Pulmonary Circulatory Dysfunction in Rats with Biliary Cirrhosis An Animal Model of the Hepatopulmonary Syndrome 1 - 3
SHIH-WEN CHANG" and NARUMI OHARA5
Patients with livercirrhosis often exhibit arterial hypoxemia caused by combinations of ventilation-perfusion mismatch and intrapulmonary shunts (1-4). Although it is believed that the abnormality in gas exchange reflects pulmonary circulatory derangements such as loss of hypoxic vasoconstriction and intrapulmonary microvasculardilatation (2, 5-7), the mechanism by which liver cirrhosis induces these pulmonary circulatory changes is unclear. In part, our ignorance of the pathogenesis of this "hepatopulmonary syndrome" (8) is due to the difficulties of pulmonary circulatory studies in humans and the lack of an animal model. Although several groups have studied the systemic and splanchnic hemodynamic changes in cirrhotic rats (9-12), no published study to date has examined the pulmonary hemodynamic changes in animals with experimental cirrhosis. In this study, we characterized the pulmonary circulatory changes in rats with biliary cirrhosis induced by chronic ligation of the common bile duct (5 to 6 wk). Surgical bile duct ligation was chosen as the cirrhosis-inducing stimulus over phenobarbital-carbontetrachloride to avoid any potential direct toxic effects of these chemicals on the pulmonary vasculature. We first compared the baseline cardiac output, systemic and pulmonary arterial pressures and resistances, and hypoxic pulmonary vasoconstriction (HPV) in awake,catheter-implanted, cirrhotic rats with the results in shamoperated control rats. As depressed systemic pressor response to angiotensin II and vasopressin have been previously documented in cirrhotic rats (11, 12), we also assessed the pulmonary pressor response to infusion of angiotensin II. Pulmonary gas exchange, assessed by mea798
SUMMARY We studied hypoxic pulmonary vasoconstriction (HPV) and pUlmonary gas exchange In unanesthetized rats with biliary cirrhosis Induced by chronic bile duct ligation (BDL) (5 to 6 wk) and compared pulmonary vascular reactivity In perfused lungs Isolated from BDL and control rats. Awake, catheter-Implanted, cirrhotic rats exhibited Increased cardiac output, normal systemic and pulmonary arterial pressures, and decreased total systemic fTSR) and pulmonary (TPR) vascular resistances In comparison with those In sham-operated control rats. HPV was markedly depressed In cirrhotic rats (percent Increase In TPR while breathing 8% O2 : 42.3 ± 13.7% In control and 0.9 ± 3.6% In cirrhotic rats, p < 0.05), and this was associated with an Increased AaP02 (control rats, 15.7 ± 1.1 mm Hgj cirrhotic rats, 23.1 ± 1.9 mm Hgj p < 0.05). In contrast, the pulmonary pressor response to angiotensin II was Intact, and the depression of HPV In cirrhotic rats was ameliorated after angiotensin II Infusion. These changes In cirrhotic rats were not due to the accompanying cholestasls since nonclrrhotlc rats with IMMtre chole8tuls had Intact HPV and normal AaP02 • Lungs Isolated from cirrhotic rats and perfused with blood from normal rats exhibited two patterns of response to hypoxia. In one group, HPV was blunted compared with that In control rats (change InpUlmonary arterial perfusion pressure attar 3% O2 : control rats, 23.2 ± 2.8 mm Hgj cirrhotic rats, 4.8 ± 1.4 mm Hgj P < 0.01).Similar to the reault In Intact rat, angiotensin-ii-Induced vasoconstriction was preserved In lungs from cirrhotic rats, and HPVIncreased significantly after angiotensin IIlnfuslon (to 17.3 ± 4.8 mm Hg). In the second group, baseline pulmonary arterial pressure progressively Increased during normoxla, and this Increase was attenuated by hypoxic ventilation (hypoxic vasodilation). We conclude that liver cirrhosis Indue.. nwerslble depression of HPV and suggest that rats with chronic BDL provide a suitable animal model for studying the pUlmonary circulatory alterations In the hepatopulmonary syndrome. AM REV RESPIR DIS 1"2; 145:711-801
suring the arterial blood gas tensions with the rats breathing room air, 8070, or 100070 oxygen, wasalso compared. Todetermine the role of cirrhosis versus cholestasis in these changes, we performed similar studies in cholestatic rats after acute bile duct ligation (10days) and compared the results with those in cirrhotic rats. Because anesthetic agents have been shown to markedly influence the systemichemodynamic variables in cirrhotic animals (10), these hemodynamic studies were performed in the unanesthetized state. Furthermore, in order to study pulmonary vascular responses without the confounding factors of altered cardiac output and systemic circulatory factors, we perfused lungs from control and cirrhotic rats ex vivo with normal blood and assessed their reactivity to alveolar hypoxia and intra-arterial angiotensin II. Our results closely parallel findings in patients with liver cirrhosis and suggest that rats with
(Received in original form April 15, 1991 and in revised form October 21, 1991) 1 From the CardiovascularPulmonary Research Laboratory, Department of Medicine, University of Colorado Health SciencesCenter, and Medical and Research Services, DenverVeterans Administration MedicalCenter, Denver, Colorado,and the Department of Medicine, NorthwesternUniversity Medical School, Chicago, Illinois. :I Supported by the Chicago Lung Association. 3 Correspondence and requests for reprints should be addressed to Shih-Wen Chang,M.D., Pulmonary Section, Northwestern University MedicalSchool, 250E. SuperiorStreet,Rm.#456, Chicago, IL 60611. 4 Recipientof Clinical InvestigatorAwardHL01966 from the National Heart, Lung, and Blood Institute. S Recipientof an Associate InvestigatorAward from the Veterans Administration. Ii Although our sample sizeis too small to allow us to confidently state that there is no difference in Pao between control and cirrhotic rats, using the formula provided on pp. 113-114 of Reference 16, the estimated sample size required to detect a 10070 differencein mean Pao with a p valueof 0.05 and statistical power of 0.80 is n = 14.
HYPOXIC PULMONARY VASOCONSTRICTION IN CIRRHOSIS
chronic bile duct ligation-induced cirrhosis provide a suitable animal model for investigating the pathogenetic mechanisms of the hepatopulmonary syndrome. Methods The following experimental protocols have been reviewed and approved by the University of Colorado Health Sciences Center Animal Care and Use Committee.
Cirrhosis Induction Weinduced biliary cirrhosis in rats by chronic ligation of the common bile duct (13).Male, Sprague-Dawley rats weighing 340 to 380 g were purchased from Harlan Industries (Indianapolis, IN) and given food and water ad libitum. Laparotomy was performed under pentobarbital anesthesia (50 mg/kg given intraperitoneally). The common bile duct was isolated and double-ligated, and sections to 6 mm were resected between the two ligatures. The abdominal incision was closed with sutures and surgical clips, and the rats were allowed to recover.In general, the hemodynamic study was performed 5 to 6 wk after bile duct ligation (BDL), and the presence of cirrhosis was documented histologically in each animal at the end of the study. Control rats received laparotomy and closure of incision similar to the bile-duct-ligated rats and were studied at a similar time point after sham surgery. In addition, to control for the effect of cholestasis on vascular reactivity, a group of rats was studied at approximately 10days after BDL (cholestatic control). At this early time point, rats with acute BDL exhibited markedly hyperbilirubinemia, but their livers showed no gross or histologic evidence of cirrhosis.
Placement of Vascular Catheters Twenty-four to 48 h prior to the hemodynamic studies, rats wereanesthetized intramuscularly with ketamine (50 mg/kg) and xylazine (5 mg/kg), and chronic indwellingcatheters were placed in the carotid artery, main pulmonary artery, and jugular veins as described previously (14). In this and our previous studies, all control rats tolerated the surgical procedures without difficulty, and surgical mortality wasless than 2070. In our early experience with the cirrhotic rat, the surgical mortality was high. However, by careful attention to anesthetic agents and surgical techniques and by limiting the amount of heparin given, we were able to decrease the surgical mortality to less than 15010.
Hemodynamic Study Protocol Awake, unanesthetized rats were studied in a small rectangular plastic chamber with a continuous flow-through of room air at 5 to 7 L/min. The catheters were irrigated with heparinized saline and connected to P23 Db transducers (Statham, Oxnard, CAl for measurements of heart rate and mean aortic and pulmonary artery pressures. Cardiac output was measured using a dye-dilution method
modified for the rat (14). Total systemic vascular resistance (TSR) and total pulmonary resistance (TPR) were calculated by dividing mean aortic and pulmonary artery pressures by cardiac index (CI = cardiac output per 100g body weight). Hypoxic challenges were performed by overflowing the study chamber with 8% O 2 for 5 to 6 min. The hypoxic pressor response (HPR) was defined as the difference between the maximal mean pulmonary artery pressure during hypoxia and the mean pulmonary artery pressure immediately preceding hypoxia. Blood gas tensions and pH in arterial blood samples (0.5ml) were measured using Corning microelectrodes (Corning Glass, Medford, MA). The AaP02 wascalculated using the modified alveolar gas equation: AaPo2 = Flo2(pB-47) - (PAco/R) - Paa2, where Flo2is the fraction of inspired O 2, PB is the barometric pressure (average, 630 mm Hg for Denver), PAeo2 is alveolar Pco, assumed to be equal to arterial Pco, (Pac02), and R is the mean respiratory quotient assumed to be 0.8 while breathing room air and 1.0while breathing 8% O2or 100% O 2. A schematic drawing of the experimental protocol is shown in figure 1. A total of eight cirrhotic, eight sham-operated control, and six cholestatic rats were studied. After the animal was placed in the study chamber, baseline hemodynamic measurements wererecorded over a 30-min period. This was followed by two sequential hypoxic challenges separated by 20 min of room-air ventilation. Before and during the second hypoxic challenge, cardiac outputs were obtained to allow calculation of the change in TPR with hypoxia. After these hypoxic challenges, angiotensin II was infused continuously in incremental doses (34 ng/min for 8 min, 68 ng/min for 8 min, and 340 ng/min for 8 min). Repeat cardiac outputs were measured during the two higher doses of angiotensin II infusion. After recovery from the high-dose angiotensin infusion (about 25 to 30 min later) a third hypoxic challenge was performed. At the indicated time points (figure 1), arterial blood gas measurements were obtained with the rat breathing room air, 8% O 2 (5 min), or 100% O 2 (8 min). At the end of the study protocol, rats were given an overdose of pentobarbital intraperitoneally (100 mg/kg), the liver and spleen were removed and weighed, and several random sections of the liver were fixed in formalin. Histologic sections of the liverswere graded without prior knowledge of the treatment groups by one of the investigators (NO), and only rats with obvious histologic changes of cirrhosis were included in the cirrhotic group. Plasma samples were sent to the chemistry laboratory for measurement of liver function tests.
Isolated Perfused Lung Study To further characterize the changes in pulmonary vascular reactivity in cirrhosis, we measured pressor responses to alveolar hypoxia and intra-arterial angiotensin II in isolated perfused lungs from eight cirrhotic and six sham-operated control rats. After pen-
tobarbital anesthesia (60 to 70 mg/kg given intraperitoneally), a plasma sample was removed from the right ventricle for measurement of liver chemistries, and the lungs were removed for ex vivo perfusion as described previously (15). Through a tracheal cannula, the lungs were ventilated with a Harvard small-animal respirator (Model 646; Harvard Apparatus Co., South Natick, MA) at 55 breaths/min with 8 em H 20-positive endexpiratory pressure. Inspired gas consisted of either 21% O 2-5% CO 2-74% N 2 (normoxia) or 3% O 2-5% CO 2-92% N 2 (hypoxia). After pulmonary artery cannulation, the lungs were perfused with 30 ml of homologous blood (removed from three healthy, ether-anesthetized donor rats via cardiac puncture) at 370 C and at a fixed rate of 0.03 ml/g body weight/min, so that changes in pulmonary artery perfusion pressure reflect changes in pulmonary vascular resistance. Effluent blood from the lungs was returned to a reservoir for recirculation. The liver and spleen were weighed, and a random section of the liver was fixed in formalin for subsequent histologic documentation of cirrhosis. After an initial20-min period of equilibration, the lungs were challenged with three sequential periods (10min each) of hypoxic ventilation separated by 10min of normoxic ventilation. Angiotensin II (0.05 J.1g) was then injected as a bolus into the pulmonary artery cannula and, 10min later, the lungs werechallenged with two more periods of hypoxic ventilation. Finally, 0.1 J.1g of angiotensin II was injected, and the experiment was terminated 10 min later. The lungs were dissected free from the mediastinal structures, weighed, and the wet lung weight-to-body-weightratios were calculated. During the second hypoxic challenge, effluent blood from the lungs was sampled for measurement of pH, Po 2, and Pco.. In a subgroup (n = 4) of perfused lungs from cirrhotic rats, the pulmonary artery perfusion pressure did not stabilize but increased steadily during the initial period of normoxic ventilation. These lungs were subjected to two brief periods (4 to 5 min) of hypoxic venillation alternated with 4 to 5 min of normoxic ventilation. The rate of increase in pulmonary artery pressure during each sequential normoxic and hypoxic period was calculated.
Statistical Analysis Data are expressed as mean ± SEM. In the conscious rat study each variable was analyzed using one-way analysis of variance and Scheffe's test for multiple comparison (16). In the isolated perfused lung study, means for control and cirrhotic groups were compared using Student's t test. Correlations were performed using standard methods (16). Differences were considered significant at p < 0.05.
Conscious Rat Hemodynamic Study The body and organ weights and plasma chemistries in the three groups of rats are
CHANG AND OHARA
24·48 hrs before study
Plasma for liver chemistries
Liver weight and histology Spleen weight
Fig. 1. Whole-animal hemodynamic study protocol. Approximate time points (in minutes) are indicated. Except during the indicated periods, the rats were studied while breathing room air. ABG = arterial blood gas; All = angiotensin II. Cardiac output (indicated by asterisks) was performed by a dye-dilution technique.
shown in table 1. The cirrhotic rats were studied at 38 ± 1 days (mean ± SEM) after BDL, and control rats at 39 ± 2 days after sham surgery. At this time point, hepatosplenomegaly was observed in the cirrhotic group, and plasma alka-
line phosphatase and total bilirubin were markedly elevated. Histologically, livers of rats with chronic BDL showed evidence of micronodular cirrhosis (not shown). The cholestatic control rats were studied at 10 ± 0.4 days after BDL, at
TABLE 1 BODY AND ORGAN WEIGHTS AND PLASMA CHEMISTRIES* Rats Control (n = 8) BW1 , 9 BWz, 9 Liver weight, 9 Spleen weight, 9 Glucose, mgldl SGPT, UlL Alkaline phosphatase, UlL Albumin, gldl Total bilirubin, mgldl
366 428 13.8 0.74 153 158 280 3.0 0.2
± ± ± ± ± ± ± ± ±
20 0.9 0.03 6 28 20 0.1 0.0
Cirrhotic (n = 8) 359 368 23.0 1.90 109 357 1,263 2.5 7.6
± ± ± ± ± ± ± ± ±
Cholestatic (n = 6)
9 12t 1.2t 0.13t 14t 126 255t 0.2 0.6t
356 328 15.1 0.77 145 201 832 2.7 9.5
± ± ± ± ± ± ± ± ±
10 10t 0.6* 0.06* 10 31 51t 0.1
Definition of abbreviations: BW, = body weight at the time of bile duet ligation or sham surgery; BW2 = body weight at the time of hemodynamic study. • Values are mean :t: SEM. t p < 0.05, different from control value. :I: p < 0.05, different from cirrhotic value.
TABLE 2 BASELINE HEMODYNAMIC DATA AND HYPOXIC PRESSOR RESPONSE* Rats Control (n "" 8) HR, min- l Pao, mm Hg Ppa, mm Hg CI, mVmin/100 9 BW TSR, mm Hglmin/100 g BW/L TPR, mm Hglminl100 9 BW/L HRP-1, mm Hg HRP-2, mm Hg HPR-3, mm Hg
343 ± 14 119.6 18.7 33.2 3,694 577 13.1 12.3 11.0
± 4.0 ± 0.7 ± 1.8 ± 270 ± 22 ± 1.9 ± 2.0 ± 1.7
Cirrhotic (n = 8) 366 108.6 18.3 42.7 2,628 455 4.8 3.6 4.7
± 17 ± 6.8 ± 1.4 ± 2.8t ± 264t ± 42t ± 0.6t ± 1.2t ± 1.1t
Cholestatic (n "" 6) 392 ± 11 117.2 ± 2.3 20.3 ± 1.4 33.9 ± 1.8* 3,502 ± 211 670 ± 14.1 ± 3.4i 11.9 ± 2.ei 11.8 ± 2.9
Definition of abbreviations: HR • heart rate; Pao mean aortic pressure; Ppa mean pumonary artery pressure; CI • cardiac index; TSR total systemic resistance; TPR • total pulmonary resistance; HPR • hypoxic pressure response. • Values are mean :t: SEM. t p < 0.05. different from control value. p < 0.05, different from cirrhotic value.
which time the liver and spleen were not enlarged, but the plasma alkaline phosphatase and total bilirubin were significantly elevated (table 1).In fact, the plasma total bilirubin level in these rats was higher than that in cirrhotic rats. Histologically, marked bile ductular proliferation without fibrosis was observed in sections of the livers from rats with acute BDL (not shown). The baseline hemodynamic data in the three groups of awake, unanesthetized rats are shown in table 2. No difference was observed in heart rate and mean aortic and mean pulmonary artery pressures in these rats." The cirrhotic rats had increased cardiac outputs and decreasedtotal systemic and pulmonary vascular resistances, reflecting systemic and pulmonary vasodilation. These hemodynamic changes cannot be attributed to BDL-induced cholestasis since the hemodynamic variables in cholestatic rats were similar to those in shamoperated control rats. HPV was elicited in awake rats by flooding the study chamber with 8070 O2 • The pulmonary arterial hypoxic pressor response in cirrhotic rats was markedly depressed in comparison with that in both control and acute cholestatic rats (table 2). Because cardiac outputs may be variably affected by hypoxia, we assessed HPV by calculating hypoxiainduced change in TPR. In figure 2, the total pulmonalry resistances for each rat are shown during both normoxia and after 5 min of hypoxic ventilation. On the average, the increases in TPR during hypoxia were 42.3 ± 13.7070 in control rats, 0.9 ± 3.60/0 in cirrhotic rats (p < 0.05 from control), and 23.8 ± 9.30/0 in cholestatic rats (not significantly different from control rats). In two cirrhotic rats, hypoxic pulmonary vasodilation (> 10070 decrease in TPR) was observed (figure 2). The changes in mean aortic arterial pressure with angiotensin II infusion are shown in figure 3. At each dose of angiotensin II, the systemic pressor response in cirrhotic rats was lower than that in either control rats or rats with acute BDL, and the difference was statistically significant at the two lower doses of angiotensin II (figure 3). On the other hand, the pulmonary pressor responses to angiotensin II weremore variable and did not differ among the three groups of rats (data not shown). Similar results were observed when the data were expressedas changes in systemicand pulmonary vascular resistance units (now shown). After angiotensin II infusion,
HYPOXIC PULMONARY VASOCONSTRICTION IN CIRRHOSIS