CN115399293A - Method for constructing arrhythmic cardiomyopathy animal model - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
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Abstract
The invention discloses a method for constructing an arrhythmic cardiomyopathy animal model. The construction method comprises the following steps: taking rat, injecting Ach and CaCl into tail vein according to dosage of 1ml/kg 2 Mixing the medicinal solutions, and injecting once a day for 8-10 days; the Ach and CaCl 2 The mixture ratio of the mixed liquid medicine is as follows: each milliliter of the mixed liquid medicine contains 66 mug of Ach and 10mg of CaCl 2 . The disease expression of the animal model obtained by the invention completely accords with the clinical characteristics of the arrhythmic cardiomyopathy, and fills the gap that the animal model of the arrhythmic cardiomyopathy is lacked in the industry at present. And the method has short molding period, simple and convenient operation and high molding rate, can be used for the research of pathological mechanism exploration, medicine research and development, acupuncture and moxibustion and other non-medicine treatments of the disease and has higher academic value.
Description
Technical Field
The invention relates to the technology related to the creation of animal models, in particular to a method for constructing an arrhythmic cardiomyopathy animal model.
Background
Arrhythmic cardiomyopathy (AIC) is a cardiomyopathy caused by arrhythmia and characterized by dysfunction of left ventricular contraction due to a rapid and/or irregular heart rhythm (e.g., atrial fibrillation, ventricular premature beats, etc.). Atrial fibrillation is the most common cause of adult arrhythmic cardiomyopathy, and 25% to 60% of patients with atrial fibrillation have a degree of arrhythmic cardiomyopathy that is of increasing concern, but the prevalence and incidence may still be underestimated. Many patients with paroxysmal or persistent Atrial Fibrillation (AF) may develop cardiomyopathy, which is associated with left ventricular dysfunction. AIC is partially or completely reversible after treatment to cause arrhythmia, but the underlying mechanisms of AIC have not yet been fully elucidated. The current therapies for controlling arrhythmic cardiomyopathy are limited, and the related research starts late, wherein one of the difficulties is that no model animal suitable for the arrhythmic cardiomyopathy exists at present. Therefore, the construction of an animal model which accords with the clinical characteristics of the arrhythmic cardiomyopathy has important significance for researching the diseases.
Disclosure of Invention
The invention provides a rat animal model with arrhythmia cardiomyopathy consistent with clinical characteristics and a construction method thereof in order to overcome the technical defects.
The rat animal model is constructed by the following method: taking rats, injecting Ach and CaCl into tail vein according to dosage of 1ml/kg 2 Mixing the medicinal solutions, and injecting once a day for 10 days; the proportion of the ACh and CaCl2 mixed liquid medicine is as follows: each milliliter of the mixed liquid medicine contains 66 mu g of Ach and 10mg of CaCl 2 。
The method comprises the steps of anaesthetizing before tail vein injection, which specifically comprises the following steps: rats were anesthetized with isoflurane induction for 4 minutes, then maintained at 2% isoflurane and injected tail vein while maintaining the rats in an anesthetic state.
The model animal of the present invention is preferably an SD rat.
Compared with the prior art, the invention has the following technical advantages:
1. tests prove that the invention passes through the specific proportion of ACh-CaCl 2 The administration induces the atrial fibrillation of rats in AIC group and changes such as cardiac function damage, motor ability reduction, ventricular morphological abnormality and the like, and the changes can be completely or partially reversed by the antiarrhythmic medicament amiodarone, thereby completely meeting the definition and diagnostic standard of AIC. The disease expression of the obtained animal model completely accords with the clinical characteristics of the arrhythmic cardiomyopathy. Fills the gap of the lack of arrhythmia cardiomyopathy animal models in the industry at present.
2. The invention has short molding cycle, simple operation and high molding rate, can be used for the research of pathological mechanism exploration, medicine research and development, acupuncture non-medicine treatment and the like of the arrhythmia myocardial diseases, and has higher academic value.
3. The invention applies the drug modeling to successfully prepare the arrhythmic cardiomyopathy animal model for the first time. The model animal adopts SD rat for scientific research, and has the advantages of low cost, fast propagation, physiological condition similar to that of human, etc.
Drawings
FIG. 1 is a diagram of the results of the echocardiography index evaluation AIC model cardiac morphology and function tests in this example; (A) is a flow chart of the modeling and treatment procedure; (B) is a representation image of an echocardiography examination; the study was divided into 3 groups, from left to right in turn, control group, AIC group and AIC + Amio group. (C) Is a graph showing changes in ejection fraction (% EF), (D) is a graph showing changes in fractional rat foreshortening rate (% FS), (E) is a graph showing changes in systolic Left Ventricular Inside Diameter (LVIDs), (F) is a graph showing changes in diastolic left ventricular inside diameter (LVIDd), (G) is a graph showing changes in systolic Left Ventricular Anterior Wall (LVAWs), (H) is a graph showing changes in diastolic left ventricular anterior wall (LVAWd), (I) is a graph showing changes in systolic Left Ventricular Posterior Wall (LVPWs), and (J) is a graph showing changes in diastolic left ventricular posterior wall (LVPWd). N =12 for each group, p <0.05, p <0.01 compared to the control group; compared to the model group, # p <0.05, # p <0.01.
FIG. 2 is a graphical representation of the ECG evaluation AIC model rat atrial fibrillation test. (A) is a modeling process and a schematic diagram; (B) The diagram shows the normal rat and atrial fibrillation, the left diagram is the electrocardiogram of the normal rat, the right diagram is the electrocardiogram of the atrial fibrillation rat, F waves appear, p waves disappear, and RR intervals are uneven. (C) The amplitude of p-wave in AIC group is compared and shown in a graph, and compared with a control group, the amplitude of p-wave in AIC group is reduced, and n =6 in the control group; AIC group n =10. (D) For comparative illustration of RR intervals, AIC group was longer than control group RR intervals with control group n =6; aicgoup n =10. (E) For the graphic representation of the situation of the incidence rate of atrial fibrillation statistics, recording an electrocardiogram in 30s after administration on the 10 th day after the model building, wherein n =8 in a control group and the incidence rate of atrial fibrillation is 0%; AIC group n =15, incidence 80%; (F) For comparative illustration of duration of atrial fibrillation in rats, n =8 in the control group and n =15 in the aic group. All data were tested using t-test and expressed as Mean ± SEM. * p <0.05, p <0.01.
FIG. 3 is a schematic representation of the rotarod test. (A) is a speed schedule diagram of an accelerated spin test; (B) Statistical plots of the rate of decline were obtained for the Control (CTL), AIC and AIC + Amio groups. (C) is a statistical graph of the descent speeds of the groups. And (D) is a graph showing the walking distance of the rats before falling. Each group N =8. P <0.05, p <0.01 compared to control.
FIG. 4 shows ACh-CaCl 2 AF myocardial morphology immunohistochemical evaluation test results are shown in the figure. And (A) is a HE staining schematic diagram of a control group and an AF group. (B) is an illustration of staining of the HE in a ventricular cross-section. And (C) is a statistical map of the ventricular anterior wall thickness. (D) is a statistical plot of ventricular posterior wall thickness. And (E) is a statistical map of the cross-sectional area of the ventricles. (F) is a statistical plot of the atrial cross-sectional area. All data are presented as mean ± SEM, and the AF group was compared to the control group using t-test. * P<0.05,P<0.01。
Detailed Description
In order that the invention may be better understood, the invention will now be further described with reference to the following examples.
1. Model construction and test method
(1) Experimental animals and groups: male SD rats of 8-10 weeks old, 250g ± 25g, from the animal testing center of traditional Chinese medicine university, guangzhou. The experimental animals were kept in SPF-rated animal rooms of the southern China acupuncture research center during the study, and the rats were maintained in a 12-hour light/dark cycle (light was lit from 7 am to 7 pm), at a temperature of 23. + -. 2 ℃ and a relative humidity of 50% + -5%. Food and water were freely available in standard cages (42X 26X 15 cm) per 4-5 cages. The padding is replaced two to three times a week, and the cage is cleaned and disinfected two to three times a week. And finally, after the experiment is finished, a high-concentration carbon dioxide asphyxiator is used for enabling SD male rats to be euthanized. SD rats with normal electrocardiogram and echocardiogram are selected, and are randomly divided into 2 groups after adaptive feeding for 1 week, wherein 8 control groups and 10 model groups.
(2) Establishing a rat arrhythmic cardiomyopathy model: anesthetized rats were induced with 4% isoflurane for 4 minutes and then maintained anesthetized rats with 2% isoflurane, and the rats were placed in prone position on foam and animal heating pads. The rat tail was then wiped with alcohol to dilate the vein. Then, the vein is found from the near end to the far end according to the two sides of the Chinese character pin. After a mixed solution of Ach and CaCl2 (66. Mu.g +10mg/ml,1 ml/kg) was drawn out using a 1ml syringe, the mixture was injected into the tail vein. The above process is performed once a day for 10 days.
(3) And (3) electrocardiogram measurement: the rat is induced and anesthetized by using 4% isoflurane for 4 minutes, then the rat is maintained to be anesthetized by keeping 2% isoflurane, the rat is fixed on an animal heating pad in a supine position, the limbs of the rat are fixed by using an adhesive tape, after the rat breathes and has a stable heartbeat, a needle electrode carried by a powerlab instrument is connected into a powerlab multi-lead physiological recorder in a standard II lead (right upper limb and left lower limb) mode, and the electrocardiogram (5 mV range, sampling rate: 1 k/s) is recorded. After 5 minutes recording using labchart8.0 software, P-wave, RR-interval, atrial fibrillation f-waveforms were recorded on the electrocardiogram of each group of rats.
(4) And (3) cardiac ultrasonic detection: after isoflurane is used for inducing anesthesia (the concentration is 4 percent, and the ventilation volume is 1L/min), the rat is fixed on an ultrasonic operating table in a supine position, the anesthesia (the concentration is 2 percent, and the ventilation volume is 0.4L/min) is maintained by using a mask, and the thermostat is opened to maintain the body temperature of the rat at about 37 ℃, so that the animal is ensured to be in the optimal physiological state. The depilatory cream was applied evenly to the rat breast with a cotton swab, and after waiting for 2-3 minutes, the hair was wiped off using moist absorbent paper, leaving the rat breast skin fully exposed. The couplant is evenly smeared on the skin of the chest of the rat, and then the ultrasonic probe is slowly attached to the heart part, and the imaging result is observed. The probe is horizontally placed at the position of a sternum handle, the long axis of the left ventricle is positioned by using the B Mode, the short axis of the left ventricle is positioned by clockwise rotating the probe by 30-45 degrees according to the determined position, and the periodic contraction and relaxation conditions of ventricular muscles are observed and recorded by using the M-Mode. The data for 3-5 cardiac cycles were selected for analysis, and the systolic and diastolic anterior and posterior wall and inside diameter lengths were measured using the system measurement lines and averaged to calculate the left anterior ventricular wall end systolic thickness (LVAWs), left anterior ventricular wall end diastolic thickness (LVAWd), left posterior ventricular end systolic thickness (LVPWs) and left posterior ventricular wall end diastolic thickness (LVPWd), left ventricular end systolic diameter (LVIDs), left ventricular end diastolic diameter (LVIDd), left ventricular ejection fraction (EF,%) and short axis shortening rate (FS,%).
(5) Rotating rod test
Locomotor ability and coordination were assessed by using an accelerated rotating bar test. Rats were trained to walk on the bar for 3 days at constant low speed rotation (4 rpm), with a test once a day (YLS-4C, shanghai bioWill, inc., china). For the accelerated tests (4-40 rpm, over 300 seconds), each test was run for 3 trials on the test day, with 30 minutes intervals between trials. The time at which the rats fell off the rods was automatically recorded using the JLBehv-RRTR rotarod analysis system.
(6) HE staining
Rat hearts were stored in 4% paraformaldehyde fixing solution and fixed for 24h. The tissue is removed from the interstitial fluid and then dehydrated, paraffin embedded and sectioned. And after the completion, HE staining is carried out on the paraffin section. HE staining procedure was as follows: 1) Paraffin sections were deparaffinized and covered with water. 2) Sections were stained with Harris hematoxylin for 3min and rinsed with water. 3) An appropriate amount of alcohol containing 1% hydrochloric acid was dropped on the slices, and after differentiation for several seconds, the slices were washed clean with water. 4) The solution was rewound with 1% ammonia and washed clean with water. 5) The sections were stained with eosin stain for 2min. 6) The sections were dehydrated to make them transparent and sealed with neutral glue.
2. Results of the study
(1) And evaluating the heart morphology and function of the AIC model through an echocardiogram index. The modeling and treatment flow chart of the experiment is shown in fig. 1 (a), and the study is divided into 6 groups, namely, a Control group, an AIC group and an AIC + Amio group from left to right. As a result, as shown in fig. 1 (B-J), the AIC group rats exhibited a decrease in the ejection blood fraction (EF), the short axis shortening rate (FS) of the left ventricle, the left ventricle anterior wall end-systolic thickness (LVAWs), the left ventricle posterior wall end-systolic thickness (LVPWs), and the left ventricle posterior wall end-diastolic thickness (LVPWd), and an increase in the left ventricle inner diameter end-systolic Length (LVIDs), without affecting the left ventricle anterior wall end-diastolic thickness (LVAWd) and the left ventricle inner diameter end-diastolic length (LVIDd), compared to the CTL group. Timely administration of amiodarone as a rhythm control drug can reverse EF, FS and other cardiac function parameters and partially recover LVIDs, LVAWs and other cardiac morphological parameters. The above results indicate that ACh-CaCl2 administration induces cardiac functional impairment and cardiac morphological abnormalities in rats in the AIC group, which are completely or partially reversed by the antiarrhythmic drug amiodarone, meeting the definition and diagnostic criteria for AIC.
(2) Arrhythmia performance evaluation in AIC model rats. Tail vein injection of ACh-CaCl2 was performed on rats in the model group for 10 consecutive days, and electrocardiogram detection was performed while tail vein injection was performed on the tenth day of model creation, and the modeling process and the principle flow are shown in FIG. 2 (A), so as to capture the arrhythmia occurrence of AIC rats. The appearance of f-wave and the disappearance of p-wave in the electrocardiogram were used as the signs of the occurrence of atrial fibrillation, and the disappearance of f-wave and the appearance of p-wave were used as the signs of sinus rhythm recovery, and the results are shown in fig. 2 (B). P-wave amplitude was significantly reduced in the AIC group compared to the CTL group, as in fig. 2 (C), and RR interval was increased as in fig. 2 (D). The AF group was prompted for abnormal electrical activity in the atria and affected their ventricular rate. As shown statistically in FIG. 2 (E), the CTL group exhibited no spontaneous atrial fibrillation while the AIC group exhibited spontaneous atrial fibrillation. The above results are combined to determine tail vein injection calcium chloride (CaCl) 2 10 mg/kg) and acetylcholine (Ach, 66 mu g/kg), and the arrhythmia cardiomyopathy model can be successfully established by repeatedly administering the mixture for 10 days. The duration of atrial fibrillation in the rats tested is shown in FIG. 2 (F).
(3) Rod rotation test for evaluating the motor ability and coordination of AIC model rat
The decline of motor ability and coordination caused by cardiac insufficiency was further evaluated by the rotarod test. As shown in fig. 3 (a-D), the dropping time, the traveling distance and the average speed of the AIC-made model group were decreased as compared with the control group, and the difference between the above-mentioned indexes was highly significant in the two groups. The above results are consistent with the clinical manifestations of "weakness" in patients with AIC and reduced heart load capacity.
(4) Immunohistochemical staining was used to evaluate ACh-CaCl2 model heart morphology. As shown in FIGS. 4 (A-F), HE staining revealed that the AF group was significantly thinner in both anterior and posterior walls of the heart, and that the atrial and ventricular areas were significantly increased, but there was no evidence of fibrosis in the myocardial tissue, as compared to the control group. The above results are consistent with congestive expansion of the heart, morphological changes in the heart, and no or only mild fibrosis in the heart in patients with AIC.
Claims (3)
1. A method for constructing an arrhythmic cardiomyopathy animal model is characterized by comprising the following steps:
taking rats, injecting Ach and CaCl into tail vein according to dosage of 1ml/kg 2 Mixing the medicinal solutions, and injecting once a day for 10 days; the Ach and CaCl 2 The mixture ratio of the mixed liquid medicine is as follows: each milliliter of the mixed liquid medicine contains 66 mug of Ach and 10mg of CaCl 2 。
2. The method of claim 1, further comprising a step of anesthesia prior to tail vein injection, in particular: rats were anesthetized with isoflurane induction for 4 minutes, and then tail vein injection was performed while maintaining the rat anesthetic state with isoflurane maintained at 2%.
3. The method of claim 1 or 2, wherein the rat is an SD rat.
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