Effect of cold aerobic perfusion on nonparenchymal cell viability of rat livers

Abstract We investigated the effect of oxygen supply on hepatic cellular viability during cold perfusion storage of rat livers- A perfluoro- N -methyldecahydroisoquinoline (FMIQ) emulsion is used as an oxygen carrier. The composition of the perfusate containing 20 w/v% FMIQ is essentially the same as the University of Wisconsin (UW) solution except for the exclusion of hydroxyethyl starch. Rat livers were perfused at 4°C for up to 24 h with either UW solution (group I, oxygenated; group II, unoxygenated) or FMIQ solution (group III, oxygenated; group IV, unoxygenated). After perfusion storage, the livers were reperfused with warm (37 °C) oxygenated or cold (4 °C) unoxygenated Krebs-Henseleit bicarbonate buffer, and nuclear trypan blue uptake was measured as the index of cell death. With warm oxygenated reperfusion, there remained less than 2% noviable parenchymal cells up to 24 h, regardless of perfusate or oxygenation. In UW-perfused livers, the proportion of nonviable nonprenchymal cells (NPC) increased progressively regardless of oxygenation, the values in groups I and II in the periportal field at 24 h being 39.9 ± 4.7% (mean ± SD) and 36.5 ± 4.2%, respectively. By contrast, in FMIQ-perfused livers, dye uptake by NPC was significantly reduced with oxygenation (16.9 ± 5.7% and 39.4%± 9.1% at 24 h in groups III and IV; P < 0.001). With cold unoxygenated reperfusion, livers in groups I, II, and IV showed a significant decrease of nonviable NPC, while those in group III showed no significant changes. These data indicate that oxygen supply during perfusion storage of the liver may ameliorate lethal injury to NPC precipitated during reperfusion.     

N-乙酰半胱氨酸对鼠肝冷缺血/再灌注损伤的保护作用

目的 为提高肝移植手术后供肝存活率,研究抗氧化剂Ｎ－乙酰半胱氨酸（ＮＡＣ）预处理是否能减少移植肝的冷缺血／再灌注损伤。方法 从大鼠肠系膜上静脉分别注入ＮＡＣ１５mg/kg或５％葡萄糖1ml,15min后取肝,以４℃ＵＷ液保存４８h,再将鼠肝置于离体灌注仪上循环灌流2h。取各时点样本测转氨酶、乳酸、酸性磷酸酶和ＡＴＰ。使用谷胱甘肽合成抑制剂ＢＳＯ处理组,在取肝前2h经用腔注射ＢＳＯ3mmol/kg。结果 ＮＡＣ可以减少鼠肝冷缺血／再灌注后ＡＳＴ、ＡＬＴ和ＬＤＨ释放,提高胆汁分泌量。ＮＡＣ抑制离体鼠肝酸性磷酸酶释放。用谷胱甘肽合成抑制剂ＢＳＯ预处理后,ＮＡＣ减少ＡＳＴ、ＡＬＴ和ＬＤＨ释放,提高胆汁产量的作用依然存在。结论 ＮＡＣ可抑制鼠肝冷缺血／再灌注损伤,其保护作用与直接抑制枯否细胞激活有关,而与促肝细胞谷胱甘肽合成作用无关。     

The effect of controlled reperfusion in porcine hearts submitted to three hours of cold global ischemia

Abstract. At present, many investigations of myocardial function following ischemic insults concentrate on the modalities of reperfusion rather than on the mode of preservation. In this study, we tried to define the effect of reperfusion using warm blood cardioplegia (WBC) after medium-term (3 h) cold global ischemia, as required in cardiac transplantation. Twenty-one porcine hearts were harvested after preservation with cold cardioplegia (St. Thomas Hospital solution) and topical cooling. Normothermic reperfusion with blood was initiated after 3 h of ischemia utilizing a special extracorporeal pump circuit. Twelve hearts served as controls (group A), while substrate-enriched WBC was applied during the initial 20 min of reperfusion in nine hearts (group B). Hearts in both groups were then studied for myocardial function and metabolism under both working and nonworking conditions for a maximum of 180 min. In the nonworking mode, left ventricular dp/dt was significantly higher in group B than in group A at 15 min (2201 ± 785 mm Hg/sec vs 1515 ± 732 mm Hg/sec) and at 180min (1730 ± 471 mm Hg/sec vs 836 ± 147 mm Hg/sec; P <0. 05). After 3 h, lactate production was significantly higher in group A (371 ± 45mg/dl) than in group B (108 ± 44 mg/dl; P < 0. 05). Creatine kinase release into the coronary sinus was also significantly elevated in group A at-15min (2807 ± 1478 IU/l vs 1148 ± 1272 IU/l; P < 0. 05). Similarly, the hemodynamic data obtained under working conditions in group B were superior to those in group A. We conclude that following 3 h of cold global ischemia, reperfusion with WBC improves myocardial function and metabolism. Cautious application in clinical heart transplantation is recommended.     

The calcium channel blocker nisoldipine minimizes the release of tumor necrosis factor and interleukin-6 following rat liver transplantation

Kupffer cells, when activated, release toxic cytokines such as tumor necrosis factor (TNF), which can cause tissue injury. Takei et al. have reported that nisoldipine, a calcium channel blocker which decreases phagocytotic activity by Kupffer cells, also diminishes liver and lung injury and dramatically improves survival following liver transplantation [27]. Therefore, we studied the effect of nisoldipine on the time course of TNF and interleukin-6 (IL-6) release following cold storage and liver transplantation in the rat. Livers were stored under survival and non-survival conditions in cold Euro-Collins solution in the presence or absence of nisoldipine (1.4 µM). After storage, the effluent was collected for determination of cytokines. The liver was then transplanted orthotopically and serum was collected at various time intervals for up to 5 h. In the effluent, TNF levels were very low in both the control and nisoldipine-treated groups and IL-6 was not measurable. Furthermore, when livers were stored under survival conditions and transplanted (liver stored in the cold for 4 h), serum TNF (2 U/ml) and IL-6 (350 U/ml) values were minimal in both the control and nisoldipine-treated groups. In contrast, when livers were stored under non-survival conditions and transplanted (liver stored in the cold for 10 h), TNF levels increased to 15 ± 2 U/ml, 150 min after graft reperfusion, an increase which was prevented by nisoldipine (6.5 U/ml). Serum IL-6 levels were also elevated 300 min after transplantation in livers stored for 10 h. Nisoldipine also reduced the release of this cytokine. Serum transaminases (SGOT) were elevated to values around 2000 U/l 5 h following transplantation. In the nisoldipine-treated group, values were lower between 60 and 300 min. In the lung, interstitial and alveolar edema and cellular infiltration were detectable 5 h post-operatively and were diminished by nisoldipine. These data confirmed that TNF and IL-6 release were minimal following cold storage and transplantation of livers stored under survival conditions, but were elevated transiently after transplantation under non-survival conditions. Nisoldipine prevented cytokine release, most likely by blocking the activation of Kupffer cells, which may explain how it decreases liver and lung injury very early following liver transplantation.     

Impact of ischemic preconditioning on dynamics of homing of endothelial progenitor cells after renal ischemia reperfusion injury

Objectives To investigate the impact of ischemic preconditioning (IPC) on dynamics of homing of endothelial progenitor cells (EPCs) after renal ischemia reperfusion injury (IR). Methods Sixty male Sprague-Dawley rats were randomly divided into three groups after right-side kidney nephrectomy: for sham-operated rats, lumbotomy without vascular clamping was performed; IR rats were clamped renal blood vessels for 40 minutes while IPC rats were pre-treated with 15 min ischemia and 10 min reperfusion. At 3, 12, 24 h, and 3 days after reperfusion, the pool of circulating, kidneys, lungs and spleens were harvested. The extent of renal injury was assessed by biochemical and histological examination. The dynamics of homing of EPCs was observed by flow cytometry. Results The rats in IPC group exhibited significant improvements in renal function and morphology. Compared with IR group and sham group, the number of EPCs in blood was increased in the IPC group at 12 h and 24 h after reperfusion (P＜0.05). The number of EPCs in kidney was increased at all times point in the IPC group and IR group as compared to the sham group (P＜0.05. In addition, EPCs number was increased in IPC group compared with the IR group at 12 h and 24 h [(11.36±0.66)% vs (6.37±0.69)%, (6.31±0.70)% vs (4.40±0.60)%, all P＜0.05]. Compared with IR group and sham group, the number of EPCs in the lung was increased in the IPC groups at 12 h after reperfusion [(2.95±0.66)% vs (1.78±0.59)%, (1.66±0.61)%, all P＜0.05]. The number of EPCs in spleen was increased in the IPC group at 72 h as compared with the IR group and sham group [(0.55±0.06)% vs (0.34±0.07)%, (0.31±0.06)%, all P＜0.05]. Conclusions Endogenous EPCs may home to injured kidney after IPC. EPCs can also gather in the lungs and spleen.     

体外持续肝脏灌注常温保存和HTK液低温保存无心跳供肝效果的比较

目的 比较应用组氨酸-色氨酸-酮戊二酸(HTK)液低温保存和体外持续肝脏灌注(ECLP)系统常温保存无心跳供肝的效果.方法 按保存方法不同将供肝随机分为A组和B组:供肝切取后,A组用HTK液在低温下保存10 h;B组用ECLP系统在常温下用稀释的自体血液持续灌注10 h.两组供肝再经过60 min冷缺血期后,连接ECLP系统用稀释的自体血液再灌注4 h.观察再灌注后1、2、3、4 h四个时间点的胆汁分泌量,门静脉和肝动脉的压力,肝脏耗氧率的变化,灌注液中丙氨酸转氨酶(ALT)、乳酸脱氢酶(LDH)、葡萄糖水平以及灌注后供肝的常规病理和超微病理变化.结果 B组再灌注后1、2、3、4 h时间点的胆汁分泌量,门静脉和肝动脉的压力,灌注液中ALT、LDH和葡萄糖水平,以及2、3、4 h时间点的耗氧率与A组比较,差异均有统计学意义(P＜0.05或P＜0.01);B组供肝的病理损害程度较A组轻.结论 供肝切取后10 h内,利用ECLP系统持续灌注常温保存比用HTK液单纯低温保存在维持无心跳供肝的功能和生理活性方面效果更好.     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

目的 比较Celsior(CS)液与UW液对大鼠无心跳供者(NHBD)供肝的保存效果.方法 选取健康雄性SD大鼠作为肝移植的供、受者.通过阻断大鼠主动脉和膈上下腔静脉10 min的方法,制备和获取NHBD供肝,并采用不同的器官保存液灌注和冷保存供肝.随机将受者分为4组.CS8 h组:受者采用经CS液灌注和冷保存8 h的供肝移植;UW8 h组:受者采用经UW液灌注和冷保存8 h的供肝移植;CS16 h组:受者采用经CS液灌注和冷保存16 h的供肝移植;UW16 h组:受者采用经UW液灌注和冷保存16 h的供肝移植.受者门静脉开放前、开放后1、3及6 h,取各组受者的静脉血检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、内皮素1(ET-1)、白细胞介素1(IL-1)及肿瘤坏死因子α(TNF-α)水平;观察和比较各组受者的胆汁生成量、移植肝组织病理学改变及术后7 d内的存活率.结果 NHBD供肝经UW液灌注后呈"花斑"状,肝叶边缘灌注不良,经CS液灌注后肝叶边缘灌注良好.CS8 h组和UW8 h组受者的胆汁生成量分别为(0.21±0.01)ml和(0.10±0.02)ml(P<0.05).门静脉开放后1、3及6 h,CS8 h组受者的血清ALT及AST水平明显低于UW8 h组(P<0.05),门静脉开放后1、3h,CS8 h组受者的血清ET-1、IL-1及TNF-α水平均明显低于UW8 h组(P<0.05);CS8 h组受者移植肝肝窦扩张、门静脉充血及炎症细胞浸润等病理学改变明显轻于UW8 h组,CS8 h组和UW8 h组受者术后7 d的存活率分别为58.3%和25.0%(P<0.05).CS16 h组和UW16 h组受者各时点的胆汁分泌量、血清ALT、AST、ET-1、IL-1及TNF-α水平的比较,差异均无统计学意义(P>0.05),两组受者均在术后3 d内死亡,两组受者移植肝组织病理学改变无明显差异.结论 CS液对大鼠NHBD供肝的保存效果优于UW液,这可能与UW液较CS液粘稠及CS液能够减少枯否细胞的激活有关;NHBD供肝的冷保存时间不宜超过16 h.     

[beta ]-Galactosidase as a marker of ischemic injury and a mechanism for viability assessment in porcine liver transplantation

Glycohydrolases are a group of enzymes contained predominantly within lysosomes, which are released during Kupffer cell activation or death. One of these, [beta ]-galactosidase, has been proposed as a marker of ischemia-reperfusion injury in the liver because Kupffer cell activation represents a primary event in the injurious reperfusion cascade. In this study, we compared B-galactosidase with more traditional indicators of liver injury and function in a porcine model of liver preservation. Porcine livers were allocated into two groups: group C (n = 5), preserved in University of Wisconsin solution by standard cold storage for 24 hours, and group W (n = 5), perfused with oxygenated autologous blood on an extracorporeal circuit for 24 hours. Both groups were subsequently tested on the circuit during a 24-hour reperfusion phase. The perfusate was sampled for levels of [beta ]-galactosidase, as well as traditional markers of liver injury and function. A sharp increase in [beta ]-galactosidase levels was seen on reperfusion of cold preserved livers to a level of 1,900 IU/mL. This contrasted dramatically with normothermically preserved livers, in which the level never exceeded 208 IU/mL (P = .002). [beta ]-Galactosidase levels showed much earlier and greater increases compared with transaminase levels in livers injured by ischemia. A rapid elevation in [beta ]-galactosidase levels corresponded well with poor liver function and more liver injury. Measurement of [beta ]-galactosidase is a simple test that quantifies ischemia-reperfusion injury of preserved livers. It is more sensitive than transaminases, with faster and larger increases in levels after ischemic injury. It can be useful in assessing the viability of a liver during machine preservation. (Liver Transpl 2002;8:21-26.)     

Protection by pentoxifylline against graft failure from storage injury after orthotopic rat liver transplantation with arterialization

Destruction of the endothelial cell lining and activation of Kupffer cells after reperfusion limits the safe storage of livers for transplantation surgery. Tumor necrosis factor-alpha (TNF) release by activated Kupffer cells may contribute to graft failure from storage injury. Accordingly, we evaluated whether pentoxifylline, which suppresses macrophage TNF release, would improve graft survival after orthotopic rat liver transplantation with arterialization. Livers from syngeneic Lewis rats were stored for 12–24 h in cold UW solution. Prior to implantation, the livers were flushed with cold Ringer's solution or warm Carolina rinse solution B. With either rinse, pentoxifylline treatment of graft recipients significantly improved graft survival. Combined use of pentoxifylline (50 mg/kg for 5 days) and Carolina rinse solution doubled the safe storage time to 24 h. Acidotic pH and antioxidants were essential components of Carolina rinse solution that acted synergistically with pentoxifylline. Pentoxifylline was also shown to suppress TNF release by lipopolysaccharide (LPS)-stimulated cultured rat Kupffer cells. Thus, pentoxifylline may protect against primary non-function and failure of grafts from storage injury by suppressing excessive TNF release by activated Kupffer cells. However, neutralization of TNF with excess anti-TNF antibody did not improve survival. This may mean that depletion of TNF is as deleterious as excess TNF production. Alternatively, other Kupffer cell secretions [e.g., interleukin-1 (IL-1), interleukin-6 (IL-6) and other cytokines] may be involved in the pathogenesis of graft failure. In conclusion, pentoxifylline could protect against graft failure from storage injury.     

Protective effects of ulinastatin against ischemia-reperfusion injury

We investigated the protective effect of urinary trypsin inhibitor (ulinastatin: UTI) in vitro, in relation to the neutrophil activity in hepatic ischemia/reperfusion (I/R) injury. The rat liver was removed and preserved in cold Ringer's lactate solution for 60 min, followed by 120 min of reperfusion with oxygenated perfusate. The rats were divided into four groups (n = 8 in each group). The livers were perfused with Krebs-Henseleit (K-H) solution containing no additives in group 1, 50,000 U/kg of UTI in group 2, 3.5 x 10(6) of neutrophils in group 3, and both neutrophils and UTI in group 4. In group 3, the AST and ALT levels were always higher than those in other three groups at any point evaluated (P < 0.01) and the LDH levels were observed to be significantly higher than those in other three groups at 0, 5, 10, 60, and 90 min after reperfusion (P < 0. 01). These increase were suppressed by additional pretreatment with UTI in group 4. The bile flow during reperfusion was significantly suppressed in group 3 compared to that of group 4, at both 30 (P < 0. 01) and 60 (P < 0.05) min after reperfusion. The MPO activity after reperfusion in group 3 also significantly increased compared to other three groups (P < 0.01). These data thus suggest that UTI ameliorated the ischemia/reperfusion injury in vitro by inhibiting of neutrophil accumulation in the postischemic liver.     

Evaluation of microperfusion disturbances in the transplanted liver after Kupffer cell destruction using GdCl3: an experimental porcine study

Background

Organ function after liver transplantation is determined by ischemia-reperfusion injury. Destruction of Kupffer cells with gadolinium chloride (GdCl₃) has been shown to have a possible preventive effect on the extent of this injury, which can be extrapolated by analyzing the distribution of hepatic microperfusion. The aim of this study was to evaluate the protective effect of GdCl₃ on disturbances of microperfusion in the transplanted liver.

Methods

Landrace pigs were randomly divided into three groups. In the control group (CG; n = 6) a mapping of the native liver was conducted. For mapping, the four hepatic liver lobes were named from right to left with A to D and every lobe was divided into three vertical segments (cranial, medial, and caudal). In each of these 12 areas, microperfusion was quantified using a thermodiffusion probe (TD [mL/100 g/min]). The other two groups were considered as transplanted treated group (TTG; n = 10) and transplanted nontreated group (TnTG; n = 10). The TTG received an infusion of 20 mg/kg GdCl₃ intravenously 24 hours before organ harvesting. Then standardized orthotopic liver transplantation was performed. In TnTG, standardized orthotopic liver transplantation was carried out without prior GdCl₃ injection. In the recipients, the microperfusion of transplanted livers were mapped in both TnTG and TTG, in two different time points (1 hour [n = 5] and 24 hours (n = 5]) after reperfusion.

Results

A significant reduction of macrophages in the TTG livers in comparison to the CG and TnTG livers was observed (P < .05). However, the number of macrophages in CG and TnTG livers showed no significant difference (P > .05). Regarding liver microperfusion, in TnTG, a marked heterogeneity was detected in the livers after reperfusion. Significant differences between liver lobes (horizontal planes; P = .032) and vertical layers of intralobar liver parenchyma (P = .029) were observed. The same pattern was seen in TTG livers after reperfusion and a significant difference between horizontal (P = .024) and vertical layers (P = .018) of liver tissue were observed. Comparing intralobar regional flow data between vertical planes 24 hours after reperfusion still showed a prominent variation of hepatic tissue perfusion in TnTG livers (P = .028). Within the same horizontal layers, no significant differences between lobes were measured anymore (P = .16). Contrary to TnTG, in TTG, a homogenous pattern of regional liver tissue perfusion was recorded 24 hours after reperfusion. Comparison of TD data on the liver regions showed no significant microperfusion differences in either horizontal (P = .888) or vertical (P = .841) layers.

Conclusions

Application of GdCl₃ resulted in a significant reduction of Kupffer cells. Twenty four hours after transplantation microperfusion showed a homogeneous pattern, which constituted an earlier and better recovery of the transplanted liver. Therefore, destruction of Kupffer cells reduced ischemia-reperfusion injury and seemed to be responsible for the early recovery of microperfusion disturbances and thus for an improvement of graft function.     

High concentrations of adenosine are needed in Carolina rinse to prevent activation of Kupffer cells

Abstract Adenosine is a key component of Carolina rinse solution, which was developed to prevent an 0,-dependent reperfusion injury following liver transplantation. Kupffer cells are activated following liver transplantation, an effect minimized by adenosine. In this study, the concentration of adenosine was varied in Carolina rinse, Ringer's, and Ringer's containing hydroxyethyl starch. Following 24 h of cold storage in University of Wisconsin cold storage solution, colloidal carbon uptake was measured to assess phagocytosis by Kupffer cells. Carbon uptake was elevated significantly in this model from values of 136 ± 15 in unstored control livers to 187 ± 25 mg/g per hour following a rinse with 10 ml of Ringer's solution. Phagocytosis was reduced about 50% when 0.1 m M adenosine was added to the Ringer's solution; however, addition of up to 1 m M adenosine in Carolina Rinse did not prevent elevated phagocytosis. In contrast, values were reduced significantly by addition of 5–10 m M adenosine to Carolina Rinse or Ringer's containing hydroxyethyl starch. Thus, the higher concentrations of adenosine needed to reduce Kupffer cell activation can best be explained by binding of adenosine to hydroxyethyl starch.     

Methyl palmitate prevents Kupffer cell activation and improves survival after orthotopic liver transplantation in the rat

The purpose of this study was to determine whether prevention of Kupffer cell activation following orthotopic liver transplantation improves postoperative survival. First, particle phagocytosis by Kupffer cells was monitored continuously from the uptake of colloidal carbon by the perfused liver. Unstored livers took up carbon at rates of around 150 mg/g per hour, whereas storage for 24 h in Euro-Collins solution nearly doubled values to about 290 mg/g per hour. Treatment of rats with methyl palmitate, an inhibitor of phagocytosis by Kupffer cells, reduced carbon uptake to about one-third to one-half of control values in unstored and stored livers, respectively. Oxygen uptake, which was increased about 25% in stored and unstored livers by infusion of colloidal carbon, was only increased 5%–10% in both groups following treatment with methyl palmitate, suggesting that Kupffer cell activation was prevented by methyl palmitate. In livers transplanted after storage for 6 h in Euro-Collins solution (nonsurvival conditions), control rats survived only about 12 h, while treatment with methyl palmitate increased survival time significantly — more than threefold — to about 40 h. These data are consistent with the hypothesis that activation of Kupffer cells following cold ischemic storage and reperfusion is an early event involved in liver graft failure. Present address: Department of Trauma Surgery, University of Saarland, W-6650 Homburg/S., Federal Republic of Germany     

Delayed pharmacological pre-conditioning effect of mitochondrial ATP-sensitive potassium channel opener on neurologic injury in a rabbit model of spinal cord ischemia

Background: Diazoxide, pharmacological openers of mitochondrial ATP-sensitive potassium channels have been shown to induce early pre-conditioning in the spinal cord. Here, the authors investigated whether diazoxide also induce delayed pre-conditioning and thereby reduce neurologic complications using a rabbit model of spinal cord ischemia.
Methods: Infrarenal blood flow was interrupted for 20 min in 21 rabbits. Non-treated control animals received no pre-treatment. Diazoxide (5 mg/kg) were given 48 h before 20 min ischemia in the 48-h DZ group, whereas 15-min DZ group received diazoxide (5 mg/kg) 15 min before 20-min ischemia. Neurological functions were evaluated using Johnson scores for 3 days after reperfusion, after which, spinal cords were procured for hematoxylin and eosin staining for cell counting.
Results: Johnson scores revealed a marked improvement in both the diazoxide-treated groups vs. the non-treated control group at 3, 24, 48, and 72 h after reperfusion ( P <0.01). The histologic changes were proportional to the Johnson scores, with better preservation of motor neuron numbers in the animals of the 48-h DZ and 15-min DZ group relative to the non-treated controls (81±12, 90±10, 50±23 motor neurons, respectively, P <0.01). No difference was found between the 48-h DZ group and 15-min DZ group with respect to the Johnson scores or neuron numbers.
Conclusions: The study demonstrates that pre-treatment with diazoxide 48 h before ischemia, induce delayed pharmacological pre-conditioning, thereby significantly improving clinical neurologic scores and histologic findings in this animal model.     

Methyl palmitate prevents Kupffer cell activation and improves survival after orthotopic liver transplantation in the rat

Abstract. The purpose of this study was to determine whetherprevention of Kupffercell activation following orthotopic liver transplantation improves postoperative survival. First, particle phagocytosis by Kupffer cells was monitored continuously from the uptake of colloidal carbon by the perfused liver. Unstored livers took up carbon at rates of around 150 mg/g per hour, whereas storage for 24 h in Euro-Collins solution nearly doubled values to about 290 mg/g per hour. Treatment of rats with methyl palmitate, an inhibitor of phagocytosis by Kupffer cells, reduced carbon uptake to about one-third to one-half of control values in unstored and stored livers, respectively. Oxygen uptake, which was increased about 25% in stored and unstored livers by infusion of colloidal carbon, was only increased 5%-10% in both groups following treatment with methyl palmitate, suggesting that Kupffer cell activation was prevented by methyl palmitate. In livers transplanted after storage for 6 h in Euro-Collins solution (nonsurvival conditions), control rats survived only about 12 h, while treatment with methyl palmitate increased survival time significantly - more than threefold - to about 40 h. These data are consistent with the hypothesis that activation of Kupffer cells following cold ischemic storage and reperfusion is an early event involved in liver graft failure.     

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.     

血红素氧合酶-1减轻大鼠移植肝缺血再灌注损伤的作用及机制

目的 探讨血红素氧合酶-1(HO-1)减轻大鼠移植肝缺血再灌注损伤的作用及其机制.方法 选择近交系雄性SD大鼠作为肝移植的供、受者;采用单纯随机方法将48只SD大鼠随机分为对照组、抑制组和诱导组(每组供、受者各8只).对照组:供肝不用任何药物处理;抑制组:在获取供肝前24 h,经供者腹腔注射HO-1抑制剂锌原卟啉20 mg/kg进行预处理;诱导组:在获取供肝前24 h,经供者腹腔注射HO-1诱导剂钴原卟啉5 mg/kg进行预处理.获取供肝后,在4℃UW液中冷保存24 h.肝移植前检测供肝HO-1的表达水平;肝移植后6 h采血并获取移植肝标本,分离培养枯否细胞;检测受者的肝功能;检测枯否细胞培养上清中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的含量;观察移植肝组织病理学表现以及枯否细胞CD14 mRNA的表达水平和蛋白含量测定.结果 移植前诱导组供肝HO-1的表达水平明显增高.移植后诱导组血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)含量明显降低;移植肝组织病理学损伤减轻;枯否细胞培养上清中TNF-α和IL-6含量减少;而且枯否细胞上的CD14 mRNA表达水平和蛋白含量也明显低于抑制组.结论 诱导供肝HO-1表达上调可能抑制了枯否细胞的激活,从而减轻大鼠移植肝缺血再灌注损伤.     

肝脏低温保存-再灌注期间肝细胞凋亡及其与Bax基因蛋白相关性的研究

目的　研究临床肝移植过程中供肝低温保存-再灌注期间肝细胞凋亡及其相关机制。方法　应用细胞凋亡原位末端标记法并结合电镜观察,检测低温保存时间分别为0(A组)、3(B组)、6(C组)、9(D组)h的4组兔肝脏在低温保存-再灌注过程中肝细胞凋亡情况。同时,对各组肝脏再灌注前后肝细胞内Bax基因蛋白表达进行测定。结果　A、B、C、D各组肝脏于低温保存后的再灌注60min时,组织内可见明显的肝实质细胞凋亡；其凋亡细胞数量(1×10