Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.
Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 μL instillate volume can be introduced into mouse lung.
1. Anesthesia
2. Preparation of Surgical Area
3. Surgical Procedure
4. Sutures.
5. Animal Recovery
6. Representative Results
Intratracheal instillation of fluorescent compound, Cy5.5 conjugated to dextran, shows successful instillation of fluorescent compound in B6 mouse lung (Figure 1). The fluorescent dye is primarily targeted to the lung and is evenly distributed. Fluorescent signal in the gastrointestinal tract immediately following the procedure indicates that the esophagus was inadvertently intubated during the procedure (Figure 2). Investigators may also verify targeted delivery of compounds into the lung, and check distribution of the instillate, following the procedure by inflating the lungs and preparing lung tissue slices (2; 3) or fixed histological samples.
Figure 1. Fluorescent Cy5.5 signal co-registered with X-ray image in successfully instilled mouse lung. 1.4 mg Cy5.5 Dextran (at a concentration of 10 μg/μL) was instilled into the trachea of B6 mouse lung. Cy5.5 signal was detected using a 675 nm excitation filter and a 695 nm emission filter.
Figure 2. Co-registration of fluorescent signal shown with X-ray overlay in poorly instilled mouse. Inappropriate intubation of the esophagus during tracheal instillation protocol results in Cy5.5 delivery to the intestines.
Intratracheal instillations have been used in several various studies to evaluate the toxicity of test compounds (6), induce alveolar lung injury (4; 7), replace surfactants (8), as well as alter gene expression via delivery of small oligonucleotides directly into the lung (5). We are currently exploring the use of instilling 1) fluorescent redox-sensitive material into the lung in order to measure oxidative stress in vivo under normal and pathophysiological conditions, as well as 2) instilling fluorescent indicators with pharmacological compounds, DNA vectors, viruses, and/or microRNA to assess the effect of each instillate on lung fluid transport.
There are several advantages of introducing foreign material into the lung using an instillation protocol over inhalation exposure (reviewed in (1)). The primary benefits of using an instillation protocol includes limiting exposure to toxic, carcinogenic, or radioactive compounds. Additionally, solids as well as liquid material, can be introduced into the lung. Arguably, the greatest drawback to using the tracheal instillation approach is that the introduction of the instillate is invasive and nonphysiological.
This work was supported by NIH K99 HL09222601 and the S&R Foundation Ryaji Uneo Award awarded to MNH.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Isoflurane | Webster Veterinary Sterling , MA | 14043-220-05 | ||
Xylazine | Lloyd Laboratories Shenandoah, IA | New Animal Drug Application #139-236 | ||
Ketamine | Bioniche Pharma Lake Forest, IL | 67457-001-10 | ||
Nair | Available at drug stores | |||
1mL SubQ Syringe | BD Syringe Franklin Lakes, NJ | 309597 | 26 5/8 G | |
4-0 Nylon Suture | Ethicon San Lorenzo, Puerto Rico | 1894G.S30 |