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Mast Cells Are Not Involved in the Ischemia-Reperfusion Injury in Perfused Rat Liver
Journal of Surgical Research 174, 114–119 (2012) doi:10.1016/j.jss.2010.11.900
Mast Cells Are Not Involved in the Ischemia-Reperfusion Injury in Perfused Rat Liver Toshishige Shibamoto, M.D.,*,1 Mikihiro Tsutsumi, M.D.,† Yuhichi Kuda, B.Sc.,* Chieko Ohmukai, B.Sc.,* Wei Zhang, M.Sc.,*,‡ and Yasutaka Kurata, M.D.* *Department of Physiology II and †Department of Gastroenterology, Kanazawa Medical University, Uchinada Ishikawa, Japan; and ‡Department of Pathophysiology, Medical College of Qinghai University, Xining, China Originally submitted June 18, 2010; accepted for publication November 16, 2010
Background. It is reported that mast cells are involved in ischemia-reperfusion (I/R) injury of several organs such as intestine, heart, and brain in rats. However, the roles of mast cells are not known in rat hepatic I/R injury. We determined using genetically mast cell deficient (Ws/Ws) rats whether mast cells participate in the genesis of hepatic I/R injury. Methods. Isolated livers from male Ws/Ws rats (n [ 6), their wild type D/D rats (n [ 6), and Sprague Dawely (SD) rats (n [ 12) were perfused portally with diluted blood (Hct 8%) at a constant blood flow. Ischemia was induced at room temperature by occlusion of the inflow line of the portal vein for 1 h, followed by 1-h reperfusion in a recirculating manner. The preand post-sinusoidal resistances were determined by measuring the portal venous pressure (Ppv), hepatic venous pressure, blood flow and the sinusoidal pressure, which was assessed by the double occlusion pressure (Pdo). Liver injury was assessed by blood alanine aminotransferase (ALT) levels, bile flow rate and histology of the livers. Results. In the D/D group, liver injury occurred after reperfusion; blood ALT levels increased from 19 ± 4 (SD) to 71 ± 18 and 135 ± 30 (IU/L) at 30 and 60 min, respectively, and bile flow decreased to 51% ± 6% of the baseline at 60 min after reperfusion. Histologic examination revealed marked hepatic degeneration. Similar changes were observed in the Ws/Ws rats and the SD rats (n [ 6), and there were no significant differences in the variables among the Ws/Ws, D/D, and SD groups. In any ischemia groups, immediately after reperfusion, Ppv substantially, but Pdo only slightly, increased, followed by a return towards the baseline, 1 To whom correspondence and reprint requests should be addressed at Department of Physiology II, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. E-mail: shibamo@ kanazawa-med.ac.jp.
0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved.
indicating a predominant increase in pre-sinusoidal resistance over post-sinusoidal resistance. Liver weight significantly increased at 60 min after reperfusion. In the control SD rats without I/R (n [ 6), no significant changes were observed in the variables. Conclusions. I/R injury occurs in the absence of hepatic mast cells in the isolated perfused rat liver model of I/R injury. Ó 2012 Elsevier Inc. All rights reserved. Key Words: hepatic circulation; double occlusion pressure; Ws/Ws; hepatic vascular resistance. INTRODUCTION
Hepatic ischemia-reperfusion injury is commonplace in liver surgery, particularly in hepatic transplantation, hepatic resection, and trauma [1]. Although the mechanisms of ischemia-reperfusion injury are numerous and complex, mast cells have recently been reported to be responsible for the ischemia-reperfusion (I/R) injury of organs such as the heart [2], brain [3], and intestine [4]. However, the roles of mast cells are not known in hepatic I/R injury in rats. We previously reported that the I/R intervention could produce hepatic injury of isolated Sprague Dawley (SD) rat livers perfused bivascularly via both the hepatic artery and the portal vein [5]. This hepatic I/R injury was characterized by decreased bile production, and increased perfusate concentration of hepatocellular enzyme of alanine aminotransferase (ALT), as well as biphasic liver weight gain [5]. On the other hand, we previously demonstrated that hepatic mast cells are involved in acute hepatic pathophysiology. We reported, by using genetically mast cell deficient (Ws/Ws) rats and their wild type (þ/þ) rats, that hepatic mast cells play a crucial role in hepatic anaphylaxis in isolated perfused rat livers [6]. These results
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provide the rationale to investigate the causative factor of the hepatic mast cells in the I/R injury of the isolated perfused rat liver preparation. Therefore, in the present study, we determined, using isolated portally perfused livers excised from Ws/Ws, þ/þ, and SD rats, whether mast cells are involved in the genesis of hepatic I/R injury. MATERIALS AND METHODS Animals Six male Ws/Ws rats (234 6 15 g), six male þ/þ rats (230 6 14 g), and 12 male SD rats (285 6 17 g) were purchased from Japan SLC (Shizuoka, Japan). Ws/Ws rats have a 12-base deletion in the tyrosine kinase domain of the c-kit cDNA, and are deficient in mast cells, whereas þ/þ rats have normal number of these cells [7]. Indeed, few mast cells are found in the livers of Ws/Ws rats [8]. All rats were maintained at 23 C and under pathogen-free conditions on a 12/12 h dark/light cycle and allowed food and water ad libitum. The present experiments were approved by the Animal Research Committee of Kanazawa Medical University.
Isolated Perfused Rat Liver Preparation Each rat was anesthetized with pentobarbital sodium (50 mg$kg–1, i.p.) and mechanically ventilated with room air. The basic methods for isolated perfused rat livers were described previously [9]. In brief,
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a polyethylene tube was placed in the right carotid artery. After laparotomy, the hepatic artery was ligated and the bile duct was cannulated with a polyethylene tube. After intra-arterial heparinization (500 U$kg–1), 8–9 mL of blood was withdrawn through the carotid arterial catheter. The intra-abdominal inferior vena cava (IVC) above the renal veins was ligated, and the portal vein was cannulated with a stainless steel cannula (2.1 mm i.d., 2.5 mm o.d.) for portal perfusion. After thoracotomy, the supra-diaphragmatic IVC was cannulated through a right atrium incision with the same size stainless cannula, and then portal perfusion was begun. The liver was rapidly excised, suspended from an isometric transducer (TB-652T; Nihon-Kohden, Tokyo, Japan) and weighed continuously throughout the experimental period. The livers were perfused at a constant flow rate in a recirculating manner via the portal vein. The perfusing blood (40 mL) diluted with Krebs solution was oxygenated in the reservoir by bubbling with 95% O2 and 5% CO2, and was pumped from the reservoir through a heat exchanger (37 C). The portal venous pressure (Ppv) and hepatic venous pressure (Phv) were measured with pressure transducers. Portal flow rate (Qpv) was measured with an electromagnetic flow meter (MFV 1200; Nihon-Kohden), and the flow probe was positioned in the inflow line. Bile was collected drop by drop in a small tube suspended from the force transducer for determination of the bile flow rate [9]. The hepatic vascular pressures, Qpv, liver weight and bile weight were monitored continuously and displayed through a thermal physiograph. Outputs were also digitized by the analog-digital converter at a sampling rate of 100 Hz. These digitized values were displayed and recorded using a personal computer for later determination of Pdo. Hepatic hemodynamic parameters were observed for at least 20 min after the start of perfusion until an isogravemetric state (no weight gain or loss) was obtained by adjusting Qpv and the height of the reservoir at a Phv of 0–1 cmH2O.
FIG. 1. Representative recordings of portal venous pressure, hepatic venous pressure, portal flow, liver weight, and bile weight after reperfusion following 1-h ischemia in the þ/þ group.
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After the baseline measurement, the perfused livers from Ws/Ws (n ¼ 6), þ/þ (n ¼ 6), and SD (n ¼ 6) rats were exposed to ischemia, which was induced by occlusion of the portal inflow line for 1 h followed by 1 h reperfusion. For the time-matched control group, the livers from SD rats (n ¼ 6) were perfused without ischemia for 2 h. The pump speed for portal perfusion, once determined at baseline, was not changed throughout the experimental period. The hepatic sinusoidal pressure was measured as the double occlusion pressure (Pdo) [10], which was the equilibrated pressure of Ppv and Phv when both the inflow and outflow lines were simultaneously and instantaneously occluded for 13 s with the solenoid valves. In each experimental group, Pdo was measured at baseline before ischemia, and 1.5, 3, 10, 20, 30, 40, 50, and 60 min after reperfusion. In the control group, Pdo was measured at the corresponding time after 60 min observation. At 60 min after reperfusion, liver specimens were fixed in a 10% buffered formalin solution and embedded in paraffin. Sections were made at 4 mm and stained with hematoxylin and eosin (HE). Liver injuries were evaluated by three parameters: (1) blood concentration of ALT, as determined with a commercially available kit (Wako Pure Chemical Industries, Osaka, Japan) by spectrophotometric method; (2) bile flow rate, which could indicate hepatocellular activity; (3) liver weight gain at the late phase of reperfusion, which could indicate the hepatocellular swelling [4]. Finally, histology of the livers was also examined. The total portal-hepatic venous (Rt), pre- (Rpre) and postsinusoidal (Rpost) resistances were calculated by the following equations: Rt ¼ ðPpv PhvÞ=Qpv
(1)
Rpre ¼ ðPpv PdoÞ=Qpv
(2)
Rpost ¼ ðPdo PhvÞ=Qpv
(3)
Data Analysis All results are expressed as the means 6 SD. Statistical analyses were performed with repeated measures analysis of variance, and a P value <0.05 was considered significant. When a significant difference was obtained, post hoc analysis was performed with the Bonferroni post-test method.
RESULTS
Figure 1 shows a recording example of variables during 60 min after reperfusion following 1 h ischemia in the þ/þ group. Figure 2 shows the summarized data of Ppv, Pdo, and segmental vascular resistances of Rpre and Rpost. In the þ/þ group, Ppv increased steeply from the pre-ischemia baseline of 6.8 6 0.7 to 18.8 6 2.8 cmH2O at 1.5 min after the start of reperfusion, and then it retuned to baseline levels at the end of the experimental period. Phv (0.6 6 0.1 cmH2O) did not change because of the constant flow perfusion (Qpv, 42 6 3 mL/min/10 g liver). Double occlusion maneuver revealed that Pdo increased slightly, but significantly, from the baseline of 2.1 6 0.4 to 4.3 6 0.5 cmH2O at 10 min, as shown in Figure 1. Based on these hepatic pressures, Rpre predominantly increased over Rpost within 10 min after reperfusion, as shown in Figure 2. The changes in these hemodynamic variables were similarly observed in the Ws/Ws and SD rats. 0.8
30
*
20
*
15
* 10
Rpre
Ppv
(cmH2O)
*
*
*
5
(mmHg/ml/min/10g liver)
0.7
25
*
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* *
0.3 0.2
* *
0.0 0
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*
*
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0.8
8
* *
* Rpost
4
2
(mmHg/ml/min/10g liver)
0.7
* 6
Pdo
0.5
0.1
0
(cmH2O)
*
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0.6 0.5 0.4 0.3 0.2 0.1
0
0.0 0
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3
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Time after reperfusion (min)
60
0
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3
Time after reperfusion (min)
FIG. 2. Time course changes in portal venous pressure (Ppv), double occlusion pressure (Pdo), and pre-sinusoidal resistance (Rpre), and post-sinusoidal resistance (Rpost) in the Ws/Ws (closed circle, n ¼ 6), þ/þ (open circle, n ¼ 6), SD ischemia (closed triangle, n ¼ 6), and SD control (open triangle, n ¼ 6) groups. Values are means 6 SD. *P < 0.05 versus baseline.
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SHIBAMOTO ET AL.: MAST CELLS IN RAT HEPATIC ISCHEMIA-REPERFUSION
A Liver weight change (g)
3
2
1
*
* * *
*
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*
*
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-1 0
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Bile flow (% of baseline)
During reperfusion, liver weight increased at the end of the experiment period in any groups after reperfusion, although it also increased immediately after reperfusion in the SD rats, as shown in Figure 3A. The liver injury was evidenced firstly by increased liver enzyme release into blood after reperfusion: In the þ/þ group, blood ALT levels increased from 19 6 4 to 71 6 18 and 135 6 30 (IU/L) at 30 and 60 min, respectively, after reperfusion (Fig. 3B). Bile flow decreased to 51% 6 6% of the baseline at 60 min after reperfusion (Fig 3C), which was another finding of liver injury. Similar changes in ALT levels and bile flow rate were observed in the Ws/Ws and SD rats, and there were no significant differences in the variables among the Ws/Ws, þ/þ, and SD groups (Fig. 3). In the control SD rats without I/R, no significant changes were observed in the variables studied (Figs. 2 and 3). Liver histology showed marked hepatic degeneration with hepatic necrosis and slightly disruption of lobular architecture in the SD ischemia, Ws/Ws, and þ/þ groups (Fig. 4B, C, and D, respectively), while there was no significant hepatic degeneration except for sinusoidal distention in SD control rats without I/R (Fig. 4A).
80
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* * *
0
0
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* * *
* * *
* * *
* * *
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* * 10
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DISCUSSION
C
300 250 200
ALT (IU/L)
In the present study, we found that the hepatic damages, as demonstrated by increased blood ALT levels, reduced bile flow rates, and histopathological findings, were observed similarly in isolated perfused livers from the mast cell deficient Ws/Ws, the control þ/þ, and SD rats. Liver weight gain was also observed at the late phase in the presence of normal sinusoidal pressure, a finding consistent with the damage of the perfused livers [5]. However, there were no significant differences in the I/R-induced liver damages among the groups. We therefore conclude that mast cells are not involved in I/R injury of isolated perfused rat livers. This study is the first to determine the roles of mast cells in hepatic I/R injury of rat. We previously reported the hepatic I/R injury in isolated SD rat livers perfused bivascularly via the hepatic artery and portal vein with diluted blood (Hct of 3.3%) [5]. This injury was characterized by the transient increase in vascular resistance with Rpre being greater than Rpost, the biphasic liver weight gain at the early and late phase, and the hepatocellular damages evidenced by increased blood hepatic enzyme level and reduced bile production. These findings were consistently observed in the present SD rat livers perfused via the portal vein with blood (higher Hct of 8%). Similar I/Rinduced changes were also detected in the livers from the Ws/Ws and þ/þ rats. Thus, we demonstrated that
150
* * *
* * *
100 50 0 -50 0
10
20
30
40
50
60
Time after reperfusion (min) FIG. 3. Time course changes in liver weight change (A), bile flow (B), and blood alanine aminotransferase (ALT) levels (C), in the Ws/Ws (closed circle, n ¼ 6), þ/þ (open circle, n ¼ 6), SD ischemia (closed triangle, n ¼ 6), and SD control (open triangle, n ¼ 6) groups. Values are means 6 SD. *P < 0.05 versus baseline.
there were no significant differences in I/R damages among the rats with and without mast cells. The rationale for studying the roles of mast cells in I/R injury is based on the following evidence. First, I/R maneuver can evoke activation of mast cells in several tissue. An increased flux of oxidants, which has been previously observed at the onset of reperfusion, may be responsible for the mast cell activation [11]: the administration of antioxidants can block the histamine
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FIG. 4. Liver histology of the SD control (A), SD ischemia (B), Ws/Ws (C), and þ/þ (D) groups (HE, 3 400). C ¼ central vein, P ¼ portal region.
release from the post-ischemic organ [12, 13]. Second, in rat, a mast cell stabilizer, which blocked mast cell degranulation and inhibited chemical mediator release, prevented I/R-induced damage in hearts [14, 15] and intestines [16], possibly via inhibition of TNF-a release [17]. In rat livers, mast cells are abundantly distributed around hepatic vessels [18, 19]. There are at least two types of mast cells, the mucosal type of mast cells (MMC) and the connective tissuetype of mast cells. In rats, staining of mast cells in liver sections with alcian blue/safranin indicates that these cells are predominantly of the mucosal subtype [18], with Collins and co-workers reporting that <7% of hepatic mast cells had the staining characteristics of connective tissue mast cells [20]. These MMC are the local source of large quantities of chemical mediators, including histamine [12, 13], leukotrienes [21], and plateletactivating factor [22], which have been postulated to play important functions in the pathogenesis of I/Rinduced tissue injury. The present study suggests that mast cells are not involved in the rat hepatic I/R injury. One of the reasons for the absence of a role of mast cells, in contrast to the I/R injuries of other tissues [2–4], may be related to the isolated perfused rat liver preparation, in which mast cells might have been inactive.
However, this possibility seems unlikely. We previously demonstrated, using similar isolated perfused livers from þ/þ and Ws/Ws rats, that hepatic mast cells could evoke substantial anaphylactic hepatic venoconstriction: 90% of hepatic venoconstriction was attributable to intrahepatic mast cells, as evidenced by the antigen-induced increases in Ppv of 13.4 and 1.4 cmH2O in the þ/þ and Ws/Ws rats, respectively [6]. A more plausible explanation is that the mechanisms, other than the mast cell-related one, such as reperfusion-induced reactive oxygen species, one of the first elements formed at the initial stage of I/R injury [1], might have overwhelmed and masked the possible detrimental action of mast cells in the hepatic I/R injury. In the isolated perfused livers of the present study, the hepatic artery was not perfused. Hepatic arterial perfusion with normally oxygenated blood would improve the metabolic milieu of the liver. However, the perfusing blood was well oxygenated by bubbling with 95% O2 and 5% CO2, which provided perfusate oxygen tension of 290 mmHg [9]. Thus the isolated livers were perfused with hyperoxic blood, rather than hypoxic blood, and oxygenation was well done. In conclusion, the I/R injuries, assessed by blood ALT levels, bile flow rate, and histology, were similarly
SHIBAMOTO ET AL.: MAST CELLS IN RAT HEPATIC ISCHEMIA-REPERFUSION
observed in isolated blood-perfused livers derived from genetically mast cell deficient Ws/Ws rats, wild type þ/þ rats, and SD rats. These results suggest that mast cells are not involved in rat hepatic I/R injury. ACKNOWLEDGMENTS This work was supported by a Grant for Collaborative Research from Kanazawa Medical University (C2010-1) and a Grant-in-Aid for Scientific Research (20592131) from the Ministry for Education, Culture, Science, and Technology of Japan.
REFERENCES 1. Montalvo-Jave EE, Escalante-Tattersfield T, OrtegaSalgado JA, et al. Factors in the pathophysiology of the liver ischemia-reperfusion injury. J Surg Res 2008;147:153. 2. Frangogiannis NG, Youker KA, Rossen RD, et al. Cytokines and the microcirculation in ischemia and reperfusion. J Mol Cell Cardiol 1998;30:2567. 3. Lindsberg PJ, Strbian D, Karjalainen-Lindsberg ML. Mast cells as early responders in the regulation of acute blood-brain barrier changes after cerebral ischemia and hemorrhage. J Cereb Blood Flow Metab 2010;30:689. 4. Andoh A, Kimura T, Fukuda M, et al. Rapid intestinal ischemiareperfusion injury is suppressed in genetically mast celldeficient Ws/Ws rats. Clin Exp Immunol 1999;116:90. 5. Ling YQ, Shibamoto T, Honda T, et al. Increased sinusoidal pressure is associated with early liver weight gain in ischemiareperfusion injury in isolated perfused rat liver. J Surg Res 2000;88:70. 6. Shibamoto T, Shimo T, Cui S, et al. The roles of mast cells and Kupffer cells in rat systemic anaphylaxis. Am J Physiol Regul Integr Comp Physiol 2007;293:R2202. 7. Niwa Y, Kasugai T. Anemia and mast cell depletion in mutant rats that are heterozygous at ‘‘white spotting (Ws)’’ locus. Blood 1991;258:G329. 8. Sugihara A, Tsujimura T, Fujita Y, et al. Evaluation of role of mast cells in the development of liver fibrosis using mast celldeficient rats and mice. J Hepatol 1999;30:859.
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9. Shibamoto T, Cui S, Ruan Z, et al. Hepatic venoconstriction is involved in anaphylactic hypotension in rats. Am J Physiol Heart Circ Physiol 2005;289:H1436. 10. Yamaguchi Y, Shibamoto T, Hayashi T, et al. Hepatic vascular response to anaphylaxis in isolated canine liver. Am J Physiol Regul Integr Comp Physiol 1994;267:R268. 11. Blum H, Summers JJ, Schnall MD, et al. Acute intestinal ischemia studies by phosphorous nuclear magnetic resonance spectroscopy. Ann Surg 1986;204:83. 12. Boros M, Kaszaki J, Nagy S. Oxygen free radical induced histamine release during intestinal ischemia and reperfusion. Eur Surg Res 1989;21:297. 13. Boros M, Kaszaki J, Nagy S. Histamine release during intestinal ischemia-reperfusion: Role of irons and hydrogen peroxide. Circ Shock 1991;35:174. 14. Keller AM, Clancy RM, Barr ML, et al. Acute reoxygenation injury in the isolated rat heart: Role of resident cardiac mast cells. Circ Res 1988;63:1044. 15. Parikh V, Singh M. Cardiac mast cell stabilization and cardioprotective effect of ischemic preconditioning in isolated rat heart. J Cardiovasc Pharmacol 1998;31:779. 16. Kanwar S, Kubes P. Mast cells contribute to ischemiareperfusion-induced granulocyte infiltration and intestinal dysfunction. Am J Physiol 1994;267:G316. 17. Gilles S, Zahler S, Welsch U, et al. Release of TNF-a during myocardial reperfusion depends on oxidative stress and is prevented by mast cell stabilizers. Cardiovasc Res 2003;60:608. 18. Armbrust T, Batusic D, Ringe B, et al. Mast cells distribution in human liver disease and experimental rat liver fibrosis. Indications for mast cell participation in development of liver fibrosis. J Hepatol 1997;26:1042. 19. Rioux KP, Sharkey KA, Wallace JL, et al. Hepatic mucosal mast cell hyperplasia in rats with secondary biliary cirrhosis. Hepatology 1996;23:88. 20. Collins AM, Leach S, Payne J, et al. A role for the hepatobiliary system in IgE-mediated intestinal inflammation in the rat. Clin Exp Allergy 1999;29:262. 21. Lehr HA, Guhlmann A, Nolte D, et al. Leukotrienes as mediators in ischemia-reperfusion injury in a microcirculation model in the hamster. J Clin Invest 1991;87:2036. 22. Kubes P, Ibbotson G, Russell J, et al. Role of platelet-activating factor in ischemia/reperfusion-induced leukocyte adherence. Am J Physiol 1990;259:G300.
Mast Cells Are Not Involved in the Ischemia-Reperfusion Injury in Perfused Rat Liver Toshishige Shibamoto, M.D.,*,1 Mikihiro Tsutsumi, M.D.,† Yuhichi Kuda, B.Sc.,* Chieko Ohmukai, B.Sc.,* Wei Zhang, M.Sc.,*,‡ and Yasutaka Kurata, M.D.* *Department of Physiology II and †Department of Gastroenterology, Kanazawa Medical University, Uchinada Ishikawa, Japan; and ‡Department of Pathophysiology, Medical College of Qinghai University, Xining, China Originally submitted June 18, 2010; accepted for publication November 16, 2010
Background. It is reported that mast cells are involved in ischemia-reperfusion (I/R) injury of several organs such as intestine, heart, and brain in rats. However, the roles of mast cells are not known in rat hepatic I/R injury. We determined using genetically mast cell deficient (Ws/Ws) rats whether mast cells participate in the genesis of hepatic I/R injury. Methods. Isolated livers from male Ws/Ws rats (n [ 6), their wild type D/D rats (n [ 6), and Sprague Dawely (SD) rats (n [ 12) were perfused portally with diluted blood (Hct 8%) at a constant blood flow. Ischemia was induced at room temperature by occlusion of the inflow line of the portal vein for 1 h, followed by 1-h reperfusion in a recirculating manner. The preand post-sinusoidal resistances were determined by measuring the portal venous pressure (Ppv), hepatic venous pressure, blood flow and the sinusoidal pressure, which was assessed by the double occlusion pressure (Pdo). Liver injury was assessed by blood alanine aminotransferase (ALT) levels, bile flow rate and histology of the livers. Results. In the D/D group, liver injury occurred after reperfusion; blood ALT levels increased from 19 ± 4 (SD) to 71 ± 18 and 135 ± 30 (IU/L) at 30 and 60 min, respectively, and bile flow decreased to 51% ± 6% of the baseline at 60 min after reperfusion. Histologic examination revealed marked hepatic degeneration. Similar changes were observed in the Ws/Ws rats and the SD rats (n [ 6), and there were no significant differences in the variables among the Ws/Ws, D/D, and SD groups. In any ischemia groups, immediately after reperfusion, Ppv substantially, but Pdo only slightly, increased, followed by a return towards the baseline, 1 To whom correspondence and reprint requests should be addressed at Department of Physiology II, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. E-mail: shibamo@ kanazawa-med.ac.jp.
0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved.
indicating a predominant increase in pre-sinusoidal resistance over post-sinusoidal resistance. Liver weight significantly increased at 60 min after reperfusion. In the control SD rats without I/R (n [ 6), no significant changes were observed in the variables. Conclusions. I/R injury occurs in the absence of hepatic mast cells in the isolated perfused rat liver model of I/R injury. Ó 2012 Elsevier Inc. All rights reserved. Key Words: hepatic circulation; double occlusion pressure; Ws/Ws; hepatic vascular resistance. INTRODUCTION
Hepatic ischemia-reperfusion injury is commonplace in liver surgery, particularly in hepatic transplantation, hepatic resection, and trauma [1]. Although the mechanisms of ischemia-reperfusion injury are numerous and complex, mast cells have recently been reported to be responsible for the ischemia-reperfusion (I/R) injury of organs such as the heart [2], brain [3], and intestine [4]. However, the roles of mast cells are not known in hepatic I/R injury in rats. We previously reported that the I/R intervention could produce hepatic injury of isolated Sprague Dawley (SD) rat livers perfused bivascularly via both the hepatic artery and the portal vein [5]. This hepatic I/R injury was characterized by decreased bile production, and increased perfusate concentration of hepatocellular enzyme of alanine aminotransferase (ALT), as well as biphasic liver weight gain [5]. On the other hand, we previously demonstrated that hepatic mast cells are involved in acute hepatic pathophysiology. We reported, by using genetically mast cell deficient (Ws/Ws) rats and their wild type (þ/þ) rats, that hepatic mast cells play a crucial role in hepatic anaphylaxis in isolated perfused rat livers [6]. These results
114
SHIBAMOTO ET AL.: MAST CELLS IN RAT HEPATIC ISCHEMIA-REPERFUSION
provide the rationale to investigate the causative factor of the hepatic mast cells in the I/R injury of the isolated perfused rat liver preparation. Therefore, in the present study, we determined, using isolated portally perfused livers excised from Ws/Ws, þ/þ, and SD rats, whether mast cells are involved in the genesis of hepatic I/R injury. MATERIALS AND METHODS Animals Six male Ws/Ws rats (234 6 15 g), six male þ/þ rats (230 6 14 g), and 12 male SD rats (285 6 17 g) were purchased from Japan SLC (Shizuoka, Japan). Ws/Ws rats have a 12-base deletion in the tyrosine kinase domain of the c-kit cDNA, and are deficient in mast cells, whereas þ/þ rats have normal number of these cells [7]. Indeed, few mast cells are found in the livers of Ws/Ws rats [8]. All rats were maintained at 23 C and under pathogen-free conditions on a 12/12 h dark/light cycle and allowed food and water ad libitum. The present experiments were approved by the Animal Research Committee of Kanazawa Medical University.
Isolated Perfused Rat Liver Preparation Each rat was anesthetized with pentobarbital sodium (50 mg$kg–1, i.p.) and mechanically ventilated with room air. The basic methods for isolated perfused rat livers were described previously [9]. In brief,
115
a polyethylene tube was placed in the right carotid artery. After laparotomy, the hepatic artery was ligated and the bile duct was cannulated with a polyethylene tube. After intra-arterial heparinization (500 U$kg–1), 8–9 mL of blood was withdrawn through the carotid arterial catheter. The intra-abdominal inferior vena cava (IVC) above the renal veins was ligated, and the portal vein was cannulated with a stainless steel cannula (2.1 mm i.d., 2.5 mm o.d.) for portal perfusion. After thoracotomy, the supra-diaphragmatic IVC was cannulated through a right atrium incision with the same size stainless cannula, and then portal perfusion was begun. The liver was rapidly excised, suspended from an isometric transducer (TB-652T; Nihon-Kohden, Tokyo, Japan) and weighed continuously throughout the experimental period. The livers were perfused at a constant flow rate in a recirculating manner via the portal vein. The perfusing blood (40 mL) diluted with Krebs solution was oxygenated in the reservoir by bubbling with 95% O2 and 5% CO2, and was pumped from the reservoir through a heat exchanger (37 C). The portal venous pressure (Ppv) and hepatic venous pressure (Phv) were measured with pressure transducers. Portal flow rate (Qpv) was measured with an electromagnetic flow meter (MFV 1200; Nihon-Kohden), and the flow probe was positioned in the inflow line. Bile was collected drop by drop in a small tube suspended from the force transducer for determination of the bile flow rate [9]. The hepatic vascular pressures, Qpv, liver weight and bile weight were monitored continuously and displayed through a thermal physiograph. Outputs were also digitized by the analog-digital converter at a sampling rate of 100 Hz. These digitized values were displayed and recorded using a personal computer for later determination of Pdo. Hepatic hemodynamic parameters were observed for at least 20 min after the start of perfusion until an isogravemetric state (no weight gain or loss) was obtained by adjusting Qpv and the height of the reservoir at a Phv of 0–1 cmH2O.
FIG. 1. Representative recordings of portal venous pressure, hepatic venous pressure, portal flow, liver weight, and bile weight after reperfusion following 1-h ischemia in the þ/þ group.
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After the baseline measurement, the perfused livers from Ws/Ws (n ¼ 6), þ/þ (n ¼ 6), and SD (n ¼ 6) rats were exposed to ischemia, which was induced by occlusion of the portal inflow line for 1 h followed by 1 h reperfusion. For the time-matched control group, the livers from SD rats (n ¼ 6) were perfused without ischemia for 2 h. The pump speed for portal perfusion, once determined at baseline, was not changed throughout the experimental period. The hepatic sinusoidal pressure was measured as the double occlusion pressure (Pdo) [10], which was the equilibrated pressure of Ppv and Phv when both the inflow and outflow lines were simultaneously and instantaneously occluded for 13 s with the solenoid valves. In each experimental group, Pdo was measured at baseline before ischemia, and 1.5, 3, 10, 20, 30, 40, 50, and 60 min after reperfusion. In the control group, Pdo was measured at the corresponding time after 60 min observation. At 60 min after reperfusion, liver specimens were fixed in a 10% buffered formalin solution and embedded in paraffin. Sections were made at 4 mm and stained with hematoxylin and eosin (HE). Liver injuries were evaluated by three parameters: (1) blood concentration of ALT, as determined with a commercially available kit (Wako Pure Chemical Industries, Osaka, Japan) by spectrophotometric method; (2) bile flow rate, which could indicate hepatocellular activity; (3) liver weight gain at the late phase of reperfusion, which could indicate the hepatocellular swelling [4]. Finally, histology of the livers was also examined. The total portal-hepatic venous (Rt), pre- (Rpre) and postsinusoidal (Rpost) resistances were calculated by the following equations: Rt ¼ ðPpv PhvÞ=Qpv
(1)
Rpre ¼ ðPpv PdoÞ=Qpv
(2)
Rpost ¼ ðPdo PhvÞ=Qpv
(3)
Data Analysis All results are expressed as the means 6 SD. Statistical analyses were performed with repeated measures analysis of variance, and a P value <0.05 was considered significant. When a significant difference was obtained, post hoc analysis was performed with the Bonferroni post-test method.
RESULTS
Figure 1 shows a recording example of variables during 60 min after reperfusion following 1 h ischemia in the þ/þ group. Figure 2 shows the summarized data of Ppv, Pdo, and segmental vascular resistances of Rpre and Rpost. In the þ/þ group, Ppv increased steeply from the pre-ischemia baseline of 6.8 6 0.7 to 18.8 6 2.8 cmH2O at 1.5 min after the start of reperfusion, and then it retuned to baseline levels at the end of the experimental period. Phv (0.6 6 0.1 cmH2O) did not change because of the constant flow perfusion (Qpv, 42 6 3 mL/min/10 g liver). Double occlusion maneuver revealed that Pdo increased slightly, but significantly, from the baseline of 2.1 6 0.4 to 4.3 6 0.5 cmH2O at 10 min, as shown in Figure 1. Based on these hepatic pressures, Rpre predominantly increased over Rpost within 10 min after reperfusion, as shown in Figure 2. The changes in these hemodynamic variables were similarly observed in the Ws/Ws and SD rats. 0.8
30
*
20
*
15
* 10
Rpre
Ppv
(cmH2O)
*
*
*
5
(mmHg/ml/min/10g liver)
0.7
25
*
0.4
* *
0.3 0.2
* *
0.0 0
1.5
3
10
20
30
40
50
60
0
1.5
3
10
20
*
*
10
20
30
40
50
60
30
40
50
60
0.8
8
* *
* Rpost
4
2
(mmHg/ml/min/10g liver)
0.7
* 6
Pdo
0.5
0.1
0
(cmH2O)
*
0.6
0.6 0.5 0.4 0.3 0.2 0.1
0
0.0 0
1.5
3
10
20
30
40
50
Time after reperfusion (min)
60
0
1.5
3
Time after reperfusion (min)
FIG. 2. Time course changes in portal venous pressure (Ppv), double occlusion pressure (Pdo), and pre-sinusoidal resistance (Rpre), and post-sinusoidal resistance (Rpost) in the Ws/Ws (closed circle, n ¼ 6), þ/þ (open circle, n ¼ 6), SD ischemia (closed triangle, n ¼ 6), and SD control (open triangle, n ¼ 6) groups. Values are means 6 SD. *P < 0.05 versus baseline.
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SHIBAMOTO ET AL.: MAST CELLS IN RAT HEPATIC ISCHEMIA-REPERFUSION
A Liver weight change (g)
3
2
1
*
* * *
*
*
*
*
* *
* *
* * *
0
-1 0
B
1.5
10
3
20
30
50
40
60
100
Bile flow (% of baseline)
During reperfusion, liver weight increased at the end of the experiment period in any groups after reperfusion, although it also increased immediately after reperfusion in the SD rats, as shown in Figure 3A. The liver injury was evidenced firstly by increased liver enzyme release into blood after reperfusion: In the þ/þ group, blood ALT levels increased from 19 6 4 to 71 6 18 and 135 6 30 (IU/L) at 30 and 60 min, respectively, after reperfusion (Fig. 3B). Bile flow decreased to 51% 6 6% of the baseline at 60 min after reperfusion (Fig 3C), which was another finding of liver injury. Similar changes in ALT levels and bile flow rate were observed in the Ws/Ws and SD rats, and there were no significant differences in the variables among the Ws/Ws, þ/þ, and SD groups (Fig. 3). In the control SD rats without I/R, no significant changes were observed in the variables studied (Figs. 2 and 3). Liver histology showed marked hepatic degeneration with hepatic necrosis and slightly disruption of lobular architecture in the SD ischemia, Ws/Ws, and þ/þ groups (Fig. 4B, C, and D, respectively), while there was no significant hepatic degeneration except for sinusoidal distention in SD control rats without I/R (Fig. 4A).
80
60
* 40
* *
* 20
* * *
0
0
1.5
* * *
* * *
* * *
* * *
* * *
* * 10
3
20
30
40
50
60
DISCUSSION
C
300 250 200
ALT (IU/L)
In the present study, we found that the hepatic damages, as demonstrated by increased blood ALT levels, reduced bile flow rates, and histopathological findings, were observed similarly in isolated perfused livers from the mast cell deficient Ws/Ws, the control þ/þ, and SD rats. Liver weight gain was also observed at the late phase in the presence of normal sinusoidal pressure, a finding consistent with the damage of the perfused livers [5]. However, there were no significant differences in the I/R-induced liver damages among the groups. We therefore conclude that mast cells are not involved in I/R injury of isolated perfused rat livers. This study is the first to determine the roles of mast cells in hepatic I/R injury of rat. We previously reported the hepatic I/R injury in isolated SD rat livers perfused bivascularly via the hepatic artery and portal vein with diluted blood (Hct of 3.3%) [5]. This injury was characterized by the transient increase in vascular resistance with Rpre being greater than Rpost, the biphasic liver weight gain at the early and late phase, and the hepatocellular damages evidenced by increased blood hepatic enzyme level and reduced bile production. These findings were consistently observed in the present SD rat livers perfused via the portal vein with blood (higher Hct of 8%). Similar I/Rinduced changes were also detected in the livers from the Ws/Ws and þ/þ rats. Thus, we demonstrated that
150
* * *
* * *
100 50 0 -50 0
10
20
30
40
50
60
Time after reperfusion (min) FIG. 3. Time course changes in liver weight change (A), bile flow (B), and blood alanine aminotransferase (ALT) levels (C), in the Ws/Ws (closed circle, n ¼ 6), þ/þ (open circle, n ¼ 6), SD ischemia (closed triangle, n ¼ 6), and SD control (open triangle, n ¼ 6) groups. Values are means 6 SD. *P < 0.05 versus baseline.
there were no significant differences in I/R damages among the rats with and without mast cells. The rationale for studying the roles of mast cells in I/R injury is based on the following evidence. First, I/R maneuver can evoke activation of mast cells in several tissue. An increased flux of oxidants, which has been previously observed at the onset of reperfusion, may be responsible for the mast cell activation [11]: the administration of antioxidants can block the histamine
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FIG. 4. Liver histology of the SD control (A), SD ischemia (B), Ws/Ws (C), and þ/þ (D) groups (HE, 3 400). C ¼ central vein, P ¼ portal region.
release from the post-ischemic organ [12, 13]. Second, in rat, a mast cell stabilizer, which blocked mast cell degranulation and inhibited chemical mediator release, prevented I/R-induced damage in hearts [14, 15] and intestines [16], possibly via inhibition of TNF-a release [17]. In rat livers, mast cells are abundantly distributed around hepatic vessels [18, 19]. There are at least two types of mast cells, the mucosal type of mast cells (MMC) and the connective tissuetype of mast cells. In rats, staining of mast cells in liver sections with alcian blue/safranin indicates that these cells are predominantly of the mucosal subtype [18], with Collins and co-workers reporting that <7% of hepatic mast cells had the staining characteristics of connective tissue mast cells [20]. These MMC are the local source of large quantities of chemical mediators, including histamine [12, 13], leukotrienes [21], and plateletactivating factor [22], which have been postulated to play important functions in the pathogenesis of I/Rinduced tissue injury. The present study suggests that mast cells are not involved in the rat hepatic I/R injury. One of the reasons for the absence of a role of mast cells, in contrast to the I/R injuries of other tissues [2–4], may be related to the isolated perfused rat liver preparation, in which mast cells might have been inactive.
However, this possibility seems unlikely. We previously demonstrated, using similar isolated perfused livers from þ/þ and Ws/Ws rats, that hepatic mast cells could evoke substantial anaphylactic hepatic venoconstriction: 90% of hepatic venoconstriction was attributable to intrahepatic mast cells, as evidenced by the antigen-induced increases in Ppv of 13.4 and 1.4 cmH2O in the þ/þ and Ws/Ws rats, respectively [6]. A more plausible explanation is that the mechanisms, other than the mast cell-related one, such as reperfusion-induced reactive oxygen species, one of the first elements formed at the initial stage of I/R injury [1], might have overwhelmed and masked the possible detrimental action of mast cells in the hepatic I/R injury. In the isolated perfused livers of the present study, the hepatic artery was not perfused. Hepatic arterial perfusion with normally oxygenated blood would improve the metabolic milieu of the liver. However, the perfusing blood was well oxygenated by bubbling with 95% O2 and 5% CO2, which provided perfusate oxygen tension of 290 mmHg [9]. Thus the isolated livers were perfused with hyperoxic blood, rather than hypoxic blood, and oxygenation was well done. In conclusion, the I/R injuries, assessed by blood ALT levels, bile flow rate, and histology, were similarly
SHIBAMOTO ET AL.: MAST CELLS IN RAT HEPATIC ISCHEMIA-REPERFUSION
observed in isolated blood-perfused livers derived from genetically mast cell deficient Ws/Ws rats, wild type þ/þ rats, and SD rats. These results suggest that mast cells are not involved in rat hepatic I/R injury. ACKNOWLEDGMENTS This work was supported by a Grant for Collaborative Research from Kanazawa Medical University (C2010-1) and a Grant-in-Aid for Scientific Research (20592131) from the Ministry for Education, Culture, Science, and Technology of Japan.
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