This protocol demonstrates that it is possible to instill research material into the trachea of neonatal rat pups, five-days-old, by direct visualization. This method improves the reliability of administration of research material or drug into the trachea by direct visualization. By avoiding injection into the trachea, this method reduces pain, and more importantly, bleeding into the trachea, allowing improved survival.
The development of fine skills is required to perform this protocol, including appropriate positioning of the animal, correct use of operating autoscope to visualize the vocal cords, and efficacy of administration into the lungs. Demonstrating the procedure will be Tara Sudhadevi, a postdoctoral fellow from my laboratory, and Alison Ha, a graduate student. Before beginning the experiment, confirm a loss of tail and pedal reflex in an anesthetized five-day-old rat pup and use laboratory labeling tape to restrain the pup in the supine position on a flat platform inclined at 45 degrees.
Use blunt forceps to gently pull the tongue to one side, and with the help of a two-millimeter diameter otoscope speculum, gently hold the tongue in place so that the larynx is visible. Using the operating otoscope with the correct sized speculum, locate the vocal cords through the magnifying lens of the otoscope. For intratracheal instillation, load a one-milliliter syringe fitted with a long-angled, gel-loading pipette tip with 0.9 cubic centimeters of air, followed by 30 to 50 microliters of the substance of interest.
Bend the pipette tip to a 30-degree angle and insert the loaded pipette tip through the speculum to about two millimeters beyond the vocal cords. Push the piston of the syringe to administer the substance of interest and the air through the speculum into the vocal cords. When all the substance has been delivered, place the pup on a 38-degree-Celsius integrated circulating fluid heating pad with monitoring until regular respiration, before reuniting the pup with the dam.
To evaluate the distribution of the substance within a lung tissue sample, wipe the chest and abdomen of the euthanized rat pups with 70%ethyl alcohol and use sterile technique to remove the lungs according to standard protocols for their visual assessment. In this study, the efficacy of the intra-tracheal instillation technique was assessed by visual examination of the lung tissue collected from a five-day-old rat pup instilled with Evans blue dye. The instillation was nearly 100%efficient, as the dye spread into the lung immediately upon administration and distributed across all the lobes of the lung tissue.
The dye was not observed within any tissues outside of the lungs, confirming the ability of the technique to be used for the selective delivery of a therapeutic of interest to the lungs. Placing the animal initially at the correct angle over a firm surface is essential for the success of the next step of visualizing the vocal chords using the operating autoscope.
