Pseudomonas aeruginosa Induced Lung Injury Model

The overall goal of this procedure is to mimic pneumonia, lung injury conditions in the mouse by pseudomonas aerogen administration. This is accomplished by first preparing pseudomonas at the appropriate concentration for injection. In the second step, the mouse trachea is carefully exposed and then the bacteria are administered to the lungs by intra tracheal injection.

In the final step, the disease progression in the mouse is monitored carefully. Ultimately, lung inflammation, cell death changes in the alveolar barrier and lung repair can be studied in the pseudomonas aerogen injected mouse lung. The implications of this technique extend toward the therapy of acute lung injury caused by pneumonia.

As this model mimics the processes following pneumonia To create quats of p Aerogen, PA 1 0 3 for installation begin by transporting a cryo vial of the bacteria from minus 80 degrees Celsius, storage to the biosafety level two or BSL two laboratory in a rack in a box lined with 70%ethanol wetted paper towels. Then under a BSL two hood streak, the P aerogen PA 1 0 3 bacterial colonies onto sheep blood agar plates, and grow out the colonies in a 37 degrees Celsius incubator. After approximately 15 hours, use a bacteria loop to transfer the colonies into five milliliters of PBS.

Then serially dilute the bacteria in more PBS to one to 10, to the four, to one to 10 to the seven concentrations plate out the dilution on fresh sheep blood agar plates. Then after about another 15 hours incubation, calculate the colony forming units for each plate to determine the bacteria concentration. Next, Eloqua the appropriate amount of bacteria in 0.5 milliliters of PBS in 1.5 milliliters, sterile screw cap cryo vials, and then place the vials sealed in a cryo vial rack on disinfectant laden paper towels in a snap lid box in a BSL two cabinet, gently cover the animal's eyes with veto ointment working.

Quickly shave the incision and clean the skin by using alternating swabs of alcohol and povidone iodine three times and confirm sedation by toe. Pinch, restrain the mouse on a surgical board after treating the skin with a local anesthetic. Next, make a small five millimeter incision at the midline of the animal's neck and use blunt forceps to gently move the muscle to access the trachea.

When the trachea has been exposed, draw up to one times 10 to the five colony forming units of the bacteria in 20 to 30 microliters of PBS into a one milliliter disposable syringe equipped with a 27 gauge needle. Then insert the needle into the trachea and slowly inject the solution. Ensure that the animal gasps, which usually indicates that the solution has reached the lung.

Now aseptically close the wound with six zero monofilament sterile sutures and administer the postoperative analgesia. Then dispose of the syringes and needles in the appropriate biohazard sharps container after each animal has been injected. How's the myers singly in clean cages in a warm environment?

Checking on the animals every 30 minutes until they regain consciousness and start to move. Treat the surgical instruments with chlorine dioxide disinfectant for 15 minutes. Then rinse and return the tools to the lab for further sterilization as necessary.

House the mice in the BSL two animal facility throughout the experiment and then after exsanguination under anesthesia, collect the lung tissue from each animal under a BSL two hood. Finally, if further processing is desired, transfer the samples in sealed tubes placed on a rack on disinfectant laden paper towels in a snap lid box to the appropriate processing facility. In this representative experiment at 24 to 72 hours post P aerogen injection, an increased cellularity can be observed in the lung sections.

By 96 hours, the lung has started to recover and by seven days, normal alveoli morphology is largely restored. Tunnel staining of lung sections prepared 24 hours post P aerogen installation exhibit cell death within the alveoli cells to study the repair process. In this injury, model BRDU can be injected into the mice three days after p aerogen administration.

As illustrated, p aerogen treated animals exhibit hyper proliferation in the lung tissue compared to control treated animals. To study the changes in barrier, permeability and inflammation post-injury, bronchoalveolar lavage or bowel fluid can be collected at different time points. Post p aerogen injection, for example, the protein concentration within the bowel is significantly increased at 48 hours after bacterial installation indicating epithelial barrier leakiness the number of cells.

The bowel are also increased significantly at this time, suggesting an inflammatory response at four to five days post P aerogen injection, both the bowel protein level and cell number start to decrease suggesting recovery. Furthermore, a level of macrophage inflammatory protein two, a cytokine involved in neutrophil attraction significantly increases at 48 hours and then returns to basal levels at 96 hours. In lung lysates without p aerogen only a small number of monocytes are observed in the bowel fluid.

In contrast, a large number of neutrophils are present in the bowel, isolated at 48 hours after injection. By 96 hours, the cell composition of the bowel returns to control levels Following this procedure. Other methods like histological, sectioning, or bronchoalveolar lavage collection can be performed to answer additional questions such as, which cells or factors are involved in inflammation and repair after acute lung injury?