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Liver slice culture as a model for lipid metabolism in fish.

ABSTRACT: Hepatic lipid metabolism is traditionally investigated in vitro using hepatocyte monocultures lacking the complex three-dimensional structure and interacting cell types essential liver function. Precision cut liver slice (PCLS) culture represents an alternative in vitro system, which benefits from retention of tissue architecture. Here, we present the first comprehensive evaluation of the PCLS method in fish (Atlantic salmon, Salmo salar L.) and validate it in the context of lipid metabolism using feeding trials, extensive transcriptomic data, and fatty acid measurements. We observe an initial period of post-slicing global transcriptome adjustment, which plateaued after 3 days in major metabolic pathways and stabilized through 9 days. PCLS fed alpha-linolenic acid (ALA) and insulin responded in a liver-like manner, increasing lipid biosynthesis gene expression. We identify interactions between insulin and ALA, where two PUFA biosynthesis genes that were induced by insulin or ALA alone, were highly down-regulated when insulin and ALA were combined. We also find that transcriptomic profiles of liver slices are exceedingly more similar to whole liver than hepatocyte monocultures, both for lipid metabolism and liver marker genes. PCLS culture opens new avenues for high throughput experimentation on the effect of "novel feed composition" and represent a promising new strategy for studying genotype-specific molecular features of metabolism.

SUBMITTER: Harvey TN

PROVIDER: S-EPMC6753922 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Liver slice culture as a model for lipid metabolism in fish.

Harvey Thomas N TN Sandve Simen R SR Jin Yang Y Vik Jon Olav JO Torgersen Jacob S JS

PeerJ 20190917

Hepatic lipid metabolism is traditionally investigated in vitro using hepatocyte monocultures lacking the complex three-dimensional structure and interacting cell types essential liver function. Precision cut liver slice (PCLS) culture represents an alternative in vitro system, which benefits from retention of tissue architecture. Here, we present the first comprehensive evaluation of the PCLS method in fish (Atlantic salmon, Salmo salar L.) and validate it in the context of lipid metabolism usi ...[more]

PMID: 31576253

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Project description:In organotypic hippocampal slice cultures, principal neurons form aberrant excitatory connections with other principal cells in response to slicing induced deafferentation, similar to mechanisms underlying epileptogenesis in posttraumatic epilepsy. To investigate the consequences of this synaptogenesis, the authors recorded field-potential activity from area CA3 during perfusion with the complete growth medium used during incubation. At 7 days in vitro, slice cultures only displayed multiunit activity. At 14 days in vitro, the majority displayed population bursts reminiscent of interictal-like spikes, but sustained synchronous activity was rare. Band-pass filtering of interictal discharges revealed fast ripple-like complexes, similar to in vivo recordings. Spontaneous ictal-like activity became progressively more prevalent with age: at 21 days in vitro, 50% of organotypic hippocampal slice cultures displayed long-lasting, ictal-like discharges that could be suppressed by phenytoin, whereas interictal activity was not suppressed. The fraction of cultures displaying ictal events continually increased with incubation time. Quantification of population spike activity throughout epileptogenesis using automatic detection and clustering algorithms confirmed the appearance of interictal-like activity before ictal-like discharges and also revealed high-frequency pathologic multiunit activity in slice cultures at 14 to 17 days in vitro. These experiments indicate that interictal-like spikes precede the appearance of ictal-like activity in a reduced in vitro preparation. Epileptiform activity in cultures resembled in vivo epilepsy, including sensitivity to anticonvulsants and steadily increasing seizure incidence over time, although seizure frequency and rate of epileptogenesis were higher in vitro. Organotypic hippocampal slice cultures comprise a useful model system for investigating mechanisms of epileptogenesis as well as developing antiepileptic and antiepileptogenic drugs.

Project description:The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-κB and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-κB signaling. Moreover, MLN4924 abrogated TNF-induced NF-κB signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics.

Dataset Information

Liver slice culture as a model for lipid metabolism in fish.

Publications

Liver slice culture as a model for lipid metabolism in fish.

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