All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Adult Intracerebral Inoculation of Zika Virus
- Inject adult mice intraperitoneally with ketamine hydrochloride (80-100 mg/kg) and xylazine (5-10 mg/kg) diluted in sterile saline to induce anesthesia. Inject buprenorphine-SR (0.5-1.0mg/kg) subcutaneously to induce analgesia. Toe/tail pinch the mouse to ensure its anesthetic state, and subsequently mount it on the stereotaxic instrument (with a heating pad) to immobilize the head during surgery.
- Add ophthalmic ointment to the eyes to prevent the eyes of the mouse from drying out during surgery. Shave the scalp starting behind the eyes to the beginning of the ears. Sterilize the exposed skin with iodine and 70% ethanol (EtOH) three times. Alternate the iodine and alcohol wipes, wipe in a circular motion away from the surgical site.
- Re-check the anesthetic depth prior to incising the skin. Using a scalpel, make a 0.5 cm incision along the medial sagittal line of the head in the sterilized location, exposing bregma. Using a rolling motion with a cotton-tipped applicator, clear the surface of the skull of meningeal tissues, while simultaneously pushing the skin away from the injection sites.
- Starting from bregma, align the surgical drill bit and identify the injection sites using the following coordinates: anteroposterior (AP): -0.5 mm, mediolateral (ML): +/-1.5 mm. Using the control foot pedal, drill into skull slowly as to only drill bone until a hole has been cleared for the needle.
- Replace the drill with the microinjector pump and gas-tight syringe (10 µL volume) on the stereotaxic. Leaving a small air bubble between the saline solution and the virus, draw ~ 4-5 µL of virus. Determine the device type code for the specific syringe volume (ie, D for 10 μL volume syringe). Lower the needle to dorsoventral (DV): -1.5 mm from the brain surface. To inject 1 µL of virus, program a rate of 10 nL/s, injecting approximately 1 µL over 1.5 min.
- When the injection finishes, wait 30 s, and remove the needle in 0.5 mm increments, waiting 30 s after each turn. This reduces backflow and leakage of the injected virus.
- When the injection(s) are finished, use forceps to loosen the skin and recover the exposed skull. Suture the scalp back together using 4.0 sutures and remove the mouse from the stereotaxic instrument. Place the mouse in a cage partially resting on a heating pad to allow the mouse to escape to a non-heated area if needed and monitor while they recover (1 - 2 h) from anesthesia.
