Attenuation of acute phase shear stress by somatostatin improves small-for-size liver graft survival.

The major concern of living donor liver transplantation is small-for-size graft injury at the early phase after transplantation. Novel therapeutic strategies should be developed. To investigate the protective effect of somatostatin related to hemodynamic stress on small-for-size liver graft injury, we applied a treatment regimen of low-dose somatostatin in a rat orthotopic liver transplantation model using small-for-size grafts (median, 38.7%; range, 35-42%). Somatostatin was given at 5 minutes before total hepatectomy and immediately after reperfusion in the recipient (20 microg/kg). Graft survival, portal hemodynamics, intragraft gene expression and hepatic ultrastructural changes were compared between the rats with or without somatostatin treatment. Seven-day graft survival rates in the somatostatin treatment group were significantly improved compared to the control group (66.7% vs. 16.7%, P = 0.036). In the treatment group, portal pressure and hepatic surface blood flow were significantly decreased within the first 30 minutes after reperfusion, whereas in the control group, transient portal hypertension and excessive hepatic blood flow were observed. Intragraft expression (both messenger RNA and protein) of endothelin-1 was significantly downregulated accompanied with upregulation of heme oxygenase-1 and A20. Better preservation of liver function was found in the treatment group. Hepatic ultrastructure, especially the integrity of sinusoids, was well protected in the treatment group. In conclusion, low-dose somatostatin rescues small-for-size grafts from acute phase injury in liver transplantation by attenuation of acute-phase shear stress that resulted from transient portal hypertension.     

Fat-Derived Hormone Adiponectin Combined with FTY720 Significantly Improves Small-for-Size Fatty Liver Graft Survival

Owing to the discrepancy between organ donation and the demand for liver transplantation, expanding the liver donor pool is of vital importance. However, marginal liver grafts, such as small-for-size and/or fatty grafts, were associated with primary graft nonfunction or poor function. Therefore, novel combination therapies to rescue small-for-size fatty liver grafts should be investigated. In this study, we applied a combination therapy using a fat-derived hormone adiponectin (anti-steatosis) plus immunomodulator FTY720 (anti-inflammatory) in a rat liver transplantation model using small-for-size fatty liver grafts, and investigated the underlying protective mechanism such as anti-steatosis, intra-graft energy metabolism, hepatic microcirculatory changes, cell signaling cascades for survival, apoptosis and inflammation. The current study demonstrated that even a single treatment of adiponectin or FTY720 improved the 7-day graft survival from 0% to 62.5% (p = 0.001). The combination therapy significantly increased the 7-day graft survival rate to 100% by remarkable attenuation of graft steatosis and acute phase inflammatory response, significant activation of cell survival Akt pathway and maintenance of intra-graft adenosine triphosphate metabolism and improvement of hepatic microcirculation. In conclusion, the fat-derived hormone adiponectin combined with FTY720 might be a novel combination drug therapy for prevention of small-for-size fatty liver graft injury.     

基质金属蛋白酶-9 与小体积移植肝早期损伤的相关性研究

目的 观察不同体积肝移植术后早期移植肝内基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的表达及活化特点,探讨MMP-2和MMP-9在小体积移植肝早期损伤中的作用机制. 方法 随机将108只SD大鼠分成3组,每组36只.分别为:全肝(100%肝体积)移植组、半肝(50%肝体积)移植组和小体积肝(25%肝体积)移植组.分别检测移植肝再灌注后0.5、6、12、24、48、72 h的肝功能及移植肝组织中丙二醛(MDA)和髓过氧化物酶(MPO)浓度,观察移植肝组织病理学特征,并运用双抗夹心酶联免疫吸附试验(ELISA)、实时定量聚合酶链反应(PCR)、明胶酶谱和免疫组织化学方法检查移植肝中MMP-2和MMP-9的表达情况. 结果 与全肝和半肝移植组比较,小体积肝移植组再灌注后6～24 h MMP-9表达明显升高;而且MMP-9活化和表达增高伴随着移植肝功能损害和严重的缺血再灌注损伤.MMP-9早期表达都集中在移植肝门静脉周围,与门静脉高灌注密切相关. 结论 MMP-9表达升高是小体积移植肝早期重要的致损伤因素;肝移植术后门静脉高灌注可能是触发MMP-9活化和表达的重要原因.     

FK 409 ameliorates small-for-size liver graft injury by attenuation of portal hypertension and down-regulation of Egr-1 pathway

OBJECTIVE: To investigate whether low-dose nitric oxide donor FK 409 could attenuate small-for-size graft injury in liver transplantation using small-for-size grafts. SUMMARY BACKGROUND DATA: The major concern of live donor liver transplantation is small-for-size graft injury at the early phase after transplantation. Novel therapeutic strategies should be investigated. METHODS: We employed a rat orthotopic liver transplantation model using small-for-size (40%) graft. FK 409 was given at 30 minutes before graft harvesting (2 mg/kg) to the donor and immediately after reperfusion (1 mg/kg) to the recipient (FK group). Graft survival, intragraft genes expression, portal hemodynamics, and hepatic ultrastructural changes were compared between the 2 groups. RESULTS: Seven-day graft survival rates in the FK group were significantly improved compared with those of rats not receiving FK 409 (control group; 80% versus 28.6%, P = 0.018). In the FK group, portal pressure was significantly decreased within the first 60 minutes after reperfusion whereas in the control group, transient portal hypertension was observed. Intragraft expression (both mRNA and protein) of early growth response-1, endothelin-1, endothelin-1 receptor A, tumor necrosis factor-alpha, macrophage-inflammatory protein-2, and inducible nitric oxide synthase was significantly down-regulated accompanied with up-regulation of heme oxygenase-1, A20, interferon-gamma-inducible protein-10, and interleukin-10 during the first 24 hours after reperfusion. Hepatic ultrastructure, especially the integrity of sinusoids was well protected in the FK group. CONCLUSIONS: Low-dose FK 409 rescues small-for-size grafts in liver transplantation by attenuation of portal hypertension and amelioration of acute phase inflammatory response by down-regulation of Egr-1, together with prior induction of heat shock proteins.     

表达HGF的骨髓间充质干细胞促进小移植肝早期肝再生

目的 建立大鼠小体积肝移植模型,输注表达人肝细胞生长因子(human hepatocyte growth factor,hHGF)的骨髓间充质干细胞(mesenchymal stem cells,MSCs),研究其在移植早期对小移植肝促再生作用.方法 将已建立的表达hHGF和绿色荧光蛋白(green fluorescence protein,GFP)的MSCs,分别命名为HGF/MSCs,GFP/Mscs.建立大鼠30%肝移植模型.受体分为4组,实验组输注5×106HGF/MSCs;对照组则分别输注相同体积的生理盐水(PS),5×106 GFP/MSCs或1.0×109 pfu含hHGF的重组腺病毒液(Ad-HGF).分别于术后1,3,5,7 d各组随机抽取5只大鼠处死.取血检测血清ALT和hHGF.记录移植物湿重.取肝组织检测hHGF、c-met表达,以及肝细胞凋亡和增殖活性.另每组15只,分组同上,用于观察生存期.结果 PS组大鼠7 d生存率33.3%;组织学及血清学检查示术后肝脏损伤重,汇管区单核细胞浸润多;而实验组大鼠7 d生存率为73.3%.肝脏损伤轻,炎性细胞浸润少;实验组移植肝再生较PS组明显增加.结论 大鼠部分肝移植后,输注HGF/MSCs能够保护小体积移植肝,促进小移植肝再生,提高7 d生存率.     

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.     

缺血预处理对大鼠小体积供肝的保护作用及机制

目的探讨缺血预处理（IPC）对大鼠小体积供肝的保护作用及其机制。方法120只SD大鼠随机分为3组（每组20对）：无热缺血组（NWI）、缺血再灌注组（WI）和缺血预处理组（IPC）。用双袖套法建立大鼠小体积肝移植模型。各组10只受体大鼠于术前1d、术后1、2、3、5d取血，用自动生化分析仪检测AST和ALT。NWI组于供肝灌注前及植入后0．5、1、2、3h，WI组于热缺血前及植入后0．5、1、2、3h，IPC组于IPC前、IPC后及植入后0．5、1、2、3h取肝组织，用硝酸还原法检测其NO浓度。结果IPC可降低大鼠小体积肝移植术后血清AST和ALT浓度，提高再灌注早期肝脏组织NO的浓度，降低再灌注晚期肝脏组织NO的浓度（P〈0．05）。结论NO在大鼠肝脏的缺血再灌注损伤中可能具有双重作用。IPC对大鼠小体积供肝的缺血再灌注损伤有保护作用。其机制可能是通过促进供肝再灌注后早期NO合成，改善肝脏微循环，同时抑制供肝再灌注后晚期NO合成，减轻过量NO的损伤作用，从而保护移植肝脏功能。     

Distinct Mechanism of Small‐for‐Size Fatty Liver Graft Injury—Wnt4 Signaling Activates Hepatic Stellate Cells

In this study, we aimed to investigate the significance of hepatic stellate cells (HSCs) activation in small‐for‐size fatty liver graft injury and to explore the underlying molecular mechanism in a rat liver transplantation model. A rat orthotopic liver transplantation model using fatty grafts (40% of fatty changes) and cirrhotic recipients was applied. Intragraft gene expression profiles, ultrastructure features and HSCs activation were compared among the rats received different types of grafts (whole vs. small‐for‐size, normal vs. fatty). The distinct molecular signature of small‐for‐size fatty graft injury was identified by cDNA microarray screening and confirmed by RT‐PCR detection. In vitro functional studies were further conducted to investigate the direct effect of specific molecular signature on HSCs activation. HSCs activation was predominantly present in small‐for‐size fatty grafts during the first 2 weeks after transplantation, and was strongly correlated with progressive hepatic sinusoidal damage and significant upregulation of intragraft Wnt4 signaling pathway. In vitro suppression of Wnt4 expression could inhibit HSC activation directly. In conclusion, upregulation of Wnt4 signaling led to direct HSC activation and subsequently induced small‐for‐size fatty liver grafts injury. Discovery of this distinct mechanism may lay the foundation for prophylactic treatment for marginal graft injury in living donor liver transplantation.     

20%小体积的大鼠肝脏移植模型

目的 应用显微外科技术建立20%小体积移植物的大鼠原位肝脏移植模型.方法 原位移植建立20%小体积大鼠肝脏移植模型.雄性Lewis大鼠40只,供体20只,受体20只.供肝经门静脉用4℃ UW液灌注.肝上下腔静脉用端端吻合连续缝合的方法.肝下下腔静脉和门静脉分别用套管方法固定.套叠缝合法重建肝动脉.胆管重建采用内支架管端端连接的方法.观察移植物的存活率.免疫组化检测肝细胞摄取溴脱氧尿核苷的情况.结果 共施行肝脏移植手术20例,移植手术成功率为100%.20%小体积肝脏移植物的存活率为93.8%(＞14 d).组织学检查移植后的肝脏组织结构良好.移植术后72 h溴脱氧尿核苷染色阳性的肝细胞计数明显增多.结论 20%小体积大鼠肝脏移植物可启动完成移植后的肝脏再生.显微外科技术是移植模型成功的关键.该模型稳定性强,适合于部分肝脏移植领域的基础研究.     

Protective effect of adenosine A2A receptor activation in small-for-size liver transplantation

The aim of the present study was to investigate the potential role of adenosine A(2A) receptor (A(2A)R) activation in small-for-size liver transplantation. A rat orthotopic liver transplantation model was performed by using 40% (range: 36-46%) liver grafts. Recipients were given either saline (control group) or CGS 21680 (2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride, a selective A(2A)R agonist), or CGS 21680+ ZM 241385 (a selective A(2A)R antagonist) immediately after reperfusion for 3 h. Compared with control group, CGS 21680 used at both low dose (0.05 microg/kg/min) and high dose (0.5 microg/kg/min) increased the survival rate from 16.7% (2/12) to 83.3% (10/12) and 66.7% (8/12), respectively. These effects correlated with improved liver function and preserved hepatic architecture. CGS 21680 effectively decreased neutrophil infiltration, suppressed pro-inflammatory (TNF-alpha, IL-1beta and IL-6) expression, promoted expression of antiapoptotic molecules, and inhibited apoptosis. The effects of CGS 21680 were prevented when ZM 241385 was co-administrated. In conclusion, the present study showed that A(2A)R activation alleviated portal hypertension, suppressed inflammatory response, reduced apoptosis, and potentiated the survival of small-for-size liver grafts. Our findings provide the rationale for a novel therapeutic approach using A(2A)R activation to maximize the availability of small-for-size liver grafts.     

大鼠40％小体积肝移植模型建立及移植肝的病理观察

目的建立稳定的大鼠40％小体积肝移植模型，并观察术后移植肝功能与病理的变化。方法采用改良二袖套法建立大鼠40％小体积肝移植模型，观察1w生存率，并于术后1、2、4、7天检测肝功能、移植肝组织学变化和免疫组化法检测肝细胞的增殖细胞核抗原（PCNA）。结果手术成功率97．5％，1w生存率60％，无肝期平均（124±2．5）min，丙氨酸转氨酶（ALT）、总胆红素（TB）术后即第1天即明显增高，第2天最高，以后逐渐降低。组织学检查术后2天即可见较多二倍体和多倍体肝细胞。免疫组化检测移植肝PCNA表达于术后第2天最高。结论通过技术改进，简化了手术操作，提高了手术成功率及模型的稳定性。大鼠小体积移植肝仍具有较强的再生能力。     

Hemodynamic interaction between portal vein and hepatic artery flow in small-for-size split liver transplantation

In split-liver transplantation, the entire portal flow is redirected through relatively small-for-size grafts. It has been postulated that excessive portal blood flow leads to graft injury. In order to elucidate the mechanisms of this injury, we studied the hemodynamic interactions between portal vein- and hepatic artery flow in an experimental model in pigs. Six whole pig liver grafts were implanted in Group 1 ( n=6) and six whole liver grafts were split into right and left grafts and transplanted to Groups 2 ( n=6) and 3 ( n=6), respectively. The graft-to-recipient liver volume ratio was 1:1, 2:3 and 1:3 in Groups 1, 2 and 3, respectively. Portal vein- and hepatic artery flows were measured with an ultrasonic flow meter at 60,120 and 180 min after graft reperfusion. Portal vein pressure was also recorded at the same time intervals. Graft function was assessed at 3,6h and 12h, and morphological changes at 12h after reperfusion. Following reperfusion, portal vein flow showed an inverse relationship to graft size, while hepatic artery flow was reduced proportionately to graft size. The difference was significant among the three groups ( P<0.05). Portal vein pressure was significantly higher in group 3, compared to groups 1 and 2 ( P<0.05). Hepatic artery buffer response was significantly higher in Group 3, compared to Groups 1 and 2 in relation to pre-occlusion values ( P<0.05). Split-liver transplantation, when resulting in small-for-size grafts, is associated with portal hypertension, diminished arterial flow, and graft dysfunction. Arterial flow impairment appears to be related to increased portal vein flow.     

Exogenous biliverdin ameliorates ischemia-reperfusion injury in small-for-size rat liver grafts

OBJECTIVE: This study sought to investigate the protective potential of exogenous biliverdin (BV) for small-for-size rat liver transplants. METHODS AND RESULTS: We employed a rat orthotopic liver transplantation model using small-for-size grafts. BV (50 mumol/kg, intravenously) given to the recipient immediately before reperfusion increased 7-day survival rates (90% vs 40% in controls) and significantly diminished hepatocyte injury, as compared with a control group. These effects correlated with improved liver function and preserved hepatic architecture. BV adjuvant increased antioxidant ability, suppressed proinflammatory tumor necrosis factor-alpha expression, down-regulated proapoptotic molecules (cytochrome C and caspase-3), and inhibited most apoptotic cells. After reperfusion, there was a significant increase of c-Jun NH(2)-terminal kinase (JNK) activation and AP-1 binding ability. BV treatment effectively repressed JNK/AP-1 activation, indicating that a beneficial effect of BV treatment may be related to suppression of the JNK/AP-1 pathway. CONCLUSIONS: BV treatment alleviated ischemia-reperfusion injury at least in part via inhibition of the proinflammatory and proapoptotic JNK/AP-1 pathway. Our findings provide a rationale for a novel therapeutic approach using BV to maximize the availability of small-for-size liver grafts.     

Effects of hepatic CYP3A4 activity on disposition of micafungin in liver transplant recipients with markedly small-for-size grafts

Micafungin, the first candin antifungal drug developed in Japan, has a significant therapeutic effect against deep-seated mycoses caused by Candida or Aspergillus. Little is known, however, about the optimal dosage or disposition of micafungin in patients with severe hepatic impairment. Nine liver transplant recipients (5 males and 4 females) were enrolled in this study. In 1 recipient with a markedly small-for-size graft (ratio of graft volume to standard liver volume at the time of transplantation: 25.9%), the areas under the plasma concentration-time curves up to 12 hours postdose (AUC(0-12 h)) at doses of 50 and 100 mg/d were 79.38 and 601.17 mug.h/mL, respectively. The corresponding elimination half-life (T(1/2)) values were 16.01 and 75.75 hours, and saturated elimination was observed only at the dose of 100 mg/d. The mean urinary ratio of 6beta-hydroxycortisol to cortisol (6beta-OHF/F) in the small-for-size graft recipient was significantly (P < .05) lower than that in the other recipients. In conclusion, graft size was an important factor affecting disposition of micafungin. For liver transplant recipients with markedly small-for-size grafts, the optimal dosage of micafungin to reach and maintain therapeutic plasma levels is estimated to be 50 mg/d.     

Antiinflammatory properties of IL-10 rescue small-for-size liver grafts.

The present study aims to investigate the potential therapeutic role of interleukin-10 (IL-10) in small-for-size liver transplantation. A syngenic rat orthotopic liver transplantation model was performed using either whole or 40% liver volume of Lewis rats as grafts according to the experimental design. IL-10 was given to the 40% grafts right after reperfusion, and also at 24 and 48 hours after transplantation. When no treatment was given, less than 40% of the small-for-size grafts survived indefinitely, whereas IL-10 treatment could increase the long-term survival rate of the small-for-size grafts to 80%. The 40% grafts presented with extensive areas of necrosis and increased number of apoptotic cells at the early phases after reperfusion. In addition, upregulation of plasma protein carbonyl content (PCC) levels was also detected in the 40% graft group. IL-10 treatment suppressed the upregulation of allograft inflammatory factor-1 (AIF-1) on macrophages in the 40% grafts, and at the same time, decreased the levels of plasma PCC, and improved the histology and function of the 40% grafts. The expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-alpha, and caspase 9 in the 40% grafts were upregulated after reperfusion, whereas the augmentation could be suppressed by the administration of IL-10. Finally, IL-10 culture could block AIF-1-mediated NO production and downregulate the expression of iNOS and TNF-alpha in a macrophage cell line. In conclusion, IL-10 rescued the small-for-size liver grafts by its antiinflammatory properties, through inhibition of AIF-1 mediated proinflammatory and proapoptotic activities of the macrophages during the early period after ischemia/reperfusion.     

两种不同方法的大鼠小体积肝移植模型建立的比较

目的　探索一种大鼠小体积肝移植模型简单有效的建立方法。方法　选取体重相近的SD大鼠作为供受体 ,采用体外和体外两种方法切除部分肝叶以获取小体积供肝 ,按Kamada两套管法做供肝植入。比较两种方法的技术并发症。结果　体外法在术后肝断面出血、渗血 ,肝后下腔静脉狭窄和继发血栓 ,术中因出血引起的低血压休克 ,相近肝叶静脉误扎引起的回流障碍等并发症发生方面较体内法多。两者总的并发症发生率分别为 35 % (6 / 16 )和 5 % (4 / 78) (P <0 0 5 ) ,而两种方法在无肝期 ,冷缺血时间及总的手术时间上无统计学差异。结论　选取体重相近的供受体进行体内切除减体积是大鼠小体积肝移植模型建立的可行有效方法     

大鼠小体积肝脏移植后早期肝内细胞因子的变化

目的 研究不同冷缺血条件下大鼠小体积肝移植(30%标准体积)后早期肿瘤坏死因子α(TNF-α)及白细胞介素6(IL-6)的变化,及其与肝脏再生的关系.方法 建立Lewis大鼠30%标准体积的原位肝移植模型.根据供肝在UW液中冷保存时间的不同,将受者分为3组:冷缺血1 h组、冷缺血8 h组和冷缺血16 h组,每组均为20只.观察受者存活情况至术后第7天,并分别在移植肝恢复血流后90 min、1 h、2 h、4 h和7 d收集样本,检测移植肝组织中TNF-α和IL-6表达情况,肝细胞DNA的合成情况,进行移植肝的形态学观察.结果 大鼠肝移植手术成功率均为100%.移植后第7天,冷缺血1 h和8 h组受鼠的存活率均为100%.冷缺血16 h组受鼠的存活率较低,移植后第7天无受鼠存活.冷缺血1 h组TNF-α和IL-6的表达水平较低,冷缺血8 h组和冷缺血16 h组TNF-α和IL-6的表达则高于冷缺血1 h组(F=58.81和F=184.12,P<0.05).冷缺血8 h组和冷缺血16 h组间TNF-α和IL-6的表达的差异无统计学意义.冷缺血1 h组增殖细胞数目明显高于冷缺血8 h组,差异有统计学意义(t=5.59,P<0.05).移植术后24h,冷缺血1 h组移植肝有轻度的组织学损伤;冷缺血8 h组移植肝有轻度的窦状隙扩张和轻度的炎症;冷缺血16 h组移植肝有局部淤血,存在肝细胞崩解和坏死等改变.结论 在小体积肝移植后早期,TNF-α和IL-6的上调表达对肝脏再生有重要意义.不同冷缺血时间的小体积肝脏移植物内存在早期启动肝脏再生的信号.