This study investigates the immune response in zebrafish larvae by injecting lipopolysaccharides (LPS) into the brain. The resulting neuroinflammation is characterized by the activation of microglia and recruitment of neutrophils, leading to neuronal death.
Take anesthetized zebrafish larvae in a dish lined with agarose gel and ensure that the brain faces upward for injection.
Position the dish under a microscope to visualize the brain ventricles, which are fluid-filled cavities within the developing brain.
Take a microinjection needle containing a solution of lipopolysaccharides, or LPS, a bacterial component to trigger an immune response.
Position the needle above the brain tectum, a region containing neurons and resident immune cells, particularly microglia.
Puncture the brain with the needle and inject the LPS solution into the ventricle.
Microglia in the surrounding tissue binds to LPS through toll-like receptors.
The binding triggers the release of pro-inflammatory cytokines, which recruit neutrophils to the site, leading to neuroinflammation.
Neutrophils generate bursts of reactive oxygen species and release proteolytic enzymes, resulting in neuronal death.
Transfer the neuroinflammation-induced larvae into a culture medium to maintain them for downstream assays.
Set up the micromanipulator so that the needle tip of the microinjection apparatus is in the same field of vision as the larvae on high magnification. Melt a solution of 2% agarose in double-distilled water using a microwave. Pour the molten agarose into a plastic dish. Use a plastic transfer pipette to transport the anesthetized larvae to the center of a 2% agarose-coated plastic dish.
Orient the mounted larvae with the brain-side side up for needle access. Adjust the magnification of the microscope so that the brain ventricular structure of zebrafish is clearly displayed in the field of vision. Place the needle carefully above the brain tectum. Clean the brain area with 70% ethanol, and puncture the skin of the zebrafish brain with the needle tip slowly using the micromanipulator.
Press the foot pedal to eject one nanoliter of 1X PBS or different concentrations of lipopolysaccharide. Transfer the larvae to a clean E3 medium immediately after the injection.
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