time to avoid damaging the pterygopalatine artery. The insertion depth of the suture was about 1 cm to block the middle cerebral artery. After the insertion was completed, the slipknot of the common carotid artery was tightened again to fix the suture, and the occlusion state was maintained for 1 hour.

[0081] Control group: The salivary glands were bluntly dissected, and then two surgical cotton threads were placed above the common carotid artery. One was placed at the bifurcation of the common carotid artery and tied, and the other was placed at the proximal end of the common carotid artery and ligated, and the left carotid artery was cut off.

[0082] (6) Suture

[0083] Removal and suturing of suture plugs in the ischemic stroke group (model group): While maintaining the anesthesia of the mice, slowly withdraw the inserted suture plug and tighten the slipknot again at the bifurcation of the common carotid artery to ensure normal blood flow recovery. Subsequently, use a surgical needle and suture thread to finely suture the neck incision layer by layer. After the suturing is completed, use iodine tincture and alcohol to disinfect the surgical area. Place the mouse on the experimental table, and use the laser speckle blood flow imager again to monitor the changes in brain blood flow to ensure the successful construction of the cerebral ischemia model. After confirming that the model is successful, turn off the small animal anesthesia equipment and place the mouse on a heating pad until it is fully awake.

[0084] Control group: While maintaining the anesthesia of the mice, surgical needles and sutures were used to suture the neck incision layer by layer. After the suturing was completed, the surgical area was disinfected with iodine and alcohol. The mice were placed on the experimental table, and the laser speckle blood flow imager was used again to monitor the changes in brain blood flow to ensure the successful construction of the cerebral ischemia model. After confirming that the model was successful, the small animal anesthesia equipment was turned off and the mice were placed on a heating pad until they were fully awake.

[0085] (7) Behavioral assessment: 3 hours after modeling, behavioral scores were performed on C57 mice after modeling, where the behavioral scores were scored according to the Zea Longa 5-point scoring standard: see Table 1. The results are shown in Table 2.

[0086] Table 1: Neurological impairment scores

[0087]

[0088] Among them, a score of 1-4 points is considered a successful modeling (although a score of 5 is theoretically considered a successful modeling, the mice with a score of 5 died and the experiment could not be continued. Therefore, the present invention uses a score of 1-4 points as the standard for successful modeling).

[0089] Table 2: Scores of ischemic stroke group and control group

[0090]

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

[0092] (9) Preparation before inducing atrial fibrillation: On the third day after modeling, the mice were removed and weighed. Subsequently, 10% chloral hydrate (350 mg / kg) was administered by intraperitoneal injection with a 1 ml syringe for anesthesia. When there was no obvious reaction when the mouse limbs were clamped with hemostatic forceps, the anesthesia was confirmed to be successful. The mice were then placed on the experimental table and erythromycin ointment was applied to their eyes to protect their eyes. The blood flow in the mouse brain was monitored and recorded using a laser speckle blood flow imager to ensure that the brain was still in an ischemic state. The mice were fixed on the operating table in a supine position, and the limbs were fixed in sequence with medical tape. The hind limbs were fixed first, and then the mouse's lower jaw was straightened with a wire, the body was straightened and fixed with hemostatic forceps, and then the forelimbs were fixed. After the fixation was completed, the operating table was raised and the head and neck of the mouse were illuminated with a table lamp. The tongue was gently dragged to one side with forceps, and the tongue was gently pushed toward the abdomen with hemostatic forceps or straight forceps to ensure sufficient operating field. The beating white bright spot is observed as the tracheal mark, and the cannula is inserted parallel to the trachea, and the metal core is quickly pulled out after insertion. Then the operating table is flattened and the ventilator is connected to confirm that the chest rise and fall is synchronized with the ventilator and the peripheral circulation of the mouse's limbs is good to confirm that the intubation is successful.

[0093] (10) ECG recording and esophageal electrode adjustment: insert a sterilized acupuncture needle into the subcutaneous part of the mouse's limbs to a depth of about 0.5 cm, and connect it with an electrocardiogram electrode and fix it firmly with an adhesive tape. According to the supine position of the mouse, connect the ECG lead wire and the second lead to the acupuncture needle respectively. Start the BL-420N biosignal acquisition and analysis system software, select channels 1 to 3, select aVR as the lead, set the sampling frequency to 1kHz or 2kHz, 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 ground wire needs to be adjusted to minimize external clutter interference. Connect the 4.5F6-pole esophageal electrode catheter to the adapter box, and connect the second lead to the adapter box to record the ECG signal of the esophageal electrode catheter. Before inserting the esophageal electrode, soak it with saline and insert it into the mouse along the esophagus. The specific insertion depth should be fine-tuned according to the changes in the ECG signal, and the correct placement should be confirmed by atrial capture. At this time, fix the esophageal electrode to prevent displacement.

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

[0095] (i) Threshold stimulation measurement: The mice that had been normally raised for 3 days were stimulated with pulse current, the current frequency of the pulse current being 1.1 times the basal heart rate of the mice (calculated as heart beats per minute / 60) (for example, if the basal heart rate of the mice is 600 beats per minute, the current frequency of the pulse current is 600÷60×1.1 Hz=11 Hz, and so on). Each stimulation lasted for 5 seconds, and the pulse current stimulation intensity was gradually increased from 0.3 mA, by 0.1 mA each time, until each P wave was replaced by a stimulation wave, thereby obtaining the threshold stimulation current intensity.

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

[0097] (12) Experimental data collection: The number of AF inductions and duration of AF in C57 mice were recorded. The total duration of AF was the cumulative value of the duration of each AF occurrence.

[0098] 2. Experimental results:

[0099] Table 3 lists whether atrial fibrillation was successfully induced in each C57 mouse in the 1-hour middle cerebral artery ischemia 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 This is a statistical analysis chart of the frequency of atrial fibrillation in the ischemic stroke group and the control group.

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

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

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

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

[0105]

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

[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 workbench, a laser speckle imaging head, and a connecting arm that can be flexibly moved in the horizontal and vertical directions. The imaging head emits a laser beam with a wavelength of 785nm and is equipped with a 12-bit charge-coupled device (CCD) camera. The system captures the laser speckle image through a CCD camera and installs a filter 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 mixture, and its body temperature was maintained at 37°C ± 0.5°C by heating pad. The mouse skull was exposed and cleaned by incising the midline skin. During image acquisition, tampons were used to keep the exposed area clean and dry, and the mouse brain was ensured to be perpendicular to the irradiation light. The focus of the CCD camera was adjusted as needed to obtain clear imaging.

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

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

[0111] Relative cerebral blood flow change = (mean blood flow index on the injured side - mean blood flow index on the contralateral side) / mean blood flow index on the contralateral side

[0112] 5) Results

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

[0114] Figure 4 This is a statistical analysis chart of the relative cerebral blood flow changes between mice in the ischemic stroke group (referred to as the cerebral ischemia group) and mice in the control group.

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

[0116] 6) Result analysis

[0117] This experiment recorded the changes in cerebral blood flow in the 1-hour middle cerebral artery ischemia model mouse group before and after surgery. 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 established.

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

[0119] 1) After C57 mice were deeply anesthetized with 4% chloral hydrate, the head was cut with scissors, the skull was opened through the foramen magnum and the brain tissue was removed.

[0120] 2) The brain tissue was quickly frozen in a -20°C refrigerator for 20 minutes. Then, the brain tissue was placed on a pre-cooled brain slice mold and cut into slices with a thickness of 2 mm.

[0121] 3) Immerse the slices in 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution and incubate at 37°C for 30 minutes. Gently shake every 5 minutes to ensure uniform staining. After incubation, wash the slices in PBS buffer solution and take photos after removal.

[0122] 4) Use Image J software to measure the infarct area of ​​each slice and the whole brain area, and calculate the ratio of infarct volume. The calculation formula of infarct volume ratio is:

[0123] Infarct volume percentage = (total infarct area / total brain area) × 100%.

[0124] 5) Results

[0125] Figure 5 This is a comparison of the cerebral infarction area between the ischemic stroke group mice and the control group mice.

[0126] Figure 6 This is the statistical analysis result of the infarct volume between the ischemic stroke group mice and the control group mice.

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

[0128] 6) Result analysis

[0129] Compared with the control group, obvious cerebral infarction area was observed in brain tissue sections of the model mice group with middle cerebral artery ischemia for 1 hour under TTC staining, indicating that significant cerebral ischemic damage occurred in the mice in the model group.

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

[0131] Masson staining method

[0132] 1) The embedded C57 mouse myocardial tissue was sliced ​​continuously with a thickness of 4 μm. After slicing, the specimen was placed in a 60°C oven for 2 hours. Then, the slices were soaked in environmentally friendly dewaxing solution I for 20 minutes, then soaked in environmentally friendly dewaxing solution II for 20 minutes, then soaked in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each, and finally soaked in 75% alcohol for 5 minutes and washed with tap water.

[0133] 2) Thawing and fixation of frozen sections: Take the frozen sections out of the -20°C refrigerator, return them to room temperature, fix them with tissue fixative for 15 minutes, and then rinse the sections with running water.

[0134] 3) Place the sections in a dye solution mixed with Masson B and Masson C in equal proportions, soak for 1 minute, and then rinse with tap water. Then use differentiation solution to differentiate for a few seconds, and then wash with tap water.

[0135] 4) Place the sections in Masson D solution for 6 minutes and then rinse with tap water.

[0136] 5) Place the sections in Masson E solution for 1 minute.

[0137] 6) Without washing, drain slightly and then place the slices directly into Masson F solution for 2 to 30 seconds.

[0138] 7) The slices were rinsed and differentiated with 1% acetic acid, and then dehydrated in two cylinders of anhydrous ethanol.

[0139] 8) Transparency and sealing: Soak the slices in the third cylinder of anhydrous ethanol for 5 minutes, then soak them in xylene for 5 minutes for transparency, and finally use neutral gum to seal the slices.

[0140] 9) Use a microscope to perform microscopic examination, collect and analyze images.

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

[0142] 11) Results

[0143] Figure 7 This is a comparison of the Masson staining results of the left atrium of the atria of the ischemic stroke group mice and the control group mice.

[0144] 12) Result analysis

[0145] The results showed that after three days of normal feeding, the atrial cardiomyocytes of the mice in the model group that had undergone 1 hour of middle cerebral artery ischemia appeared blue after Masson staining compared with the control group, indicating that obvious myocardial fibrosis occurred in the atrial part.

[0146] Example 5: HE staining

[0147] HE staining experimental method

[0148] 1) Dewaxing of paraffin sections: Soak the sections in environmentally friendly dewaxing solution I for 10 minutes, then in environmentally friendly dewaxing solution II and environmentally friendly dewaxing solution III for 10 minutes each. Then, soak the sections in anhydrous ethanol I, II and III for 5 minutes each, and finally wash with distilled water.

[0149] 2) Thawing and fixation of frozen sections: Take the frozen sections out of the -20°C refrigerator, return them to room temperature, fix them with tissue fixative for 15 minutes, and then rinse with running water.

[0150] 3) Pretreatment: Immerse the sections in high-definition constant stain pretreatment solution for 1 minute.

[0151] 4) Hematoxylin staining: Place the sections in hematoxylin staining solution for 3-5 minutes, then rinse with tap water. Use differentiation solution for differentiation, rinse again with tap water, use bluing solution for bluing, and finally rinse with running water.

[0152] 5) Eosin staining: Dehydrate the sections in 95% alcohol for 1 minute, and then stain them in eosin solution for 15 seconds.

[0153] 6) Dehydration and sealing: Soak the slices in anhydrous ethanol I, II, and III for 2 minutes each, and then soak them in n-butanol I and II for 2 minutes each. Then soak the slices in xylene I and II for 2 minutes each. Place the slide rack in a fume hood to dry, and then seal the slides with neutral gum. Observe the tissue morphology of the slices under an optical microscope. The cell nucleus appears blue and the cytoplasm is stained red.

[0154] 7) Microscopic examination and image acquisition: Observe tissue morphology under a microscope and collect 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 This is a comparison of the HE staining results of the left atrium of the hearts of mice in the ischemic stroke group and the control group.

[0158] Fig. 9This is a comparison chart of the proportion of left atrial fibrous tissue in the left atrium of the heart of mice in the ischemic stroke group and mice in the control group.

[0159] 9) Result analysis

[0160] HE staining results showed that the nuclei of the ischemic stroke group were larger than those of the control group, indicating that the infiltration of inflammatory cells in the left atrium of the heart of the ischemic stroke mice was higher than that of the control group, and the inflammation was more severe; and Fig. 9 It can be seen that there is a statistically significant difference between the ischemic stroke group and the control group.

[0161] Example 6: CD68 immunohistochemistry experiment

[0162] CD68 Immunohistochemistry Experimental Methods

[0163] 1) Dewaxing of paraffin sections: Soak the sections in environmentally friendly dewaxing solutions I, II, and III for 10 minutes each, then in anhydrous ethanol I, II, and III for 5 minutes each, and finally wash with distilled water.

[0164] 2) Antigen repair: Place the slices in EDTA pH 8.0 hot repair solution for 30 minutes. During the process, prevent the repair solution from evaporating excessively and avoid drying of the slices. After natural cooling, place the slices in PBS (pH 7.4) and wash 3 times on a decolorizing shaker, 5 minutes each time. (The repair solution and repair conditions are determined according to the tissue type)

[0165] 3) Blocking endogenous peroxidase: Place the slices in a 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Then place the slices in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each time.

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

[0167] 5) Primary antibody reaction: After shaking off the excess blocking solution, add the CD68 antibody prepared in proportion to the slices, place the slices flat in a humidified box, and incubate at 4°C overnight.

[0168] 6) Secondary antibody reaction: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time. After drying the sections, add goat anti-rabbit IgG secondary antibody labeled with HRP (horseradish peroxidase) in the tissue 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 3 times, 5 minutes each time. After drying, add freshly prepared DAB color development solution to the slices and control the color development time under a microscope. The positive signal appears brown-yellow. Rinse the slices with tap water to terminate the color development reaction.

[0170] 8) Nuclear counterstaining: Place the sections in hematoxylin staining solution for counterstaining for about 3 minutes, then wash with tap water. Use differentiation solution for a few seconds, rinse with tap water again, then use blueing solution to blue, and wash with running water.

[0171] 9) Dehydration and sealing: Soak the slices in 75% alcohol and 85% alcohol for 5 minutes each, then in anhydrous ethanol I and II for 5 minutes each, and then soak in n-butanol and xylene for 5 minutes each for transparent treatment. Take out the slices and let them dry slightly, then use sealing glue to seal the slices.

[0172] 10) Microscopic examination: Observe the sections and interpret the results under a white light microscope.

[0173] 11) Results

[0174] Fig.10 This is a comparison of the CD68 immunohistochemistry results in the left atrium of the heart of mice in the ischemic stroke group and the control group.

[0175] Fig.11 This is a comparison of the expression results of CD68 in the left atrium of the heart of mice in the ischemic stroke group and the control group under a 40x microscope.

[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) Result analysis:

[0178] The results of CD68 immunohistochemistry analysis showed that the CD68 protein in the left atrium of the heart of the ischemic stroke group was significantly increased compared with the control group, and the number of macrophages on the surface was significantly increased. The inflammation of the left atrium of the heart of the ischemic stroke group was more severe.

[0179] Comparative Example 1-Comparative Example 4: Pulse Current Frequency Investigation

[0180] Referring to the method of Example 1, the 40 Hz pulse current frequency in step (ii) was adjusted to the pulse current frequency in Table 4, and the remaining operations were the same as in Example 1, and the number of successful model construction and atrial fibrillation induction rate of different pulse current frequencies were detected. The results are shown in Table 4.

[0181] Table 4: Model construction success rate at 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, which has unexpected technical effects.

[0184] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for establishing a mouse model of newly-onset atrial fibrillation due to ischemic stroke, characterized in that: The following steps were performed on the mice: (1) Treatment of cerebral ischemia; (2) behavioral assessment; (3) Mice that showed successful modeling based on behavioral evaluation were kept normally for 3 days; (4) Measuring the threshold stimulation of mice after being raised normally for 3 days in step (3): Stimulating the mice after being raised normally for 3 days in step (3) with pulse current, wherein the current frequency of the pulse current is 1.1 times the basal heart rate of the mice, each stimulation lasts for 5 seconds, and the pulse current stimulation intensity gradually increases from 0.3 mA, each time by 0.1 mA, until each P wave of the mouse electrocardiogram is replaced by the stimulation wave, thereby obtaining the threshold stimulation current intensity; (5) Programmed electrical stimulation to induce atrial fibrillation: The mice were stimulated five times with a pulse current of 2 times the threshold stimulation current intensity and a current frequency of 40 Hz, each lasting 15 to 30 seconds, with a stimulation interval of 3 minutes, and the interval was kept consistent each time.

2. The method according to claim 1, wherein the cerebral ischemia treatment in step (1) comprises occluding the carotid artery of the mouse for 45 minutes to 1 hour.

3. According to the establishment method according to any one of claims 1-2, the behavioral assessment comprises: 3 hours after the treatment in step (1), the mice were subjected to behavioral assessment using the Zea Longa 5-point scoring standard. in, Normal activity was scored as 0, indicating no signs of neurological damage; The inability to fully extend the contralateral forelimb during the tail-lift test was scored as 1 point, indicating a mild focal injury; Rotation to the contralateral side during walking was scored as 2 points, indicating a moderate focal injury; Falling to the contralateral side at rest was scored as 3 points, indicating severe focal injury; No spontaneous activity with impaired consciousness is 4 points, indicating extremely severe focal injury; Death is 5 points; A score of 1 to 4 was considered a successful model.

4. According to the establishment method according to any one of claims 1 to 3, the steps (4) and (5) use an esophageal electrode catheter for stimulation.

5. According to the establishment method according to any one of claims 1 to 4, the stimulation current in step (4) and step (5) adopts a square wave with a wave width of 1 ms.

6. According to the establishment method according to any one of claims 1 to 5, the stimulation in step (4) and step (5) adopts S1S1 stimulation mode.

7. According to the establishment method according to any one of claims 1 to 5, the step (5) comprises: The stimulation intensity was 2 times the threshold, and the frequency was 40 Hz, with 5 stimulations, each lasting 15 seconds, and the stimulation interval was 3 minutes, keeping the interval consistent each time.

8. The establishment method according to any one of claims 1 to 7, wherein the mouse is a C57 mouse.

9. Use of the mouse model of newly-onset atrial fibrillation after ischemic stroke constructed by the method according to any one of claims 1 to 8 in the study of the mechanism of cerebrovascular disease and heart disease and / or in the preparation of drugs for preventing or treating diseases related to newly-onset atrial fibrillation after ischemic stroke.

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