Intracranial pressure observations in a canine model of acute liver failure supported by a bioartificial liver support system

Abstract:  Life-threatening, severely elevated intracranial pressure (ICP) is a common feature of acute liver failure (ALF). Perfusion with a bioartificial liver may serve to mitigate rising ICP. A retrospective analysis of ICP measurements in a canine ALF model prospectively supported with a bioartificial liver support system (BLSS) is presented. Animals are divided into two groups based upon care provided: (i) standard medical care ( n  = 6); and (ii) standard medical care plus BLSS support ( n  = 9). Nonparametric analysis with respect to ICP, arterial NH₃, lactate, and supportive-care parameters found BLSS-supported animals evidenced significantly less metabolic acidosis than unsupported animals. Analysis of variance/linear regression for direct dependence of ICP on arterial NH₃, lactate, and supportive care parameters irrespective of care found ICP was uncorrelated with any measured factor ( P  > 0.06 for all factors). Lack of correlation of ICP with the considered parameters indicates that none of these factors are predictive of the extent of ICP elevation in the d -galactosamine canine model. Blood chemistry and supportive care factors that are correlated with and predictive of ICP elevation remain to be identified.     

急性肝功能衰竭猪应用同种体外肝脏灌注技术支持的实验研究

目的 评价体外肝脏灌注（extracorporeal liver perfusion,ECLP）技术应用于急性肝功能衰竭（acute liver failure,ALF）短暂替代治疗的疗效和可行性。方法 实验动物均为健康普通长白猪,体重20～30kg。按随机数字表法随机分为3组：肝衰组（n=5）,通过结扎肝脏血供和门体分流制备肝衰模型,肝衰模型制成8h后处死取标本;肝衰＋ECLP组（n=5）,受体为肝衰猪,供肝阻断血供后迅速切取,连接ECLP开始灌注,ECLP灌注时间为4h,肝衰模型制成8h后处死取标本;正常肝＋ECLP组（n=4）,受体为正常猪,灌注方法同肝衰＋ECLP组。观察受体一般情况、肝脏和脑组织病理变化,检测受体血常规、血生化、血凝、血氨和TNF等指标。结果 肝衰组PT、AST、TNF、血氨、RBC和HCT值明显高于肝衰＋ECLP组（P〈0．05）;正常肝＋ECLP组的FIB、AST、TNF和血氨的变化明显低于肝衰＋ECLP组（P〈0．05）。病理学检查显示,肝衰组受体的脑组织出现广泛的脑水肿,表现为神经元细胞问隙明显增宽,并有多数神经元细胞死亡;肝衰＋ECLP组受体的脑组织也出现有脑水肿的表现,并有少数神经元细胞死亡,但较肝衰组轻;正常肝＋ECLP组的脑组织病理检查基本正常。肝衰组和肝衰＋ECLP组的受体肝脏病理检查可见大片肝细胞坏死,而正常肝＋ECLP组受体的肝脏病理检查基本正常。结论 同种ECLP能有效地改善肝衰受体的体内环境,缓解症状．尤其是这种技术能缓解肝衰受体的脑水肿,而脑水肿可能是肝衰受体最主要的致死原因,但ECLP也有其自身的并发症存在。总之,同种ECLP技术是一种有效而且可行的ALF短暂替代治疗方案。     

Standardization of Ischemic Hepatic Failure in Pigs as a Model for Bioartificial Liver Assessment

Purpose. A standard protocol of ischemic liver failure in pigs was examined to establish a system for assessing the efficacy of a bioartificial liver, based on clinical practice. Methods. The portal blood flow was extracorporeally bypassed into the cervical jugular vein, using a centrifugal blood pump. The portal vein and hepatic artery were then ligated. Results. The maintenance protocol was established as follows: (1) the concentration of the inhaled anesthetic was decreased by 0.2% when the systolic blood pressure was 100mmHg; (2) the volume of an infusion containing 5% glucose was increased to 10ml/kg per hour when central venous pressure was 5mmHg; (3) 20ml of 50% glucose was injected intravenously when the blood glucose was 50mg/dl; (4) 2000 units of heparin was injected intravenously when the activated clotting time was 150s; (5) sodium bicarbonate was given when the blood pH was 7.3; (6) tidal volume was increased by 1ml/kg when the pCO₂ was 80mmHg; (7) oxygen was increased by 25% when the pO₂ was 100mmHg. No vasopressors were used in the experiment. Conclusion. Our protocol reduced the operating time and minimized the risk of data deviation that can arise from variations in operating techniques and individual animal conditions. This experimental model is also easy to use as a bridge to transplantation.     

肝细胞移植治疗急性肝功能衰竭的免疫排斥研究

目的研究肝细胞腹腔移植治疗急性肝功能衰竭的免疫排斥反应。方法用胶原酶灌注法分离幼猪及BALB/c和C57BL/6小鼠肝细胞;用腹腔注射CCl4的方法建立C57BL/6急性肝功能衰竭模型;将实验动物分为同基因移植组、同种异基因移植组和异种移植组。各组动物肝细胞移植入急性肝功能衰竭小鼠腹腔内,观察受体小鼠存活情况,比较各组动物T细胞亚群、免疫球蛋白水平和细胞因子的变化。结果①受体小鼠存活情况:同基因移植组为8/10,同种异基因移植组为6/10,异种移植组为3/10,同基因移植组和同种异基因移植组受体的生存情况明显好于异种移植组(P<0.05)。②T细胞亚群变化:移植后7 d内,同基因移植组外周血中CD4+和CD8+T细胞均无明显变化,同种异基因移植组CD4+T细胞于移植后3 d时达高峰(P<0.05),而CD8+T细胞7 d内无明显变化,异种移植组CD4+和CD8+T细胞移植后7 d内均明显升高,且于移植后3 d时达高峰(P<0.05)。③免疫球蛋白水平:同基因移植组血清免疫球蛋白IgM和IgG水平在移植后0.5、1和3 d时未见明显变化,同种异基因移植组和异种移植组的IgM在移植后1 d时达高峰(P<0.05),而IgG在移植后3 d时达高峰(P<0.05)。④血清细胞因子水平:移植后7 d,同种异基因移植组和异种移植组的IFN-γ、TNF-α及IL-2血清浓度明显高于同基因移植组(P<0.05);异种移植组的IL-6血清浓度明显高于同基因移植组和同种异基因移植组(P<0.05)。结论无论同种还是异种肝细胞移植,初期均发生了强烈的CD4+及CD8+T细胞参与的细胞免疫和较强体液免疫反应。     

A Novel Liver Parenchyma Transection Technique Using Locking Straight Rigid Ties. An Experimental Study in Pigs

ABSTRACT

Introduction: Technological advances have led to the development of many devices used in liver resections. However, no single transection tool is uniformly considered to be better than the others. This study aimed to develop an effective, fast, and cost-efficient technique for hepatic parenchymal transection. Materials and Methods: A liver parenchyma compression device in the form of a locking straight rigid tie (LoStRiT) was newly developed. Twelve pigs were distributed into two groups. The control group (?n = 6) comprised animals that underwent hepatectomy using the standard Kelly-clysis technique. The study group (n = 6) comprised animals that underwent hepatectomy using sequential LoStRiT mechanisms. The transection speed, blood loss, and biloma formation were recorded. Results: The mean parenchymal transection speed was 1.27 ± 0.27 cm²/min for the control group and 2.39 ± 0.56 cm²/min for the LoStRiT group (?p = .003). The mean blood loss per kilogram of body weight was 9.8 ± 5.2 ml/kg for the control group and 3.9 ± 0.9 ml/kg for the LoStRiT group (?p = .040). No bilomas were identified. Conclusion: LoStRiT hepatectomy appears to be effective, fast, and reproducible in a porcine model of liver resection. Further development of this novel and potentially cost-efficient technique includes construction of the device using absorbable materials.     

Semipermeable Hollow Fiber Membranes in Hepatocyte Bioreactors: A Prerequisite for a Successful Bioartificial Liver?

Abstract: Recent studies have shown that liver support systems based on viable hepatocytes can prolong life in animal models of acute liver failure. Now the time has come to elucidate the design characteristics that are essential to construct an efficient bioreactor. The gold standard remains the intact liver. Despite the very high cell density in this organ, individual cell perfusion is guaranteed resulting in low diffusional gradients which are essential for optimal mass transfer. These conditions are not met in bioreactors based on hollow fiber membranes. Moreover, the semipermeable membranes can foul and act as a diffusional barrier between the hepatocytes and the blood or plasma of the recipient. We devised a novel bioreactor for use as a bioartificial liver that does not include hollow fiber membranes for blood or plasma perfusion. The device is based on an integral oxygenator and a nonwoven polyester matrix material for hepatocyte culture as small aggregates. The efficacy of this original design was tested in rats with liver ischemia. Preliminary results show statistically significantly improved survival; life was prolonged 100% compared to the control experiments.     

闭合型双循环生物人工肝支持系统治疗犬急性肝功能衰竭实验研究

目的探讨和评价闭合型双循环生物人工肝支持系统(CBC-BALSS)在治疗犬急性肝功能衰竭模型过程中的稳定性、安全性和有效性。方法建立犬急性肝功能衰竭模型(门腔分流联合胆总管离断),采用CBC-BALSS进行支持治疗。20只模型犬分为两组CBC-BALSS治疗组(n=11);无肝细胞CBC-BALSS对照组(n=9)。治疗时限6h。检测实验犬血氨、生化全套、凝血因子(FactorⅦ)、支/芳氨基酸(BCAA/AAA)、单乙基甘氨酸二甲苯胺(monoethylglycinexylidide,MEGX)和细胞循环路生化全套、肝细胞密度和数量。结果CBC-BALSS细胞回路细胞悬液总体积200ml,肝细胞的总数1×1010个、密度5×107/ml、活率98%左右。治疗中16只犬的生命体征平稳,在治疗30min内均出现一过性低血压;2只转流开始15min出现过敏反应;1只转流中因上消化道出血死亡;1只因穿刺部位出血死亡。模型治疗前血氨、ALT、TBil/DBil、白蛋白、FactorⅦ和BCAA/AAA分别达150mmol/L、400U/L、80/55mmol/L、35g/L、20%和1.6;CBC-BALSS治疗6h后,血氨、TBil/DBil下降均显著低于对照组;ALT存在下降趋势且在第6小时差异有统计学意义;白蛋白、FactorⅦ和BCAA/AAA在所有时段、组间差异均无统计学意义。在治疗1h和2h,MEGX差异有统计学意义,治疗组MEGX比对照组提前2h达最高点。治疗15~30min后,双循环路压力至115mmHg趋于平稳,且在±5mmHg波动。在治疗过程中,治疗组细胞循环路ALT显著性升高;组间细胞循环路TBil/DBil变化差异无统计学意义,而两组在各时间点均显著性升高;白蛋白变化无统计学意义。结论CBC-BALSS治疗犬急性肝功能衰竭过程中,安全、有效、稳定且代谢支持作用明显。     

人工肝支持系统在肝衰竭和肝脏移植中的临床应用研究

目的评价人工肝支持系统（ALSS）在肝衰竭和肝脏移植中的作用。方法对44例肝衰竭患者（包括12例行肝移植者）进行分子吸附循环系统（MARS）或血浆置换治疗，检测治疗前、后各种有毒物质及细胞因子等的改变并进行比较。结果44例肝衰竭患者，经ALSS治疗后临床症状及体征明显改善；血总胆红素、总胆汁酸、谷丙转氨酶、肌酐、尿素氮、血氨及内毒素水平明显降低（P〈0．05）；血清NO和TNF-α、IL-4、IL-6水平亦明显降低（P〈0．05）；治疗前、后红细胞、白细胞、血小板无明显变化（P〉0．05）。30例重型乙肝肝衰竭患者存活18例，存活率为60．0％；6例肝脏移植术前急性肝衰竭患者均成功接受肝脏移植；6例肝脏移植术后急性肝衰竭患者存活2例；2例胰十二指肠切除术后急性肝衰竭者，死亡1例。结论ALSS通过全面清除肝衰竭患者体内毒素、NO和细胞因子，对肝衰竭有肯定的治疗作用；并对等待肝脏移植的肝衰竭患者发挥过渡性桥梁作用。     

The Effects of Serum from Patients with Acute Liver Failure on the Growth and Metabolism of Hep G2 Cells

In many bioartificial liver systems currently being designed and evaluated for use in fulminant hepatic failure, direct contact is required between the patient's blood and the liver cells in the device. The efficacy of such devices will be influenced by the interaction of fulminant hepatic failure (FHF) patient serum with the cells. We have found that FHF serum inhibits the growth rate and the synthesis of DNA, RNA, and protein; disturbs glutathione homeostasis; and induces morphological changes in cultured human Hep G2 cells. These interactions should influence the design of bioartificial liver devices based on proliferating cell lines and indicate the requirement to pretreat FHF patient plasma to reduce the toxin load.     

Bioartificial Liver Support System Using Porcine Hepatocytes Entrapped in a Three-Dimensional Hollow Fiber Module with Collagen Gel: An Evaluation in the Swine Acute Liver Failure Model

The perfusion culture system of hepatocytes entrapped in a three-dimensional hollow fiber module with collagen gel is expected to realize an effective bioartificial liver (BAL) support system. The BAL module contained 5.4 billion porcine hepatocytes, which is approximately 7% of the total liver, supplied adequate metabolic functions, and improved the survival of the experimental acute liver failure swine. Improvement of the prothrombin time by the BAL treatment was not significant; however, the bleeding tendency was suppressed, which resulted in a significantly prolonged survival time of the animals in the BAL treatment group. A drastic up-regulation of the expression of the mRNA for plasminogen activator inhibitor 1 (PAI-1), which is known as an inhibitor for fibrinolysis, may explain the suppression of the bleeding tendency. These results support the efficacy of our BAL system for the treatment of acute liver failure.     

加味茵陈汤防治大鼠急性肝功能衰竭的实验研究

目的:探索中药加味茵陈汤对急性肝功能衰竭大鼠的防治效果。方法:加味茵陈汤汤剂(2g/mL),Wistar大鼠D-氨基半乳糖腹腔内一次注射(剂量1200mg/kg),分别以不同剂量中药灌胃(A组45g/kg;B组30g/kg;C组15g/kg),对照组(D组)给予相同体积生理盐水。观察一般情况改变及生存时间,于给药前、给药后24h、48h、72h取血测定血氨、AST、ALT及TBil,并取肝脏进行病理检查。结果:随中药剂量增加模型大鼠中位生存时间延长,A、B组与D组间比均有统计学差异(P<0.05)。C组及D组血氨、AST、ALT及TBil水平明显高于A、B两组,且D组高于C组(P<0.05)。A、B两组大鼠肝脏病理结果改善,均较D组坏死轻。结论:中药加味茵陈汤可以延长D-氨基半乳诱导大鼠急性肝功能衰竭模型的生存时间,改善TBiL及血氨等肝功能指标,减轻肝脏损伤的病理改变。     

Combination of Plasma Exchange and Continuous Hemofiltration as Temporary Metabolic Support for Patients with Acute Liver Failure

Abstract: The combination of plasma exchange and continuous hemofiltration was applied to patients with acute liver failure, and its clinically advantageous effects were evaluated. This procedure was found to be a powerful approach in decreasing patient morbidity; however, the effect was temporary and the patients finally died. Efforts to eliminate the hepatitis virus, which is the main etiology of hepatic necrosis in Japan, are urgent to salvage patients with fulminant liver failure.     

Hepatocyte Attachment on Biodegradable Modified Chitosan Membranes: In Vitro Evaluation for the Development of Liver Organoids

Extracellular matrix structures including glycosaminoglycans play a critical role in cell attachment, differentiation, and morphogenesis. We evaluated chitosan ([1 → 4] linked 2-amino-2-deoxy-β- D -glucan) as a biomaterial for hepatocyte attachment because of its structural similarity to glycosaminoglycans. Freshly isolated rat and fetal porcine hepatocytes were seeded on chitosan membranes that had been previously blended with collagen, gelatin, or albumin to improve biocompatibility and surface roughness. The optimal cell density and attachment kinetics were quantified. The metabolic activity was investigated by measuring daily urea and total protein secretion by the cells for 2 weeks. While collagen blended-chitosan membranes provided a good attachment surface for rat hepatocytes, albumin and gelatin blended chitosan membranes were superior for fetal porcine hepatocyte attachment. The optimal attachment was maintained with membranes of medium molecular weight ( M _r = 750,000 daltons) chitosan, at 3–4 × 10 ⁴ cells/cm² after 3 h of incubation. In vitro experiments demonstrated that fetal porcine hepatocytes survived at least 14 days when seeded on the chitosan-albumin matrix, demonstrating that this biomaterial can provide suitable cell attachment scaffolds for creating liver tissue organoids.     

Percutaneous Treatment of Liver Failure and Acute Mesenteric Ischaemia

Introduction

Synchronous embolism to the superior mesenteric artery (SMA) and coeliac axis (CA) is a rare disease.

Report

A 67-year-old man with atrial fibrillation developed acute liver failure due to an embolic occlusion of the CA and SMA, with a severe coagulation disorder. He was successfully managed with percutaneous stent placement and an exploratory laparotomy was not needed. He remains symptom-free 1 year after the procedure, and duplex follow-up showed stent patency.

Conclusion

Endovascular techniques in patients with liver failure, no signs of peritonism, early diagnosis and high operative risk seem feasible and should be used if possible, as first-line option.     

Outcomes of Severe Pregnancy‐Related Liver Disease: Refining the Role of Transplantation

Severe liver disease in pregnancy is generally considered to have a favorable prognosis. The limited data available have not yielded disease‐specific prognostic criteria or guidance on who should undergo liver transplantation (LT). We retrospectively evaluated 54 admissions with pregnancy‐related liver disease to (1) evaluate if any admission parameters were associated with death and/or transplantation and (2) identify maternal complications. Eighteen had acute fatty liver of pregnancy and 32 had hypertension/eclampsia related disease. Seven patients (13%) died and four (7%) underwent LT. Survival rates were 43/48 if not listed for LT and 4/6 if listed. Of the four transplanted, three survived. Patients who died and/or underwent LT were more likely to have encephalopathy (p = 0.04) and hyperlactaemia (p = 0.03). Serum lactate was the best discriminant (ROC AUC 0.84). An admission lactate greater than 2.8mg/dL had 73% sensitivity and 75% specificity for predicting death or LT. The addition of encephalopathy to this parameter increased sensitivity and specificity to 90% and 86%, respectively. The King's College criteria were not effective in predicting outcome. This study confirms the overall favorable prognosis in pregnancy‐related liver failure but indicates that elevated lactate levels in the presence of encephalopathy best identify patients at greatest risk of death or LT.     

Liver Repopulation: A New Concept of Hepatocyte Transplantation

Hepatocyte transplantation has been recognized as an alternative strategy for organ transplantation because the supply of donor livers is limited. However, in conventional hepatocyte transplantation, only 1%–10% of the liver replaced with transplanted hepatocytes. Recently a novel concept termed “liver repopulation” has been established, where the whole recipient liver can be replaced by a small number of donor hepatocytes. To induce liver repopulation, growth advantage of the donor hepatocytes against the host liver seems to be required according to the data of previous studies. Additionally, various cell sources, including bone marrow cells and other stem cells, could potentially be used as donor cells for liver repopulation. In this article, we discuss recent progress and future perspectives of this emerging technology.     

Early Evidence of Bone Marrow Dysfunction in Children with Indeterminate Fulminant Hepatic Failure Who Ultimately Develop Aplastic Anemia

In children, aplastic anemia (AA) is a common complication associated with fulminant hepatic failure (FHF). The objective of this study was to determine whether specific pretransplantation clinical and laboratory characteristics can be used to distinguish between patients with FHF who are at higher risk of developing AA. We performed a retrospective case-control study to evaluate the clinical and laboratory characteristics of those patients who presented with evidence of FHF and eventually developed aplastic anemia. We identified nine patients with AA, and all had the indeterminate form of FHF and underwent liver transplantation (LTx). The AA patients were compared with a control group of 47 patients with indeterminate FHF that underwent transplantation and did not develop AA. We found that males were over-represented in the group of patients that developed AA (p = 0.01). Furthermore, during the pretransplant period, the AA group had a significantly lower white count (p = 0.005), absolute lymphocyte count (p = 0.004), and platelet count (p = 0.019) when compared with controls. We conclude that evidence of early bone marrow dysfunction is apparent before liver transplantation and the development of AA in a subset of patients with the indeterminate form of FHF.     

骨髓间充质干细胞经脾移植治疗大鼠急性肝功能衰竭的实验研究

目的 探讨大鼠骨髓间充质干细胞（MSCs）经脾移植治疗急性肝功能衰竭的疗效,并观察MSCs在体内的迁徙情况。方法 收集1只SD雄性大鼠胫骨及股骨的骨髓,采用密度梯度离心联合贴壁培养法分离、纯化及扩增雄性SD大鼠的骨髓MSCs,再行免疫组化染色以观察第4代骨髓MSCs的表面标志物。联合应用D-氨基半乳糖和肿瘤坏死因子-α （TNF-α）建立24只雌性大鼠急性肝功能衰竭模型,将其随机分为2组：实验组（n=12）大鼠于造模后24 h行骨髓MSCs脾内移植;空白对照组（n=12）仅于脾内注射0.5 mL生理盐水。2组大鼠于移植后均取血检测丙氨酸氨基转移酶（ALT）、总胆红素（TBIL）及白蛋白（ALB）水平,采用PCR法检测大鼠肝脏组织中Y性别决定区基因（SRY基因）的表达,并行HE染色观察肝脏组织的病理学改变。结果 第4代MSCs表达CD44和CD29,但不表达CD34。MSCs移植72 h及以后,实验组存活5只大鼠（41.7%）,空白对照组存活3只大鼠（25.0%）,2组大鼠的存活率比较差异有统计学意义（P＜0.05）,实验组较高。将雄性大鼠的骨髓MSCs移植于雌性大鼠的脾内后,在雌性大鼠肝脏中能检测到SRY基因的表达;且HE染色结果显示,实验组大鼠的肝功能在移植后4周明显改善。移植后与空白对照组比较,实验组各时点的ALT和TBIL水平均较低（P＜0.05）;移植后1周和2周,实验组的ALB水平均高于空白对照组（P＜0.05）。结论 骨髓MSCs经脾内移植后迁徙并定居于受损的肝脏内,可替代肝细胞的功能。     

Acute Oxalate Nephropathy: A New Etiology for Acute Renal Failure Following Nonrenal Solid Organ Transplantation

Acute renal insufficiency (ARI) is a frequent complication of nonrenal solid organ transplantation and may be responsible for an unfavorable outcome, particularly if dialysis is required. The etiology of post-transplantation ARI is poorly understood, with only isolated clinical cases being reported, most imputed to drug toxicity. We report here, the first three observations of irreversible ARI associated with acute oxalate nephropathy (AON) in the course of nonrenal organ transplants: a lung transplant and a lung-liver transplant in two patients with mucoviscidosis, and a cardiac transplant. The diagnosis of AON was made histologically. In all three cases, the ARI supervened after prolonged consumption of antibiotics capable of interfering with the colonic flora, and leading to enteric hyperoxaluria. The recognition of AON as a cause of post-transplantation, ARI underlines hyperoxaluria and digestive hyperabsorption of oxalate as specific risk factors for AON and should permit better posttransplant care of these patients.