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.     

Intermittent Ischemic Preconditioning Protects Against Hepatic Ischemia-Reperfusion Injury and Extensive Hepatectomy in Steatotic Rat Liver

ABSTRACT

Background: Hepatic steatosis causes severe liver damage and has deleterious effects when associated with ischemia-reperfusion mechanisms. Ischemic preconditioning (IPC) protects lean liver against prolonged ischemia by improving micro-circulation and reducing lipid peroxidation. We investigated the effect of intermittent IPC on liver ischemia-reperfusion injury (IRI) and extensive hepatectomy in severe hepatic steatosis. Methods: Severe hepatic steatosis was performed by 12–14 weeks of choline-free diet in 108 Wistar rats. We induced 30-minute ischemia-reperfusion manipulations and extensive hepatectomy with or without prior IPC in steatotic livers and after 6 and 24 hours of reperfusion blood transaminases, and IL6, TNFα, NO and Lactate in blood and liver tissue were measured. Results: Steatotic rats subjected to hepatic ischemia-reperfusion alone after extensive hepatectomy, showed severe liver damage with significantly increased values of AST, ALT, TNFα and Lactate and significantly reduced IL6 and NO, while no one rat survived for more than 29 hours. On the contrary, steatotic rats subjected to intermittent IPC, 24 hours before ischemia-reperfusion, presented increased 30-day survival (67%), lower values of AST, ALT, TNFα and Lactate, and increased IL6 and NO levels. Simple and intermittent IPC manipulations, 1 hour before the IRI and extended hepatectomy, did not prolong survival more than 57 and 98 hours, respectively. Simple IPC, 24 hours before IRI and extended hepatectomy had the lowest possible survival (16.7%).Conclusions: Hepatic steatosis and IRI after major liver surgery largely affect morbidity and mortality. Intermittent IPC, 24 hours before IRI and extensive hepatectomy, presents higher 30-day survival and improved liver function parameters.     

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.     

Impaired hepatic regeneration by ischemic preconditioning in a rat model of small-for-size liver transplantation

OBJECTIVE: Graft size is one of the major risk factors in adult-to-adult living donor liver transplantation and rapid regeneration is an essential post-operative requirement. Ischemic preconditioning (IPC) has been shown to be an effective strategy in the reduction of hepatic ischemia-reperfusion injury and stimulation of liver regeneration. This study was designed to evaluate the effects of IPC on liver regeneration in small-for-size liver grafts. METHODS: We employed a rat orthotopic liver transplantation model using small-for-size (30%) grafts, in the presence or absence (control) of IPC (10 min of ischemia followed by 15 min of reperfusion). Survival rate, graft injury, hepatocellular proliferation, cell cycle progression, Stat3 activation, as well as TNF-alpha and IL-6 expression were assessed. RESULTS: IPC significantly enhanced the extent of graft injury and hindered hepatic regeneration in small-for-size liver grafts. The 7-day survival rate was also reduced by IPC, but failed to reach statistical significance. IPC did not affect TNF-alpha levels, but significantly decreased the elevation of IL-6 after reperfusion. These findings were correlated with down-regulation of cyclin E and cyclin D1, and decreased numbers of PCNA-positive nuclei in IPC grafts. These results were inconsistent with Stat3 activation, as P-Stat3 exhibited a stronger and prolonged pattern of expression in the IPC group, compared to controls. CONCLUSIONS: Ischemic preconditioning may impair liver regeneration in small-for-size liver grafts by decreasing IL-6 and blunting cell cycle progression, through a mechanism at least partially independent of Stat3.     

Ischemic preconditioning impairs liver regeneration in extended reduced-size livers

OBJECTIVE: To evaluate the effect of ischemic preconditioning (IPC) in an experimental setting of extended liver resection with 30 minutes of inflow occlusion in rats. SUMMARY BACKGROUND DATA: IPC has been proven an effective strategy against hepatic ischemia-reperfusion injury in both animal and human studies. However, decreased protective effects in terms of transaminase levels were found in patients with larger resection volume, questioning the benefit of IPC in case of small liver remnants. METHODS: Rats undergoing 90% hepatectomy under strict inflow occlusion for 30 minutes were subjected to either receive or not receive an IPC period (5 minutes of ischemia followed by 30 minutes of reperfusion). In addition to 10-day survival rate, laser Doppler flowmetry of hepatic blood flow and fluorescence microscopic analysis of the hepatic microcirculation were performed to assess the effect of IPC on initial microvascular reperfusion of liver remnants after 90% resection. Moreover, regeneration capacity of livers undergoing IPC and 70% resection was studied over 7 days by means of histology and immunohistochemistry. RESULTS: Ten-day survival of rats which underwent IPC and 90% hepatectomy was 0 out of 10 animals versus 1 out of 10 animals without IPC. Hemodynamic and microcirculatory analysis revealed signs of hyperperfusion during initial reperfusion of preconditioned liver remnants in 90% hepatectomized animals. In addition to increased transaminase levels, IPC impaired hepatic proliferative response after 70% organ resection, as indicated by both a significant reduction in mitotic figures and Ki-67 nuclear staining of hepatocytes, as well as a decrease in restitution of liver mass. CONCLUSIONS: Though portal hypertension reflecting shear stress has been reported to trigger liver regeneration, remnant liver tissue after major hepatectomy may not benefit from hyperperfusion-induced trigger for cell cycle entry but is rather dominated from hyperperfusion-induced local organ injury. Further studies are required to finally judge on the harmfulness of IPC in extended liver resection.     

血晶素对大鼠70%肝脏切除后再生的影响

目的 探讨血晶素(Hemin)对大鼠70%肝脏切除术后肝脏再生的影响.方法 复制大鼠70%肝脏切除模型,随机分成血晶素治疗组和生理盐水对照组,在术后第1天、第2天、第3天和第7天测定比较肝重/体重、血清肿瘤坏死因子α(TNF-α)、肝脏组织中血晶素加氧酶1(HO-1)含量及肝细胞核增殖蛋白抗原(PCNA)表达指数.结果 术后第7天治疗组肝重/体重明显高于对照组(P<0.05),第3天开始肝细胞 PCNA表达指数较对照组升高,差异有统计学意义(P<0.01).治疗组术后肝脏组织中HO-1浓度比对照组高,血清TNF-α的含量比对照组低(P<0.05).结论 血晶素大鼠肝脏70%切除术后肝脏再生的速度明显提高,这可能跟HO-1表达升高有关.     

Ischemic preconditioning and liver tolerance to warm or cold ischemia: experimental studies in large animals

BACKGROUND: In the rodent, ischemic preconditioning (IPC) has been shown to improve the tolerance of the liver to ischemia-reperfusion under normothermic or hypothermic conditions. The aim of the present study was to test this hypothesis in a dog model, which may be more relevant to the human. METHODS: Beagle dogs were used in two distinct animal models of hepatic warm ischemia and orthotopic liver transplantation (hypothermic ischemia). IPC consisted of 10 minutes of ischemia followed by 10 minutes of reperfusion. In the first model, livers were exposed to 55 minutes prolonged warm ischemia and reperfused for 3 days (n = 6). In the second model, livers were retrieved and preserved for 48 hours at 4 degrees C in University of Wisconsin solution, transplanted, and reperfused without immunosuppression for 7 days (n = 5). In each model, nonpreconditioned animals served as controls (n = 5 in each group). Also, isolated dog hepatocytes were subjected to warm and cold storage ischemia-reperfusion to model the animal transplant studies using IPC. RESULTS: In the first model (warm ischemia), IPC significantly decreased serum aminotransferase activity at 6 and 24 hours post-reperfusion. After 1 hour of reperfusion, preconditioned livers contained more adenosine triphosphate and produced more bile and less myeloperoxidase activity (neutrophils) relative to controls. In the second model (hypothermic preservation), IPC was not protective. Finally, IPC significantly attenuated hepatocyte cell death after cold storage and warm reperfusion in vitro. CONCLUSIONS: IPC is effective in large animals for protecting the liver against warm ischemia-reperfusion injury but not injury associated with cold ischemia and reperfusion (preservation injury). However, the IPC effect observed in isolated hepatocytes suggests that preconditioning for preservation is theoretically possible.     

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.     

二氮嗪预处理抑制大鼠肝缺血再灌注所致细胞凋亡的延迟保护作用

目的：探讨二氮嗪(DE)预处理模拟缺血预处理(IP)抑制肝缺血再灌注(I/R)损伤所致细胞凋亡的延迟保护(DP)作用及其可能机制。方法：大鼠随机分为5组:IP组以肝缺血5min作I/R预处理;DE组静脉注射DE 作I/R预处理;DE+5-HD组在DE组基础上再予静脉注射5-HD作预处理;对照组(C组)仅以等量生理盐水作预处理;假手术组（S组）仅行2次开腹手术，不作其他处理。4个预处理组均在24h后行肝缺血1h再灌注3h。切取肝组织用免疫组化法检测Bcl-2蛋白表达及用TUNEL法检测肝细胞凋亡,并观察显微结构变化。结果： C组肝细胞凋亡指数(AI)明显高于S组(P<0.01),光镜与电镜下肝脏结构损伤明显;IP组与DE组Bcl-2蛋白表达指数(BI)高于C组(P<0.01),AI明显低于C组(P<0.05),组织损伤也轻于C组;而DE+5-HD组BI低于DE组(P<0.01),AI则高于DE组(P<0.05)。结论：使用DE预处理能模拟IP抗大鼠I/R损伤所致肝细胞凋亡的DP作用,可能系通过诱导肝细胞Bcl-2蛋白表达上调而发挥抗凋亡作用。     

Advantage of ischemic preconditioning for hepatic resection in pigs

BACKGROUND: Ischemic preconditioning (IP) and intermittent inflow occlusion (IO) have provided beneficial outcomes in hepatic resection. However, comparison of these two procedures against warm hepatic ischemia-reperfusion injury has not been studied enough. MATERIALS AND METHODS: Pigs that had undergone 65% hepatectomy were subjected to Control (120 min continuous ischemia, n = 6), IP (10 min ischemia and 10 min reperfusion, followed by 120 min continuous ischemia, n = 6), and IO (120 min ischemia in the form of eight successive periods of 15 min ischemia and 5 min reperfusion, n = 6). We evaluated hepatocyte injury by aspartate aminotransferase, lactate dehydrogenase and hepaplastin test, hepatic microcirculation by hepatic tissue blood flow (HTBF) and endothelin (ET)-1, inflammatory response by tumor necrosis factor-alpha (TNF-alpha), and histopathology after reperfusion. RESULTS: IP prevented hepatocyte injury, HTBF disturbance, and hepatocyte necrosis in histopathology as well as IO. These two groups showed significantly better outcomes than Control. IP produced significantly less ET-1 and TNF-alpha than IO. CONCLUSIONS: IP ameliorated hepatic warm ischemia-reperfusion injury. Furthermore, IP gained more advantages in preventing chemokine production such as ET-1 and inflammatory response over IO. IP could take the place of IO for hepatectomy.     

Beneficial effect of deletion variant of hepatocyte growth factor for impaired hepatic regeneration in the ischemically damaged liver

This study was conducted to determine the influence of hepatic ischemia and reperfusion (HIR) injury on liver regeneration and the effect of the deletion variant of hepatocyte growth factor (dHGF) under these conditions. Male Sprague-Dawley rats were subjected to 60 minutes of total hepatic ischemia, and two-thirds hepatectomy was performed just before reperfusion. Animals received intravenous administration of either vehicle buffer (vehicle control group) or dHGF (1 mg/kg) (HGF group) at the end of the period of hepatic ischemia and again 6 hours after reperfusion. At 8 hours after hepatectomy, plasma HGF levels in the vehicle control group were significantly lower than those in the nonischemic controls. Plasma aspartate transaminase levels in the vehicle control group reached 3,462 +/- 1,039 IU/L, but levels in the HGF group were significantly inhibited to 1,849 +/- 605 IU/L. The relative liver weight in the vehicle control group was significantly greater than in the HGF group, a finding that was implicated in focal liver necrosis with sinusoidal congestion. Less histological damage was observed in the HGF group. Twenty-four hours after hepatectomy, an increase in the relative liver weight in nonischemic controls and in the HGF group was higher than that in vehicle control group, and the 5-bromo-2?deoxyuridine (BrdU) labeling index in the HGF group was 23% versus 18% in the nonischemic controls. Administration of dHGF significantly improved the 7-day survival to 82% versus 40% in the vehicle control group. dHGF has potential benefit as a pharmacological agent to ameliorate impairment of the hepatic microcirculation and to potentiate a regeneration response in the ischemically damaged liver after hepatectomy and/or liver transplantation.     

Preventive Effect of Fibroblast Growth Factor and Hepatocyte Growth Factor on Ceramide‐Induced Dysfunction Human Small Intestinal Cells

Aim:  To investigate whether TNF‐ is necessary for hepatocyte proliferation, we study liver regeneration after partial hepatectomy in mice lacking TNF receptor‐1.
Methods:  TNF receptor type‐1 knockout mice and wild‐type mice were subjected to two‐thirds partial hepatectomy (PHx). Liver regeneration was evaluated by assessing liver weights and Ki67 immunohistochemistry. Riken cDNA microarray analysis was performed on liver samples from mice undergoing PHx to compare clearly differentiated mouse PHx models (TNFR‐1 knockout mice‐K group, and wild type mice‐W group).
Results:  The cumulative survival after PHx in K group was lower than in W group. The mortality rate in K group during the first 3 days after PHx was higher (33%) than in W group. The time to regain the liver weight in K group was 14 days and 7 days in W group. The plasma IL‐6 levels in K type at 3 hr was significantly higher than in W group. The Ki67 expression in K group at 4 days was lower than in W group. LPS, Toll like receptor 4 precursor and MAPK 8 interacting protein in K group was higher than in W group. For cell cycle‐regulated genes, cyclin D1, NFB light chain and TNF receptor super family membrane 1a in K group was lower than in W group.
Conclusions:  Lack of TNF‐ signaling through TNF receptor type 1 suppresses liver regeneration after partial hepatectomy in spite of enhancement of LPS–JNK pathway, no TNF‐a and IL‐6 pathway.     

FR167653 improves survival and pulmonary injury after partial hepatectomy under ischemia/reperfusion in rats.

BACKGROUND: FR167653 is a potent suppressant of production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta, which play an important role in hepatic and pulmonary injury due to ischemia/reperfusion of the liver and in liver regeneration after hepatectomy. We examined the effects of FR167653 on hepatectomy under ischemia/reperfusion in rats. METHODS: After initial 15-min ischemia and 5-min reperfusion, 70% hepatectomy was performed during the second 15-min ischemia period in FR167653-treated (FR group) and saline-treated (saline group) rats. The survival rate, relative liver weight, TNF-alpha, IL-1 beta, DNA synthesis rate of the remnant liver, and histological change and adhesion molecule (ICAM-1) of the lung were examined. Serum glutamic pyruvic transaminase and hepatic malondialdehyde were also measured. RESULTS: Expressions of TNF-alpha and IL-1 beta in the remnant liver were significantly inhibited in the FR group compared to the saline group. The survival was significantly better and pulmonary damage was less in the FR group after hepatectomy under ischemia/reperfusion. ICAM-1 expression of the lung was not altered after hepatectomy and was not significantly different between the two groups. Liver regeneration and injury were not significantly different between the two groups. CONCLUSION: FR167653 does not affect liver injury and regeneration after hepatectomy under ischemia/reperfusion, while it ameliorates pulmonary injury and improves the survival.     

肝切除联合肝动脉切除对梗阻性黄疸大鼠肝细胞再生和凋亡的影响

目的 探索在梗阻性黄疸时,不同范围肝切除联合肝动脉切除对肝细胞再生和凋亡的影响.方法 155只雄性SD大鼠行胆总管结扎制备梗阻性黄疽模型,5 d后二次手术分为:胆肠再通内引流组;肝切除(42%、70%)联合胆肠再通内引流组;肝切除(42%,70%)联合肝固有动脉切除、胆肠再通内引流组.动态观察二次手术后24 h、72 h、7 d肝组织HGF、bcl-2 mRNA含量及蛋白表达、肝细胞增殖和凋亡指数的变化,并统计各组死亡率.结果 高胆红素血症、行胆肠冉通内引流的同时,大鼠肝切除或肝切除联合肝动脉切除后,肝再生均受抑制,凋亡增多;较之肝切除组和42%肝切除联合肝固有动脉切除组.70%肝切除联合肝固有动脉切除组术后肝组织HGF、bcl-2 mRNA含量显著减少,肝细胞再生明显受抑而凋亡显著增多,死亡率显著增高(P<0.05).结论 高胆红素血症时,肝切除量是影响大鼠肝切除联合肝动脉切除实施安全性的重要因素,42%肝切除联合肝固有动脉切除、胆肠再通内引流,对肝细胞再生和凋亡影响较小,安全町行;70%肝切除联合肝动脉切除、胆肠再通内引流后肝细胞再生显著受抑制,凋亡增多,死亡率高,应避免实施.     

缺血预处理对大鼠肝脏部分切除术后残留肝脏的保护作用及其机制的研究

目的　探讨缺血预处理 (ischemicpreconditioning ,IPC)对大鼠肝脏部分切除术后残留肝脏的保护作用及其机制。方法　 5 0只雄性SD大鼠随机均分为假手术 (Sham)、单纯热缺血 (warmis chemia ,WI)、IPC、IPC L arginine(NO供体 )和WI NAME(NO合成酶抑制剂 ) 5组。术前、术后 1、2、3d检测血清AST和ALT ,术前、IPC后、热缺血后 0 5、1、2、3h检测肝脏组织NO浓度。AST和ALT用自动生化分析仪检测 ,NO用硝酸还原法检测。结果　WI、IPC、IPC L arginine及WI NAME组术后AST和ALT均高于Sham组 (P <0 0 5或P <0 0 1) ,IPC和WI NAME组术后AST和ALT低于WI和IPC L arginine组 (P <0 0 5 )。WI、IPC、IPC L arginine及WI NAME组术后 0 5h肝脏组织NO浓度开始升高(P <0 0 5或P <0 0 1) ,IPC和WI NAME组术后肝脏组织NO浓度低于WI和IPC L arginine组 (P <0 0 5 )。结论　IPC对大鼠肝脏部分切除术后残留肝脏的缺血再灌注损伤有保护作用。IPC通过抑制大鼠热缺血再灌注肝脏产生NO ,减少NO所诱发的肝脏组织细胞的凋亡或坏死 ,保护肝脏功能。     

Preconditioning protects against ischemia/reperfusion injury of the liver

Ischemic preconditioning (IPC) of an organ may induce protection against the injury caused by longer duration of ischemia and subsequent reperfusion. In a standardized model of such injury in the rat liver, we used the following protocol to investigate whether adenosine played a role in IPC by preventing its enzymatic degradation by dipyridamole pretreatment according to the following protocol: group 1, non-ischemic control rats; group 2, ischemic control rats subjected to 60 minutes of ischemia by clamping of the common hepatic artery followed by 60 minutes of reperfusion; group 3, IPC with 10 minutes of ischemia followed by 15 minutes of reperfusion, prior to the ischemia/reperfusion period as in group 2; group 4, pharmacologic preconditioning with administration of dipyridamole prior to the ischemia/reperfusion period as in group 2. Peripheral liver blood flow was significantly reduced during clamping (groups 2 to 4). After unclamping, blood flow was still reduced in the ischemic rats (group 2) but had returned to preclamp values in the animals that had been subjected to ischemic (group 3) or pharmacologic (group 4) preconditioning. Liver cell injury was significantly increased in the ischemia group (group 2) only. In our experimental model of ischemia/reperfusion injury in the rat liver, we found an equally beneficial effect with ischemic and pharmacologic preconditioning. Adenosine appears to be a crucial factor in IPC.     

Protective effect of ischemic preconditioning against intermittent warm-ischemia-induced liver injury

BACKGROUND: Although ischemic preconditioning (IPC) has been reported to protect the liver from injury when subjected to continuous hepatic ischemia, whether IPC protects rat livers against ischemia-reperfusion (I/R) injury after intermittent ischemia has not been elucidated. MATERIALS AND METHODS: Five groups of Wistar rats were subjected to intermittent hepatic ischemia (I) comprising 15-min ischemia and 5-min reperfusion three times with or without prior IPC (10-min ischemia and 10-min reperfusion), 45-min continuous ischemia (C) with or without IPC, and sham operation. Serum transaminase and lactic acid levels, hepatic tissue energy charges, and hepatic blood perfusion were measured after reperfusion. Plasma tumor necrosis factor-alpha (TNF-alpha) levels were determined after reperfusion for 120 min. Histological and apoptotic findings were evaluated after reperfusion for 180 min. RESULTS: IPC significantly reduced serum transaminase levels after continuous and intermittent ischemia (IPC + C, 1107 vs C, 2684 IU/l; IPC + I, 708 vs I, 1859 IU/l). After hepatic ischemia without IPC, apoptosis and necrosis with increased plasma TNF-alpha levels were observed. IPC protected livers from injury by interfering with the increase in plasma TNF-alpha (IPC + I, 27.6 vs I, 64.8 pg/ml; IPC + C, 21.6 vs C, 49.3 pg/ml). This resulted in the attenuation of hepatic necrosis after continuous ischemia and significantly reduced necrosis and apoptosis after intermittent ischemia. CONCLUSIONS: IPC exerts a greater protective effect against hepatic I/R injury after intermittent hepatic ischemia than after continuous hepatic ischemia.     

缺血或药物预处理对大鼠供肝缺血再灌注损伤的抑制作用

目的　探讨缺血预处理 (IPC)或阿霉素预处理 (DPC ,模拟IPC)对大鼠供肝延迟性保护作用的发生机制。方法　将供鼠分为 3组。IPC组 :供鼠采用肝脏预先缺血 10min后再开放 ;DPC组 :供鼠经静脉注射阿霉素 (1mg/kg体重 ) ;对照组 :供鼠用等量生理盐水注射。观察各组预处理后血红素氧化酶 1(HO 1)和热休克蛋白 70 (HSP70 )含量 ;建立上述各组大鼠原位肝移植模型 ,并设假手术对照组 ,观察肝移植后各组对供肝缺血再灌注损伤的影响。结果　IPC组HO 1、HSP70含量分别于预处理 12h和 2 4h达到高峰 ;IPC和DPC组预处理 2 4h ,诱导的HSP70、HO 1含量差异无显著性 (P >0 .0 5 )。对照组肝移植后 6h ,肝组织中ICAM 1mRNA表达和内皮细胞ICAM 1分子表达明显增强 ,髓过氧化物酶 (MPO)活性增高 ,血清中天冬氨酸转氨酶 (AST)、丙氨酸转氨酶 (ALT)、乳酸脱氢酶 (LDH)及肝组织湿重 /干重 (W/D)水平明显升高 ,和假手术组相比 ,差异有显著性 (P <0 .0 1)。IPC或DPC组肝移植后减弱了ICAM 1mRNA和蛋白表达及MPO活性 ,AST、ALT、LDH及W/D的水平亦明显降低 ,与对照组比较 ,差异有显著性 (P <0 .0 5 )。结论　IPC的延迟保护作用是通过降低中性粒细胞的粘附浸润来实现的 ,这与IPC诱导生成HSP70和HO 1有关。DPC可以模拟IPC的延迟性保护     

缺血预处理对大鼠肝脏缺血/再灌注损伤的延迟保护作用及机制

目的 研究缺血预处理(IPC)对大鼠肝脏缺血/再灌注损伤的延迟保护作用,并探讨线柱体ATP敏感性钾通道(mitoKATP通道)在这种保护机制中的作用. 方法 SD大鼠随机分为5组(每组8只).IPC组以肝缺血5 min作预处理;DE组以静脉注射mitoKATP通道选择性开放剂二氮嗪(DE)作为预处理;IPC+5-HD组是在IPC组基础上再予静注mitoKATP通道特异性阻滞剂5-hydroxydecanoate(5-HD)进行预处理;对照组(C组)仅以静注等量生理盐水作为预处理;上述4组均在预处理24 h后行肝缺血1 h再灌注3 h,缺血方式均为70%肝脏热缺血.假手术组(S组)仅行两次开腹手术,不作其它处理.完成预定实验操作后取血用于血清谷丙转氨酶(ALT)与乳酸脱氢酶(LDH)检测,切取肝组织用于测定超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、湿重/干重(W/D)及观察显微及超微结构变化. 结果 C组ALT,LDH,MDA及W/D值明显高于S组(P<0.01),而SOD活性明显低于S组(P<0.01),肝脏的显微及超微结构损伤明显;IPC组与DE组的各项肝损伤指标均明显好于C组(P<0.05及P<0.01);IPC+5-HD组的肝损伤指标均差于IPC组(P<0.05及P<0.01). 结论 缺血预处理对正常大鼠肝脏I/R损伤具有延迟保护作用,肝细胞mitoKATP通道的开放在其中发挥了重要作用,作用途径可能与诱导肝脏SOD活性增加,改善肝组织微循环,减轻肝脏水肿有关.     

广泛肝切除联合肝固有动脉切除对正常大鼠及梗阻性黄疸大鼠的影响

目的 探索在正常及高胆红素血症情况下,大鼠70%肝切除联合肝固有动脉切除对肝功能、肝细胞能量代谢以及肝再生和细胞凋亡的影响.方法 雄性成年SD大鼠133只,将其中40只分为2组,每组20只,均行胆总管-十二指肠插管桥接,同时行70%肝切除或70%肝切除联合肝固有动脉切除.另87只行胆总管结扎制备梗阻性黄疸模型.5 d后手术分为70%联合肝切除胆肠再通内引流组,及70%肝切除联合肝固有动脉切除、胆肠再通内引流2组.动态观察术后24 h、72 h、7 d肝功能和肝细胞能量代谢、肝组织HGF和bcl-2 mRNA含量及其蛋白表达、肝细胞增殖指数和凋亡指数的变化,并统计各组死亡率.另取6只作为假手术组,测定术后0 h肝功能和肝细胞能量指标.结果 正常大鼠能够耐受70%肝切除联合肝动脉切除,术后肝细胞能量代谢和肝功能迅速恢复正常,肝再生良好.高胆红素血症时,大鼠术后肝再生受抑制,细胞凋亡增多.较之70%肝切除组,70%肝切除联合肝固有动脉切除组对肝细胞能量代谢的影响更为显著,术后肝功能恶化,肝组织HGF和Bcl-2 mRNA含量显著减少,肝再生明显受抑制,细胞凋亡增多,死亡率显著增高(P<0.05).结论 正常大鼠70%肝切除联合肝动脉切除术后肝再生不受影响,高胆红素血症时,70%肝切除联合肝动脉切除的大鼠死亡率高,因此术前引流减黄应是必要的措施.