Generation of a Mouse Model of Experimental Cerebral Malaria Using Host Mosquito-Derived Sporozoites

Overview

This video demonstrates the generation of an experimental mouse model of cerebral malaria. Sporozoites isolated from Plasmodium berghei-infected female mosquitoes are injected into a restrained mouse's tail veins, leading to blood-brain barrier disruption and brain inflammation, causing cerebral malaria. This infected mouse model offers a valuable tool to study in vivo microvascular pathology associated with cerebral malaria.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Infection

1. 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.
2. Collect female mosquitoes from their cage 17 to 22 days after the blood meal. Place them on ice to anesthetize them.
3. 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.
4. 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.
5. 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.
6. 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.
7. Centrifuge for 3 min at 1,000 x g and 4 °C to purify the sporozoites from the remaining tissue.
8. Pipette the supernatant, which contains the sporozoites (SPZ), to a new centrifuge tube and count the purified sporozoites in a Neubauer hemocytometer.
9. Adjust the concentration of purified sporozoites to 10,000/mL by adding phosphate-buffered saline.
10. 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.
11. Once daily, check blood-stage parasitemia on blood smears from day 3 onwards after the SPZ infection.
12. Assess the mice once daily with the Rapid Murine Coma and Behavior Scale (RMCBS) score, starting from day 5 after the sporozoite injection.
13. 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.

Materials

Name	Company	Catalog Number	Comments
Isoflurane	Baxter	1001747	for anesthesia
Dotarem	Guebert	1086923	Gd-DTPA contrast agent; 0.5mmol/ml
Amira (Image Processing Program)	FEI Group		Version Amira 5.3.2
MATLAB	The MathWorks, Inc.,		Release 2012b
FDT toolbox	FMRIB's Software Library