Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders. Hence, it is of great interest to research and develop alternative in vivo neuroinflammation models to facilitate studies of pathological development. The technique described here is an excellent tool to better understand pathological changes in the brain via in vivo imaging, and for quickly and efficiently evaluating possible anti-neuroinflammatory drugs.

Prepare for the injection by pulling glass capillaries using a micropipette puller following the five-step protocol for glass capillary tube pulling. Open the tip of the needle to the appropriate size at an angle using forceps. Fill the needle with 0.1 milliliters of mineral oil to ensure that there are no bubbles.

Remove the screw cap of the steel needle of the microinjection apparatus. Align the glass needle hole with the steel needle, then tighten the screw cap. Mount the loaded needle microinjection apparatus in a micromanipulator.

Adjust the position of the microinjection apparatus so that the micromanipulator can flexibly move the apparatus under the microscope. Discharge an appropriate volume of paraffin oil required to make the steel needle enter the capillary glass tube. Drop the injection solution consisting of PBS or lipopolysaccharide on a glass slide sterilized with 70%ethanol and adjust the microinjection apparatus so that the tip is inserted into the liquid drop.

Load approximately two microliters of injection solution into the needle. 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 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. After 24 hours, collect the larvae for microscopic imaging, locomotive behavioral assay, and determination of other indicators.

Prepare 1.5%low-melt agarose solution in E3 medium and heat it in a microwave to form a clear liquid. Use a clean plastic transfer pipette to transport the larvae to a glass slide and remove as much water as possible. Use a plastic transfer pipette to add a drop of 1.5%low-melt agarose to the larvae.

Use a one milliliter syringe needle to orient the larvae. Wait until the agarose cools down and solidifies before starting imaging. At 24 hours after lipopolysaccharide brain ventricular injection, proceed with determining gene expression markers.

Use two one milliliter syringes to separate the head portions of the larvae without the eye and yolk sac regions. Homogenize the head portion with 200 microliters of RNA extraction reagent using a tissue grinder at 11, 000 rotations per minute for five seconds, then perform RNA extraction using the chloroform isopropanol method. Air dry the RNA pellet for five to 10 minutes, then add 30 microliters of RNAse-free water to dissolve the RNA pellet.

Synthesize cDNA using reverse transcriptase with random primers as described in the text manuscript. Next, perform real-time PCR on a qPCR system using a commercially available RT-qPCR kit to determine the expression of the target genes. At 24 hours after lipopolysaccharide brain ventricular injection, transfer the zebrafish larvae to the wells of a 96-well square microplate individually.

Add 300 microliters of E3 medium to each well and then incubate the larvae for four hours to acclimatize to the testing plate. Transfer the larvae loaded microplate to the zebrafish tracking box. Turn on the light source and incubate the larvae in the testing box for 30 minutes to acclimatize to the environment.

Monitor and record the zebrafish behavior using an automated video tracking system. Record 12 sessions of five minutes each for individual zebrafish larvae and define the total distance as described in the text manuscript. After treatment for 24 hours, one to five milligrams per milliliter of lipopolysaccharide ventricular brain injection induced a significant loss of RA neurons in the brain of TgEtvmat2:GFP larval zebrafish compared to the control and sham groups.

The transgenic line elavl3:mCherry zebrafish demonstrated significant changes in the fluorescence integrated density of larval brain neurons when injected with 2.5 to five milligrams per milliliter lipopolysaccharide, while the one milligram per milliliter lipopolysaccharide injection showed no effect. Further, five milligrams per milliliter lipopolysaccharide induced a locomotion deficiency and decreased the total distance of zebrafish movement over a 60-minute tracking period. The nitrous oxide production and mRNA expression of pro-inflammatory cytokines in the head of zebrafish larvae were increased on 2.5 to five milligrams per milliliter lipopolysaccharide treatment compared to the expression in the control and sham groups.

The injection of one to five milligrams per milliliter lipopolysaccharide demonstrated the recruitment of neutrophils into the larval zebrafish brain, resulting in a significant increase in the number of neutrophils in the Tgmpo:eGFP zebrafish brain region. The opening size of needles and amount of injection force for microinjection along with the head separation from the eyes and body of zebrafish is crucial for this procedure.