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ORIGINAL ARTICLE |
Department of Cardiothoracic Surgery, Jinling Hospital, Clinical Medicine School of Nanjing University, Nanjing, China
Hua Jing, MD, Tel: +86 025 80860075, Fax: +86 025 84819984, Email: hjjinghua{at}gmail.com, Department of Cardiothoracic Surgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing, China.
ABSTRACT
An experimental model of cardiopulmonary bypass in rats with pulmonary hypertension is necessary to understand underlying mechanisms and develop protective strategies. Male Sprague-Dawley rats were randomly divided into a sham group, cardiopulmonary bypass group, pulmonary hypertension group, and pulmonary hypertension with cardiopulmonary bypass group. Both groups with pulmonary hypertension received a subcutaneous injection of monocrotaline 60 mg · kg–1 on day 0. Cardiopulmonary bypass was instituted in one of them 21 days later. The sham and pulmonary hypertension control groups underwent cannulation only. Cardiopulmonary bypass was conducted for 60 min at a flow rate of 100 mL · kg–1 · min–1. Hemodynamic investigations, blood gas analysis, interleukin-6, tumor necrosis factor-
, and survival studies were performed subsequently. Time-dependent increases of serum interleukin-6 and tumor necrosis factor- were found after cardiopulmonary bypass in both groups. This model allows the study of multiple organ pathophysiological processes after cardiopulmonary bypass in rats with pulmonary hypertension, as well as the evaluation of possible protective strategies.
Key Words: Cardiopulmonary Bypass • Disease Models • Animal • Hypertension • Pulmonary • Rats • Systemic Inflammatory Response Syndrome
INTRODUCTION
Pulmonary hypertension (PH) is characterized by raised pulmonary arterial (PA) pressure, defined as mean PA pressure >25 mm Hg at rest or >30 mm Hg during exercise.1 PH may lead to decreased functional capacity, right ventricular (RV) failure, and early death.2 Although the prevalence of PH in patients with congenital heart disease is unknown, it has been suggested that 10% of all adult patients with congenital heart disease develop PH sooner or later.3 In congenital heart disease, PH may develop as a consequence of systemic-to-pulmonary shunting, which increases pulmonary blood flow. This leads to increased PA pressure, endothelial dysfunction, and increased vascular resistance. Cardiopulmonary bypass (CPB) is an essential component of conventional cardiac surgery and may be used in many other surgical procedures.4 CPB has been universally used in patients with chronic thromboembolic PH undergoing pulmonary thromboendarterectomy, and in those with PH undergoing lung transplantation or surgical correction of congenital heart disease. Despite excellent improvements, the manifestations of post-perfusion syndrome, such as pulmonary and renal dysfunction, neurological and gastrointestinal injury, increased interstitial fluid, and susceptibility to infections, have been continually reported.5,6 The underlying mechanism is probably multifactorial, including surgical trauma, anesthetic effects and muscle paralysis, increase in capillary permeability, and the impact of the CPB apparatus.6,7 To clarify the pathophysiological processes, numerous experimental studies have been conducted.8,9 However, to our knowledge, no small animal model of CPB in PH has been described so far. The current models do not adequately represent the mechanical and histological changes in animals undergoing CPB. To advance the field, additional research in a suitable rodent model with good survivability appears to be warranted. We successfully established a rat model of CPB in PH, based on an existing model of CPB.10
MATERIAL AND METHODS
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 85–23, revised 1996). The local ethics committee approved the experimental protocol and animal care methods. Male Sprague-Dawley rats weighting 450 to 550 g were used. Twenty rats were randomly allocated into 2 groups of 10 on day 0: a sham group that underwent cannulation for CPB only, and a CPB group that had CPB on day 21. Another 30 rats were given PH by injecting monocrotaline subcutaneously at a dose of 60 mg · kg–1 into the dorsum of the neck on day 0. These 30 rats were randomly allocated to 2 groups: a PH group of 20 had cannulation for CPB only, and 10 received CPB on day 21 (PH + CPB group). All animals were bred for 3 weeks before the procedure. Ten rats in the PH group had PA pressure measured on day 21, followed by exsanguination; the other 20 rats were exsanguinated 2 weeks after the operation. We modified our rat model of CPB described previously.10 Rats were anesthetized by intraperitoneal administration of butylene (60 mg · kg–1), and anesthesia was maintained with additional butylene. When a surgical level of anesthesia was achieved, the rats were secured supine. They were intubated with a 16G catheter (Abbott, Hoofddorp, The Netherlands) used as a tracheal tube, and mechanically ventilated (inspired O2 fraction, 40%; frequency, 30 min; peak pressure, 15 cm H2O) to achieve normocapnia (PaCO2 35–45 mm Hg). All subsequent procedures were performed under aseptic conditions.
The right femoral artery was cannulated for arterial pressure monitoring (24G heparinized Teflon catheter) and to collect samples for arterial blood gas analysis (GEM Premier 3000, USA). Mean arterial pressure was recorded during the experiment. The homolateral femoral vein was cannulated with a 20G catheter for blood and fluid replacement. After administration of heparin 250 U · kg–1, a multi-orifice 16G catheter was inserted into the right atrium through the right jugular vein for venous outflow. Tail artery cannulation was achieved with a 22G catheter that served as the arterial infusion line for the CPB circuit. The mini-CPB circuit comprised a 10-mL venous reservoir, a specially designed membrane oxygenator (gas exchange surface area of 0.05 m2, Micro-1; Kewei Medical Instrument, Guangzhou, China), and a roller pump (BT00-300M; Lange, Shanghai, China). All components were connected with polyethylene tubing. The blood was drained from the right atrium via a jugular vein catheter to a 10-mL sterile open reservoir by gravity and siphon. The relatively large venous tube (4 mm) and the high siphon level (30 cm) overcame the resistance of drain flow and produced adequate venous return. The total assembly dynamic priming volume approximated 4 mL. We used 4 circuits in each CPB group, and each circuit was used twice. The reused circuits were sterilized with ethylene oxide before operations. Body core temperature was monitored with a rectal probe, and kept at 37°C–38°C with a heat lamp placed around the animal and the CPB equipment. The CPB circuit was primed with 10 mL consisting of 1 mL heparin 250 U · kg–1, 8 mL synthetic colloid, and 1 mL sodium bicarbonate solution. The flow rate was gradually increased to 100 mL · kg–1 · min–1 and maintained for 60 min. Throughout the experiment, mean arterial pressure was maintained at 60–80 mm Hg. After 60 min of normothermic bypass, ventilation was restarted (inspired O2 fraction = 1) and the extra-corporeal circuit was disconnected. The right jugular vein was de-cannulated as soon as CPB was terminated. According to the blood pressure, some of the remaining priming volume was infused through the tail artery. The tail and femoral artery catheters were removed, and the incisions were sutured 4 h after CPB. The animals remained ventilated for another 60 min, and recovered in an oxygen-enriched environment thereafter. The rats were given water and food 6 h after the operation, and they were monitored for 24 h postoperatively. The sham group underwent the same cannulation procedures, except for connection to the extracorporeal circuit and discontinuation of ventilation. For PA pressure measurement, the rats were anesthetized, intubated, and arterial pressure was monitored as in the other animals. After administration of heparin 250 U · kg–1, a catheter was inserted into the PA to measure PA pressure, and the shape of the pressure tracing was displayed on a polygraph (RM-6000, Nihon Konden, Tokyo, Japan). The PA pressures of the other 40 rats were measured 24 h after CPB, before they were sacrificed.
Blood samples were obtained immediately after heparinization (T0), at the end of CPB (T1), and at 1, 2, 4, and 24 h after the operation (T2–T5). A 0.3-mL blood sample was taken for blood gas analysis. At the same time, arterial oxygen tension-to-inspired O2 fraction ratio and respiratory index were calculated. Respiratory index was calculated as the difference between alveolar and arterial oxygen tension gradients, divided by arterial oxygen tension. Plasma was separated by centrifuging at 4°C for 10 min, and stored at –70°C until analyzed for interleukin-6 (IL-6) and tumor necrosis factor- (TNF-). After 2 weeks of follow-up, the rats were exsanguinated, cardiopulmonary tissues were excised, and the wet weights of the RV, left ventricle including septum (LV + S), lungs, and lung-to-body weight ratio were measured. The RV– to-LV + S ratio was used as an index of RV hypertrophy.
Statistical analysis was performed using SPSS 13.0 software. Results are reported as mean ± standard deviation. Comparisons between groups were analyzed by one-way or two-way repeated-measures analysis of variance. Time-dependent changes and post-hoc comparisons were performed using the Tukey test. All p values less than 0.05 were considered statistically significant.
RESULTS
No thrombosis or hemorrhage occurred in rats with or without CPB. There was one delayed death in the CPB group and 2 delayed deaths in the PH + CPB group (2 and 5 days postoperatively). These rats failed to resume normal eating and drinking patterns. The 2-week follow-up period was uneventful for the surviving rats. Table 1
gives the physiological parameters of all animals after CPB. Because of the injection of mono-crotaline, PH rats demonstrated less weight gain, a higher RV– to-LV + S ratio, and higher mean PA pressure compared to sham group animals. Table 2 shows the physiologic data of all animals over the study period. Mean arterial pressure remained stable at each time point throughout the experiments. The results of blood gas analysis at different times fell within our acceptable range in all groups, indicating good gas exchange using the mini-oxygenator. In the CPB and PH + CPB groups, PaO2 values during and after CPB were significantly lower than baseline (p < 0.001), whereas PaCO2 remained stable. In the sham and PH groups, there was no change in PaO2 and PaCO2. The hematocrit decreased significantly in the CPB and PH + CPB groups during the perfusion process, but did not change in the sham and PH groups. This hemodilution followed by a return to near baseline values was due to the nature of CPB. The pH values in the CPB and PH + CPB groups decreased, and this group showed a tendency to develop metabolic acidosis. As time passed, PaO2 recovered gradually, and autonomous respiration resumed. Figure 1 shows the variation in serum levels of IL-6 and TNF-; both increased after termination of CPB, and there were significant time-dependent changes in the CPB and PH + CPB groups, but no time-dependent changes were found in the sham and PH groups.
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Pulmonary hypertension is caused by various patho-physiological mechanisms and characterized by a progressive increase in pulmonary vascular resistance due to vascular cell proliferation and obliteration of pulmonary microvasculature, leading to severe PH, right-sided heart failure, and early death.1–3 Cardiac surgeons often encounter patients with PH in congenital or rheumatic heart disease. Research focusing on cardioprotective strategies during cardiac surgery has been hindered by the lack of a suitable small animal model permitting CPB with good survivability. Survival studies have been performed using dogs or pigs, but they are limited due to sample size and costs. Although a number of rat CPB models have been described, none included PH animals.10–12 Thus we developed a CPB model with consistent long-term (>72 h) survival for studies of PH in rats.
Monocrotaline is a phytotoxin derived from the seeds of Crotalaria spectabilis, which is widely used to provoke endothelial injury in the pulmonary vascular tree. This has been useful in studying many features of PH including the extracellular matrix and growth factors. Monocrotaline is activated in vivo by mixed-function oxidases in the liver to form the reactive bifunctional cross-linked compound, monocrotaline pyrrole. Monocrotaline has a selective toxic effect on pulmonary vessels, without an effect on systemic vessels, which can stimulate vascular smooth muscle cell proliferation and hyper-contraction, enhance apoptosis, increase macrophage infiltration, and decrease endothelium-dependent relaxation. In-vitro treatment of endothelial cells with monocrotaline pyrrole showed covalent binding to cytosolic and cytoskeletal proteins and to DNA. Subcutaneous injection of monocrotaline (60–80 mg · kg–1) in rats leads to increased muscularization and development of marked PH after 4 weeks.13 Monocrotaline can increase muscularization in precapillary pulmonary arterioles, and induce RV hypertrophy. A strong inflammatory reaction develops, with endothelial cell death in small arterioles, a reduction in the number of peripheral arteries, and a twofold increase in the ratio of alveoli to arteries.14 Both monocrotaline and increased pulmonary blood flow increase muscularization in precapillary pulmonary arterioles, but the PA pressure rise and RV hypertrophy are slower and more modest in the monocrotaline model. Moreover, no vasoconstriction or polycythemia is observed following monocrotaline. However, although monocrotaline produces substantial PH and RV hypertrophy in rats, it does not reproduce the pathologic features of vascular injury seen in severe human disease (neointimal formation and plexiform lesions).
In this study, marked increases in mean PA pressure and RV hypertrophy were observed in rats 3 weeks after administration of monocrotaline. These conditions closely resemble the changes due to PH in patients. An interesting aspect of the present study was that the mean PA pressures in PH rats were milder than in previous published research.13 In the previous study, RV systolic pressure increased from 22 mm Hg in normal rats to 49 mm Hg, consistent with the development of PH; however, lower PA pressures were found in the sham group (6.17 vs. 22 mm Hg) in the present study. One explanation for these low pressures is an anesthesia-induced decrease in PA pressure, another is that we measured PA pressure 3 weeks after monocrotaline injection, while it was carried out after 4 weeks in the previous study.
In the search for a suitable model to study CPB in PH rats, we modified a rodent model that had been previously described and utilized.10 We adopted the tail artery to serve as the arterial infusion line instead of a jugular artery, to decrease possible neurological deficits after CPB. A small, specifically designed oxygenator with a surface area of 0.05 m2 was used to reduce the priming volume to 10 mL, thus abolishing the need for donor blood to prime the circuit and achieving an appropriate CPB volume. In addition, lowering of the reservoir level was observed in some animals during the experiments. It is possible that this phenomenon was due to blood sampling, fluid accumulation in the tissues, and evaporation, which could be easily corrected using additional Ringer’s solution for replacement. A multi-orifice catheter in the forepart was employed to assure the position during right atrial draining, and careful positioning of the venous outflow catheter allowed for optimal venous drainage and CPB flows consistent with the normal cardiac output in rats.
Tumor necrosis factor- is an important proinflammatory factor in the activation of other cytokines and induction of the expression of adhesive molecules in endothelial cells.15 In animal studies, TNF- was shown to increase pulmonary vascular reactivity and decrease prostacyclin production in PA smooth muscle cells.16,17 It is also increased in patients with PH.18 We documented significant differences not only between plasma levels of TNF- in the sham and CPB groups, but also between the PH and PH + CPB groups. The rise in serum TNF- was higher after CPB. There are some explanations for this discrepancy. First, ventilation continued during CPB in clinical studies, possibly reducing the hypoxia-induced inflammatory reaction. Second, inflammation might have been exacerbated because of the reused circuits in our study. Interleukin-6 is responsible for coordination of the acute phase response, and it plays a positive role in the local inflammatory reaction by amplifying leukocyte accumulation. Clinical investigation showed that plasma levels of IL-6 were associated with the severity of the inflammatory response to CPB, and it is a known independent risk factor for cardiovascular events.19 Several reports have indicated the potential role of IL-6 in severe primary PH.20 In the present study, circulating IL-6 levels showed similar changes to TNF-. However, an aspect of this study was that the maximum level of IL-6 was detected 2 h after CPB, earlier than TNF-.
This model has several potential pitfalls. During the bypass processes, the heart kept beating at 210–280 beats · min –1 as arterial pressure remained pulsatile and must contribute to the blood pressure. Lung perfusion persisted during CPB. In addition, this study did not systematically investigate the systemic inflammatory response in CPB and PH + CPB rats in detail. However, due to its minimal invasiveness, reproducibility, and the ease of recovery, this model will not only facilitate further research to elucidate mechanisms of CPB and organ dysfunction but also to evaluate novel approaches to improved organ protection in PH rats. Medium- or long-term post-CPB multiple organ pathophysiological processes, possible protective strategies, CPB time, and histological outcomes in PH rodents can be assessed more accurately than with other CPB models. Major advantages of this model include its overall feasibility and cost effectiveness. In the field of myocardial and pulmonary protection, rodent models retain an important role in research.
REFERENCES
Asian Cardiovasc Thorac Ann 2009;
17:285-290
© 2009 by SAGE Publications
DOI: 10.1177/0218492309104775
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