Establishment method and application of traumatic hemorrhagic sudden cardiac arrest rat model

Abstract

The invention discloses an establishment method and application of a traumatic hemorrhagic sudden cardiac arrest rat model, a rat is used as an experimental subject, a researcher can accurately adjust the hemorrhagic degree in an animal model by controlling the bloodletting speed and amount to better control experimental conditions, the repeatability and reliability of experimental results are ensured, and the method has the advantages of being high in operability and high in reliability. In the model building process, not only is the factor of blood loss considered, but also tissue damage caused by laparotomy is combined, physiological changes under the combined action of multiple factors after trauma are reflected more comprehensively, and the complexity and authenticity of the model are improved; the method has the advantages that the operation is simple, the experiment cost is lower, the repeatability is high, the scale and frequency of cardiac arrest research experiments are not limited, the experiment result has higher reliability and generalizability, and the method not only can be used for basic medical research and exploration of the pathophysiology mechanism of traumatic hemorrhagic shock, but also is suitable for pharmacological research, and has a wide application prospect. The effectiveness of new drugs or therapies for improving prognosis is assessed.

Description

Technical Field

[0001] The invention relates to a traumatic hemorrhagic cardiac arrest rat model technology, in particular to a method for establishing a traumatic hemorrhagic cardiac arrest rat model and application thereof. Background Art

[0002] In modern wars and daily accidents, traumatic blood loss is one of the main causes of death in patients with non-immediate fatal injuries, and the resulting cardiac arrest increases the difficulty of treatment. In order to better understand the pathophysiological mechanism of traumatic hemorrhagic cardiac arrest and develop effective prevention and treatment methods, it is very necessary to establish a stable and reliable animal model.

[0003] Currently, the commonly used animal models of cardiac arrest include: a pig model in which external jugular vein bleeding causes hypovolemic shock of varying severity leading to cardiac arrest, a cardiopulmonary resuscitation model for rats with type 2 diabetes mellitus produced by percutaneous epicardial electrical stimulation to induce fibrillation, a pig model in which tension pneumothorax pressure causes cardiac arrest by injecting gas after chest catheterization, a pig model in which pulmonary embolism causes cardiac arrest by injecting thrombus through the external jugular vein, and a pig model in which ventricular fibrillation cardiac arrest is induced by electrical stimulation after controlled hemorrhagic shock.

[0004] Although these cardiac arrest animal models can simulate the clinical course of cardiac arrest, they have various limitations. The pig models of hypovolemic shock of different severity to cardiac arrest caused by external jugular vein bleeding, the pig models of cardiac arrest caused by tension pneumothorax pressure after gas injection after chest tube placement, the pig models of cardiac arrest caused by pulmonary embolism caused by thrombus injection through the external jugular vein, and the pig models of ventricular fibrillation cardiac arrest induced by electrical stimulation after controlled hemorrhagic shock are relatively expensive to conduct experiments on large animals such as pigs, including the cost of purchasing animals, feeding, anesthesia, and subsequent treatment, resulting in low reproducibility and limiting the scale and frequency of experiments. Although external jugular vein bleeding can simulate different degrees of hypovolemic shock by precisely controlling the amount of bloodletting, and the operation is relatively simple, in actual clinical practice, cardiac arrest caused by hemorrhagic shock is often mainly arterial bleeding, and is accompanied by other complex physiological changes, such as tissue damage, etc. It is difficult to fully simulate these complex situations simply by bleeding. Although electrical stimulation is also a relatively controllable method, it requires precise placement of electrodes on the epicardium, which is difficult to operate. In addition to inducing ventricular fibrillation, electrical stimulation may also have other adverse effects on the heart, such as myocardial damage or electrophysiological changes. These side effects make the experimental results unreliable and may interfere with the study of the mechanism of cardiac arrest. The method of percutaneous epicardial electrical stimulation to induce fibrillation is used to make a cardiopulmonary resuscitation model for rats with type 2 diabetes. It is simple to operate, low cost, does not limit the scale and frequency of the experiment, and has high repeatability. However, in actual clinical practice, there are very few patients with cardiac arrest caused by electrical stimulation, and only a small proportion of patients with cardiac arrest caused by pulmonary embolism and tension pneumothorax. It cannot be used for the study of cardiac arrest caused by arterial bleeding and has poor applicability. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for establishing a rat model of traumatic hemorrhagic cardiac arrest and its application which is simple to operate, has low experimental cost, high repeatability, does not impose restrictions on experimental scale and frequency, and has experimental results with high reliability and generalizability.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a method for establishing a rat model of traumatic hemorrhagic cardiac arrest, comprising the following steps:

[0007] Step 1, after weighing the rat, place it on the operating table, fix the rat's limbs, and then induce anesthesia by intraperitoneal injection of pentobarbital. The injection dose of pentobarbital is 30 mg / kg body weight, and the body temperature of the rat is maintained at 38.0±0.5℃ using a temperature-controlled blanket;

[0008] Step 2, establish endotracheal intubation for the rat, and use a rat ventilator to assist mechanical ventilation, expose the carotid artery of the surgical rat at the neck, insert a 20F arterial catheter into the carotid artery of the rat, and connect the 20F arterial catheter at the carotid artery of the rat to a dynamic blood pressure detection device and an electrocardiogram monitoring device, and record the initial vital signs of the rat through the dynamic blood pressure detection device and the electrocardiogram monitoring device;

[0009] Step 3, surgically exposing the right femoral artery of the rat, and inserting a 22F arterial catheter into the right femoral artery of the rat for bleeding at a uniform rate during the period of blood loss in the rat;

[0010] Step 4: surgically expose the left femoral vein of the rat, and insert a 22F intravenous catheter into the left femoral vein of the rat for fluid replacement, drug administration, and blood transfusion;

[0011] Step 5, make a 5 cm incision in the middle of the rat's abdomen and perform laparotomy. After the laparotomy is completed, the opening is sutured with continuous silk thread;

[0012] Step 6, conduct blood loss intervention, the specific process is: take the current time as the experimental start time 0min, record it as R0, start timing, and bleed the rat at the same time through the 22F arterial catheter at the right femoral artery at a rate of 2ml / kg / min, and store the blood for reinfusion until the MAP blood pressure of the rat is monitored to drop to 10mmHg, stop bleeding, maintain the MAP blood pressure of the rat at 10mmHg for at least 10s, and determine it as cardiac arrest, at which time mechanical ventilation is suspended and maintained for 180 seconds;

[0013] Step 7, restart mechanical ventilation, and start intravenous injection of epinephrine at a dose of 20ug / kg body weight through the 22F intravenous catheter in the left femoral vein of the rat. After the injection is completed, continue to press the rat's heart with bare hands at 200 times / minute. At the same time, while injecting the first dose of calcium chloride solution through the 22F intravenous catheter in the left femoral vein of the rat, half of the blood stored for reinfusion is infused into the rat through the 22F arterial catheter in the right femoral artery of the rat within 5 minutes, and pay real-time attention to the changes in the rat's blood pressure from the beginning of the infusion. When the rat's MAP blood pressure>50mmHg and the pulse pressure difference>30mmHg When the pressure is on, stop pressing the rat's heart and observe whether there is ROSC. If there is no ROSC and the pressing time has not reached 10 minutes, continue pressing. If there is no ROSC and the pressing time has reached 10 minutes, stop pressing. If there is ROSC, stop pressing. From the beginning of pressing the rat's heart with 200 times / minute, every time the time reaches an integer multiple of 3 minutes, inject epinephrine once through the 22F intravenous catheter at the left femoral vein of the rat at a dose of 20ug / kg body weight until the pressing is stopped. When the pressing is stopped, if half of the blood stored for reinfusion has not been infused, continue infusing at the original speed until it is completed.

[0014] Step 8, infuse the remaining half of the blood to be returned into the rat through the 22F arterial catheter at the right femoral artery of the rat within 30 minutes, and record the timing time when the infusion is completed as R1, R1 is the timing time from the start time R0 of the experiment to the current time, at this time, blood is sampled from the rat for blood gas measurement through the 22F arterial catheter at the right femoral artery, if the free calcium is lower than 1.0 mmol / L, continue to push the second dose of calcium chloride solution through the 22F venous catheter at the left femoral vein of the rat; then, within 1 hour, infuse the same amount of normal saline as the amount of bloodletting of the rat in step 6 through the 22F venous catheter at the left femoral vein of the rat, and record the time when the normal saline infusion is completed as R2, R2 is the timing time from the start time R0 of the experiment to the current time, at this time, blood is sampled from the rat for blood gas measurement again through the 22F arterial catheter at the right femoral artery of the rat, if the alkali excess is If the base surplus is greater than or equal to -10 and the free calcium is greater than or equal to 1.0 mmol / L, directly proceed to step 9; if the base surplus is less than -10 and the free calcium is greater than or equal to 1.0 mmol / L, continue to infuse 0.5 times the amount of bloodletting in step 6 through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9; if the base surplus is greater than or equal to -10 and the free calcium is less than 1.0 mmol / L, then push the third dose of calcium chloride solution through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9; if the base surplus is less than -10 and the free calcium is less than 1.0 mmol / L, continue to infuse 0.5 times the amount of bloodletting in step 6 through the 22F intravenous catheter at the left femoral vein of the rat, and push the third dose of calcium chloride solution through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9;

[0015] Step 9, remove the 20F arterial catheter at the rat's carotid artery, the 22F arterial catheter at the rat's right femoral artery, and the 22F venous catheter at the rat's left femoral vein, and suture the wounds at the rat's carotid artery, the rat's right femoral artery, and the rat's left femoral vein, complete the experiment, and return the rat to the cage after keeping it warm with a temperature-controlled blanket;

[0016] Step 10: 24 hours after the experiment, the rats were evaluated and scored using mNSS (modified rat neurological deficit score), and then the rats were euthanized, and tissue specimens and blood samples were taken for testing to obtain experimental results.

[0017] Furthermore, in step 2, the parameters of the rat ventilator are set to a tidal volume of 8 ml / kg, a respiratory rate of 70 times / minute, and an inspired oxygen concentration of 21%. In step 7, when mechanical ventilation is restarted, the tidal volume, respiratory rate and inspired oxygen concentration of the rat ventilator remain unchanged from those in step 2.

[0018] Furthermore, in step 7, when the first dose of calcium chloride solution is injected, the dosage of the calcium chloride solution is 7.5 mg / kg body weight, the injection time is 10 s, and the volume concentration of the calcium chloride solution is 1%.

[0019] Furthermore, in step 8, the second dose of calcium chloride solution is equal in amount and concentration to the first dose of calcium chloride solution, and the push injection time is 10 s; the third dose of calcium chloride solution is equal in amount and concentration to the first dose of calcium chloride solution, and the push injection time is 10 s.

[0020] Furthermore, before the experiment, the rats were not allowed to eat or drink.

[0021] A method for establishing a traumatic hemorrhagic cardiac arrest rat model is used in drug screening and emergency measure evaluation.

[0022] Compared with the prior art, the advantages of the present invention are that in the process of establishing a rat model of traumatic hemorrhagic cardiac arrest, rats are used as experimental subjects, the operation is simple, the experimental cost is low, the repeatability is high, and there is no restriction on the scale and frequency of cardiac arrest research experiments. At the same time, by controlling the speed and amount of bloodletting, researchers can accurately adjust the degree of blood loss in the animal model to better control the experimental conditions and ensure the repeatability and reliability of the experimental results. In addition, in the process of model establishment, not only the factor of blood loss is considered, but also the tissue damage caused by laparotomy is combined, which more comprehensively reflects the physiological changes under the joint action of multiple factors after trauma, thereby improving The complexity and authenticity of the model are improved, and the experimental results have high reliability and generalizability. Therefore, the traumatic hemorrhagic cardiac arrest rat model established by the present invention can better simulate the situation of human traumatic hemorrhagic shock, including acute massive bleeding and subsequent cardiac dysfunction, which is of great significance for understanding pathophysiological processes and testing new treatment methods. It can not only be used for basic medical research and exploring the pathophysiological mechanism of traumatic hemorrhagic shock, but also is suitable for pharmacological research and evaluating the effectiveness of new drugs or therapies in improving prognosis, creating good model conditions for domestic and foreign peers to conduct basic and preclinical research on traumatic hemorrhagic cardiac arrest. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the traumatic hemorrhagic cardiac arrest rat model of the present invention;

[0024] Figure 2 This is a graph showing changes in blood pressure of rats in a sham operation group in an embodiment of the method for establishing a rat model of traumatic hemorrhagic cardiac arrest of the present invention;

[0025] Figure 3 This is a graph showing changes in blood pressure of rats in a saline resuscitation group in an embodiment of the method for establishing a rat model of traumatic hemorrhagic cardiac arrest of the present invention;

[0026] Figure 4 This is a graph showing changes in blood pressure of rats in a blood transfusion resuscitation group in an embodiment of the method for establishing a rat model of traumatic hemorrhagic cardiac arrest of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the accompanying drawings.

[0028] The invention discloses a method for establishing a traumatic hemorrhagic cardiac arrest rat model, which is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0029] Embodiment 1: Figure 1 As shown, a method for establishing a rat model of traumatic hemorrhagic cardiac arrest comprises the following steps:

[0030] Step 1. Weigh rats that were not fasted or deprived of water before the experiment, place them on the operating table, fix their limbs, and then induce anesthesia by intraperitoneal injection of pentobarbital at a dose of 30 mg / kg body weight. Use a temperature-controlled blanket to maintain the body temperature of the rats at 38.0±0.5°C.

[0031] Step 2, establish endotracheal intubation for the rat, and use a rat ventilator to assist mechanical ventilation, expose the carotid artery of the surgical rat at the neck, insert a 20F arterial catheter into the carotid artery of the rat, and connect the 20F arterial catheter at the carotid artery of the rat to a dynamic blood pressure detection device and an electrocardiogram monitoring device, and record the initial vital signs of the rat through the dynamic blood pressure detection device and the electrocardiogram monitoring device;

[0032] Step 3, surgically exposing the right femoral artery of the rat, and inserting a 22F arterial catheter into the right femoral artery of the rat for bleeding at a uniform rate during the period of blood loss in the rat;

[0033] Step 4: surgically expose the left femoral vein of the rat, and insert a 22F intravenous catheter into the left femoral vein of the rat for fluid replacement, drug administration, and blood transfusion;

[0034] Step 5, make a 5 cm incision in the middle of the rat's abdomen and perform laparotomy. After the laparotomy is completed, the opening is sutured with continuous silk thread;

[0035] Step 6, conduct blood loss intervention, the specific process is: take the current time as the experimental start time 0min, record it as R0, start timing, and bleed the rat at the same time through the 22F arterial catheter at the right femoral artery at a rate of 2ml / kg / min, and store the blood for reinfusion until the MAP blood pressure of the rat is monitored to drop to 10mmHg, stop bleeding, maintain the MAP blood pressure of the rat at 10mmHg for at least 10s, and determine it as cardiac arrest, at which time mechanical ventilation is suspended and maintained for 180 seconds;

[0036] Step 7, restart mechanical ventilation, and start intravenous injection of epinephrine at a dose of 20ug / kg body weight through the 22F intravenous catheter in the left femoral vein of the rat. After the injection is completed, continue to press the rat's heart with bare hands at 200 times / minute. At the same time, while injecting the first dose of calcium chloride solution through the 22F intravenous catheter in the left femoral vein of the rat, half of the blood stored for reinfusion is infused into the rat through the 22F arterial catheter in the right femoral artery of the rat within 5 minutes, and pay real-time attention to the changes in the rat's blood pressure from the beginning of the infusion. When the rat's MAP blood pressure>50mmHg and the pulse pressure difference>30mmHg When the pressure is on, stop pressing the rat's heart and observe whether there is ROSC. If there is no ROSC and the pressing time has not reached 10 minutes, continue pressing. If there is no ROSC and the pressing time has reached 10 minutes, stop pressing. If there is ROSC, stop pressing. From the beginning of pressing the rat's heart with 200 times / minute, every time the time reaches an integer multiple of 3 minutes, inject epinephrine once through the 22F intravenous catheter at the left femoral vein of the rat at a dose of 20ug / kg body weight until the pressing is stopped. When the pressing is stopped, if half of the blood stored for reinfusion has not been infused, continue infusing at the original speed until it is completed.

[0037] Step 8, infuse the remaining half of the blood to be returned into the rat through the 22F arterial catheter at the right femoral artery of the rat within 30 minutes, and record the timing time when the infusion is completed as R1, R1 is the timing time from the start time R0 of the experiment to the current time, at this time, blood is sampled from the rat for blood gas measurement through the 22F arterial catheter at the right femoral artery, if the free calcium is lower than 1.0 mmol / L, continue to push the second dose of calcium chloride solution through the 22F venous catheter at the left femoral vein of the rat; then, within 1 hour, infuse the same amount of normal saline as the amount of bloodletting of the rat in step 6 through the 22F venous catheter at the left femoral vein of the rat, and record the time when the normal saline infusion is completed as R2, R2 is the timing time from the start time R0 of the experiment to the current time, at this time, blood is sampled from the rat for blood gas measurement again through the 22F arterial catheter at the right femoral artery of the rat, if the alkali excess is If the base surplus is greater than or equal to -10 and the free calcium is greater than or equal to 1.0 mmol / L, directly proceed to step 9; if the base surplus is less than -10 and the free calcium is greater than or equal to 1.0 mmol / L, continue to infuse 0.5 times the amount of bloodletting in step 6 through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9; if the base surplus is greater than or equal to -10 and the free calcium is less than 1.0 mmol / L, then push the third dose of calcium chloride solution through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9; if the base surplus is less than -10 and the free calcium is less than 1.0 mmol / L, continue to infuse 0.5 times the amount of bloodletting in step 6 through the 22F intravenous catheter at the left femoral vein of the rat, and push the third dose of calcium chloride solution through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9;

[0038] Step 9, remove the 20F arterial catheter at the rat's carotid artery, the 22F arterial catheter at the rat's right femoral artery, and the 22F venous catheter at the rat's left femoral vein, and suture the wounds at the rat's carotid artery, the rat's right femoral artery, and the rat's left femoral vein, complete the experiment, and return the rat to the cage after keeping it warm with a temperature-controlled blanket;

[0039] Step 10: 24 hours after the experiment, the rats were evaluated and scored using mNSS (modified rat neurological deficit score), and then the rats were euthanized, and tissue specimens and blood samples were taken for testing to obtain experimental results.

[0040] In this embodiment, rats are used as experimental subjects, which is simple to operate, has low experimental costs, high repeatability, and will not limit the scale and frequency of cardiac arrest research experiments. At the same time, by controlling the speed and amount of bloodletting, researchers can accurately adjust the degree of blood loss in the animal model to better control the experimental conditions and ensure the repeatability and reliability of the experimental results. In addition, in the process of model establishment, not only the factor of blood loss is considered, but also the tissue damage caused by laparotomy is combined, which more comprehensively reflects the physiological changes under the joint action of multiple factors after trauma, improves the complexity and authenticity of the model, and the experimental results have high reliability and generalizability. Therefore, the traumatic hemorrhagic cardiac arrest rat model can better simulate the situation of human traumatic hemorrhagic shock, including acute massive bleeding and subsequent cardiac dysfunction, which is of great significance for understanding pathophysiological processes and testing new treatment methods. It can not only be used for basic medical research and exploring the pathophysiological mechanism of traumatic hemorrhagic shock, but also is suitable for pharmacological research to evaluate the effectiveness of new drugs or therapies in improving prognosis, creating good model conditions for domestic and foreign peers to conduct basic and preclinical research on traumatic hemorrhagic cardiac arrest.

[0041] Embodiment 2: This embodiment is basically the same as Embodiment 1, except that: in this embodiment, the parameters of the ventilator in step 2 are set to a tidal volume of 8 ml / kg, a respiratory rate of 70 times / minute, and an inspired oxygen concentration of 21%. In step 7, when mechanical ventilation is restarted, the tidal volume, respiratory rate, and inspired oxygen concentration remain unchanged from step 2.

[0042] In this embodiment, when the first dose of calcium chloride solution is injected in step 7, the dose of calcium chloride is 7.5 mg / kg body weight, the injection time is 10 s, and the volume concentration of the calcium chloride solution is 1%;

[0043] In this embodiment, in step 8, the second dose of calcium chloride solution is equal in amount and concentration to the first dose of calcium chloride solution, and the push injection time is 10 s. The third dose of calcium chloride solution is equal in amount and concentration to the first dose of calcium chloride solution, and the push injection time is 10 s.

[0044] In this example, 30 SD rats were prepared, each weighing about 400 g. The 30 SD rats were divided into three groups according to different intervention measures, with 10 rats in each group. The three groups were: a sham operation group, a saline resuscitation group, and a blood transfusion resuscitation group.

[0045] Experiments were performed on 10 SD rats in the sham operation group, saline resuscitation group and transfusion resuscitation group, respectively. Among them, the 10 SD rats in the sham operation group were subjected to experimental operations only according to steps 1 to 4, and the 10 SD rats in the transfusion resuscitation group were subjected to experimental operations according to the same steps 1-7. The experimental operations for the 10 SD rats in the saline resuscitation group were mostly the same as those for the 10 SD rats in the transfusion resuscitation group, with the only difference being that in step 7, what was infused into the 10 SD rats in the saline resuscitation group through the 22F arterial catheter at the right femoral artery of the rats within 5 minutes was not half of the blood stored for transfusion, but an equal amount of normal saline.

[0046] During the experimental operation, the blood pressure changes of each SD rat were detected in real time. The blood pressure change data of a SD rat in the sham operation group are as follows: Figure 2 As shown in Figure 2, the blood pressure changes of one SD rat that did not survive in the saline resuscitation group were as follows: Figure 3 As shown in Figure 2, the blood pressure changes of a SD rat in the transfusion resuscitation group were as follows: Figure 4 shown.

[0047] Figure 2 In the preparation phase from -20min to 0min and the experimental phase from 0 to 110min, the blood pressure of SD rats in the sham operation group remained relatively stable. Figure 2 It can be seen that the anesthesia, catheterization, and mechanical ventilation operations from step 1 to step 4 had no significant effect on the blood pressure of SD rats.

[0048] Figure 3 During the preparation phase from -20min to 0min, the mean arterial pressure of SD rats was relatively stable. After the bloodletting model was established, the blood pressure of SD rats dropped rapidly. The bloodletting reached the standard of cardiac arrest at 16min. Saline resuscitation and cardiac compression were started at 19min. After 10min of compression, the spontaneous heart rate did not recover. At 28min, the SD rats were declared dead and the experiment was terminated. Analysis Figure 3 It can be seen that the blood pressure of SD rats in the saline resuscitation group did not increase when their spontaneous heart rate did not recover.

[0049] Figure 4During the preparation period from -20min to 0min, the mean arterial pressure of SD rats was relatively stable. After the bloodletting model was established, the blood pressure of SD rats dropped rapidly. The bloodletting reached the standard of cardiac arrest at 17min. Blood transfusion resuscitation and cardiac compression were started at 20min. After 3 minutes of compression, the spontaneous heart rate was restored. After the compression was stopped, blood transfusion was continued, and the blood pressure gradually returned to normal levels. Analysis Figure 4 It can be seen that after blood transfusion, resuscitation and compression, the blood pressure of SD rats can gradually return to normal after the spontaneous heart rate is restored.

[0050] The experimental data statistics of 10 SD rats in the sham operation group, saline resuscitation group and blood transfusion resuscitation group are shown in Table 1:

[0051] Table 1 Experimental data statistics

[0052]

[0053] Analysis of the data in Table 1 shows that the 24-hour survival rate of SD rats in the blood transfusion resuscitation group was significantly higher than that in the saline resuscitation group. The 24-hour neurological function and liver and kidney function of the blood transfusion resuscitation group were also better than those of the saline resuscitation group.

[0054] In summary, in the rat model of traumatic hemorrhagic cardiac arrest, the effect of blood transfusion resuscitation was significantly better than saline resuscitation, which is consistent with clinical reality and can fully simulate the rescue scenario of TCA patients in clinical practice.

[0055] The present invention also discloses the application of the method for establishing the above-mentioned traumatic hemorrhagic cardiac arrest rat model in drug screening and emergency measure evaluation. The method for establishing the above-mentioned traumatic hemorrhagic cardiac arrest rat model can not only be used for basic medical research and exploration of the pathophysiological mechanism of traumatic hemorrhagic shock, but is also suitable for pharmacological research and evaluation of the effectiveness of new drugs or therapies in improving prognosis, thus creating good model conditions for domestic and foreign peers to conduct basic and preclinical research on traumatic hemorrhagic cardiac arrest.

Claims

1. A method for establishing a rat model of traumatic hemorrhagic cardiac arrest, characterized in that The following steps are involved: Step 1, after weighing the rat, place it on the operating table, fix the rat's limbs, and then induce anesthesia by intraperitoneal injection of pentobarbital. The injection dose of pentobarbital is 30 mg / kg body weight, and the body temperature of the rat is maintained at 38.0±0.5℃ using a temperature-controlled blanket; Step 2, establish endotracheal intubation for the rat, and use a rat ventilator to assist mechanical ventilation, expose the carotid artery of the surgical rat at the neck, insert a 20F arterial catheter into the carotid artery of the rat, and connect the 20F arterial catheter at the carotid artery of the rat to a dynamic blood pressure detection device and an electrocardiogram monitoring device, and record the initial vital signs of the rat through the dynamic blood pressure detection device and the electrocardiogram monitoring device; Step 3, surgically exposing the right femoral artery of the rat, and inserting a 22F arterial catheter into the right femoral artery of the rat for bleeding at a uniform rate during the period of blood loss in the rat; Step 4: surgically expose the left femoral vein of the rat, and insert a 22F intravenous catheter into the left femoral vein of the rat for fluid replacement, drug administration, and blood transfusion; Step 5, make a 5 cm incision in the middle of the rat's abdomen and perform laparotomy. After the laparotomy is completed, the opening is sutured with continuous silk thread; Step 6, conduct blood loss intervention, the specific process is: take the current time as the experimental start time 0min, record it as R0, start timing, and bleed the rat at the same time through the 22F arterial catheter at the right femoral artery at a rate of 2ml / kg / min, and store the blood for reinfusion until the MAP blood pressure of the rat is monitored to drop to 10mmHg, stop bleeding, maintain the MAP blood pressure of the rat at 10mmHg for at least 10s, and determine it as cardiac arrest, at which time mechanical ventilation is suspended and maintained for 180 seconds; Step 7, restart mechanical ventilation, and start intravenous injection of epinephrine at a dose of 20ug / kg body weight through the 22F intravenous catheter in the left femoral vein of the rat. After the injection is completed, continue to press the rat's heart with bare hands at 200 times / minute. At the same time, while injecting the first dose of calcium chloride solution through the 22F intravenous catheter in the left femoral vein of the rat, half of the blood stored for reinfusion is infused into the rat through the 22F arterial catheter in the right femoral artery of the rat within 5 minutes, and pay real-time attention to the changes in the rat's blood pressure from the beginning of the infusion. When the rat's MAP blood pressure>50mmHg and the pulse pressure difference>30mmHg When the pressure is on, stop pressing the rat's heart and observe whether there is ROSC. If there is no ROSC and the pressing time has not reached 10 minutes, continue pressing. If there is no ROSC and the pressing time has reached 10 minutes, stop pressing. If there is ROSC, stop pressing. From the beginning of pressing the rat's heart with 200 times / minute, every time the time reaches an integer multiple of 3 minutes, inject epinephrine once through the 22F intravenous catheter at the left femoral vein of the rat at a dose of 20ug / kg body weight until the pressing is stopped. When the pressing is stopped, if half of the blood stored for reinfusion has not been infused, continue infusing at the original speed until it is completed. Step 8, infuse the remaining half of the blood to be returned into the rat through the 22F arterial catheter at the right femoral artery of the rat within 30 minutes, and record the timing time when the infusion is completed as R1, R1 is the timing time from the start time R0 of the experiment to the current time, at this time, blood is sampled from the rat for blood gas measurement through the 22F arterial catheter at the right femoral artery, if the free calcium is lower than 1.0 mmol / L, continue to push the second dose of calcium chloride solution through the 22F venous catheter at the left femoral vein of the rat; then, within 1 hour, infuse the same amount of normal saline as the amount of bloodletting of the rat in step 6 through the 22F venous catheter at the left femoral vein of the rat, and record the time when the normal saline infusion is completed as R2, R2 is the timing time from the start time R0 of the experiment to the current time, at this time, blood is sampled from the rat for blood gas measurement again through the 22F arterial catheter at the right femoral artery of the rat, if the alkali excess is If the base surplus is greater than or equal to -10 and the free calcium is greater than or equal to 1.0 mmol / L, directly proceed to step 9; if the base surplus is less than -10 and the free calcium is greater than or equal to 1.0 mmol / L, continue to infuse 0.5 times the amount of bloodletting in step 6 through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9; if the base surplus is greater than or equal to -10 and the free calcium is less than 1.0 mmol / L, then push the third dose of calcium chloride solution through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9; if the base surplus is less than -10 and the free calcium is less than 1.0 mmol / L, continue to infuse 0.5 times the amount of bloodletting in step 6 through the 22F intravenous catheter at the left femoral vein of the rat, and push the third dose of calcium chloride solution through the 22F intravenous catheter at the left femoral vein of the rat, and then proceed to step 9; Step 9, remove the 20F arterial catheter at the rat's carotid artery, the 22F arterial catheter at the rat's right femoral artery, and the 22F venous catheter at the rat's left femoral vein, and suture the wounds at the rat's carotid artery, the rat's right femoral artery, and the rat's left femoral vein, complete the experiment, and return the rat to the cage after keeping it warm with a temperature-controlled blanket; Step 10: 24 hours after the experiment, the rats were evaluated and scored using mNSS (modified rat neurological deficit score), and then the rats were euthanized, and tissue specimens and blood samples were taken for testing to obtain experimental results.

2. The method for establishing a rat model of traumatic hemorrhagic cardiac arrest according to claim 1, characterized in that In the step 2, the parameters of the rat ventilator are set to a tidal volume of 8 ml / kg, a respiratory rate of 70 times / minute, and an inspired oxygen concentration of 21%. In the step 7, when mechanical ventilation is restarted, the tidal volume, respiratory rate and inspired oxygen concentration of the rat ventilator remain unchanged from those in the step 2.

3. The method for establishing a rat model of traumatic hemorrhagic cardiac arrest according to claim 1, characterized in that In step 7, when the first dose of calcium chloride solution is injected, the dosage of the calcium chloride solution is 7.5 mg / kg body weight, the injection time is 10 s, and the volume concentration of the calcium chloride solution is 1%.

4. The method for establishing a rat model of traumatic hemorrhagic cardiac arrest according to claim 3, characterized in that In step 8, the second dose of calcium chloride solution is equal in amount and concentration to the first dose of calcium chloride solution, and the push injection time is 10 seconds. The third dose of calcium chloride solution is equal in amount and concentration to the first dose of calcium chloride solution, and the push injection time is 10 seconds.

5. The method for establishing a rat model of traumatic hemorrhagic cardiac arrest according to claim 1, characterized in that Before the experiment, the rats were not allowed to eat or drink.

6. Use of the method for establishing a traumatic hemorrhagic cardiac arrest rat model according to any one of claims 1 to 5 in drug screening and emergency measure evaluation.

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