Non-Alcoholic Steatohepatitis: Methods and Protocols (Methods in Molecular Biology, 2455)

✍ Scribed by Devanand Sarkar (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 363
• Category: Library

✦ Synopsis

This volume contains a comprehensive collection of laboratory protocols used by researchers to analyze varied aspects of non-alcoholic steatohepatitis (NASH). The chapters in this book cover topics such as methods for histological diagnosis of NASH; the purpose of generating an in vivo NASH model; protocols for isolating hepatocytes and Kupffer cells, bone marrow derived macrophages, and adipocytes; techniques to develop human pluripotent stem cells-derived liver organoids; single-cell and RNA-sequencing; and a description of how to extract exosomes and exosomal miRNAs from mesenchymal stem cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Thorough and practical, Non-Alcoholic Steatohepatitis: Methodsand Protocols is a valuable resource for new and experiences investigators studying NASH, and serves as an essential reference on NASH for basic and clinical researchers and students.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Histopathological Diagnosis of Nonalcoholic Steatohepatitis (NASH)
1 Introduction
2 Pathogenesis of NAFLD
3 Prevalence and Incidence
4 Risk Factors
5 Clinical History
6 Natural History
7 Clinical Diagnosis of NAFLD
7.1 Serologic Evaluation
7.2 Noninvasive Assessment of NAFLD and Steatohepatitis
7.3 The Role and Indication of Liver Biopsy in NAFLD
8 Pathology of NAFLD and NASH
8.1 Gross Features
8.2 Microscopic Features
8.3 Steatosis
8.4 Steatohepatitis
8.5 Lobular Inflammation
8.6 Other Histopathologic Features of NAFLD
9 Fibrosis and Architectural Remodeling: The Potential Complications of NASH
10 Pathology of Pediatric NAFLD/NASH
11 Conclusion
References
Chapter 2: Generation of a Diet-Induced Mouse Model of Nonalcoholic Fatty Liver Disease
1 Introduction
2 Material
2.1 Mice
2.2 Diet
2.3 Glucose and Insulin Tolerance Tests (GTT, ITT)
2.4 Blood Collection (See Note 2)
2.5 Tissues Sample Collection
2.6 Histological Analysis
3 Methods
3.1 Mice and Diets
3.2 Glucose and Insulin Tolerance Tests (GTT, ITT)
3.3 Blood Collection and Analysis
3.4 Liver Sample Collection and Processing
3.5 Histological Analysis (See Note 15)
4 Notes
References
Chapter 3: Hydrodynamic Injection for Developing NASH Model
1 Introduction
2 Materials
2.1 Preparation of Plasmid-Saline Solution
2.2 Hydrodynamic Tail Vein Injection
3 Methods
3.1 Preparation of Plasmid-Saline Solution
3.2 Preparation of Syringes
3.3 HTVi
4 Notes
References
Chapter 4: Measurement of Hepatic Lipids
1 Introduction
2 Materials
3 Methods
3.1 Lipid Extraction
3.2 Separation
3.3 Isolating Lipids
3.4 Quantification of Lipids
3.5 Cholesterol Assay
3.6 NEFA Assay
3.7 Phospholipid Assay
3.8 Triglyceride Assay
3.9 Calculations
4 Notes
References
Chapter 5: Measurement of Fatty Acid Oxidation in Mammalian Cells
1 Introduction
1.1 Overview of Current Protocols
2 Materials and Equipment
2.1 Cell Culture
2.2 Animals
2.3 3H-Palmitic Acid Tracing to Quantify FAO
2.4 Seahorse to Measure FAO-Driven Oxygen Consumption
3 Methods
3.1 3H-Palmitic Acid Tracing to Quantify FAO in Cultured Cells
3.2 3H-Palmitic Acid Tracing to Quantify FAO In Vivo
3.3 Seahorse to Measure FAO-Driven Oxygen Consumption
3.4 Statistical Considerations
4 Notes
References
Chapter 6: Measurement of In Vivo VLDL and Chylomicron Secretion
1 Introduction
2 Materials
2.1 Animals
2.2 Chemicals
2.3 Reagents and Consumables
2.4 Plasma Triglyceride (TG) Measurement Kits
2.5 Equipment
3 Methods
3.1 Monitoring In Vivo Chylomicron Formation/Secretion
3.2 Monitoring In Vivo Hepatic VLDL Secretion
4 Notes
References
Chapter 7: Isolation and Culture of Mouse Hepatocytes and Kupffer Cells (KCs)
1 Introduction
2 Materials
2.1 Liver Perfusion
2.1.1 Reagents
2.1.2 Equipment and Consumables
2.2 Hepatocyte Culture
2.2.1 Reagents
2.2.2 Equipment and Consumables
2.3 Kupffer Cell Isolation and Culture
2.3.1 Reagents
2.3.2 Equipment and Consumables
3 Methods
4 Notes
References
Chapter 8: Mouse Bone Marrow Cell Isolation and Macrophage Differentiation
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment and Consumables
3 Methods
4 Notes
References
Chapter 9: Purification and Isolation of Hepatic Stellate Cells
1 Introduction
2 Materials
2.1 Isolation of Quiescent Human HSCs
2.2 Isolation of Rodent HSCs
3 Methods
3.1 Isolation of Quiescent Human HSCs
3.2 Isolation of Rodent HSCs
4 Notes
References
Chapter 10: Isolation of the Stromal Vascular Fraction from Adipose Tissue and Subsequent Differentiation into White or Beige ...
1 Introduction
2 Materials
2.1 Reagents and Equipment
2.2 Optional Reagents and Equipment
2.3 Reagents for SVF Isolation
2.4 Reagents for Adipocyte Differentiation
2.5 Reagents for Quantitative Real-Time PCR (qRT-PCR)
3 Methods
3.1 Murine Fatpad Isolation
3.2 SVF Isolation
3.3 Gelatin Coating of Culture Plates
3.4 Pre-adipocyte Expansion
3.5 White Adipocyte Differentiation
3.6 Beige Adipocyte Differentiation
3.7 Confirmation of Adipocyte Differentiation by qRT-PCR
4 Notes
References
Chapter 11: Culture of Mouse Liver Ductal Organoids
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Plastic Supplies
3 Methods
3.1 Preparation of Reagents and Materials
3.2 Organoid Initiation
3.3 Passaging Organoids
3.4 Organoid Cryopreservation
3.5 Organoid Recovery
3.6 Transfection in Hepatic Organoids
3.7 Organoid Staining
3.8 Whole-Mount Staining
3.9 Paraffin Embedding
4 Notes
References
Chapter 12: Development of a Scalable Three-Dimensional Culture of Human Induced Pluripotent Stem Cells-Derived Liver Organoids
1 Introduction
2 Materials
2.1 Agarose Micro-well Mold Array Preparation and Human Embryoid Bodies (hEBs) Formation
2.2 Hepatic Differentiation Procedure
2.3 Organoids Morphology Assessment
2.4 Immunofluorescence Assay
2.5 Gene Expression Assay
2.6 Albumin, AFP Secretion Assays
2.7 Detoxification Activity
2.8 Equipment
3 Methods
3.1 Agarose Micro-well Arrays Mold Preparation and Embryoid Bodies (hEBs) Formation
3.2 Hepatic Differentiation Procedure
3.3 Organoid Morphology Assessment
3.4 Immunofluorescence Assay
3.5 Gene Expression Assay
3.6 Albumin, AFP Secretion Assays
3.7 Detoxification Activity
4 Notes
References
Chapter 13: Chromatin Immunoprecipitation Assay in Primary Mouse Hepatocytes and Mouse Liver
1 Introduction
2 Materials
2.1 Cross-Linking
2.2 Sonication
2.3 Immunoprecipitation
2.4 Elution and Reverse Cross-Linking
2.5 Purification of DNA Fragments and qPCR
2.6 Equipment
3 Methods
3.1 Cross-Linking in Primary Hepatocytes
3.2 Cross-Linking in Mouse Liver Tissues
3.3 Sonication
3.4 Reverse Cross-Linking and Determination of Chromatin Size and Concentration
3.5 Chromatin Pre-clearing and Immunoprecipitation
3.6 Washing, Eluting, and Reverse Cross-Linking
3.7 PCR Quantification
4 Notes
References
Chapter 14: Analysis of Liver Responses to Non-alcoholic Steatohepatitis by mRNA-Sequencing
1 Introduction
2 Materials
2.1 Equipment
2.2 Plastics
2.3 Reagents and Solutions
3 Methods
3.1 Tissue Homogenization
3.2 Collection of Cultured Cells
3.3 RNA Extraction from Tissue or Isolated Cells
3.4 Preparation of RNA-Sequencing Library from Messenger RNA
3.4.1 Purification and Fragmentation of polyA+ mRNA
3.4.2 First Strand cDNA Synthesis
3.4.3 Second Strand cDNA Synthesis
3.4.4 Adenylation of 3′ Ends
3.4.5 Ligation of Anchors (See Note 11)
3.4.6 Amplification of Library
3.4.7 Cleanup of Library
3.4.8 Checking Library Quality
3.4.9 Dilution of Library to the Starting Concentration
3.5 Computational Analysis of Sequencing Data
4 Notes
References
Chapter 15: Single Cell RNA Sequencing in NASH
1 Introduction
2 Materials
2.1 Isolation of Hepatic Immune Cells
2.2 Sorting for Myeloid Cells
2.3 GEM Generation and Barcoding
2.4 Library Construction
2.5 Sequencing on Illumina NextSeq 500
2.6 Bioinformatic Analysis
3 Methods
3.1 Isolation of CD45+ Myeloid Immune Cells from Mouse Liver
3.1.1 Isolation of Non-parenchymal Cells Using Nycodenz
3.1.2 Staining and Fluorescent-Activated Cell Sorting for Myeloid Cell Fraction
3.2 Loading the 10x Chip and GEM Generation
3.3 Post GEM-RT Cleanup and cDNA Amplification
3.3.1 Emulsion Break Up and Cleanup with DynaBeads
3.3.2 cDNA Amplification and SPRIselect Cleanup
3.4 Library Construction
3.4.1 Fragmentation, End Repair, and A-Tailing
3.4.2 Double Sided Size Selection
3.4.3 Adaptor Ligation and Cleanup
3.4.4 Index PCR and Double Sided Size Selection
3.4.5 Quality Control and Library Quantification
3.5 Sequencing Libraries
3.6 Bioinformatic Analysis
3.6.1 Extraction of Expression Levels Using Cell Ranger
3.6.2 Preprocessing of Filtered and Raw Expression Matrices
3.6.3 Cluster Identification
3.6.4 Assessment of Differential Expression and Functionally Enriched Categories
4 Notes
References
Chapter 16: Computational Pipeline for Next-Generation Sequencing (NGS) Studies in Genetics of NASH
1 Introduction
1.1 Genetics of NAFLD and The Use of Genomic Strategies
1.2 Introduction to High-Throughput Sequencing
2 Materials
2.1 File Formats
2.1.1 FASTA and FASTQ
2.1.2 SAM & BAM
2.1.3 VCF and BCF
2.2 Other File Formats
2.2.1 Indexes
2.2.2 TXT, CSV, and TSV
2.2.3 BED and GFF
2.3 List of Tools
3 Methods
3.1 Installing Software Dependencies with Bioconda
3.2 Alignment
3.3 Quality Control
3.4 Variant Calling
3.5 Variant Representation
3.6 Association Testing
3.7 Public Databases
3.7.1 Variant Effect Predictor
3.7.2 LitVar
4 Notes
References
Chapter 17: Metabolic Phenotyping in Mice with NASH Using Indirect Calorimetry
1 Introduction
2 Materials
2.1 Animals
2.2 Indirect Calorimetry System
2.3 Calibration Gases
3 Methods
3.1 Calorimetry Preparation and Experiment Running
3.2 Data Analysis and Interpretation
4 Notes
References
Chapter 18: Analysis of Insulin Resistance in Nonalcoholic Steatohepatitis
1 Introduction
2 Materials
2.1 Establishing the Mouse Model of NAFLD
2.2 Reagents and Equipment for IPGTT and IPITT Assays
2.3 Reagents and Equipment for Western Blot or IP-Western Blot Analysis
3 Methods
3.1 IPGTT Assay
3.2 IPITT Assay
3.3 IP-Western Blot Analysis
3.4 Western Blot Analysis
4 Notes
References
Chapter 19: Assessment of Lipotoxic Endoplasmic Reticulum (ER) Stress in Nonalcoholic Steatohepatitis (NASH)
1 Introduction
2 Materials
2.1 Isolation and Treatment of Primary Mouse Hepatocytes
2.2 RNA and Protein Analyses
3 Methods
3.1 Isolation of Primary Mouse Hepatocytes
3.2 Treatment with Palmitic Acid
3.3 Extraction of RNA and Quantitative Real-Time PCR
3.4 Capillary Electrophoresis
3.5 Extraction of Protein and Western Blot Analysis
4 Notes
References
Chapter 20: Ex Vivo Dual-Hit Method for Inflammasome Activation in Liver
1 Introduction
2 Materials
2.1 Reagents
2.2 Media
2.3 Equipment
3 Methods
3.1 Preparation Prior to the Experiment
3.2 Preparation of Media and Peristaltic Pump
3.3 Preparation of Mice, Cannulation, and Perfusion
3.4 Digestion and Collection of Liver
3.5 Isolation of KC
3.6 Isolation of Primary Hepatocytes
3.7 Hepatocyte Counting and Plating
3.8 Inflammasome Activation
3.9 To Elucidate the Role of Cytokines upon Inflammasome Activation and Its Effect on Hepatocytes
4 Notes
References
Chapter 21: In Vivo Analysis of Necrosis and Ferroptosis in Nonalcoholic Steatohepatitis (NASH)
1 Introduction
2 Materials
2.1 Liver Injury Models
2.2 Preparation of Cryosections from Mouse Liver
2.3 Oil Red O Staining
2.4 Picro-Sirius Red Staining
2.5 Detection of Necrotic Cells In Vivo
2.6 Immunohistochemistry
2.7 Ferroptosis Inhibitor
3 Methods
3.1 Liver Injury Models
3.1.1 CCl4-Induced Liver Injury
3.1.2 Choline-Deficient, Ethionine-Supplemented (CDE) Diet-Induced Steatohepatitis
3.2 Histological Analyses of Liver
3.2.1 Preparation of Cryosections from Liver
3.2.2 Oil Red O Staining
3.2.3 Picro-Sirius Red Staining
3.3 In Vivo Necrosis Assay
3.3.1 Detection of Necrotic Cells by PI Injection
3.3.2 Immunohistochemistry
3.3.3 Administration of Ferroptosis Inhibitor
4 Notes
References
Chapter 22: Analysis of the Sphingolipidome in NAFLD
1 Introduction
1.1 Sphingolipid Metabolism
1.2 The Roles of Sphingolipids in NAFLD/NASH
1.2.1 Lipid Metabolism
1.2.2 Insulin Resistance
1.2.3 Inflammation
1.2.4 Fibrosis
2 Materials
2.1 Induction of NASH in C57BL/6J Mice
2.2 Preparation of Liver Tissue
2.3 Isolation of Primary Mouse Hepatocytes
2.4 Lipid Extraction (Bligh-Dyer)
2.5 Normalization by Phosphate
2.6 Lipid Analysis
3 Methods
3.1 Induction of NASH in C57BL/6J Mice
3.2 Preparation of Liver Tissue
3.3 Isolation of Primary Mouse Hepatocytes
3.4 Extraction of Sphingolipids
3.5 Normalization by Phosphate
3.6 Lipid Analysis
4 Notes
References
Chapter 23: Bile Acid Profiling in Mouse Biofluids and Tissues
1 Introduction
2 Materials
2.1 Chemicals and Reagents
2.2 Equipment
3 Methods
3.1 Mouse Tissues and Biofluids Collection
3.2 Preparation of BA Standards for LC/MS
3.2.1 Preparation of BA Standard Solutions
3.2.2 Preparation of Serum/Plasma/Urine Tissue Matrix
3.2.3 Preparation of Gallbladder (GB) Tissue Matrix
3.2.4 Preparation of Liver Tissue Matrix
3.2.5 Preparation of Tissue Matrix from Small Intestine
3.2.6 Preparation of Colon Tissue Matrix
3.3 Preparation of BA Standards Using Tissue Matrix
3.4 Preparation of Tissues from Experimental Mice for BA Extraction
3.4.1 Preparation of Serum/Plasma/Urine Samples
3.4.2 Preparation of Bile Samples
3.4.3 Preparation of Liver Homogenate
3.4.4 Preparation of Small Intestine Homogenate
3.4.5 Preparation of Colon Homogenate
3.5 Extraction of BAs from Biofluids and Tissue Homogenates
3.6 BA Profile Analysis by Ultra Performance Liquid Chromatography System (UPLC)/Electrospray Ionization (ESI)/Ion Trap Mass S...
4 Notes
References
Chapter 24: Preparation and Characterization of a Liver Targeted, Poly(amidoamine) Based, Gene Delivery System
1 Introduction
2 Materials
2.1 Synthesis of PAMAM-PEG-Gal
2.2 Characterization of PAMAM-PEG-Gal
2.3 Preparation of Nanoplexes of PAMAM-PEG-Gal and pDNA
2.4 Characterization of Nanoplexes of PAMAM-PEG-Gal and pDNA
2.4.1 Size and Surface Charge Measurements
2.4.2 Gel Electrophoresis
2.4.3 Transmission Electron Microscope (TEM) Analysis
3 Methods
3.1 Synthesis of PAMAM-PEG-Gal
3.1.1 Buffers and Stock Solutions
3.1.2 Conjugation of the Gal Carboxylic Group to the PEG Amine Group to Form PEG-Gal
3.1.3 Conjugation of the PEG-Gal Free Carboxylic Group to the PAMAM Amine Group to Form PAMAM-PEG-Gal
3.2 Characterization of PAMAM-PEG-Gal
3.2.1 1H NMR Spectroscopy
3.2.2 Mass Spectrometry
3.3 Preparation of Nanoplexes of PAMAM-PEG-Gal and pDNA
3.3.1 Preparation of Stock Solutions
3.3.2 Calculation of N/P Ratios
3.3.3 Preparation of Nanoplexes
3.4 Characterization of Nanoplexes of PAMAM-PEG-Gal and pDNA
3.4.1 Size and Surface Charge Measurements
3.4.2 Electrophoretic Mobility
3.4.3 Surface Morphology Using TEM
4 Notes
References
Chapter 25: Extraction of Exosomes and Exosomal miRNA from Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 Culture Medium for Human Bone Marrow Stromal Cells (hBMSCs)
2.2 Materials for Ultracentrifugation
2.3 miRNA Extraction from Exosomes
3 Methods
3.1 Prepare Exosome-Depleted Culture Medium
3.2 Culture hBMSCs and Collect Conditioned Culture Medium
3.3 Isolate hBMSC Exosomes Through Sequential Ultracentrifugation
3.4 Extract RNA/miRNA from Exosomes
4 Notes
References
Chapter 26: Generation of Adenovirus for In Vitro and In Vivo Studies of Hepatocytes
1 Introduction
2 Materials
2.1 Adenovirus Generation
2.1.1 Generating Recombinant Adenoviral Constructs Via Homologous Recombination
2.1.2 Adenovirus Generation in 293 Cells
2.2 Adenovirus Propagation
2.3 Adenovirus Purification and Titration
2.4 Hepatocyte Infection In Vitro and In Vivo with Adenovirus
2.5 Equipment
3 Methods
3.1 Adenoviral Construct Generation Through Homologous Recombination In Vitro and in E. coli
3.1.1 Using BLOCK-iT U6 Adenoviral RNAi Expression System (In Vitro)
3.1.2 Using AdTrack System in BJ5183-AD (E. coli)
3.2 Adenovirus Generation in 293AD Cells
3.2.1 One-Step Generation of Recombinant Adenovirus from Shuttle Plasmids in 293 Cells Using RAPAd CMV Viral System
3.2.2 Adenovirus Generation from Recombinant Adenoviral Plasmids
3.3 Adenovirus Propagation in 293AD cells
3.4 Adenovirus Purification Via CsCl Discontinuous Gradient Centrifugation
3.5 Adenovirus Titration
3.5.1 Viral Particle Optical Absorption
3.5.2 End-Point Dilution
3.6 Infection of Primary Mouse Hepatocytes and Mouse Liver
3.6.1 Infection of Primary Mouse Hepatocytes (See Note 17)
3.6.2 Infection of Mouse Liver (See Note 33)
4 Notes
References
Correction to: Measurement of Fatty Acid Oxidation in Mammalian Cells
Index