This study investigates the effects of Zika virus on neural progenitor cells (NPCs) and glial cells in the mouse brain. The methodology involves a precise surgical procedure to inject the virus, followed by an analysis of the resulting cellular responses and neurodegeneration.
Take an anesthetized mouse secured in a stereotaxic frame. A heating pad maintains its body temperature.
Shave and sterilize the scalp, make a midline incision to expose the bregma, a landmark on the skull to identify the injection location, and drill a small hole.
Inject a Zika virus suspension into the brain tissue. Slowly withdraw the needle to prevent backflow.
Suture the scalp and transfer the mouse to a cage for recovery.
The virus reaches the subventricular zone, an area rich in neural progenitor cells, or NPCs, and glial cells.
The virus binds to NPC receptors and is endocytosed. Viral replication triggers a cellular stress response, inducing apoptosis.
Microglia, the resident immune cells, engulf the virus and produce oxidative bursts that damage surrounding tissue. They also release cytokines and neurotoxic factors, which recruit cytotoxic T-cells to kill the virus-infected cells.
The resulting depletion of NPCs and glial cells reflects virus-induced neurodegeneration.
Immobilize the head of an anesthetized adult mouse in the stereotaxic instrument and place a heating pad under the mouse.
After a toe or tail pinch to ensure a surgical plane of anesthesia, shave the scalp starting behind the eyes to the beginning of the ears. Sterilized the exposed skin with iodine and 70% ethanol. After making a 0.5-centimeter incision along the medial sagittal line of the head in the sterilized location to expose bregma, clear the surface of the skull of meningeal tissues while simultaneously pushing the skin away from the injection sites.
Align the surgical drill bit with bregma and then, identify the injection site by the stereotaxic coordinates. Using the control foot pedal, slowly drill into the skull until the hole is clear for the needle. Replace the drill bit with the microinjector pump and gas-tight 10-microliter syringe.
Leave a small air bubble between the saline solution and the virus, and draw up 4 to 5 microliters of virus. Lower the needle to 1.5 millimeters below the brain surface from the brain surface, and inject 1 microliter of virus at a rate of 10 nanometers per second, injecting the 1 microliter over 1.5 minutes. After removing the needle from the brain in increments as before, use forceps to loosen the skin and recover the exposed skull. Once the scalp has been sutured with 4-O sutures, remove the mouse from the stereotax and monitor on a heating pad until recovered.
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