■在肝硬化中,星形细胞肿胀被认为是氨神经毒性导致肝性脑病(HE)的主要机制。神经元功能障碍在HE中的作用尚不清楚。我们旨在探讨高氨血症对神经元和星形胶质细胞的原代共培养物以及肝硬化大鼠急性脑切片中线粒体功能的影响。
■对于星形胶质细胞和神经元的原代共培养,应用低浓度(1和5μM)的NH4Cl。在胆管结扎(BDL)诱导的肝硬化大鼠中,已知诱导高氨血症和最小HE的模型,研究了急性脑切片。一组BDL大鼠每天用氨清除剂鸟氨酸苯乙酸盐(OP;0.3g/kg)处理两次。线粒体膜电位变化的荧光测量(Δφm),胞质和线粒体活性氧(ROS)的产生,脂质过氧化(LP)率,和细胞活力使用共聚焦显微镜进行。
■用NH4Cl处理的神经元培养物表现出线粒体功能障碍,ROS生产过剩,细胞活力降低(27.8±2.3%和41.5±3.7%,分别)与未经处理的培养物(15.7±1.0%,两者p<0.0001)。BDL导致脑LP增加(p=0.0003)和胞质ROS产生(p<0.0001),通过OP恢复(两者p<0.0001)。线粒体功能在BDL中严重受损,导致ΔΦm超极化,从而过度消耗三磷酸腺苷,并增加线粒体ROS的产生。OP的管理恢复了ΔΦm。在BDL动物中,在海马区观察到神经元丢失,部分被OP阻止。
■我们的结果阐明了肝硬化中的低度高氨血症可严重影响脑线粒体功能。在高氨血症条件下观察到深神经元损伤,这是部分可逆的OP。这指向HE发展的新机制。
■高氨血症的影响,肝硬化患者的常见发现,在这项研究中,对大脑线粒体功能进行了研究。结果表明,在患者中常见的浓度的氨诱导严重的线粒体功能障碍,有害氧分子的过量生产,以及大鼠脑细胞中神经元的深度损伤和死亡。这些发现指出了肝衰竭中氨诱导的脑损伤的新机制和潜在的新治疗靶标。
UNASSIGNED: In cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonaemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging.
UNASSIGNED: To primary cocultures of astrocytes and neurons, low concentrations (1 and 5 μM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (Δψm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy.
UNASSIGNED: Neuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction, and reduced cell viability (27.8 ± 2.3% and 41.5 ± 3.7%, respectively) compared with untreated cultures (15.7 ± 1.0%, both p <0.0001). BDL led to increased cerebral LP (p = 0.0003) and cytosolic ROS generation (p <0.0001), which was restored by OP (both p <0.0001). Mitochondrial function was severely compromised in BDL, resulting in hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate and augmentation of mitochondrial ROS production. Administration of OP restored Δψm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP.
UNASSIGNED: Our results elucidate that low-grade hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development.
UNASSIGNED: The impact of hyperammonaemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules, and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets.