Axon Regeneration: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Defining Selective Neuronal Resilience and Identifying Targets for Neuroprotection and Axon Regeneration Using Sing...
1 Introduction
1.1 Basic Strategy
1.2 Perturbation Model
1.3 RGC Purification and scRNA-Seq
2 Materials
2.1 Mouse Strains
2.2 Perfusion and Retina Dissection (See Note 1)
2.3 Single-Cell Dissociation and RGC Purification
2.3.1 Stock Solutions for Cell Dissociation and RGC Purification
2.3.2 Working Solutions for Cell Dissociation and RGC Purification
2.4 Immunohistochemistry (IHC) and Imaging
2.4.1 Working Solutions for IHC
2.5 FACS and QC
3 Methods
3.1 RGC Single-Cell Dissociation for scRNA-Seq
3.2 FACS
3.3 Validation of Enrichment (See Note 14)
3.4 Single-Cell Sequencing Library Preparation
4 Notes
References
Chapter 2: Defining Selective Neuronal Resilience and Identifying Targets of Neuroprotection and Axon Regeneration Using Singl...
1 Introduction
2 Materials
2.1 Python Packages
2.2 scRNA-seq Datasets
3 Methods
3.1 Initializing iGraphBoost with the Adult RGC Atlas
3.2 Read in Injured RGCs as AnnData Objects
3.3 iGraphBoost Overview and Classification of 0dpc RGCs
3.3.1 Step 1: Supervised Classification
3.3.2 Step 2: Nearest-Neighbor Voting
3.4 iGraphBoost Classification of 0.5dpc, 1dpc, and 2dpc RGCs
3.5 iGraphBoost Classification of 4d, 7d, and 14d RGCs
3.6 Identifying Genes Correlated with Resilience and Susceptibility
4 Notes
References
Chapter 3: Retinal Ganglion Cell Axon Fractionation
1 Introduction
2 Materials
3 Methods
3.1 Anti-CTB Magnetic Bead Preparation Prior to Optic Nerve Dissection
3.2 Optic Nerve Isolation and Tissue Dissociation
3.3 Conjugated CTB Binding and Magnetic Separation
3.4 Verification of RGC Separation with Immunodetection
4 Notes
References
Chapter 4: Analysis of Immediate Early Gene Expression Levels to Interrogate Changes in Cortical Neuronal Activity Patterns up...
1 Introduction
2 Materials
3 Methods
3.1 Monocular Enucleation
3.2 Sample Preparation
3.3 Labeling and Purification of the Probe
3.4 Quantifying the Amount of Labeled Oligo Probes
3.5 Tissue Fixation
3.6 Hybridization
3.7 Rinsing and Preparation for Film Exposure
3.8 Film Development
3.9 Analysis
4 Notes
References
Chapter 5: Noncoding Regulatory RNAs: Isolation and Analysis of Neuronal Circular RNAs and MicroRNAs
1 Introduction
2 Materials
2.1 Reagents and Equipment for Working with RNA
2.2 RNA Isolation
2.3 Quantification of circRNAs
2.4 Quantification of miRNAs
3 Methods
3.1 RNA Isolation from Brain Tissues
3.2 circRNA cDNA Synthesis
3.3 circRNA qRT-PCR, Primers´ Design, and Validation
3.4 circRNA Quantification by qRT-PCR
3.5 miRNA-Specific cDNA Synthesis
3.6 miRNA-Specific Quantification by qRT-PCR
4 Notes
References
Chapter 6: Transneuronal Delivery of Cytokines to Stimulate Mammalian Spinal Cord Regeneration
1 Introduction
2 Materials
2.1 Animals and Animal Care
2.2 Surgical Tools for Crush
2.3 Tools and Supplies for Intracortical Injection
2.4 Viruses
2.5 Validation
3 Methods
3.1 T8 Spinal Cord Crush
3.2 Intracortical Injections
3.3 Validation Steps
3.3.1 Tissue Preparation
3.3.2 Immunostaining
3.3.3 Crush Quality: Spared Axons
3.3.4 Crush Quality: Lesion Size
3.3.5 Intracortical Injection Quality: Virus Efficacy
3.3.6 Intracortical Injection Quality: Layer V Transduction
3.3.7 Intracortical Injection Quality: Transneuronal hIL-6 Delivery
4 Notes
References
Chapter 7: Epigenomic Profiling of Dorsal Root Ganglia upon Regenerative and Non-regenerative Axonal Injury
1 Introduction
1.1 Peripheral and Central Axonal Injury and Sciatic DRG Dissection
1.2 Total RNA Extraction from DRG Tissue
1.3 RNA-Seq Library Preparation
1.4 Chromatin Immunoprecipitation from DRG Tissue
1.5 ChIP-Seq Library Preparation
1.6 ATAC-Seq from DRG Tissue
2 Materials
2.1 Common Materials
2.2 Sciatic DRG Axonal Injury and Dissection
2.3 Total RNA Extraction from DRG Tissue
2.4 RNA-Seq Library Preparation
2.5 Chromatin Immunoprecipitation from DRG Tissue
2.6 ChIP-Seq Library Preparation
2.7 ATAC-seq Library Preparation from DRG Tissue
3 Methods
3.1 Peripheral and Central Axonal Injury and Sciatic DRG Dissection
3.1.1 Animal Preparation
3.1.2 Sciatic Nerve Axotomy Surgery
3.1.3 Dorsal Column Axotomy Surgery
3.1.4 Sciatic DRG Dissection
3.2 Total RNA Extraction from DRG Tissue and RNA-Seq
3.3 RNA Preparation
3.3.1 mRNA Purification and Fragmentation
3.3.2 First Strand cDNA Synthesis
3.3.3 Second Strand cDNA Synthesis
3.3.4 End Repair
3.3.5 3′ Ends Adenylation
3.3.6 Adapter Ligation
3.3.7 PCR Library Amplification
3.4 Chromatin Immunoprecipitation from DRG Tissue
3.4.1 Chromatin Preparation
3.4.2 Immunoprecipitation
3.4.3 Washing, Elution, and Cross-Linking Reversal
3.4.4 DNA Purification and Quantification
3.5 ChIP-Seq Library Preparation
3.6 ATAC-Seq from DRG Tissue
3.6.1 Cell Preparation and Transposition Reaction
3.6.2 Library Amplification
3.6.3 Library Purification
4 Notes
References
Chapter 8: Profiling Locally Translated mRNAs in Regenerating Axons
1 Introduction
2 Materials
2.1 DRG Cell Culture and Transfections
2.2 Study of Local Translation Using FRAP
2.3 Fluorescence Recovery After Photobleaching (FRAP) and Data Analysis
3 Methods
3.1 Preparation of Glass Bottom Dishes
3.2 DRG Culture
3.3 FRAP
3.4 Study Effect of External Environment on Local Translation Using FRAP
3.5 Quantification of FRAP Data
4 Notes
References
Chapter 9: Analysis of Axonal Regrowth and Dendritic Remodeling After Optic Nerve Crush in Adult Zebrafish
1 Introduction
2 Materials
2.1 Optic Nerve Crush (ONC)
2.2 Biocytin-Soaked Gelfoam Clot Preparation and Application
2.3 Perfusion and Tissue Dissection
2.4 Cryopreservation, Embedding, and Cryostat Sectioning
2.5 Embedding and Vibratome Sectioning
2.6 Hematoxylin and Eosin (H&E) Staining
2.7 Diaminobenzidine Tetrahydrochloride (DAB) Staining
2.8 Immunofluorescent Staining
2.9 Microscopy and Quantification
3 Methods
3.1 Optic Nerve Crush (ONC)
3.2 Biocytin-Soaked Gelfoam Clot Preparation and Application for Anterograde and Retrograde Tracing
3.3 Transcardial Perfusion and Tissue Dissection and Fixation
3.4 Cryopreservation, Embedding, and Cryostat Sectioning
3.5 Embedding and Vibratome Sectioning
3.6 DAB Staining
3.7 H&E Staining
3.8 Fluorescent Stainings
3.9 Microscopic Visualization and Quantification of the Biocytin-Traced RGCs
3.10 Microscopic Visualization and Quantification of Axonal Regeneration in the Optic Nerve
3.11 Microscopic Visualization and Quantification of Tectal Reinnervation
3.12 Microscopic Visualization and Quantification of Dendritic Degeneration/Recovery Using IPL Thickness Measurements
3.13 Microscopic Visualization and Quantification of Synaptic and Dendritic Degeneration/Recovery Using Fluorescent Stainings
4 Notes
References
Chapter 10: Assaying Optic Nerve Regeneration in Larval Zebrafish
1 Introduction
2 Materials
2.1 RGC Axon Transection in Larval Zebrafish
2.2 Immunostaining Tg(isl2b:GFP) Larvae
2.3 Using Lipophilic Dyes to Label RGC Axons
3 Methods
3.1 RGC Axon Transection in Larval Zebrafish
3.2 Immunostaining Tg(isl2b:GFP) Larvae (See Note 17)
3.3 Using Lipophilic Dyes to Label RGC Axons
4 Notes
References
Chapter 11: Surgical Methods in Postmetamorphic Xenopus laevis: Optic Nerve Crush Injury Model
1 Introduction
2 Materials
3 Methods
3.1 Anesthetization
3.2 Ventral or Buccal Optic Nerve Crush Injury
3.3 Dorsal Optic Nerve Crush Injury
3.4 Recovery and Postsurgical Care
4 Notes
References
Chapter 12: Generating Widespread and Scalable Retinal Lesions in Adult Zebrafish by Intraocular Injection of Ouabain
1 Introduction
2 Materials
2.1 Solutions
2.2 Supplies
2.3 Equipment
3 Methods
3.1 Preparation of the Workspace
3.2 Intraocular Injection of Ouabain
3.3 Recovery
4 Notes
References
Chapter 13: A Reproducible Spinal Cord Crush Injury in the Regeneration-Permissive Axolotl
1 Introduction
2 Materials
2.1 Axolotl Care
2.2 Weight Drop Apparatus for Spinal Crush Injury (Fig. 1)
2.3 Visualization of Lesion Site Using AFOG Staining
2.4 RNA Extraction
3 Methods
3.1 Apparatus Preparation
3.2 Spinal Cord Crush Injury
3.3 Paraffin Wax Tissue Processing
3.4 AFOG Staining
3.5 RNA Extraction
4 Notes
References
Chapter 14: Live Imaging of Axonal Dynamics After Laser Axotomy of Peripheral Neurons in Zebrafish
1 Introduction
2 Materials
2.1 Somatosensory Neuron Labeling
2.2 Fish Embryo Cultivation
2.3 Preventing Pigment Development
2.4 Anesthetizing Fish
2.5 Mounting Embryos for Imaging
3 Methods
3.1 Injections
3.2 Screening Embryos
3.3 Mounting
3.4 Pre-axotomy Imaging
3.5 Axotomies with a Two-Photon Microscope
3.6 Larva Recovery
3.7 Post-regeneration Imaging and Time-Lapse Movies
3.8 Peripheral Axon Arbor Tracing
3.9 Post-imaging Processing
4 Notes
References
Chapter 15: Rapid Testing of Gene Function in Axonal Regeneration After Spinal Cord Injury Using Larval Zebrafish
1 Introduction
2 Materials
2.1 CRISPR/Cas9 Microinjections
2.2 Quality Control by Restriction Fragment Length Polymorphism
2.3 Larval Spinal Cord Lesions
3 Methods
3.1 Designing sCrRNAs (See Note 11)
3.2 Microinjection of CRISPR/Cas9 gRNA (See Note 16)
3.3 Quality Control Using RFLP for Injected Larvae (See Note 24)
3.4 Spinal Cord Injury of 3-Day Postfertilization Larvae
3.5 Bridging Assay for Regenerative Success
4 Notes
References
Chapter 16: Translating Ribosome Affinity Purification (TRAP) and Bioinformatic RNA-Seq Analysis in Post-metamorphic Xenopus l...
1 Introduction
2 Materials
2.1 TRAP Supplies and Reagents
2.2 TRAP Buffers (See Note 14)
3 Methods
3.1 Preliminary Experiments
3.2 Pre-experiment Preparations for TRAP
3.3 Preparation of Affinity Matrix (2.5 h) (See Note 22)
3.4 Preparatory Steps for Tissue Collection and Homogenization (See Notes 10 and 27)
3.5 Tissue Collection and Homogenization-Work on Ice/Keep Samples Cold (See Notes 10 and 27)
3.6 Lysate Preparation-Work on Ice Unless Indicated
3.7 Immunopurification
3.8 Preparatory Steps for RNA Isolation (See Note 36)
3.9 RNA Isolation (See Notes 8 and 36)
3.10 Assessment of RNA Quantity (See Note 42)
3.11 Assessment of RNA Quality (See Note 11)
3.12 cDNA Library Construction and RNA Sequencing
3.13 Data Analysis Overview and Working Environment
3.14 Sequence File Preparation and Quality Control Filtering
3.15 Mapping RNA-Seq Reads to the Genome
3.16 Higher-Order Analysis
4 Notes
References
Chapter 17: LCM-Seq for Retinal Cell Layer-Specific Responses During Optic Nerve Regeneration
1 Introduction
2 Materials
2.1 Zebrafish Optic Nerve Crush Surgery, Tissue Dissection, and Cryosectioning
2.2 Cresyl Violet Staining
2.3 Laser Capture Microdissection
2.4 RNA Purification and Quality Control
3 Methods
3.1 Zebrafish Optic Nerve Crush Surgery
3.2 Tissue Dissection
3.3 Cryosectioning
3.4 Cresyl Violet Staining
3.5 Laser Capture Microdissection
3.6 RNA Purification
4 Notes
References
Chapter 18: Profiling Dynamic Changes in DNA Accessibility During Axon Regeneration After Optic Nerve Crush in Adult Zebrafish
1 Introduction
2 Materials
2.1 Adult Optic Nerve Crush and Retina Dissection (See Note 1)
2.2 Retina Dissociation and Cell Sorting
2.3 ATAC-Seq Library Preparation and Quality Assessment
3 Method
3.1 Adult Optic Nerve Crush
3.2 Retina Dissection
3.3 Retina Dissociation into Single-Cell Suspensions
3.4 Fluorescence-Activated Cell Sorting (FACS) of GFP-Positive RGCs
3.5 Nuclear Isolation and Transposase Reaction
3.5.1 Transposed DNA Cleanup
3.6 Library Amplification
3.6.1 ATAC-Seq Library Cleanup
3.7 Library Quality Control
3.7.1 NanoDrop Directions
3.7.2 Fluorometric Quantitation Platform Directions
3.7.3 Size Selection Using Chip-Based Capillary Electrophoresis Platform Directions
4 Notes
References
Chapter 19: Quantitative Proteomics of Nervous System Regeneration: From Sample Preparation to Functional Data Analyses
1 Introduction
2 Materials
2.1 Acquisition of Tissue Samples
2.2 Protein Extraction
2.3 Cleanup and Tryptic Digestion
2.4 iTRAQ 8-Plex Labeling
2.5 Strong Cation-Exchange Fractionation of iTRAQ Labeled Peptides
2.6 Reversed-Phase Liquid Chromatography (RPLC)-Electrospray Ionization (ESI)-Tandem Mass Spectrometry (MS/MS) Analysis
2.7 Software Packages for Identification, Quantification, Differential Abundance Analysis, and Functional Analysis of Proteins
3 Methods
3.1 Acquisition of Tissue Samples
3.2 Protein Extraction
3.3 Cleanup and Tryptic Digestion
3.4 iTRAQ 8-Plex Labeling
3.5 Strong Cation-Exchange Fractionation of iTRAQ Labeled Peptides
3.6 Reversed-Phase Liquid Chromatography (RPLC)-Electrospray Ionization (ESI)-Tandem Mass Spectrometry (MS/MS) Analysis
3.7 Protein Identification and Quantification
3.8 Differential Protein Abundance Analysis
3.9 Finding the Human or Mouse Ortholog of Your Gene List Using an Ortholog Converter Tool (See Note 28)
3.10 Finding the Human or Mouse Ortholog by Transforming Your Identifiers to Their Official Gene Symbols (See Note 31)
3.11 Obtaining All Functional Annotations for Your Gene Sets
3.12 Functional Enrichment Using the g:Profiler Tool (See Note 35)
3.13 Cytoscape and STRING Protein-Protein Network
3.14 Generating Venn Diagrams
4 Notes
References
Chapter 20: Live Cell Imaging of Dynamic Processes in Adult Zebrafish Retinal Cross-Section Cultures
1 Introduction
2 Materials
2.1 Chemicals/Solutions
2.2 Dissection Tools/Equipment
2.3 Culturing
2.4 Confocal Microscopy
3 Methods
3.1 Light Damage Paradigm
3.2 Preparations on the Day of Culturing
3.3 Retinal Isolation, Mounting, and Culturing
3.4 Imaging Interkinetic Nuclear Migration with an Inverted Confocal Microscope (See Note 32)
3.5 Z-Intensity Correction to Overcome Pixel Intensity Loss in Deeper Z-Layers (See Note 52)
4 Notes
References
Chapter 21: PCNA Staining of Retinal Cryosections to Assess Microglial/Macrophage Proliferation
1 Introduction
2 Materials
2.1 Antigen Retrieval
2.2 Immunostaining of Cryosections
2.3 Microscopy
3 Methods
3.1 Heat-Mediated Antigen Retrieval in Citrate Buffer
3.2 Immunofluorescent Staining with Antibodies to Detect PCNA and Microglia
3.3 Imaging and Analysis
4 Notes
References
Chapter 22: Drosophila Laser Axotomy Injury Model to Investigate RNA Repair and Splicing in Axon Regeneration
1 Introduction
1.1 RNA Repair and Axon Regeneration
1.2 Drosophila Sensory Neuron Injury Model
2 Materials
2.1 Equipment
2.2 Reagents
3 Methods
3.1 Collection of Larvae for Injury
3.2 Laser Axotomy
3.3 Post-injury Imaging
3.4 Axon Regeneration Quantification
3.5 Nociceptive Behavior Test
4 Notes
References
Chapter 23: Assessing Rewiring of the Retinal Circuitry by Electroretinogram (ERG) After Inner Retinal Lesion in Adult Zebrafi...
1 Introduction
2 Materials
2.1 Solutions
2.2 Supplies
2.3 Equipment
3 Methods
3.1 Dark Adaptation and Recording Preparation
3.2 Preparation of Zebrafish for Recording
3.3 Light Stimulation and ERG Recording
3.4 Post-ERG Acquisition Steps
4 Notes
References
Chapter 24: Analysis of Visual Recovery After Optic Nerve Crush in Adult Zebrafish
1 Introduction
2 Materials
2.1 DLR Test Method 1
2.2 DLR Test Method 2
2.3 OKR Test
3 Methods
3.1 DLR Method 1 Test Subject Preparation
3.2 DLR Method 1 Testing and Quantification
3.3 DLR Method 2 Test Subject Preparation
3.4 DLR Method 2 Testing and Quantification
3.5 OKR Test Equipment Setup
3.6 OptoMotry Software
3.7 OKR Test Habituation
3.8 OKR Testing
4 Notes
References
Index