Construction method of left bundle branch conduction block rat animal model

Abstract

The invention relates to the technical field of animal model construction, in particular to a method for establishing a left bundle branch block rat animal model. The construction method specifically comprises the step of injecting absolute ethyl alcohol into a left bundle branch walking area under the endocardium of the left ventricular surface of the upper segment of the ventricular septum of a rat. According to a left bundle branch conduction block (LBBB) rat model constructed by puncturing and ablating the left bundle branch under ultrasonic guidance, QRS waves are obviously widened on an electrocardiogram, V5 lead R waves are large in width and have incisura, ventricular septal movement abnormality (flickering sign) is shown on an echocardiogram, the LBBB rat model conforms to clinical electrocardiogram and ultrasonic characteristics of the LBBB, and the LBBB rat model has the advantages of being simple in structure and convenient to use. The obtained animal model is clinically fit, and the blank that the left bundle branch conduction block small animal model is lacked in the industry at present is filled. The modeling method designed by the invention is simple and convenient to operate and high in repeatability, harm to animals is extremely reduced, and the modeling method can be used for research in the aspects of pathological mechanism exploration, drug research and development and the like of left bundle branch conduction block, and has relatively high academic value.

Description

Technical Field

[0001] The invention relates to the technical field of animal model construction, and in particular to a method for constructing a left bundle branch block rat animal model. Background Art

[0002] Left bundle branch block (LBBB) is a common arrhythmia in clinical practice. Its onset is insidious and is often discovered during physical examinations or routine electrocardiograms for other diseases. Its duration is difficult to trace. In recent years, cardiac surgeries such as transcatheter aortic valve replacement (TAVR) and left ventricular outflow tract patency for obstructive hypertrophic cardiomyopathy have been continuously carried out, and the incidence of acquired LBBB has gradually increased. Studies have shown that the incidence of LBBB caused by TAVR surgery can be as high as 65%. Many studies have found that LBBB is not a benign arrhythmia. It can lead to a decrease in left ventricular ejection fraction, induce left ventricular remodeling, and increase the risk of malignant arrhythmias or sudden cardiac death.

[0003] At present, most of the relevant studies on LBBB are retrospective studies, which mainly focus on analyzing the imaging characteristics of the disease, the effects of drugs or pacing treatment, etc., and fail to deeply explore the pathological changes and mechanisms of the disease. One of the fundamental reasons is that animal experiments have not been fully utilized. In the past, animal models of LBBB were mainly based on large animals such as pigs or dogs. The establishment of large animal models requires that the animals be anesthetized, intubated and maintained by a ventilator, and then the femoral artery of the animal is punctured to insert a sheath, and then the ablation catheter is inserted through the sheath. Under the guidance of X-ray, it passes through the descending aorta, aortic arch, ascending aorta in turn, and then retrogradely crosses the aortic valve. Under the guidance of electrical mapping, the left bundle branch is thermally ablated. This process is extremely difficult to operate, requires a lot of manpower and material resources, and has extremely high requirements for the modeling environment, equipment and materials. There are no reports of LBBB models in small animals. This is because the instruments and consumables used in the modeling process of large animals, such as navigation and visualization technology surface electrodes, electrophysiological mapping instruments, sheaths and ablation catheters, are mostly instruments used in human surgery. They can only be used to establish models in large animals that are closer to the human body size, and cannot be applied to the establishment of LBBB models in small animals. In addition, the anatomical variation of the left bundle branch is large, and there is a lack of accurate positioning methods in vivo except for electrical mapping technology. In addition, the cultivation cost of large animals is high, and their gene annotation and antibody resources are relatively imperfect. The above factors together constitute obstacles to in-depth research on the occurrence and development mechanism of LBBB cardiomyopathy. Therefore, constructing a small animal model that meets the clinical characteristics of left bundle branch block is of great significance for studying such diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a method for establishing a left bundle branch block rat animal model, which can establish a rat animal model that is more suitable for clinical practice at low cost, effectively and simply, with high repeatability, and can minimize the damage to the cardiovascular system during the establishment of the animal model and alleviate the pain of the rat.

[0005] To achieve this purpose, the present invention provides a method for establishing a rat animal model of left bundle branch block, wherein the method is to puncture the ventricular septum of the rat through the body surface under ultrasound guidance, and then inject anhydrous ethanol to ablate the left bundle branch to establish a rat animal model of left bundle branch block. The specific technical scheme is as follows:

[0006] The invention provides a method for constructing a left bundle branch block rat animal model, comprising the following steps: puncturing the left bundle branch course area under the left ventricular surface endocardium of the upper segment of the ventricular septum of the rat under real-time ultrasound guidance and injecting anhydrous ethanol to obtain a left bundle branch block rat model.

[0007] Wherein, the rats are 6-8 week old SD rats.

[0008] Among them, the puncture, the needle insertion point is the chest wall, and the specific needle insertion point is the intersection of the midpoint of the lower edge of the ultrasound probe and the chest wall.

[0009] Wherein, the needle used for the puncture is a 30G syringe needle with a length of 25 mm. Preferably, the needle used for the injection is also a 30G syringe needle with a length of 25 mm.

[0010] The injection frequency of the anhydrous ethanol is 1-2 times, and the injection dose each time is 0.02-0.03 mL. Preferably, the total injection dose is 0.02-0.06 mL. The injection site of the anhydrous ethanol is the left bundle branch course area under the endocardium of the left ventricular surface of the ventricular septum;

[0011] Wherein, the rats are anesthetized and then punctured to the left bundle branch area under the endocardium of the left ventricle on the upper part of the ventricular septum to inject anhydrous ethanol. The anesthesia is first induced by 3.5%-5% v / v isoflurane for 3-5 minutes, and then maintained at 1.5%-2% v / v isoflurane to maintain the anesthetic state of the rats; the flow rate of the isoflurane is 700-1000mL / min. Preferably, the rats are first induced by 5% v / v isoflurane for 5 minutes, and then maintained at 2% v / v isoflurane to maintain the anesthetic state of the rats; the flow rate of the isoflurane is 1000mL / min.

[0012] The rat is placed on an operating table for ultrasound and puncture; the operating table is tilted 5-15 degrees clockwise, and the ultrasound probe used for ultrasound is tilted 40-50 degrees counterclockwise. Preferably, the operating table is tilted 15 degrees clockwise, and the ultrasound probe used for ultrasound is tilted 45 degrees counterclockwise.

[0013] Wherein, the ultrasound real-time guidance uses the parasternal long-axis right ventricular focused section.

[0014] Preferably, an electrocardiogram is collected synchronously during the above-mentioned modeling process.

[0015] In a second aspect, the present invention provides an application of a rat model constructed by the construction method described in the first aspect in studying the mechanism of left bundle branch block.

[0016] In a third aspect, the present invention provides the use of a rat model constructed by the construction method described in the first aspect in screening products for preventing or treating left bundle branch block.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0018] 1. Experimental verification shows that the LBBB rat model constructed by ultrasound-guided puncture and ablation of the left bundle branch in the present invention shows significantly widened QRS waves and wide and notched R waves in lead V5 on the electrocardiogram, and abnormal ventricular septal motion (scintillation sign) on the echocardiogram, which is consistent with the electrocardiogram and ultrasound characteristics of LBBB in clinical practice. This shows that the obtained animal model is in line with clinical practice and fills the gap in the current lack of small animal models of left bundle branch block in the industry;

[0019] 2. The present invention is easy to operate, highly repeatable, and has minimal harm to animals. It can be used for the exploration of the pathological mechanism of left bundle branch block cardiomyopathy, drug development, and other research, and has high academic value;

[0020] 3. The present invention uses surface puncture ablation modeling to successfully prepare a small animal model of left bundle branch block for the first time. The model animal adopts SD rats commonly used in scientific research, which have the characteristics of low cost, fast reproduction, and physiological conditions close to humans. At the same time, rat gene annotation and antibody resources are very rich, and gene editing technology for rats is also relatively developed. Therefore, the animal model prepared by the present invention has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0022] Figure 1 Schematic diagram of the rat modeling position. Figure 1 A is the main view; Figure 1 B is a top view.

[0023] Figure 2 Ultrasound-guided section and ultrasound display images during puncture used for rat modeling. Figure 2A is the parasternal long-axis right ventricular focused section used for rat modeling, RV. right ventricle; IVS. interventricular septum; LV. left ventricle; MV. mitral valve; LA. left atrium; AV. aortic valve; AO. aorta; Figure 2 B is an ultrasound image of puncturing the ventricular septum during rat modeling. The red arrow indicates the location of the needle, which is the injection site of anhydrous ethanol.

[0024] Figure 3 To establish a graphical representation of the results for the electrocardiographic evaluation of LBBB model rats, Figure 3 A is the electrocardiogram of rats before and immediately after modeling. The left picture is the electrocardiogram of rats before modeling, and the right picture is the electrocardiogram of LBBB rats after modeling. The QRS wave is widened, and the R wave in lead V5 is wide and notched (indicated by the black arrow); Figure 3 B is a comparison of the QRS wave width before and after modeling. The QRS wave width is significantly wider than before modeling, n = 6. Compared with before modeling, ***p < 0.0005.

[0025] Figure 4 The results of echocardiographic evaluation of the LBBB rat model were shown in Figure 2. Figure 4 A is the M-mode ultrasound image of the ventricular septum of rats before modeling, RV. right ventricle; IVS. ventricular septum; LV. left ventricle; Figure 4 B is an M-mode ultrasound image of the ventricular septum immediately after modeling. After modeling, weakened ventricular septal pulsation and abnormal ventricular septal motion (scintillation sign) can be seen. The white arrow indicates scintillation of the ventricular septum.

[0026] Figure 5 This is a schematic diagram of constructing the LBBB rat animal model described in the present invention. DETAILED DESCRIPTION

[0027] In order to better understand the present invention, the present invention is further described below in conjunction with embodiments.

[0028] Example 1 Model construction and testing method

[0029] 1. Experimental Animals

[0030] Eight SD rats aged 6-8 weeks, 200 g ± 20 g, were obtained from Shanghai Bikaikoyi Biotechnology Co., Ltd. The experimental animals were kept in the SPF animal room of the Experimental Animal Center of Nanjing University during the study. The rats were maintained in a 12-hour light / dark cycle, a temperature of 23 ± 2 °C, and a relative humidity of 50% ± 5%. Three rats were kept in standard cages (545 × 395 × 200 mm) per cage, with free access to food and water. The bedding was changed 2-3 times a week, and the cages were cleaned and disinfected 2-3 times a week. Echocardiograms and electrocardiograms were evaluated before the operation to exclude organic heart disease and arrhythmias. After one week of adaptive feeding, baseline electrocardiograms and echocardiograms were collected, and then the model was established.

[0031] 2. Construction of LBBB rat animal model

[0032] The method for constructing the LBBB rat animal model includes the following steps:

[0033] (1) First, the rats were anesthetized with 5% isoflurane for 5 minutes, and then the rats were anesthetized with 2% isoflurane (isoflurane flow rate was 1000 mL / min).

[0034] (2) Place the rat in supine position on a heated rat operating table equipped with a small animal physiological information detection module, fix the limbs with tape, and apply coupling agent to the sensor electrodes on the rat operating table.

[0035] (3) After the rat skin was prepared, the BL-420S bio-function experimental system electrocardiogram was connected, and the operating table was tilted 5-15° clockwise to the right ( Figure 1 ), wipe the rat's chest with iodine cotton balls for disinfection and then apply coupling agent.

[0036] (4) Probe MS250 is tilted 40-55° counterclockwise to the left and placed on the right edge of the rat's sternum, with the probe mark pointing to the rat's head. The right ventricle (RV), aorta (AO), and interventricular septum (IVS) are clearly displayed on the ultrasound image. Figure 1 B and Figure 2 A. The angle can be adjusted accordingly according to the actual situation of the rat's body position and heart orientation (in this embodiment, the operating table is actually tilted 15° clockwise to the right, and the probe MS250 is actually tilted 45° counterclockwise to the left).

[0037] (5) Take a 30G injection needle (length 25 mm), lubricate the tube with heparin, and puncture the left bundle branch under the endocardium of the left ventricle on the upper part of the ventricular septum under ultrasound guidance. The specific insertion point is the junction of the midpoint of the lower edge of the probe and the chest wall. Inject anhydrous ethanol for ablation. Figure 2 B). Each injection of 0.02-0.03mL, injection 1-2 times. In the present embodiment, 8 rats all adopted this modeling method, of which 2 rats were injected once (each injected with 0.03mL), and the remaining 6 rats were injected twice (of which 1 rat was injected with 0.02mL / time, and 5 rats were injected with 0.03mL / time). The operation was synchronized and recorded throughout the electrocardiogram for real-time monitoring. The electrocardiogram standard for successful preparation of the LBBB model is: sinus rhythm, QRS widening, V5 lead R wave wide, thick and blunt or accompanied by notches. The electrocardiogram can change immediately after injection. After observing for 1-2min, if the electrocardiogram maintains the LBBB morphology, it indicates that the modeling is successful. The electrocardiogram may return to normal during observation, and if it is recovered, a second injection is performed. If the electrocardiogram does not change or cannot maintain the LBBB morphology after two injections, it is determined to be a modeling failure.

[0038] 3. Echocardiographic Image Acquisition

[0039] Echocardiogram collection: Echocardiograms were collected before and immediately after modeling. Rats were first anesthetized with 3.5%-5% v / v isoflurane for 5 minutes, and then maintained with 1.5%-2% v / v isoflurane to maintain anesthesia. The rats were placed in a supine position on a heated rat operating table equipped with a small animal physiological information detection module, and the limbs were fixed with tape, and coupling agent was applied to the sensing electrode sheet of the rat operating table. After the rat chest was prepared, it was wiped and disinfected with iodine cotton balls and coupling agent was applied. Dynamic transthoracic echocardiogram images of rats were collected using a small animal ultrasound machine, and electrocardiograms were recorded synchronously. The parasternal long-axis right ventricular focused section M-mode ultrasound images were retained. The ultrasound images were completed by the same operator and stored on a mobile hard disk.

[0040] 4. ECG image acquisition and analysis

[0041] (1) ECG image acquisition: ECG images were collected before, immediately after, and one week after modeling. Rats were first anesthetized with 3.5%-5% v / v isoflurane for 5 minutes, and then maintained with 1.5%-2% v / v isoflurane to maintain anesthesia. The rats were placed in a supine position on a heated rat operating table equipped with a small animal physiological information detection module, and their limbs were fixed with tape. The BL-420S biofunction experimental system was used to collect ECG images of the rats, with 4 channels collected each time, and the ECGs of leads I, II, III, avL, avR, avF, V1, and V5 were collected.

[0042] (2) ECG image analysis: Use the system's built-in measurement tools to measure heart rate and QRS wave width. Perform three measurements each time and take the average value.

[0043] Example 2 Modeling Results Evaluation

[0044] 1. Evaluate the establishment of LBBB model through electrocardiogram indicators

[0045] like Figure 3As shown, after LBBB modeling, the rat electrocardiogram showed widened QRS wave, and V5 lead was wide and visible notch, which was the typical performance of LBBB electrocardiogram. Compared with baseline, the rat QRS wave width immediately after modeling was significantly greater than before modeling. It can be determined that the model was successfully established. Embodiment 1 A total of 8 rats were used, and 6 of them maintained LBBB morphology after modeling (successful modeling), 1 rat electrocardiogram did not change (anhydrous ethanol injection 2 times, 0.03mL / time), 1 rat electrocardiogram showed second degree type II atrioventricular block (anhydrous ethanol injection 2 times, 0.03mL / time), and the recent success rate was 75%. In the first week of follow-up, 2 electrocardiograms of 6 LBBB rats returned to normal (anhydrous ethanol injection 2 times, 0.03mL / time), 4 rat electrocardiograms maintained LBBB morphology, and the long-term success rate was 50%.

[0046] 2. Evaluation of the establishment of the LBBB model by echocardiography

[0047] like Figure 4 As shown in the figure, the M-mode ultrasound image of the ventricular septum of rats after modeling showed abnormal ventricular septal motion (scintillation sign), which is a unique echocardiographic manifestation of LBBB.

[0048] The modeling method and modeling evaluation results of the present invention are as follows Figure 5 As shown, Figure 5 The ventricular septum flicker mentioned in the text is the abnormal ventricular septum movement (scintillation sign).

[0049] Comparative Example 1 Selection of Ultrasound-Guided Sections

[0050] The operation was the same as in Example 1, except that the ultrasound-guided section was the parasternal left ventricular long-axis section. After the operation, it was found that the model could not be successfully established. To explore the reason, the injection site was marked under the guidance of the parasternal left ventricular long-axis section using subagar blue. After the rat was dissected, it was confirmed that the site of puncture injection under the guidance of the section was not the ventricular septum, but the anterior wall of the left ventricle. The rat was dissected and found that the orientation of the rat heart was slightly different from that of the human heart, which was manifested as the rat heart rotated counterclockwise by about 20-30° compared with the human heart.

[0051] Selection of needle used for puncture in comparative example 2

[0052] The operation was the same as in Example 1, except that a 25G syringe needle with a length of 16 mm was used. During the puncture, it was found that rats were very prone to a large amount of pericardial effusion, leading to cardiac tamponade. A 30G syringe needle with a length of 10 mm was then used. It was found that moderate or more pericardial effusion was not likely to occur during the puncture, but the syringe was not long enough to puncture the left ventricular side of the ventricular septum.

[0053] Comparative Example 3 Selection of Anhydrous Ethanol Injection Volume and Injection Times

[0054] The operation is the same as in Example 1, except that 0.01 mL / time of anhydrous ethanol is injected. It is found that due to the small injection volume and limited damage range, the left bundle branch block caused by injection often recovers to normal in about 1 minute, so repeated punctures and injections are required. Repeated punctures and too many punctures can easily lead to moderate or more pericardial effusion in rats, and even cardiac tamponade and death. The modeling success rate is extremely low. Anhydrous ethanol injection may damage the surrounding myocardial tissue, and excessive injection volume may cause serious damage to the ventricular septum, introducing experimental confounding factors.

[0055] The present invention provides a method and idea for constructing a rat animal model of left bundle branch block. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for constructing a left bundle branch block rat animal model, characterized in that: The drug was obtained by puncturing the left bundle branch area under the endocardium of the left ventricle on the upper segment of the ventricular septum of rats under the guidance of real-time ultrasound and injecting anhydrous ethanol.

2. The construction method according to claim 1, characterized in that: The rats are 6-8 week old SD rats.

3. The construction method according to claim 1, characterized in that: The puncture is performed at the chest wall.

4. The construction method according to claim 1, characterized in that: The puncture was performed using a 30G syringe needle with a length of 25 mm.

5. The construction method according to claim 1, characterized in that: The injection frequency of the anhydrous ethanol is 1-2 times, and the injection dosage of each time is 0.02-0.03 mL.

6. The construction method according to claim 1, characterized in that: The rats were anesthetized and then punctured to the left bundle branch area under the endocardium of the left ventricle on the upper part of the ventricular septum to inject anhydrous ethanol. The specific method of anesthesia was as follows: firstly, 3.5%-5% v / v isoflurane was used to induce anesthesia in the rats for 3-5 minutes, and then 1.5%-2% v / v isoflurane was maintained to maintain the anesthesia state of the rats; Wherein, the flow rate of isoflurane is 700-1000 mL / min.

7. The construction method according to claim 1, characterized in that: The rats were placed on an operating table for the ultrasound and puncture; the operating table was tilted 5-15 degrees clockwise, and the ultrasound probe used for ultrasound was tilted 40-50 degrees counterclockwise.

8. The construction method according to claim 1, characterized in that: The ultrasound real-time guidance uses a parasternal long-axis right ventricular focused section.

9. Use of the rat animal model constructed by the construction method according to any one of claims 1 to 8 in screening products for preventing or treating left bundle branch block disease.

10. Use of the rat animal model constructed by the construction method according to any one of claims 1 to 8 in studying the disease mechanism of left bundle branch block.

Images