Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Summary

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.

Abstract

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Introduction

Experimental animal models are indispensable in basic immune research. Administration of whole bacteria or microbial components has been frequently used in small animal models to induce local or systemic inflammation¹. Lipopolysaccharide (LPS, or bacterial endotoxin) is a cell wall component and surface antigen of gram-negative bacteria (e.g., Enterobacteriaceae, Pseudomonas spp., or Legionella spp.). The thermostable and large molecule (molecular weight 1-4 x 10⁶ kDa) consists of a lipid moiety (Lipid A), core region (oligosaccharide), and an O polysaccharide (or O antigen). Lipid A, with its hydrophobic fatty acid chains, anchors the molecule into a bacterial membrane and mediates (upon degradation of bacteria) the immunological activity and toxicity of LPS. Following binding to the LPS binding protein (LBP), LPS:LBP complexes ligate the CD14/TLR4/MD2 receptor complex located on the surface of many cell types, inducing a strong proinflammatory reaction with NF-κB nuclear translocation and subsequent upregulation of cytokine expression².

Acute lung injury (ALI) is defined as acute hypoxemic respiratory failure with bilateral pulmonary edema in the absence of heart failure³. Airway administration of LPS is a common way to induce pulmonary inflammation and ALI^4,5,6,7. Although the sterile substance is not suitable to study pharmacologic interventions in infectious diseases, mechanistic immunological questions may be answered with adequate precision. Instillation of LPS into the trachea induces a robust inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier within hours to days, depending on the LPS dosage^3,6,7.

The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by intratracheal LPS instillation. The model has been validated by assessing cytokine expression, neutrophil granulocyte invasion, and intra-alveolar albumin leakage as previously described⁸. 

Protocol

This animal protocol was approved by the local committee for animal care (LANUV, Recklinghausen, Germany; protocol no. 84-02.04.2015) and was performed in accordance with the National Institutes of Health guidelines for the use of live animals (NIH publication No. 85-23, revised 1996).

1. ALI induction

1. Use adult C57BL/6 mice at ages of about 10-12 weeks. House the animals in individually ventilated cages with free access to water and standard rodent chow. However, it is possible to perform this approach on younger animals and with other mice strains.
2. Store LPS (Escherichia coli O111:B4) in aliquots in concentrations of 5 mg/mL at -20 °C. For intratracheal instillation, dilute LPS in sterile phosphate-buffered saline (PBS) to a final concentration of 2,000 µg/mL.
3. Weigh the mouse. Inject ketamine [120 mg/kg mouse bodyweight (BW)] and xylazine (16 mg/kg BW) intraperitoneally (lower one-third of the abdomen, paramedian), and wait until the onset of anesthesia.
4. Check the depth of anesthesia by inducing a tactile stimulus. In case of insufficient anesthesia, repeat the injection with ketamine (30 mg/kg BW) and xylazine (4 mg/kg BW).
5. Place the mouse in prone position on a temperature-controlled table to maintain a body temperature of 37 °C.
6. Apply sterile ophthalmic lubricant to prevent desiccation of corneas under anesthesia.
7. Lift the head and hook incisors on a horizontal bar positioned approximately 5 cm above the table while the forepaws remain in close contact with the table. Super-extend the neck in a 90° angle relative to the table (Figure 1). Hold the tongue with forceps to straighten the throat for easier intubation conditions.
8. Cut a 22 gauge (G) venous catheter to a length of 20 mm. Gently insert the catheter in the vertical direction along the tongue’s root. Place a cold-light source on the skin above the larynx to help visualize the vocal chords and aim for the trachea. If resistance of the larynx occurs, retract the catheter a few millimeters before advancing again.
9. Insert the catheter approximately 10 mm into the trachea. Ensure that the insertion is not too deep as this will result in unilateral instillation of fluid into the right or left main bronchus.
10. Inject LPS (5 µg/g BW) diluted in PBS using a pipette [injected volume depends on mouse bodyweight (e.g., 20 g bodyweight → use 50 µL of LPS solution)].
    NOTE: The mouse will typically respond with coughing or gasping to proper instillation of fluid into the trachea.
11. Connect a syringe and add a bolus of 50 μL air to assure that complete liquid volume is distributed in the lungs. Slowly remove the catheter.
12. Keep the mouse's upper body in an upright position for 30 s to avoid leakage of the fluid from the trachea.
13. In sham-operated animals, inject 50 µL of sterile PBS intratracheally instead of LPS.
14. Inject buprenorphine hydrochloride 0.08 mg/kg BW subcutaneously into the loose skin over the neck immediately following ALI induction and every 12 h thereafter, during the first 48 h.
15. Maintain a body temperature of 37 °C until full awareness is regained by keeping the mouse on the warming pad.
16. Transfer the mouse into an individually ventilated cage with free access to food and water. Monitor the mouse regularly. Decreasing body temperature and respiratory depression indicates proper induction of ALI. 

2. Blood sampling, bronchoalveolar lavage, organ harvesting

NOTE: Timing of euthanization depends on the scientific issue addressed. Usually, it is performed 12-72 h following LPS instillation^3,4,9,10. Severity of ALI can be determined clinically by regular observation of body temperature and respiratory distress symptoms¹¹.

1. Induce anesthesia by placing the mouse in a chamber flooded with isoflurane. Use 3 vol% isoflurane with an oxygen flow of 1 L/min. Ensure deep narcosis by inducing a tactile stimulus. In case of insufficient depth of anesthesia, increase isoflurane up to 5 vol%.
2. Sacrifice the mouse in deep anesthesia by atlanto-occipital dislocation.
3. Fix the mouse with tape on an operation table and shortly disinfect the fur over the abdomen with 70% ethanol. Open the abdominal cavity carefully in the median line with scissors and tweezers. Remove parts of the intestine to achieve access to the vena cava inferior (IVC) right to the vertebral column and the abdominal aorta.
4. Locate the kidney veins and insert a bent 23 G canula connected to a 1 mL syringe into the IVC directly below the confluence of the veins. Aspirate 250 µL of blood and transfer into a 1.5 mL tube filled with 20 µL of 0.5 M ethylenediaminetetraacetic acid (EDTA) solution. Shake gently to facilitate EDTA mixing and put the tube on ice.
5. For bronchoalveolar lavage (BAL), prepare three 1 mL syringes with 0.5 mL of sterile PBS and 0.1 mL of air each. Shortly disinfect the fur of the throat with 70% ethanol and carefully expose the trachea with scissors and tweezers. Mobilize the trachea and wrap around a suture.
6. Perform BAL: Puncture the trachea using micro-scissors and insert a 22 G venous catheter cut to a length of 20 mm. Fix the catheter with the suture and instillate 0.5 mL of sterile PBS and 0.1 mL of air. Aspirate the fluid after 60 s. Repeat the procedure with the additional two syringes and collect the whole aspirate in a 15 mL tube on ice.
7. Carefully open the thorax with scissors and tweezers to harvest the lungs. Cut the diaphragm along the costal margin and cut through the ribs with two lateral incisions. Carefully avoid puncturing the lungs. Lift the sternum cranially and fix or remove it.
8. Prepare two 10 mL syringes with 37 °C warm PBS (without calcium and magnesium). Make a small incision into the left ventricle. Puncture the right ventricle with a 26 G canula and flush the pulmonary circulation with the prewarmed PBS. Be aware of the lungs turning pale during the procedure. 
9. Remove the right lobe of the lungs and cut it in two halves. Snap-freeze them in liquid nitrogen, followed by long-term storage at -80 °C for further gene expression and protein analysis.
10. Remove the whole left lung and homogenize it in a 48 well plate by mincing the tissue with scissor and tweezer. Incubate the tissue in 2 mL of digestion buffer [RPMI 1640 with 10% fetal calf serum (FCS) and 0.1% NaN₃, collagenase I (1 mg/mL), and DNase II (7 mg/mL)] at 37 °C for 60 min. Perform further homogenization by careful pipetting of the lung tissue pieces up and down.

3. Tissue preparation for FACS analysis

1. Prepare fresh FACS buffer (Table 1): always use calcium- and magnesium-free PBS to reduce cation-dependent cell-to-cell adhesion and prevent clumping. Supplement with FCS (1%) to protect cells from apoptosis, prevent non-specific staining, and prevent cells from sticking to the FACS tubes. Include EDTA (0.5 mM) to prevent cation-based cell-to-cell adhesion when working with sticky and adherent cells like macrophages. Add sodium azide (0.1%), as it prevents bacterial contamination and photobleaching of fluorochromes and blocks antibody shedding.
2. Transfer the blood samples (step 2.4) into 5 mL FACS tubes and gently mix the blood with 2 mL of red blood cell lysis buffer. Put the tubes on ice and terminate the reaction after 2 min by adding 2 mL of ice-cold PBS. Centrifuge the samples for 5 min at 400 x g and discard the supernatant. Resuspend the cell pellet with 60 µL of FACS buffer and process for subsequent FACS staining according to previously described protocols¹².
   NOTE: Timing of euthanization of the mouse influences leukocyte count as part of the systemic inflammation. Therefore, it is recommended to adjust the cell number to 1 x 10⁶ cells/60 µL in this step to achieve the best staining results for flow cytometry analysis.
3. Centrifuge BAL fluid (step 2.6) for 5 min at 400 x g. Aspirate the supernatant and freeze it in liquid nitrogen, followed by long-term storage at -80 °C for further protein analysis. Resuspend BAL cell pellet with 2 mL of cold FACS buffer, then transfer the suspension into a 5 mL FACS tube using a 100 µm mesh filter to restrain hairs.
4. Again, centrifuge the sample for 5 min at 400 x g. Resuspend the pellet with 60 µL of FACS buffer and process for subsequent FACS staining according to previously described protocols¹².
   NOTE: Timing of euthanization of the mouse influences leukocyte count in BAL as part of the inflammation. Therefore, it is recommended to adjust the cell number to 1 x 10⁶ cells/60 µL in this step to achieve the best staining results for flow cytometry analysis.
5. Transfer the digested left lung tissue (step 2.10) into a 5 mL FACS tube using a 100 µL mesh filter to extract clumps and terminate the digestion process by adding 2 mL of ice-cold FACS buffer. Centrifuge the sample for 5 min at 400 x g. Discard the supernatant and resuspend the pellet with 60 µL of FACS buffer and process for subsequent FACS staining according to previously described protocols¹².
   NOTE: Timing of euthanization of the mouse influences leukocyte count in lung tissue as part of the inflammation. Therefore, it is recommended to adjust the cell number to 1 x 10⁶ cells/60 µL to achieve the best staining results for flow cytometry analysis.
6. For FACS analysis, incubate cells with CD16/CD32 antibody at 4 °C for 15 min to block non-specific binding of immunoglobulin to the Fc receptors. Add 20 µL of blocking solution to 1 x 10⁶ cells in 60 µL in a 5 mL tube.
7. Meanwhile, prepare a master mix with FACS buffer and antibodies as described in Table 2.
8. After blocking, do not wash the cells. Add 20 µL of antibody master mix per sample to obtain a final volume of 100 µL. Incubate the samples for 20 min in the dark at 4 °C.
9. Wash each sample with 1 mL of FACS buffer and centrifuge for 5 min at 400 x g. Discard the supernatant and resuspend the pellet with FACS buffer to the appropriate cell concentration for FACS measurements.
   NOTE: A cell number of 1 x 10⁶ cells/500 µL is suggested to achieve the best immune phenotyping results in FACS analysis with this protocol. However, it is recommended that antibodies have to be titrated individually.
10. If required, add live/dead staining prior to the surface staining using specific commercially available kits⁸.
11. Finally, add fixed numbers of commercially available fluorochrome-coupled calibration beads (3 x 10⁵ beads in 20 µL of FACS buffer) to each sample to determine absolute cell numbers¹². The gating strategy for blood, BAL, and tissue cells is shown in Figure 2.

Results

The described approach to induce ALI in mice was validated by assessing cytokine expression, neutrophil granulocyte infiltration, and alveolo-capillary barrier disruption 24 h and 72 h after LPS instillation. PBS-injected animals served as control. Intratracheal LPS administration induced a robust pulmonary proinflammatory response. Expression of TNF-α in lung tissue was significantly upregulated, reaching a sustained and more than 50-fold increase compared to the control animals [RQ (TNF-α/18s); 24 h: 53.7 (SD = 11.6); ...

Discussion

Minimal invasiveness, simple handling, and good reproducibility are key features of the presented approach to induce ALI in a small rodent model. The use of LPS instead of whole bacteria in animal models has advantages. It is a stable and pure compound and can be stored in lyophilized form until use. It is a potent stimulant for innate immune responses via the TLR4 pathway, and its biological activity may readily be quantified, facilitating the titration of disease severity with good reproducibility. Moreover, the use of...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 ml syringes	BD, Franklin Lakes, NJ, USA	300013
10 ml syringes	BD, Franklin Lakes, NJ, USA	309110
Anti-CD115 (c-fms) APC	Thermo Fisher, Waltham, MA, USA	17-1152-80
Anti-CD11b (M1/70) - FITC	Thermo Fisher, Waltham, MA, USA	11-0112-81
Anti-CD45 (30-F11) - eF450	Thermo Fisher, Waltham, MA, USA	48-0451-82
Anti-F4/80 (BM-8) - PE Cy7	Thermo Fisher, Waltham, MA, USA	25-4801-82
Anti-Gr1 (RB6-8C5)	BD Biosciences, Franklin Lakes, NJ, USA	552093
Anti-Ly6C (HK1.4) PerCP-Cy5.5	Thermo Fisher, Waltham, MA, USA	45-5932-82
Anti-Ly6G (1A8) APC/Cy7	Bio Legend, San Diego, CA	127623
Buprenorphine hydrochloride	Indivior UK Limited, Berkshire, UK
C57BL/6 mice, female, 10 - 12 weeks old	Charles River, Wilmongton, MA, USA
CaliBRITE APC-beads (6µm)	BD Biosciences, Franklin Lakes, NJ, USA	340487
Canula 23 gauge 1''	BD, Franklin Lakes, NJ, USA	300800
Canula 26 gauge 1/2''	BD, Franklin Lakes, NJ, USA	303800
Cell strainer 70 µm	BD Biosciences, Franklin Lakes, NJ, USA	352350
Collagenase Type I	Sigma-Aldrich, St. Louis, MO, USA	1148089
Deoxyribonuclease II	Sigma-Aldrich, St. Louis, MO, USA	D8764
Dulbecco's Phosphate Buffered Saline (PBS), sterile	Sigma-Aldrich, St. Louis, MO, USA	D8662
Dulbecco’s Phosphate Buffered Saline (PBS), without calcium chloride and magnesium chloride, sterile	Sigma-Aldrich, St. Louis, MO, USA	D8537
Ethylenediaminetetraacetic acid (EDTA) solution	Sigma-Aldrich, St. Louis, MO, USA	E7889
FACS tubes, 5 ml	Sarstedt, Nümbrecht, Germany	551579
Fetal calf serum (FCS)	Sigma-Aldrich, St. Louis, MO, USA	F2442
Forceps	Fine Science Tools, Heidelberg, Germany	11049-10
Isoflurane	Baxter, Unterschleißheim, Germany
Ketamine hydrochloride	Serumwerk Bernburg, Bernburg, Germany
Lipopolysaccharides (LPS) from Escherichia coli O111:B4	Sigma-Aldrich, St. Louis, MO, USA	L2630
LIVE/DEAD Fixable Dead Cell Green Kit	Thermo Fisher, Waltham, MA, USA	L23101
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™), Clone 2.4G2	BD, Franklin Lakes, NJ, USA	553141
Red blood cell lysis buffer	Thermo Fisher, Waltham, MA, USA	00-4333-57
RPMI-1640, with L-glutamine and sodium bicarbonate	Sigma-Aldrich, St. Louis, MO, USA	R8758
Scissors	Fine Science Tools, Heidelberg, Germany	14060-09
Sodium azide (NaN3)	Sigma-Aldrich, St. Louis, MO, USA	S2002
Spring scissors	Fine Science Tools, Heidelberg, Germany	15018-10
Tissue forceps	Fine Science Tools, Heidelberg, Germany	11021-12
Tubes	Eppendorf, Hamburg, Germany	30125150
Venous catheter, 22 gauge	B.Braun, Melsungen, Germany	4268091B
Xylazine hydrochloride	Serumwerk Bernburg, Bernburg, Germany