Name: noninvasive intratracheal lipopolysaccharide instillation in mice
Uploaded: 2023-03-31T13:40:00
Description: Watch this Scientific Journal Video about Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice at JoVE.com

This work was supported by the National Natural Science Foundation of China (No.: 81903902), the China Postdoctoral Science Foundation (No.: 2019M663457), the Sichuan Science and Technology Program (No.: 2020YJ0172), and the Xinglin Scholar Research Premotion Project of Chengdu University of TCM (No.: QJRC2022053).

The animal experiment protocol was reviewed and approved by the Management Committee of Chengdu University of Traditional Chinese Medicine (Record No. 2021-11). Male C57/BL mice (20-25 g, 6-8 weeks old) were used for the present study. The mice were kept in an animal chamber and were free to drink and eat during the experiment.
1. Preparation
<ol>
	<li>Ensure that the intubation platform consists of a base, a riser, a paper clip, two rubber bands, and some strings. Take a st...

<ol>
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Initially, we looked inside the oral cavity to find the location of the trachea19. However, during this process, we discovered that the trachea of C57/BL mice is narrow, which makes it difficult to find the correct location by this method without the help of equipment such as an endoscope20. Upon further exploration, we found that the light from the intubator lamp could penetrate the surface of the body, allowing the operator to determine the position of the cannula<sup cla...

Acute lung injury (ALI) is a common clinical syndrome. Under a variety of pathogenic factors, the disruption of the physiological barrier of the lung epithelial cells and vascular endothelial cells leads to increased alveolar permeability, thereby causing decreased lung compliance, pulmonary edema, and severe hypoxemia1. Acute respiratory distress syndrome (ARDS) is the most severe form of ALI. Uncontrolled inflammation and oxidative stress damage are considered to be the main causes of ALI and the more severe ARDS2. When alveolar epithelial cells are directly injured due to trauma, the inflammatory response chain of alveolar macrophages is activated, leading to inflammation in the lung3. Globally, there are more than 3 million patients with acute ARDS per year, and they account for approximately 10% of intensive care unit admissions; additionally, the mortality rate in severe cases is as high as 46%4,5,6. Therefore, there is a need to establish a suitable animal model of ALI to study its pathogenesis. The mouse is the most commonly used experimental animal in the study of ALI since its respiratory tract can simulate the human respiratory tract well for ALI studies. Furthermore, ALI manifests as massive inflammatory cell infiltration, increased pulmonary vascular permeability, and pulmonary edema. The changes in inflammatory cytokines in serum and the lung dry-wet weight ratio reflect the degree of ALI7.
At present, the main methods for modeling LPS-induced ALI in mice include intranasal and surgical tracheal intubation8,9. Here, we propose a new method to deliver LPS into the trachea via noninvasive oropharyngeal intubation. This method uses an illuminated intubator to find the trachea of the mouse and then delivers LPS into the trachea and lung. This method delivers LPS to the lungs more accurately than the intranasal method of delivery. Compared with surgical tracheal intubation, this method does not require surgery, avoids causing wounds, and reduces pain in mice10. Therefore, this method can be used to establish a more convincing mouse model of ALI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lipopolysaccharide</td>
			<td>MERK</td>
			<td>L4130</td>
			<td>LPS</td>
		</tr>
		<tr>
			<td>Microliter Syringes</td>
			<td>SHANGHAI GAOGE INDUSTRY AND TRADE CO., LTD</td>
			<td>10028505008124</td>
			<td>To deliver LPS</td>
		</tr>
		<tr>
			<td>Mouse cannula</td>
			<td>RWD Life Science</td>
			<td>803-03008-00</td>
			<td>Mouse cannula</td>
		</tr>
		<tr>
			<td>Mouse intubation kit</td>
			<td>RWD Life Science</td>
			<td>903-03027-00</td>
			<td>Including a base, a riser, a intubator, a surgical forceps and some strings</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Biosharp life science</td>
			<td>BS-XG-03</td>
			<td>To verify the success of intubation</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Beijing Chemical Co., China</td>
			<td>20220918</td>
			<td>To anesthetize mice</td>
		</tr>
	</tbody>
</table>

noninvasive intratracheal lipopolysaccharide instillation in mice

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of acute lung injury or acute inflammation. The current most commonly used methods in acute lung injury mouse models are an intraperitoneal injection of LPS and tracheostomy for the tracheal infusion of LPS. However, the former method lacks lung targeting and damages other organs, and the latter method induces operative trauma, infection risk, and a low survival rate. Here, we recommend a noninvasive oropharyngeal endotracheal intubation method for LPS instillation in mice. In this method, LPS is noninvasively introduced into the trachea through the oropharyngeal cavity to be instilled into the lung with the help of an apparatus for endotracheal intubation. This method not only assures lung targeting but also avoids damage and the risk of death in the animals. We expect that this approach will become widely used in the field of acute lung injury.

The proposed method for LPS instillation in mice was verified by evaluating the expression of the inflammatory cytokine TNF-&#945; and the lung dry-wet weight ratio 12 h after LPS instillation. There were four groups in the experiment: blank control (without any treatment), surgical intubation16, intranasal17,18, and noninvasive oropharyngeal intubation (n = 6). Compared with the blank control group, the se...

Watch this Scientific Journal Video about Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice at JoVE.com

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

Here, we propose a protocol for intratracheal lipopolysaccharide (LPS) delivery via noninvasive oropharyngeal endotracheal intubation. This method minimizes the trauma of the surgical procedure for the animal and accurately delivers LPS to the trachea and then to the lungs.

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of ...

Our protocol proposes a new noninvasive delivery method of LPS from the trachea to the lung, more accurately. People who are trying this method for the first time should put much effort into finding the position of the trachea. To prepare the intubation platform, pass the string through the two holes and tie its ends to the small protrusions at the top of the riser.Next, attach two rubber bands to each end of the paperclip and tape them to the riser's back. Finally, fix the riser to the base at a 90-degree angle. Assemble the cannula onto a cannula pen, and turn on the pen's light.Next, disinfect small surgical forceps and Pasteur pipette using 70%alcohol. To prepare the test compound solution, add three milligrams of lipopolysaccharide, or LPS, in one milliliter of pH 7.2 PBS. Next, dissolve 10 milligrams of pentobarbital sodium in one milliliter of normal saline, and filter the solution using a 0.45-micrometer syringe filter.Place the anesthetized mouse on the intubation platform. Then fix the top front teeth with the string, and the two forefeet with the rubber bands. Using tweezers, pull the tongue out and hold it.Push the cannula slowly along the mouth to the mandibular epiglottis. Locate the trachea with the cannula pen's light and slowly insert the cannula into it. Then withdraw the intubation pen, leaving the cannula inside.Insert the Pasteur pipette into the cannula joint and press the head. After successful endotracheal intubation, using a flat-head microsyringe, instill the prepared LPS solution at three milligrams per kilogram dose through the cannula. Once finished, take out the microsyringe and cannula.Remove the mouse from the intubation platform and place it in a separate cage to recover. Observe the respiratory state of the mouse until it recovers and regains consciousness. At 12 hours after LPS tracheal instillation, the expression of an inflammatory cytokine, TNF-alpha, and the lung dry-wet weight ratio were evaluated and compared to other instillation methods.The serum TNF-alpha levels in the noninvasive oropharyngeal intubation group were significantly increased compared to the blank control group. The lung dry-wet weight ratio was also increased, reaching the same level as in the surgical tracheal intubation group. It is important to not scratch the trachea in the process of finding the location of trachea.The technique could also be used to deliver other drugs that need to reach the lung in a noninvasive way.

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