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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 death2,3. 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 suppre....

Protocol

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 na.......

Representative Results

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.......

Discussion

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

Acknowledgements

This study was supported by grants from the Science and Technology Development Fund (FDCT) of Macao SAR (Ref. No. FDCT0058/2019/A1 and 0016/2019/AKP), Research Committee, University of Macau (MYRG2020-00183-ICMS and CPG2022-00023-ICMS), and National Natural Science Foundation of China (No. 81803398).

....

Materials

NameCompanyCatalog NumberComments
AgaroseSigma-AldrichA6361
Agarose, low gelling temperatureSigma-AldrichA9414
Drummond Nanoject III Programmable Nanoliter InjectorDrummond Scientific3-000-207
Fluorescence stereo microscopesLeicaM205 FA
GraphPad Prism softwareGraphPad SoftwareVer. 7.04
Lipopolysaccharides from Escherichia coli O111:B4Sigma-AldrichL3024
Manual micromanipulatorWorld Precision InstrumentsM3301
Mineral oilSigma-AldrichM5904
Mx3005P qPCR systemAgilent TechnologiesMx3005P
Nanovue plus spectrophotometerBiochrom80-2140-46
Nitrite concentration assay kitBeyotime BiotechnologyS0021M
Phosphate-buffered salineSigma-AldrichP4417
Programmable Horizontal Pipette PullerWorld Precision InstrumentsPMP-102
PTU (N-Phenylthiourea)Sigma-AldrichP7629
Random primersTakara3802
SuperScript II Reverse TranscriptaseInvitrogen18064014
SYBR Premix Ex Taq II kitAccurate BiologyAG11701
The 3rd Gen TgrinderTiangenOSE-Y30
Thin wall glass capillaries (4”) with filament, OD 1.5 mmWorld Precision InstrumentsTW150F-4
Tricaine (3-amino benzoic acid ethyl ester)Sigma-AldrichA-5040
TRNzol Universal reagentTiangenDP424
Zebrafish tracking boxViewPoint Behavior Technology

References

  1. Xanthos, D. N., Sandkuhler, J. Neurogenic neuroinflammation: inflammatory CNS reactions in response to neuronal activity. Nature Reviews Neuroscience. 15 (1), 43-53 (2014).
  2. Fan, L. W., Pang, Y.

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Brain VentricleMicroinjectionLipopolysaccharideNeuroinflammationNeurotoxicityZebrafishIn Vivo ModelAnti neuroinflammatory DrugsGlass CapillaryMicromanipulatorAgaroseAnesthesia

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