Spatially segregated defects and IGF1-responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome.

BACKGROUND & AIMS: Alagille syndrome (ALGS) manifests with peripheral intrahepatic bile duct (IHBD) paucity, which can spontaneously resolve. In a model for ALGS, Jag1Ndr/Ndr mice, this occurs with distinct architectural mechanisms in hilar and peripheral IHBDs. Here, we investigated region-specific IHBD characteristics and addressed whether IGF1, a cholangiocyte mitogen that is downregulated in ALGS and in Jag1Ndr/Ndr mice, can improve biliary outcomes. METHODS: Intrahepatic cholangiocyte organoids (ICOs) were derived from hilar and peripheral adult Jag1+/+ and Jag1Ndr/Ndr livers (hICOs and pICOs, respectively). ICOs were grown in Matrigel or microwell arrays, and characterized using bulk RNA sequencing, immunofluorescence, and high throughput analyses of nuclear sizes. ICOs were treated with IGF1, followed by analyses of growth, proliferation, and death. CellProfiler and Python scripts were custom written for image analyses. Key results were validated in vivo by immunostaining. RESULTS: Cell growth assays and transcriptomics demonstrated that Jag1Ndr/Ndr ICOs were less proliferative than Jag1+/+ ICOs. IGF1 specifically rescued survival and growth of Jag1Ndr/Ndr pICOs. Jag1Ndr/Ndr hICOs were the least proliferative, with lower Notch signalling and an enrichment of hepatocyte signatures and IGF uptake/transport pathways. In vitro (Jag1Ndr/Ndr hICOs) and in vivo (Jag1Ndr/Ndr hilar portal tracts) analyses revealed ectopic HNF4a+ hepatocytes. CONCLUSIONS: Hilar and peripheral Jag1Ndr/Ndr ICOs exhibit differences in Notch signalling status, proliferation, and cholangiocyte commitment which may result in cholangiocyte-to-hepatocyte transdifferentiation. While Jag1Ndr/Ndr pICOs can be rescued by IGF1, hICOs are unresponsive, perhaps due to their hepatocyte-like state and/or expression of IGF transport components. IGF1 represents a potential therapeutic for peripheral bile ducts.

Inhibition of Oxidative Stress and the NF-κB Pathway by a Vitamin E Derivative: Pharmacological Approach against Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder. In this study, PD was induced via (ip) injection of haloperidol (1 mg/kg/day). Animals were divided into seven groups (n = 70). Group I received the vehicle carboxymethylcellulose (CMC; 0.5%), group II was treated with designated 1 mg/kg haloperidol, and group III received the standard drug Sinemet (100 mg/kg), while groups IV-VII received a tocopherol derivative (Toco-D) at dose levels of 5, 10, 20, and 40 mg/kg, respectively, via the oral route. All groups received haloperidol for 23 consecutive days after their treatments except the control group. The improvement in locomotor activity and motor coordination was evaluated by using behavioral tests. Oxidative stress markers, neurotransmitters, and monoamine oxidase B (MAO-B) as well as NF-κB levels in the whole brain were measured. mRNA expression analysis of α-synuclein was carried out using the PCR technique. Toco-D at 20 mg/kg showed the maximum improvement in locomotor activity. The levels of antioxidant enzymes and neurotransmitters were also increased by the treatment with Toco-D. Inflammatory cytokine levels and mRNA expression of α-synuclein were decreased by Toco-D in treated animals. This study concluded that Toco-D might be effective in the improvement of locomotor activity and motor coordination in haloperidol-induced PD.

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis.

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or "DUCT". DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.