Neural Repair: Methods and Protocols (Methods in Molecular Biology, 2616) 9781071629253, 9781071629260, 1071629255

Table of contents :
Preface
Contents
Contributors
Part I: Stroke Models and Surgical Interventions
Chapter 1: Rodent Stroke Models to Study Functional Recovery and Neural Repair
1 Introduction
2 Models of Ischemic Stroke for Chronic Outcomes
3 Concluding Remarks
References
Chapter 2: Subcortical White Matter Stroke in the Mouse: Inducing Injury and Tracking Cellular Proliferation
1 Introduction
2 Materials
2.1 EdU Administration
2.2 Induction of WMS
2.3 EdU Labeling
3 Methods
3.1 Administration of EdU
3.2 Induction of WMS
3.3 Labeling of EdU
4 Notes
References
Chapter 3: A Low-Budget Photothrombotic Rodent Stroke Model
1 Introduction
2 Materials
2.1 Animals
2.2 Equipment
2.3 Surgical Instruments
2.4 Reagents and Supplies
3 Methods
3.1 Aseptic Preparation of the Animal
3.2 Surgery
3.3 Post-Surgical Care
3.4 Further Experimentation and Outcome Measures
4 Notes
References
Chapter 4: Photothrombotic Model to Create an Infarct in the Hippocampus
1 Introduction
2 Materials
2.1 Photothrombotic Surgery
2.1.1 Materials and Reagents
2.1.2 Equipment
2.1.3 Laser Setup
2.2 2,3,5-Triphenyltetrazolium Chloride (TTC) Staining
2.2.1 Materials and Reagents
2.2.2 General Equipment
3 Methods
3.1 Hippocampal Photothrombosis
3.1.1 Preparation of Reagents and Work Area
3.1.2 Photothrombotic Surgery
3.2 TTC (2,3,5-Triphenyltetrazolium Chloride) Staining
3.2.1 Tissue Collection and Staining
3.2.2 Imaging of TTC-Stained Sections
4 Notes
References
Chapter 5: Bilateral Carotid Artery Stenosis and Cerebral Blood Flow Outcomes
1 Introduction
2 Materials
2.1 Equipment and Instruments
2.2 Supplies
3 Methods
3.1 Autoclave Surgery Materials
3.2 Surgical Preparation of Mice
3.3 Pre-BCAS Surgery, CBF Measurement
3.4 BCAS Surgery and Post-BCAS CBF Measurement
4 Notes
References
Chapter 6: Internal Carotid Artery Stenosis: A Surgical Mouse Model to Study Moyamoya Syndrome
1 Introduction
2 Materials
2.1 Animals and Surgical Supplies
3 Methods
3.1 Pre-ICAS Preparations
3.2 ICAS Procedure
4 Notes
References
Chapter 7: Modeling Distal Middle Cerebral Artery Occlusion in Neonatal Rodents with Magnetic Nanoparticles or Magnetized Red ...
1 Introduction
2 Materials
2.1 The SIMPLE Model
2.2 The SIMPLeR Model
3 Methods
3.1 SIMPLE Model
3.2 SIMPLeR Model
4 Notes
References
Part II: In Vivo and Post-mortem Imaging of Recovery Mechanisms
Chapter 8: In Vivo Imaging of the Structural Plasticity of Cortical Neurons After Stroke
1 Introduction
1.1 Optical Window on the Mouse Brain Cortex
1.2 In Vivo Two-Photon Imaging of Neuronal Plasticity
1.3 Photothrombotic Stroke
2 Materials
2.1 Optical Window on the Mouse Brain Cortex
2.2 In Vivo Two-Photon Imaging of Neuronal Plasticity
2.3 Photothrombotic Stroke
2.4 Image Processing and Analysis
3 Methods
3.1 Optical Window on the Mouse Brain Cortex
3.2 In Vivo Two-Photon Imaging of Neuronal Plasticity
3.3 Photothrombotic Stroke
3.4 Image Processing and Analysis
4 Notes
References
Chapter 9: Measurement of Uninterrupted Cerebral Blood Flow by Laser Speckle Contrast Imaging (LSCI) During the Mouse Middle C...
1 Introduction
2 Materials
2.1 Animals
2.2 Equipment for Modifying Bench Top
2.3 Laser Speckle Contrast Imaging System
2.4 Surgical Equipment and Supplies
3 Methods
3.1 Mounting the Inverted Laser Speckle Contrast Imaging System
3.2 Mounting the Head Frame to the Skull
3.3 Preparation of Skull to Improve Clarity for Imaging
3.4 Procedure for LSCI with the Middle Cerebral Artery Occlusion and Reperfusion (MCAo/R) Model
3.5 Modified Procedure for LSCI and MCAo/R Procedure with Aged Mice
4 Notes
References
Chapter 10: Multi-exposure Speckle Imaging for Quantitative Evaluation of Cortical Blood Flow
1 Introduction
2 Materials
2.1 Illumination Optics
2.2 Image Acquisition
2.3 Post-processing
3 Methods
3.1 System Setup
3.1.1 Optical Components
3.1.2 Electrical Components
3.2 Laser Alignment
3.3 MESI Calibration
3.4 MESI Acquisition
3.5 Shutdown
3.6 Data Processing
3.6.1 Calculating Speckle Contrast
3.6.2 Imaging and Extracting Quantitative Flow Information
4 Notes
References
Chapter 11: Wide-Field Optical Imaging in Mouse Models of Ischemic Stroke
1 Introduction
1.1 Animals and Imaging Contrasts, Housing, and Animal Preparation
1.2 Wide-Field Optical Imaging System: Overview
1.3 Imaging Protocol Considerations During Anesthesia
1.4 Imaging Protocols: Acclimation for Awake Imaging
1.5 Imaging Protocols: Evoked Responses
1.6 Imaging Protocols: Resting-State Imaging
1.7 WFOI Data Processing
1.8 WFOI Data Analysis
2 Materials
2.1 Animal Preparation for Serial Wide-Field Optical Imaging
2.2 Wide-Field Optical Imaging System
2.2.1 LEDS and Filters
2.2.2 Imaging System
2.2.3 Control Hardware
2.2.4 Head-Fixing Apparatus
2.3 Imaging Protocols: Anesthesia
2.4 Imaging Protocols: Acclimation for Awake
2.5 Imaging Protocols: Forepaw-Evoked Response Imaging
2.6 Imaging Protocols: Whisker-Evoked Response Imaging
2.7 Imaging Protocols: Resting State
2.8 WFOI Data Processing and Analysis
3 Methods
3.1 Animal Preparation for Serial Wide-Field Optical Imaging
3.2 Wide-Field Optical Imaging System
3.2.1 To Build the System of LEDs
3.2.2 Head Mounting Setup of the Imaging System (a Picture of Which Is Shown in Fig. 3)
3.3 Imaging Protocols: Anesthesia
3.4 Imaging Protocols: Acclimation for Awake Imaging
3.5 Imaging Protocols: Forepaw-Evoked Response Imaging
3.6 Imaging Protocols: Whisker-Evoked Response Imaging
3.7 Imaging Protocols: Resting-State Imaging
3.8 WFOI Processing
3.8.1 OIS Processing
3.8.2 Fluorescence Processing and Hemodynamic Correction
3.9 WFOI Analysis
4 Notes
References
Chapter 12: Post-mortem Magnetic Resonance Imaging of Degenerating and Reorganizing White Matter in Post-stroke Rodent Brain
1 Introduction
2 Materials
2.1 Transcardial Perfusion-Fixation
2.2 Preparation of Sample for Scanning
2.3 MR Scanning
3 Methods
3.1 Transcardial Perfusion-Fixation of Rat and Mouse Brains
3.2 Preparation of Sample for Scanning
3.3 MR Scanning
3.4 Analysis of Diffusion MRI Data
3.4.1 Pre-processing
3.4.2 Registration to Rodent Brain Atlas
3.4.3 Analysis of Diffusion Parameters
4 Notes
References
Untitled
Part III: Methods to Identify Molecular and Immune Mechanisms Supporting Recovery
Chapter 13: Quantitative Spatial Mapping of Axons Across Cortical Regions to Assess Axonal Sprouting After Stroke
1 Introduction
1.1 Overview of Methods
2 Materials
2.1 Axonal Tracer
2.2 Surgical Reagents
2.3 Surgical Equipment
2.4 Histology Reagents
2.5 Microscopy and Analysis
3 Methods
3.1 Animal Surgery
3.2 Histology
3.3 Microscopy and Semi-automated Axonal Tracing
3.4 Analysis
4 Notes
References
Chapter 14: Quantitative Evaluation of Cerebral Microhemorrhages in the Mouse Brain
1 Introduction
2 Materials
2.1 Mouse Brain Preparation
2.2 H & E Staining
2.3 Prussian Blue Staining
2.4 Cerebral Microhemorrhage Imaging and Quantification
3 Methods
3.1 Mouse Brain Preparation
3.2 Slide Preparation
3.3 H & E Staining
3.4 Prussian Blue Staining
3.5 Imaging and Quantification of H & E and Prussian Blue Staining
4 Notes
References
Chapter 15: In Vivo Evaluation of BBB Integrity in the Post-stroke Brain
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Solutions
3.2 Quantitative Assays
3.2.1 Administration of Tracers, Cardiac Perfusion, and Serum and Brain Collection
3.2.2 Homogenization and Centrifugation
3.2.3 Fluorescence Measurement and Quantification
3.2.4 Example of Quantitation Calculation
3.3 Morphometric Assays
3.3.1 Section Slide Preparation
3.3.2 IHC-F Staining
4 Notes
References
Chapter 16: High-Resolution RNA Sequencing from PFA-Fixed Microscopy Sections
1 Introduction
2 Materials
2.1 Tissue Isolation
2.1.1 Dissection Equipment
2.1.2 General Equipment
2.1.3 Media and Reagents
2.2 Lysis and Reverse Cross-Link
2.3 Purify and Elute mRNA
2.3.1 General Equipment
2.3.2 Media and Reagents
2.4 RNA-seq with Smart-seq2
3 Methods
3.1 Brain Tissue Isolation via Microdissection
3.2 Lyse Tissue and Reverse Cross-Link
3.3 Purify and Elute mRNA
3.4 RNA-seq with Smart-seq2
4 Notes
References
Chapter 17: FACS to Identify Immune Subsets in Mouse Brain and Spleen
1 Introduction
2 Materials
2.1 Tissue Collection/Brain Perfusion
2.2 Spleen Processing
2.3 Brain Processing
2.4 Fluorescently Activated Cell Sorting (FACS Staining)
3 Methods
3.1 Tissue Collection
3.2 Spleen Processing
3.3 Brain Processing
3.4 FACS Staining of Mouse Brain and Spleen
3.5 Compensation Controls of Single Antibody Stain
3.5.1 Cells for Compensation
3.5.2 Beads for Compensation
4 Notes
References
Chapter 18: A Guide on Analyzing Flow Cytometry Data Using Clustering Methods and Nonlinear Dimensionality Reduction (tSNE or ...
1 Introduction
2 Materials
3 Methods
3.1 FlowAI
3.2 Gating Target Population and Concatenation
3.3 Using FlowSOM
3.4 Visualization in Two-Dimensional Space Using tSNE or UMAP
4 Notes
References
Chapter 19: Co-culturing Immune Cells and Mouse-Derived Mixed Cortical Cultures with Oxygen-Glucose Deprivation to In Vitro Si...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Oxygen-Glucose Deprivation
2.3 Cell Viability Assays
3 Methods
3.1 Cortex Isolation
3.2 Cell Culture
3.3 Co-culture MCC with B Cells
3.4 Assays for Cell Viability
3.4.1 General Cell Viability with the MTT Assay
3.4.2 Neuronal Health Assessment
4 Notes
References
Part IV: Behavioral Methods for Quantifying Functional Recovery
Chapter 20: Assessing Depression and Cognitive Impairment Following Stroke and Neurotrauma: Behavioral Methods for Quantifying...
1 Introduction
1.1 Barnes Maze Test
1.2 Novel Object Recognition Test (NORT)
1.3 Sucrose Preference Test Methods
1.4 Three-Chambered Sociability Approach Test/Social Interaction
1.5 Burrowing Test
2 Materials
2.1 Barnes Maze Test
2.2 Novel Object Recognition Test
2.3 Sucrose Preference Test Methods
2.4 Three-Chambered Sociability Approach Test/Social Interaction
2.5 Burrowing Test: Protocol for Mice
2.6 Burrowing Test: Protocol for Rats
3 Methods
3.1 Barnes Maze Test
3.1.1 Acclimation
3.1.2 Testing Spatial Learning and Memory
3.1.3 Data Analyses
3.2 Novel Object Recognition Test
3.2.1 Acclimation
3.2.2 Testing Novel Object Recognition
3.2.3 Data Analysis
3.3 Sucrose Preference Test Methods
3.3.1 Preparation
3.3.2 Acclimation
3.3.3 Testing Sucrose Preference
3.3.4 Data Analysis
3.4 Three-Chambered Sociability Approach Test/Social Interaction
3.4.1 Acclimation
3.4.2 Testing for Social Interaction
3.4.3 Data Analysis
3.5 Burrowing Test: Protocol for Mice
3.5.1 Preparation
3.5.2 Testing Burrowing (Baseline and Experiment)
3.5.3 Data Analysis
3.6 Burrowing Test: Protocol for Rats
3.6.1 Preparation
3.6.2 Acclimation
3.6.3 Testing Burrowing
3.6.4 Data Analysis
4 Notes
References
Chapter 21: Use of an Automated Mouse Touchscreen Platform for Quantification of Cognitive Deficits After Central Nervous Syst...
1 Introduction
2 Materials
3 Methods
3.1 Pre-experiment Preparation
3.1.1 Mouse Preparation
3.1.2 Food Restriction
3.1.3 Habituation to Reward
3.2 Daily Session Protocol
3.2.1 Mouse Habituation to Touchscreen Testing Environment
3.2.2 Chamber Setup: Loading Reward
3.2.3 Chamber Setup: Testing Touchscreen, Reward Magazine, and IR Beam
3.2.4 Chamber Setup: Load Session Schedules and Load Mice into Chambers
3.2.5 Chamber Setup: Between Subjects
3.2.6 Chamber and Room Cleaning: At End of Day
3.3 Pretraining
3.3.1 Preparing for Pretraining
3.3.2 Running Pretraining
3.3.3 Pretraining Data Collection
3.4 Paired Associates Learning (PAL)
3.4.1 PAL Task-Specific Training
3.4.2 PAL Test
3.4.3 PAL Data Collection
3.5 Location Discrimination Reversal (LDR)
3.5.1 LDR Train
3.5.2 LDR Test
3.5.3 LDR Data Collection
3.6 Autoshaping (AUTO)
3.6.1 AUTO-Specific Training
3.6.2 AUTO-Specific Testing
3.6.3 AUTO Data Collection
3.7 Extinction (EXT)
3.7.1 EXT-Specific Training (Acquisition of Stimulus-Response)
3.7.2 EXT-Specific Testing
3.7.3 EXT Data Collection
3.8 Troubleshooting
4 Notes
References
Chapter 22: Using Operant Reach Chambers to Assess Mouse Skilled Forelimb Use After Stroke
1 Introduction
2 Materials
3 Methods
3.1 Hardware Setup
3.2 Calibration and Testing (Initial Setup and Weekly)
3.3 Training-Group Phase (5 Consecutive Days, 1 Week)
3.4 Training: Individual Phase (5 Sessions per Week, up to 3 Weeks)
3.5 Individual Baseline Measurement (3 Consecutive Days)
3.6 Post-stroke Assessment and Post-stroke Rehabilitation
4 Notes
References
Chapter 23: The Finer Aspects of Grid-Walking and Cylinder Tests for Experimental Stroke Recovery Studies in Mice
1 Introduction
2 Materials
2.1 Animals
2.2 Equipment
2.3 Supplies
3 Methods
3.1 Preparation of Animals
3.2 Execution of the Grid-Walking and Cylinder Tests
3.3 Data Acquisition and Analysis
4 Notes
References
Chapter 24: Performing Enriched Environment Studies to Improve Functional Recovery
1 Introduction
2 Materials
2.1 Animals
2.2 Cages
2.2.1 Rat Cages
2.2.2 Mouse Cages
2.3 Components of Cages
2.3.1 Cage Interior
2.3.2 Food and Water Supply
3 Methods
3.1 Design of EE Experiments
3.2 Duration of the Experiments
3.3 Preparation of Cages
3.4 Animals´ Allocation into Cages
3.5 Daily Checkups
3.6 Assessment of Neurological Function After EE
3.6.1 Effects of Multimodal Stimulation by EE
3.6.2 Behavior Assessment
4 Notes
References
Part V: Neurotherapeutics and Functional Recovery
Chapter 25: Clinically Applicable Experimental Design and Considerations for Stroke Recovery Preclinical Studies
1 Introduction
2 Starting Considerations
3 Rehabilitation
4 Start of Therapy
5 Sequence and Pairing of Therapies
6 Dose and Duration of Therapy
7 Outcome Measures
8 Concluding Remarks
References
Chapter 26: Hydrogels and Nanoscaffolds for Long-Term Intraparenchymal Therapeutic Delivery After Stroke
1 Introduction
2 Materials
2.1 General Equipment
2.2 Chemicals and Reagents
2.2.1 Chitosan/β-Glycerophosphate Hydrogel
2.2.2 PVA-Tyramine Hydrogel
2.2.3 BioTime Hydrogel
2.2.4 Hydrogel Injection Surgery
3 Methods
3.1 Preparation of Hydrogels
3.1.1 Chitosan/β-Glycerophosphate Hydrogel
3.1.2 PVA-Tyramine Hydrogel
3.1.3 BioTime Hydrogel
3.2 Embedding of Therapeutics into Hydrogels
4 Notes
References
Chapter 27: Reverse Translation to Develop Post-stroke Therapeutic Interventions during Mechanical Thrombectomy: Lessons from ...
1 Introduction
2 BACTRAC Methods Relevant to Reverse Translation
2.1 Clinical Data Relevant to Identifying Mechanism(S) of Injury and Repair
2.2 Methods to Isolate Thrombus
2.3 Methods to Isolate Peri-Infarct and Systemic Arterial Blood Samples
2.4 Methods to Analyze Human Specimens Collected During Thrombectomy
3 Creating a BACTRAC-Relevant Animal Model of Stroke
3.1 Conclusion
4 Notes
References
Chapter 28: Methods to Study Drug Uptake at the Blood-Brain Barrier Following Experimental Ischemic Stroke: In Vitro and In Vi...
1 Introduction
2 Materials
2.1 Transwell Permeability Assay
2.2 Cellular Uptake Assay
2.3 In Situ Brain Perfusion
2.3.1 Perfusion Equipment and Surgical Instruments
2.3.2 Perfusion Media
3 Methods
3.1 Transwell Permeability Assay
3.2 Cellular Uptake Assay
3.3 In Situ Perfusion
3.3.1 Perfusion Preparation
3.3.2 Surgery and Perfusion
3.3.3 Data Analysis
4 Notes
References
Chapter 29: Gene Silencing in the Brain with siRNA to Promote Long-Term Post-Stroke Recovery
1 Introduction
2 Materials
2.1 siRNA Formulations
2.2 Materials for siRNA Delivery
2.3 Materials for Evaluating siRNA Efficiency and Toxicity
2.4 Materialsfor Infarct Assessment
3 Methods
4 Notes
References
Part VI: Models of Comorbidities
Chapter 30: Diabetic Rodent Models for Chronic Stroke Studies
1 Introduction
1.1 Overview of Animal Models of Diabetes
1.2 Spontaneous Diabetic Animal Models
1.2.1 AKITA Mice
1.2.2 Lepob/ob Mice and Leprdb/db Mice
1.2.3 KK Mice
1.2.4 BB Rats
1.2.5 OLETF Rats
2 Materials
2.1 Equipment
2.2 Personal Protective Equipment
2.3 Materials and Reagents
3 Methods
3.1 Preparation of Stock Citrate Buffer
3.2 Working Solution (STZ-Buffer)
3.3 Preparation of Streptozotocin Solution
3.4 Streptozotocin Injection
3.5 Follow-Up
4 Notes
References
Chapter 31: Use of Conventional Cigarette Smoking and E-Cigarette Vaping for Experimental Stroke Studies in Mice
1 Introduction
2 Materials
2.1 Animals
2.2 Conventional and E-Cigarettes
2.3 Smoking Equipment
2.4 Materials for Experimental Stroke
3 Methods
3.1 Generation of Cigarette Smoke and Chronic Animal Exposure
3.2 Induction of Stroke in Mice After Chronic Cigarette Smoke Exposure
3.3 Basic Outcome Measures
3.3.1 Behavioral Tests
3.3.2 Open-Field Test
3.3.3 Terminal Procedure
4 Notes
References
Chapter 32: Middle Cerebral Artery Occlusion in Aged Animal Model
1 Introduction
2 Materials
3 Methods
3.1 Preparation
3.2 Occlusion
3.3 Reperfusion
3.4 Postoperative Care
4 Notes
References
Chapter 33: Acute Ischemic Stroke by Middle Cerebral Artery Occlusion in Rat Models of Diabetes: Importance of Pre-op and Post...
1 Introduction
2 Materials
2.1 Animals
2.2 Diet
2.3 Chemicals
2.4 Tools
3 Methods
3.1 Diabetes Induction
3.2 Blood Clot Preparation
3.3 Nylon Suture
3.4 Pre-op Care and Preparation (Fig. 1, See Note 8)
3.5 Operation Procedures (See Note 10)
3.6 Post-op Care (Fig. 2)
3.7 Surgery Evaluation
4 Notes
References
Chapter 34: The DOCA-Salt Model of Hypertension for Studies of Cerebrovascular Function, Stroke, and Brain Health
1 Introduction
2 Materials
2.1 Pellet Preparation (See Notes 1 and 2)
2.2 Surgical Instruments and Setup (See Note 3)
2.3 Drinking Water
3 Methods
3.1 Pellet Preparation (If Using Pre-purchased Pellets, Proceed to 3.2)
3.2 Subcutaneous Implantation of DOCA Pellet
3.3 Post-Surgical Monitoring and Supply of 0.15 M NaCl in Drinking Water
4 Notes
References
Index