Neuromuscular Assessments of Form and Function (Neuromethods, 204)

Preface to the Series
Preface
Contents
Contributors
Chapter 1: Estimation of Lean Soft Tissue by Dual-Energy X-Ray Absorptiometry as a Surrogate for Muscle Mass in Health, Obesit...
	1 Introduction
	2 Materials
		2.1 Maintenance
			2.1.1 Daily Quality Assurance
			2.1.2 Weekly Cleaning
			2.1.3 Annually - GE-Authorized Maintenance
		2.2 Measurement
	3 Methods
		3.1 Maintenance
			3.1.1 Daily Quality Assurance
		3.2 Measurement
		3.3 Analysis
	4 Notes
	References
Chapter 2: Analysis of Skeletal Muscle Mass from Preexisting Computerized Tomography (CT) Scans
	1 Introduction
	2 Materials
	3 Methods
		3.1 CT Image Analysis
		3.2 Landmarking
		3.3 Manual Segmentation
		3.4 Determining Body Composition Measures
	4 Notes
	References
Chapter 3: Imaging Skeletal Muscle by Magnetic Resonance Imaging (MRI)
	1 Introduction
		1.1 A Primer on MRI Principles
		1.2 Image Processing and Software
		1.3 Measurement of Skeletal Muscle Volume
			1.3.1 Suggested Scan Card
			1.3.2 Software Tools for Volume Estimation
		1.4 Measurement of Muscle to Fat Ratio
			1.4.1 Suggested Scan Card
			1.4.2 Software Tools for Muscle to Fat Ratio
		1.5 Measurement of Skeletal Muscle Fibers
			1.5.1 Suggested Scan Card
			1.5.2 Software Tools for Fiber Measurements
		1.6 Looking to the Future of MRI of Muscle Mass
	2 Notes
	References
Chapter 4: Imaging of Skeletal Muscle Mass: Ultrasound
	1 Introduction: Muscle Ultrasound
		1.1 Ultrasound Measurements of Skeletal Muscle
			1.1.1 Muscle Dimensions
		1.2 Muscle Thickness (MT)
		1.3 Anatomical Cross-Sectional Area (ACSA)
		1.4 Muscle Volume Reconstruction
			1.4.1 Muscle Architecture
	2 Materials
	3 Methods
		3.1 Identification of Muscle Regions of Interests and Scan Sites
		3.2 Muscle Thickness (MT): Methodological Aspects
		3.3 Anatomical Cross-Sectional Area (ACSA): Methodological Aspects
		3.4 Muscle Volume Reconstruction: Methodological Aspects
		3.5 Muscle Architecture: Methodological Aspects
	4 Notes
		4.1 Muscle Thickness (MT): Notes
		4.2 Anatomical Cross-Sectional Area (ACSA): Notes
		4.3 Muscle Volume Reconstruction: Notes
		4.4 Muscle Architecture: Notes
		4.5 Applications of Ultrasound to Assess Muscle Mass
			4.5.1 Hypertrophy
			4.5.2 Atrophy
		4.6 Aging
		4.7 Spaceflight
	References
Chapter 5: Measures of Neuromuscular Function
	1 Introduction
	2 Assessments of Neuromuscular Function
		2.1 Strength Testing
		2.2 Dynamometer Testing
		2.3 Force Plates
		2.4 Evoked Contractions
	3 Conclusions
	4 Notes
	References
Chapter 6: Neuromuscular Function: High-Density Surface Electromyography
	1 Introduction
	2 Materials
		2.1 HDsEMG Amplifiers
		2.2 HDsEMG Electrodes
		2.3 Skin Preparation and Electrode Placement
			2.3.1 HDsEMG Electrode Cleaning
	3 Methods
		3.1 Tasks
		3.2 Target Muscle
		3.3 EMG Signal Preprocessing Steps
		3.4 HDsEMG Motor Unit Decomposition
		3.5 Analysis of Motor Unit Firing Data
		3.6 Motor Unit Firing Characteristics
		3.7 Muscle Fiber Properties
		3.8 Motor Unit Longitudinal Tracking
	4 Conclusion
	5 Notes
		5.1 Signal Saturation
		5.2 Electrode Repositioning
		5.3 Short Circuits Between Channels
		5.4 Disconnected Channels
		5.5 Long Signals: Nonstationarity/Stationarity
		5.6 Population Under Study
	References
Chapter 7: Neuromuscular Function: Intramuscular Electromyography
	1 Introduction
	2 Materials
		2.1 Concentric Needle Electrode
		2.2 Acquisition Equipment
	3 Methods
		3.1 Needle Insertion
			3.1.1 Signal Decomposition
			3.1.2 iEMG Parameters/Features
			3.1.3 Concluding Remarks
	4 Notes
	References
Chapter 8: Magnetic Resonance Quantification of Muscle Phosphocreatine Resynthesis Kinetics During Exercise Recovery: An In Vi...
	1 Introduction
	2 Materials
		2.1 mDIXON Scans
		2.2 31P MR-Spectroscopy
		2.3 In-Bore Exercise
	3 Methods
	4 Notes
		4.1 Note 1: Use of a Nonlocalized 31P Sequence
		4.2 Note 2: Muscle Acidosis Complicates the Interpretation of 31P-Derived PCr Resynthesis Data During Exercise Recovery
		4.3 Note 3: Consistent Muscle PCr Depletion Is Important to Achieve If the Comparison of PCr Recovery Kinetics Is a Study Obje...
	References
Chapter 9: Immunohistochemistry, Microscopy, and Image Analysis of Human Muscle Biopsies: Muscle Fiber Denervation as a Workin...
	Abbreviations
	1 Introduction
		1.1 Models of Muscle Denervation
		1.2 Methods for Studying Muscle Denervation
	2 Methods and Materials
		2.1 Percutaneous Muscle Biopsy and Tissue Preservation
		2.2 Immunohistochemistry
		2.3 A: Day 1
		2.4 B: Day 2
		2.5 Fluorescent Widefield Microscopy
		2.6 Quantitative and Qualitative Analyses
		2.7 A: Quantification of Denervated Fibers
		2.8 B: Step-by-Step Guide
		2.9 C: Serial Cross-Sections
		2.10 List of Materials
	3 Notes
	4 Conclusions
	References
Chapter 10: Stable Isotope Tracer Methods for the Measure of Skeletal Muscle Protein Turnover
	1 Introduction
		1.1 Importance and Value of Protein Turnover Measures
		1.2 Stable Isotopes
		1.3 Mass Spectrometry
	2 Material and Reagents
		2.1 Stable Isotope Tracers-Storage and Preparation-Substrate Specific and D2O
		2.2 Reagents Required for Preparation of Amino Acid and Keto Acid Derivatives-Precursor Enrichment and Concentration
		2.3 Reagents Required for Derivatization of Amino Acid Hydrolysates for IRMS Analyses
		2.4 Reagents Required for Derivatization of Amino Acid Hydrolysates as Methoxy-Carbonyl-Methyl Ester (MCME)-An Alternative Der...
		2.5 Reagents for Muscle Fractionation and Processing
	3 Methods for Measuring Protein Synthesis
		3.1 Precursor-Product Relationship-Measurement of Protein Synthesis-A Typical Labeled AA Constant Infusion Approach
		3.2 Choice of Precursor Pool
		3.3 Sample Collection (Fluid/Tissue) Preparation and Storage
		3.4 Preparation of Plasma for Measurement of the Plasma Precursor Enrichment and Amino Acid Concentration
		3.5 Preparation of Muscle for Measurement of the Intracellular Precursor Amino Acid Enrichment
		3.6 Preparation of Muscle Fractions for the Measurement of Tracer AA Incorporation, i.e., the Product
		3.7 Use of Dowex Resin to Purify Free Amino Acids or to Hydrolyze Protein Fractions
		3.8 Determination of Enrichment of the Product by Isotope Ratio Mass Spectrometry
		3.9 Mass Spectrometric Analyses
	4 Measurement of Protein Synthesis-A D2O Approach
		4.1 Saliva Sample Collection and Analysis by TCEA-IRMS for Precursor Enrichment-D2O Only
		4.2 Mass Spectrometry Analytical Requirements for D2O-Based Tracer Studies
	5 Summary and Comparison of Both Primed Constant Infusion and D2O Approaches
	6 Notes
	References
Chapter 11: Ex Vivo Human Single Muscle Fibers: An Insightful Approach to Skeletal Muscle Function
	1 Three Main Fiber Types in Human Skeletal Muscles
	2 Single-Fiber Historical Overview
	3 Methods for Experiments on Human Single Muscle Fibers
		3.1 The Biopsy
		3.2 Sarcolemma Permeabilization and Sarcolemma Removal
		3.3 Methods for Sample Storage
		3.4 Single Muscle Fiber Dissection
		3.5 Setup, Solutions, and Protocols for Mechanical Experiments
		3.6 Biochemical and Molecular Analysis on Single Fibers
	4 What Can Experiments on Human Single Muscle Fibers Tell Us?
	5 Conclusions
	References
Chapter 12: Myokines, Measurement, and Technical Considerations
	1 Introduction
	2 Myokines: Characterization and Biological Function(s)
		2.1 Defining Myokines
		2.2 Biological Functions of Myokines
	3 Primary Approaches Toward Myokine Analyses
		3.1 Primary Techniques for Targeted Myokine Analysis
			3.1.1 Real-Time PCR (qPCR)
			3.1.2 Immunoassays
				ELISA
				Western Blot
				Antibody Arrays
			3.1.3 Immunohistochemistry (IHC)
		3.2 Approaches Toward Untargeted Myokine Discovery
	4 Methodology for Omics-Driven Myokine Prediction
		4.1 Protein Sequence-Based Prediction of Candidate Myokine Targets
			4.1.1 Obtaining Protein Sequences for Input into Prediction Tools
				Ensembl
				UniProt
			4.1.2 Signal Peptide Prediction via SignalP
			4.1.3 Prediction of Nonclassically Secreted Proteins Using SecretomeP
			4.1.4 Prediction of Protein Subcellular Localization Using DeepLoc
			4.1.5 Prediction of Transmembrane Helices in Proteins Using TMHMM
			4.1.6 Prediction of Exosome-Secreted Proteins Using ExoPred
		4.2 Putative Myokine Screening via the Human Protein Atlas
			4.2.1 Human Protein Atlas-Defined Human Secretome
			4.2.2 Using the Human Protein Atlas to Establish Skeletal Muscle Specificity
	5 General Myokine Experimental Considerations
		5.1 In Vivo and In Vitro Sampling
		5.2 Quantification of Myokine Gene Versus Protein Changes
	6 Summary: Road Map for Myokine Discovery and Analyses
	References
Chapter 13: Skeletal Muscle Satellite Cell Physiology and Function: Complimentary In Vitro and In Vivo Models and Methods
	1 Introduction
		1.1 Skeletal Muscle Development
		1.2 Key Milestones in Skeletal Muscle Research
		1.3 The Satellite Cell
		1.4 Example Satellite Cell Models
		1.5 Stretch and Load
		1.6 Electrical Stimulation
		1.7 Introduction to the Study of Satellite Cells In Vivo
		1.8 Immunohistochemistry Considerations
		1.9 Histological Analyses
		1.10 Identifying Satellite Cells and Myonuclei in Histological Muscle Samples
	2 Materials
		2.1 Adult Human Satellite Cell Isolation
			2.1.1 Preparation and Biopsy Harvest
			2.1.2 Biopsy Transfer
			2.1.3 Human Primary Cell Isolation
			2.1.4 Cell Expansion and Storage
			2.1.5 Cell Loading
			2.1.6 Electrical Stimulation
		2.2 Histological Preparation
			2.2.1 Histological Immunofluorescence
			2.2.2 Single Extracted Muscle Fibers Immunofluorescence
	3 Methods (See Subheading 2.1 for Materials Specific to Satellite Cell Isolation and Stimulation)
		3.1 Adult Human Satellite Cell Isolation
			3.1.1 Preparation and Biopsy Harvest/Transfer
			3.1.2 Human Primary Cell Isolation
			3.1.3 Cell Trypsinization for Expansion and Storage
			3.1.4 Resuscitation of Cryopreserved Cells
			3.1.5 Cell Loading/Stretching (Fig. 4)
				For Mechanical Loading of Cells in Monolayer
				For Bioengineering and Loading of 3D Skeletal Muscle Using Fibrin as the Extracellular Matrix
				Mechanical Loading of Bioengineered Skeletal Muscle
			3.1.6 Electrical Stimulation
		3.2 Histological Preparation (for Materials, See Subheading 2.2)
		3.3 Single Fiber Extraction
			3.3.1 Immunohistochemical Labeling of Single Fibers
	References
Chapter 14: Using the Model Organism Caenorhabditis elegans to Explore Neuromuscular Function
	1 Introduction
		1.1 Caenorhabditis elegans
		1.2 C. elegans Musculature
	2 Materials
		2.1 Reagents
		2.2 Obtaining Nematodes
		2.3 Microscopes and Cameras
	3 Methods
		3.1 C. elegans Husbandry
		3.2 Preparing Worms for Microscopy
		3.3 Detailed Assessments of Neuromuscular Function
			3.3.1 Sarcomere Morphology
			3.3.2 Mitochondrial Morphology
		3.4 Mobility Assays
		3.5 Burrowing Assay
		3.6 Nematode Electropharyngeograms (EPGs)
		3.7 Oxygen Consumption
		3.8 Other Considerations
	4 Notes
	References
Chapter 15: Methodologies to Quantify Skeletal Muscle Blood Flow/Perfusion
	1 Introduction
	2 Methods
		2.1 Doppler Ultrasound
		2.2 Xenon-133
		2.3 Near-Infrared Spectroscopy
		2.4 Thermodilution
		2.5 Contrast-Enhanced Ultrasound
	3 A Focus on CEUS
		3.1 Principles of Contrast Agents
		3.2 Setup and Operation of CEUS for Recording from Skeletal Muscle
		3.3 Analysis of CEUS Data
		3.4 Step-By-Step Guide for the Assessment of Microvascular Blood Flow with CEUS
			3.4.1 Ultrasound Machine Setup
			3.4.2 Participant Preparation
			3.4.3 Sonovue Preparation
			3.4.4 CEUS Measurement Protocol
			3.4.5 Data Handling and Analysis
	4 Applications in Neuromuscular Research
	5 Conclusion
	6 Notes
	References
Index