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).

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
<ol>
	<li>Generate zebrafish embryos (200-300 embryos per mating) by natural pairwise mating as previously reported28.</li>
	<li>Prepare egg water (E3) medium stock (60&#215;) by dissolving 34.8 g of NaCl, 1.6 g of KCl, 5.8 g of CaCl2&#183;2H2O, and 9.78 g of MgCl2&#183;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&#215;), dilute the stock 60 times in ddH2O. Store at 28.5 &#176;C when not in use.</li>
	<li>Use a plastic transfer pipette to transfer the zebrafish embryos to a dish containing E3 medium. Raise the embryos at 28.5 &#176;C in an incubator and monitor their development and health. Remove dead embryos and replace unclean E3 medium with fresh medium daily.</li>
	<li>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&#215; 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.</li>
	<li>Maintain the zebrafish embryos under the above conditions until embryos reach the desired developmental larval stage.</li>
</ol>
2. Preparing for microinjection
<ol>
	<li>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).</li>
	<li>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).</li>
	<li>Fill the needle with 0.1 mL of mineral oil to ensure that there are no bubbles.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Drop the injection solution (such as 1&#215; 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 &#181;L of injection solution into the needle.</li>
	<li>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).</li>
</ol>
3. Mounting zebrafish for microinjections
NOTE: Zebrafish brain development occurs within 3 dpf and matures at 5 dpf with a well-developed central nervous system31,32. Therefore, 5 dpf larvae are already suitable for studying LPS-mediated neuronal damage as well as behavioral and inflammatory responses.
<ol>
	<li>Inject the zebrafish larvae within approximately 30 min of reaching the stage of interest, to maintain the consistency of injection timing.</li>
	<li>To anesthetize the zebrafish larvae, combine tricaine with clean fish tank water (final concentration of 0.02% w/v tricaine).</li>
	<li>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 &#176;C for up to 2 weeks.</li>
	<li>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.</li>
</ol>
4. Injecting the brain ventricle
<ol>
	<li>Adjust the magnification of the microscope so that the brain ventricular structure of zebrafish is clearly displayed in the field of vision (Figure 2B).</li>
	<li>Place the needle carefully above the brain tectum (Figure 2C).</li>
	<li>Puncture the skin of the zebrafish brain with the needle tip slowly using the micromanipulator.</li>
	<li>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.</li>
</ol>
5. Imaging
<ol>
	<li>Prepare 1.5% low-melt agarose solution in E3 medium and heat it in a microwave to form a clear liquid.</li>
	<li>Use a clean plastic transfer pipette to transport the larvae to a glass slide and remove as much water as possible.</li>
	<li>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.</li>
	<li>Use a 1 mL syringe needle to orient the larvae. Wait until the agarose cools down and solidifies before starting imaging.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
6. Determination of gene expression markers
<ol>
	<li>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).</li>
	<li>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).</li>
	<li>Extract RNA from the larval head portions.
	<ol>
		<li>For RNA extraction, homogenize the head portion with 200 &#181;L 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.</li>
		<li>Perform RNA extraction using the chloroform-isopropanol method. To do so, add 40 &#181;L 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 &#176;C.</li>
		<li>Transfer the aqueous phase to a fresh tube (about 100 &#181;L) and add 100 &#181;L of isopropanol. Incubate for 10 min and then centrifuge at 12,000 x g for 10 min at 4 &#176;C. Remove the supernatant.</li>
		<li>Add 200 &#181;L of 75% ethanol to wash the RNA pellet. Vortex the samples briefly and then centrifuge at 7500 x g for 5 min at 4 &#176;C. Discard the supernatant and wash the RNA pellet again.</li>
		<li>Air dry the RNA pellet for 5-10 min, then add 30 &#181;L 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).</li>
	</ol>
	</li>
	<li>Synthesize cDNA from the RNA extracted in step 6.3, using reverse transcriptase with random primers (see Table of Materials).
	<ol>
		<li>Add 1 &#181;L of random primers, 1 &#181;L of dNTPs, 1 &#181;g of RNA, and distilled water (total volume to 12 &#181;L) to a nuclease-free tube. Heat the mixture to 65 &#176;C for 5 min and quick chill on ice.</li>
		<li>Add 1 &#181;L of 0.1 M DTT, 1 &#181;L of RNase inhibitor, 4 &#181;L of 5&#215; first-strand buffer, and 1 &#181;L of M-MLV reverse transcriptase (total volume: 20 &#181;L) to the tube. Mix gently and incubate the tube at 25 &#176;C for 2 min, followed by 42 &#176;C for 50 min. Inactivate the reaction by heating at 70 &#176;C for 15 min.</li>
	</ol>
	</li>
	<li>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-1&#946;, IL-6, and TNF-&#945;). The reaction mixture (20 &#181;L) contained 10 &#181;L of SYBR green, 0.4 &#181;L of ROX reference dye, 0.8 &#181;L each of the forward and reverse primers, 6 &#181;L of ddH2O, and 2 &#181;L of template cDNA (1 &#181;g). Perform PCR amplification under the conditions: 95 &#176;C for 30 s, followed by 40 cycles of 95 &#176;C for 5 s and 60 &#176;C for 34 s, and with a dissociation stage of 95 &#176;C for 15 s, 60 &#176;C for 60 s, and 95 &#176;C for 15 s.</li>
	<li>Normalize the mRNA levels of the target genes to that of a housekeeping gene, elongation factor 1 &#945; (Ef1&#945;). Calculate the expression levels for each target gene by the 2&#8722;&#916;&#916;CT method33. The primer sequences of each gene are described in Table 2.</li>
	<li>For the determination of nitric oxide, homogenize the head portion (obtained in step 6.2) in 100 &#181;L 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 &#176;C. Collect the lysates and perform nitrite concentration assay34 using a commercially available kit (see Table of Materials).</li>
</ol>
7. Zebrafish locomotive behavioral assay
<ol>
	<li>At 24 h after LPS brain ventricular injection, transfer the zebrafish larvae to the wells of a 96-well square microplate individually. Add 300 &#181;L of E3 medium to each well and then incubate the larvae for 4 h to acclimate to the testing plate.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
8. Statistical analysis
<ol>
	<li>Perform statistical analyses using standard analysis software (see Table of Materials).</li>
	<li>Perform statistical analysis of differences between two groups using ordinary one-way ANOVA. Calculate Pearson&#39;s correlation coefficient to assess the strength of correlations. P &#60; 0.05 was considered significant in all analyses.</li>
</ol>

The authors declare no competing financial interest.

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</s...

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-&#945;, IL-1&#946;, and IL-6) are secreted into the extracellular space and act as crucial components in neuron death and the suppression of neurogenesis4,5,6.
Microinjection of inflammation mediators (such as IL-1&#946;, L-arginine, and endotoxins) into brain can cause neuronal cell reduction and neuroinflammation7,8,9. 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 diseases10,12,13,14,15. 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 processes17,18,19. 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 imaging19,20,21,22,23. 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 response24,25,26,27. 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.

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

brain ventricular microinjections of lipopolysaccharide into larval zebrafish to assess neuroinflammation and neurotoxicity

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-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) 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-1&#946;, and TNF-&#945; 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.

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...

Watch this Scientific Journal Video about Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity at JoVE.com

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

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.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

brain-ventricular-microinjections-lipopolysaccharide-into-larval

Research

JoVE Journal

Neuroscience

3.2K Views.  University of Macau, Avenida da Universidade. 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. 

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

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Immunology and Infection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Biology

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage (ICH). Modelling ICH pre-clinically has proven difficult, and current rodent models poorly recapitulate the spontaneous nature of human ICH. Therefore, there is an urgent requirement for alternative pre-clinical methodologies for study of disease mechanisms in ICH and for potential drug discovery.
The use of zebrafish represents an increasingly popular approach for translational research, primarily due to a number of advantages they possess over mammalian models of disease, including prolific reproduction rates and larval transparency allowing for live imaging. Other groups have established that zebrafish larvae can exhibit spontaneous ICH following genetic or chemical disruption of cerebrovascular development. The aim of this methodology is to utilize such models to study the pathological consequences of brain hemorrhage, in the context of pre-clinical ICH research. By using live imaging and motility assays, brain damage, neuroinflammation and locomotor function following ICH can be assessed and quantified.
This study shows that key pathological consequences of brain hemorrhage in humans are conserved in zebrafish larvae highlighting the model organism as a valuable in vivo&#160;system for pre-clinical investigation of ICH. The aim of this methodology is to enable the pre-clinical stroke community to employ the zebrafish larval model as an alternative complementary model system to rodents.

Here we present a protocol to quantify brain injury, locomotor deficits and neuroinflammation following bleeding in the brain in zebrafish larvae, in the context of human intracerebral hemorrhage (ICH).

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage ...

Inducing Neuroinflammation in Zebrafish Larvae with a Lipopolysaccharide Injection

Source: He, Y. et al. <a href="https://app.jove.com/v/64313">Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity.</a> J. Vis. Exp. (2022).
The video demonstrates the microinjection of lipopolysaccharides (LPS) into the brains of zebrafish larvae to induce neuroinflammation. The larvae are positioned on an agarose-lined dish, the needle is inserted into the brain ventricles, and the LPS is injected. The injected LPS triggers an immune response that leads to neuroinflammation.

Wywoływanie zapalenia nerwów u larw danio pręgowanego za pomocą wstrzyknięcia lipopolisacharydu

1. Preparing for microinjection 

	Prepare for the injection by pulling glass capillaries using a micropipette puller (see Table of Materials), following ...

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

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Wywoływanie zapalenia nerwów u larw danio pręgowanego za pomocą wstrzyknięcia lipopolisacharydu