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

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 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 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.......

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

Materials

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