Our protocol proposes a new noninvasive delivery method of LPS from the trachea to the lung, more accurately. People who are trying this method for the first time should put much effort into finding the position of the trachea. To prepare the intubation platform, pass the string through the two holes and tie its ends to the small protrusions at the top of the riser.
Next, attach two rubber bands to each end of the paperclip and tape them to the riser's back. Finally, fix the riser to the base at a 90-degree angle. Assemble the cannula onto a cannula pen, and turn on the pen's light.
Next, disinfect small surgical forceps and Pasteur pipette using 70%alcohol. To prepare the test compound solution, add three milligrams of lipopolysaccharide, or LPS, in one milliliter of pH 7.2 PBS. Next, dissolve 10 milligrams of pentobarbital sodium in one milliliter of normal saline, and filter the solution using a 0.45-micrometer syringe filter.
Place the anesthetized mouse on the intubation platform. Then fix the top front teeth with the string, and the two forefeet with the rubber bands. Using tweezers, pull the tongue out and hold it.
Push the cannula slowly along the mouth to the mandibular epiglottis. Locate the trachea with the cannula pen's light and slowly insert the cannula into it. Then withdraw the intubation pen, leaving the cannula inside.
Insert the Pasteur pipette into the cannula joint and press the head. After successful endotracheal intubation, using a flat-head microsyringe, instill the prepared LPS solution at three milligrams per kilogram dose through the cannula. Once finished, take out the microsyringe and cannula.
Remove the mouse from the intubation platform and place it in a separate cage to recover. Observe the respiratory state of the mouse until it recovers and regains consciousness. At 12 hours after LPS tracheal instillation, the expression of an inflammatory cytokine, TNF-alpha, and the lung dry-wet weight ratio were evaluated and compared to other instillation methods.
The serum TNF-alpha levels in the noninvasive oropharyngeal intubation group were significantly increased compared to the blank control group. The lung dry-wet weight ratio was also increased, reaching the same level as in the surgical tracheal intubation group. It is important to not scratch the trachea in the process of finding the location of trachea.
The technique could also be used to deliver other drugs that need to reach the lung in a noninvasive way.
