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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Infection
- Infect Anopheles stephensi mosquitoes with Plasmodium berghei ANKA by feeding them for 15 min on a gametocytemic mouse. Keep the infected mosquitoes at 80% humidity and 21 °C.
- Collect female mosquitoes from their cage 17 to 22 days after the blood meal. Place them on ice to anesthetize them.
- Using forceps, place three to four mosquitoes on a glass slide covered with a drop of cold RPMI medium. Place the slide under a microscope.
- Using forceps, carefully stretch the mosquito between the head and body. Isolate the salivary gland using a syringe and needle. Repeat this procedure with the remaining mosquitoes.
- Collect the salivary glands from the glass slide by sucking them up with a glass pipette and collecting them in a 1.5 mL centrifuge tube.
NOTE: Depending on the infection rates, usually 8,000 to 15,000 infectious sporozoites can be obtained per salivary gland.
- For approximately 3 min, smash the isolated salivary glands within the centrifuge tube with a small, plastic stick to isolate the sporozoites from the salivary gland tissue.
- Centrifuge for 3 min at 1,000 x g and 4 °C to purify the sporozoites from the remaining tissue.
- Pipette the supernatant, which contains the sporozoites (SPZ), to a new centrifuge tube and count the purified sporozoites in a Neubauer hemocytometer.
- Adjust the concentration of purified sporozoites to 10,000/mL by adding phosphate-buffered saline.
- Inject a total of 1,000 sporozoites (0.1 mL) into the tail veins of inbred C57BL/6 mice to initiate infection. To facilitate the injections, place the C57BL/6 mice in a restrainer and put the tails into warm (approximately 37 °C) water to assist with the visualization of the tail veins.
NOTE: The injection itself is a short procedure that can be performed within a few seconds.
- Once daily, check blood-stage parasitemia on blood smears from day 3 onwards after the SPZ infection.
- Assess the mice once daily with the Rapid Murine Coma and Behavior Scale (RMCBS) score, starting from day 5 after the sporozoite injection.
- Assess the mice with magnetic resonance imaging (MRI) imaging according to the Rapid Murine Coma and Behavior Scale (RMCBS) score and the research question to be addressed.