In vivo and ex vivo effects of propofol on myocardial performance in rats with obstructive jaundice Names of Authors

Background The responsiveness of “jaundiced heart” to propofol has not been fully elucidated. The purpose of this study aims to evaluate the effect of propofol on myocardial performance in rats with obstructive jaundice. Methods Male Sprague-Dawley rats (n=40) were randomly allocated into two groups. Twenty received bile duct ligation (BDL) whilst same number of rats underwent sham operation. Seven days after the surgery, three concentrations of propofol were administered in vivo and ex vivo (Langendorff) preparations. Heart rate (HR), left ventricular end-systolic pressure (LVESP) left ventricular end-diastolic pressure (LVEDP), maximal rate for left ventricular pressure rising and declining (±dP/dtmax) were measured. Performance of the rat hearts toward propofol was examined using above indexes of cardiac function. Results Impaired basal cardiac function was observed in the BDL isolated hearts, whereas indexes of basal cardiac function, LVESP and ±dP/dt, in vivo were significantly higher than those receiving sham operations. With low or intermediate concentrations of propofol, these indexes of cardiac function in both groups were within the normal physiological range and the responsiveness to propofol was unaffected by bile duct ligation. While propofol of the highest concentration was administered, significant decline in cardiac function was found in BDL group. Conclusion The basal cardiac performance was better in vivo but worse in ex vivo in the BDL group than in the sham controls. Propofol of low and intermediate concentration appears not to have significant negative effect in cardiac function of rats with obstructive jaundice. Background The association between obstructive jaundice and postoperative multiple organ dysfunction syndrome is a well known clinical phenomenon.[1-3] Cardiovascular instability of cholestatic patients, caused by depression of myocardial performance and defective vascular reactivity, were considered to be major mechanisms in the pathophysiology of multiple organ dysfunction syndrome.[4-5] In 1986, Green[5] found impaired left ventricular performance in dogs with cholemia, and this hepatic cardiomyopathy was called “jaundiced heart”[4]. The “jaundiced heart” was also characterized by defective vascular reactivity that has been attributed to altered beta-adrenergic receptor signaling function,[6] and the downregulation of cardiac beta-adrenoceptor density and affinity and membrane fluidity.[7] Propofol is a widely used intravenous anesthetic both in the induction and maintenance of anesthesia. It is characterized by rapid onset and offset of drug effect, and fast elimination from the body.[8-9] However, propofol may also induce cardiovascular depression, manifested mainly by a decrease of arterial blood pressure,[10] which is attributed to inhibition of the sympathetic nervous system[11] and negative inotropic effect[12] with reduction in preload[13] With this background, there was still need to clarify whether propofol would affect myocardial performance in rats with obstructive jaundice when compared with that of normal rats. In present study, the rat model of 7-days bile duct ligation (BDL) was used to evaluate the effect of propofol on cardiac function in vivo. Moreover, an ex vivo experiment was used to evaluate the direct effects of propofol on isolated heart of BDL rats in order to exclude vaso-dilative effect of propofol and related neurohormonal adaptation in vivo.[14-15] Methods Animals This study was approved by the Institutional Animal Care and Use Committee, the Second Military Medical University. Male Sprague-Dawley rats, weighing 250-300 g, were housed in a temperature and humidity-controlled environment with a 12-h light dark cycle. The rats received unlimited access to water and food. The food was removed from the cage 16 hours prior to the experiments. Procedures Forty rats were randomly allocated into two study groups. Twenty received bile duct ligation (BDL group) whilst twenty underwent sham operation (SO group), according to Lee’s[16] methods. Rats were anesthetized with pentobarbital sodium (40 mg kg intraperitoneal (i.p.)). After opening of the abdominal cavity, the bile duct was ligated at a point proximal to the hilus and a point immediately distal to the entry of the bile duct into the duodenum. The bile duct was severed between the ligatures. Sham operation consisted of all procedures and manipulations except for the ligation and severing the bile duct. Blood sampling for the assessment of levels of total bilirubin (TBIL) and alanine aminotransferase (ALT) was collected at 7 days after BDL and sham operation. In vivo experiment The in vivo experiments consisted of 20 rats, 10 receiving BDL and 10 sham operation. Also they were randomized from the in vivo group. On the seventh postoperative day, rats were anesthetized with sodium pentobarbital (40 mg kg i.p.). All the surgical procedures were performed under sterile conditions. After tracheotomy, tracheal intubation, and mechanical ventilation was provided with a Harvard Apparatus Rodent Respirator (Harvard Apparatus, Boston, MA), with room air at 60–70 breaths per min. Body temperature was maintained using a heating pad under the animal to keep the rectal temperature at 36±1°C. A 1-cm midline incision was made in the neck, and the right common carotid artery was exposed. A polyethylene cannula was inserted through the artery to the left ventricle. The cannula was connected to a pressure transducer (Rm6240B type, Chengdu Instrument Corp, China) to monitor the intraventricular pressure. A cannula connected to a microinfusion pump (Graseby 3500, Graseby Medical Ltd Watford, U.K.) via a three-port switch, was placed in the left jugular vein for propofol administration. After a period of 30 min stabilization, rats received an intravenous injection of propofol (Diprivan 2%, AstraZeneca UK Ltd, U.K.) The dose range used in our study encompassed the ED50 (effective dose 50%) of propofol reported by Carmichael et al.[17] At escalating doses of 300, 600 and 900 μg•kg•min, infusion of each dose was completed constantly within a period of 15 minutes. Dose-response curves of heart rate (HR), left ventricular end-systolic pressure (LVESP) left ventricular enddiastolic pressure (LVEDP), maximal rate for left ventricular pressure rising and declining (±dP/dtmax) were constructed. The dose-response curves toward changes in heart rate and the indexes of cardiac function were determined at the point of maximum response to each dose of the propofol. Ex vivo experiment In this section of experiments, the Langendorff heart preparation (ML870B2 ADInstruments Ltd, Shanghai, China) was used to evaluate propofol effects on cardiac performance in 10 BDL and 10 sham operation rats. Animals were anesthetized with sodium pentobarbital (40 mg kg i.p.). The heart was removed from the thoracic cavity and placed in ice-cold Krebs-Henseleit bicarbonate buffer (pH 7.4) containing heparin sodium. The heart was rapidly mounted on a Langendorff perfusion system via the aorta. A cardiac catheter, with a latex saccule at the tip, was inserted from a cut in the left auricle left atrium to the left ventricle. The saccule was filed with water so that the left ventricular end-diastolic pressure was maintained at 68 mmHg. The other end of the catheter was connected to a pressure transducer (MLT844, Shanghai, China). The average surrounding pressure was 70-80 mmHg. The perfusion medium was a modified Krebs-Henseleit bicarbonate buffer gassed with 95% O2 and 5% CO2 at atmospheric pressure and maintained at 37.5 ̊C. The ionic composition of the buffer was composed of NaCl 118 mmol l, KCl 4.7 mmol l , KH2PO4 1.2 mmol l, NaHCO3 25mmol l, CaCl2 2.5mmol l, glucose 11.0 mmol l, EDTA-Na2 0.5 mmol l, MgSO4 1.2 mmol l. Variables being recorded included heart rate (HR), left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure(LVEDP), maximal rate for left ventricular pressure rising and declining(±dP/dtmax). Effects of propofol were tested at the following concentrations: 12.5, 25, and 50μmol l using a method described by Jacob et al. [18] In the 3-day bile duct ligated (BDL) rats, Jacob[18] showed the impaired basal cardiac contractility and reduced left intraventricular pressure using two preparations of pithed rats and isolated functioning hearts. And then each drug concentration was tested for a period of 15 min. When the aortic flow and left ventricular pressure stabilized, data were acquired for 30 consecutive seconds prior to the drug testing. Statistical methods Data analysis was performed with a personal computer statistical software package (Prism version 4.0; Graph-Pad Software, San Diego, CA). Responses of cardiac function to propofol were expressed as percentage of the baseline values and the data was presented as mean ± SEM. Statistical analysis was performed with Student’s unpaired t-test or Chi-square test. Two-way repeated measure analysis of variance followed by the Bonferroni test, a multiple-comparisons correction, was done as a post hoc test for all the in vivo and ex vivo myocardial performance data. A P value of less than 0.05 was considered statistically significant. Results Serum total bilirubin(TBIL)and alanine transaminase (ALT) in the BDL group were significantly higher than the control group receiving sham operation as table 1 (P<0.01). In vivo cardiac function The baseline contractility and lusitropy (e.g., LVESP and ±dP/dtmax) were significantly higher in the BDL group than in the sham controls (p<0.05) while LVEDP did not differ significantly between the two study groups (table 2). At infusion rate between 300-600 μg•kg -1 •min -1 , propofol decreased HR, LVESP, and ±dP/dtmax, but not LVEDP. The dose-dependent decrease in HR, LVESP and ±dP/dtmax was similar between the BDL and the SO group, but not LVEDP as figure 1. At the dose of 900 μg•kg -1 •min -1 , the inhibitory effects of propofol on cardiac performance were significantly greater in the BDL group in comparison to that in the sham controls (p<0.05, figure 1). Ex vivo cardiac function The baseline measures of LVESP, ±dP/dt, LVEDP and HR were significantly lower in the BDL group than in the sham controls (p<0.05, table 3). At concentrations of 12.5 and 50 μmol l, propofol significantly decreased HR, LVESP, and ±dP/dtmax with increase in LVEDP. The dose-dependent decrease in response in the BDL group was similar to the SO group. The magnitude of such effects were more pronounced in BDL group and became significant compared with SO group at the highest dose of 50 μmol l(p<0.01, figure 2). Discussion Although our current study could not confirm impaired cardiac performance in a rat model of obstructive jaundice due to the inconsistent results between in vivo and isolated heart, an important and novel finding of our experiments is that the inhibitory effects of propofol on the heart were comparable in the BDL groups vs. the rats receiving sham operation at low and intermediate doses. At the highest dose or infusion rate of propofol tested in our experiments, more significant effects on cardiac function were demonstrated in rats with obstructive jaundice. The dose range used in our in vivo and in ex vivo study all encompassed the concentrations might uncollected to those used clinically.[17, 19-20] The present study did not confirm our hypothesis, which suggested propofol might significantly depress myocardial performance with obstructive jaundice compared with that of normal conditions. The discrepancy may be attributed to a number of factors. Firstly, most experimental and human studies have shown that propofol can induce cardiovascular depression that was dose-dependent. Furthermore, clinically relevant concentrations of propofol does not cause significant depression on myocardial contractility,and existing evidence suggests that the pharmacological properties may partly be due to a simultaneous enhanced sensitivity of the myofilaments to Ca 2+ despite of reducing uptake of Ca 2+ into the sarcoplasmic reticulum.[21-22] Moreover, propofol has long been known as a cardioprotective agent, and it may protect the myocardium from injury such as the ischemia–reperfusion.[23-24] A newly reported mechanism of the protective action of propofol on myocardium is the inhibition of the mitochondrial permeability transition pore.[23, 25] In addition, it is well established that retention and accumulation of bile acids is a causative factor for “jaundiced heart”.[5] Bile acids have negative chronotropic and inotropic effects on the heart by damage to mitochondria.[12, 26] Therefore, we proposed these aforementioned factors, especially a simultaneous enhanced sensitivity of the myofilaments to Ca2+ and the inhibition of the mitochondrial permeability transition pore, may exert counteraction against the adverse effect of cardiovascular depression, which account for the minimal responsiveness to propofol in low and intermediate concentration in BDL. Another interesting observation in this study was the opposite results of basal cardiac contractility in vivo and in ex vivo of BDL. In the ex vivo experiments, the hearts from the BDL rats exhibited impaired contractility and a reduction in the maximum pressure in the left ventricle, while in vivo experiments, BDL significantly increased LVSEP, +dP/dtmax and -dP/dtmax. The mechanism underlying this phenomenon was unclear, but may involve neural or humoral adaptations. Dabagh[27] reported increased concentration of norepinephrine (NE) and epinephrine (E) during the development of cholestasis. Poo[28] found elevated renin and angiotensin II at 1 week after establishment of obstructive jaundice in rats. In our in vivo experiments, increased LVSEP 、 +dP/dtmax , -dP/dtmax, were associated with myocardial contractility, which may imply the up-regulation of the sympathetic nervous system. In addition, LVEDP which directly relates to the cardiac preload might remain unchanged possibly not owing to the depletion of body fluid in obstructive jaundice.[29-30] but the occurrence of the lower heart rate may partly be due to the bile acids impaired cardiac conductive function by affecting calcium uptake from the sarcoplasmic reticulum.[31-32] Recent research has focused on the over production of nitric oxide, which results in bradycardia in BDL.[33-34] Despite the enhanced cardiac contractility in vivo, our experiment showed, at high dose, propofol produced more significant inhibition on cardiac function in BDL rats, thus unmasking the latent cardiac dysfunction in BDL rats. These results was consistent with the previous studies though the blood pressure could be normal, BDL rats were more hemodynamic instability[5]. Conclusions In conclusion, the present experimental study demonstrated that BDL impaired cardiac function in ex vivo but enhanced cardiac performance in vivo. The depression of cardiac parameters with propofol administrated at low and intermediate doses was not exaggerated by obstructive jaundice. At high dose, however, propofol causes exaggerated cardiac depression in jaundiced rats. Our study suggested that propofol appeared to be a viable anesthetic agent in patients with obstructive jaundice at low and intermediate doses. Competing interests The authors declare that they have no competing interests. Authors' contributions H.R: data mining and analysis in fulfillment of his MD thesis. L.Y and Z.L: ideal , experimental design and part of animal studies. C.C and K.T: conception and help with statistics. C.C: conception writing of the paper. J.S and W.C: ex vivo experiment W.Y: ideal, experimental design and part of animal studies All authors read and approved the final version of the manuscript. H.R , L.Y and Z.L contributed equally to this work. 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