Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Summary

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.  In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.  This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

Abstract

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.  However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.  Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.  It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Introduction

Direct pulmonary administration of experimental agents in mice allows for the study of lung immune responses, acute lung injury, and lung fibrosis. Direct pulmonary administration is typically performed via intratracheal (IT) instillation, as described previously^1,2,3. However, this approach is nonselective, affecting both lungs in a nontargeted and often asymmetric fashion.  Experimental modeling of lung injury may benefit from the ability to selectively target one specific lung, allowing for use of the contralateral lung as a control. Conversely, accurate modeling of human diffuse lung diseases benefits from symmetric distribution of experimental agents to the bilateral lung parenchyma.

The overall goal of this report is to describe a method for selective delivery of experimental agents to the left or right lung of a mouse (Figure 1). This intrabronchial (IB) administration approach allows for unilateral treatment of a mouse lung and can be easily adapted to ensure equal delivery of an agent to the bilateral mainstem bronchi. By using IB administration to deliver larger doses of experimental agents to the larger right lung and smaller volumes to the smaller left lung (i.e., dose-adjusted IB administration), demonstrated in this report is an improvement in the homogeneity of pulmonary delivery of experimental agents, optimizing the model of diffuse lung injury in mice. As such, this report may hold value for investigators seeking to either unilaterally administer experimental agents to mice or improve the symmetry of drug deposition in both lungs.

Protocol

All animal protocols have been approved by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC). All procedures described below (sections 4–7) have been optimized using both male and female C57BL/6 mice. This approach has been validated using mice ranging from 19–40 g in body weight.

1. Creation of platform for IB administration

1. Bend the bookend from the original 90° angle between the basal wing and standing wing to 70° (Figure 2A).
2. Drill a hole at the center-top of the standing wing of the metal bookend (Figure 2A).
3. Drill a hole of identical size at the corresponding position of the plastic board. Drill two smaller holes inferiorly and laterally (Figure 2A).
4. Drape a 4:0 silk suture between these small holes in the plastic board (Figure 2A).
5. Place the hook and loop tape on the edge of the plastic board (Figure 2A).
6. Assemble the plastic board to the metal bookend with the screw (Figure 2B). Ensure that the screw-nut is sufficiently tight to hold the board in position while allowing for adjustment of angle, if necessary.
7. Ensure that rotation of the plastic board is clockwise and counterclockwise, moving freely.
   NOTE: Clockwise motion is represented in this report as a (+) degree rotation, and counterclockwise is represented as a (-) degree rotation.
8. Use an angle protractor to position the plastic board at +30°, +86°, -30°, and -74° and mark them on the bookend, respectively.

2. Creation of extended catheters for IB agent administration

1. Make a right angle cut with a sharp blade on the tip of an original 22 G catheter (25 mm, see Table of Materials) (Figure 3, step 1a).
2. Bevel (~50°–60°) the tip of the other original catheter (25 mm) with the blade, then cut off at right angle from the hub (Figure 3, step 1b).
3. Glue the two catheters at their blunt ends with a slightly less than 180° angle (Figure 3, step 2).
4. Blunt the beveled tip by melting with a low temperature cautery (see Table of Materials).
5. Polish the extended catheter with “0” size sandpaper on the glued area and the beveled tip of the extended catheter (Figure 3, step 3).
6. Mark on the extended catheter with different colors at 25 mm, 30 mm, and 35 mm (Figure 3, step 3).
7. Indicate the bevel side of the extended catheter by labeling its hub with marker.
8. Rinse the extended catheter with DI water, following by flushing inside of the catheter with 70% ethanol. Airdry the catheter.
9. Sterilize with UV light for 10 min before use.

3. Pre-procedure preparation

1. Make all administered agents in a biological safety hood under sterile technique.
2. Clean the workplace with 70% ethanol.
3. Sterilize all surgical tools with 70% ethanol.
4. Fix the base of the work platform to the bench immediately in front of the researcher by affixing C-clamps to the basal wing of the bookend.
5. Generate several makeshift spirometers, which are devices that will allow for detection of tidal airflow in mice. Briefly, deposit 60 µL of sterilized saline into a 1 mL syringe (plunger removed) with a gel loading tip.
   NOTE: The deposited drop of saline occludes the barrel and moves upwards and downwards when exposed to tidal ventilation³.
6. Loosely attach the hub of a 22 G extended catheter to the makeshift spirometer.
7. Place each of the glass droppers to each side of the platform for ease of access.
8. Connect the isoflurane induction chamber to the rodent anesthesia machine (see Table of Materials) in an isoflurane-compatible biological safety cabinet.

4. Non-operative IT intubation approach

1. Anesthetize a C57BL/6 mouse (male or female, 8–10 weeks, ~25 g) with oxygen (2 L/min) and 5% isoflurane (see Table of Materials) in an induction chamber for 4 min.
2. Aspirate the experimental agent to be delivered (e.g., Evans blue dye or FITC-dextran, as demonstrated in Figure 4) into two pipettes, then place them to each side of the platform during sedation.
3. Ensure a respiratory rate of approximately 24–30 breaths/min before removing the mouse from the anesthesia induction chamber.
   NOTE: Isoflurane anesthesia typically lasts for 4 min, sufficient for all IB procedures. If the operator is not proficient with the technique, ketamine/xylazine (80 mg/kg and 10 mg/kg intraperitoneally, see Table of Materials) may be used for more prolonged anesthesia.
4. Suspend the mouse by its incisors on the draped suture line in the supine position. Secure the mouse with two to three pieces of hook and loop the tape loosely, avoiding restriction of ventilation.
5. Turn on the LED fiber optic illuminator (see Table of Materials, Figure 2C).
6. Position the operator behind the platform (dorsal to the mouse).
7. Orient the gooseneck of the illuminator so that it illuminates the larynx area through the skin. The distance between the mouse and light source is 2–3 cm (Figure 2C).
8. Confirm the depth of anesthesia with a toe/paw pinch before performing all procedures below.
9. Hold the sterile forceps with the dominant hand, then draw the tongue out of the oral cavity with the forceps.
10. Hold the sterile depressor with the nondominant hand, then flatten the root of the tongue with the depressor to expose the oropharynx widely. The forceps can then be released, freeing the dominant hand.
11. Use the dominant hand to intubate the extended catheter into the trachea via the oral cavity (Figure 2C).
12. Confirm placement by observing if the bubble in the syringe moves up and down with each breath.
13. Additional details of IT intubation have been published previously³. Total procedure time, excluding anesthesia, lasts 10–15 s for a well-trained operator.

5. Non-operative IB intubation and delivery approaches

1. IB approach to selective lobar cannulation of the distal right lung
   1. After performing IT cannulation (step 4.11), rotate the plastic board +30° (Figure 4A).
   2. Hold the hub of the catheter and guide it naturally in parallel to the mouse midline, extending it to weight-based depths as described in Table 1.
      NOTE: The resistance at these depths should be noted. At this point, the mouse will become slightly tachypneic, as explained in the representative results. For an experienced operator, approximately 90% of attempts will successfully cannulate the right lung (with tachypnea noted).
   3. Deliver 20 µL of 0.3% Evans blue dye (EBD, see Table of Materials) with a gel loading tip.
   4. Dispense 1–2 aliquots (0.1 mL each) of air by using the glass dropper.
      NOTE: This ensures clearance of the residual EBD solution (or experimental agents) from inside of the catheter.
   5. Withdraw the catheter, then maintain the mouse position for 30 s.
   6. Place the animal on a warming blanket until it regains consciousness. Recovery is typically complete within 2 min.
2. IB approach to selective segmental cannulation of the distal left lung
   1. After performing IT cannulation (step 4.11), rotate the plastic board -74° (Figure 4B).
   2. Hold the hub of the catheter and apply gentle pressure to advance the catheter into the left mainstem bronchus, while placing modest pressure both downwards (90°) and towards the bookend. At depths noted in Table 1, the operator should note resistance as the lower segments of the left lung are engaged. If tachypnea occurs, withdraw the catheter to the 20–25 mm position, and reattempt.
   3. After cannulating the left lower lung segments, a change in position is required to allow gravitational assistance for agent administration. Rotate the plastic board -30° (Figure 4B).
   4. Deliver 40 µL of 0.3% EBD with a gel loading tip.
      NOTE: It is feasible to deliver a larger volume of agent because the left lung has only one lobe.
   5. Dispense 1–2 aliquots (0.1–0.3 mL each) of air using the glass droppers.
      NOTE: This ensures clearance of any residual EBD (or experimental agents) from inside of the catheter.
   6. Withdraw the catheter, then maintain the mouse position for 30 s.
   7. Place the animal on a warming blanket until it regains consciousness. Recovery is typically complete within 2 min.
3. Adaptation of IB administration to allow delivery of agent to entirety of left or right lung
   NOTE: If the operator seeks to deliver agents not to a specific right lung lobe or left lung segment, but rather to the entire lung (right or left lung), the catheter should be slightly withdrawn to the respective mainstem bronchi, as follows.
   1. Right entire lung administration
      1. After step 4.11, rotate the plastic board +30° (Figure 5A).
      2. Hold the hub of the catheter and guide it naturally in parallel to the mouse midline, reaching it to depths necessary for right sided distal lobar cannulation (Table 1).
      3. Confirm appearance of the tachypnea sign.
      4. Rotate the mouse -74° to enable gravity assistance for agent delivery (Figure 5B).
      5. Withdraw the catheter to a position that corresponds to the takeoff of the right mainstem bronchus (Table 1). Ensure that the bevel of the catheter faces downward (Figure 5B).
      6. Deliver 30 µL of 0.3% EBD with a gel loading tip to the right lung.
      7. Dispense 1–2 aliquots (0.1–0.3 mL each) of air using a glass dropper.
      8. Withdraw the catheter, then maintain the mouse position for 30 s. Place the animal on a warming blanket until it regains consciousness. Recovery is typically complete within 2 min.
   2. Left entire lung administration
      1. After step 4.11, rotate the plastic board -74° (Figure 6A). Alternatively, rotation may occur after step 5.3.1.8 by withdrawing the catheter to the trachea, enabling bilateral IB agent administration.
      2. Hold the hub of the catheter and apply gentle pressure to advance the catheter into the left mainstem catheter, while placing modest pressure both downwards (90°) and towards the bookend. Depth of intubation is guided by Table 1.
      3. Confirm the no tachypnea sign.
      4. Rotate the mouse +86° to allow for gravity assistance with agent administration.
      5. Withdraw the catheter to the left mainstem bronchus (the same distances as the right lung are sufficient, Table 1) and rotate the bevel of the catheter faces downward (Figure 6B).
      6. Deliver 30 µL of 0.3% EBD with a gel loading tip to the left lung.
      7. Dispense 1–2 aliquots (0.1–0.3 mL each) of air using a glass dropper.
      8. Withdraw the catheter, then maintain the mouse position for 30 s. Place the animal on a warming blanket until it regains consciousness. Recovery is typically complete within 2 min.

6. Use of sequential IB cannulation approaches to deliver dose-adjusted volumes of agent to each lung

1. IT administration group
   1. Perform IT cannulation as described in steps 4.1–4.11.
   2. Deliver 60 µL of 0.05% FITC-dextran (see Table of Materials) with a gel loading tip (Figure 1B).
   3. Dispense 1–2 aliquots (0.1–0.3 mL each) of air using the glass droppers.
   4. Keep the position for 60 s and allow for mouse recovery as described above.
2. Symmetric bilateral IB administration
   1. Perform steps 5.3.1.1–5.3.1.8 (right lung) and steps 5.3.2.1–5.3.2.8 (left lung).
   2. Administer equal volumes (30 µL) of 0.05% FITC-dextran (or an experimental agent) to each side of the lung.
3. Dose-adjusted bilateral IB administration
   1. Perform step 5.3.1.1–5.3.1.8 (right lung) and steps 5.3.2.1–5.3.2.8 (left lung).
   2. Administer larger volume (40 µL) of 0.05% FITC-dextran to the larger right lung, and a smaller volume (20 µL) of 0.05% FITC-dextran to the smaller left lung. In lieu of FITC-dextran, an experimental agent can be administered.

7. Use of Dose-Adjusted IB administration to improve symmetry of single dose bleomycin (BLM)-induced lung injury

1. BLM administration groups
   1. Dose-adjusted IB-BLM (1.2 mg/kg, see Table of Materials) administration group: 60 µL (20 µL for left lung and 40 µL for right lung, respectively) of BLM solution were delivered to mice (n = 5). Controls (n = 5) received similar volumes of saline.
      NOTE: Refer to steps 5.3.1 and 5.3.2.
   2. IT administration group: 60 µL of BLM solution were delivered to mice with IT administration techniques.
      NOTE: Refer to steps 6.1.1–6.1.4.
2. Lung function measurement
   1. On day 21 after BLM or saline, anesthetize mice with an intraperitoneal (IP) injection of ketamine (160 mg/kg) and xylazine (32 mg/kg).
   2. After confirming depth of anesthesia by paw/toe pinch, perform a tracheostomy with an 18 G cannula (see Table of Materials).
   3. Connect mice to the ventilator and measure respiratory mechanics as previously described⁴.
3. Lung tissue collection and processing
   1. Following measurement of pulmonary mechanics, euthanize the anesthetized mice by cardiac puncture.
   2. Open the chest wall and induce bilateral pneumothoraces.
   3. Inflate lungs with 1% low melt agarose (40 °C)⁵ in PBS at a consistent pressure (42 cm H₂O).
   4. Cut four to five pieces of the lung along the long axis transversely, fix in 10% formalin, and embed in paraffin.
   5. Cut 5 µm sections and stain with Masson’s trichrome to visualize collagen deposition.

8 Post-procedural care

1. At the end of survival procedures, place the animal on a warming blanket until it regains consciousness. Recovery is typically complete within 2 min.

Results

Selective IB intubation targets specific lobes (right lung) or basilar segments (left lung).

IB administration of EBD to the right lung was performed as described in section 5.1. After completion of the experiment, mice were administered a lethal dose of intraperitoneal ketamine/xylazine, and lungs were harvested for demonstration of EBD distribution (Figure 4A, right). Gross appear...

Discussion

Lung injury has been classically modeled in rodents using IT administration of injurious agents such as BLM⁶. Such IT administration, however, only leads to patchy injury, reflecting the nontargeted nature of lung delivery with this approach⁷. These limitations of modeling lung injury are instructive challenges faced when attempting the IT delivery of non-injurious experimental agents, such as drugs, siRNA, or cellular therapies.

In this report, ...

Materials

Name	Company	Catalog Number	Comments
22 G shielded IV Catheter	BD	381423
Bleomycin	Enzo life sciences	BML-AP302-0010
Compact Mini rodent anesthesia machine	DRE Veterinary	9280
Evans blue dye	Sigma-Aldrich	E2129
FITC-dextran	Sigma-Aldrich	FD150
Isoflurane	Piramal Critical Care	NDC
LED-30W Fiber Optic Dual Gooseneck Lights Microscope Illuminator	AmScope	LED-30W
Low temperature cautery with fine tip	Bovie	AA02
Precisionglide needle, 18G x 1"	BD	305195	Beveled tip, 12 mm in length
Xylazine	AKORN	NDC 59399-110-20
Zatamine	VetOne	NDC 13985-702-10	Ketamine