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

The overall goal of this procedure is to demonstrate the use of oropharyngeal intratracheal, LPS administration and bronchoalveolar lavage to evaluate the host immune response in mice. This is accomplished by first inoculating the mouse with LPS using oropharyngeal administration. In the second step, a tracheal cannula is inserted bronchoalveolar lavage fluid, or is then collected through the cannula for assessing the local inflammatory response.

In the final step, the lungs are inflated with formalin for histopathological assessment. Ultimately, the cytokine profile of the bowel and the histopathology of the lung can be analyzed to evaluate the host immune response to LPS. The main advantage of this technique over existing methods like intranasal administration and surgical intratracheal administration, is that this technique is relatively simple to perform and allows accurate dosing and lung deposition.

To intratracheally, inject the LPS first. Suspend an anesthetized mouse on an intubation. Stand by its front incisors, ensuring that the animal is securely restrained and that the mouth and tongue are accessible.

Gently secure the tongue with straight forceps, then grasp the tongue with angled forceps and gently pull the muscle out of the mouth until a slight resistance is felt while holding the tongue. In this position, administer a 50 microliter dose of LPS into the back of the throat and immediately cover the animal's nostrils with a gloved finger for five to 10 breaths to collect the bath after sacrifice. Next, make a horizontal incision across the length of the peritoneal cavity and a vertical incision from the peritoneal cavity to the lower jaw of the mouse.

Then grasp both sides of the incision with the forceps and gently pull the skin away from the underlying peritoneal and thoracic cavities. Next, make a large incision along the length of the peritoneal cavity from the genitals to the sternum, taking care to avoid cutting the diaphragm. Then gently shift the intestines within the peritoneal cavity to allow access to one of the kidneys.

Cut the renal vein leading to the kidney to serve as a drainage point for perfusion. Then nick the diaphragm with scissors, taking care to avoid the lungs and heart. To expose the left side of the heart, use a 10 milliliter syringe equipped with a 27 gauge needle to manually perfuse the heart with PBS.

The remaining blood should drain from the portal vein incision. Then use blunt, blunt scissors to carefully cut the rib cage along the sternum. Taking care to avoid cutting the heart and lungs.

Gently isolate the heart and lungs, and then carefully cut each section of the rib cage with the blunt, blunt scissors as close to the spine as possible. After fully removing both rib sections, separate the salivary glands to expose the trachea and carefully cut the muscles overlying the trachea without cutting the trachea itself. Now separate the collarbone and gently grasp the thymus, lifting it away from the heart.

Then remove the thymus with scissors to care to avoid cutting the heart or lungs. Next, use angled sharp, sharp scissors to make a small horizontal incision large enough to firmly secure a cannula in the trachea, one to two rings below the larynx. Then insert a cannula so that the tapered end extends two to three tracheal rings.

Below the incision, pass a silk suture between the trachea and the esophagus, pulling the suture tight on the cannula to secure it. Then fill a one milliliter syringe with HBSS and gently insert its lure into the cannula using a slow but constant motion. Next, inject approximately 900 microliters of the HBSS into the trachea to inflate the lungs, and then immediately with draw the HBSS, dispense the recovered valve into a 15 milliliter conical tube.

After repeating the lavage two more times to collect approximately three milliliters of valve. Store the valve on ice to dissect the lungs For histopathological analysis, insert a 10 milliliter syringe into a lung inflation stand and attach the tubing to the lure, the lure to the stop cock and the stop cock to the syringe, confirm the stopcock is in the closed position, and then fill the syringe with neutral buffered formalin solution. When the syringe is completely filled with fixative, place an absorbent towel under the open end of the tubing and open the stop cock to allow the fixative to fill the tubing.

Once the fixative begins flowing from the tubing, immediately close the stop cock and refill the syringe to the top with fixative. Then gently passes Second piece of suture thread under the trachea, approximately one to two tracheal rings. Below the end of the cannula, tie a single loose knot in the suture and then insert the tubing from the mouse inflation.

Stand into the cannula. Open the stop cock and allow the lungs to fill by gravity inflation. And then once the lungs reach their maximum inflation level, pull the suture tight and tie a second knot.

Close the stop cock and remove the tubing from the cannula, and then grasp the suture with forceps. Firmly pull the suture down towards the thoracic cavity and gently pull the cannula back towards the mouse's nose to remove the cannula. Next, gently lift the trachea away from the neck cavity and use blunt scissors to sever the trachea coddle to the tide suture.

Once the trachea is free from the underlying tissues, begin to pull the trachea away from the mouse and gently lift the lungs out of the thoracic cavity. Then carefully cut the connective tissue holding the lungs in the thoracic cavity with a steady upward force and continue pulling the trachea away from the mouse's body, taking care not to cut the lungs. Finally, fix the lungs in 10 milliliters of buffered formin.

In models of LPS exposure, both body temperature and weight, our typical surrogate markers of animal morbidity and disease progression, LPS challenge will cause a significant decrease in body temperature within the first six hours as illustrated in the graph, which gradually increases back to baseline over the course of 24 hours. Body weight, however, steadily decreases over the course of 24 hours when it will peak and gradually recover over the next 48 to 72 hours. Thus, body temperature is a more appropriate marker to evaluate the early stages of inflammation initiation.

Whereas body weight is more reliable during later stages of pathogenesis and recovery, airway LPS exposure results in a significant influx of leukocytes. The lungs within the first six hours cells associated with the host innate immune response can be observed in the valve valve. Cellularity then continues to increase over the next 24 to 48 hours when the immune response peaks and subsequently enters a period of inflammation resolution By 24 hours, a significant number of neutrophils are present in the lungs and can be observed in the valve following differential staining.

Valve cellularity assessments provide a robust and quantifiable technique for characterizing the cells associated with the host immune response in the lungs. This increase is consistent with a significant influx of neutrophils into the airways, blood vessels and lung parenchyma. Lung histopathology can be effectively evaluated using a semi quantitative scoring system as can be observed in the image.

LPS induces a significant increase in perivascular, peri bronchiolar and parenchymal inflammation. LPS administration induces high levels of local and systemic pro-inflammatory mediators, including several cytokines associated with the innate immune response as detected by Eliza Common cytokines that are upregulated in the lungs following LPS administration include TNF, alpha IL one beta, and IL six. Systemic cytokine levels can also be evaluated in the serum using the same techniques as demonstrated for the bowel assessments.

The gravity displacement method of fixation facilitates optimal histopathology evaluation conditions compared to uninflated lungs or manual inflation lungs fixed by gravity. Inflation demonstrate uniform features, allowing accurate scoring, reduced alveolar damage, enhanced visualization, and higher resolution Evaluations of inflammation, manual inflation of the lungs typically results in non-uniform areas of inflation. These areas are often partially inflated, resulting in tissue collapse that is commonly mistaken as pathological features by novice reviewers.

Manual inflation also results in areas of the lungs that are overinflated, resulting in extensively damaged alveolar spaces. Uninflated lungs demonstrate collapsed alveolar spaces and areas that are difficult to resolve and evaluate without extensive training. Likewise, due to the organ being collapsed, this technique does not allow visualization of the lungs as they appear in situ.

After watching this video, you should have a good understanding of how to induce and assess LPS mediated acute lung inflammation in mice using a simple non-surgical oropharyngeal and tracheal administration procedure and tissue harvest.