An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain

Samenvatting

Source: Kawasaki, H., et al. Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain. J. Vis. Exp. (2016).

This video demonstrates the intracerebroventricular injection process for studying murine cytomegalovirus (MCMV) distribution in the neonatal mouse brain. MCMV, engineered to express an enhanced green fluorescent protein (EGFP) for visibility, is injected into the lateral ventricle of a mouse pup's brain. The injected virus circulates through the cerebrospinal fluid, infecting cells in the periventricular region, including the marginal zone, choroid plexus, and subventricular zone.

Protocol

1. ICV Injection of MCMV and Fluorescent Microbeads into Neonatal Mice

1. Maintain normal pregnant ICR mice in a temperature-controlled facility under a 12 hr light/dark cycle. The neonates are designated as P 0.5 on the day of birth.
2. Sterilize a 10-µl syringe and a 27 G needle with 70% alcohol.
3. Load the injection solution (5 µl) containing MCMV or fluorescent microbeads into the needle by carefully pulling the plunger of the syringe.
4. Restrain neonatal mouse (P 0.5) by putting the mouse on ice for 3 - 4 min. Once the animal is under anesthesia, use the toe-pinch response method to determine the depth of anesthesia.
5. Mark the injection site with a non-toxic laboratory pen at a location approximately 0.7 - 1.0 mm lateral to the sagittal suture and 0.7 - 1.0 mm caudal from the neonatal bregma (Figure 1A).
6. Insert the needle 2 mm deep, perpendicular to the skull surface at the marked injection site. For reference, mark 2 mm from the tip of needle with a non-toxic maker.
7. Slowly inject 5 µl of MCMV (approximately 5 × 10⁵ PFU) into the lateral ventricle without opening the scalp (Figure 1B).
8. In another group of mice, inject a 5 µl solution containing fluorescent microbeads (0.1 - 0.3 µm, approximately 5.75 × 10⁸ particles) by the same method.
9. Slowly remove the needle 10 - 20 sec after discontinuing the plunger movement to prevent backflow.
10. To recover, keep the mice for 5 - 10 min in a warm container until movement and general responsiveness are restored.
11. Harvest the brains at a range of time points (3, 12, 24, 48, and 72 hr) post-injection.

Resultaten

Figure 1: Injection Sites of the Neonatal Mouse. (A, B) ICV injection of 5 µl MCMV (approximately 5 × 10⁵ PFU) or of 5 µl microbeads (approximately 5.75 × 10⁹ particles). (A) The injection site i...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
SPHERO TM Fluorescent Polystyrene Nile Red 0.04-0.06	Spherotech, Inc.	FP-00556-2
SPHERO TM Fluorescent Polystyrene Nile Red 0.1-0.3	Spherotech, Inc.	FP-0256-2
SPHERO TM Fluorescent Polystyrene Nile Red 1.7-2.2	Spherotech, inc.	FP-2056-2
10% mouse serum	DAKO	X0910
C57BL/6 mouse	SLC, Inc.
ICR mouse	SLC, Inc.
Modified Microliter Syringes (7000 Series)	Hamilton company
35-gauge needle	Saito Medical
pEGFP-N1 vector	Clontech	#6085-1