Retinal Ganglion Cells: Methods and Protocols (Methods in Molecular Biology, 2708)

Preface
Contents
Contributors
Chapter 1: Isolation and Culture of Primary Retinal Ganglion Cells from Rodent Retina
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Slides
		3.2 Dissection of the Retina
		3.3 Dissociation of the Retina
		3.4 RGC Separation
		3.5 Plating Cell Suspension
		3.6 Assessment of RGC Culture for Neuroprotection and Regeneration
	4 Notes
	References
Chapter 2: Purification of Retinal Ganglion Cells from Differentiation Through Adult via Immunopanning and Low-Pressure Flow C...
	1 Introduction
	2 Materials
		2.1 Dissection, Immunopanning, and FACS
		2.2 RGC Growth Media
	3 Methods
		3.1 Preparation of Plating Surfaces, Solutions, and Panning Dishes
		3.2 Preparation of Solutions and Plates the Day of Dissection
		3.3 Dissection of Pups
		3.4 Dissection of Embryos
		3.5 Digestion of Retinas
		3.6 Macrophage Panning
		3.7 Immunopanning of RGCs with Thy1.2
		3.8 Low-Pressure Sorting of GFP-Tagged RGCs
		3.9 Plating and Growth of RGCs
	4 Notes
	References
Chapter 3: Preparation of Retinal Explant Cultures
	1 Introduction
	2 Materials
		2.1 Instruments Required
		2.2 Cell Culture
	3 Methods
		3.1 Retinal Wholemounts
	4 Notes
	References
Chapter 4: Gene Gun DiOlistic Labelling of Retinal Ganglion Cells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Bullet Preparation
		3.2 Flatmount Retina Dissection
		3.3 DiOlistics Labelling
		3.4 Confocal Microscopy
		3.5 Retinal Ganglion Cell Reconstruction
		3.6 Sholl Analysis
	4 Notes
	References
Chapter 5: Differentiation of Human Embryonic/Induced-Pluripotent Stem Cells to Retinal Ganglion Cells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Maintenance and Passaging of Human ESC/iPSC
		3.2 Differentiation of ESC/iPSC
	4 Notes
	References
Chapter 6: Laser-Induced Ocular Hypertension in a Mouse Model of Glaucoma
	1 Introduction
	2 Materials
	3 Methods
	4 Note
	References
Chapter 7: Silicone Oil-Induced Ocular Hypertension Glaucoma Model (SOHU) in Rodent and Nonhuman Primate
	1 Introduction
	2 Materials
		2.1 Mouse
			2.1.1 Glass Micropipette for Intracameral Injection
			2.1.2 Paracentesis Needle
			2.1.3 SO Injector
			2.1.4 Irrigation System for SO Removal
			2.1.5 Drainage System for SO Removal
			2.1.6 Optokinetic Response
			2.1.7 Spectral-Domain Optical Coherence Tomography (SD-OCT) Imaging and Fundus Fluorescein Angiography (FFA)
			2.1.8 Pattern Electroretinogram (PERG) Recording
		2.2 Rhesus Macaque Monkey
	3 Methods
		3.1 Intracameral SO Injection
			3.1.1 Mouse
			3.1.2 Mouse High Frequency (HF) and No Dilation (ND) SOHU Glaucoma Models
			3.1.3 Rhesus Macaque
		3.2 SO Removal
			3.2.1 Mouse
			3.2.2 Rhesus Macaque
		3.3 IOP Measurement
			3.3.1 Mouse
			3.3.2 Rhesus Macaque
		3.4 In vivo Imaging and Visual Function Assays in Mice
			3.4.1 Opto-Kinetic Response (OKR)
			3.4.2 Spectral-Domain Optical Coherence Tomography (SD-OCT) Imaging
			3.4.3 Fundus Fluorescein Angiography (FFA)
			3.4.4 Pattern Electroretinogram (PERG) Recording
	4 Notes
	References
Chapter 8: Microbead-Induced Ocular Hypertension in a Rodent Model of Glaucoma
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 9: Viral Vector-Induced Ocular Hypertension in Mice
	1 Introduction
	2 Materials
		2.1 Viral Vectors (Ad5 or Lenti)
		2.2 Intraocular Injection and Induction of Ocular Hypertension
		2.3 IOP Measurement and Anterior Segment Examination
		2.4 Aqueous Humor Dynamics in Live Mouse Eyes
	3 Methods
		3.1 Animal Husbandry
		3.2 Anesthesia
			3.2.1 Injection of Anesthetic Cocktail
			3.2.2 Inhalation Anesthesia
		3.3 Intraocular Injection of Vector
			3.3.1 Intravitreal (ivt) Injection
			3.3.2 Intracameral Injection
			3.3.3 Slow Intracameral Infusion
		3.4 Measurement of IOP
			3.4.1 Conscious Measurement of Intraocular Pressure (IOP)
			3.4.2 IOP Measurement under Anesthesia
		3.5 Measurement of Aqueous Humor Dynamics
		3.6 Examination of Anterior Segment
	4 Notes
	References
Chapter 10: Optic Nerve Crush Injury in Rodents to Study Retinal Ganglion Cell Neuroprotection and Regeneration
	1 Introduction
	2 Materials
		2.1 Instruments
		2.2 Analgesia and Anesthesia
	3 Methods
		3.1 Optic Nerve Crush Injury
	4 Notes
	References
Chapter 11: Optical Coherence Tomography: Imaging Visual System Structures in Mice
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Equipment Setup and Pre-imaging Preparation
		3.2 Mouse Preparation
		3.3 Imaging Acquisition
		3.4 Data Analysis
	4 Notes
	References
Chapter 12: Confocal Scanning Laser Ophthalmoscopy to Image Retinal Ganglion Cells in Real-Time
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Recipes
	3 Methods
		3.1 Animal Preparation for cSLO Imaging
		3.2 Image Acquisition
		3.3 Data Analysis
	4 Notes
	References
Chapter 13: In Vivo Detection of Retinal Ganglion Cell Stress in Rodents with DARC
	1 Introduction
	2 Materials
		2.1 Anesthesia
		2.2 DARC
		2.3 Pupil-Dilating Eye Drops
		2.4 cSLO
		2.5 Pipette
		2.6 Eye Care
	3 Methods
		3.1 Pupil Dilation
		3.2 Anesthesia
		3.3 Baseline cSLO Imaging
		3.4 Image Acquisition
		3.5 Administration of DARC
		3.6 Assessment of DARC Signal
	4 Notes
	References
Chapter 14: Measuring the Full-Field Electroretinogram in Rodents
	1 Introduction
	2 Materials
		2.1 Electroretinography
	3 Methods
		3.1 Laboratory and Electrode Preparation
		3.2 Animal Preparation
		3.3 Signal Acquisition
		3.4 Signal Analysis
	4 Notes
	References
Chapter 15: Evaluation of Retinal Ganglion Cell via Visual Evoked Potential
	1 Introduction
	2 Materials
		2.1 Additional Materials If Using Goat
		2.2 Additional Materials If Using Rhesus Macaque (Inc. All Above)
	3 Methods
		3.1 FVEP in Rodents
		3.2 FVEP in Goat
		3.3 PVEP in Rhesus Macaque
	4 Notes
	References
Chapter 16: High-Throughput Binocular Pattern Electroretinograms in the Mouse
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 17: Intravitreal Injection of AAV for the Transduction of Mouse Retinal Ganglion Cells
	1 Introduction
		1.1 Retinal Ganglion Cells
		1.2 Recombinant Adeno-Associated Viral Vectors to Transduce RGCs
		1.3 Considerations of AAVs for Preclinical Examination
		1.4 Intravitreal Injection of AAVs
	2 Materials
		2.1 AAV Preparation
		2.2 Anesthetic and Eye Drops
		2.3 Equipment for AAV Administration
		2.4 Equipment for Ocular Collection
	3 Methods
		3.1 AAV Working Solution Preparation
		3.2 Preparing the Injection Device
		3.3 Hamilton Needle Cleaning and Storage
		3.4 Preparing the Animal for Intravitreal Injection
		3.5 Performing the Intravitreal Injection
		3.6 Typical Study Duration for Stable Expression
		3.7 Collecting AAV Expressing Mouse Ocular Tissue
		3.8 Preparation of Retinal Wholemounts
		3.9 Preparation of Retinal Cross-Sections
		3.10 Immunohistochemistry
		3.11 Assessment of RGC Transduction
	4 Notes
	References
Chapter 18: Methods to Identify Rat and Mouse Retinal Ganglion Cells in Retinal Flat-Mounts
	1 Introduction
	2 Materials
		2.1 RGC Retrograde Tracing
		2.2 Immunodetection of RGCs and Their Subtypes in Flat-Mount Retinas
	3 Methods
		3.1 Tracing of RGCs for Their Identification
			3.1.1 Tracing from the ON
			3.1.2 Tracing from Both SCi
		3.2 Immunodetection of RGCs and Their Main Subtypes in Flat-Mounted Retinas
			3.2.1 Permeabilization
			3.2.2 Incubation with Primary Antibody
			3.2.3 Incubation with Secondary Antibody
	4 Notes
	References
Index