Laser‐Mediated Microlesions in Mouse Neocortex to Investigate Neuronal Degeneration and Regeneration

Johanna Jackson1, Alison J. Canty1, Lieven Huang2, Vincenzo De Paola3

1 These authors contributed equally to this work, 2 Australian Regenerative Medicine Institute, Monash University, Clayton, 3 MRC Clinical Sciences Centre, Imperial College London, London
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.24
DOI:  10.1002/0471142301.ns0224s73
Online Posting Date:  October, 2015
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In vivo two‐photon (2P) imaging enables neural circuitry to be repeatedly visualized in both normal conditions and following trauma. This protocol describes how laser‐mediated neuronal microlesions can be created in the cerebral cortex using an ultrafast laser without causing a significant inflammatory reaction or compromising the blood‐brain barrier. Furthermore, directives are provided for the acute and chronic in vivo imaging of the lesion site, as well as for post‐hoc analysis of the lesion site in fixed tissue, which can be correlated with the live imaging phase. © 2015 by John Wiley & Sons, Inc.

Keywords: microlesion; live imaging; laser ablation; axotomy; 2‐photon microscopy; glial scar; axons; synapses; cortex

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Table of Contents

  • Introduction
  • Basic Protocol 1: Two‐Photon (2P) Imaging of Neuronal Structures in Mouse Cerebral Cortex
  • Basic Protocol 2: Pre‐Lesion Imaging
  • Basic Protocol 3: In Vivo Lesioning
  • Basic Protocol 4: Lesioning in Organotypic Brain Slices
  • Basic Protocol 5: Post‐Lesion Imaging
  • Basic Protocol 6: Post‐Mortem Imaging
  • Basic Protocol 7: Immunohistochemistry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Two‐Photon (2P) Imaging of Neuronal Structures in Mouse Cerebral Cortex

  • Mice
  • Isoflurane (Abbott)
  • Oxygen
  • 100 mg/ml ketamine (10‐ml stock solution)
  • 20 mg/ml xylazine (50‐ml stock solution)
  • Sterile saline solution
  • Sterile lubricant eye ointment (e.g., Vaseline or Lacrilube, Allergan)
  • Antiseptic solution (e.g., Iodine 10% in saline or Videne, Ecolab)
  • Induction chamber
  • Inhalational anesthesia vaporizer
  • Heating pad
  • Two‐photon (2P) microscope powered by an ultrafast laser (e.g., Coherent Chameleon Ultra II), 40×, 0.8‐NA water immersion objective or similar
  • Head fixation device (custom‐made) or stereotaxic frame
  • Cotton swabs (150‐mm length, wood shaft, Medical Wire)
  • Light‐proof cage

Basic Protocol 2: Pre‐Lesion Imaging

  • In vivo imaging set up on 2P microscope stage (see protocol 1)
  • Dextran‐Texas Red (mol. wt. 10,000; Life Technologies, cat. no. D1828)
  • Sterile saline
  • Image acquisition software (PrairieView, ScanImage, or similar)

Basic Protocol 3: In Vivo Lesioning

For Materials, see Basic Protocols protocol 11 and protocol 22.

Basic Protocol 4: Lesioning in Organotypic Brain Slices

  • Organotypic slice cultures
  • Tyrode's salt solution (see recipe or Sigma)
  • 35‐mm petri dishes

Basic Protocol 5: Post‐Lesion Imaging

For Materials, see protocol 1.

Basic Protocol 6: Post‐Mortem Imaging

  • Paraformaldehyde (see recipe), ice cold
  • Phosphate buffer saline (PBS) (see recipe)
  • Sodium azide, optional
  • Agarose, optional
  • Dissection instruments
  • Small dissecting dish
  • Paperclip, optional
  • Additional reagents and equipment see protocol 1
CAUTION: Sodium azide (NaN 3) is poisonous; wear gloves.

Basic Protocol 7: Immunohistochemistry

  • Dissected brains (see protocol 6)
  • Sucrose
  • PBS (see recipe)
  • Cresyl violet, optional
  • Sodium azide
  • Ketamine/xylazine
  • 2.5% glutaraldehyde (EM‐grade; Electron Microscopy Sciences, cat. no. 16200)
  • Paraformaldehyde (see recipe)
  • Glycerol, optional
  • Nail polish
  • Resin
  • Tangential vibratome or cryostat
  • Fine brush or glass stick
  • 24‐well dishes
  • Glass petri dishes
  • Glass slides and coverslips
  • Standard microscope with camera
  • Adobe Photoshop software
  • 2P microscope
  • Parafilm
  • 1.0‐ml microcentrifuge tubes
CAUTION: Sodium azide (NaN 3) is poisonous; wear gloves.
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Literature Cited

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