Cardiopulmonary bypass (CPB) is an essential component of conventional cardiac surgery. However, the procedure is still associated with frequent manifestation of postperfusion syndromes such as clinical signs of pulmonary and renal dysfunction, neurological and gastrointestinal injury, coagulation disorders, and hemolysis, increase in interstitial fluid, and susceptibility to infections.1
The underlying mechanisms are probably multifactorial, including surgical trauma, anesthetic effects and muscle paralysis, increase in capillary permeability, and impacts of the CPB apparatus. In order to clarify the pathophysiology, numerous experimental studies have been developed, but mostly with larger animal models, such as dogs, sheep, pigs, and rabbits.2
Due to the similar size and anatomical structure with humans, the large animal models were initially used for CPB studies. However, they were limited by of the need for trained human and material resources. The rat gradually replaced large animals for various reasons, including reduced cost and experimental devices, feasibility for single-person studies, availability of genetic and protein analysis and so forth.3
Previous rodent models were not relevant to clinical application. After summarizing the results of previous studies, we developed an improved version, according to our own conditions. We hope to establish a novel, convenient and effective model in rats, with the following advantages: (1) precise control of temperature and depth of anesthesia, (2) good stability and reproducibility, (3) similarity to clinical cardiac surgery, and (4) long-term survival. We established a clinically relevant model of CPB with deep hypothermic circulatory arrest (DHCA) in the rat. Our CPB circuit has several novel features, including a small priming volume, active cooling/rewarming processes, vacuum-assisted venous drainage, peripheral cannulation without thoracotomy or sternotomy, and an accurate means of monitoring peripheral tissue oxygenation. The small animal model provides a simple experimental system for future studies that use CPB and DHCA.4
Male Wistar rats from the National Animal Center (Beijing China), weighing 250–300 g, were used in all experiments. All animals received humane care in compliance with the Guide for the care and use of laboratory animals published by the National Institutes of Health (NIH publication 83–23, revised 1996). The study protocol was approved by the Laboratory Animal Ethics Committee of Capital Medical University.
Anesthesia and preoperative preparation
Rats were anesthetized with intraperitoneal administration of ketamine (50 mg/kg) and chlorpromazine (2 mg/kg). In order to reduce respiratory secretion, atropine (0.1 mg/kg) was also added to the agents. Following anesthesia, rats were placed in a supine position. A polyethylene cannula (internal diameter 3 mm) was inserted into the trachea and the lungs were mechanically ventilated with a small animal ventilator (Rodent Respirator, TKR-200C, China). The tidal volume was approximately 10 ml/kg. The respiratory rate was 60 breaths per minute. The respiration concentration of O2 was 100%. Ventilation was adjusted to maintain an arterial carbon dioxide tension (PaCO2) of 35–45 mmHg. Anesthesia was maintained throughout the experiment with additional doses of intravenous ketamine.
All subsequent procedures were performed under aseptic conditions. The left femoral artery was cannulated by a 22-gauge heparin-coated Teflon catheter to monitor systemic arterial pressure (SAP) and to collect arterial blood for arterial blood gas analysis (blood gas analyzer, GEM Premier 3000, USA). The homolateral femoral vein was cannulated with a 20-gauge catheter for blood and fluid replacement. Following administration of heparin (500 U/kg), a 16-gauge catheter, modified to a multisided-orifices cannula in the forepart, was inserted into the right jugular vein and advanced to the right atrium. This position resulted in excellent drainage, which supported high-flow (>100 ml•kg-1•min-1) perfusion for complete CPB. Subsequently, the left carotid artery was exposed and cannulated with a 22-gauge catheter placed into the aortic arch, which served as the arterial perfusion line for the extracorporeal circuit. A nontransthoracic rat model of CPB is shown in Figure 1.
The entire CPB circuit consisted of a reservoir (5 ml syringe), a membrane oxygenator with a priming volume less than 4 ml (Kewei Medical Instrument Inc, Shenzhen, China), a Stöckert twin-roller pump (Stöckert, Munich, German), a heat exchanger with a priming volume less than 3 ml (Xijing Medical Instrument Inc, Xi'an, China), all of which were connected via silicon tubes (outer diameter about 4 mm, inner diameter about 3 mm, length about 60 cm). The difference in height between the venous cannula tip and the venous reservoir blood level was about 35 cm. During the CPB period, the heater/cooler (Maquet Critical Care AB, Solna, Sweden) was used to control the temperature of the rats. The priming solution, composed of 6% HES130/0.4 (Fresenius Cabi Pharmaceutical Limited, Beijing, China) and 125 IU heparin, was less than 12 ml. A schematic diagram is shown in Figure 1.
Rats were randomized to CPB with DHCA and the control groups and both included 10 rats. The experimental protocol, including blood and crystalloid fluid administration, anesthesia, orotracheal intubation, ventilation, cannulation, and heparinization were identical in both groups. In the DHCA group, a rectal temperature of 18°C was achieved over 40 minutes, using CPB assisted cooling. At this temperature, rats still showed a heart rate in the 30 seconds. If the heart rate was not 0, we further cooled to induce cardiac arrest by echocardiogram (typically 14°C). The circuit was then turned off and rats were left in a DHCA state for 15 minutes. Rats were rewarmed to 34°C to 35°C over a period of 36 to 42 minutes using CPB-assisted rewarming, a heating blanket, and a heating lamp, ensuring that the temperature gradient between the CPB circuit and body core did not exceed 10°C. Simultaneously, 0.1 mEq of sodium bicarbonate and 0.14 mEq of calcium chloride were administered. After full rewarming, the remaining priming volume was reinfused and animals were weaned from CPB.
Physiologic measurements and analysis
The core and circuit temperature were continuously monitored with a dual temperature monitor. During CPB, the blood flow and venous saturation were continuously monitored. Targeted CPB flow was 100 to 120 ml•kg-1•min-1, corresponding to about 70% of normal cardiac output.
The data were expressed as mean ± standard deviation (SD). Data for non-repetitive measurements were analyzed by one-way analysis of variance (ANOVA) for betweengroup comparison. Data at different time points were analyzed by variance analysis of repeated measures. All analyses were performed using SPSS 15.0 software for windows (SPSS Inc, Chicago, IL, USA). P values of less than 0.05 were considered statistically significant.
All experiments were processed without incident and all animals survived the operation. Neither thrombosis nor hemorrhage occurred in rats with or without CPB.
Blood gas analysis
The average values of blood gas parameters, immediately before the initiation of the cooling process (baseline) and at various stages of the procedure (i.e., during cooling at 20 and 40 minutes and during rewarming at 30, 60 and 90 minutes) are shown in Table 1. Mean hematocrit was significantly lower in the CPB group compared withSham due to hemodilution associated with priming. Hematocrit remained stable in the sham group throughout the experiments.
Table 2 displays the parameters of weight and vital signs. In intergroup comparisons, statistical differences were observed in all parameters (i.e., during cooling at 20 and 40 minutes and during rewarming at 30, 60 and 90 minutes).
In this study, we successfully established a novel rat model of cardiopulmonary bypass for deep hypothermic circulatory arrest without blood priming.
Despite the decline in the hematocrit (Hct) and hemoglobin (Hb) caused by the lack of blood in the priming solution, low level lactate and adequate oxygenation in the CPB group demonstrate sufficient systemic blood supply. We achieved the goal of priming without blood using the model.5
CPB is one of the major technological advances in medicine allowing the cardiac surgeon to operate in controlled conditions.6,7 While modern CPB may have made rapid advances, it is associated with repeated side effects.8 CPB has been shown to be associated with systemic inflammatory response leading to postoperative organ dysfunction and postperfusion syndromes.9 The study of bypass pathophysiology and the testing of novel therapeutic strategies were more important than the CPB technology itself in our recent clinical investigation.10 A proper preclinical bypass animal model was needed for ethical reasons, which prevent the utilization of experimental procedures in the clinical setting.11 Earlier experimental studies to induce CPB have been limited to large animals. Unfortunately, large animal models are increasingly expensive, complex, tedious, labor-intensive, entailing sophisticated surgical expertise, and associated with short recovery intervals.12
Open heart surgery with CPB induces a systemic inflammatory response, due to surgical trauma, exposure of blood to foreign surfaces, ischemia-reperfusion injury, mechanical shear stress, hemodilution, and hypothermia.13 Extreme hemodilution with a relatively large priming volume and an inflammatory response caused by contact with the synthetic surface of the CPB circuit are thought to be the main mechanisms for water retention and tissue edema, which can result in postoperative organ dysfunction, especially in immature neonatal organs.14
All the changes were consistent with clinical results. Further, we studied and analyzed some pivotal sections of the model, as follows.1
The roller pump, the most commonly used source of power for CPB clinically, was used in our CPB circuit. The roller pump, however, can cause hemolysis, Therefore, the small pump head adopted in our study not only inherits the advantages of the peristaltic pump, but truly reflects the clinical process. With all these modifications, we were able to achieve a flow rate of 100–150 ml·kg-1· min-1, which is relevant to clinically used CPB circuits.15
Animal model with DHCA
Our circuit has several innovative elements, including vacuum-assisted venous drainage, peripheral cannulation (without thoracotomy or sternotomy), active heating and cooling with a heat exchanger, a novel small membrane oxygenator, lack of need for intubation, a venous oximeter, and ultrasonic flow meter. In our experiments, we minimized surgical trauma and improved the postoperative survival rate.10 After cessation of CPB, the cannula was removed from the right jugular vein that was then ligated.
Miniaturization of the membrane oxygenator represents the major limiting factor in the creation of a successful CPB model in small animals. Previous models required high priming volumes to achieve acceptable hematocrit concentrations during the experiment. Our study used a miniature novel oxygenator, modified venous cannula, and reduced length of connection tubing, which allowed minimization of the priming volume, thus improving postoperative outcomes.2
Venous drainage tube
Currently, the 16-gauge catheter is the most commonly used in the rat model. We consider that efficient venous drainage needs: (a) a soft tube to avoid chronic venous insufficiency, (b) the inner diameter of the tube around 2 mm, and (c) the tip of the tube containing several lateral apertures. This study used a 22-gauge silicon tube from a venous blood sampling needle with a diameter of nearly 2 mm, achieving a satisfactory flow rate and increased success rate.16
Based on the principle of miniaturization and availability, we created a novel rat model of cardiopulmonary bypass for deep hypothermic circulatory arrest without blood priming. Depth of anesthesia, and body temperature were controlled precisely. The parameters of hemodynamics and blood gas analysis proved the effectiveness of the model and its relevance in the injury mechanisms underlying CPB with DHCA. Further studies are needed for short-term and long-term neurological dysfunction after DHCA.
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