Inducing Local Demyelination in the Optic Nerve Using Lysolecithin

研究方案

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.    

1. Optic Nerve Injection

1. Anesthetize the animal with an intraperitoneal injection of ketamine (75 mg/kg) and medetomidine (0.5 mg/kg).
   NOTE: Following induction of anesthesia, observe the withdrawal reflexes (pinch test, corneal and palpebral reflex, etc.) and their absence as an indication to begin surgery. Continually monitor animals throughout the surgery and administer the additional anesthetic drug (10% of the initial ketamine dose per top-up) if the reflexes are present. Adult rats (>12 weeks) are used in the experiments.
2. Shave the skin of the surgical area. Place the animal on a warming pad (37 °C) to maintain body temperature during the surgery. Prepare skin through topical application of povidone-iodine. Apply surgical draping. Apply topical ophthalmic ointment to prevent corneal dryness under general anesthesia. Maintain asepsis by using sterile instruments.
3. Make a 1- to 1.5-cm incision in the skin above the orbit of a randomly selected eye. Open the subcutaneous tissue to reach the orbital cavity using fine iris scissors. Open the conjunctiva and anterior Tenon's capsule under the operating microscope.
4. Retract the extraocular muscles and intraorbital lacrimal glands to expose approximately 3 mm length of the optic nerve. Open the dura and arachnoid matter layers around the optic nerve longitudinally using an ophthalmic blade.
5. Insert the glass pipette into the optic nerve at a distance of 2 mm posterior to the globe. The glass micropipette is attached to a Hamilton syringe.
6. Inject 1% lysolecithin (0.4 - 1.0 µl, with 0.02% Evan's Blue which doesn't have effects on myelination) slowly into the nerve approximately over a period of 30 sec.
7. Suture the skin incision. Apply antibiotic ointment to prevent infection. Fellow eyes can be served as internal controls for electrophysiology recordings.
8. Place the animals on a warming pad to recover from anesthesia.

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材料

Name	Company	Catalog Number	Comments
Ketamine 100 mg/ml (Ketamil)	Troy Laboratories	AC 116
Medetomidine 1 mg/ml (Domitor)	Pfizer	sc-204073
Homoeothermic blanket system	Harvard Apparatus	NC9203819
Hamilton syringe	Hamilton	87930
Lysolecithin	Sigma	L4129
Evan's blue	Sigma	E2129