This article describes a method for inducing focal demyelination in the mouse brain using lysophosphatidylcholine (LPC). The procedure involves precise stereotaxic injections into the white matter region, allowing researchers to study the effects of demyelination on neuronal function.
Begin with an anesthetized mouse secured in a stereotaxic apparatus with its head shaved.
Disinfect the scalp and make an incision to expose the skull.
Clean the skull to remove the connective tissue layer.
Using the reference point on the skull, mark the target site and drill a hole.
Take a syringe containing a solution of lysophosphatidylcholine, or LPC, fixed on the stereotaxic apparatus.
Determine the coordinates for two injection points in the white matter region of the brain and position the syringe accordingly.
Inject the LPC solution and keep the needle in place for some time after injection at each site.
Suture the incision and let the animal recover.
At the injection site, LPC interacts directly with the myelin, disrupting its structure and causing its breakdown.
This causes local neuronal dysfunction, thus establishing a focal demyelination model.
Begin by connecting a 32-gauge needle to a five-microliter syringe. Withdraw 5 microliters of LPC solution for the preparation of the injection. Next, confirm the depth of anesthesia of the mouse by the lack of toe-pinch reflex, while the breathing is smooth. Position the mouse in a stereotaxic frame with the dorsal side up, and secure the head with a nose cone and tooth clamp.
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. Disinfect the head skin by wiping with iodophor. Then, use scalpel to cut a small incision to about 1.5 centimeters along the midline of the scalp to expose the skull. 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. Ensure the horizontal positioning of the animal's and locate the corpus callosum. Once the injection site is determined, record the coordinates and make a mark with a marker on the skull. Gently drill the marked site with a skull drill. Take care to avoid any bleeding.
Slowly move the needle to the given coordinates. To induce corpus callosum demyelination, inject 2 microliters of LPC solution at each injection site at a rate of 0.4 microlitres per minute. After injection, keep the needle in each site for an additional 10 minutes. Finally, suture the skin with a 4-0 suture and wait for the animal to wake up within 10 minutes.
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