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.

Representative 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 appearance of the lung demonstrates that 90% of attempts cannulated the small posterior lobe of the right lung, while 10% of attempts targeted the inferior lobe. It is speculated that the small volumes of these lobes explains the compensatory tachypnea of the mouse during distal cannulation (to maintain minute ventilation through the catheter).

IB administration of EBD to the left lung was performed as described in section 5.2. 100% of attempts target the inferior segments of the left lung (Figure 4B). In contrast to right-sided intubation, no tachypnea occurs with this engagement, reflecting intubation (and ventilation) of the larger left lung segments.

Adaptation of the selective IB cannulation technique can target the entire left or right lung.

Once IB cannulation is performed, withdrawal of the IB catheter (and changes in mouse positioning, as detailed in section 5.3) can be used to improve delivery of agents to all lobes of the right lung (and all segments of the left lung). Instillation of EBD solution to the right lung (section 5.3.1) successfully targeted all right lobes, as demonstrated in Figure 5C. Instillation of EBD solution to the left lung (section 5.3.2) successful targeted all left segments (Figure 6C).

IT administration or symmetric IB administration yields asymmetric lung parenchymal agent concentrations, which can be corrected by IB dose-adjustment.

Mice underwent bilateral administration of 30 µL of 0.05% FITC-dextran to the left lung and 30 µL of 0.05% FITC-dextran to the right lung as described in section 6.2. Alternatively, mice received 60 µL of 0.05% FITC-dextran intratracheally as per section 6.1. At the end of the experiment, mice were euthanized via terminal anesthesia overdose (ketamine/xylazine). Lungs were immediately harvested and homogenized. FITC-fluorescence (quantified by optical density) was measured with 96-well plate reader. Date were analyzed with student’s t-test for two-group comparisons.

As detailed in Figure 7, both IT (Figure 7A) and symmetric IB administration (Figure 7B) of FITC-dextran led to asymmetric lung parenchymal FITC fluorescence, with greater relative concentrations (normalized to weight) noted in the left lung. This suggests that asymmetric lung delivery of experimental agents after IT administration is not a consequence of asymmetric presentation of these agents to each mainstem bronchus. Rather, it was hypothesized that equal mainstem delivery (as ensured by symmetric IB administration) was diluted by differences in lung weights/mass, as observed in Table 2.

To overcome these differences in symmetric delivery, 40 µL of 0.05% FITC-dextran was administered to the larger right lung and 20 µL to the smaller left lung, as per section 6.3. This “dose-adjusted IB administration” improved the symmetry of lung parenchymal agent delivery (Figure 8A). Despite this correction, however, we observed persistent heterogeneity within different lobes of the right lung (Figure 8B).

BLM-induced lung injury in different delivery systems:

To demonstrate that dose-adjusted IB administration of experimental agents can improve modeling of diffuse lung disease, we administered BLM (a mouse model of fibrosing lung injury) either intratracheally or via dose-adjusted IB administration, as per section 7. As expected with this model of injury, both IT and IB injections of BLM led to lung injury and systemic illness (with loss of weight). This systemic illness resolved in 7 days. 21-day mortality was 20% (1/5) in the IT group and 0% (0/5) in the dose-adjusted IB group.

21 days after IT- or IB-BLM administration, mice were harvested for lung histology. As demonstrated in representative histologic images (Figure 9A),

To determine if this improved left-right homogeneity of fibrotic lung injury is physiologically relevant, it was observed that dose-adjusted IB administration of BLM imparted a more consistent loss of inspiratory capacity (IC) and respiratory system compliance (Crs), as well as a concordant increase in respiratory system elastance (Ers) (Figure 9B).

Figure 1: Anatomy of mouse airway cannulation. (A) A mouse airway cast was made by inflating a mouse lung (harvested from a 25 g mouse) with silicon elastomer. (B) Catheter placement for standard IT administration. (C) Catheter placement for IB administration. Please click here to view a larger version of this figure.

Figure 2: Setup for the work platform. (A) A metal bookend (90° angle) is bent to 70°. A screw hole is placed in the top midline to anchor a movable (80 mm x 150 mm). Hook and loop tape and a suspending suture are placed to allow positioning of an anesthetized mouse on the board. (B) The plastic board is anchored with a screw on the metal bookend. The screw is sufficiently loose to allow rotation of the board in a clockwise (+) or counterclockwise (-) direction. (C) An anesthetized mouse is positioned using with hook and loop tape (0.75” W) for IT/IB agent administration. A suture is passed under the mouse incisors to allow head stabilization. The operator is positioned at the dorsal aspect of the mouse, and the neck is illuminated via a goose neck lamp. Please click here to view a larger version of this figure.

Figure 3: Creation of customized catheters for IB administration. (Step 1) To enable sufficient catheter length to engage the mainstem bronchi, two catheters are combined. (Step 2) Catheters are connected at a slight angle, facilitating selective intubation to the mainstem bronchi. (Step 3) Furthermore, the distal catheter tip is beveled, allowing better directional control of airway instillation. Please click here to view a larger version of this figure.

Figure 4: Approach for selective right/left lung lobar cannulation and administration. (A) To target the right lung, the plastic board is rotated +30°, improving ease of selectively engaging the right mainstem bronchus. The catheter is advanced (per distances proposed in Table 1) to selectively engage right sided lobes. 20 µL of 0.3% EBD was administered. In ~90% of attempts, the posterior lobe is cannulated. The remaining 10% of attempts engage the inferior lobe. (B) To target the left lung, the plastic board is first rotated -74° for left mainstem engagement. After successful intubation of the catheter, rotation is then decreased to -30° to allow for gravity to assist with agent delivery. To prove selective engagement of the left side, 40 µL of 0.3% EBD was delivered. This approach consistently (100% of attempts) targeted left lung basilar segments. Please click here to view a larger version of this figure.

Figure 5: Symmetric administration approach to unilaterally deliver agents to the entire right lung. (A) Right-side IB intubation was performed at +30°, identical to selective right lung lobar cannulation (Figure 4A). (B) The plastic board was then rotated to -74° to allow for gravity assistance during agent administration. The catheter tip is then withdrawn to depths detailed in Table 1, corresponding to the right mainstem bronchus. The bevel of the tip is positioned downwards by rotating the catheter hub. (C) 30 µL of EBD was delivered at -74°, proving diffuse right lung administration of EBD. Please click here to view a larger version of this figure.

Figure 6: Symmetric administration approach to unilaterally administer agents to the entire left lung. (A) Left-sided IB intubation was performed at -74°, identical to selective left lung lobar cannulation (Figure 4B). (B) After a successful intubation, the plastic board was then rotated +86° to allow for gravity assistance during agent administration. The catheter tip is then withdrawn to depth detailed in Table 1. The bevel of the tip is shifted downwards by rotating the catheter hub. (C) 30 µL of EBD was delivered with gel loading tip, proving diffuse left lung administration of EBD. Please click here to view a larger version of this figure.

Figure 7: IT administration of experimental agents is equally delivered to mainstem bronchi, yet leads to different lung parenchymal concentrations. (A) IT administration of 0.05% FITC-dextran (60 µL) imparted higher fluorescence in the left lung, suggesting uneven lung concentrations of delivered agent. (B) This unequal lung parenchymal fluorescence persists even when equal volumes of 0.05% FITC-dextran (30 µL) are administered to each mainstem bronchus. This persistent parenchymal imbalance, despite equal right/left mainstem delivery, suggests that differences in lung agent concentrations reflect dilution in the larger right lung (n = 10 per group). Please click here to view a larger version of this figure.

Figure 8: Improved homogeneity of agent deposition by dose-adjusted IB administration. (A) Asymmetry of lung parenchymal delivery is improved when a greater proportion of agent (40 µL of 0.05% FITC-dextran) is administered to the larger right lung and a lesser proportion of agent (20 µL of 0.05% FITC-dextran) to the smaller left lung. (B) Despite this improved left-right symmetry, there remains lobar heterogeneity of agent deposition (black: superior lobe; yellow: middle lobe; blue: inferior lobe; green: posterior lobe; red: left lung). Please click here to view a larger version of this figure.

Figure 9: Improvement of mouse BLM-induced lung fibrosis model using dose-adjusted IB administration. (A) IT administration of BLM (1.2 mg/kg in 60 µL solution) induces left-side predominant lung injury/fibrosis 21 days later, consistent with higher lung concentrations of agent in this smaller lung. Left-right symmetry improves by adjusting the volume of BLM to each side of lung: 40 µL of the solution is administered to the larger right lung and 20 µL of the solution is administered to the smaller left lung. I: inferior lobe, M: middle lobe, S: superior lobe, P: posterior lobe. Images represent lobes from a single, representative mouse. (B) Consistent with improved symmetry of distribution, the dose-adjusted IB administration of BLM improves the physiologic modeling of lung fibrosis, with more representative increases in respiratory system elastance (Ers) and decreases in inspiratory capacity (IC) and dynamic respiratory system compliance (Crs). Please click here to view a larger version of this figure.

Body weight (g)	Number of mice tested	Catheter depth (mm) for Selective cannulation		Catheter depth (mm) for whole lung cannulation
		Right lung	Left lung
15 - 19	17	37	38	26
20 - 25	22	38	39	27
25 - 30	29	39	40	28
> 30	11	40	41	31

Table 1: Suggested depth of catheter insertion. Predicted catheter depths necessary to selectively cannulate the distal and proximal lungs were empirically determined using C57BL/6 mice of various weights (total = 79 mice).

Body weight (g)	Number of mice tested	Ratio of lung weights
14 - 10	25	2.01 ± 0.16
20 - 25	35	1.88 ± 0.27
25 - 30	15	1.88 ± 0.27
> 30	6	2.03 ± 0.09

Table 2: Right:left lung weight ratios. Differences in lung weights observed in 81 C57BL/6 mice demonstrate rationale for corrected IB drug administration. Lungs were dissected and weighed after a lethal dose of ketamine and xylazine.

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, we describe the direct IB administration of experimental agents. This approach offers two distinct benefits over classic approaches to IT administration. Firstly, the approach allows for selective unilateral administration to one lung, allowing for sparing of the contralateral lung. This approach is useful for the selective administration of drugs into a unilaterally injured lung (e.g., ischemia-reperfusion injury⁸), avoiding nonspecific effects in the uninjured lung. Furthermore, directed administration of tumor cells can be used to distinguish primary tumor growth from contralateral, metastatic spreading^9,10.

Secondly, the report details a previously unrecognized benefit of IB administration. As detailed in Figure 7A, IT administration relatively concentrates experimental agents within the smaller left lung. This asymmetry can be corrected by administering a relatively larger volume of agent to the larger right lung (Table 2), while delivering a smaller volume to the smaller left lung (Figure 8A). The relevance of this dose-adjusted IB administration to BLM-induced fibrotic lung injury was demonstrated here. Dose adjustment mitigates the injury to the left lung (which received less BLM), while increasing injury to the right lung (Figure 9A). This increased symmetry coincides with decreased variability of lung injury, as quantified by measurements of pulmonary function (Figure 9B).

There are several critical steps in the protocol, including the need to have a stand capable of easily and repeatedly altering animal positioning (i.e., rotation). More critical is the ability to determine when selective lung cannulation has been achieved. As described in section 4.12, use of a spirometer (in which a water column demonstrates tidal ventilation) ensures successful tracheal cannulation³. Observation of mouse tachypnea is consistent with distal right lung segment cannulation, while the absence of dyspnea (despite feeling resistance with catheter insertion) suggested cannulation of the left lung. Using these non-operative localization techniques, the operator should be able to accurately guide IB cannulation and experimental agent deposition.

This approach has several limitations. The IT model of agent delivery is attractive in its simplicity. It requires a moderate degree of practice and technical skill, although a skilled operator can still rapidly perform this technique within the window of isoflurane anesthesia. The additional technical skill/practice required, however, may be easily offset by the benefit of this approach in experiments that prioritize either selective agent/siRNA/cell delivery or increased homogeneity of agent deposition. An additional limitation to this method is the uncertainty regarding length of catheter insertion. As detailed in Table 1, 79 male and female mice were measured to estimate the depth of catheter insertion necessary for selective IB cannulation. These data serve as a resource to guide the operator to perform our protocol. However, we cannot confidently extrapolate our resource to other mouse strains (including knockout mice) or morbidly obese mice. In addition, we have not measured if there are differences in airspace volume (lobar, segmental) that vary based on weight. As such, it is possible that large mice may be able to accommodate larger instillation volumes with IB administration. Thus, the operator should perform an initial optimization/troubleshooting step (using EBD instillation) to ensure that our technique is well-adapted to the desired mouse model.

In summary, this report describes a novel IB technique that can be used to selectively administer experimental agents to a single lung or adapted to ensure symmetric distribution throughout both lungs. These benefits justify the marginal increase in complexity in comparison to standard IT techniques.

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