This work was funded by NHLBI grant HL125371 to E.P.S. and by DOD (CDMRP) grant W81XWH-17-1-0051 to Y.Y.

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&#8211;7) have been optimized using both male and female C57BL/6 mice. This approach has been validated using mice ranging from 19&#8211;40 g in body weight.
1. Creation of platform for IB administration
<ol>
	<li>Bend the bookend from the original 90&#176; angle between the basal wing and standing wing to 70&#176; (Figure 2A).</li>
	<li>Drill a hole at the center-top of the standing wing of the metal bookend (Figure 2A).</li>
	<li>Drill a hole of identical size at the corresponding position of the plastic board. Drill two smaller holes inferiorly and laterally (Figure 2A).</li>
	<li>Drape a 4:0 silk suture between these small holes in the plastic board (Figure 2A).</li>
	<li>Place the hook and loop tape on the edge of the plastic board (Figure 2A).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use an angle protractor to position the plastic board at +30&#176;, +86&#176;, -30&#176;, and -74&#176; and mark them on the bookend, respectively.</li>
</ol>
2. Creation of extended catheters for IB agent administration
<ol>
	<li>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).</li>
	<li>Bevel (~50&#176;&#8211;60&#176;) 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).</li>
	<li>Glue the two catheters at their blunt ends with a slightly less than 180&#176; angle (Figure 3, step 2).</li>
	<li>Blunt the beveled tip by melting with a low temperature cautery (see Table of Materials).</li>
	<li>Polish the extended catheter with &#8220;0&#8221; size sandpaper on the glued area and the beveled tip of the extended catheter (Figure 3, step 3).</li>
	<li>Mark on the extended catheter with different colors at 25 mm, 30 mm, and 35 mm (Figure 3, step 3).</li>
	<li>Indicate the bevel side of the extended catheter by labeling its hub with marker.</li>
	<li>Rinse the extended catheter with DI water, following by flushing inside of the catheter with 70% ethanol. Airdry the catheter.</li>
	<li>Sterilize with UV light for 10 min before use.</li>
</ol>
3. Pre-procedure preparation
<ol>
	<li>Make all administered agents in a biological safety hood under sterile technique.</li>
	<li>Clean the workplace with 70% ethanol.</li>
	<li>Sterilize all surgical tools with 70% ethanol.</li>
	<li>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.</li>
	<li>Generate several makeshift spirometers, which are devices that will allow for detection of tidal airflow in mice. Briefly, deposit 60 &#181;L 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.</li>
	<li>Loosely attach the hub of a 22 G extended catheter to the makeshift spirometer.</li>
	<li>Place each of the glass droppers to each side of the platform for ease of access.</li>
	<li>Connect the isoflurane induction chamber to the rodent anesthesia machine (see Table of Materials) in an isoflurane-compatible biological safety cabinet.</li>
</ol>
4. Non-operative IT intubation approach
<ol>
	<li>Anesthetize a C57BL/6 mouse (male or female, 8&#8211;10 weeks, ~25 g) with oxygen (2 L/min) and 5% isoflurane (see Table of Materials) in an induction chamber for 4 min.</li>
	<li>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.</li>
	<li>Ensure a respiratory rate of approximately 24&#8211;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.</li>
	<li>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.</li>
	<li>Turn on the LED fiber optic illuminator (see Table of Materials, Figure 2C).</li>
	<li>Position the operator behind the platform (dorsal to the mouse).</li>
	<li>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&#8211;3 cm (Figure 2C).</li>
	<li>Confirm the depth of anesthesia with a toe/paw pinch before performing all procedures below.</li>
	<li>Hold the sterile forceps with the dominant hand, then draw the tongue out of the oral cavity with the forceps.</li>
	<li>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.</li>
	<li>Use the dominant hand to intubate the extended catheter into the trachea via the oral cavity (Figure 2C).</li>
	<li>Confirm placement by observing if the bubble in the syringe moves up and down with each breath.</li>
	<li>Additional details of IT intubation have been published previously3. Total procedure time, excluding anesthesia, lasts 10&#8211;15 s for a well-trained operator.</li>
</ol>
5. Non-operative IB intubation and delivery approaches
<ol>
	<li>IB approach to selective lobar cannulation of the distal right lung
	<ol>
		<li>After performing IT cannulation (step 4.11), rotate the plastic board +30&#176; (Figure 4A).</li>
		<li>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).</li>
		<li>Deliver 20 &#181;L of 0.3% Evans blue dye (EBD, see Table of Materials) with a gel loading tip.</li>
		<li>Dispense 1&#8211;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.</li>
		<li>Withdraw the catheter, then maintain the mouse position for 30 s.</li>
		<li>Place the animal on a warming blanket until it regains consciousness. Recovery is typically complete within 2 min.</li>
	</ol>
	</li>
	<li>IB approach to selective segmental cannulation of the distal left lung
	<ol>
		<li>After performing IT cannulation (step 4.11), rotate the plastic board -74&#176; (Figure 4B).</li>
		<li>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&#176;) 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&#8211;25 mm position, and reattempt.</li>
		<li>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&#176; (Figure 4B).</li>
		<li>Deliver 40 &#181;L 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.</li>
		<li>Dispense 1&#8211;2 aliquots (0.1&#8211;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.</li>
		<li>Withdraw the catheter, then maintain the mouse position for 30 s.</li>
		<li>Place the animal on a warming blanket until it regains consciousness. Recovery is typically complete within 2 min.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Right entire lung administration
		<ol>
			<li>After step 4.11, rotate the plastic board +30&#176; (Figure 5A).</li>
			<li>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).</li>
			<li>Confirm appearance of the tachypnea sign.</li>
			<li>Rotate the mouse -74&#176; to enable gravity assistance for agent delivery (Figure 5B).</li>
			<li>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).</li>
			<li>Deliver 30 &#181;L of 0.3% EBD with a gel loading tip to the right lung.</li>
			<li>Dispense 1&#8211;2 aliquots (0.1&#8211;0.3 mL each) of air using a glass dropper.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Left entire lung administration
		<ol>
			<li>After step 4.11, rotate the plastic board -74&#176; (Figure 6A). Alternatively, rotation may occur after step 5.3.1.8 by withdrawing the catheter to the trachea, enabling bilateral IB agent administration.</li>
			<li>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&#176;) and towards the bookend. Depth of intubation is guided by Table 1.</li>
			<li>Confirm the no tachypnea sign.</li>
			<li>Rotate the mouse +86&#176; to allow for gravity assistance with agent administration.</li>
			<li>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).</li>
			<li>Deliver 30 &#181;L of 0.3% EBD with a gel loading tip to the left lung.</li>
			<li>Dispense 1&#8211;2 aliquots (0.1&#8211;0.3 mL each) of air using a glass dropper.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Use of sequential IB cannulation approaches to deliver dose-adjusted volumes of agent to each lung
<ol>
	<li>IT administration group
	<ol>
		<li>Perform IT cannulation as described in steps 4.1&#8211;4.11.</li>
		<li>Deliver 60 &#181;L of 0.05% FITC-dextran (see Table of Materials) with a gel loading tip (Figure 1B).</li>
		<li>Dispense 1&#8211;2 aliquots (0.1&#8211;0.3 mL each) of air using the glass droppers.</li>
		<li>Keep the position for 60 s and allow for mouse recovery as described above.</li>
	</ol>
	</li>
	<li>Symmetric bilateral IB administration
	<ol>
		<li>Perform steps 5.3.1.1&#8211;5.3.1.8 (right lung) and steps 5.3.2.1&#8211;5.3.2.8 (left lung).</li>
		<li>Administer equal volumes (30 &#181;L) of 0.05% FITC-dextran (or an experimental agent) to each side of the lung.</li>
	</ol>
	</li>
	<li>Dose-adjusted bilateral IB administration
	<ol>
		<li>Perform step 5.3.1.1&#8211;5.3.1.8 (right lung) and steps 5.3.2.1&#8211;5.3.2.8 (left lung).</li>
		<li>Administer larger volume (40 &#181;L) of 0.05% FITC-dextran to the larger right lung, and a smaller volume (20 &#181;L) of 0.05% FITC-dextran to the smaller left lung. In lieu of FITC-dextran, an experimental agent can be administered.</li>
	</ol>
	</li>
</ol>
7. Use of Dose-Adjusted IB administration to improve symmetry of single dose bleomycin (BLM)-induced lung injury
<ol>
	<li>BLM administration groups
	<ol>
		<li>Dose-adjusted IB-BLM (1.2 mg/kg, see Table of Materials) administration group: 60 &#181;L (20 &#181;L for left lung and 40 &#181;L 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.</li>
		<li>IT administration group: 60 &#181;L of BLM solution were delivered to mice with IT administration techniques. 
		NOTE: Refer to steps 6.1.1&#8211;6.1.4.</li>
	</ol>
	</li>
	<li>Lung function measurement
	<ol>
		<li>On day 21 after BLM or saline, anesthetize mice with an intraperitoneal (IP) injection of ketamine (160 mg/kg) and xylazine (32 mg/kg).</li>
		<li>After confirming depth of anesthesia by paw/toe pinch, perform a tracheostomy with an 18 G cannula (see Table of Materials).</li>
		<li>Connect mice to the ventilator and measure respiratory mechanics as previously described4.</li>
	</ol>
	</li>
	<li>Lung tissue collection and processing
	<ol>
		<li>Following measurement of pulmonary mechanics, euthanize the anesthetized mice by cardiac puncture.</li>
		<li>Open the chest wall and induce bilateral pneumothoraces.</li>
		<li>Inflate lungs with 1% low melt agarose (40 &#176;C)5 in PBS at a consistent pressure (42 cm H2O).</li>
		<li>Cut four to five pieces of the lung along the long axis transversely, fix in 10% formalin, and embed in paraffin.</li>
		<li>Cut 5 &#181;m sections and stain with Masson&#8217;s trichrome to visualize collagen deposition.</li>
	</ol>
	</li>
</ol>
8 Post-procedural care
<ol>
	<li>At the end of survival procedures, place the animal on a warming blanket until it regains consciousness. Recovery is typically complete within 2 min.</li>
</ol>

<ol>
	<li>MacDonald, K. D., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+simple+method+of+mouse+lung+intubation.">An improved simple method of mouse lung intubation.</a> Journal of Applied Physiology. 106 (3), 984-987 (2009).</li><li>Thomas, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotracheal+intubation+in+mice+via+direct+laryngoscopy+using+an+otoscope.">Endotracheal intubation in mice via direct laryngoscopy using an otoscope.</a> Journal of Visualized Experiments. (86), (2014).</li><li>Vandivort, T. C., An, D., Parks, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+Method+for+Rapid+Intubation+of+the+Trachea+in+Mice.">An Improved Method for Rapid Intubation of the Trachea in Mice.</a> Journal of Visualized Experiments. (108), 53771 (2016).</li><li>McGovern, T. K., Robichaud, A., Fereydoonzad, L., Schuessler, T. F., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+respiratory+system+mechanics+in+mice+using+the+forced+oscillation+technique.">Evaluation of respiratory system mechanics in mice using the forced oscillation technique.</a> Journal of Visualized Experiments. (75), e50172 (2013).</li><li>Halbower, A. C., Mason, R. J., Abman, S. H., Tuder, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+infiltration+improves+morphology+of+cryostat+sections+of+lung.">Agarose infiltration improves morphology of cryostat sections of lung.</a> Laboratory Investigation. 71 (1), 149-153 (1994).</li><li>Thrall, R. S., McCormick, J. R., Jack, R. M., McReynolds, R. A., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleomycin-induced+pulmonary+fibrosis+in+the+rat:+inhibition+by+indomethacin.">Bleomycin-induced pulmonary fibrosis in the rat: inhibition by indomethacin.</a> American Journal of Pathology. 95 (1), 117-130 (1979).</li><li>Matute-Bello, G., Frevert, C. W., Martin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+acute+lung+injury.">Animal models of acute lung injury.</a> American Journal of Physiology Lung Cellular and Molecular Physiology. 295 (3), L379-L399 (2008).</li><li>Del Sorbo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+Administration+of+Small+Interfering+RNA+Targeting+Fas+Reduces+Lung+Ischemia-Reperfusion+Injury.">Intratracheal Administration of Small Interfering RNA Targeting Fas Reduces Lung Ischemia-Reperfusion Injury.</a> Criticial Care Medicine. 44 (8), e604-e613 (2016).</li><li>McLemore, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+intrapulmonary+model+for+orthotopic+propagation+of+human+lung+cancers+in+athymic+nude+mice.">Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice.</a> Cancer Research. 47 (19), 5132-5140 (1987).</li><li>Vertrees, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+human+to+murine+orthotopic+xenotransplanted+lung+cancer+model.">Development of a human to murine orthotopic xenotransplanted lung cancer model.</a> Journal of Investigative Surgery. 13 (6), 349-358 (2000).</li></ol>

The authors declare that they have no competing financial interests.

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, ...

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 previously1,2,3. However, this approach is nonselective, affecting both lungs in a nontargeted and often asymmetric fashion.&#160; 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:739px;" width="739">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">22 G shielded IV Catheter</td>
			<td style="width:139px;">BD</td>
			<td style="width:140px;">381423</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Bleomycin</td>
			<td style="width:139px;">Enzo life sciences</td>
			<td style="width:140px;">BML-AP302-0010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Compact Mini rodent anesthesia machine&#160;</td>
			<td style="width:139px;">DRE Veterinary</td>
			<td style="width:140px;">9280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Evans blue dye</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:140px;">E2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">FITC-dextran</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:140px;">FD150</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Isoflurane</td>
			<td style="width:139px;">Piramal Critical Care</td>
			<td style="width:140px;">NDC&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">LED-30W Fiber Optic Dual Gooseneck Lights Microscope Illuminator</td>
			<td style="width:139px;">AmScope</td>
			<td style="width:140px;">LED-30W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low temperature cautery with fine tip&#160;</td>
			<td style="width:139px;">Bovie</td>
			<td style="width:140px;">AA02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Precisionglide needle, 18G x 1&#34;</td>
			<td style="width:139px;">BD</td>
			<td style="width:140px;">305195</td>
			<td>Beveled tip, 12 mm in length&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Xylazine</td>
			<td style="width:139px;">AKORN</td>
			<td style="width:140px;">NDC 59399-110-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Zatamine</td>
			<td>VetOne</td>
			<td style="width:140px;">NDC 13985-702-10&#160;</td>
			<td>Ketamine</td>
		</tr>
	</tbody>
</table>

direct intrabronchial administration to improve the selective agent deposition within the mouse lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; 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.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; 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.

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...

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

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; 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.

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

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13.3K Views.  University of Colorado Denver. 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.

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

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

Design, Fabrication, and Administration of the Hand Active Sensation Test (HASTe)

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of outcome measures of upper extremity motor performance has grown. However, clinicians and researchers lack practical, quantitative measures of the hand&#8217;s natural role as a receptor of the environment. The Hand Active Sensation Test (HASTe), developed by Williams and colleagues in 2006, is a valid and reliable measure of haptic performance. Though not available commercially, the HASTe can be fabricated from inexpensive materials, and it has been used successfully to identify impairments in haptic touch in individuals with stroke. (Williams, 2006). This paper presents the methods of design and fabrication of the HASTe testing kit, as well as a visual screen to be used during administration, and instructions for the tests administration and scoring.

Design, Fabrication, and Administration of the Hand Active Sensation Test HASTe

The Hand Active Sensation Test (HASTe) is a valid and reliable measure of haptic performance, which has been used successfully to identify impaired haptic touch in individuals with stroke. The purpose of this paper is to describe the design, fabrication and administration of the HASTe.

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of ...

2-Methacryloyloxyethyl Phosphorylcholine Polymer Treatment of Complete Dentures to Inhibit Denture Plaque Deposition

Removable dentures made of poly (methyl methacrylate) (PMMA) are prone to bacterial adherence and dental plaque formation, which is called denture plaque. Denture plaque-associated infection is a source of serious dental and medical complications in the elderly. 2-Methacryloyloxyethyl phosphorylcholine (MPC) is a well-known biomedical material that exhibits marked antithrombogenicity and tissue compatibility because of its high resistance to protein adsorption and cell adhesion. Therefore, MPC polymer coatings are suggested to have the potential to inhibit plaque deposition on the surface of PMMA dentures. However, coating MPC polymer on the surface of a PMMA denture is a complex procedure that requires specialized equipment, which is regarded as a major barrier to its clinical application. 
Here, we introduce a new MPC polymer treatment procedure that uses poly (MPC-co-BMA-co-MPAz) (PMBPAz) to prevent denture plaque deposition on removable dentures. This procedure enables the MPC coating of PMMA denture surfaces in a simple and stable manner that is resistant to various chemical and mechanical stresses due to the MPC layer of PMBPAz that is covalently bound to the PMMA surface by ultraviolet light irradiation. In addition, the procedure does not require any specialized equipment and can be completed by clinicians within 2 min. We applied this procedure in a clinical setting and demonstrated its clinical utility and efficacy in inhibiting plaque deposition on removable dentures.

Removable poly(methyl methacrylate) (PMMA) dentures are prone to bacterial adherence and plaque formation. Denture plaque-associated infection is a source of serious dental and medical complications in the elderly. This paper introduces a novel protocol to treat PMMA dentures with 2-methacryloyloxyethyl phosphorylcholine polymer, poly(MPC-co-BMA-co-MPAz), to suppress plaque deposition on PMMA dentures.

Removable dentures made of poly (methyl methacrylate) (PMMA) are prone to bacterial adherence and dental plaque formation, which is called denture plaque. ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...