266
Journal of Hepatology, 1992; 16:266-272 01992 ElsevierScientificPublishers Ireland Ltd. All rights reserved. 0168-8278/92/$05.00
HEPAT 01139
Gut-derived substances in activation of hepatic macrophages after partial hepatectomy in rats Satoshi Mochida, Yasuhiko Ohta, Itsuro Ogata and Kenji Fujiwara First Department of Internal Medicine, Faculty of Medicine, Universityof Tokyo, Tokyo, Japan (Received31 January 1991)
When liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was performed in rats 24 h after twothirds liver resection, there were marked deposits of formazan converted from nitro blue tetrazolium in hepatic macrophages throughout the liver, indicating macrophage activity. The extent of the deposits was significantly reduced when perfusion was performed following oral administration of polymyxin B sulfate, a non-absorbable bacteriocidal agent against gram-negative bacilli which can also bind endotoxin lipopolysaccharides. Polymyxin B sulfate administration also attenuated the derangements of SGPT and the histological liver injury provoked by endotoxin administration after partial hepatectomy. These results suggests that gut-derived substances sensitive to polymyxin B sulfate may contribute to activation of hepatic macrophages after partial hepatectomy in rats. Key words: Liver resection; Polymyxin B sulfate; Nitro blue tetrazolium; Phorbol myristate acetate; Endotoxin
The function of the reticuloendothelial system has been shown to decrease after partial hepatectomy due to a reduction in the number of Kupffer cells (1,2). We demonstrated that hepatic macrophages are activated after partial hepatectomy and may contribute to the onset of massive hepatic necrosis following endotoxin administration in rats (3,4). When hepatic macrophages are exposed to foreign substances from the gut such as endotoxin and bacilli (5,6), these macrophages can be activated, and become a major barrier to prevent the outflow of these substances into the systemic circulation. It is well known that endotoxemia often occurs after partial hepatectomy (7,8). Thus, it is possible that the excessive amount of gut-derived substances in the remaining liver after partial hepatectomy leads to activation of hepatic macrophages. In the present work, we studied the possibility to elucidate the mechanisms of activation of hepatic macrophages after partial hepatectomy in rats.
Materials and Methods Experimental design
Male Sprague-Dawley rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) weighing 170-190 g were fed a commercial pelleted diet and water ad libitum in a cage at 22___2°C under normal lighting conditions throughout experiments. They received 250000 U/kg body wt. of polymyxin B sulfate (PMB; Pfizer Pharmaceuticals Inc., Tokyo, Japan) as a 50000 U/ml solution in water (PMB group) or the same volume of water (control group) through an esophageal tube everyday for 7 days. Experiment I Rats underwent two-thirds liver resection according to the method of Higgins and Anderson (9) between 9:00 and 11:00 a.m. under light ether anesthesia 24 h after the final dose of PMB, and were subjected to liver perfusion with nitro blue tetrazolium (NBT) and phorbol 12-myristate 13-acetate (PMA) 24 h after operation.
Correspondence to: Kenji Fujiwara, M.D., First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo,
Bunkyo-ku,Tokyo 113,Japan.
HEPATICMACROPHAGESAFTER LIVERRESECTION
Experiment II Rats underwent partial hepatectomy 24 h after the final dose of PMB. They received an intravenous injection of 200 ~g/kg body wt. of endotoxin (Escherichia coli 026: B6; Difco, Detroit, MI, U.S.A.) as a 100 I~g/ml solution in saline 48 h after operation, and were sacrificed 5 h later under ether anesthesia. At the time of sacrifice, blood was collected through the inferior cavai vein with an empty plastic syringe and a plastic syringe containing I:10 volume of 3.8% sodium citrate solution to prepare serum and plasma, respectively, for determinations of liver function tests, and the liver was excised for histological examination. Experiment III Blood was collected 24 h after the final dose of PMB from the rats given PMB for 7 days through both the inferior caval vein and portal vein under ether anesthesia to determine serum concentrations of PMB and endotoxin, respectively.
Procedure for formazan detection after liver perfusion with N B T and PMA Liver perfusion with NBT and PMA and formazan detection were based on the procedure previously described (10,11). After injection of 200 units of heparin (Novo Industri A/S, Copenhagen, Denmark) into the inferior vena cava under anesthesia with an intraperitoileal injection of pentobarbital sodium, the liver was perfused successively with Ca z+- and MgZ+-free Hanks' balanced salt solution [HBSS(-)] for 5 min, Eagle's MEM with 0.5 p.g/ml PMA (Sigma Chemical Company, St. Louis, MO, U.S.A.) for 5 rain, 0.05% NBT (Grade lII, Sigma Chemical Company) in MEM with PMA for 10 min, and finally HBSS (-) for 5 min. The liver was then excised for histological examination. The perfusion was performed at a flow rate of 7 ml/min, at 37°C with continuous Oz bubbling. All the perfusate contained 20mM HEPES (pH 7.4). Formazan detection was performed under a light microscope on specimens from the excised liver fixed with 20% formalin, embedded in paraffin, and counterstained with Kernechtrot. The extent of formazan deposition in hepatic macrophages was classified using a 5-degree score as shown in Figure I: in grades 1, 3 and 5, the deposits corresponded to the maximum degree seen in periportal and midzonal Kupffer cells in normal liver, in hepatic macrophages in centrilobular necrotic areas 3 days after carbon tetrachloride (CC14) intoxication and in hepatic macrophages throughout the liver 6 days after intravenous injection of heat-killed Corynebacterium parvum, respectively, according to the previous observations (10,1 I). Grades 2 and 4 were intermediate to grades I and 3, and 3 and 5, respectively. Grading of experimental
267 samples was determined by the maximum extent of formazan deposition in the observation fields.
Determination for liver function tests and histological examination SGPT activity was determined in sera using a commercial kit (Iatron Laboratories Inc., Tokyo, Japan). Prothrombin time (PT) was measured in plasma using SIMPLASTIN (Division of Warner Lambert Co., NJ, U.S.A.) and a fibrometer (Division of Beckton, Dickinson and Company, Cockeysville, MD, U.S.A.). Hepatic histology on a light microscope was studied in formalin-fixed and paraffin-embedded specimens of the liver stained with the hematoxylin/eosin or Masson's trichrome. All slides were evaluated blindly by one of the authors. The extent of liver injury was graded according to the area of coagulative necrosis in hepatic lobules as shown in Figure 2: grade 0, no coagulative necrosis; grade 1, coagulative necrosis covering less than 10%; grade 2, 10-50%; grade 3, more than 50%. Determination of serum PMB and endotoxin concentrations Serum PMB concentration was measured from bacteriocidal activity against Bordetella bronchiseptica (ATCC 4617) (12,13). Serum endotoxin concentration was measured chromogenically using a commercial kit (ENDOSPECY-test; Seikagaku Kogyo Co., Ltd., Tokyo, Japan) (14).
Results
Effect of P M B on Jormazan deposition in hepatic macrophages after liver pelfusion with N B T and PMA in partially hepatectomized rats Formazan deposition was observed in hepatic macrophages t.hroughout the liver in the control group. Its extent was grades 3 and 4. Although the extent did not differ throughout the liver in grade 4 (Fig. 3a), it was less in hepatic macrophages in the centrilobular areas than in those in the periportal and midzonal areas in grade 3. PMB-treated rats showed grades 2 and 3; the distribution of formazan deposition in grade 3 was similar to that seen in control rats showing grade 3, and the deposition in grade 2 was observed only in hepatic macrophages in the periportal and midzonal areas (Fig. 3b). Table 1 gives the summary of the extent of formazan deposition in hepatic macrophages in the PMB-treated and control groups. Although 3 of 6 rats showed grade 4 in the control group, there were no such rats in the PMB-treated group.
268
S. M O C H I D A et al.
Fig. !.
HEPATIC MACROPHAGES A F T E R LIVER RESECTION
.~-,~
269
:?.~ -,~,-....~.~
.
~.~
'
~2~,~
..~....
~'~
,, ~
-~-~),
Fig. 2. Light micrographs showing coagulative necrosis after endotoxin administration in partially hepatectomized rats. (a) Grade I covering less than 10% of hepatic Iobules. (b) Grade 2 covering 10-50% of the Iobules. (c) Grade 3 covering more than 50% of the lobules. HE stain.
Effect of P M B on liverfunction tests and histological extent of liver injury in partially hepatectomized rats after endotoxin administration In the control group, 9 of 12 rats showed coagulative necrosis in the midzonal areas. Its extent varied from grades I to 3. In contrast, such necrosis was observed only in 5 of 16 rats in the PMB-treated group, and the extent was grade I in all these 5 rats. S G P T values were aLso significantly lower in the PMB-treated group than in the control group, as shown in Table 2. Serum P M B and endotoxin concentrations Serum concentration of PMB was not detectable (less than 2.5 U/ml) in 10 rats given PMB. Mean serum concentration (+ SD; n = 9) of endotoxin in the portal vein was 4.5 -I- 1.3 and 6.9 _+ 3.7 pg/ml for PMB-treated FABLE I I~ffects of oral administration of polymyxin B sulfate (PMB) on formazan deposition in hepatic macrophages after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate in partially hcpatectomized rats
Groups
No. of rats
Extent of formazan deposition (grades) I
2
3
4
5
Control PMB-treated
6 6
0 0
0 4
3 "~
3 0
0 0*
Figures refer to the number of rats. *p < 0.05• vs. control group, by the Mann-Whitney test.
rats and control rats, respectively (p = 0.09, by Student's t-test).
Discussion In the present study, we showed that oral administration of PMB attenuated formazan deposits in rats after liver perfusion with NBT and PMA following partial hepatectomy. Abnormalities of liver function tests and the histological extent of liver injury were also improved in rats given endotoxin after partial hepatectomy. In order to reduce the amount of gut-derived substances, PMB was used, since this substance, a cyclic cationic polypeptide, has both strong bacteriological action on gram-negative bacilli (15) and binds endotoxin lipopolysaccharides (16-19). PMB is hardly absorbed in the gut when orally administered. In the present study, however,'serum PMB concentrations were measured to exclude any possible direct action of gut-absorbed PMB on hepatic macrophages, since PMB inhibits phospholipid-sensitive Ca 2÷ -dependent protein kinase (20) which is essential for stimulating macrophages (21). Since no serum PMB was detectable in normal rats given the dose used for partially hepatectomized rats, it can be assumed that the effect of orally administered PMB affected the gut.
'4 Fig. 1. Light micrographs showing formazan deposition in hepatic macrophages after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate. (a) Grade I in a normal rat. Faint formazan deposition is observed in Kupffer cells in the periportal and midzonal areas. (bl Grade 3 in a carbon-tetrachloride-intoxicated rat. Marked formazan deposition is seen in hepatic macrophages in centrilobular necrotic areas. (el Grade 5 in a Corynehacterium parrum-treated rat. Extensive formazan deposition is seen in hepatic macrophages throughout the liver. Kernechtrot stain.
270
S. M O C H I D A et al.
Fig. 3. Light micrographs showing formazan deposition in hepatic macrophages in partially hepatectomized rats after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate. (a) Grade 4 in a control rat. Marked formazan deposition is observed in hepatic macrophages throughout the liver. (b) Grade 2 in a PMB-treated rat. Faint formazan deposition is seen in hepatic macrophages in periportal and midzonal areas. Kernechtrot stain. TABLE 2 Effects of oral administration of polymyxin B sulfate (PMB) on SGPT values, prothrombin time (PT) and the histological extent of liver injury in partially hepatectomized rats 5 h after endotoxin administration Groups
No. of rats
SGPT (Karmen units)
PT (s)
Control
12
PMB-treated
16
697 a +672 249** +191
15.8 +5.8 12.6" +3.8
Extent of liver injury (grades) 0
1
2
3
3
4
3
2b
I1
5
0
0"**
Walues are mean __+ SD. b Figures refer to number of rats. *p < 0.I0, **p < 0.05 vs. control group, by Student's t-test. ***p < 0.01 vs. control group, by Mann-Whitney test.
To clarify the relationship between the activation of hepatic macrophages and the existence of gut-derived substances, we performed liver perfusion with NBT and PMA, and observed liver damage which developed after endotoxin administration in partially hepatectomized rats. As previously reported (10,11), the effect of liver perfusion with NBT and PMA indicates in situ the stimulatory state of hepatic macrophages based on their ability to produce superoxide anions and the cellular sites of its production. With this method, only slight formazan deposits were found in periportal and midzo-
nal Kupffer cells in normal rats (10). Although increased formazan deposition was observed in hepatic macrophages in both CCl4-treated and heat-killed Corynebacterium parvum-treated rats, the cellular sites for superoxide production were different in these models (11). After CCl4-intoxication, hepatic macrophages in centrilobular necrotic areas can mainly generate superoxide anions inside the cells such as in phagosomes by PMA stimulation, which suggests that these macrophages are at the responsive stage (11). In contrast, hepatic macrophages throughout the liver in Corynebac-
HEPATIC MACROPHAGES AFTER LIVER RESECTION
271
terium parvum-treated rats can be considered at the primed stage, since they can release large a m o u n t s of superoxide anions outside the cells (11). We also demonstrated that from 24 to 72 h after partial hepatectomy most hepatic macrophages are at the primed stage with increased ability to release superoxide anions outside the cells by stimuli (3,4). The stimulatory stages of hepatic macrophages are determined by the cellular sites of superoxide production as estimated by the extent of attenuation of formazan deposition following the addition of superoxide dismutase to the perfusate (11). F o r m a z a n deposits in hepatic macrophages at the primed stage after liver perfusion with only N B T and PMA, however, are more extensive than when macrophages are at the responsive stage. Thus, in the present experiment, we determined the stimulatory stages of hepatic macrophages based on the extent of formazan deposition after liver perfusion with N B T and PMA. According to our 5-degree score, hepatic macrophages in grades 2 and 3 were considered at the responsive stage and those in grade 4 and 5 at the primed stage. For another parameter of activation of hepatic macrophages, we used liver d a m a g e following endotoxin administration in partially hepatectomized rats, since hepatic macrophages at the primed stage can contribute to the development of massive hepatic necrosis due to intravascular coagulation after endotoxin administration (3,4,22). W h e n liver perfusion with N B T and P M A was performed in partially hepatectomized r a t s , there were marked formazan deposits in hepatic macrophages
t h r o u g h o u t the liver, and these macrophages were considered at the primed stage in 50% of 6 partially hepatectomized rats. The extent of these deposits was significantly reduced by pretreatment with oral administered PMB, and hepatic macrophages were at the responsive stage in all rats. In these rats, formazan deposits in hepatic macrophages in the centrilobular areas were less extensive than in the periportai areas, suggesting that gut-derived substances reached to the centrilobular areas to a lesser degree via the portal vein. The extent of liver injury following endotoxin administration after partial hepatectomy was also attenuated in PMB-treated rats. F r o m these results, we can reasonably assume that activation of hepatic macrophages was inhibited by oral administration of PMB, and that gut-derived substances sensitive to P M B m a y contribute to the activation of macrophages after partial hepatectomy. Although serum concentrations of endotoxin in the portal vein tended to be lower in PMB-treated rats than in control rats, this difference was not statistically significant. Gutderived substances other than endotoxin related to PMB-sensitive bacilli may cause activation and should be further investigated. In conclusion, since gut-derived substances may contribute to the activation of hepatic macrophages after partial hepatectomy, oral administration of P M B may help prevent acute liver failure after liver resection.
References
The significance of activation of reticuloendothelial function on hepatectomy. Tohoku J Exp Med 1987; 153: 123-32. 9 Higgins GM, Anderson RM. Experimental pathology of the liver. !. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol 1931; 12: 186-202. 10 Ogata I, Mochida S, Fujiwara K. Formazan formation in hepatic macrophages after nitro blue tetrazolium perfusion into rat liver. Biomed Res 1988;9: 113-7. II Mochida S, Ogata I, Ohta Y, Yamada S, Fujiwara K. In situ evaluation of the stimulatory state of hepatic macrophages based on their ability to produce superoxide anions in rats. J Pathol 1989; 158: 67-71. 12 Kunin CM, Bugg A. Recovery of tissue bound polymyxin B and colistimethate. Proc Soc Exp Biol Med 1971; 137: 786-90. 13 Craig WA, Kunin CM. Dynamics of binding and release of the polymyxin antibiotics by tissues. J Pharmacol Exp Ther 1973; 184: 757-65. 14 Ohbayashi T, Tamura H, Tanaka Set al. A new chromogenic endotoxin-specific assay using recombined Limulus coagulation enzymes and its clinical applications. Clin Chim Acta 1985; 149: 55-65. 15 Newton BA. The properties and mode of action of polymyxin. Bacteriol Rev 1956; 20: 14-27. 16 Rifkind D, Palmer J. Neutralization of endotoxin in chick embryos by antibiotics. J Bacteriol 1966;92: 815-9.
I Gross K, Katz S, Dunn SP, Cikrit D, Rosenthal R, Grosfeld JL. Bacterial clearance in the intact and regenerating liver. J Pediatr Surg 1985; 20: 320-33. 2 Nakagawa K, Ouchi K, Matsubara S, Hashimoto L, Yajima Y. Activation of reticuloendothelial function for prevention of endotoxemia after hepatectomy in cirrhotic patients. Tohoku J Exp Med 1987; 153: 133-6. 3 Mochida S, Ogata I, Hirata, K, Ohta Y, Yamada S, Fujiwara K. Provocation of massive hepatic necrosis by endotoxin after partial hepatectomy in rats. Gastroenterology 1990;99: 771-7. 4 Fujiwara K, Ogata 1, Mochida S et al. Activated Kupffer cells as a factor of massive hepatic necrosis after liver resection. Hepatogastroenterology 1990; 37: 194-7. 5 Ravin HA, Rowley D, Jenkins C, Fine J. On the absorption of bacterial endotoxin from the gastro-intestinal tract of the normal and shocked animal. J Exp Med 1960; 112: 783-92. 6 Jacob A1, Goldberg PK, Bloom N, Degenshein GA, Kozinn PJ. Endotoxin and bacteria in portal blood. Gastroenterology 1977; 72: 1268-70. 7 Cornell RP. Restriction of gut-derived endotoxin impairs DNA synthesis for liver regeneration. Am J Physiol 1985; 249: 563-9. 8 Nakagawa K, Ouchi K, Matsubara S, Hashimoto L, Yajima Y.
Acknowledgement We thank Ms. K a y o k o Naiki for technical assistance.
272 17 Corrigan JJ, Bell BM. Comparison between the polymyxins and gentamicine in preventing endotoxin-induced intravascular coagulation and leukopenia. Infect Immunity 1971; 4: 563-6. 18 Craig WA, Turner JH, Kunin CM. Prevention of the generalized Schwartzman reaction and endotoxin lethality by polymyxin B localized in tissues. Infect Immun 1974; 10: 287-92. 19 Ingoldby CJH. The value of polymyxin B in endotoxaemia due to experimental obstructive jaundice and mesenteric ischaemia. Br J Surg 1980; 67: 565-7. 20 Mazzei G J, Katoh N, Kuo JF. Polymyxin B is a more selective
S. MOCHIDA et al. inhibitor for phospholipid-sensitive CaZ+-dependent protein kinase than for calmodulin-sensitive Caa--dependent protein kinase. Biochem Biophys Res Commun 1982; 109:1129-33. 21 Somers SD, Weiel JE, Hamilton TA, Adams DO. Phorbol esters and calcium ionophore.can prime murine peritoneal macrophages for tumor cell destruction. J lmmunol 1986; 136: 4199-205. 22 Yamada S, Ogata I, Hirata K, Mochida S, Tomiya T, Fujiwara K. lntravascular coagulation in the development of massive hepatic necrosis induced by Corynebacterium parvum and endotoxin in rats. Scand J Gastroenterol 1989; 24: 293-8.
Journal of Hepatology, 1992; 16:266-272 01992 ElsevierScientificPublishers Ireland Ltd. All rights reserved. 0168-8278/92/$05.00
HEPAT 01139
Gut-derived substances in activation of hepatic macrophages after partial hepatectomy in rats Satoshi Mochida, Yasuhiko Ohta, Itsuro Ogata and Kenji Fujiwara First Department of Internal Medicine, Faculty of Medicine, Universityof Tokyo, Tokyo, Japan (Received31 January 1991)
When liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was performed in rats 24 h after twothirds liver resection, there were marked deposits of formazan converted from nitro blue tetrazolium in hepatic macrophages throughout the liver, indicating macrophage activity. The extent of the deposits was significantly reduced when perfusion was performed following oral administration of polymyxin B sulfate, a non-absorbable bacteriocidal agent against gram-negative bacilli which can also bind endotoxin lipopolysaccharides. Polymyxin B sulfate administration also attenuated the derangements of SGPT and the histological liver injury provoked by endotoxin administration after partial hepatectomy. These results suggests that gut-derived substances sensitive to polymyxin B sulfate may contribute to activation of hepatic macrophages after partial hepatectomy in rats. Key words: Liver resection; Polymyxin B sulfate; Nitro blue tetrazolium; Phorbol myristate acetate; Endotoxin
The function of the reticuloendothelial system has been shown to decrease after partial hepatectomy due to a reduction in the number of Kupffer cells (1,2). We demonstrated that hepatic macrophages are activated after partial hepatectomy and may contribute to the onset of massive hepatic necrosis following endotoxin administration in rats (3,4). When hepatic macrophages are exposed to foreign substances from the gut such as endotoxin and bacilli (5,6), these macrophages can be activated, and become a major barrier to prevent the outflow of these substances into the systemic circulation. It is well known that endotoxemia often occurs after partial hepatectomy (7,8). Thus, it is possible that the excessive amount of gut-derived substances in the remaining liver after partial hepatectomy leads to activation of hepatic macrophages. In the present work, we studied the possibility to elucidate the mechanisms of activation of hepatic macrophages after partial hepatectomy in rats.
Materials and Methods Experimental design
Male Sprague-Dawley rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) weighing 170-190 g were fed a commercial pelleted diet and water ad libitum in a cage at 22___2°C under normal lighting conditions throughout experiments. They received 250000 U/kg body wt. of polymyxin B sulfate (PMB; Pfizer Pharmaceuticals Inc., Tokyo, Japan) as a 50000 U/ml solution in water (PMB group) or the same volume of water (control group) through an esophageal tube everyday for 7 days. Experiment I Rats underwent two-thirds liver resection according to the method of Higgins and Anderson (9) between 9:00 and 11:00 a.m. under light ether anesthesia 24 h after the final dose of PMB, and were subjected to liver perfusion with nitro blue tetrazolium (NBT) and phorbol 12-myristate 13-acetate (PMA) 24 h after operation.
Correspondence to: Kenji Fujiwara, M.D., First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo,
Bunkyo-ku,Tokyo 113,Japan.
HEPATICMACROPHAGESAFTER LIVERRESECTION
Experiment II Rats underwent partial hepatectomy 24 h after the final dose of PMB. They received an intravenous injection of 200 ~g/kg body wt. of endotoxin (Escherichia coli 026: B6; Difco, Detroit, MI, U.S.A.) as a 100 I~g/ml solution in saline 48 h after operation, and were sacrificed 5 h later under ether anesthesia. At the time of sacrifice, blood was collected through the inferior cavai vein with an empty plastic syringe and a plastic syringe containing I:10 volume of 3.8% sodium citrate solution to prepare serum and plasma, respectively, for determinations of liver function tests, and the liver was excised for histological examination. Experiment III Blood was collected 24 h after the final dose of PMB from the rats given PMB for 7 days through both the inferior caval vein and portal vein under ether anesthesia to determine serum concentrations of PMB and endotoxin, respectively.
Procedure for formazan detection after liver perfusion with N B T and PMA Liver perfusion with NBT and PMA and formazan detection were based on the procedure previously described (10,11). After injection of 200 units of heparin (Novo Industri A/S, Copenhagen, Denmark) into the inferior vena cava under anesthesia with an intraperitoileal injection of pentobarbital sodium, the liver was perfused successively with Ca z+- and MgZ+-free Hanks' balanced salt solution [HBSS(-)] for 5 min, Eagle's MEM with 0.5 p.g/ml PMA (Sigma Chemical Company, St. Louis, MO, U.S.A.) for 5 rain, 0.05% NBT (Grade lII, Sigma Chemical Company) in MEM with PMA for 10 min, and finally HBSS (-) for 5 min. The liver was then excised for histological examination. The perfusion was performed at a flow rate of 7 ml/min, at 37°C with continuous Oz bubbling. All the perfusate contained 20mM HEPES (pH 7.4). Formazan detection was performed under a light microscope on specimens from the excised liver fixed with 20% formalin, embedded in paraffin, and counterstained with Kernechtrot. The extent of formazan deposition in hepatic macrophages was classified using a 5-degree score as shown in Figure I: in grades 1, 3 and 5, the deposits corresponded to the maximum degree seen in periportal and midzonal Kupffer cells in normal liver, in hepatic macrophages in centrilobular necrotic areas 3 days after carbon tetrachloride (CC14) intoxication and in hepatic macrophages throughout the liver 6 days after intravenous injection of heat-killed Corynebacterium parvum, respectively, according to the previous observations (10,1 I). Grades 2 and 4 were intermediate to grades I and 3, and 3 and 5, respectively. Grading of experimental
267 samples was determined by the maximum extent of formazan deposition in the observation fields.
Determination for liver function tests and histological examination SGPT activity was determined in sera using a commercial kit (Iatron Laboratories Inc., Tokyo, Japan). Prothrombin time (PT) was measured in plasma using SIMPLASTIN (Division of Warner Lambert Co., NJ, U.S.A.) and a fibrometer (Division of Beckton, Dickinson and Company, Cockeysville, MD, U.S.A.). Hepatic histology on a light microscope was studied in formalin-fixed and paraffin-embedded specimens of the liver stained with the hematoxylin/eosin or Masson's trichrome. All slides were evaluated blindly by one of the authors. The extent of liver injury was graded according to the area of coagulative necrosis in hepatic lobules as shown in Figure 2: grade 0, no coagulative necrosis; grade 1, coagulative necrosis covering less than 10%; grade 2, 10-50%; grade 3, more than 50%. Determination of serum PMB and endotoxin concentrations Serum PMB concentration was measured from bacteriocidal activity against Bordetella bronchiseptica (ATCC 4617) (12,13). Serum endotoxin concentration was measured chromogenically using a commercial kit (ENDOSPECY-test; Seikagaku Kogyo Co., Ltd., Tokyo, Japan) (14).
Results
Effect of P M B on Jormazan deposition in hepatic macrophages after liver pelfusion with N B T and PMA in partially hepatectomized rats Formazan deposition was observed in hepatic macrophages t.hroughout the liver in the control group. Its extent was grades 3 and 4. Although the extent did not differ throughout the liver in grade 4 (Fig. 3a), it was less in hepatic macrophages in the centrilobular areas than in those in the periportal and midzonal areas in grade 3. PMB-treated rats showed grades 2 and 3; the distribution of formazan deposition in grade 3 was similar to that seen in control rats showing grade 3, and the deposition in grade 2 was observed only in hepatic macrophages in the periportal and midzonal areas (Fig. 3b). Table 1 gives the summary of the extent of formazan deposition in hepatic macrophages in the PMB-treated and control groups. Although 3 of 6 rats showed grade 4 in the control group, there were no such rats in the PMB-treated group.
268
S. M O C H I D A et al.
Fig. !.
HEPATIC MACROPHAGES A F T E R LIVER RESECTION
.~-,~
269
:?.~ -,~,-....~.~
.
~.~
'
~2~,~
..~....
~'~
,, ~
-~-~),
Fig. 2. Light micrographs showing coagulative necrosis after endotoxin administration in partially hepatectomized rats. (a) Grade I covering less than 10% of hepatic Iobules. (b) Grade 2 covering 10-50% of the Iobules. (c) Grade 3 covering more than 50% of the lobules. HE stain.
Effect of P M B on liverfunction tests and histological extent of liver injury in partially hepatectomized rats after endotoxin administration In the control group, 9 of 12 rats showed coagulative necrosis in the midzonal areas. Its extent varied from grades I to 3. In contrast, such necrosis was observed only in 5 of 16 rats in the PMB-treated group, and the extent was grade I in all these 5 rats. S G P T values were aLso significantly lower in the PMB-treated group than in the control group, as shown in Table 2. Serum P M B and endotoxin concentrations Serum concentration of PMB was not detectable (less than 2.5 U/ml) in 10 rats given PMB. Mean serum concentration (+ SD; n = 9) of endotoxin in the portal vein was 4.5 -I- 1.3 and 6.9 _+ 3.7 pg/ml for PMB-treated FABLE I I~ffects of oral administration of polymyxin B sulfate (PMB) on formazan deposition in hepatic macrophages after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate in partially hcpatectomized rats
Groups
No. of rats
Extent of formazan deposition (grades) I
2
3
4
5
Control PMB-treated
6 6
0 0
0 4
3 "~
3 0
0 0*
Figures refer to the number of rats. *p < 0.05• vs. control group, by the Mann-Whitney test.
rats and control rats, respectively (p = 0.09, by Student's t-test).
Discussion In the present study, we showed that oral administration of PMB attenuated formazan deposits in rats after liver perfusion with NBT and PMA following partial hepatectomy. Abnormalities of liver function tests and the histological extent of liver injury were also improved in rats given endotoxin after partial hepatectomy. In order to reduce the amount of gut-derived substances, PMB was used, since this substance, a cyclic cationic polypeptide, has both strong bacteriological action on gram-negative bacilli (15) and binds endotoxin lipopolysaccharides (16-19). PMB is hardly absorbed in the gut when orally administered. In the present study, however,'serum PMB concentrations were measured to exclude any possible direct action of gut-absorbed PMB on hepatic macrophages, since PMB inhibits phospholipid-sensitive Ca 2÷ -dependent protein kinase (20) which is essential for stimulating macrophages (21). Since no serum PMB was detectable in normal rats given the dose used for partially hepatectomized rats, it can be assumed that the effect of orally administered PMB affected the gut.
'4 Fig. 1. Light micrographs showing formazan deposition in hepatic macrophages after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate. (a) Grade I in a normal rat. Faint formazan deposition is observed in Kupffer cells in the periportal and midzonal areas. (bl Grade 3 in a carbon-tetrachloride-intoxicated rat. Marked formazan deposition is seen in hepatic macrophages in centrilobular necrotic areas. (el Grade 5 in a Corynehacterium parrum-treated rat. Extensive formazan deposition is seen in hepatic macrophages throughout the liver. Kernechtrot stain.
270
S. M O C H I D A et al.
Fig. 3. Light micrographs showing formazan deposition in hepatic macrophages in partially hepatectomized rats after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate. (a) Grade 4 in a control rat. Marked formazan deposition is observed in hepatic macrophages throughout the liver. (b) Grade 2 in a PMB-treated rat. Faint formazan deposition is seen in hepatic macrophages in periportal and midzonal areas. Kernechtrot stain. TABLE 2 Effects of oral administration of polymyxin B sulfate (PMB) on SGPT values, prothrombin time (PT) and the histological extent of liver injury in partially hepatectomized rats 5 h after endotoxin administration Groups
No. of rats
SGPT (Karmen units)
PT (s)
Control
12
PMB-treated
16
697 a +672 249** +191
15.8 +5.8 12.6" +3.8
Extent of liver injury (grades) 0
1
2
3
3
4
3
2b
I1
5
0
0"**
Walues are mean __+ SD. b Figures refer to number of rats. *p < 0.I0, **p < 0.05 vs. control group, by Student's t-test. ***p < 0.01 vs. control group, by Mann-Whitney test.
To clarify the relationship between the activation of hepatic macrophages and the existence of gut-derived substances, we performed liver perfusion with NBT and PMA, and observed liver damage which developed after endotoxin administration in partially hepatectomized rats. As previously reported (10,11), the effect of liver perfusion with NBT and PMA indicates in situ the stimulatory state of hepatic macrophages based on their ability to produce superoxide anions and the cellular sites of its production. With this method, only slight formazan deposits were found in periportal and midzo-
nal Kupffer cells in normal rats (10). Although increased formazan deposition was observed in hepatic macrophages in both CCl4-treated and heat-killed Corynebacterium parvum-treated rats, the cellular sites for superoxide production were different in these models (11). After CCl4-intoxication, hepatic macrophages in centrilobular necrotic areas can mainly generate superoxide anions inside the cells such as in phagosomes by PMA stimulation, which suggests that these macrophages are at the responsive stage (11). In contrast, hepatic macrophages throughout the liver in Corynebac-
HEPATIC MACROPHAGES AFTER LIVER RESECTION
271
terium parvum-treated rats can be considered at the primed stage, since they can release large a m o u n t s of superoxide anions outside the cells (11). We also demonstrated that from 24 to 72 h after partial hepatectomy most hepatic macrophages are at the primed stage with increased ability to release superoxide anions outside the cells by stimuli (3,4). The stimulatory stages of hepatic macrophages are determined by the cellular sites of superoxide production as estimated by the extent of attenuation of formazan deposition following the addition of superoxide dismutase to the perfusate (11). F o r m a z a n deposits in hepatic macrophages at the primed stage after liver perfusion with only N B T and PMA, however, are more extensive than when macrophages are at the responsive stage. Thus, in the present experiment, we determined the stimulatory stages of hepatic macrophages based on the extent of formazan deposition after liver perfusion with N B T and PMA. According to our 5-degree score, hepatic macrophages in grades 2 and 3 were considered at the responsive stage and those in grade 4 and 5 at the primed stage. For another parameter of activation of hepatic macrophages, we used liver d a m a g e following endotoxin administration in partially hepatectomized rats, since hepatic macrophages at the primed stage can contribute to the development of massive hepatic necrosis due to intravascular coagulation after endotoxin administration (3,4,22). W h e n liver perfusion with N B T and P M A was performed in partially hepatectomized r a t s , there were marked formazan deposits in hepatic macrophages
t h r o u g h o u t the liver, and these macrophages were considered at the primed stage in 50% of 6 partially hepatectomized rats. The extent of these deposits was significantly reduced by pretreatment with oral administered PMB, and hepatic macrophages were at the responsive stage in all rats. In these rats, formazan deposits in hepatic macrophages in the centrilobular areas were less extensive than in the periportai areas, suggesting that gut-derived substances reached to the centrilobular areas to a lesser degree via the portal vein. The extent of liver injury following endotoxin administration after partial hepatectomy was also attenuated in PMB-treated rats. F r o m these results, we can reasonably assume that activation of hepatic macrophages was inhibited by oral administration of PMB, and that gut-derived substances sensitive to P M B m a y contribute to the activation of macrophages after partial hepatectomy. Although serum concentrations of endotoxin in the portal vein tended to be lower in PMB-treated rats than in control rats, this difference was not statistically significant. Gutderived substances other than endotoxin related to PMB-sensitive bacilli may cause activation and should be further investigated. In conclusion, since gut-derived substances may contribute to the activation of hepatic macrophages after partial hepatectomy, oral administration of P M B may help prevent acute liver failure after liver resection.
References
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