Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine

Abstract

Source: Pang, X., et al. A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice. J. Vis. Exp. (2022)

This video demonstrates the generation of a focal demyelination mouse model using a two-point Lysophosphatidylcholine (LPC) injection. The skull of an anesthetized mouse is exposed. The injection coordinates are determined, and a hole is drilled to enable LPC injections at targeted sites. LPC induces focal demyelination of neurons via inflammation, while a two-point injection ensures its sustainability.

Protocol

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

1. Lysophosphatidylcholine (LPC) solution preparation

1. Dissolve 25 mg of LPC powder (see Table of Materials) with 250 µL of chloroform and methanol mixed solution (1:1) to make a 10% LPC solution and transfer it to a 500 µL centrifuge tube.
    NOTE: If the LPC is not completely dissolved, place the centrifuge tube in an ultrasonic cleaner and ultrasonicate at 40 kHz for ~1 h to obtain a uniform solution.
2. Divide the solution into 3 µL/tube and store at −80 °C.
    NOTE: The solution can be stored for ~2 years.
3. Before surgery, dilute the solution (step 1.2) with 27 µL of 0.9% Sodium chloride (NaCl) solution, and keep the solution in a constant temperature water bath at 37 °C.
    ​NOTE: Prepare the solution just before starting the injection.

2. Surgical preparation

1. Use a 32 G, 2-inch needle to connect to a 5 µL syringe (see Table of Materials). Ensure that the microliter syringe is unobstructed. Withdraw 5 µL of LPC solution to prepare the injection.
2. Anesthetize the mouse in an induction chamber connected to an isoflurane vaporizer with 3% isoflurane mixed with 100% oxygen at a rate of about 0.3 L/min.
3. Confirm the depth of anesthesia by the lack of toe-pinch reflex while the breathing is smooth.
4. Shave the head of the mouse between the ears using an electric shaver. Apply lubricating eye drops to prevent corneal dryness during the procedure.
5. Then, position the mouse in a stereotaxic frame (see Table of Materials) with the dorsal side up, and secure the head with a nose cone and tooth clamp. Maintain anesthesia with 1.2%-1.6% isoflurane through its nose.
   NOTE: Isoflurane concentration can be adjusted according to the respiratory status of the mice.

3. Surgical procedure

NOTE: Animals are placed on a heating pad during all procedures.

1. Fix the mouse to the stereotaxic apparatus with bilateral ear bars. Ensure that the ear bars are level and the head is horizontal and stable.
2. Disinfect the skin on the head by wiping it several times with iodophor, followed by alcohol in a circular motion. Then, a scalpel is used to cut a small incision of about 1.5 cm along the midline of the scalp to expose the skull.
3. Wipe the skull with a cotton swab dipped in 1% hydrogen peroxide until the bregma, lambda, and posterior fontanelle are exposed. Place the syringe on the stereotaxic apparatus.
    NOTE: Use hydrogen peroxide with care and avoid touching the surrounding tissues.
4. Ensure horizontal positioning of the animal's head (both front and rear and left and right).
   1. Adjust the Z-axis knob of the stereotaxic frame so that the needle tip and the skull just touch without bending, then measure the Z-axis coordinate. Check the Z coordinates of bregma and posterior fontanelle.
   2. Adjust the ear bar so that the difference between the Z coordinates of bregma and posterior fontanelle is no more than 0.02 mm. Then, follow the same method to measure the Z coordinates of the corresponding positions on the left and right sides of the midline. Adjust the ear bar to ensure the left and right are at the same level.
5. Locate the corpus callosum. Set the XYZ origin to bregma.
    NOTE: The first injection site is 1.0 mm lateral to the bregma, 2.4 mm deep, and 1.1 mm anterior. The second injection site is 1.0 mm lateral to the bregma, 2.1 mm deep, and 0.6 mm anterior. For example, the coordinate of the bregma is (0,0,0). Measure the Z coordinate of the corresponding location marked as (-1, 1.1, X) and (-1, 0.6, Y). It can be determined the first injection site coordinate of the corpus callosum is (-1, 1.1, −[X + 2.4]), and the second injection site is (-1, 0.6, −[Y + 2.1]).
6. Once the injection site is determined, make a mark with a sterile marker on the skull and record the coordinates.
7. Gently drill the marked site with a skull drill (see Table of Materials). Take care to avoid any bleeding.
8. Slowly move the needle to the given coordinates and start the injection. To induce corpus callosum demyelination, inject 2 µL of LPC solution (step 1.2) at each injection site (step 3.5.) at a rate of 0.4 µL/min.
9. After injection, keep the needle in each site for an additional 10 min.
    ​NOTE: Ensure that the interval of two injections is no more than 20 min.
10. Suture the skin with a 4-0 suture and wait for the animal to wake up within 10 min. Administer analgesics postoperatively in accordance with institutional animal care regulations.
     NOTE: The recommended time for euthanasia can be determined according to the purpose of the experiment.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-α-Lysophosphatidylcholine from egg yolk	Sigma-Aldrich	L4129-25MG
32 gauge needle	HAMILTON	7762-05
10 μl syringe	HAMILTON	80014
High speed skull drill	strong,korea	strong204
Drill	Hager & Meisinger, Germany	REF 500 104 001 001 005
Matrx Animal Aneathesia Ventilator	MIDMARK	VMR
Portable Stereotaxic Instrument for Mouse	Reward	68507
Micro syringe	Reward	KDS LEGATO 130
Isoflurane	VETEASY
Suture	FUSUNPHARMA	20152021225
Adult C57BL/6 male and female mice	Hunan SJA Laboratory Animal Co. Ltd