Pseudomonas aeruginosa Induced Lung Injury Model

Summary

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Abstract

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Introduction

Lungs are exposed to environmental pathogens and are susceptible to inflammation and injury^1-3. During pathological conditions such as pneumonia or Adult Respiratory Distress Syndrome (ARDS), pathogens as well as inflammatory factors released by leukocytes induce injury and death of alveolar cells^1-3. It is important to develop animal models of acute lung injury to facilitate the study of pathology of injury as well as mechanism of repair.

Currently, most people use hyperoxia and bleomycin induced mouse lung injury models⁴. However, the mechanisms of hyperoxia caused injury are not the same as most common lung injuries that occur during pneumonia or ARDS⁵. Bleomycin induced acute injury is rare in a clinical context⁴. Here we report a mouse lung injury model using intra-tracheal injection of P. aeruginosa ^6,7. This model is clinically relevant, and mimics the processes that happen following pneumonia⁸.

As an opportunistic, nosocomial pathogen of immunocompromised individuals, P. aeruginosa typically infects the pulmonary tract, urinary tract, burns, wounds, and also causes other blood infections⁶. The bacteria release virulence factor exotoxin A, multiply and trigger immune responses⁶. Intra-trachael administration of P. aeruginosa reflects the situation in human exposure to the bacteria which cause pneumonia and the pathology is likely to be different from the recently reported influenza virus H1N1 induced lung injury model⁹. Since P. aeruginosa is an opportunistic pathogen, it is relatively safe to handle as compared to some of the more virulent pathogens.  Here we used intra-tracheal injection to administer the bacteria because we observed that this method introduced more bacteria into the distal alveoli region of the lung compared with some other procedures such as using a catheter via mouth.

Compared with other acute lung injury models, the P. aeruginosa model described here is suitable for studying lung injury induced by bacteria and by excessive inflammation. Unlike other animal models that use P. aeruginosa to induce sepsis^10,11, here we use intra-tracheal injection of these bacteria to induce localized acute lung injury.

Protocol

The animal experiments were approved by the Animal Care Committee and Institutional Biosafety Committees of the University of Illinois at Chicago.

NOTE: All procedures involving pseudomonas should be performed with Biosafety Level 2 (BSL2) practices, which include but are not limited to: mask, eye protection, gown or jumpsuit, and double gloves. Work in certified Biosafety Cabinet. Treat instruments in contact with bacteria with bleach or chlorine dioxide based disinfectant. Use a sealed box for transport samples.

1. P. aeruginosa Culture and Growth

1. Store P. aeruginosa PA103 as a bacterial stock in a screw cap cryovial at -80 °C.
2. Place the vial in a rack in a box with 70% ethanol wetted paper towels and take to the Biosafety Level 2 (BSL2) laboratory.
3. Streak the bacteria onto sheep blood agar plates and grow at 37 °C for ~15 hr in an incubator.
4. In a BSL2 hood, scratch the bacteria from plate with bacteria loop and resuspend in 5 ml PBS.
5. Store the stock at 4 °C for up to 3 months. However, determine the titer every 2 weeks.
6. Serially dilute the bacteria in PBS (usually from 1:10⁴ to 1:10⁷) and plate out the known dilution on sheep blood agar plates.
7. Incubate the plates for ~15 hr. Count colonies and calculate Colony Forming Unit (CFU) to determine bacteria concentration.
8. Resuspend the appropriate concentrations (~5 x 10³ CFU/µl) in 0.5 ml PBS in 1.5 ml sterile screw cap cryovials. Seal the cryovials and place them in a cryovial rack on disinfectant laden paper towels in a snap lid box.
9. Transport the box to BSL2 animal facility. Once there, open the box in the BSL2 cabinet.

2. P. aeruginosa Instillation

1. Perform survival surgery aseptically (sterile gloves, sterile instruments, and aseptic techniques). Use a sterile drape to provide a working surface for sterile (autoclaved) surgical instruments. Sterilize instruments using a hot bead sterilizer between each tracheal instillation procedure.
2. Weigh mice prior to anesthetization. Anesthetize mice with ketamine (100 mg/kg), xylazine (5 mg/kg), in 0.1 - 0.2 ml PBS intraperitoneally (i.p.). Determine the effectiveness of the anesthetic by non-responsiveness to toe pinch. Use a vet ointment on eyes to prevent dryness under anesthesia.
3. Restrain mice on a surgical board in BSL2 cabinet.
4. Identify the area for the cut down. Shave this area and prepare the skin using alternating alcohol and povidone iodine swabs 3 times.
5. Treat the incision area with local anesthetic (lidocaine) as this anesthesia is sufficient for a minor surgery such as a skin incision to access the trachea.
6. Make a small incision (approximately 5 mm) at the midline of the neck, and use blunt forceps to gently move the muscle for access to the trachea. Expose the trachea by surgical dissection.
7. Draw bacteria solution into a 1 ml disposable syringe with 27 G needle. For each mouse, administer 20-30 µl of bacteria at the appropriate concentration (up to 10⁵ CFU each mouse).
8. Insert needle into the trachea. Inject solution slowly into the tracheal.
9. Ensure that the animal gasps, which usually indicates that solution has reached into the lung.
10. Close the wound with sterile sutures (6-0 monofilament) under aseptic conditions.
11. Use 0.1 mg/kg buprenorphine by subcutaneous injection as post-op analgesia to control post-surgical pain.
12. Ensure that the time required from initial anesthetic induction to incision closure is less than 15 min.
13. After injection, dispose of syringes and needles in an appropriate biohazard sharps container.
14. Treat the surgical instruments with chlorine dioxide based disinfectant for 15 min, rinse and return to the lab for sterilization and reuse as necessary.
15. House the mice singly in clean cages at warm environment.
16. Check on the animal every 30 min until it regains consciousness and starts to move; and later at 12 hr intervals for the first 3 days post-surgery.
17. Keep the mice in the animal BSL2 facility throughout the experiment. Ensure that only tissue contained in sealed box leaves the animal BSL2 facility. If the mice express any of the moribund behaviors (defined as respiratory distress, lethargy, failure to ambulate in response to gentle stimulation) at any point in the study, euthanize the mice by CO₂ inhalation from a bottled source followed by cervical dislocation.
18. Euthanize the experimental subject by exsanguination under anesthesia.
    1. Collect lung tissue from euthanized mice. Carry out these procedures in a BSL2 hood. If needed, transfer the sample using sealed tubes placed in a rack on disinfectant laden paper towels in a snap lid box for further process.

Results

Starting from 24 - 72 hr post P. aeruginosa injection, increased cellularity was observed in lung sections (Figure 1A-D). The lung started to recover from 96 hr post injury (Figure 1E). At 7 days post P. aeruginosa, normal alveoli morphology was largely restored (Figure 1F). Tunnel staining using lung sections prepared at 24 hr post P. aeruginosa showed cell death in alveoli cells (Figure 1G-I). In order to study the repair pro...

Discussion

The Pseudomonas mouse lung injury model that we describe here mimics the whole process of inflammation, lung injury, repair, and resolution that occur following acute lung injury or pneumonia. It has unique advantages comparing with several other injury models in that it is clinically relevant and relatively safe and easy to handle.

The critical step in the procedure is that the injection of bacteria solution needs to be very slow. If injection is too fast, the mice are likely to die by choke....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthetic: Ketamin, xylazine, lidocaine, buprenorphine			pharmaceutical grade
27 G needle	Fisher	1482648
syringe	Fisher	14823434	1 ml
scissors	Fine Science tools
forceps	Fine Science tools
suture	Fisher	19-037-526
Eye gauge, glove, gown
Biosafety Cabinet
chlorine dioxide based disinfectant			Clidox
sheep blood agar plates	Medex supply	HL-1160