An Improved Method for Rapid Intubation of the Trachea in Mice

Podsumowanie

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Streszczenie

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

Wprowadzenie

In spite of some anatomical and physiological differences,¹ murine models continue to be invaluable for modeling human biology and disease pathogenesis.² From a husbandry standpoint, mice are easy to handle, have a low breeding time, an accelerated lifespan, and are relatively inexpensive to house. With the development of diverse genetic strains and strategies (e.g., conditional knock-outs, reporter mice, lineage-tracing approaches, etc.), as well as the wide range of available reagents (e.g., antibodies, recombinant proteins, inhibitors, etc.), mice have become an essential model vertebrate organism to uncover human homeostasis and disease processes.³

Mice have been especially valuable for studying pulmonary conditions, including acute lung injury (ALI) and pulmonary fibrosis.⁴ ALI in humans can be caused by trauma, injury, or sepsis and is characterized by epithelial and endothelial leak (i.e., edema), inflammation, and nascent fibrosis. In many patients, ALI progresses to its severe form, acute respiratory distress syndrome (ARDS), which often results in fibrosis and death due to respiratory failure.^5,6 Pulmonary fibrosis is a progressive, fatal pathology characterized by the excess deposition of extracellular matrix, most notably type I collagen, leading to impaired lung function.^7,8 Administration of bleomycin (BLM) is the most widely used and best characterized model for inducing ALI and fibrosis in experimental animals.⁹ Although BLM-induced pulmonary fibrosis in rodents does not recapitulate fully the human fibrotic phenotypes,¹⁰ mouse studies with this model have led to the discovery of many important factors influencing the onset and progression of disease.¹¹

While the exact mechanism(s) behind BLM-induced fibrogenesis are unknown, the initiating injury is thought to arise from contact-dependent DNA strand breaks in the epithelial cells lining the conducting airways and alveoli, and in particular, type 1 pneumocytes.¹² The need for direct contact between BLM and the pulmonary epithelium highlights the importance of a robust delivery route, and these concerns are also germane to a broad range of treatments targeted to the distal airways, including recombinant proteins, antibodies, siRNA, virus, bacteria, particulates, and more. Oropharyngeal aspiration (OPA) has been widely used for this purpose¹³, but a major a shortcoming of OPA is that some portion of the delivered agent may be swallowed into the gastrointestinal tract, thereby leading to imprecision in the administered dose. Another widely used approach is transtracheal instillation, which involves tracheostomy under strong anesthesia to expose the trachea and instillation of an agent directly into the respiratory tract.¹⁴ However, not only may such a procedure be undesirable due to its invasivity, but it is also time consuming, requires a fair bit of training, and causes a potent injury to the respiratory tract.^15,16 Several protocols have been developed that involve the direct administration of agents into the trachea without the need for surgical intervention,^{16,17,18,19,20} but these methods involve extended recovery times caused by powerful anesthetics, the use of expensive equipment (i.e., otoscope/laryngoscope, commercially available procedure boards, fiber-optic wires, etc.), an excess of manipulation in the oral cavity, and uncertainty regarding the dosage.

This paper describes a relatively easy method of administration via intubation that allows a researcher to quickly, inexpensively, and reliably instill a reagent into the murine lung with limited risk of residual damage to the surrounding tissues.

Protokół

The Institutional Animal Care and Use Committees (IACUC) at the University of Washington and Cedars-Sinai Medical Center have approved the animal work necessary for these studies.

1. Preparation

1. Sterilize both the blunt end forceps and the depressor via autoclave.
2. Using a biological safety cabinet, prepare a working stock of BLM in PBS from the lyophilized powder.  Sonicate the solution for 10 min at 35 Khz to assure even mixing.
   Note: A total volume of between 30 and 45 µl is recommended to prevent pipetting variation on the low end, and suffocation with larger volumes.
3. Prepare a clean workspace that includes approximately 1 m² for the procedure itself, as well as designated locations for cages both before and after the procedure.
4. Fix the base of the procedure board to the bench immediately in front of the researcher by laying 2 or 3 strips of laboratory tape across the base and underlying bench. See Figure 1 for further specifications on creating a board.
5. Tie a single length of size 4.0 suture thread between the two positioning screws of the procedure board.
6. Generate a makeshift spirometer by removing and discarding the plunger from three 1 ml syringes, and depositing 60 µl of PBS into the top of each barrel to form an airtight seal. Secure the hub of the catheter loosely to one of the syringes and place it to one side of the board.
7. Aspirate 300 µl of air into a 1 ml syringe and place it to one side of the board.
8. Cut an additional piece of tape approximately 6 inches in length and place to one side. This will be used to secure the animal to the board in step 2.4.
9. Set up an isoflurane chamber. Attach O₂, isoflurane, and vacuum to the appropriate ports on both the exposure chamber and the clearance vacuum. Alternatively, administer anesthetic in an isoflurane-compatible biological safety cabinet.

2. Intubation

1. Anesthetize the mouse with isoflurane in the chamber until it loses consciousness and respiration slows to an appropriate rate. A typical exposure includes 4% isoflurane and 2% O₂ for 3 to 4 min, and the ideal outcome is 2 to 2.5 min of sedation. This corresponds to a respiration rate of 1 breath every 2 sec.
2. While waiting for sedation to set in, aspirate between 30 and 45 µl of BLM into a pipettor and place to one side.
3. When ready, suspend the sedated mouse by its upper incisors from the thread attached to the positioning screws of the procedure platform. Make sure that the animal's dorsum lies flat against the platform surface.
4. Being careful not to restrict ventilation, place a piece of tape loosely across the lower (caudal) portion of the thoracic cavity, just above the diaphragm. Placement should be tight enough to maintain proper alignment during the procedure, but not so tight that it restricts respiration.
5. Turn on the illuminator to between 80% and 100% intensity and orient the gooseneck so that it is 1 to 2 cm from the surface of the skin, near the solar plexus. Periodically check the tip of the gooseneck for heat to ensure that it does not injure the mouse.
6. Standing behind the platform, use the sterile, blunt end forceps to locate the tongue. Being careful to avoid the lower incisors, gently grip and draw the tongue out of the oral cavity.
7. Using the remaining hand, insert the depressor and use it to flatten the tongue against the floor of the oral cavity. Release the forceps, but leave the depressor in place for the next two steps.
8. Orient the light so that the trachea is visible by guiding the gooseneck proximally from the level of the solar plexus until it reaches the level of the mainstem bronchi.
   Note: The trachea can be easily distinguished by the action of respiration, which causes the emitted light to fluctuate in intensity. When correctly positioned, this structure will be discernable in the axial plane as a centrally located pin of light with minimal ambient light in the oral cavity itself.
9. Angle the syringe so that it follows the natural path of the trachea, and lower the 22-G catheter tip, with the attached syringe containing the droplet, straight into the lumen. The PBS bubble will begin to rise and fall with each breath upon successful placement.
   Note: This action may be delayed by several seconds as a result of deep sedation.
10. Feed the catheter in an additional 5 mm. Remove the tongue depressor.
11. Shift the syringe to the opposite hand, and gripping the hub, gently remove the syringe.
12. Deposit between 30 and 45 µl of BLM into the center of the interior of the catheter hub, attach the second syringe and dispense 300 µl of air into the hub.
13. Replace the second syringe with the first containing the bubble of PBS. The bubble will continue to rise and fall if the procedure has been performed successfully.

3. Post-procedural Care

1. Remove the catheter and tape, and place the animal in a dry warm place until it regains consciousness - typically within a couple of minutes.

Wyniki

Intubated mice were monitored daily for weight loss and distress, and sacrificed 4, 10 or 17 days later via intraperitoneal injection of 2.5% 2,2,2-tribromoethanol. Bronchoalveolar lavage (BAL) was collected in three washes of PBS as described elsewhere ²¹, and the right lung was fixed in 10% formalin, paraffin embedded, and stained with Masson's Trichrome by the University of Washington Histology and Imaging Core ²².

Dyskusje

In instances where aerosolization is impractical due to limited reagent availability, safety, or cost, direct tracheal administration is a superior method for delivery of exogenous agents into the lungs.¹⁶ Transtracheal instillation has been widely used to accomplish this; however, as with all surgical intervention, it also carries with it the potential for complications caused by the procedure itself, and not necessarily the agent being instilled.¹³ For these reasons, it has become...

Materiały

Name	Company	Catalog Number	Comments
Bleomycin For Injection, 30 units/vial	APP Pharmaceuticals, LLC	103720	For best results, BLM should be suspended in PBS, aliquoted, and stored as single use lyophilzed aliquots
Blunt End Forceps	N/A	N/A
Tongue Depressor (i.e. bent Valleylab Blade Electrode, 2.4")	Covidien	E1551G	Before use, create a 45 degree bend 1.5 cm  from the blade tip. A suitable depressor can also be created from any metal implement of similar dimensions.
Exel Safelet Catheter 22G X 1"	Exel International	26746
1 mL Slip-tip Disposable Tuberculin Syringe (200/sp, 1600/ca)	BD	309659
0.2ml Pipettor and Filter Tips	N/A	N/A
Fiber-Lite Illuminator. Model 181-1: Model 180 mated with Standard Dual Gooseneck illuminator	Dolan Jenner Industries, Inc.	181-1	Lower output LED illuminators are not recommended as they fail to suficiently illuminate the trachea.
Intubation Board	N/A	N/A	See Diagram 1.
Colored Label Tape: 0.5 in. Wide	Fisherbrand	15-901-15A
Oxygen	N/A	N/A
Phosphate-Buffered Saline, 1X	Corning	21-040-CV	Product should be sterile
Non-Sterile Silk Black Braided Suture Spool, 91.4 m, Size 4-0	Harvard Apparatus	517698
Table Top Anesthesia Machine Isoflurane	Highland Medical Equipment	N/A	http://www.highlandmedical.net/
Slide Top Induction Mouse Isoflurane Chamber	MIP / Anesthesia Technologies	AS-01-0530-SM
FORANE (isoflurane, USP) Liquid For Inhalation 100 mL	Baxter	1001936040
Nanozoomer Digital Pathology system	Hamamatsu
IgM ELISA Quantification Kit	Bethyl Laboratories	E90-101