This article describes a method for injecting murine cytomegalovirus into the lateral ventricle of neonatal mice. The procedure allows for the visualization of the virus using enhanced green fluorescent protein.
Take a suspension of murine cytomegalovirus, genetically engineered to express an enhanced green fluorescent protein, allowing for visualization.
Load the suspension into a syringe with an attached needle.
Next, anesthetize a mouse pup by placing it on ice.
Identify the injection site on the pup's head.
Insert the needle perpendicular to the skull surface until it reaches the lateral ventricle, a cerebrospinal fluid, or CSF filled cavity in the brain.
Inject the suspension into the lateral ventricle.
Allow the pup to recover.
The injected virus circulates through the ventricular CSF, reaching the ependymal cells of the marginal area and the epithelial cells of the choroid plexus.
The virus attaches to surface receptors on these cells, internalizes, undergoes replication, and spreads from cell to cell, establishing an acute infection in the choroid plexus, the marginal area, and the subventricular zone.
Begin by sterilizing a 10-microliter syringe and a 27-gauge needle with 70% alcohol. Then, load 5 microliters of murine cytomegalovirus injection solution or fluorescent microbeads into the needle by carefully pulling the plunger of the syringe.
After anesthetizing and immobilizing a P0.5 neonatal mouse by placing it on ice for three to four minutes, use the toe-pinch response method to determine the depth of anesthesia. Mark the injection site on the anesthetized mouse with a nontoxic laboratory pen at a location approximately 0.7 to 1.0 millimeters lateral to the sagittal suture, and 0.7 to 1.0 millimeters caudal from the neonatal bregma. This diagram shows the site in more detail.
For reference, mark 2 millimeters from the tip of the needle with a nontoxic marker. Next, insert the needle 2 millimeters deep, perpendicular to the skull surface at the marked injection site. Then, slowly inject 5 microliters of murine cytomegalovirus into the lateral ventricle without opening the scalp.
In another group of mice, inject a 5-microliter solution containing fluorescent microbeads by the same method. After injection, allow the needle to remain in place for 10 to 20 seconds to prevent backflow. Then, slowly remove the needle. Allow the injected animals to recover for 5 to 10 minutes in a warm container until movement and general responsiveness are restored.
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