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reperfusion in rats
Gadolinium pretreatment decreases survival and impairs liver regeneration after partial hepatectomy under ischemia/reperfusion in rats Masato Watanabe, MD, Kazuo Chijiiwa, MD, Nobuhisa Kameoka, MD, Koji Yamaguchi, MD, Syoji Kuroki, MD, and Masao Tanaka, MD, Fukuoka, Japan
Background. Modulation of Kupffer cell functions by treatment with gadolinium chloride protects the liver against reperfusion injury. However, its effect on liver regeneration after hepatectomy under ischemia/reperfusion has not been studied. Using a common clinical ischemia/reperfusion technique, we examined the effect of gadolinium on liver regeneration after hepatectomy in rats. Methods. After an initial 15-minute ischemia and 15-minute reperfusion, 70% hepatectomy was performed during the second 15-minute ischemia period in gadolinium-pretreated (gadolinium group) and saline solution—pretreated (control group) rats. The 24-hour survival rate, relative liver weight, DNA synthesis rate, and hepatic adenosine triphosphate level were examined immediately after hepatectomy and on postoperative days (PODs) 1, 2, 3, and 7. Serum levels of total bilirubin, glutamic pyruvic transaminase, and endotoxin were also measured. Results. The 24-hour survival rate was significantly lower in the gadolinium group (67%) than in the control group (100%). On POD 1, the relative liver weight and DNA synthesis rate were significantly lower in the gadolinium group than in the control group. On POD 1, serum total bilirubin and endotoxin levels were significantly higher in the gadolinium group than in the control group. Immediately after hepatectomy, the hepatic adenosine triphosphate level was significantly lower in the gadolinium group than in the control group. Conclusions. Under ischemia/reperfusion, gadolinium pretreatment impairs liver regeneration and energy status after hepatectomy and decreases postoperative survival. (Surgery 2000;127:456-63.) From the Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
LIVER REGENERATION AFTER HEPATECTOMY is mediated by growth factors and cytokines mainly released from the nonparenchymal cells such as Kupffer cells. Gadolinium chloride has been reported to inhibit phagocytosis of the Kupffer cells selectively by interfering with calcium-dependent cell surface interactions.1,2 Most studies have focused on the effect of gadolinium on either hepatic ischemia/reperfusion liver injury3,4 or liver regenSupported by a Grant-in-Aid (No. 09671318 for K. Chijiiwa) from the Ministry of Education, Science, and Culture, Japan. Accepted for publication November 19, 1999. Reprint requests: Kazuo Chijiiwa, MD, PhD, FACS, Chief, Hepatobiliary and Pancreatic Surgery, Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Copyright © 2000 by Mosby, Inc. 0039-6060/2000/$12.00 + 0 doi:10.1067/msy.2000.104744
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eration after hepatectomy without any ischemic procedure,5 and the findings have suggested improvement of reperfusion injury and regeneration of the liver, respectively. In a lipopolysaccharide (LPS)-responsive mouse strain, however, suppression of the Kupffer cells by gadolinium impaired the regeneration of the liver after hepatectomy.6 The effect of gadolinium treatment on liver regeneration after hepatectomy under ischemia/reperfusion has not been examined. Clinically, hepatic resection is usually performed under the total or partial exclusion of the blood inflow to the liver to control bleeding during parenchymal dissection. Liver regeneration is disturbed after hepatectomy under ischemia/reperfusion by Pringle’s maneuver in rats.7 If gadolinium pretreatment improves ischemia/reperfusion injury,3,4 the liver regeneration rate after hepatectomy under the ischemia/reperfusion technique should be improved to a similar extent to that after
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B
Fig 1. A, Changes in relative liver weight of the remnant liver after 70% hepatectomy. The relative liver weight (grams liver/100 g body weight) in the gadolinium group (Gd) was lower than that in the control group on POD 1. All values are expressed as means ± SEM of 6 rats. B, The hepatic DNA synthesis rate after 70% hepatectomy. The DNA synthesis rate in the gadolinium group was lower than that in the control group on POD 1. Values are means ± SEM of 6 rats in each group.
hepatectomy without the ischemia/reperfusion procedure. We hypothesized that modulation of Kupffer cell function by gadolinium treatment would have favorable effects on liver regeneration after hepatectomy with Pringle’s maneuver. Because the liver is a vital organ, it must maintain metabolic and other functions throughout the regenerative process. To keep organ viability, the hepatic level of adenosine triphosphate (ATP) is important. ATP is necessary for various vital functions of the liver, including regeneration. In the present study, we examined the effects of gadolinium pretreatment on survival rate, liver regeneration rate, and concentration of ATP in the liver after hepatectomy under ischemia/reperfusion. In addition, the concentrations of the hyaluronic acid and endotoxin in the serum were measured. MATERIAL AND METHODS Animals. Male 8-week-old Wistar rats (Kyudo Co, Ltd, Fukuoka, Japan), weighing 240 to 310 g were
used as the experimental animals. The rats were kept in a temperature-controlled room with a 12hour light-dark cycle and acclimated for at least 7 days before use. All animals had free access to water and standard laboratory diet (Oriental Yeast Co, Ltd, Tokyo, Japan). Before hepatectomy, diet was withheld overnight, but the free access to water was continued. The rats were randomly divided into 2 groups: gadolinium and control groups. In the gadolinium group, gadolinium (7 mg/kg body weight) dissolved in saline solution (3.5 mg/mL) was intravenously administered through the penile vein at 48 and 24 hours before hepatectomy. The dose and timing of the gadolinium treatment was generally used and similar to those in a previous study,3,6,8 which showed a 47% to 67% reduction in phagocytic activity of Kupffer cells.3,8 In the control group, the same volume of physiologic saline solution without gadolinium was administered in the same manner. This experiment was reviewed by the Committee on the Ethics of Animal Experiments in
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A
B
Fig 2. The liver regeneration rate assessed by the relative liver weight of the remnant liver (A) and the hepatic DNA synthesis rate (B) on POD 1 after hepatectomy. In both the gadolinium-treated (Gd) and nontreated rats, the ischemia/reperfusion (I/R) procedure itself decreased the relative liver weight and DNA synthesis rate. The gadolinium treatment itself also lowered DNA synthesis rate on POD 1 after hepatectomy. Values are means ± SEM of 6 rats in each group.
Graduate School of Medical Sciences, Kyushu University, and performed under the control of the Guideline for Animal Experiment in Kyushu University and The Law (No. 105) and Notification (No. 6) of the Japanese Government. Surgical procedure. All surgical procedures were performed in the morning (9-12 AM) under light ether anesthesia. After midline laparotomy, the portal vein, hepatic artery, and bile duct were clamped with a vascular clip (Mizuho Ikakogyo Co, Ltd, Tokyo, Japan) for 15 minutes and then reperfused for 15 minutes. During a second 15 minutes of ischemia, left lateral and median lobes were excised (70% hepatectomy) according to the procedure of Higgins and Anderson.9 This procedure mimicked hepatectomy in clinical fields, where Pringle’s maneuver (15 minutes of ischemia and 5
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minutes of reperfusion) is repeatedly performed to control bleeding during parenchymal dissection. All the animals received 10% glucose solution and standard diet ad libitum after the operation. All rats in the control group survived although some of the gadolinium-treated group died shortly after hepatectomy. For the survival study, 50 rats (control group, 20 rats; gadolinium group, 30 rats) were used to examine the 24-hour survival rate. In an analytic study, a total of 74 rats (control group, 29 rats; gadolinium group, 45 rats) were used. The animals that survived hepatectomy (day 0) and survivors on days 1, 2, 3, and 7 after hepatectomy were subjected to the analyses described later. To determine the DNA synthesis rate of the liver, tritiated thymidine (Amersham Life Science, Little Chalfont, England; specific activity, 25 Ci/mmol) was injected intraperitoneally at a dose of 0.4 µCi/g of body weight 60 minutes before the animals were killed. The abdomen was opened, and a small portion of the liver (right lateral lobe) that weighed less than 100 mg was immediately excised, frozen in liquid nitrogen, and stored at –80°C for analysis of hepatic ATP levels. The remaining liver was weighed, and a portion was used to determine the DNA synthesis rate by the incorporation rate of tritiated thymidine into DNA. Arterial blood was withdrawn simultaneously, and the serum was used to determine concentrations of endotoxin and hyaluronic acid and for liver function tests. Relative liver weight. The remaining liver was weighed, and the relative liver weight was calculated as the actual liver weight/100 g body weight. Hepatic DNA synthesis rate. The DNA fraction of the liver tissue was extracted by the method of Schneider.10,11 Briefly, the tissue was homogenized in 4 volumes of cold 1.15% potassium chloride, and 2.5 mL of ice-cold 10% trichloroacetic acid (TCA) solution was added. After vigorous stirring, the mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded, and the sediment was washed successively with 10% TCA solution, 5 mL of 76% ethanol, 5 mL of 95% ethanol, and 10 mL of ethanol/ether (3/1, vol/vol). The sediment was suspended in 3% potassium hydroxide solution. The suspension was incubated at 37°C for 60 minutes, neutralized, and centrifuged. The supernatant was discarded, and the sediment was washed with 5 mL of 5% TCA and heated at 90°C for 15 minutes, with occasional shaking. After cooling and centrifugation at 3000 rpm for 10 minutes, 1 mL of the supernatant was subjected to a liquid scintillation counter (Alga Liquid Scintillation system, LSC-703, Tokyo, Japan) after the addition of 10 mL of Ready Protein
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Fig 3. The concentration of ATP in the liver tissue before (before hepatectomy and ischemia/reperfusion) and after (just after hepatectomy under ischemia/reperfusion) 70% hepatectomy. In both groups, the hepatic ATP level was decreased just after hepatectomy under ischemia/reperfusion. The hepatic ATP level on POD 0, just after hepatectomy, was lower in the gadolinium group (Gd) than in the control group. All values are means ± SEM of 6 rats.
(Beckman Co, Ltd, Fullerton, Calif). The DNA concentration in the supernatant was also determined. Three milliliters of diphenylamine reagent (0.75 g diphenylamine in 50 mL glacial acetic acid and 1.14 mL concentrated H2SO4) was added to 2 mL of the supernatant, and the mixture was heated at 100°C for 10 minutes. After gently cooling to room temperature, absorbance at 600 nm was measured. Calf thymus DNA (Sigma Co, Ltd, St Louis, Mo) was used as a standard. The incorporation of [3H]-thymidine into DNA was calculated as disintegrations per minute per microgram DNA. Liver function tests. Serum levels of total bilirubin and glutamic pyruvic transaminase (GPT) were analyzed with the use of an automated multichannel analyzer (736-40 Automatic Analyzer; Hitachi, Ltd, Tokyo, Japan). The serum hyaluronic acid level has been used as an index of endothelial damage of the liver after cold preservation/reperfusion12 and graft viability after liver transplantation.13 Serum concentrations of hyaluronic acid were measured with the use of the sandwich-binding protein assay method previously reported by us.14 Adenine nucleotide concentrations in the liver. The concentrations of ATP in the liver were measured as previously reported by our laboratory.15 Briefly, the frozen liver tissues were lyophilized overnight, and samples that weighed 10 to 15 mg each were homogenized in 1 mL of 0.5 N perchloric acid. The homogenate was centrifuged for 5 minutes at 10,000 × g at 4°C. After addition of 0.05 mL of 3 mmol potassium hydroxide to 0.5 mL of supernatant, the samples were recentrifuged for 10 minutes under the same condition. The supernatants were run through a
microfilter (0.22 µm pore size; Japan Millipore Co, Ltd, Tokyo, Japan) and 3 µL per sample of the filtrate was used for high performance liquid chromatography on an anion exchange column, DEAE-2SW (4.6 × 250 mm; Tosoh Co, Ltd, Tokyo, Japan), equilibrated with 0.2 mmol phosphate buffer, pH 6.0, to determine concentrations of ATP. The level of ATP was expressed as micromole per gram liver tissue. Standard ATP was purchased from Sigma Co, Ltd. Endotoxin measurement. Sterile endotoxin-free disposable ware was used to prevent contamination of blood samples. Arterial blood was aspirated aseptically from the abdominal aorta. The concentration of endotoxin was determined by an endotoxin-specific assay with the use of recombined limulus coagulation enzymes (Seikagakukogyo Co, Tokyo, Japan). Statistical analysis. Significance of survival rate was assessed with the Log-rank test. All data are expressed as means ± SEM. The statistical differences between groups were determined by analysis of variance using the Mann-Whitney U test. A probability value less than .05 was considered significant. RESULTS Survival rate after hepatectomy. All 20 animals in the control group survived for 24 hours after hepatectomy under the ischemia/reperfusion, although 10 of 30 rats in the gadolinium group died. The 24-hour survival rate in the gadolinium group was significantly lower than that in the control group (67% vs 100%; P < .01). Because the gadolinium pretreated rats showed cyanosis and histopathologic study at necropsy showed pulmonary edema, the primary reasons for death seemed to be pulmonary failure.
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Fig 4. A, Changes in serum endotoxin concentrations after 70% hepatectomy. The endotoxin level was higher in the gadolinium group (Gd) than in the control group on POD 1. B, Serum endotoxin concentrations on POD 1 after hepatectomy in both the gadolinium-treated and nontreated rats with and without ischemia/reperfusion (I/R). Hepatectomy under ischemia/reperfusion resulted in a higher endotoxin level than that without ischemia/reperfusion in both the gadolinium-treated and gadoliniumnontreated rats. All values are means ± SEM of 6 rats.
Body weight and liver function tests. The body weight was not significantly different between the 2 groups throughout the study (Table I). The serum level of total bilirubin was elevated to its maximal value on POD 2 in the control group and on POD 1 in the gadolinium group. Mean serum total bilirubin level was significantly higher in the gadolinium group than in the control group on POD 1 (Table I). Mean serum glutamic-pyruvic transaminase levels were higher in the gadolinium group than in the control group on PODs 0 and 1, but the differences were not significant (Table I). The values declined thereafter in both groups. Serum concentrations of hyaluronic acid in the 2 groups were not significantly different (Table I). Liver regeneration. Liver regeneration, assessed by the relative liver weight and DNA synthesis rate,
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was significantly impaired in the gadolinium group. The relative liver weight in the gadolinium group was significantly lower than that in the control group on POD 1 (1.376% ± 0.072% vs 1.685% ± 0.028%; P < .02; Fig l, A). Liver weight was restored slowly in the gadolinium group. The DNA synthesis rate was significantly lower in the gadolinium group than in the control group on POD 1 (9.297 ± 1.508 dpm/µg of DNA vs 91.357 ± 45.346 dpm/µg of DNA; P < .02) but became higher on POD 2 (129.302 ± 20.982 dpm/µg of DNA vs 67.786 ± 5.716 dpm/µg of DNA; P < .02; Fig l, B). DNA synthesis reached its peak by POD 3 in both groups and then declined. No significant difference in DNA synthesis rate was observed between the 2 groups on PODs 3 and 7. In a separate experiment, the effect of ischemia/reperfusion on liver regeneration was examined on POD 1 in both groups (Fig 2). When liver regeneration after hepatectomy was compared by the presence or absence of ischemia/reperfusion in gadolinium-treated rats, the relative liver weight and DNA synthesis rate without ischemia/reperfusion were significantly higher than those with ischemia/reperfusion on POD 1 (relative liver weight, 1.774% ± 0.066% vs 1.376% ± 0.072%; P < .02; DNA synthesis rate, 127.057 ± 25.582 dpm/µg of DNA vs 9.297 ± 1.508 dpm/µg of DNA; P < .01). In gadolinium-nontreated rats, the relative liver weight and DNA synthesis rate without ischemia/reperfusion were also significantly higher than those in a group with ischemia/reperfusion (relative liver weight, 1.930% ± 0.101% vs 1.685% ± 0.028%; P < .05; DNA synthesis rate, 323.568 ± 38.539 dpm/µg of DNA vs 91.537 ± 45.346 dpm/µg of DNA; P < .02). The ischemia/reperfusion procedure itself significantly decreased the relative liver weight and DNA synthesis rate. In rats without ischemia/reperfusion, the gadolinium pretreatment significantly lowered DNA synthesis rate on POD 1 after hepatectomy (127.057 ± 25.582 dpm/µg of DNA vs 323.568 ± 38.539 dpm/µg of DNA; P < .01; Fig 2, B). Hepatic energy status. The concentrations of ATP in the liver before ischemia/reperfusion and hepatectomy procedures were statistically similar in the gadolinium and control groups (8.848 ± 0.342 µmol/g vs 8.538 ± 0.745 µmol/g of dry liver; Fig 3). Just after hepatectomy under ischemia/reperfusion (POD 0), the hepatic ATP level was significantly lower than that before hepatectomy and ischemia/reperfusion in both groups (gadolinium group, P < .01; control group, P < .05), but the decline was more profound in the gadolinium group. Immediately after hepatectomy on POD 0, the concentration of ATP in the liver was signifi-
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Surgery Volume 127, Number 4 Table I. Body weight and liver function tests
Body weight (g) Control Gadolinium Total bilirubin (mg/dL) Control Gadolinium Glutamic-pyruvic transaminase (IU/L) Control Gadolinium Hyaluronic acid (ng/mL) Control Gadolinium
0
1
275 ± 5 269 ± 8
270 ± 8 273 ± 7
0.33 ± 0.10 0.40 ± 0.16
Days after hepatectomy 2
3
7
261 ± 4 251 ± 5
279 ± 6 267 ± 8
300 ± 9 293 ± 5
0.33 ± 0.12 1.07 ± 0.36*
0.63 ± 0.22 1.05 ± 0.52
0.30 ± 0.10 0.15 ± 0.04
0.16 ± 0.09 0.06 ± 0.02
802 ± 135 999 ± 239
209 ± 47 465 ± 226
157 ± 42 89 ± 15
91 ± 15 55 ± 4
76 ± 13 54 ± 1
58 ± 24 60 ± 16
209 ± 53 194 ± 60
60 ± 16 119 ± 36
79 ± 35 115 ± 38
38 ± 22 19 ± 4
Values are means ± SEM (n = 6). *P
< .05, vs control.
cantly lower in the gadolinium group than in the control group (4.149 ± 0.416 µmol/g vs 6.607 ± 0.323 µmol/g of dry liver; P < .01; Fig 3). The hepatic ATP level was statistically similar in both groups on POD 1, and the value became essentially the same as that before ischemia/reperfusion in each group. Serum endotoxin concentration. The serum endotoxin level reached its maximum on POD 1 in both groups, and the value was significantly higher in the gadolinium group than in the control group on POD 1 (315.12 ± 33.99 pg/mL vs 214.56 ± 13.21 pg/mL; P < .05; Fig 4, A). The serum endotoxin level decreased on PODs 2 and 3, and no further significant differences were found between 2 groups (Fig 4, A). The serum endotoxin levels on POD 1 after hepatectomy with and without ischemia/reperfusion were determined in another experiment. In gadolinium-treated and gadolinium-nontreated rats, hepatectomy with ischemia/reperfusion resulted in a significantly higher endotoxin level on POD 1 than that without ischemia/reperfusion (gadolinium-treated, 151.84 ± 40.99 pg/mL vs 315.12 ± 33.99 pg/mL; P < .02; gadolinium-nontreated, 20.60 ± 6.35 pg/mL vs 214.56 ± 13.21 pg/mL; P < .01; Fig 4, B). Even without ischemia/reperfusion, the serum endotoxin level on POD 1 after hepatectomy was significantly (P < .02) increased by gadolinium treatment (Fig 4, B). DISCUSSION Liver regeneration assessed by relative liver weight and DNA synthesis rate after hepatectomy under ischemia/reperfusion was significantly impaired by gadolinium pretreatment. On POD 1 after hepatectomy, the serum concentrations of
total bilirubin and endotoxin were significantly higher in the gadolinium group than in the control group. The hepatic ATP level just after hepatectomy (POD 0) under ischemia/reperfusion was significantly lower in the gadolinium group than in the control group. The gadolinium pretreatment significantly worsened the 24-hour survival rate. These findings indicate that gadolinium pretreatment has disadvantages for regeneration and energy status of the liver after hepatectomy under ischemia/reperfusion, thus leading to a higher mortality rate. Gadolinium selectively inhibits Kupffer cell phagocytosis by interfering with calcium-dependent cell surface interactions.1,2 Gadolinium decreases phagocytic activity of Kupffer cells.3,8 Gadolinium reduces hepatic bacterial clearance but does not decrease the activity of bacterial killing.16 The use of gadolinium has been shown to have beneficial effects on liver injury and liver regeneration. Treatment with gadolinium attenuated endotoxin-induced liver injury after partial hepatectomy and improved the 24-hour survival rate.17 Pretreatment with gadolinium increased liver regeneration that was assessed by the incorporation rate of [3H]-thymidine into DNA and mitotic activity of the hepatocytes.5 In contrast, gadolinium pretreatment in an LPS-responsive mouse has been reported to reduce the proliferating cell nuclear antigen labeling index of hepatocytes and impair liver regeneration after hepatectomy.6 However, these studies have examined animals after hepatectomy without any ischemia/reperfusion procedures. In our experiment, the 24-hour survival rate after hepatectomy under ischemia/reperfusion was significantly lower in the gadolinium group than in the control group. The pretreatment with gadolinium deteriorated liver function and liver regeneration after hepatectomy
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under ischemia/reperfusion. This is the first report that shows an inhibitory effect of the gadolinium pretreatment on liver regeneration after partial hepatectomy under ischemia/reperfusion. Because hepatectomy is usually carried out under ischemia/reperfusion in clinical practice, a similar maneuver was used in the current study. There are 2 aspects of ischemia/reperfusion injury of the liver. One is the injury caused by ischemia and portal congestion during the interruption of hepatic blood inflow, and the other is the reperfusion injury caused by pooled portal blood in the ischemic liver.18,19 The ischemia/reperfusion procedure itself has been shown to significantly reduce the DNA synthesis rate and recovery of liver weight 24 hours after partial hepatectomy.7 Van Deventer et al20 suggested that endotoxins in the portal blood that were translocated from the intestine are one of the factors inhibiting liver regeneration. Although we did not measure the endotoxin level in the portal vein, we did observe an increase in its level in systemic blood, which supports their finding. The present study confirms that the ischemia/reperfusion procedure itself significantly decreases liver regeneration (relative liver weight and DNA synthesis rate) 24 hours after hepatectomy. Our pretreatment procedure with gadolinium (timing and dosage) was essentially the same as those used in previous reports,3,6,8,21 where the reduction in Kupffer cell phagocytosis was noted in rats and mice that had been intravenously administrated 7 mg/kg of gadolinium for 2 days. Under these conditions, phagocytic activity and plasma tumor necrosis factor-α (TNFα) levels were decreased after ischemia/reperfusion in rats,3 and serum levels of cytokine-induced neutrophil chemoattractant were decreased after ischemia/reperfusion in rats.22 Moreover, liver regeneration after hepatectomy has been reported to be not affected by endotoxin in gadolinium-pretreated rats17 or to be impaired in LPS-responsive strain mice.6 However, hepatectomy was performed without ischemia/reperfusion in these reports. Our results indicate that the treatment with gadolinium itself increased the serum endotoxin level and worsened the DNA synthesis rate on POD 1 after hepatectomy without ischemia/reperfusion. As a result, liver regeneration was significantly inhibited after hepatectomy under ischemia/reperfusion in the gadolinium group. The Kupffer cells, which are exposed to portal vein circulation, remove harmful substances from the portal blood, such as intestinal endotoxins and bacteria. When the portal endotoxin level exceeds the phagocytic abilities of the Kupffer cells, endotoxin spills out from the Kupffer cells and directly injures the hepatocytes.23 In our present study, the serum endotoxin level on POD 1 in the gadolinium
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group was significantly higher than that in the control group. It is likely that the Kupffer cells pretreated with gadolinium could not remove the excessive endotoxins translocated from the intestines after hepatectomy under ischemia/reperfusion. Even without ischemia/reperfusion, the serum endotoxin levels were significantly higher in the gadoliniumtreated rats on POD 1 after hepatectomy than in the gadolinium-nontreated rats. The primary reason for high mortality rates in the gadolinium group was respiratory failure, which is consistent with the report that endotoxins induce acute respiratory failure.24 Direct organ damage by gadolinium may be another possible explanation for poor survival in the gadolinium group. Spencer et al25 have shown that gadolinium treatment causes mineral deposition in capillary beds (particularly lung and kidney) and injuries to the liver and spleen. Thus a reduction in the capacity of Kupffer cells to remove endotoxin or direct organ damage by gadolinium may explain the high mortality rate, hyperbilirubinemia, and retarded liver regeneration in the gadolinium group in our present study. Another possible factor is the release of proinflammatory cytokines from Kupffer cells because cytokines such as TNF-α and IL-6 are important for liver regeneration.26 TNF-α and IL-6 regulate liver regeneration through the induction of transcription factors such as Stat3 and NF-κΒ.27 Effects of gadolinium on serum TNF-α and IL-6 levels are controversial. Gadolinium causes sustained overexpression of TNF messenger RNA and transient overexpression of circulating TNF protein after hepatectomy; both TNF-inducible cytokines (IL-1, IL-6) are also relatively overexpressed.28 On the other hand, it has been reported that serum TNF-α and IL-6 levels are reduced in the gadolinium-pretreated rats after hepatectomy.3,6 The inhibition of hepatic regeneration in our gadolinium group might have been caused by the depletion of cytokines in the Kupffer cells by the gadolinium treatment. Measurement of proinflammatory cytokines, such as TNF-α and IL-6, is necessary to elucidate the exact mechanism. Extensive hepatectomy induces acute metabolic stress on the remaining liver, causing a marked decrease in energy levels.29 The energy balance is maintained by compensatory enhancement of mitochondrial phosphorylative activity when the metabolic load is maximal on the remnant liver.30 The production of ATP is necessary for both liver regeneration and organ viability, and an ATP deficit causes organ failure. After cold preservation for a short time, mitochondrial ATPase activity of the reperfused liver, a key enzyme of ATP synthesis, has been reported to be decreased by the treat-
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Surgery Volume 127, Number 4 ment with gadolinium.31 In the present study, the hepatic ATP level in the gadolinium group just after hepatectomy was significantly lower than that in the control group. The reason may be ascribed to the decreased mitochondrial ATPase activity. The lower concentration of ATP in the gadolinium group may explain the high mortality rate within 24 hours after hepatectomy in the gadolinium group, because hepatic ATP levels before hepatectomy and the ischemia/reperfusion procedures were not different between the 2 groups. CONCLUSION Pretreatment with gadolinium did not improve the hepatic ATP level and liver regeneration after hepatectomy under ischemia/reperfusion and, in fact, led to a high mortality rate.
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The authors thank Ms Keiko Nishimura for her technical assistance. REFERENCES 1. Lazar G. The reticuloendothelial-blocking effect of rare earth metals in rats. J Reticuloendothel Soc 1973;13:231-7. 2. Husztik E, Lazar G, Parducz A. Electron microscopic study of Kupffer-cell phagocytosis blockade induced by gadolinium chloride. Br J Exp Pathol 1980;61:624-30. 3. Suzuki S, Nakamura S, Sakaguchi T, Ochiai H, Konno H, Baba S, et al. Alteration of reticuloendothelial phagocytic function and tumor necrosis factor-alpha production after total hepatic ischemia. Transplantation 1997;64:821-7. 4. Bremer C, Bradford BU, Hunt KJ, Knecht KT, Connor HD, Mason RP, et al. Role of Kupffer cells in the pathogenesis of hepatic reperfusion injury. Am J Physiol 1994;267:G630-6. 5. Rai RM, Yang SQ, McClain C, Karp CL, Klein AS, Diehl AM. Kupffer cell depletion by gadolinium chloride enhances liver regeneration after partial hepatectomy in rats. Am J Physiol 1996;270:G909-18. 6. Shiratori Y, Hongo S, Hikiba Y, Ohmura K, Nagura T, Okano K, et al. Role of macrophages in regeneration of liver. Dig Dis Sci 1996;41:1939-46. 7. Foschi D, Castoldi L, Lesma A, Musazzi M, Benevento A, Trabucchi E. Effects of ischaemia and reperfusion on liver regeneration in rats. Eur I Surg 1993;159:393-8. 8. Kamei T, Callery MP, Flye MW. Kupffer cell blockade prevents induction of portal venous tolerance in rat cardiac allograft transplantation. J Surg Res 1990;48:393-6. 9. Higgins GM, Anderson RM. Experimental pathology of the liver: I. Restoration of the liver oh the white eat following partial surgical removal. Arch Pathol 1931;12:186-202. 10. Schneider WC. Phosphorus compounds in animal tissues: I. Extraction and estimation of desoxypentose nucleic acid and pentose nucleic acid. J Biol Chem 1945;161:293-303. 11. Schneider WC. Phosphorus compounds in animal tissues: III. A comparison of methods for the estimation of nucleic acids. J Biol Chem 1946;164:747-51. 12. Wang L, Zhao D, Suehiro T, Boros P, Miller CM. Assessment of damage and recovery of sinusoidal endothelial cell function by in vivo hyaluronic acid uptake in coldpreserved and transplanted rat livers. Transplantation 1996;62:1217-21. 13. Suehiro T, Boros P, Emre S, Sheiner P, Guy S, Schwartz ME,
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et al. Assessment of liver allograft function by hyaluronic acid and endothelin levels. J Surg Res 1997;73:123-8. Chijiiwa K, Watanabe M, Hachiya Y, Kameoka N, Tanaka M. Serum hyaluronic acid level reflects volume and ATP levels of the liver after extended hepatectomy with and without preoperative portal vein occlusion. J Surg Res 1997;72:107-11. Chijiiwa K, Kameoka N, Saeki S, Komura M, Yamaguchi K, Kuroki S, et al. Functional contribution of preoperative portal vein occlusion to hepatectomy: with special reference to hepatic energy charge and DNA synthesis after hepatectomy in rats. Arch Surg 1996;131:779-84. Klein A, Zhadkewich M, Margolick J, Winkelstein J, Bulkley G. Quantitative discrimination of hepatic reticuloendothelial clearance and phagocytic killing. J Leukoc Biol 1994;55:248-52. Suzuki S, Nakamura S, Serizawa A, Sakaguchi T, Konno H, Muro H, et al. Role of Kupffer cells and the spleen in modulation of endotoxin-induced liver injury after partial hepatectomy. Hepatology 1996;24:219-25. Nitta N, Yamamoto S, Ozaki N, Morimoto T, Mori K, Yamaoka Y, et al. Is the deterioration of liver viability due to hepatic warm ischemia or reinflow of pooled-portal blood in intermittent portal triad cross-clamping? Res Exp Med 1988;188:341-50. Zhou W, McCollum MO, Levine BA, Olson MS. Inflammation and platelet-activating factor production during hepatic ischemia/reperfusion. Hepatology 1992;16:1236-40. van Deventer SJ, ten Cate JW, Tytgat GN. Intestinal endotoxemia: clinical significance. Gastroenterology 1988;94:825-31. Callery MP, Kamei T, Flye MW. Kupffer cell blockade increases mortality during intra-abdominal sepsis despite improving systemic immunity. Arch Surg 1990;125:36-41. Hisama N, Yamaguchi Y, Ishiko T, Miyanari N, Ichiguchi O, Goto M, et al. Kupffer cell production of cytokine-induced neutrophil chemoattractant following ischemia/reperfusion injury in rats. Hepatology 1996;24:1193-8. Pagani R, Portole, MT, Diaz-Laviada I, Municio AM. Morphological damage induced by Escherichia coli lipopolysaccharide in cultured hepatocytes: localization and binding properties. Br J Exp Pathol 1988;69:537-49. Perkowski SZ, Sloane PJ, Spath JA Jr, Elsasser TH, Fisher JK, Gee MH. TNF-alpha and the pathophysiology of endotoxininduced acute respiratory failure in sheep. J Appl Physiol 1996;80:564-73. Spencer AJ, Wilson SA, Batchelor J, Reid A, Rees J, Harpur E. Gadolinium chloride toxicity in the rat. Toxicol Pathol 1997;25:245-55. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276:60-6. Taub R. Liver regeneration 4: transcriptional control of liver regeneration. FASEB J 1996;10:413-27. Rai RM, Loffreda S, Karp CL, Yang SQ, Lin HZ, Diehl AM. Kupffer cell depletion abolishes induction of interleukin-10 and permits sustained overexpression of tumor necrosis factor alpha messenger RNA in the regenerating rat liver. Hepatology 1997;25:889-95. Kamiyama Y, Ozawa K, Honjo I. Changes in mitochondrial phosphorylative activity and adenylate energy charge of regenerating rabbit liver. J Biochem 1976;80:875-81. Kurokawa T, Nonami T, Sugiyama S, Ozawa T, Takagi H. Effects of long-acting superoxide dismutase on liver metabolism after major hepatic resection in rats with cirrhosis. Eur Surg Res 1991;23:65-72. Shibuya H, Ohkohchi N, Seya K, Satomi S, Mori S. Modulation of mitochondrial ATP synthesis and lipid peroxidation by Kupffer cells in liver grafts. Transplant Proc 1996;28:321-3.
Background. Modulation of Kupffer cell functions by treatment with gadolinium chloride protects the liver against reperfusion injury. However, its effect on liver regeneration after hepatectomy under ischemia/reperfusion has not been studied. Using a common clinical ischemia/reperfusion technique, we examined the effect of gadolinium on liver regeneration after hepatectomy in rats. Methods. After an initial 15-minute ischemia and 15-minute reperfusion, 70% hepatectomy was performed during the second 15-minute ischemia period in gadolinium-pretreated (gadolinium group) and saline solution—pretreated (control group) rats. The 24-hour survival rate, relative liver weight, DNA synthesis rate, and hepatic adenosine triphosphate level were examined immediately after hepatectomy and on postoperative days (PODs) 1, 2, 3, and 7. Serum levels of total bilirubin, glutamic pyruvic transaminase, and endotoxin were also measured. Results. The 24-hour survival rate was significantly lower in the gadolinium group (67%) than in the control group (100%). On POD 1, the relative liver weight and DNA synthesis rate were significantly lower in the gadolinium group than in the control group. On POD 1, serum total bilirubin and endotoxin levels were significantly higher in the gadolinium group than in the control group. Immediately after hepatectomy, the hepatic adenosine triphosphate level was significantly lower in the gadolinium group than in the control group. Conclusions. Under ischemia/reperfusion, gadolinium pretreatment impairs liver regeneration and energy status after hepatectomy and decreases postoperative survival. (Surgery 2000;127:456-63.) From the Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
LIVER REGENERATION AFTER HEPATECTOMY is mediated by growth factors and cytokines mainly released from the nonparenchymal cells such as Kupffer cells. Gadolinium chloride has been reported to inhibit phagocytosis of the Kupffer cells selectively by interfering with calcium-dependent cell surface interactions.1,2 Most studies have focused on the effect of gadolinium on either hepatic ischemia/reperfusion liver injury3,4 or liver regenSupported by a Grant-in-Aid (No. 09671318 for K. Chijiiwa) from the Ministry of Education, Science, and Culture, Japan. Accepted for publication November 19, 1999. Reprint requests: Kazuo Chijiiwa, MD, PhD, FACS, Chief, Hepatobiliary and Pancreatic Surgery, Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Copyright © 2000 by Mosby, Inc. 0039-6060/2000/$12.00 + 0 doi:10.1067/msy.2000.104744
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eration after hepatectomy without any ischemic procedure,5 and the findings have suggested improvement of reperfusion injury and regeneration of the liver, respectively. In a lipopolysaccharide (LPS)-responsive mouse strain, however, suppression of the Kupffer cells by gadolinium impaired the regeneration of the liver after hepatectomy.6 The effect of gadolinium treatment on liver regeneration after hepatectomy under ischemia/reperfusion has not been examined. Clinically, hepatic resection is usually performed under the total or partial exclusion of the blood inflow to the liver to control bleeding during parenchymal dissection. Liver regeneration is disturbed after hepatectomy under ischemia/reperfusion by Pringle’s maneuver in rats.7 If gadolinium pretreatment improves ischemia/reperfusion injury,3,4 the liver regeneration rate after hepatectomy under the ischemia/reperfusion technique should be improved to a similar extent to that after
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A
B
Fig 1. A, Changes in relative liver weight of the remnant liver after 70% hepatectomy. The relative liver weight (grams liver/100 g body weight) in the gadolinium group (Gd) was lower than that in the control group on POD 1. All values are expressed as means ± SEM of 6 rats. B, The hepatic DNA synthesis rate after 70% hepatectomy. The DNA synthesis rate in the gadolinium group was lower than that in the control group on POD 1. Values are means ± SEM of 6 rats in each group.
hepatectomy without the ischemia/reperfusion procedure. We hypothesized that modulation of Kupffer cell function by gadolinium treatment would have favorable effects on liver regeneration after hepatectomy with Pringle’s maneuver. Because the liver is a vital organ, it must maintain metabolic and other functions throughout the regenerative process. To keep organ viability, the hepatic level of adenosine triphosphate (ATP) is important. ATP is necessary for various vital functions of the liver, including regeneration. In the present study, we examined the effects of gadolinium pretreatment on survival rate, liver regeneration rate, and concentration of ATP in the liver after hepatectomy under ischemia/reperfusion. In addition, the concentrations of the hyaluronic acid and endotoxin in the serum were measured. MATERIAL AND METHODS Animals. Male 8-week-old Wistar rats (Kyudo Co, Ltd, Fukuoka, Japan), weighing 240 to 310 g were
used as the experimental animals. The rats were kept in a temperature-controlled room with a 12hour light-dark cycle and acclimated for at least 7 days before use. All animals had free access to water and standard laboratory diet (Oriental Yeast Co, Ltd, Tokyo, Japan). Before hepatectomy, diet was withheld overnight, but the free access to water was continued. The rats were randomly divided into 2 groups: gadolinium and control groups. In the gadolinium group, gadolinium (7 mg/kg body weight) dissolved in saline solution (3.5 mg/mL) was intravenously administered through the penile vein at 48 and 24 hours before hepatectomy. The dose and timing of the gadolinium treatment was generally used and similar to those in a previous study,3,6,8 which showed a 47% to 67% reduction in phagocytic activity of Kupffer cells.3,8 In the control group, the same volume of physiologic saline solution without gadolinium was administered in the same manner. This experiment was reviewed by the Committee on the Ethics of Animal Experiments in
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A
B
Fig 2. The liver regeneration rate assessed by the relative liver weight of the remnant liver (A) and the hepatic DNA synthesis rate (B) on POD 1 after hepatectomy. In both the gadolinium-treated (Gd) and nontreated rats, the ischemia/reperfusion (I/R) procedure itself decreased the relative liver weight and DNA synthesis rate. The gadolinium treatment itself also lowered DNA synthesis rate on POD 1 after hepatectomy. Values are means ± SEM of 6 rats in each group.
Graduate School of Medical Sciences, Kyushu University, and performed under the control of the Guideline for Animal Experiment in Kyushu University and The Law (No. 105) and Notification (No. 6) of the Japanese Government. Surgical procedure. All surgical procedures were performed in the morning (9-12 AM) under light ether anesthesia. After midline laparotomy, the portal vein, hepatic artery, and bile duct were clamped with a vascular clip (Mizuho Ikakogyo Co, Ltd, Tokyo, Japan) for 15 minutes and then reperfused for 15 minutes. During a second 15 minutes of ischemia, left lateral and median lobes were excised (70% hepatectomy) according to the procedure of Higgins and Anderson.9 This procedure mimicked hepatectomy in clinical fields, where Pringle’s maneuver (15 minutes of ischemia and 5
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minutes of reperfusion) is repeatedly performed to control bleeding during parenchymal dissection. All the animals received 10% glucose solution and standard diet ad libitum after the operation. All rats in the control group survived although some of the gadolinium-treated group died shortly after hepatectomy. For the survival study, 50 rats (control group, 20 rats; gadolinium group, 30 rats) were used to examine the 24-hour survival rate. In an analytic study, a total of 74 rats (control group, 29 rats; gadolinium group, 45 rats) were used. The animals that survived hepatectomy (day 0) and survivors on days 1, 2, 3, and 7 after hepatectomy were subjected to the analyses described later. To determine the DNA synthesis rate of the liver, tritiated thymidine (Amersham Life Science, Little Chalfont, England; specific activity, 25 Ci/mmol) was injected intraperitoneally at a dose of 0.4 µCi/g of body weight 60 minutes before the animals were killed. The abdomen was opened, and a small portion of the liver (right lateral lobe) that weighed less than 100 mg was immediately excised, frozen in liquid nitrogen, and stored at –80°C for analysis of hepatic ATP levels. The remaining liver was weighed, and a portion was used to determine the DNA synthesis rate by the incorporation rate of tritiated thymidine into DNA. Arterial blood was withdrawn simultaneously, and the serum was used to determine concentrations of endotoxin and hyaluronic acid and for liver function tests. Relative liver weight. The remaining liver was weighed, and the relative liver weight was calculated as the actual liver weight/100 g body weight. Hepatic DNA synthesis rate. The DNA fraction of the liver tissue was extracted by the method of Schneider.10,11 Briefly, the tissue was homogenized in 4 volumes of cold 1.15% potassium chloride, and 2.5 mL of ice-cold 10% trichloroacetic acid (TCA) solution was added. After vigorous stirring, the mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded, and the sediment was washed successively with 10% TCA solution, 5 mL of 76% ethanol, 5 mL of 95% ethanol, and 10 mL of ethanol/ether (3/1, vol/vol). The sediment was suspended in 3% potassium hydroxide solution. The suspension was incubated at 37°C for 60 minutes, neutralized, and centrifuged. The supernatant was discarded, and the sediment was washed with 5 mL of 5% TCA and heated at 90°C for 15 minutes, with occasional shaking. After cooling and centrifugation at 3000 rpm for 10 minutes, 1 mL of the supernatant was subjected to a liquid scintillation counter (Alga Liquid Scintillation system, LSC-703, Tokyo, Japan) after the addition of 10 mL of Ready Protein
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Fig 3. The concentration of ATP in the liver tissue before (before hepatectomy and ischemia/reperfusion) and after (just after hepatectomy under ischemia/reperfusion) 70% hepatectomy. In both groups, the hepatic ATP level was decreased just after hepatectomy under ischemia/reperfusion. The hepatic ATP level on POD 0, just after hepatectomy, was lower in the gadolinium group (Gd) than in the control group. All values are means ± SEM of 6 rats.
(Beckman Co, Ltd, Fullerton, Calif). The DNA concentration in the supernatant was also determined. Three milliliters of diphenylamine reagent (0.75 g diphenylamine in 50 mL glacial acetic acid and 1.14 mL concentrated H2SO4) was added to 2 mL of the supernatant, and the mixture was heated at 100°C for 10 minutes. After gently cooling to room temperature, absorbance at 600 nm was measured. Calf thymus DNA (Sigma Co, Ltd, St Louis, Mo) was used as a standard. The incorporation of [3H]-thymidine into DNA was calculated as disintegrations per minute per microgram DNA. Liver function tests. Serum levels of total bilirubin and glutamic pyruvic transaminase (GPT) were analyzed with the use of an automated multichannel analyzer (736-40 Automatic Analyzer; Hitachi, Ltd, Tokyo, Japan). The serum hyaluronic acid level has been used as an index of endothelial damage of the liver after cold preservation/reperfusion12 and graft viability after liver transplantation.13 Serum concentrations of hyaluronic acid were measured with the use of the sandwich-binding protein assay method previously reported by us.14 Adenine nucleotide concentrations in the liver. The concentrations of ATP in the liver were measured as previously reported by our laboratory.15 Briefly, the frozen liver tissues were lyophilized overnight, and samples that weighed 10 to 15 mg each were homogenized in 1 mL of 0.5 N perchloric acid. The homogenate was centrifuged for 5 minutes at 10,000 × g at 4°C. After addition of 0.05 mL of 3 mmol potassium hydroxide to 0.5 mL of supernatant, the samples were recentrifuged for 10 minutes under the same condition. The supernatants were run through a
microfilter (0.22 µm pore size; Japan Millipore Co, Ltd, Tokyo, Japan) and 3 µL per sample of the filtrate was used for high performance liquid chromatography on an anion exchange column, DEAE-2SW (4.6 × 250 mm; Tosoh Co, Ltd, Tokyo, Japan), equilibrated with 0.2 mmol phosphate buffer, pH 6.0, to determine concentrations of ATP. The level of ATP was expressed as micromole per gram liver tissue. Standard ATP was purchased from Sigma Co, Ltd. Endotoxin measurement. Sterile endotoxin-free disposable ware was used to prevent contamination of blood samples. Arterial blood was aspirated aseptically from the abdominal aorta. The concentration of endotoxin was determined by an endotoxin-specific assay with the use of recombined limulus coagulation enzymes (Seikagakukogyo Co, Tokyo, Japan). Statistical analysis. Significance of survival rate was assessed with the Log-rank test. All data are expressed as means ± SEM. The statistical differences between groups were determined by analysis of variance using the Mann-Whitney U test. A probability value less than .05 was considered significant. RESULTS Survival rate after hepatectomy. All 20 animals in the control group survived for 24 hours after hepatectomy under the ischemia/reperfusion, although 10 of 30 rats in the gadolinium group died. The 24-hour survival rate in the gadolinium group was significantly lower than that in the control group (67% vs 100%; P < .01). Because the gadolinium pretreated rats showed cyanosis and histopathologic study at necropsy showed pulmonary edema, the primary reasons for death seemed to be pulmonary failure.
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B
Fig 4. A, Changes in serum endotoxin concentrations after 70% hepatectomy. The endotoxin level was higher in the gadolinium group (Gd) than in the control group on POD 1. B, Serum endotoxin concentrations on POD 1 after hepatectomy in both the gadolinium-treated and nontreated rats with and without ischemia/reperfusion (I/R). Hepatectomy under ischemia/reperfusion resulted in a higher endotoxin level than that without ischemia/reperfusion in both the gadolinium-treated and gadoliniumnontreated rats. All values are means ± SEM of 6 rats.
Body weight and liver function tests. The body weight was not significantly different between the 2 groups throughout the study (Table I). The serum level of total bilirubin was elevated to its maximal value on POD 2 in the control group and on POD 1 in the gadolinium group. Mean serum total bilirubin level was significantly higher in the gadolinium group than in the control group on POD 1 (Table I). Mean serum glutamic-pyruvic transaminase levels were higher in the gadolinium group than in the control group on PODs 0 and 1, but the differences were not significant (Table I). The values declined thereafter in both groups. Serum concentrations of hyaluronic acid in the 2 groups were not significantly different (Table I). Liver regeneration. Liver regeneration, assessed by the relative liver weight and DNA synthesis rate,
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was significantly impaired in the gadolinium group. The relative liver weight in the gadolinium group was significantly lower than that in the control group on POD 1 (1.376% ± 0.072% vs 1.685% ± 0.028%; P < .02; Fig l, A). Liver weight was restored slowly in the gadolinium group. The DNA synthesis rate was significantly lower in the gadolinium group than in the control group on POD 1 (9.297 ± 1.508 dpm/µg of DNA vs 91.357 ± 45.346 dpm/µg of DNA; P < .02) but became higher on POD 2 (129.302 ± 20.982 dpm/µg of DNA vs 67.786 ± 5.716 dpm/µg of DNA; P < .02; Fig l, B). DNA synthesis reached its peak by POD 3 in both groups and then declined. No significant difference in DNA synthesis rate was observed between the 2 groups on PODs 3 and 7. In a separate experiment, the effect of ischemia/reperfusion on liver regeneration was examined on POD 1 in both groups (Fig 2). When liver regeneration after hepatectomy was compared by the presence or absence of ischemia/reperfusion in gadolinium-treated rats, the relative liver weight and DNA synthesis rate without ischemia/reperfusion were significantly higher than those with ischemia/reperfusion on POD 1 (relative liver weight, 1.774% ± 0.066% vs 1.376% ± 0.072%; P < .02; DNA synthesis rate, 127.057 ± 25.582 dpm/µg of DNA vs 9.297 ± 1.508 dpm/µg of DNA; P < .01). In gadolinium-nontreated rats, the relative liver weight and DNA synthesis rate without ischemia/reperfusion were also significantly higher than those in a group with ischemia/reperfusion (relative liver weight, 1.930% ± 0.101% vs 1.685% ± 0.028%; P < .05; DNA synthesis rate, 323.568 ± 38.539 dpm/µg of DNA vs 91.537 ± 45.346 dpm/µg of DNA; P < .02). The ischemia/reperfusion procedure itself significantly decreased the relative liver weight and DNA synthesis rate. In rats without ischemia/reperfusion, the gadolinium pretreatment significantly lowered DNA synthesis rate on POD 1 after hepatectomy (127.057 ± 25.582 dpm/µg of DNA vs 323.568 ± 38.539 dpm/µg of DNA; P < .01; Fig 2, B). Hepatic energy status. The concentrations of ATP in the liver before ischemia/reperfusion and hepatectomy procedures were statistically similar in the gadolinium and control groups (8.848 ± 0.342 µmol/g vs 8.538 ± 0.745 µmol/g of dry liver; Fig 3). Just after hepatectomy under ischemia/reperfusion (POD 0), the hepatic ATP level was significantly lower than that before hepatectomy and ischemia/reperfusion in both groups (gadolinium group, P < .01; control group, P < .05), but the decline was more profound in the gadolinium group. Immediately after hepatectomy on POD 0, the concentration of ATP in the liver was signifi-
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Surgery Volume 127, Number 4 Table I. Body weight and liver function tests
Body weight (g) Control Gadolinium Total bilirubin (mg/dL) Control Gadolinium Glutamic-pyruvic transaminase (IU/L) Control Gadolinium Hyaluronic acid (ng/mL) Control Gadolinium
0
1
275 ± 5 269 ± 8
270 ± 8 273 ± 7
0.33 ± 0.10 0.40 ± 0.16
Days after hepatectomy 2
3
7
261 ± 4 251 ± 5
279 ± 6 267 ± 8
300 ± 9 293 ± 5
0.33 ± 0.12 1.07 ± 0.36*
0.63 ± 0.22 1.05 ± 0.52
0.30 ± 0.10 0.15 ± 0.04
0.16 ± 0.09 0.06 ± 0.02
802 ± 135 999 ± 239
209 ± 47 465 ± 226
157 ± 42 89 ± 15
91 ± 15 55 ± 4
76 ± 13 54 ± 1
58 ± 24 60 ± 16
209 ± 53 194 ± 60
60 ± 16 119 ± 36
79 ± 35 115 ± 38
38 ± 22 19 ± 4
Values are means ± SEM (n = 6). *P
< .05, vs control.
cantly lower in the gadolinium group than in the control group (4.149 ± 0.416 µmol/g vs 6.607 ± 0.323 µmol/g of dry liver; P < .01; Fig 3). The hepatic ATP level was statistically similar in both groups on POD 1, and the value became essentially the same as that before ischemia/reperfusion in each group. Serum endotoxin concentration. The serum endotoxin level reached its maximum on POD 1 in both groups, and the value was significantly higher in the gadolinium group than in the control group on POD 1 (315.12 ± 33.99 pg/mL vs 214.56 ± 13.21 pg/mL; P < .05; Fig 4, A). The serum endotoxin level decreased on PODs 2 and 3, and no further significant differences were found between 2 groups (Fig 4, A). The serum endotoxin levels on POD 1 after hepatectomy with and without ischemia/reperfusion were determined in another experiment. In gadolinium-treated and gadolinium-nontreated rats, hepatectomy with ischemia/reperfusion resulted in a significantly higher endotoxin level on POD 1 than that without ischemia/reperfusion (gadolinium-treated, 151.84 ± 40.99 pg/mL vs 315.12 ± 33.99 pg/mL; P < .02; gadolinium-nontreated, 20.60 ± 6.35 pg/mL vs 214.56 ± 13.21 pg/mL; P < .01; Fig 4, B). Even without ischemia/reperfusion, the serum endotoxin level on POD 1 after hepatectomy was significantly (P < .02) increased by gadolinium treatment (Fig 4, B). DISCUSSION Liver regeneration assessed by relative liver weight and DNA synthesis rate after hepatectomy under ischemia/reperfusion was significantly impaired by gadolinium pretreatment. On POD 1 after hepatectomy, the serum concentrations of
total bilirubin and endotoxin were significantly higher in the gadolinium group than in the control group. The hepatic ATP level just after hepatectomy (POD 0) under ischemia/reperfusion was significantly lower in the gadolinium group than in the control group. The gadolinium pretreatment significantly worsened the 24-hour survival rate. These findings indicate that gadolinium pretreatment has disadvantages for regeneration and energy status of the liver after hepatectomy under ischemia/reperfusion, thus leading to a higher mortality rate. Gadolinium selectively inhibits Kupffer cell phagocytosis by interfering with calcium-dependent cell surface interactions.1,2 Gadolinium decreases phagocytic activity of Kupffer cells.3,8 Gadolinium reduces hepatic bacterial clearance but does not decrease the activity of bacterial killing.16 The use of gadolinium has been shown to have beneficial effects on liver injury and liver regeneration. Treatment with gadolinium attenuated endotoxin-induced liver injury after partial hepatectomy and improved the 24-hour survival rate.17 Pretreatment with gadolinium increased liver regeneration that was assessed by the incorporation rate of [3H]-thymidine into DNA and mitotic activity of the hepatocytes.5 In contrast, gadolinium pretreatment in an LPS-responsive mouse has been reported to reduce the proliferating cell nuclear antigen labeling index of hepatocytes and impair liver regeneration after hepatectomy.6 However, these studies have examined animals after hepatectomy without any ischemia/reperfusion procedures. In our experiment, the 24-hour survival rate after hepatectomy under ischemia/reperfusion was significantly lower in the gadolinium group than in the control group. The pretreatment with gadolinium deteriorated liver function and liver regeneration after hepatectomy
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under ischemia/reperfusion. This is the first report that shows an inhibitory effect of the gadolinium pretreatment on liver regeneration after partial hepatectomy under ischemia/reperfusion. Because hepatectomy is usually carried out under ischemia/reperfusion in clinical practice, a similar maneuver was used in the current study. There are 2 aspects of ischemia/reperfusion injury of the liver. One is the injury caused by ischemia and portal congestion during the interruption of hepatic blood inflow, and the other is the reperfusion injury caused by pooled portal blood in the ischemic liver.18,19 The ischemia/reperfusion procedure itself has been shown to significantly reduce the DNA synthesis rate and recovery of liver weight 24 hours after partial hepatectomy.7 Van Deventer et al20 suggested that endotoxins in the portal blood that were translocated from the intestine are one of the factors inhibiting liver regeneration. Although we did not measure the endotoxin level in the portal vein, we did observe an increase in its level in systemic blood, which supports their finding. The present study confirms that the ischemia/reperfusion procedure itself significantly decreases liver regeneration (relative liver weight and DNA synthesis rate) 24 hours after hepatectomy. Our pretreatment procedure with gadolinium (timing and dosage) was essentially the same as those used in previous reports,3,6,8,21 where the reduction in Kupffer cell phagocytosis was noted in rats and mice that had been intravenously administrated 7 mg/kg of gadolinium for 2 days. Under these conditions, phagocytic activity and plasma tumor necrosis factor-α (TNFα) levels were decreased after ischemia/reperfusion in rats,3 and serum levels of cytokine-induced neutrophil chemoattractant were decreased after ischemia/reperfusion in rats.22 Moreover, liver regeneration after hepatectomy has been reported to be not affected by endotoxin in gadolinium-pretreated rats17 or to be impaired in LPS-responsive strain mice.6 However, hepatectomy was performed without ischemia/reperfusion in these reports. Our results indicate that the treatment with gadolinium itself increased the serum endotoxin level and worsened the DNA synthesis rate on POD 1 after hepatectomy without ischemia/reperfusion. As a result, liver regeneration was significantly inhibited after hepatectomy under ischemia/reperfusion in the gadolinium group. The Kupffer cells, which are exposed to portal vein circulation, remove harmful substances from the portal blood, such as intestinal endotoxins and bacteria. When the portal endotoxin level exceeds the phagocytic abilities of the Kupffer cells, endotoxin spills out from the Kupffer cells and directly injures the hepatocytes.23 In our present study, the serum endotoxin level on POD 1 in the gadolinium
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group was significantly higher than that in the control group. It is likely that the Kupffer cells pretreated with gadolinium could not remove the excessive endotoxins translocated from the intestines after hepatectomy under ischemia/reperfusion. Even without ischemia/reperfusion, the serum endotoxin levels were significantly higher in the gadoliniumtreated rats on POD 1 after hepatectomy than in the gadolinium-nontreated rats. The primary reason for high mortality rates in the gadolinium group was respiratory failure, which is consistent with the report that endotoxins induce acute respiratory failure.24 Direct organ damage by gadolinium may be another possible explanation for poor survival in the gadolinium group. Spencer et al25 have shown that gadolinium treatment causes mineral deposition in capillary beds (particularly lung and kidney) and injuries to the liver and spleen. Thus a reduction in the capacity of Kupffer cells to remove endotoxin or direct organ damage by gadolinium may explain the high mortality rate, hyperbilirubinemia, and retarded liver regeneration in the gadolinium group in our present study. Another possible factor is the release of proinflammatory cytokines from Kupffer cells because cytokines such as TNF-α and IL-6 are important for liver regeneration.26 TNF-α and IL-6 regulate liver regeneration through the induction of transcription factors such as Stat3 and NF-κΒ.27 Effects of gadolinium on serum TNF-α and IL-6 levels are controversial. Gadolinium causes sustained overexpression of TNF messenger RNA and transient overexpression of circulating TNF protein after hepatectomy; both TNF-inducible cytokines (IL-1, IL-6) are also relatively overexpressed.28 On the other hand, it has been reported that serum TNF-α and IL-6 levels are reduced in the gadolinium-pretreated rats after hepatectomy.3,6 The inhibition of hepatic regeneration in our gadolinium group might have been caused by the depletion of cytokines in the Kupffer cells by the gadolinium treatment. Measurement of proinflammatory cytokines, such as TNF-α and IL-6, is necessary to elucidate the exact mechanism. Extensive hepatectomy induces acute metabolic stress on the remaining liver, causing a marked decrease in energy levels.29 The energy balance is maintained by compensatory enhancement of mitochondrial phosphorylative activity when the metabolic load is maximal on the remnant liver.30 The production of ATP is necessary for both liver regeneration and organ viability, and an ATP deficit causes organ failure. After cold preservation for a short time, mitochondrial ATPase activity of the reperfused liver, a key enzyme of ATP synthesis, has been reported to be decreased by the treat-
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Surgery Volume 127, Number 4 ment with gadolinium.31 In the present study, the hepatic ATP level in the gadolinium group just after hepatectomy was significantly lower than that in the control group. The reason may be ascribed to the decreased mitochondrial ATPase activity. The lower concentration of ATP in the gadolinium group may explain the high mortality rate within 24 hours after hepatectomy in the gadolinium group, because hepatic ATP levels before hepatectomy and the ischemia/reperfusion procedures were not different between the 2 groups. CONCLUSION Pretreatment with gadolinium did not improve the hepatic ATP level and liver regeneration after hepatectomy under ischemia/reperfusion and, in fact, led to a high mortality rate.
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