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

Summary

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.

Abstract

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.

Introduction

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 cells^1,2,3,4.

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 infection^10, and viral infection such as SARS-CoV-2^11,12,13,14. 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 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 scenario^23,24,25.

Protocol

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 study^26,27,28. 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

1. Animal weight measurement and LPS dose calculation
   1. Weigh each animal using a calibrated balance to calculate the precise LPS dose. Select Wistar rats weighing between 140-160 g for the study.
   2. 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 µg/g body weight)^29,30 is used.
2. Anaesthesia induction and critical timing
   1. 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.
   2. 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.

2. Endotracheal instillation of LPS

1. 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 in a semi-recumbent position, suspended by incisors on a solid supporting platform.
2. 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.
3. 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.
4. LPS administration: Draw 100 µL 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.
5. 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.
6. Key considerations
   1. Visual acuity: Ensure optimal alignment of the laryngoscope for accurate LPS delivery. Use magnification tools to enhance precision and efficacy.
   2. Grasp the tongue carefully and hold it against the lower jaw or edge of the mouth to stabilize it.

3. Sample collection

1. Perform bronchoalveolar lavage (BAL) as described in earlier studies, with minor modifications^31,32,33. Following tracheotomy, gently infuse 2 mL of cold PBS into the lungs and retrieve the fluid.
2. Centrifuge the collected lavage fluid at 5000 x g for 5 min at 4 °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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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