DNA polymerases and Ki-67 nuclear antigen are induced in correlation with the resected mass of rat liver up to 90%

Background and aims: We studied the regeneration potential by measuring induction of DNA polymerases in the remnant rat liver after a partial hepatectomy (PHx) that is maximal but compatible with survival. Methods: The regenerating rat liver was obtained after the 90% PHx. The induction of activities of DNA polymerase α, δ, and ɛ were measured after partial purification. The Ki-67 nuclear antigen was also detected histochemically. These parameters were compared with those after both 30% and 70% PHx. Results: The 90% hepatectomy resulted in the strong inductions of DNA polymerase α, δ, and ɛ, at 48 h after operation, in association with increases in wet weight and total DNA in the remnant liver. The enzyme induction was much higher after 90% PHx than after 30% and 70% hepatectomy, in correlation with the resection volume. At 48 h after 90% hepatectomy, the Ki-67 positive cells increased up to 47.2% of hepatocytes in the remnant liver. Conclusion: The higher induction of replication enzymes by 90% hepatectomy reflects more cells entering mitogenic cell cycle, which supports the fast regeneration of the remnant liver. The number of proliferating hepatocytes is stringently controlled by an unknown mechanism sensing the mass of resected liver parenchyma. Received: 9 March 1999; In revised form: 2 August 1999 Accepted: 20 August 1999     

Anti-transforming growth factor-β1 antibody transiently enhances DNA synthesis during liver regeneration after partial hepatectomy in rats

The regulation of liver regeneration after partial hepatectomy (PHx) is complex and involves many different cytokines. We investigated the role of one of these, transforming growth factor-β1 (TGF-β1), an inhibitor of liver regeneration, in a Wistar male rat model, in which anti-TGF-β1 antibody was injected immediately or 24 h after 70% PHx. Livers from treated animals contained an increased number of cells in S phase, according to 5-bromo-2′-deoxyuridine (BrdU) labeling 36 h after PHx. Antibody administration 24 h after PHx resulted in the highest peak of proliferation; moreover, peak MIB-5 labeling was also observed at that time. However, neither residual liver-weight-to-body-weight ratios nor regeneration rates differed significantly between any of the animals. Therefore, we also measured levels of serum TGF-β1 and hepatocyte growth factor (HGF; an activator). With antibody administration at 0 or 24 h, TGF-β1 levels were diminished at 24 or 36 h as compared with levels in control rats, but then rebounded, reaching a delayed peak at 48 or 72 h after PHx, respectively. Interestingly, there were also similar trends in HGF levels. These results indicate that TGF-β1 may inhibit the G1 checkpoint, and serum TGF-β1 concentration may influence HGF to regulate liver regeneration and to maintain homeostasis of proliferation after PHx. Received: November 15, 2000 / Accepted: February 15, 2001     

Acute portal hypertension reflecting shear stress as a trigger of liver regeneration following partial hepatectomy

The concept of injury in liver regeneration after partial hepatectomy (PHx), and the reason hepatocytes that have not been directly injured regenerate, remain unclear. It is known that shear stress resulting from blood flow plays an important role in the mechanism of remodeling blood vessels, and portal pressure reflects shear stress. This study was conducted to determine whether acute portal hypertension (APH) can become a trigger of liver regeneration as shear stress following PHx in a rat model. Portal pressures became elevated immediately after 70% and 90% PHx, peaking on postoperative day (POD) 3, and thereafter decreasing in proportion to the diminution of liver regeneration. The portal pressures after 90% PHx were significantly higher than those after 70% PHx even on POD 7, while those of the portocaval (PC) shunt groups decreased following PC shunting both with and without 70% PHx. The liver/body weight (LW/BW) ratio also decreased in the PC shunt both with and even without 70% PHx. The gradient expressions of class I antigen on sinusoidal endothelial cells (SEC) were found only in the periportal area, which has the highest portal pressure in the healthy rat liver. However, after hepatectomy these expressions were detected from the periportal area to the central venous area. These results suggest that APH as shear stress following PHx may not only become a trigger of hepatocyte regeneration, but also of SEC regeneration, and that surplus APH induces liver dysfunction.     

Comparison of the stress response after laparoscopic and open cholecystectomy

Background: We designed a prospective controlled animal study to compare the stress response induced after laparoscopic and open cholecystectomy. Methods: Twelve female pigs (20–25 kg body weight) were anesthetized with ketamine, pentobarbital, and fentanyl. The animals were randomized into the following four groups: control (C), pneumoperitoneum with CO₂ at 14–15 mmHg (P), laparoscopic cholecystectomy (LC), and open cholecystectomy (OC). The average duration of the procedure in each group was 35 min. Results: Central venous pressure, mean arterial pressure, pulmonary capillary wedge pressure, and cardiac output were monitored. Measurements were recorded when animals were anesthetized (baseline), immediately before and after surgery, and thereafter every 30 min for a maximum of 3 h. White blood cell count (WBC) was determined from blood samples taken before and after 3 h of surgery. Ultrasound-guided liver biopsies were done preoperatively and after 3 h of surgery. Total RNA was isolated from the liver biopsy specimens. Steady-state mRNA levels of β-fibrinogen (β-fib), α 1-chymotrypsin inhibitor (α1-CTI), metallothionein (MT), heat shock protein 70 (Hsp70), and polyubiquitin (Ub) were detected by Northern blot/hybridization. There were no statistical differences in the hemodynamic parameters among the groups. The number of circulating neutrophils and monocytes decreased only after LC. Expression of Hsp70 was not induced after any surgical procedure, and the mRNA levels of Ub did not change after surgery. The expression of α1-CTI and β-fib (acute phase genes) were similarly increased after LC and OC. Steady-state mRNA levels of MT were slightly increased after P and LC but not after OC. Conclusion: These data indicate that there are no significant differences between LC and OC in terms of induction of the stress response. Received: 19 March 1999/Accepted: 2 July 1999/Online publication: 20 September 2000     

Blood transfusion causes deterioration in liver regeneration after partial hepatectomy in rats.

BACKGROUND: This study investigated the effects of blood transfusion on liver regeneration and function after hepatectomy in rats. METHODS: Inbred male Sprague-Dawley rats underwent a sham operation or a 70% hepatectomy (PHx) and were randomly divided into seven groups according to transfusion type: groups I and II underwent a sham operation and received saline (I) or whole blood (II). Groups III to VII underwent PHx with saline (III), whole blood (IV), irradiated/leukocyte-depleted whole blood (V), plasma (VI), or autologous blood (VII). The liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling index, serum aspartate aminotransferase, alanine aminotransferase, purine nucleoside phosphorylase (PNP) activity, hepatocyte growth factor (HGF), and activated transforming growth factor beta1 (TGF-beta(1)) were measured 6 and 24 h and 5 days after PHx. RESULTS: The liver regeneration rate and PCNA labeling index were lower in groups IV and V than in the other groups. Serum liver enzymes 6 h after PHx were worst in groups IV and V. PNP activity increased most in group IV, 6 and 24 h after PHx. The HGF values 6 h after PHx in all the transfused groups were lower than in group III. The activated TGF-beta(1) level 6 h after surgery was highest in group IV. CONCLUSION: Whole blood or irradiated/leukocyte-depleted whole blood impaired liver regeneration after PHx, probably through the production of activated TGF-beta(1) and HGF outside the liver, and plasma or autologous blood reduced the deleterious effects.     

Role of shear stress and immune responses in liver regeneration after a partial hepatectomy

This report reviews studies addressing the new concepts in liver regeneration after a partial hepatectomy (PHx). The review begins with an overview of the immunologic mechanisms of liver regeneration after PHx, especially regarding Kupffer cells and extrathymic T cells of the regenerative liver in the cell-mediated immunity, based on major histocompatibility complex I and II antigens. Attention is then devoted to “on and off” studies in liver regeneration after PHx, by hypothesizing the shear stress based on the fact that the portal flow against hepatocytes or sinusoidal endothelial cells triggers their regeneration after a partial hepatectomy and controls the volume of the regenerating liver by the stimulating the cell surface modulator (CSM) of hepatocytes and sinusoidal endothelial cells (SEC). We propose that the acute elevation of shear stress after PHx influences the adhesion between SEC and intrahepatic leukocytes. These concepts are expected to positively contribute to the future research on liver regeneration after PHx.     

Effects of ischemic preconditioning on regenerative capacity of hepatocyte in the ischemically damaged rat livers

BACKGROUND: Liver regeneration after partial hepatectomy is regulated by several factors that activate or inhibit hepatocyte proliferation. A short period of ischemia-reperfusion (IR), called ischemic preconditioning (IPC), protects the liver against subsequent sustained ischemic insults. The present study investigated the effects of IPC on liver regeneration after partial hepatectomy under IR in rats. MATERIALS AND METHODS: Male Wistar rats were subjected to 45 min of total hepatic ischemia, and 70% hepatectomy was performed just before reperfusion. Animals were pre-treated with either IPC (10/15 min) (IPC + PHx group) or not (ischemia + PHx). The survival rate, serum transaminases, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 levels, hepatocyte proliferation and histological change of the remnant liver were measured in both groups and compared with non-ischemic controls subjected to 70% hepatectomy alone (PHx group). RESULTS: The survival rate was significantly better in the IPC + PHx group than in the ischemia + PHx group. Furthermore, IPC reduced liver injury determined by liver histology and serum transaminases. There was an early rise in serum TNF-alpha and IL-6 levels in the ischemia + PHx group. Compared with non-ischemic controls, IPC significantly decreased TNF-alpha, but not IL-6 during the late (24 and 48 h) phases of reperfusion. Rats subjected to 70% hepatectomy and 45 min of hepatic ischemia showed significantly reduced hepatocyte proliferation (mitotic index, proliferating cell nuclear antigen, and relative liver weight) when compared with animals subjected to hepatectomy alone. However, hepatocyte proliferation was markedly increased in rats pretreatment with IPC when compared with ischemic controls. CONCLUSION: These results suggest that ischemic pre-conditioning ameliorates the hepatic injury associated with ischemia-reperfusion and has a stimulatory effect on liver cell regeneration that may make it valuable as a hepatoprotective modality. Il-6 appears to be key mediator in promoting regeneration after combined ischemia and hepatic resection.     

Research review: DNA polymerases as molecular markers of the regenerating capacity of hepatocytes

Hepatocyte regeneration has been widely investigated, with the mitotic index and the incorporation of [³H]thymidine being used as regeneration markers. We focused on the induction of DNA replication enzymes, particularly DNA polymerases (pol) α, δ, and ε. Using rat models, we have shown that the activity of pol α in crude liver extract well represents the regenerating capacity of hepatocytes. Using pol α as an indicator, we analyzed liver regeneration in rat models under various conditions: obstructive jaundice, external or internal biliary drainage, and the obstruction of portal vein branches. It has been revealed that the ligation of the common bile duct alone induces a certain amount of hepatocyte proliferation. It was striking that external biliary drainage suppressed regeneration capacity in cholestatic rat liver after partial hepatectomy. The strong regeneration in nonligated lobes induced by portal branch ligation was similar to the liver regeneration seen after partial hepatectomy with respect to the induction of DNA polymerases. Taken together, the aspects of DNA replication, particularly the induction of DNA polymerases, may contribute to shedding new light on the regeneration of human liver. This work was supported in part by a Grant-in-Aid for General Scientific Research and for Cancer Research from the Ministry of Education, Science and Culture, Japan, and by grants from the Uehara Memorial Foundation     

补体旁路途径的激活在肝再生过程中作用的研究

目的探讨在小鼠肝切除术后补体旁路途径的激活在肝再生过程中的作用。方法将实验动物分为3组:A组野生型C57BL/6小鼠行假手术,B组野生型C57BL/6小鼠行70%肝部分切除术(PHx)和C组补体旁路途径激活的关键蛋白-B因子缺陷(fB-/-)小鼠行70%PHx,评估小鼠术后肝脏损伤及肝再生情况。结果与A组比较,B组术后48h血清ALT和T-BIL水平均显著增高(P0.01),而C组更分别比B组高出2.2倍和3倍(P0.01)。术后48h肝脏组织经HE染色发现,A组无明显病理性改变;B组肝脏呈轻度脂肪变性,坏死少见;C组肝脏组织为中重度脂肪变性,坏死明显。通过采用肝脏重量的重建、BrdU结合率及有丝分裂指数来综合评估肝脏的再生能力,结果与A、B两组相比,C组小鼠肝脏的再生功能受到了严重损害。免疫组织化学染色检测补体C3的沉积,A组为阴性,B组为强阳性,C组为弱阳性。生存率分析,野生型小鼠术后7d存活率为100%,而fB-/-小鼠仅为58.33%,两组间差异有统计学意义(P0.01)。结论小鼠行70%PHx后可通过旁路途径激活补体系统,并参与肝脏再生,阻断补体旁路途径的激活会严重抑制肝脏的再生功能。     

BCL-2 and BAX expression and cell proliferation,after partial hepatectomy with and without ischemia,on cholestatic liver in rats: an experimental study

BACKGROUND: Liver regeneration after partial hepatectomy (PHx) is regulated by several factors that activate or inhibit hepatocyte proliferation. Apoptosis seems to play an important role in cellular proliferation and liver regeneration. This study investigates the expression apoptosis-associated genes bcl-2 and bax, and the presence of apoptosis and cell proliferation after PHx, in normal and jaundiced rats with or without superimposed ischemia. MATERIALS AND METHODS: The study included 50 male Wistar rats assigned into; five groups (10 rats each). On day 0, rats of groups II, IV, and V underwent common bile duct ligation (BDL). On day 10, total liver ischemia (TLI) (occlusion of hepatic artery and portal vein-TLI) for 30 min was performed on animals of group V. When TLI was completed, all 30 animals (of groups I, IV, and V) underwent PHx (68%). Animals of group III underwent only TLI for 30 min. Rats of groups I, IV, and V were sacrificed 24 and 48 h after PHx was completed. Rats of group II were sacrificed 10, 11, and 12 days after BDL. Rats of group III were sacrificed immediately, 24 and 48 h after TLI completion. Liver tissue was obtained and pathologic examination included: (a) H&E stain, (b) in situ hybridization (detection of bcl-2 and bax mRNA) in paraffin sections, (c) Western blot analysis for the evaluation of bcl-2 and bax protein levels, (d) in situ hybridization (TUNEL) for the detection of apoptotic bodies, and (e) immunohistochemical stains (streptavidin-biotin method) in paraffin sections to detect cells that (i) express bcl-2 and bax proteins and (ii) undergo proliferation (Ki67+ cells). Results were expressed following morphometric analysis. RESULTS: Before hepatectomy, bcl-2 (protein or mRNA) levels were higher in jaundiced rats vs controls. Furthermore, bax (protein or mRNA) levels and apoptotic body index (ABI) were higher in cholestatic livers. After hepatectomy, there was an early decrease in the protein and mRNA levels of antiapoptotic gene bcl-2 and a late increase of proapoptotic gene bax and the ABI, compared to controls. Cell proliferation of hepatocytes was lower in group V (BDL + TLI) compared to that of groups II and IV (BDL). CONCLUSIONS: This study shows that apoptosis takes place in cholestatic livers with or without superimposed ischemia and may contribute in the impaired regenerative response observed in livers of jaundiced rats after partial hepatectomy.     

Does the approach to the groin make a difference in hernia repair?

Background Laparoscopic and open preperitoneal hernia repair techniques both use the preperitoneal space. This study investigated whether the surgical approach to the inguinal canal affects outcome measures. Methods One hundred sixty patients with inguinal hernia were assigned randomly into open anterior (42), open preperitoneal (39), laparoscopic transabdominal preperitoneal (39), and laparoscopic total extraperitoneal (40) groups according to the surgical method. The peroperative serum tumor necrosis factor-α (TNF-α) levels, interleukin-6 (IL-6) levels, VAS scores at 6 and 48 h, per- and postoperative complications, and recurrence rates were determined as main variables. Results The serum IL-6 levels were 335 ± 1.8, 283 ± 1.8, 283 ± 1.4, and 269.3 ± 1.6 pg/ml in the open anterior, posterior, transabdominal preperitoneal, and total extraperitoneal groups, respectively (P < 0.01). The TNF-α levels were highest in the open anterior group. The pain scores were lower in groups undergoing the posterior approach than in the open anterior approach group. Conclusion The approach to the inguinal canal through the preperitoneal space appears to be less invasive than the transinguinal anterior approach.     

Effects of Ozone Oxidative Preconditioning on Liver Regeneration after Partial Hepatectomy in Rats

ABSTRACT

Aim: Similar protective effect of ischemic and ozone oxidative preconditioning (OzoneOP) in hepatic ischemia–reperfusion (I/R) injury was demonstrated, providing evidences that both preconditioning settings shared similar biochemical mechanisms of protection. We investigated the effects of OzoneOP on liver regeneration after 70% partial hepatectomy (PHx) in rats. Methods: Rats were divided into three groups: PHx, I/R + PHx, and OzoneOP + I/R + PHx groups. Ozone (intraperitoneal, 1.2 mg/kg) was given to rats subjected to I/R and 70% hepatectomy daily five times before operation. At 24 hr and 48 hr after resection, samples were collected for the measurement of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Moreover, liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling index, mitotic index, and histopathological examination were evaluated. Results: OzoneOP reduced liver injury determined by liver histology and serum transaminases. There was a rise in serum TNF-α and IL-6 levels in the I/R + PHx group whereas OzoneOP significantly decreased the rise in the level of TNF-α but not IL-6 on the 24 hr and 48 hr of reperfusion. Moreover, liver regeneration in OzoneOP + PHx group, as assessed by the regenerated liver weight, mitotic, and PCNA-labeling index, was significantly improved when compared to I/R + PHx group. Conclusion: These results suggest that OzoneOP ameliorates the hepatic injury associated with I/R and has a stimulatory effect on liver cell regeneration that may make it valuable as a hepatoprotective modality.     

Laparoscopic surgery and Kupffer cell activation

Background: Evidence tends to support a relative preservation of the systemic immune response with laparoscopy as compared with laparotomy. However, the role of hepatic macrophages, or Kupffer cells, in modulating this immune advantage is unknown. This study investigated the functions of Kupffer cells after either laparoscopy or laparotomy in a rat model. Methods: Rats underwent laparoscopy, laparotomy, or control operations. Kupffer cells were harvested, cultured, and stimulated with lipopolysaccharide. Culture supernatants were analyzed for tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). Cytoplasmic lysates were analyzed for activation of two mitogen-activated protein kinases (MAPKs). Results: Production of TNF-α and IL-6 was similar in laparoscopy, laparotomy, and control groups. Both laparotomy and laparoscopy showed increased activation of p38 MAPK as compared with controls. Activation of ERK1/2 was decreased during laparotomy as compared with laparoscopy. Conclusions: Although cytokine production was similar in the laparoscopy and laparotomy groups, changes in MAPK activation suggest that intracellular pathways are more affected during laparotomy than during laparoscopy. Received: 1 April 1999/Accepted: 18 August 1999/Online publication: 26 July 2000     

Metabolism of prostaglandins in porcine liver transplantation with a graft harvested after 30- and 60-minute warm ischemia

The influence of warm ischemia on the metabolism of prostaglandins was investigated using a pig liver transplantation model employing the temporary portal arterialization technique. Eighteen pigs were divided into three groups according to warm ischemia time: 0 min (group I,n=6), 30 min (group II,n=6), and 60 min (group III,n=6). During portal arterialization, the hepatic venous prostaglandin E₂ (PGE₂) level in group III (3356.0±1011.8pg/ml) was significantly higher than that in group I (831.7±182.1pg/ml;P=0.0285). The hepatic venous PGE₂ levels were significantly higher than the arterial counterparts in all groups both at the beginning and during portal arterialization. At 60 min after portal revascularization, the arterial PGE₂ level in group III (886.7±268.0pg/ml) was significantly higher than that in groups I (99.0±18.6 pg/ml;P=0.0116) and II (204.2±65.4pg/ml;P=0.0282). Neither thromboxane B₂ (TXB₂) nor 6-keto PGF_1α showed any significant differences. In conclusion, the intra-operative changes of PGE₂ thus reflected the degree of warm ischemic damage, and PGE₂ could also be released from the graft. On the other hand, the increased levels of TXB₂ and 6-keto PGF_1α were throught to have an extrahepatic origin.     

The promotion of liver regeneration in mice after a partial hepatectomy as a result of the modulation of macrophage activation by dexmedetomidine

BackgroundThis study investigates the effect of dexmedetomidine (DEX), a highly selective agonist of alpha 2-adrenergic receptors (α2-ARs), on the regulation of hepatic macrophage activation in liver regeneration.MethodsA two-thirds partial hepatectomy (PHx) mouse model was performed. DEX (25 μg/kg) or a vehicle control (saline) was injected i.p. at 30 min before and every 12 h after PHx. The expression of α2B-ARs in the liver was detected using immunofluorescence staining. The effects of DEX on liver regeneration were assessed by Ki67 staining. The gene expression of inflammatory cytokines in isolated hepatic macrophages was quantified 36 h after the PHx.Resultsα2B-ARs colocalized with hepatic macrophages after the PHx. The number of Ki67-positive hepatocytes in the mice treated with DEX was markedly increased (p < 0.05). The increases in Ki67-positive hepatocytes after treatment with DEX were inhibited in the macrophage-depleted mice. DEX treatment inhibited the expression of major pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor and elevated the expression of anti-inflammatory cytokines IL-4, IL-10, and transforming growth factor-β1 in hepatic macrophages 36 h after the PHx (p < 0.05).ConclusionsThe α2B-AR subtype is expressed in hepatic macrophages after a PHx. DEX modulates hepatic macrophage activation toward an anti-inflammatory phenotype via α2B-AR, which promotes the process of liver regeneration.     

Evaluation of antibiotic-induced nephrotoxicity in preterm neonates by determining urinary α1-microglobulin

α₁-Microglobulin (α₁-m, protein HC), a relatively low molecular weight protein of about 31,000 daltons, was measured in urine of three groups of 34 preterm neonates: group A consisted of 9 healthy preterm neonates; groups B (n = 13) and C (n = 12) consisted of preterm neonates with suspected or confirmed bacterial infections. Immediately after birth, all group B neonates were treated with ampicillin and aztreonam in combination, and all group C neonates were treated with oxacillin and amikacin in combination. To optimize amikacin administration, computerized individually tailored doses were administered. Urine samples were obtained from a short collection in sterile bags on the 1st, 4th, and 7th day after delivery in all infants. Urinary α₁-m concentrations were measured by a turbidimetric method (latex agglutination photometric immunoassay) and results were expressed as a ratio to urinary creatinine. In group A, urinary α₁-m concentrations were stable after birth. In group C, α₁-m excretion increased immediately within the 1st day of treatment, and over the 1st week of life urinary α₁-m levels were significantly higher than in group A (P = 0.033). These data support the conclusion that amikacin administration was the most important factor inducing renal tubular dysfunction in the neonates of group C. Received March 6, 1995; received in revised form and accepted January 19, 1996     

Increase in portal blood interleukin-6 soon after the commencement of digestive surgery

To determine whether cytokines produced in the operative field during digestive surgery selectively spill over into the portal blood, the changes in interleukin-6 (IL-6) levels in portal and peripheral venous blood were assayed at several points in time from the commencement of surgery until 14 days later, in 11 patients. Similar changes in the IL-6 levels were observed in the portal and peripheral blood samples; however, the IL-6 levels in the portal blood reached a maximum 6–12 h after the commencement of surgery, being earlier than in the peripheral venous blood. In fact, between 3 and 12 h after the commencement of surgery, the IL-6 levels were higher in the portal blood by 33–81 pg/ml. By 24 h or more after the commencement of surgery, the IL-6 levels did not differ significantly in the two types of blood samples. Moreover, the C-reactive protein levels 2 days after surgery were even more closely correlated to the maximum IL-6 levels in the portal blood than to those in the peripheral venous blood. These results suggest that IL-6 produced during intraabdominal digestive surgery initially enters the portal blood, being trapped by IL-6 receptors in the liver, where it may regulate the synthesis of acute-phase proteins as a hepatocyte-stimulating factor.     

Cyclosporine overcomes cold preservation/reperfusion injury of liver graft: Chemokine release and liver ultrastructure

There is a growing body of evidence that the cytokine, tumor necrosis factor-α (TNF-ga), plays an important role in the development of hepatic ischemia/reperfusion injury. We found that the immunosuppressants, cyclosporine-A (CsA), azathioprine, and FK506, have protective effects on such injury. The purpose of the present study was to elucidate mechanisms involved in these beneficial effects of the immunosuppressant, CsA, on liver injury following cold preservation and transplantation, with special reference to the suppression of TNF-α release. Rat livers were stored in Euro-Collins solution (EC) at 4°C for 6h and orthotopically transplanted. The animals allotted to two groups: group A (untreated controls) and group B (CsA pretreatment of recipients). CsA (10 mg/kg, p.o.) was given for 3 consecutive days preoperatively. CsA pretreatment of the recipients significantly improved the 2-week survival rate (0/6 for group A, 3/6 for group B;P<0.05) and this was associated with a significant decrease in serum TNF-α levels 2h posttransplantation (group A, 69.8±15.7 pg/ml; group B, 22.8±6.8; mean±SEM;n=12 each;P<0.05) and amelioration of sinusoidal endothelial injury, assessed by electron microscopy. Plasma endotoxin levels following reperfusion of the grafts were not altered by the CsA therapy. Morphologically, CsA pretreatment of the recipients did not alter activation of Kupffer cells. CsA pretreatment of the recipient aids in preventing cold preservation/reperfusion injury of the liver graft, possibly by modulating effects of TNF-α.