Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

The overall goal of this procedure is to noninvasively deliver bacteria directly into the lungs of mice. This is accomplished by first loading a 150 microliter air cushion into a gas tight syringe, followed by 50 microliters of the prepared bacterial suspension. In the second step, an anesthetized mouse is intubated with a 20 gauge catheter using a guide wire to assist the placement.

Then after confirming the success of the intubation using a simple flow meter, the bacterial inoculum is delivered through the catheter directly into the lung with a 22 gauge long blunt needle. Ultimately, intubation mediated intratracheal or omit inoculation can be used to study lower respiratory tract disease in mice, avoiding the concurrent involvement of the upper respiratory tract. The main advantage of imit over internasal inoculation methods is that this technique allows for accurate and reproducible delivery of an inoculum directly to the lungs.

Because IIT is a non-surgical technique, it also reduces the potential for complications associated with surgical intratracheal procedures. Though this method can provide a reliable mechanism to deliver bacteria to the lung to study pathogenesis, IMA can also be applied to the delivery of other reagents, such as environmental compounds to study lung damage, target delivery of therapeutics for treatment and delivery of imaging probes or SRNA to study basic lung biology. Generally, individuals new to this method will struggle with the intubation step as the step requires careful positioning of the animal and the auto scope for guidewire insertion.

Visual demonstration of this method is helpful as the initial placement of the guidewire into the trachea is conceptually difficult to describe to new researchers. 15 hours before the installation inoculate three milliliters of broth culture with a single bacterial colony and grow the culture at 37 degrees Celsius and a shaker at 200 RPM. After 15 hours, spin down one milliliter of the cells in a 1.5 milliliter micro centrifuge tube, and re suspend the palate in one milliliter of PBS.

Next, measure a one to 10 dilution of the bacterial stock suspension at an OD 600 to determine the bacterial concentration. Then dilute the bacterial cells in PBS to the desired concentration of bacteria per 50 microliters of inoculum. For the IIT installation, mice are prepared for IIT by administering a 2%lidocaine solution to a lightly anesthetized mouse by gavage needle mice.

A return to anesthesia while the lidocaine takes effect. Now draw 150 microliters of air into a 250 microliter gas type rescission syringe, equipped with a 22 gauge long blunt needle. Then advance the Teflon plunger from the 150 microliter mark to the 200 microliter mark on the syringe body to draw up 50 microliters of the bacterial inoculum when the syringe is loaded.

LA, a sedated mouse supine on an intubation platform securing the animal by the incisors on a no ring attached to a Velcro strip connected to the platform. Raise the mouse to a 45 degree incline and then use a micro cotten applicator to retract the tongue with a rolling motion with the dominant hand. Next, with the non-dominant hand, use an operating otoscope fit with an intubation specular to both maintain the tongue retraction and visualize the epiglottis.

Then holding a guide wire threaded through a 20 gauge catheter in the dominant hand, intubate the mouse to a 10 millimeter depth within the trachea and remove the otoscope. Secure the catheter with the non-dominant hand. Then briefly attach a lure connected length of one 16th clear tubing containing a colored dye to the catheter.

The dye should rapidly migrate back and forth in response to the animal's breathing when the animal has been successfully intubated. Insert the 22 gauge blunt needle into the catheter and dispense the inoculum directly into the lung in a single fluid motion. Then immediately remove the needle and catheter from the animal and return the mouse to its cage with monitoring until the animal is fully recovered from the anesthesia.

To evaluate the bacterial burden of the infected lung tissue, remove the lungs using sterile technique and place the tissue into a sterile pre weighed one ounce sample bag. After weighing the sample bag plus the lungs, add one milliliter of sterile PBS to the tissue and resell the sample bag. Then gently roll a 25 milliliter serological pipette over the sample bag to homogenize the tissue.

Remove the connected tissue through a sterile polyester wall course filter and treat the homogenate with tritton X 100 at a final concentration of 1%to liberate the bacteria. Next, use a multi-channel pipette to conduct a sixfold serial dilution of the homogenate in a UBO 96 well plate. Then use an eight well multi-channel to plate the homogenate triplicate 10 microliter aliquots onto an LBA agar plate.

Finally, incubate the plates overnight at 37 degrees Celsius and enumerate the colony forming units the following day. Deliver of the bacteria is highly efficient via this method with over 98%of the pseudomonas aerogen inoculum recovered from the lungs of the instilled animals in this representative experiment. Furthermore, IIT installation is highly reproducible regardless of the concentration of the inoculum administered.

To visualize the distribution of the instilled material via the IIT method, 50 microliters of 0.1%Kumasi brilliant blue dye can be delivered into the lungs as just demonstrated for the inoculum. Note, the distribution of the dye to all lobes of the lung Once mastered. This method can be rapidly performed with a team of two researchers in an average time of two to three minutes, including anesthesia, IMA inoculation and recovery from anesthesia.

While attempting this procedure, it is important not to make more than two failed attempts to intubate the mouse. Failed intubation may result in potential trauma from inserting the catheter into the esophagus rather than the trachea. Many respiratory pathogens, including those requiring abs L three containment can be effectively delivered by imed as this procedure can be performed as demonstrated in by safety cabinets and additionally by researchers wearing full respiratory protection including a face shield.

We have demonstrated that imit is a very accurate and efficient method for the delivery of materials directly into the lungs of mice. These properties make imit well suited for GLP type studies that require highly reproducible model systems.