The overall goal of this procedure is to observe the distribution of viral particles during the acute phase of infection in the central nervous system. This method can help answer key questions in the neurovirology field. Such as how do virus particles distribute in the immature central nervous system during the acute infection phase.
The main advantage of this technique is that the initial distribution of viral particles can be observed directly with a microscope using two simple methods. Such as intercerebroventricular and intravascular injection. Begin my sterilizing a 10 microliter syringe and a 27 gauge needle with 70%alcohol.
Then load five 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 PO.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 non toxic laboratory pen at a location approximately 0.7 to 1.0 mm lateral to the saggital suture and 0.7 to 1.0 mm cuddle from the neonatal bregma.
This diagram shows the site in more detail. For reference, mark 2 mm from the tip of the needle with a non toxic marker. Next, insert the needle 2 mm 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. For intravenous injection secure the anesthetized neonate to the transilluminator using surgical tape and then visualize the superficial temporal vein.
Next, while wearing 1.5x magnifying glasses use a 1 ml syringe with a 35 gauge needle to slowly infuse 50 microliters of murine cytomegalovirus virus into the superficial temporal vein. Again repeat the procedure with fluorescent microbeads of the appropriate size in the control group. After removing the needle use gauze containing 70%alcohol to apply pressure to the injection site until the bleeding ceases.
Give the neonate approximately five minutes in a warm container to recover before returning it to the cage. Two hours after introcerbroventricular injection in the acute infection phase green recombinant M32 EGFP MCMV particles are mainly observed in the marginal area as indicated by the arrows and seen in the magnified region. At this time the M32 EGFP MCMV particles are also seen in the choroid plexus and subventricular zone.
The MCMV genome is first detected in the subventricular zone at three hours post intracerebralventricular injection. Using fluorescent and C2 hybridization the MCV genome is visible as green spots and indicated by arrows in this magnified region. At three hours post intravenous injection the MCMV particles visible as green spots and indicated by arrows are found inside and outside of the vascular area of the parenchyma, as this increased magnification image demonstrates.
At this time the MCMV particles are still visible in the choroid plexus and subventricular zone. Using fluorescent and C2 hybridization the MCMV genome is detected in the vascular area and meninges at 12 hours post IV injection. As indicated by the arrows.
Once mastered, this technique can be done in five minutes if it is performed properly. After watching this video you should have a good understanding of how to observe the distribution of viral particles during the acute phase of infection in the central nervous system. Don't forget that working with a virus can be extremely hazardous and precaution such as wearing masks and gloves should always be taken while performing this procedure.
