Name: Non-Invasive Endotracheal Administration of Lipopolysaccharide to Induce Acute Lung Injury in Rodents (Video) | JoVE Journal
Uploaded: 2025-12-05T12:40:00.000+00:00
Duration: 4 min 10 s
Description: Learn a non-invasive endotracheal technique to induce acute lung injury in rats using lipopolysaccharide delivery. This method enables precise, repeatable dosing for studying lung repair mechanisms and testing targeted therapeutics.

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5 minutes

The corresponding author, SR, acknowledges the financial support from Ramalingaswami Fellowship (Ref. No. BT/RLF/Re-entry/13/2019), Department of Biotechnology, Government of India. The authors are thankful to Dr. Govind Yadav, Dr. Ramajayan Pandian, and CSIR-IIIM animal house staff for their support.

All Institutional and CPCSEA guidelines were followed for the care and use of animals. The animals were continuously monitored post-procedure to ensure their well-being and to comply with animal ethical guidelines of CSIR-Indian Institute of Integrative Medicine, Jammu, under Institutional Animal Ethics Committee (IAEC) approval number: 468/87/8/2025. This protocol outlines the non-invasive endotracheal instillation of lipopolysaccharide (LPS) using the direct visual delivery method, as previously demonstrated, with some methodological enhancements introduced in the current study26,27,28. The technique is optimized to induce ALI in a controlled and consistent manner, ensuring robust experimental outcomes while maintaining high standards of animal care. The reagents and the equipment used are listed in the Table of Materials.
1. Anaesthesia and LPS preparation
<ol>
	<li>Animal weight measurement and LPS dose calculation
	<ol>
		<li>Weigh each animal using a calibrated balance to calculate the precise LPS dose. Select Wistar rats weighing between 140-160 g for the study.</li>
		<li>Prepare LPS solution by diluting the lyophilized Escherichia coli LPS in sterile phosphate-buffered saline (PBS) to achieve the desired concentration. Freshly prepared LPS solution (5 &#181;g/g body weight)29,30&#160;is used.</li>
	</ol>
	</li>
	<li>Anaesthesia induction and critical timing
	<ol>
		<li>Anaesthetize the animals via intraperitoneal injection of a freshly prepared ketamine-xylazine cocktail (ketamine: 75 mg/kg and xylazine: 10 mg/kg) using 1 mL syringe. After injection, allow approximately 2-5 min for the anaesthesia to take effect, without disturbing the animals.</li>
		<li>Confirm adequate anaesthesia by ensuring the absence of spontaneous movement and a negative toe pinch reflex. LPS is administered via endotracheal instillation 5-8 min after anaesthesia induction. On the day of experiment termination, perform procedures such as tracheotomy and thoracotomy 4-7 min post anaesthesia induction. 
		NOTE: Proper anaesthetic depth is critical for successful endotracheal instillation. Over-anaesthetization may suppress the gag reflex, while under-anaesthetization may cause distress or movement during the procedure. Use freshly prepared LPS to maintain consistency across experiments. Monitor the animals closely during and after the procedure to ensure proper welfare.</li>
	</ol>
	</li>
</ol>
2. Endotracheal instillation of LPS
<ol>
	<li>Assessment of anaesthetic adequacy: Following the administration of the anaesthetic cocktail, evaluate the depth of anaesthesia by applying a firm toe pinch with forceps or fingers. If a response occurs, deeper anaesthesia is required. Upon confirmation of anaesthetized state, proceed with the instillation protocol. Position the rat&#160;in a semi-recumbent position, suspended by incisors on a solid supporting platform.</li>
	<li>Tracheal visualization: Open the mouth of the anaesthetized animal, gently grasp the tongue using forceps, and carefully insert the laryngoscope, aligning it precisely for optimal visualization of the tracheal opening. Ensure the light is properly directed to illuminate the laryngeal region, facilitating unobstructed access.</li>
	<li>Tongue manipulation: Employ curved, blunt-ended forceps to delicately grasp the tongue and gently retract it in an upward and lateral direction. 
	NOTE: Utilize handheld magnifiers to enhance the clarity of the laryngeal anatomy and ensure meticulous alignment of the instillation apparatus for enhanced precision.</li>
	<li>LPS administration: Draw 100 &#181;L of LPS solution into the syringe and attach it to a 16 G rat endotracheal tube; administer the LPS solution near the tracheal opening. Immediately after instillation, gently occlude the nostrils for 2-4 s using blunt-ended forceps to promote the aspiration of the LPS solution into the lungs through the trachea. Continuously monitor the delivery using a light source-enabled laryngoscope to confirm accurate and complete administration.</li>
	<li>Post-procedural monitoring: Carefully reposition the tongue to its normal anatomical orientation following successful LPS delivery. Transfer the animal to the cage and closely monitor for any signs of respiratory compromise or choking for at least 1 min.</li>
	<li>Key considerations
	<ol>
		<li>Visual acuity: Ensure optimal alignment of the laryngoscope for accurate LPS delivery. Use magnification tools to enhance precision and efficacy.</li>
		<li>Grasp the tongue carefully and hold it against the lower jaw or edge of the mouth to stabilize it.</li>
	</ol>
	</li>
</ol>
3. Sample collection
<ol>
	<li>Perform bronchoalveolar lavage (BAL) as described in earlier studies, with minor modifications31,32,33. Following tracheotomy, gently infuse 2 mL of cold PBS into the lungs and retrieve the fluid.</li>
	<li>Centrifuge the collected lavage fluid at 5000 x g for 5 min at 4&#160;&#176;C and use the supernatant for the detection of inflammatory markers. Collect the lungs for histopathological examination or homogenize for myeloperoxidase (MPO) activity. Keep the samples on ice during the collection process to preserve sample integrity.</li>
</ol>

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</ol>

The authors declare that they have no competing financial interests.

The endotracheal instillation method provides a non-invasive, reproducible, and targeted approach for pulmonary administration, effectively bypassing the need for surgical intervention. This method significantly reduces the risks associated with surgical trauma, such as tissue damage, while also minimizing the potential for post-operative infections and unintended organ damage that can arise from invasive procedures. By delivering agents directly to the lungs, the endotracheal route ensures precise localized treatment, e...

Acute Lung Injury (ALI) is a life-threatening inflammatory condition characterised by arterial hypoxemia, pulmonary edema, compromised endothelial-epithelial barrier integrity, and massive infiltration of immune cells into the lungs1. The pathophysiology of ALI is complex and involves the interplay and disruption of multiple overlapping and interacting pathways, such as coagulation, tissue injury, and both pulmonary and systemic inflammation. Prompt suppression of this inflammatory response is crucial for restoring tissue homeostasis. If left untreated, this rapidly progressing disorder frequently escalates to Acute Respiratory Distress Syndrome (ARDS), a more severe condition defined by refractory hypoxemia. The pathogenesis of ALI has been associated with lung injury and damage or death of alveolar epithelial cells and microvascular endothelial cells1,2,3,4.
The etiology responsible for ALI/ARDS involves a wide range of infectious and non-infectious agents, arising from both direct and indirect sources of lung injury5. Direct causes include pneumonia6, near drowning7, aspiration of gastric content8, toxin inhalation such as chlorine gas9, bacterial infection10, and viral infection such as SARS-CoV-211,12,13,14. Indirect causes originate from systemic processes that exert their influence on the lungs secondarily, which include sepsis15, trauma16, pancreatitis17, drug overdose, and blood transfusions18.
The primary goal of this method is to establish a reproducible and non-invasive model of ALI in rodents via endotracheal administration of endotoxin. The rationale for developing this technique stems from limitations of conventional delivery methods. The technique was demonstrated with the goal of achieving precise direct delivery to lung alveoli, minimizing systemic exposure, and&#160;making it a promising approach for future targeted pulmonary therapies. Intranasal instillation poses several challenges, some of which can significantly impact study outcomes, such as poor targeting to deep lung regions and respiratory variability among animals19. Other factors, such as limited dose volume and potential nasal irritation, are less critical but may still affect consistency. In comparison, endotracheal administration provides a more accurate and direct delivery to the lower respiratory tract, ensuring better lung targeting and dosing uniformity with minimum variability between animals, making this route more effective and reliable for pulmonary exposure20. The inhalation route is clinically relevant due to its non-invasive nature, but it is also associated with certain limitations in pre-clinical research, such as upper airway drug loss and reduced efficacy in targeting distal lung regions. These limitations become more pronounced in severe models, including double-hit injury or genetically modified models involving knock-in or knock-out mice. In contrast, endotracheal administration ensures more consistency for direct drug delivery to the lower respiratory tract, minimizing loss and enabling effective targeting in complex pre-clinical models as well21. Another relevant route of administration used in pre-clinical research is invasive intratracheal delivery. While it is effective, but is associated with the risk of surgical stress, infection, variation in readout, and the requirement of temperature maintenance22. In contrast, the endotracheal method offers a reliable non-invasive alternative, making it ideal for studies involving repeated assessments over extended observation periods.
This method can be applied in animal models beyond ALI, including asthma, pulmonary fibrosis, COPD, pneumonia, and infectious lung diseases. It is also relevant in pre-clinical research areas such as anaphylaxis, cardiac arrest, and cardiac arrhythmias. Notably, the endotracheal method is already established in clinical practice for the administration of certain drugs, depending on the urgency and clinical scenario23,24,25.

<table><tbody><tr><td>&amp;nbsp;Prism 8</td><td>Graphpad</td><td>https://www.graphpad.com/features</td><td></td></tr><tr><td>Bradford Reagent&amp;nbsp;</td><td>Himedia, India</td><td>ML106-500 ML&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Himedia, India&amp;nbsp;</td><td>RM525-25G</td><td></td></tr><tr><td>Eosin&amp;nbsp;</td><td>Loba Chemie, India</td><td>3660</td><td></td></tr><tr><td>Ethylene Diamine Tetra Acetic Acid (EDTA)</td><td>Loba Chemie, India</td><td>6381926</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td></td><td>-</td><td></td></tr><tr><td>Formalin 10%&amp;nbsp;</td><td>Sigma Aldrich, USA&amp;nbsp;</td><td>HT501320</td><td></td></tr><tr><td>Hematoxylin</td><td>SDFCL</td><td>74108G05</td><td></td></tr><tr><td>HEPES</td><td>Himedia, India</td><td>MB016</td><td></td></tr><tr><td>Hydrogen peroxide&amp;nbsp;&amp;nbsp;</td><td>Avra, India</td><td>7722841</td><td></td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Neon Laboratories Ltd, India</td><td>-&amp;nbsp;</td><td></td></tr><tr><td>Laryngoscope</td><td>Medical Sources Co., Ltd., China&amp;nbsp;</td><td>MS-LA500FV&amp;nbsp;</td><td></td></tr><tr><td>Lipopolysaccharide from Escherichia coli O55:B5&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sigma Aldrich, USA</td><td>L2880&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>o-Dianisidine dihydrochloride&amp;nbsp;</td><td>Sigma Aldrich, USA</td><td>D3252-5G&amp;nbsp;</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS)&amp;nbsp;</td><td>Invitrogen, USA</td><td>3002</td><td></td></tr><tr><td>Potassium dihydrogen orthophosphate</td><td>Himedia, India</td><td>IN26185530500</td><td></td></tr><tr><td>Protein inhibitor cocktail</td><td>Sigma Aldrich</td><td>P8340</td><td></td></tr><tr><td>Rat IL-1&amp;beta; ELISA Kit&amp;nbsp;</td><td>Thermofisher, USA</td><td>BMS630</td><td></td></tr><tr><td>Sodium Chloride</td><td>Himedia, India</td><td>TC046M</td><td></td></tr><tr><td>Sodium fluoride</td><td>Himedia, India</td><td>GRM7502</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>Sigma Aldrich, USA</td><td>S6508</td><td></td></tr><tr><td>Sodium Pyrophosphate</td><td>Merck, USA</td><td>MA3M722243</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma Aldrich, USA</td><td>339741</td><td></td></tr><tr><td>Triton X-100</td><td>Thermofisher, USA</td><td>T8787</td><td></td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Indian Immunologicals Ltd, India</td><td>-</td><td></td></tr></tbody></table>

non-invasive endotracheal administration of lipopolysaccharide to induce acute lung injury in rodents

Non-Invasive Endotracheal Administration of Lipopolysaccharide to Induce Acute Lung Injury in Rodents

Acute Lung Injury (ALI) is a life-threatening inflammatory condition characterized by arterial hypoxemia, alveolar edema, compromised epithelial-endothelial capillary barrier integrity, followed by infiltration of inflammatory cells into the lung tissue, often progressing to its more severe form, Acute Respiratory Distress Syndrome (ARDS), which often culminates in acute respiratory failure. The ALI mouse models, primarily induced by lipopolysaccharide (LPS), are a key tool in research aimed at understanding the mechanisms of lung inflammation and its therapeutics. Direct and indirect rodent lung injury models offer distinct advantages as well as limitations, particularly in terms of survival rate, duration of inflammation-resolution phase, risks of surgical trauma and infection. In the present study, a non-invasive endotracheal instillation technique has been used to develop a direct ALI model in rats. This technique ensures focused delivery of LPS or test material into the lungs via the endotracheal route with the aid of a basic laryngoscope. The procedure begins with anaesthetizing the rat and positioning it in a semi-recumbent posture. Subsequently, the LPS solution is administered near the tracheal opening using a syringe attached to a rat endotracheal tube. At the time of experiment termination, lung or lavage samples are collected to assess inflammatory parameters and lung architecture. This technique outperforms other models in terms of speed, specificity, and reliability, providing specific targeting with minimal tissue damage, a low risk of mortality, and negligible off-site inflammation. This method is especially valuable in pre-clinical studies of ALI, ARDS, and related conditions like anaphylaxis or cardiac arrest. Comparatively, this method integrates more with advanced imaging techniques such as intravital microscopy to monitor drug distribution and disease progression in real time.

The model was validated by measuring inflammatory markers, including IL-1β levels, MPO activity, total protein content, and histopathology at 24 h and 120 h post-LPS instillation. The study included three experimental groups: a control group (untreated) and two groups subjected to LPS instillation, with one euthanized at 24 h and the other at 120 h (following institutionally approved protocols).
Compared to the control group, the LPS-exposed group euthanized at 24 h showed a s...

In this study, a non-invasive endotracheal instillation technique is demonstrated to develop ALI in rats using a basic laryngoscope to ensure precise LPS delivery to the lungs. The LPS is instilled via an endotracheal tube using a laryngoscope. The magnitude of lung injury is evaluated at multiple time points.

Acute Lung Injury (ALI) is a life-threatening inflammatory condition characterized by arterial hypoxemia, alveolar edema, compromised ...

The scope of our research is to develop a clinically relevant, non-invasive model of acute lung injury to improve the clinical testing. Modern lung biology research integrates advanced lung models, nano carriers and aerosol technologies to optimize and study targeted drug delivery within physiologically relevant models. To begin, weigh each animal using a calibrated balance.Prepare the lipopolysaccharide solution by diluting the lyophilized Escherichia coli lipopolysaccharide in sterile PBS to achieve the desired concentration. After anesthetizing the rat, check the depth of anesthesia and position the animal in a semi recumbent position with its incisors suspended on a solid supporting platform. Then open the mouth of the anesthetized animal.Gently grasp the tongue using curved blunt ended forceps and retract it in an upward and lateral direction. Then carefully insert the laryngoscope, aligning it precisely for optimal visualization of the tracheal opening. Now, draw 100 microliters of lipopolysaccharide solution into a syringe and attach it to a 16 gauge rat endotracheal tube.Administer the lipopolysaccharide solution near the tracheal opening. Immediately after installation, gently occlude the nostrils using blunt ended forceps for two to four seconds to promote aspiration of the solution into the lungs through the trachea. Then, carefully reposition the tongue to its normal anatomical orientation following lipopolysaccharide delivery.Transfer the animal back to its cage and closely monitor for any signs of respiratory compromise or choking for at least one minute. After performing bronchoalveolar lavage and tracheotomy, gently infused two milliliters of cold PBS into the lungs and retrieve the fluid carefully. Centrifuge the collected lavage fluid at 5, 000 G for five minutes at four degrees Celsius.Use the supernatant to detect inflammatory markers. Collect the lungs for histopathological examination, or homogenize them for myeloperoxidase activity. Compared to the control group, the group euthanized 24 hours after lipopolysaccharide installation, showed a significant increase in interleukin-1 beta levels, elevated myeloperoxidase activity and higher total protein content.At 120 hours post lipopolysaccharide installation, interleukin-1 beta, myeloperoxidase activity and total protein levels had returned to near baseline values. Lung histopathology at 24 hours demonstrated disintegrated alveolar structure, swelling of the alveolar wall, hemolysis, and severe neutrophil infiltration. Lung architecture was restored at 120 hours post lipopolysaccharide installation.Using non-invasive endotracheal pulmonary delivery, we created a self-limiting acute lung injury models for testing targeted therapeutics and studying lung repair. The key benefit of this method is it's precise non-traumatic delivery, which facilitates repeated dosing in the same animal and customization for a particular research objectives. Future acute lung injury research will focus on developing targeted therapies and understanding lung injury and repair mechanism.

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Biology

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Source: Radulovic, K., et al. Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach. J. Vis. Exp. (2018)This video demonstrates a step-by-step procedure for injecting lipopolysaccharide (LPS) into mice intraperitoneally to induce systemic inflammation.

Intraperitoneal Injection of Bacterial Lipopolysaccharide in Mice to Study Systemic Immune Responses

Source: Yuan, Z., et al. Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury. J. Vis. Exp. (2018)This video demonstrates an in vivo mouse model for inducing and analyzing acute lung injury (ALI) using intraperitoneal administration of bacterial lipopolysaccharide. The inflammatory cascade triggered by alveolar macrophages and neutrophils leads to epithelial and endothelial barrier disruption, allowing fluid and immune cells to accumulate in the alveoli. Alveolar...

Lipopolysaccharide-Induced Acute Lung Injury in Mice for Exosome and Immune Analysis

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit β and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

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.

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

We provide a protocol for an experimental endotoxemic shock model in pigs by infusion of lipopolysaccharide.

Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model

This protocol describes the development of a neonatal piglet model of acute lung injury that models early pathogenic events occurring in the preterm lung, including sub-adequate surfactant quantity, hyperoxia, high-pressure ventilation, and inflammation, to facilitate understanding of molecular triggers of bronchopulmonary dysplasia and enhance therapeutic translation.

Development of a Neonatal Piglet Acute Lung Injury Model Recreating the Early Environment of Preterm Infant Lungs

Learn a non-invasive endotracheal technique to induce acute lung injury in rats using lipopolysaccharide delivery. This method enables precise, repeatable dosing for studying lung repair mechanisms and testing targeted therapeutics.

Non-Invasive Endotracheal Administration of Lipopolysaccharide to Induce Acute Lung Injury in Rodents

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