inducing neuroinflammation in zebrafish larvae with a lipopolysaccharide injection

Inducing Neuroinflammation in Zebrafish Larvae with a Lipopolysaccharide Injection

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 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.

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1. 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 the needle (thin wall glass capillaries [3.5 in] with filament, outer diameter [OD] 1.14 mm) to the appropriate size at an angle using forceps (Figure 1A).</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; phosphate-buffered saline (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 1B).</li>
</ol>
2. Mounting zebrafish for microinjections
NOTE: Zebrafish brain development occurs within 3 days post-fertilization (dpf) and matures at 5 dpf with a well-developed central nervous system. 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 double-distilled water (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>
3. 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 1B).</li>
	<li>Place the needle carefully above the brain tectum (Figure 1C).</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 the brain ventricle was injected with 1 nL of 1% Evans blue dye (diluted in PBS) are shown as an example (Figure 1D). Transfer the larvae to clean egg water (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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>Phosphate-buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall glass capillaries (4&#34;) 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>
	</tbody>
</table>

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.

Table 1: Five-step protocol for glass capillary tube pulling.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Operating time (sec)</td>
			<td>Heat level</td>
			<td>Action</td>
		</tr>
		<tr>
			<td>T1</td>
			<td>L&#62;</td>
			<td>H80-89</td>
			<td>P1L005</td>
		</tr>
		<tr>
			<td>T2</td>
			<td>1.6-3</td>
			<td>H00</td>
			<td></td>
		</tr>
		<t...

1. Preparing for microinjection 

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

Take anesthetized zebrafish larvae in a dish lined with agarose gel and ensure that the brain faces upward for injection.
Position the dish under a microscope to visualize the brain ventricles, which are fluid-filled cavities within the developing brain.
Take a microinjection needle containing a solution of lipopolysaccharides, or LPS, a bacterial component to trigger an immune response.
Position the needle above the brain tectum, a region containing neurons and resident immune cells, particularly microglia.
Puncture the brain with the needle and inject the LPS solution into the ventricle.
Microglia in the surrounding tissue binds to LPS through toll-like receptors.
The binding triggers the release of pro-inflammatory cytokines, which recruit neutrophils to the site, leading to neuroinflammation.
Neutrophils generate bursts of reactive oxygen species and release proteolytic enzymes, resulting in neuronal death.
Transfer the neuroinflammation-induced larvae into a culture medium to maintain them for downstream assays.

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Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

科研

JoVE Journal

发育生物学

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

为梭状体开发拉瓦尔斑马鱼感染模型

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

脂多糖注射液致大鼠亚急性脑 Microhemorrhages

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...

Inducing Neuroinflammation in Zebrafish Larvae with a Lipopolysaccharide Injection

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Inducing Neuroinflammation in Zebrafish Larvae with a Lipopolysaccharide Injection