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Effect of ethanolamine on liver regeneration after 90% hepatectomy in rats
Effect of Ethanolamine on Liver Regeneration After 90% Hepatectomy in Rats A. Mimuro, T. Aoki, A. Tsuchida, T. Miyashita, Y. Koyanagi, and S. Enosawa
S
URGICAL PROCEDURES, such as extended liver resection or living-related liver transplantation may be performed because normal liver has vigorous regenerative potential. However, reduced hepatic volume can produce postoperative hepatic insufficiency, which is a serious problem. After extensive liver resection for hepatic tumor or in living-related liver donors maximum volume of liver resection or thus the minimum volume of donor liver are limited.1,2 When the volume exceeds these limits, surgical treatment is not indicated. However, a new treatment that promotes postoperative liver regeneration would extend these limits. Two approaches have been attempted to promote liver regeneration. One approach prepares the host environment for liver regeneration by plasma exchange. In recent years, favorable results also have been reported using a bioartificial liver as a bridge.3– 8 The other approach is promotion of liver regeneration using hepatocyte growth factor (HGF) or ethanolamine (EA). EA is a nutrient factor derived from the small intestine. In this study, we investigated the enhancing effect of EA using a 90% partial hepatectomy (PHX) rat model. MATERIALS AND METHODS Animals and Operative Procedure Male Sprague-Dawley rats (Nihon Clea, Tokyo, Japan), weighing 300 g to 350 g, were used as experiment animals. They were supplied no food for 12 hours prior to surgery. Under ether anesthesia, the liver was resected by the method of Kubota et al.1 The median lobe and left lateral lobe were removed for 70% PHX rats and the right lateral lobe also was resected for 90% PHX. After surgery, the animals were allowed food ad lib. For 2 operative days after surgery, the animals were given 20% glucose instead of water.
Administration of BrdU and EA On the days 0, 1, 3, 5, or 7 after PHX, 100 mg/kg of BrdU (Takeda Chemical Industries, Ltd., Osaka, Japan) was administered intraabdominally. Laparotomy was performed 1 hour after administration; blood samples were taken from the inferior vena cava and the remnant liver was removed for investigation. Immediately after 90% PHX, 24 mg/kg of ethanolamine (Sigma-Aldrich Japan, Tokyo, Japan) was administered intra-abdominally every 24 hours thereafter (EA group; n ⫽ 6). For the control group (n ⫽ 6) and 70% PHX group (n ⫽ 6), the rats were given the same amount of saline intra-abdominally.
Postoperative Evaluation Blood Tests. Aspartate aminotransferase (AST), total bilirubin (T-Bil), bile acid (BA), and hepaplastin test (HPT) were measured. Weight of remnant liver: The weight of resected remnant liver was measured and calculated in terms of 300 g body weight for evaluation. Regeneration rate: The weight of remnant liver on day 7 was divided by the liver weight estimated prior to surgery. Increased Liver Weight. The percentage of increased liver weight per day over the liver weight measured previously was calculated. Labeling index: Frozen specimens were prepared from the resected liver for immunohistochemical staining using an antiBrdU monoclonal antibody (Sigma-Aldrich Japan, Tokyo, Japan). An image analyzer (SP-500F; Olympus Co., Ltd, Tokyo, Japan), detected the areas of BrdU-positive (the nuclei were stained dark brown) and-negative hepatocytes , which were measured. The rate of BrdU-positive hepatocytes was calculated as a labeling index (LI) for assessment of liver regeneration.
Statistical Analysis Mann-Whitney U test was used for the differences of each value among groups. A P value less than .05 was considered statistically significant.
RESULTS Blood Examination
In the 70% PHX group, AST, T-Bil, and BA increased and HPT decreased slightly on day 1 after surgery, and thereafter they were gradually improved. In contrast among the 90% PHX groups, the AST increased excessively on the day 1, however, it rapidly improved on day 3. T-Bil and BA also increased on day 1 and gradually improved from day 3. In addition, HPT decreased to approximately 10% on day 1 and improved up to approximately 40% on day 3, however, thereafter it was gradually decreasing from day 5. No significant differences could be noted between the EA group and the controls (Fig 1). From the Department of Surgery, Tokyo Medical University, and the Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, Tokyo, Japan. Address reprint requests to Dr A. Mimuro, Department of Surgery, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-Ku, Tokyo 160-0023, Japan. E-mail: mimuro@tokyo-med. ac.jp
0041-1345/02/$–see front matter PII S0041-1345(02)03368-7
© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710
2664
Transplantation Proceedings, 34, 2664 –2667 (2002)
EFFECT OF ETHANOLAMINE ON LIVER REGENERATION
2665
Fig 1. AST (A), BA (B), T-Bil (C), and HPT (D). Graphs of biochemical test data after PHX are shown. Although liver dysfunction in 70% PHX rat (䊐) was slight, a high degree of liver dysfunction was manifested in 90% PHX rat, yet it was quickly ameliorated. No significant differences were noted between the control group (‚) and the EA group (E).
Postoperative Change of Remnant Liver
In the 70% PHX group, the weight of the remnant liver increased steadily from day 1; the regeneration rate was 74% on day 7. On days 1, 3, 5, and 7, the increases in liver weight were 52, 19, 7, and 1% per day, respectively, which showed a decrease from the peak rate on day 1. In the 90% PHX group, the increased liver weight indicated a peak on day 3 at 62% per day in the control group and 92% per day in the EA group. On day 7, however, the rate increased again by 28% per day in the control group and 22% per day in the EA group. The regeneration rate was 64% in the control group and 62% in the EA group (Table 1).
It was similar to the pattern with the control group. There was a statistically significant difference (P ⫽ .0039) for LI between the control group and the EA group on days 1 and 3, respectively (Fig 2 and Fig 3). DISCUSSION
EA is a low molecular nutritious factor derived from the small intestine and used in the synthesis of phospholipids.9 EA has been reported to enhance the proliferation of Table 1. Weight of Remnant Liver, Increased Rate of Liver Weight, and Regeneration Rate Weight of Remnant Liver (g:Mean ⫾ SD)
LI
In the 70% PHX group, LI reached the peak at a median value of 28.4% on day 1 and decreased thereafter. On day 7, it decreased to the same value as immediately after resection. In the control group, LI reached a peak at a median value of 5.1% on day 3 and decreased thereafter. On day 7, it decreased to a median value of 1.6%, which was almost equivalent to the value immediately after resection. On the contrary, in the EA group, LI reached the peak at a median value of 30.7% on day 3 and decreased thereafter. On day 7, it decreased to a median value of 1.6%, which was almost equivalent to the value immediately after resection.
Day 0
Day 1
Day 3
Day 5
Day 7
70% PHX 3.8 ⫾ 0.8 5.8 ⫾ 0.5 7.9 ⫾ 0.6 8.9 ⫾ 0.7 9.1 ⫾ 0.4 Control group 1.2 ⫾ 0.1 1.7 ⫾ 0.3 3.7 ⫾ 0.7 4.8 ⫾ 0.8 7.6 ⫾ 1.5 EA group 1.2 ⫾ 0.1 1.6 ⫾ 0.2 4.5 ⫾ 0.8 5.1 ⫾ 1.0 7.4 ⫾ 1.4 Increased Rate of Liver Weight (%/d)
70% PHX Control group EA group
Regeneration Rate
Day 1
Day 3
Day 5
Day 7
(%)
26 21 17
19 62 92
7 15 7
1 28 22
74 64 62
2666
MIMURO, AOKI, TSUCHIDA ET AL
Fig 2. A graph of LI is shown. In 70% PHX rat (u), the value peaked on day 1 and decreased subsequently. In 90% PHX rat, the value peaked on day 3. Significant differences between the EA group (■) and the control group (䊐) on days 1 and 3 were noted (*P ⫽ .0039).
initially isolated hepatocytes in the rat increasing DNA synthesis in the 70% PHX rat.10 We investigated the enhancing effect of EA on liver regeneration in a 90% PHX rat, which is a model of hepatic insufficiency. In the 70% PHX rat, increased liver weight and LI were observed on day 1, resulting in the vigorous liver regeneration. Thereafter, both values gradually decreased returning on day 7 to the same level as before resection. Thus liver regeneration seems to almost be complete at 7 days after resection in 70% PHX rats. In 90% PHX rats, on the other hand, the increase in liver weight was slight and the LI was also low on day 1. By day 3, LI showed a maximum value and the liver weight increased. Thereafter LI decreased, however, liver weight increased again on day 7, findings that were similar between the EA group and the control group. From these results, it is concluded that: (1) liver regeneration is delayed in the 90% PHX group; (2) in the course of liver regeneration, the period of DNA synthesis (S phase) terminates around 7 days after PHX regardless of the resected volumes, and (3) in the 90% PHX group, there can be a time lag in the course of regeneration after the S phase. Causes of the delay in liver regeneration11 have been reported to be decreased protein synthesis,1 impaired remnant liver or hepatic metabolism by endogenous endotoxin,12,13 decreased reticuloendothelial system function, and decreased glycogenolysis.14 After administration of EA to the 90% PHX rat, LI showed a significant difference from the control at days 1 and 3. The LI median value (18.0%) on day 1 suggested that DNA was being vigorously synthesized in the EA group. Thus EA administration seems to promote or enhance DNA synthesis. No significant differences were observed in liver weight or in blood data, however, for two reasons: (1) EA may enhance the progression of DNA synthesis from the G0 to the S phase, or (2) operative stress or unidentified substrates in the 90% PHX host may disturb liver regeneration. Even if there is an increase in S phase
Fig 3. Control (A) and EA (B). Microscopic images of hepatic tissue (⫻40) showing immunohistological staining by anti-BrdU monoclonal antibody on day 3.
EFFECT OF ETHANOLAMINE ON LIVER REGENERATION
hepatocytes after EA administration, unless there is progression to late G2 and M phases, mature hepatocytes do not proliferate. In other words, there will be no significant differences in remnant liver weight or in blood data. In conclusion, EA seems to promote or enhance DNA synthesis during the early stage after liver surgery even in environments unfavorable for liver regeneration, such as 90% PHX. Since EA is a low molecular substance present in the human body and its safety has been established, it may be suitable as an enhancer for hepatic regenerative processes. REFERENCES 1. Kubota T, Takabe K, Meng Y, et al: J Hep Bil Pancr Surg 4:398, 1997 2. Kawasaki S, Makuuchi M, Matsunami H, et al: Transplant Proc 27:1170, 1995
2667 3. Chen SC, Hewitt WR, Watanabe FD, et al: Int J Artif Organs 19:664, 1996 4. Kamohara Y, Rozga J, Demetriou A: J Hep Bil Pancr Surg 5:273, 1998 5. Kamohara Y, Fujioka H, Eguchi S, et al: Artif Organs 24:265, 2000 6. Rozga J, Podesta L, LePage E, et al: Ann Surg 219:538, 1994 7. Bosse B, Smith MD, Gerlach J, et al: Langenbecks Arch Surg 384:588, 1999 8. Miyashita T, Enosawa S, Suzuki S, et al: Transplant Proc 32:2355, 2000 9. Sundler R, Akesson B: J Biol Chem 250:3359, 1975 10. Sasaki H, Kume H, Nemoto A, et al: Proc Natl Acad Sci 94:7320, 1997 11. Zieve L, Anderson W, Lindblad S, et al: J Lab Clin Med 105:331, 1985 12. Kawasaki Y, Miyara K, Isa T, et al: Jap J Gastroenterol Surg 22:1778, 1989 13. Ando K, Miyazaki M, Ito H, et al: Jap J Gastroenterol Surg 27:1753, 1994 14. Gaub J, Iversen J: Hepatology 4:902, 1984
S
URGICAL PROCEDURES, such as extended liver resection or living-related liver transplantation may be performed because normal liver has vigorous regenerative potential. However, reduced hepatic volume can produce postoperative hepatic insufficiency, which is a serious problem. After extensive liver resection for hepatic tumor or in living-related liver donors maximum volume of liver resection or thus the minimum volume of donor liver are limited.1,2 When the volume exceeds these limits, surgical treatment is not indicated. However, a new treatment that promotes postoperative liver regeneration would extend these limits. Two approaches have been attempted to promote liver regeneration. One approach prepares the host environment for liver regeneration by plasma exchange. In recent years, favorable results also have been reported using a bioartificial liver as a bridge.3– 8 The other approach is promotion of liver regeneration using hepatocyte growth factor (HGF) or ethanolamine (EA). EA is a nutrient factor derived from the small intestine. In this study, we investigated the enhancing effect of EA using a 90% partial hepatectomy (PHX) rat model. MATERIALS AND METHODS Animals and Operative Procedure Male Sprague-Dawley rats (Nihon Clea, Tokyo, Japan), weighing 300 g to 350 g, were used as experiment animals. They were supplied no food for 12 hours prior to surgery. Under ether anesthesia, the liver was resected by the method of Kubota et al.1 The median lobe and left lateral lobe were removed for 70% PHX rats and the right lateral lobe also was resected for 90% PHX. After surgery, the animals were allowed food ad lib. For 2 operative days after surgery, the animals were given 20% glucose instead of water.
Administration of BrdU and EA On the days 0, 1, 3, 5, or 7 after PHX, 100 mg/kg of BrdU (Takeda Chemical Industries, Ltd., Osaka, Japan) was administered intraabdominally. Laparotomy was performed 1 hour after administration; blood samples were taken from the inferior vena cava and the remnant liver was removed for investigation. Immediately after 90% PHX, 24 mg/kg of ethanolamine (Sigma-Aldrich Japan, Tokyo, Japan) was administered intra-abdominally every 24 hours thereafter (EA group; n ⫽ 6). For the control group (n ⫽ 6) and 70% PHX group (n ⫽ 6), the rats were given the same amount of saline intra-abdominally.
Postoperative Evaluation Blood Tests. Aspartate aminotransferase (AST), total bilirubin (T-Bil), bile acid (BA), and hepaplastin test (HPT) were measured. Weight of remnant liver: The weight of resected remnant liver was measured and calculated in terms of 300 g body weight for evaluation. Regeneration rate: The weight of remnant liver on day 7 was divided by the liver weight estimated prior to surgery. Increased Liver Weight. The percentage of increased liver weight per day over the liver weight measured previously was calculated. Labeling index: Frozen specimens were prepared from the resected liver for immunohistochemical staining using an antiBrdU monoclonal antibody (Sigma-Aldrich Japan, Tokyo, Japan). An image analyzer (SP-500F; Olympus Co., Ltd, Tokyo, Japan), detected the areas of BrdU-positive (the nuclei were stained dark brown) and-negative hepatocytes , which were measured. The rate of BrdU-positive hepatocytes was calculated as a labeling index (LI) for assessment of liver regeneration.
Statistical Analysis Mann-Whitney U test was used for the differences of each value among groups. A P value less than .05 was considered statistically significant.
RESULTS Blood Examination
In the 70% PHX group, AST, T-Bil, and BA increased and HPT decreased slightly on day 1 after surgery, and thereafter they were gradually improved. In contrast among the 90% PHX groups, the AST increased excessively on the day 1, however, it rapidly improved on day 3. T-Bil and BA also increased on day 1 and gradually improved from day 3. In addition, HPT decreased to approximately 10% on day 1 and improved up to approximately 40% on day 3, however, thereafter it was gradually decreasing from day 5. No significant differences could be noted between the EA group and the controls (Fig 1). From the Department of Surgery, Tokyo Medical University, and the Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, Tokyo, Japan. Address reprint requests to Dr A. Mimuro, Department of Surgery, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-Ku, Tokyo 160-0023, Japan. E-mail: mimuro@tokyo-med. ac.jp
0041-1345/02/$–see front matter PII S0041-1345(02)03368-7
© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710
2664
Transplantation Proceedings, 34, 2664 –2667 (2002)
EFFECT OF ETHANOLAMINE ON LIVER REGENERATION
2665
Fig 1. AST (A), BA (B), T-Bil (C), and HPT (D). Graphs of biochemical test data after PHX are shown. Although liver dysfunction in 70% PHX rat (䊐) was slight, a high degree of liver dysfunction was manifested in 90% PHX rat, yet it was quickly ameliorated. No significant differences were noted between the control group (‚) and the EA group (E).
Postoperative Change of Remnant Liver
In the 70% PHX group, the weight of the remnant liver increased steadily from day 1; the regeneration rate was 74% on day 7. On days 1, 3, 5, and 7, the increases in liver weight were 52, 19, 7, and 1% per day, respectively, which showed a decrease from the peak rate on day 1. In the 90% PHX group, the increased liver weight indicated a peak on day 3 at 62% per day in the control group and 92% per day in the EA group. On day 7, however, the rate increased again by 28% per day in the control group and 22% per day in the EA group. The regeneration rate was 64% in the control group and 62% in the EA group (Table 1).
It was similar to the pattern with the control group. There was a statistically significant difference (P ⫽ .0039) for LI between the control group and the EA group on days 1 and 3, respectively (Fig 2 and Fig 3). DISCUSSION
EA is a low molecular nutritious factor derived from the small intestine and used in the synthesis of phospholipids.9 EA has been reported to enhance the proliferation of Table 1. Weight of Remnant Liver, Increased Rate of Liver Weight, and Regeneration Rate Weight of Remnant Liver (g:Mean ⫾ SD)
LI
In the 70% PHX group, LI reached the peak at a median value of 28.4% on day 1 and decreased thereafter. On day 7, it decreased to the same value as immediately after resection. In the control group, LI reached a peak at a median value of 5.1% on day 3 and decreased thereafter. On day 7, it decreased to a median value of 1.6%, which was almost equivalent to the value immediately after resection. On the contrary, in the EA group, LI reached the peak at a median value of 30.7% on day 3 and decreased thereafter. On day 7, it decreased to a median value of 1.6%, which was almost equivalent to the value immediately after resection.
Day 0
Day 1
Day 3
Day 5
Day 7
70% PHX 3.8 ⫾ 0.8 5.8 ⫾ 0.5 7.9 ⫾ 0.6 8.9 ⫾ 0.7 9.1 ⫾ 0.4 Control group 1.2 ⫾ 0.1 1.7 ⫾ 0.3 3.7 ⫾ 0.7 4.8 ⫾ 0.8 7.6 ⫾ 1.5 EA group 1.2 ⫾ 0.1 1.6 ⫾ 0.2 4.5 ⫾ 0.8 5.1 ⫾ 1.0 7.4 ⫾ 1.4 Increased Rate of Liver Weight (%/d)
70% PHX Control group EA group
Regeneration Rate
Day 1
Day 3
Day 5
Day 7
(%)
26 21 17
19 62 92
7 15 7
1 28 22
74 64 62
2666
MIMURO, AOKI, TSUCHIDA ET AL
Fig 2. A graph of LI is shown. In 70% PHX rat (u), the value peaked on day 1 and decreased subsequently. In 90% PHX rat, the value peaked on day 3. Significant differences between the EA group (■) and the control group (䊐) on days 1 and 3 were noted (*P ⫽ .0039).
initially isolated hepatocytes in the rat increasing DNA synthesis in the 70% PHX rat.10 We investigated the enhancing effect of EA on liver regeneration in a 90% PHX rat, which is a model of hepatic insufficiency. In the 70% PHX rat, increased liver weight and LI were observed on day 1, resulting in the vigorous liver regeneration. Thereafter, both values gradually decreased returning on day 7 to the same level as before resection. Thus liver regeneration seems to almost be complete at 7 days after resection in 70% PHX rats. In 90% PHX rats, on the other hand, the increase in liver weight was slight and the LI was also low on day 1. By day 3, LI showed a maximum value and the liver weight increased. Thereafter LI decreased, however, liver weight increased again on day 7, findings that were similar between the EA group and the control group. From these results, it is concluded that: (1) liver regeneration is delayed in the 90% PHX group; (2) in the course of liver regeneration, the period of DNA synthesis (S phase) terminates around 7 days after PHX regardless of the resected volumes, and (3) in the 90% PHX group, there can be a time lag in the course of regeneration after the S phase. Causes of the delay in liver regeneration11 have been reported to be decreased protein synthesis,1 impaired remnant liver or hepatic metabolism by endogenous endotoxin,12,13 decreased reticuloendothelial system function, and decreased glycogenolysis.14 After administration of EA to the 90% PHX rat, LI showed a significant difference from the control at days 1 and 3. The LI median value (18.0%) on day 1 suggested that DNA was being vigorously synthesized in the EA group. Thus EA administration seems to promote or enhance DNA synthesis. No significant differences were observed in liver weight or in blood data, however, for two reasons: (1) EA may enhance the progression of DNA synthesis from the G0 to the S phase, or (2) operative stress or unidentified substrates in the 90% PHX host may disturb liver regeneration. Even if there is an increase in S phase
Fig 3. Control (A) and EA (B). Microscopic images of hepatic tissue (⫻40) showing immunohistological staining by anti-BrdU monoclonal antibody on day 3.
EFFECT OF ETHANOLAMINE ON LIVER REGENERATION
hepatocytes after EA administration, unless there is progression to late G2 and M phases, mature hepatocytes do not proliferate. In other words, there will be no significant differences in remnant liver weight or in blood data. In conclusion, EA seems to promote or enhance DNA synthesis during the early stage after liver surgery even in environments unfavorable for liver regeneration, such as 90% PHX. Since EA is a low molecular substance present in the human body and its safety has been established, it may be suitable as an enhancer for hepatic regenerative processes. REFERENCES 1. Kubota T, Takabe K, Meng Y, et al: J Hep Bil Pancr Surg 4:398, 1997 2. Kawasaki S, Makuuchi M, Matsunami H, et al: Transplant Proc 27:1170, 1995
2667 3. Chen SC, Hewitt WR, Watanabe FD, et al: Int J Artif Organs 19:664, 1996 4. Kamohara Y, Rozga J, Demetriou A: J Hep Bil Pancr Surg 5:273, 1998 5. Kamohara Y, Fujioka H, Eguchi S, et al: Artif Organs 24:265, 2000 6. Rozga J, Podesta L, LePage E, et al: Ann Surg 219:538, 1994 7. Bosse B, Smith MD, Gerlach J, et al: Langenbecks Arch Surg 384:588, 1999 8. Miyashita T, Enosawa S, Suzuki S, et al: Transplant Proc 32:2355, 2000 9. Sundler R, Akesson B: J Biol Chem 250:3359, 1975 10. Sasaki H, Kume H, Nemoto A, et al: Proc Natl Acad Sci 94:7320, 1997 11. Zieve L, Anderson W, Lindblad S, et al: J Lab Clin Med 105:331, 1985 12. Kawasaki Y, Miyara K, Isa T, et al: Jap J Gastroenterol Surg 22:1778, 1989 13. Ando K, Miyazaki M, Ito H, et al: Jap J Gastroenterol Surg 27:1753, 1994 14. Gaub J, Iversen J: Hepatology 4:902, 1984