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

Podsumowanie

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Streszczenie

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β), and human tumor necrosis factor α (TNF-α) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1β, and TNF-α in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

Wprowadzenie

Neuroinflammation has been described as a crucial anti-neurogenic factor involved in the pathogenesis of several neurodegenerative diseases of the central nervous system (CNS)¹. Following pathological insults, neuroinflammation may result in various adverse consequences, including inhibition of neurogenesis and induction of neuronal cell death^2,3. In the process underlying the response to inflammation induction, multiple inflammatory cytokines (such as TNF-α, IL-1β, and IL-6) are secreted into the extracellular space and act as crucial components in neuron death and the suppression of neurogenesis^4,5,6.

Microinjection of inflammation mediators (such as IL-1β, L-arginine, and endotoxins) into brain can cause neuronal cell reduction and neuroinflammation^7,8,9. Lipopolysaccharide (LPS, Figure 1), a pathogenic endotoxin present in the cell wall of Gram-negative bacteria, can induce neuroinflammation, exacerbate neurodegeneration, and reduce neurogenesis in animals¹⁰. LPS injection directly into the CNS of the mouse brain increased levels of nitric oxide, pro-inflammatory cytokines, and other regulators¹¹. Furthermore, stereotaxic injection of LPS into the local brain environment can induce excessive production of neurotoxic molecules, resulting in impaired neuronal function and subsequent development of neurodegenerative diseases^{10,12,13,14,15}. In the neuroscience field, live and time-course microscopic observations of cellular and biological processes in living organisms are crucial for understanding the mechanisms underlying pathogenesis and pharmacological action¹⁶. However, live imaging of mouse models of neuroinflammation and neurotoxicity is fundamentally constrained by the limited optical penetration depth of microscopy, which precludes functional imaging and live observation of developmental processes^17,18,19. Therefore, the development of alternative neuroinflammation models is of great interest to facilitate the study of pathological development, and the mechanism underlying neuroinflammation and neurodegeneration, by live imaging.

Zebrafish (Danio rerio) has emerged as a promising model to study neuroinflammation and neurodegeneration due its evolutionarily conserved innate immune system, optical transparency, large embryo clutch size, genetic tractability, and suitability for in vivo imaging^{19,20,21,22,23}. Previous protocols have either directly injected LPS into the yolk and hindbrain ventricle of larval zebrafish without mechanistic assessment, or simply added LPS to fish water (culture medium) to induce a lethal systemic immune response^24,25,26,27. Herein, we developed a protocol for microinjection of LPS into the brain ventricles, to trigger an innate immune response or neurotoxicity in the 5 days post fertilization (dpf) zebrafish larvae. This response is evidenced by neuronal cell loss, locomotory behavior deficit, increased nitrite oxide release, activation of inflammatory gene expression, and recruitment of neutrophils in the zebrafish brain at 24 h after injection.

Protokół

AB wild-type zebrafish and transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were obtained from the Institute of Chinese Medical Sciences (ICMS). Ethical approval (UMARE-030-2017) for the animal experiments was granted by the Animal Research Ethics Committee, University of Macau, and the protocol follows the institutional animal care guidelines.

1. Zebrafish embryo and larval husbandry

1. Generate zebrafish embryos (200-300 embryos per mating) by natural pairwise mating as previously reported²⁸.
2. Prepare egg water (E3) medium stock (60×) by dissolving 34.8 g of NaCl, 1.6 g of KCl, 5.8 g of CaCl₂·2H₂O, and 9.78 g of MgCl₂·6H₂O in ddH₂O to a final volume of 2 L and adjust pH to 7.2 using NaOH and HCl. To prepare working concentration of E3 medium (1×), dilute the stock 60 times in ddH₂O. Store at 28.5 °C when not in use.
3. Use a plastic transfer pipette to transfer the zebrafish embryos to a dish containing E3 medium. Raise the embryos at 28.5 °C in an incubator and monitor their development and health. Remove dead embryos and replace unclean E3 medium with fresh medium daily.
4. For zebrafish imaging experiments, use a plastic transfer pipette to collect 0-2 h post fertilization (hpf) embryos (around 200 eggs) in approximately 15 mL of 1× E3 medium containing 0.003% N-phenylthiourea (PTU).
   NOTE: PTU can inhibit the formation of melanophores during embryogenesis²⁹. Treatment of embryos with PTU during embryogenesis can improve signal detection in microscopy or expression of fluorescence³⁰.
5. Maintain the zebrafish embryos under the above conditions until embryos reach the desired developmental larval stage.

2. Preparing for microinjection

1. Prepare for the injection by pulling glass capillaries using a micropipette puller (see Table of Materials), following the five step protocol for glass capillary tube pulling (Table 1).
2. Open the tip of needle (thin wall glass capillaries [3.5 in] with filament, OD 1.14 mm) to the appropriate size at an angle using forceps (Figure 2A).
3. Fill the needle with 0.1 mL of mineral oil to ensure that there are no bubbles.
4. Remove the screw cap of the steel needle of the microinjection apparatus. Align the glass needle hole with the steel needle and then tighten the screw cap. Mount the loaded needle microinjection apparatus in a micromanipulator (see Table of Materials).
5. Adjust the position of the microinjection apparatus so that the micromanipulator can flexibly move the apparatus under the microscope. Discharge a certain volume of paraffin oil to make the steel needle enter the capillary glass tube.
6. Drop the injection solution (such as 1× PBS or 5 mg/mL LPS) on a glass slide sterilized with 70% ethanol and adjust the microinjection apparatus so that the tip is inserted into the liquid drop. Load ~2 µL of injection solution into the needle.
7. Set up the micromanipulator (fine adjustment settings of up, down, left, and right) so that the needle tip of the microinjection apparatus is in the same field of vision as the larvae on high magnification (Figure 2B).

3. Mounting zebrafish for microinjections

NOTE: Zebrafish brain development occurs within 3 dpf and matures at 5 dpf with a well-developed central nervous system^31,32. Therefore, 5 dpf larvae are already suitable for studying LPS-mediated neuronal damage as well as behavioral and inflammatory responses.

1. Inject the zebrafish larvae within approximately 30 min of reaching the stage of interest, to maintain the consistency of injection timing.
2. To anesthetize the zebrafish larvae, combine tricaine with clean fish tank water (final concentration of 0.02% w/v tricaine).
3. Melt a solution of 2% agarose in ddH₂O using a microwave. Pour the molten agarose into a plastic dish. The 2% agarose-coated plastic dish can be stored at 4 °C for up to 2 weeks.
4. Use a plastic transfer pipette to transport anesthetized larvae to the center of the 2% agarose-coated plastic dish. Orient the mounted larvae with the brain side up for needle access.

4. Injecting the brain ventricle

1. Adjust the magnification of the microscope so that the brain ventricular structure of zebrafish is clearly displayed in the field of vision (Figure 2B).
2. Place the needle carefully above the brain tectum (Figure 2C).
3. Puncture the skin of the zebrafish brain with the needle tip slowly using the micromanipulator.
4. Press the foot pedal to eject 1 nL of 1x PBS or different concentrations of LPS (1.0, 2.5, and 5.0 mg/mL) (see Table of Materials). Successful ventricle injection images at which brain ventricle was injected with 1 nL of 1% Evans blue dye (diluted in PBS) are shown as an example (Figure 2D). Transfer the larvae to clean E3 medium immediately after the injection. After 24 h, collect the larvae for microscopic imaging, locomotive behavioral assay, and determination of other indicators.

5. Imaging

1. Prepare 1.5% low-melt agarose solution in E3 medium and heat it in a microwave to form a clear liquid.
2. Use a clean plastic transfer pipette to transport the larvae to a glass slide and remove as much water as possible.
3. Use a plastic transfer pipette to add a drop of 1.5% low-melt agarose to the larvae.
   NOTE: Cool the low-melt agarose in the pipette for a while before adding so as not to injure the larvae.
4. Use a 1 mL syringe needle to orient the larvae. Wait until the agarose cools down and solidifies before starting imaging.
5. Observe and photograph the neuron region in the whole brain of Tg(ETvmat2:EGFP) and Tg(elavl3:mCherry) zebrafish, as well as the recruitment of neutrophils in the Tg(mpo:EGFP) zebrafish brain under a fluorescence stereo microscope (see Table of Materials).
   NOTE: Fluorescence microscope settings used for imaging are shown below. Tg(ETvmat2:EGFP) zebrafish brain (excitation wavelength: 450-490 nm, emission wavelength: 500-550 nm, visual magnification: 100x); Tg(elavl3:mCherry) zebrafish brain (excitation wavelength: 541-551 nm, emission wavelength: 590 nm, visual magnification: 100x); Tg(mpo:EFGP) zebrafish brain (excitation wavelength: 450-490 nm, emission wavelength: 500-550 nm, visual magnification: 63x).
6. Measure and analyze the fluorescence intensity and length of the Ra neuron region in Tg(ETvmat2:EGFP) zebrafish and the fluorescence intensity of Tg(elavl3:mCherry) zebrafish brain neurons using ImageJ software. Manually count the neutrophils in the brain region of Tg(mpo:EFGP) zebrafish.

6. Determination of gene expression markers

1. At 24 h after LPS brain ventricular injection, proceed with the determination of gene expression markers. To do so, anesthetize zebrafish larvae with 0.02% tricaine (as mentioned in step 3.2).
2. Use two 1 mL syringes to separate the head portions of the larvae (40 larvae per group) without the eye and yolk sac regions (Figure 2E).
3. Extract RNA from the larval head portions.
   1. For RNA extraction, homogenize the head portion with 200 µL of RNA extraction reagent (see Table of Materials) using a tissue grinder (see Table of Materials) at a rotation speed of 11,000 rpm for 5 s.
   2. Perform RNA extraction using the chloroform-isopropanol method. To do so, add 40 µL chloroform, shake the tubes vigorously, and incubate them at room temperature for 10 min. Centrifuge the samples at 12,000 x g for 15 min at 4 °C.
   3. Transfer the aqueous phase to a fresh tube (about 100 µL) and add 100 µL of isopropanol. Incubate for 10 min and then centrifuge at 12,000 x g for 10 min at 4 °C. Remove the supernatant.
   4. Add 200 µL of 75% ethanol to wash the RNA pellet. Vortex the samples briefly and then centrifuge at 7500 x g for 5 min at 4 °C. Discard the supernatant and wash the RNA pellet again.
   5. Air dry the RNA pellet for 5-10 min, then add 30 µL of RNase-free water to dissolve the RNA pellet. Check the purity (ratio of absorbance at 260 nm and 280 nm) and integrity of RNA using a microvolume spectrophotometer (see Table of Materials).
4. Synthesize cDNA from the RNA extracted in step 6.3, using reverse transcriptase with random primers (see Table of Materials).
   1. Add 1 µL of random primers, 1 µL of dNTPs, 1 µg of RNA, and distilled water (total volume to 12 µL) to a nuclease-free tube. Heat the mixture to 65 °C for 5 min and quick chill on ice.
   2. Add 1 µL of 0.1 M DTT, 1 µL of RNase inhibitor, 4 µL of 5× first-strand buffer, and 1 µL of M-MLV reverse transcriptase (total volume: 20 µL) to the tube. Mix gently and incubate the tube at 25 °C for 2 min, followed by 42 °C for 50 min. Inactivate the reaction by heating at 70 °C for 15 min.
5. Perform real time PCR on a qPCR System (see Table of Materials) using a commercially available RT-qPCR kit (see Table of Materials) to determine the expression of the target genes(IL-1β, IL-6, and TNF-α). The reaction mixture (20 µL) contained 10 µL of SYBR green, 0.4 µL of ROX reference dye, 0.8 µL each of the forward and reverse primers, 6 µL of ddH₂O, and 2 µL of template cDNA (1 µg). Perform PCR amplification under the conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s, and with a dissociation stage of 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s.
6. Normalize the mRNA levels of the target genes to that of a housekeeping gene, elongation factor 1 α (Ef1α). Calculate the expression levels for each target gene by the 2−ΔΔCT method³³. The primer sequences of each gene are described in Table 2.
7. For the determination of nitric oxide, homogenize the head portion (obtained in step 6.2) in 100 µL of cold PBS using a tissue grinder. Centrifuge the resultant PBS and head portion suspension at 12,000 x g for 15 min at 4 °C. Collect the lysates and perform nitrite concentration assay³⁴ using a commercially available kit (see Table of Materials).

7. Zebrafish locomotive behavioral assay

1. At 24 h after LPS brain ventricular injection, transfer the zebrafish larvae to the wells of a 96-well square microplate individually. Add 300 µL of E3 medium to each well and then incubate the larvae for 4 h to acclimate to the testing plate.
2. Transfer the larvae-loaded microplate to the zebrafish tracking box (see Table of Materials). Turn on the light source and then incubate the larvae in the testing box for 30 min to acclimate the environment.
3. Monitor and record the zebrafish behavior using an automated video tracking system. Record 12 sessions (5 min each, total 1 h) for each zebrafish. Define the total distance (consists of inactive, small, and large distances) as the distance (in mm) that each fish moved during a 60 min tracking period.

8. Statistical analysis

1. Perform statistical analyses using standard analysis software (see Table of Materials).
2. Perform statistical analysis of differences between two groups using ordinary one-way ANOVA. Calculate Pearson's correlation coefficient to assess the strength of correlations. P < 0.05 was considered significant in all analyses.

Wyniki

The workflow described here presents a new, quick, and efficient methodology for inducing LPS-mediated neuroinflammation and neurotoxicity in zebrafish larvae. In this described protocol, 5 dpf zebrafish were injected with LPS (Figure 1) into brain ventricles using a microinjector (Figure 2A-C). Successful injection into the brain ventricle site was verified using 1% Evans blue stain (Figure 2D). Th...

Dyskusje

An increasing amount of epidemiological and experimental data implicate chronic bacterial and viral infections as possible risk factors for neurodegenerative diseases³⁶. The infection triggers the activation of inflammatory processes and host immune responses³⁷. Even if the response acts as a defense mechanism, overactivated inflammation is detrimental to neurogenesis, and the inflammatory environment does not allow for the survival of newborn neurons³⁸

Materiały

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A6361
Agarose, low gelling temperature	Sigma-Aldrich	A9414
Drummond Nanoject III Programmable Nanoliter Injector	Drummond Scientific	3-000-207
Fluorescence stereo microscopes	Leica	M205 FA
GraphPad Prism software	GraphPad Software	Ver. 7.04
Lipopolysaccharides from Escherichia coli O111:B4	Sigma-Aldrich	L3024
Manual micromanipulator	World Precision Instruments	M3301
Mineral oil	Sigma-Aldrich	M5904
Mx3005P qPCR system	Agilent Technologies	Mx3005P
Nanovue plus spectrophotometer	Biochrom	80-2140-46
Nitrite concentration assay kit	Beyotime Biotechnology	S0021M
Phosphate-buffered saline	Sigma-Aldrich	P4417
Programmable Horizontal Pipette Puller	World Precision Instruments	PMP-102
PTU (N-Phenylthiourea)	Sigma-Aldrich	P7629
Random primers	Takara	3802
SuperScript II Reverse Transcriptase	Invitrogen	18064014
SYBR Premix Ex Taq II kit	Accurate Biology	AG11701
The 3rd Gen Tgrinder	Tiangen	OSE-Y30
Thin wall glass capillaries (4”) with filament, OD 1.5 mm	World Precision Instruments	TW150F-4
Tricaine (3-amino benzoic acid ethyl ester)	Sigma-Aldrich	A-5040
TRNzol Universal reagent	Tiangen	DP424
Zebrafish tracking box	ViewPoint Behavior Technology