RU2760087C1 - Method for modeling stable atrial fibrillation in animals (variants) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely experimental cardiovascular surgery. An endocardial implantation of a bipolar electrode is performed in animal. In this case, the proximal end of the electrode is installed in the right atrium with access through the jugular or subclavian vein to the target zone: the sinoatrial node, which is the focus of primary atrial excitation and determined by activation electrophysiological examination. The distal end of the bipolar electrode is carried subcutaneously into the neck area, where it is connected to an implanted pacemaker. Next, atrial stimulation is carried out, while gradually increasing voltage from 1 to 4 Volts, gradually increasing the frequency of stimulation from 300 to 450 beats/min, with the achievement of constant atrial fibrillation. After that, high-frequency stimulation of the atria with a frequency of 450 beats/min at a voltage value of 4 Volts is carried out for 30 days. In the second variant, epicardial implantation of a bipolar electrode is performed in animal. In this case, the proximal end of which is installed in the right atrium in the target zone from the epicardium, which is the focus of primary atrial excitation and determined by activation electrophysiological examination, under visual control of vision with mini-access in the 3^rd-4^th intercostal space. The distal end of the bipolar electrode is carried subcutaneously into the neck area, where it is connected to an implanted pacemaker. Next, atrial stimulation is carried out, while gradually increasing voltage from 1 to 4 Volts, gradually increasing the frequency of stimulation from 300 to 450 beats/min, with the achievement of constant atrial fibrillation. After that, high-frequency stimulation of the atria with a frequency of 450 beats/min at a voltage value of 4 Volts is carried out for 30 days.

EFFECT: group of inventions makes it possible to simulate stable atrial fibrillation (AF) in animals, with the receipt of signs characteristic of prolonged persistent atrial fibrillation in humans.

2 cl, 1 dwg, 2 ex

Description

The group of inventions relates to biology, namely to the creation of a model of persistent atrial fibrillation (AF) in large animals with the aim of further use in pharmacological studies of drugs, as a testing ground for the development and optimization of methods for treating AF, for predicting the outcomes of AF when using either a different method of treatment, to study the pathogenesis of various stages of AF development, to train surgeons in existing methods of AF treatment.

Atrial fibrillation is the most common type of supraventricular tachyarrhythmia encountered in the clinical practice of cardiologists (Krijthe BP, 2013).

At AF, chaotic electrical activity of the atria is recorded with a frequency of up to 350-700 per minute with the absence of a P wave on the ECG, in which there is no possibility of coordinated contraction of the atrial myocardium, which, as a rule, is combined with an irregular rhythm of the ventricles (Kirchhof P., 2016). According to a study carried out by the American Heart Association (Chung, 2020), atrial fibrillation is the most serious threat of all heart rhythm disorders, and by 2020 it affects more than 33 million people in the world. Factors that increase the risk of the onset and development of AF include obesity, diabetes, a sedentary lifestyle, the presence of sleep apnea, coronary heart disease, hypertension and chronic alcoholism. If a patient has at least one risk factor for the development of cardiovascular diseases (hypertension, diabetes or smoking), then the likelihood of AF increases to 38% (Chung, 2020; Staerk L, 2018). In healthy people over 45 years of age, the risk of the onset and development of AF is over 20% (Staerk L, 2018).

The percentage of people with AF increases with age: if only 0.1% under 50 years old suffer from this disease, then 4% of the population aged 60-70 years and 14% of people over 80 years old have AF. The importance of AF as a public health problem is further emphasized by its strong and independent association with an increased risk of stroke, heart failure, sudden death, cognitive dysfunction, and a decline in quality of life (Meyre P, 2020; Conen D, 2011; Thrall G, 2006).

The development of AF is accompanied by the formation of changes in the structure of the atria due to their fibrosis. Fibrotic changes occur as a result of primary hyperextension of the atrial walls. Much less often, inflammation and genetic factors can be the cause of fibrosis. Atrial overstretching can also be a consequence of increased pressure in the ventricles of the heart. The reason for this may be due to stenosis or insufficiency, most often, of the mitral (or tricuspid) valve, long-term systemic hypertension, or the phenomenon of prolonged heart failure. The cause of myocardial fibrosis can be inflammatory processes as a result of sarcoidosis or autoimmune diseases with the presence of antibodies to proteins that make up the structure of cardiomyocytes (CMC). With the death of the CMC, stromal fibers replace the formed void, and the quality of the electrical impulse will suffer. By creating an organic substrate for the emergence and consolidation of pathological circles of excitation by the re-entry mechanism. The processes of fibrosis develop gradually, and in the course of experimental studies it was shown that at the first stage, separate zones of fibrosis appear in the atrial CMC. This creates an organic substrate for the onset of AF in the form of zones that are incapable of transmitting an action potential, which can subsequently merge into larger formations in the event of progression of the underlying pathological condition (Li M, 2014; Kalogeropoulos A, 2011).

Today, the recognized "gold standard" for the treatment of atrial fibrillation is the method of radiofrequency catheter ablation (2017 HRS / EHRA / ECAS / APHRS / SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation: Executive summary. Heart Rhythm. 2017. Vol.14 (10), e445-e494). During ablation, the affected myocardium coagulates with its further transformation into scar tissue that is not capable of conducting an impulse.

However, the catheter ablation method also has disadvantages, including:

1. Low penetrating ability of the thermoelectric effect of the ablation catheter (no more than 7-8 mm);

2. High morphological heterogeneity of tissues after ablation, which in the long-term follow-up period greatly reduces the therapeutic efficacy of AF treatment;

3. The laboriousness and complexity of the procedure, which is due to overcoming the heterogeneity of the surface of the heart (sinuses, anatomical features), which leads to the impossibility of performing continuous ablation lines delimiting the target zone along the perimeter and the entire depth of the myocardium;

4. A significant percentage of intra- and postoperative complications, which is associated with the duration of the procedure, the development of heart perforations, inflammatory syndrome, with frequent deaths;

5. The socio-economic side of the complex treatment of AF is associated with high costs and can exceed 1 million rubles per person (taking into account the cost of implantable devices).

Thus, given the ever-increasing relevance of the prevention and treatment of AF in humans, the insufficient effectiveness of existing methods of AF treatment, high requirements for the experience of the arrhythmologist and the technical equipment of the arrhythmological operating room, it becomes necessary to develop a model of AF in a large animal that is as close as possible to humans in its pathophysiological , functional, clinical, morpho-histological and mass-dimensional indicators. This model is necessary for conducting an extended and in-depth study of the pathophysiological causes of the onset, course and outcome of AF, for the development and testing of new methods of treatment of AF, for the study of new pharmacological substances for the treatment of rhythm disturbances, and also as a training bench for practicing skills in the course of training doctors. methods of surgical treatment of AF.

The possibility of using a pig as a model object in biomedical experiments is predetermined by its extreme similarity to humans in a whole set of anatomical and physiological characteristics and biological properties. The similarity is noted in the morphology and functioning of internal organs and systems, in particular the cardiovascular, digestive, excretory, endocrine and nervous systems, including the brain and sense organs, in the organization of metabolic processes, etc.

A known method of electrophysiological mapping of the atria and modeling of AF in pigs by high-frequency stimulation through lamellar epicardial electrodes (Jiunn-Lee Lin, Ling-Ping Lai Chih-Shen Lin et al. Electrophysiological Mapping and Histological Examinations of the Swine Atrium with Sustained (≥24 h ) Atrial Fibrillation: A Suitable Animal Model for Studying Human Atrial Fibrillation. Cardiology 2003; 99: 78-84). The disadvantage of this method is the use of transthoracic access with the opening of the chest and the use of massive plates of needle electrodes, which are applied directly to the surface of the heart, pierce the epicardium and injure the heart. This method does not allow to provide long-term, more than 30 days, myocardial stimulation in order to obtain a relevant human model of AF.

The objective of the presented group of inventions is to develop a method for modeling persistent atrial fibrillation in large animals, which has all the electrophysiological, functional and histological signs characteristic of long-term persistent atrial fibrillation in humans.

EFFECT: developed a method for modeling stable atrial fibrillation (AF) in animals (for example, pigs) by endovascular or epicardial implantation of a bipolar electrode into a predetermined zone, followed by long-term high-frequency stimulation with an implantable pacemaker, with obtaining signs characteristic of long-term persistent atrial fibrillation in humans ...

To achieve the technical result, two variants of modeling stable atrial fibrillation (AF) in animals are used with the implantation of a bipolar electrode, one end of which is inserted into the right atrium by the endovascular method or epicardially (using thoracotomy with intercostal mini-access): 1) endocardial implantation (minimally invasive) an electrode into the right atrium with access through the jugular or subclavian vein into the target zone - the sinoatrial node, which is the focus of primary atrial excitation, determined by electrophysiological examination (EPI); 2) implantation of an epicardial bipolar electrode into the right atrium into the target zone from the side of the epicardium, which is the focus of primary excitation of the atria, determined by EFI, under visual control of vision with a mini-access in 3-4 intercostal space. Then, in both the first and second versions, the opposite end of the electrode is passed subcutaneously into the neck area and connected to an implantable pacemaker programmed in high-frequency stimulation mode, followed by programmed stimulation for 30 days.

The electrode consists of several conductive elements (wires), united by a dielectric sheath. When installing, the ends of the electrode are determined by their location relative to the heart, hereinafter referred to as the proximal end, installed in the right atrium, and distal - distant from the heart, installed in the neck. The proximal end of the electrode, made in the form of a corkscrew, is screwed into the myocardial tissue, which makes it possible to contact the conductive elements with the heart tissues, as a result of which an electrical impulse is transmitted from the pacemaker (ECS). The distal end of the electrode is equipped with a connector that connects to the pacemaker and is sealed to prevent short circuits.

Atrial stimulation is performed using an implantable pacemaker. The stimulation mode for each animal is selected individually so as to ensure that AF is achieved, but does not cause disturbances in the rhythm of ventricular contraction.

The stimulation frequency values were selected from the range in which from 350 to 700 beats / min is achieved, preferably 450 beats / min. Values less than 350 beats / min and more than 700 beats / min were not used due to the ineffectiveness of this mode of atrial contraction stimulation. The voltage amplitude values were selected from the range from 1 to 10 Volts, preferably 4 Volts. Values less than 1 Volt were ineffective and did not lead to imposition of a rhythm, values over 10 Volts were excessive and had a negative effect on the rhythm of ventricular contraction, which led to ventricular fibrillation and cardiac arrest.

Preparation for surgery includes monitoring of electrocardiography, invasive monitoring of blood pressure, monitoring of peripheral blood oxygen saturation, infusion support of blood circulation.

Implantation of a bipolar electrode in option 1 includes isolation of the jugular or subclavian vein, insertion of an introducer, holding a catheter electrode for recording electrophysiological signals, performing an electrophysiological study (EPI) of the atria, placing the proximal end of the bipolar electrode in the right atrium under fluoroscopy control. To accurately localize the focus of primary excitation of the atria (sinoatrial node), the method of activation EPI is used. Then the distal end of the electrode is passed into the preformed pacemaker bed in the neck area and connected to the implanted pacemaker, followed by programming in the high-frequency stimulation mode. At the same time, the stimulation mode is gradually increased: the voltage is gradually increased, starting from 1 Volt, bringing to 4 Volts, and the stimulation frequency starts from 300 beats / min, bringing to 450 beats / min, in order to achieve constant atrial fibrillation (AF), followed by programmed stimulation - 450 beats / min at a voltage value of 4 Volts for 30 days.

Implantation of a bipolar electrode in option 2 includes performing a minithoracotomy in 3-4 intercostal space, applying a retractor, performing a pericardotomy and exposing the surface of the right atrium. Next, the femoral vein is isolated, an introducer is installed, a catheter-electrode is held to register electrophysiological signals, and an electrophysiological study (EPI) of the atria is performed. With the help of a catheter-electrode and a system for EPI, the focus of primary atrial excitation is precisely localized, for which the method of activation EPI is used. After that, in the found area - the focus of primary excitation of the atria - from the side of the epicardium, under visual control, the proximal end of the bipolar electrode is installed. Then the distal end of the electrode is passed subcutaneously into the neck area and connected to the implanted pacemaker, followed by programming in the high-frequency stimulation mode. At the same time, the stimulation mode is gradually increased: the voltage is gradually increased, starting from 1 Volt, bringing to 4 Volts, and the stimulation frequency starts from 300 beats / min, bringing to 450 beats / min, in order to achieve constant atrial fibrillation (AF), followed by programmed stimulation - 450 beats / min at a voltage value of 4 Volts for 30 days.

Figure 1 illustrates the dynamics of the ECG before the start of stimulation (A), during the implementation of stimulation (B), after 30 days of constant stimulation after disconnecting the electrodes (C). The illustrated ECG dynamics reflects two ways of modeling AF in pigs.

The experimental work was carried out in accordance with ethical standards governing experiments on animals in accordance with international and Russian regulatory documents ("European Convention for the Protection of Vertebrate Animals used for Experiments or for Other Scientific Purposes: EST No. 123" dated March 18, 1986, Strasbourg , 1986; Order of the Ministry of Health of the Russian Federation "No. 199n" On approval of the rules of good laboratory practice "dated 01.04.2016; Interstate standard GOST 33044-2014" Principles of good laboratory practice "; Euroguide on the accommodation and care of animals used for experimental and other scientific purposes. 2007. FELASA: Federation of European Laboratory Animal Science Associations, 25 Shaftesbury Avenue, London W1D 7EG, UK).

The invention is supported by the following examples.

Example 1. A method for modeling AF by endocardial (minimally invasive) implantation of an electrode into the right atrium of animals

The study was carried out on a group of 3 mini-pigs "Minisibs" of the selection of the ICG SB RAS. Preparation for the operation was carried out in the following way. On the morning of the surgery, the veterinarian performed premedication with a combination of atropine, zoletil-100, and ketamine. The dosage in each case was chosen based on the weight of the animal. After the onset of sleep, the animal is thoroughly washed, shaved off the bristles on the ears, chest, on the inner surface of the thigh and on the tail, and weighed. On the operating table, the animal is fixed in the "supine" position. A peripheral injection catheter of a suitable size is inserted into the marginal vein of the ear, and electrocardiography probes are attached to the chest wall. A plethysmographic sensor for monitoring peripheral blood oxygen saturation was attached to the tail. To control blood pressure, an arterial catheter was inserted into the left femoral artery by a puncture method, through which an invasive pressure control system was connected. All critical vital parameters - pulse, blood pressure, throat temperature, ECG, blood oxygen saturation - were recorded in "real time" by the V26E monitoring system (Philips, Germany). Intubation of the trachea with a tube 8-9 mm in diameter was performed against the background of muscle relaxation after intravenous administration of 4 mg of adruan. The obturating cuff is inflated, the Julian Plus anesthesia-breathing apparatus (Draeger, Germany) is connected, forced ventilation is started with 60% oxygen containing 3% sevoflurane (by volume) with a frequency of 12-14 cycles per minute. Respiratory volume is selected individually, depending on the weight of the animal. Through the previously installed peripheral catheter, infusion support of blood circulation is carried out with crystalloid (Ringer's solution, isotonic solution) and / or colloidal (gelafusin, polyglucin) solutions. The volume of infusion is determined by the veterinarian according to the weight and height indicators and the data of objective monitoring of the vital functions of the experimental animal. If necessary, a soft urinary catheter is inserted to control urine output. Before the operation, a transesophageal ultrasound examination (US) is performed, the parameters of the ejection fraction (EF) of the left ventricle, the end systolic (ESR) and diastolic size (EDD) of the left ventricle (LV), and the size of the left atrium (LA) are assessed.

Implantation of electrodes and pacemaker is performed in the following way. In the position on the left side, the right jugular or subclavian vein is punctured with a needle, a wire guide is inserted and an 8Fr introducer is installed to install the electrodes. The left femoral vein is punctured with a needle, a guide wire is inserted, and another 8Fr introducer for the EFI catheter is inserted. The conductors are cleaning up. A catheter electrode for electrophysiological studies such as Navistar (Biosence Webster, USA) or similar is inserted into the introducer on the lower extremity, using the C-arm OEC9800 (GE Healthcare, USA), under fluoroscopy control, the catheter is advanced to the right atrium through the right ventricle, periodically performing a control arteriography with 5-20 ml of X-ray contrast agent to make sure the catheter is in the correct position. An activation EFI is performed and the area of primary excitation of the atria is determined. A delivery system is passed through the introducer on the neck and the proximal end of an endocardial bipolar electrode of the Plexa type (Biotronik, USA) or similar is installed in the atrium. The distal end of the electrode is passed subcutaneously into the lateral region of the neck in the region of the spine, where it was connected to an Edora-type pacemaker (Biotronik, USA) or similar, with the ability to record ECG data. The entire system is placed in the subcutaneous tissue so that tissue tension is not created, after which the access area is sutured with non-absorbable sutures and treated with an antiseptic. Then, the normal sinus rhythm of the heart is recorded, after which, using an induction wireless programmer, the pacemaker is stimulated, while the stimulation mode is gradually increased: the stimulation frequency starts from 300 beats / min, bringing to 450 beats / min, and the voltage is gradually increased, starting from 1 Volt, bringing up to 4 Volts, so as to achieve permanent atrial fibrillation (AF). The result was monitored by ECG for 30 minutes. After that, the animal is taken out of anesthesia, activated and transferred to the aviary for observation. After 30 days, the pacemaker is turned off and the stimulation is stopped; as a result, the preservation of AF was recorded by ECG for 7 days. Before removing the animal from the experiment, a transesophageal ultrasound scan is performed, during which new indicators of EF, CVR and EDC of the left ventricle, and the size of the left atrium (LA) are assessed. After removal, samples are taken for histological examination.

As a result of the study, it was shown that constant endocardial stimulation of the atria in minipigs after 30 days leads to the formation of stable atrial fibrillation. Ultrasound revealed an increase in the LA size (26 ± 4 mm and 33 ± 4 mm before and after stimulation, respectively, p = 0.009), preservation of the LV ejection fraction (72 ± 8% and 69 ± 11% before and after stimulation, respectively, p = 0.34), preservation of LVEF (28 ± 4 mm and 29 ± 5 mm before and after stimulation, respectively, p = 0.89) and LV EDS (44 ± 8 mm and 48 ± 7 mm before and after stimulation, respectively, p = 0.41). There were no lethal outcomes among experimental animals. Microscopic analysis of histological changes in the LA myocardium revealed pronounced lymphocytic infiltration, widespread myocardial fibrosis, lipomatosis of LA tissues with signs of fatty degeneration, an increase in the number of muscle fibers per unit area of the atrium by more than 40%, hypertrophy and hyperchromatosis of cardiomyocyte nuclei.

Example 2. Modeling method for "open" surgery - AF by epicardial implantation of an electrode into the right atrium of animals

The study was carried out on a group of 3 mini-pigs "Minisibs" of the selection of the ICG SB RAS. Preparation for surgery and anesthetic management were similar to those described above. Before the operation, a transesophageal ultrasound examination was performed, the parameters of EF, CVR and EDC of the left ventricle, and the size of the left ventricle were assessed.

Surgical access and operative reception are carried out in the following sequence. In the position on the left side, a right-sided minithoracotomy is performed in 3-4 intercostal space, a retractor is applied to dilute the edges of the wound, a pericardotomy is performed, the pericardial leaves are spread on holders and the surface of the atria is visualized. Next, the right jugular or subclavian vein is punctured with a needle, a guide wire is inserted and an 8Fr introducer is inserted. Then the guide is removed. A catheter electrode for electrophysiological studies such as Navistar (Biosence Webster, USA) or similar is inserted into the introducer, using a C-arm OEC9800 (GE Healthcare, USA), under fluoroscopy control, the catheter is advanced to the right atrium through the right ventricle, periodically performing control arteriography by injection 5 -20 ml X-ray contrast agent to make sure the catheter is in the correct position. An activation EPI is performed and the area of primary excitation of the atria is determined. The catheter is left in the area found with gentle pressure (or pushing) so that the tip of the catheter is contoured (defined) from the epicardium. A proximal part of a bipolar electrode of the Plexa type (Biotronik, USA) or similar is placed in this area under visual control. The distal part of the electrode is passed subcutaneously into the lateral region of the neck in the region of the spine, where it was connected to an Edora-type pacemaker (Biotronik, USA) or similar, with the ability to record ECG data. The entire system was placed in the subcutaneous tissue so that tissue tension was not created. The thoracotomy wound and other access areas are sutured with non-absorbable sutures and treated with an antiseptic. Then the normal sinus rhythm of the heart is recorded, after which, using an induction wireless programmer, the pacemaker is stimulated, while the stimulation mode is gradually increased: the stimulation frequency starts from 300 beats / min, bringing to 450 beats / min, and the voltage is gradually increased, starting from 1 volt bringing up to 4 volts, so as to achieve permanent atrial fibrillation (AF). The result is monitored by ECG for 30 minutes. After that, the animal is taken out of anesthesia, activated and moved to the aviary for observation. After 30 days, the pacemaker is turned off and the stimulation is stopped. As a result, the preservation of AF was further recorded by ECG for 7 days. Then, before removing the animal from the experiment, a transesophageal ultrasound examination is performed, during which new indicators of EF, CVR and EDD of the left ventricle, and the size of the left atrium (LA) are assessed. After hatching, samples were taken for histological examination.

As a result, it was shown that constant epicardial atrial stimulation in minipigs after 30 days leads to the formation of stable atrial fibrillation (figure 1). Ultrasound revealed an increase in the LA size (25 ± 4 mm and 30 ± 5 mm before and after stimulation, respectively, p = 0.01), preservation of the LV ejection fraction (68 ± 9% and 65 ± 10% before and after stimulation, respectively, p = 0.23), preservation of LV ESR (27 ± 3 mm and 30 ± 5 mm before and after stimulation, respectively, p = 0.66) and LV ESR (46 ± 6 mm and 50 ± 8 mm before and after stimulation resp., p = 0.78). There were no lethal outcomes among experimental animals. Microscopic analysis of histological changes in the LA myocardium revealed pronounced lymphocytic infiltration, widespread fibrosis, foci of lipomatosis with transition to fatty degeneration of tissues, an increase of more than 50% in the number of muscle fibers per unit area of the atrium, hypertrophy and hyperchromatosis of cardiomyocyte nuclei.

Claims (2)

1. A method of modeling stable atrial fibrillation in animals by endocardial implantation of a bipolar electrode, the proximal end of which is installed in the right atrium with access through the jugular or subclavian vein into the target zone - the sinoatrial node, which is the focus of primary excitation of the atria and is determined by the activation, distal end a bipolar electrode is passed subcutaneously into the neck area, where it is connected to an implanted pacemaker, then the atria are stimulated, while the voltage is gradually increased from 1 to 4 Volts, gradually increasing the frequency of stimulation from 300 to 450 beats / min, with the achievement of constant atrial fibrillation, after whereby high-frequency atrial stimulation with a frequency of 450 beats / min at a voltage of 4 Volts is carried out for 30 days. 2. A method of modeling stable atrial fibrillation in animals by epicardial implantation of a bipolar electrode, the proximal end of which is installed in the right atrium in the target zone from the epicardium, which is the focus of primary atrial excitation and determined by activation electrophysiological examination, under visual control in 3 mini-access -4 intercostal space, the distal end of the bipolar electrode is carried out subcutaneously into the neck area, where it is connected to the implanted pacemaker, then the atria are stimulated, while the voltage is gradually increased from 1 to 4 Volts, gradually increasing the frequency of stimulation from 300 to 450 beats / min, s achievement of constant atrial fibrillation, after which high-frequency atrial stimulation with a frequency of 450 beats / min at a voltage value of 4 Volts is carried out for 30 days.

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