Name: multiple intravenous bolus dosing and invasive hemodynamic assessment in a hypoxia-induced mouse pulmonary artery hypertension model
Uploaded: 2022-11-11T13:10:00
Description: Watch this Scientific Journal Video about Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model at JoVE.com

This work was supported, in part, by a Joint Biology Consortium Microgrant provided under NIH grant P30AR070253 (PYC), Cardiovascular Medical Research Education Fund (PYC), VasoRx, Inc. Fund (MS) and NIH grants HL135582 (MS), HL152197 (MS).

All animal procedures were performed under protocols approved by Yale University Institutional Animal Care and Use Committees.
1. Preparation of animals, tools, blood pressure measuring equipment, and hypoxia chamber
<ol>
	<li>Animal acclimation. 
	NOTE: Experimental animals used for this study were male, 8-week-old C57BL/6 mice weighing 25-27 g. Several factors should be considered when estimating the number of animals required for the experiment, including surgery-ass...

<ol>
	<li>McLaughlin, V. V., McGoon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+arterial+hypertension.">Pulmonary arterial hypertension.</a> Circulation. 114 (1), 1417-1431 (2006).</li><li>Hoeper, M. M., Humbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+haemodynamic+definition+of+pulmonary+hypertension:+evidence+prevails,+finally.">The new haemodynamic definition of pulmonary hypertension: evidence prevails, finally.</a> European Respiratory Journal. 53 (3), 1900038 (2019).</li><li>Chen, Y., 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=A+novel+rat+model+of+pulmonary+hypertension+induced+by+mono+treatment+with+SU5416.">A novel rat model of pulmonary hypertension induced by mono treatment with SU5416.</a> Hypertension Research. 43 (8), 754-764 (2020).</li><li>Xiong, M., 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=Mouse+model+of+experimental+pulmonary+hypertension:+Lung+angiogram+and+right+heart+catheterization.">Mouse model of experimental pulmonary hypertension: Lung angiogram and right heart catheterization.</a> Pulmonary Circulation. 11 (4), 20458940211041512 (2021).</li><li>Kmiotek, E. K., Baimel, C., Gill, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+intravenous+self+administration+in+a+mouse+model.">Methods for intravenous self administration in a mouse model.</a> Journal of Visualized Experiments. (70), e3739 (2012).</li><li>Potus, F., Martin, A. Y., Snetsinger, B., Archer, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biventricular+assessment+of+cardiac+function+and+pressure-volume+loops+by+closed-chest+catheterization+in+mice.">Biventricular assessment of cardiac function and pressure-volume loops by closed-chest catheterization in mice.</a> Journal of Visualized Experiments. (160), e61088 (2020).</li><li>Bueno-Beti, C., Hadri, L., Hajjar, R. J., Sassi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sugen+5416/hypoxia+mouse+model+of+pulmonary+arterial+hypertension.">The Sugen 5416/hypoxia mouse model of pulmonary arterial hypertension.</a> Experimental Models of Cardiovascular Diseases. 1816, 243-252 (2018).</li><li>Chen, P. Y., 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=FGF+regulates+TGF-beta+signaling+and+endothelial-to-mesenchymal+transition+via+control+of+let-7+miRNA+expression.">FGF regulates TGF-beta signaling and endothelial-to-mesenchymal transition via control of let-7 miRNA expression.</a> Cell Reports. 2 (6), 1684-1696 (2012).</li><li>Chen, P. Y., 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=Endothelial+TGF-beta+signalling+drives+vascular+inflammation+and+atherosclerosis.">Endothelial TGF-beta signalling drives vascular inflammation and atherosclerosis.</a> Nature Metabolism. 1 (9), 912-926 (2019).</li><li>Daugherty, A., Rateri, D., Hong, L., Balakrishnan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+blood+pressure+in+mice+using+volume+pressure+recording,+a+tail-cuff+method.">Measuring blood pressure in mice using volume pressure recording, a tail-cuff method.</a> Journal of Visualized Experiments. (27), e1291 (2009).</li><li>Alam, M. A., Parks, C., Mancarella, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+blood+pressure+measurement+in+freely+moving+mice+using+telemetry.">Long-term blood pressure measurement in freely moving mice using telemetry.</a> Journal of Visualized Experiments. (111), e53991 (2016).</li><li>Luo, F., 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=Invasive+hemodynamic+assessment+for+the+right+ventricular+system+and+hypoxia-induced+pulmonary+arterial+hypertension+in+mice.">Invasive hemodynamic assessment for the right ventricular system and hypoxia-induced pulmonary arterial hypertension in mice.</a> Journal of Visualized Experiments. (152), e60090 (2019).</li></ol>

Several pulmonary hypertension animal models have been established to mimic the elevated pulmonary vascular resistance events in human subjects. Among them, the mouse hypoxia-induced PAH model has been widely used for evaluating the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds to the mice. In comparison with other published intravenous (IV) injection and invasive hemodynamic assessment protocols, this method provides both visual illustration...

Pulmonary artery hypertension (PAH) is defined by a mean pulmonary artery systolic pressure greater than 20 mmHg at rest1,2. It is a progressive and fatal disease characterized by a sustained elevation in pulmonary arterial pressure, leading to right ventricle overload and ultimately death due to right ventricular failure1. Currently, there is no cure for PAH.
The use of animal models of pulmonary hypertension is important for testing the effectiveness of experimental PAH therapies. Among those models, the mouse hypoxia-induced PAH model has provided key insights into human PAH group 3 disease development3,4. Research using this model often requires the administration of compounds in mice to evaluate the novel compound&#39;s effectiveness and safety. Therefore, investigators need a detailed experimental procedure for compound dosing and hemodynamic measurements to ensure injection consistency and blood pressure measurement reproducibility from the beginning to the end.
Methods for intravenous (IV)&#160;injection and blood pressure measuring have been reported in the literature5,6. However, the methodology lacks visual illustration and detailed description. Here we illustrate the key steps for a successful IV bolus injection and accurate measurement and recording of systemic and right ventricle blood pressure. The procedures presented here are an important resource for investigators interested in the IV route of compound administration platform to develop a treatment for PAH.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-0 prolene suture pack</td>
			<td>Ethicon</td>
			<td>8698G</td>
			<td>for incision closure</td>
		</tr>
		<tr>
			<td>8-0 nylon suture pack</td>
			<td>AROSurgical Instruments</td>
			<td>T06A08N14-13</td>
			<td>for ligation</td>
		</tr>
		<tr>
			<td>Anesthesia induction chamber</td>
			<td>VETEQUIP</td>
			<td>#941444</td>
			<td>Holds the animal during anesthesia exposure</td>
		</tr>
		<tr>
			<td>Catheter Interface Cable PEC-4D</td>
			<td>Millar</td>
			<td></td>
			<td>for connecting Millar Mikro-Tip catheter to PCU-2000</td>
		</tr>
		<tr>
			<td>Charcoal canister filters</td>
			<td>VETEQUIP</td>
			<td>#931401</td>
			<td>&#160;to help remove&#160;waste anesthetic&#160;gases</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>McKesson</td>
			<td>24-106</td>
			<td>for applying pressure to the injection site to prevent bleeding</td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14059-11</td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Insulin syringe 28 G</td>
			<td>EXEL</td>
			<td>26027</td>
			<td>for jugular vein IV injection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>COVETRUS</td>
			<td>#029405</td>
			<td>for mouse anesthesia</td>
		</tr>
		<tr>
			<td>LabChart 8 Software</td>
			<td>ADInstruments</td>
			<td></td>
			<td>for data analysis</td>
		</tr>
		<tr>
			<td>Mikro-Tip Pressure Catheter SPR-1000 (1.0 F)</td>
			<td>Millar</td>
			<td></td>
			<td>for invasive blood pressure measurement</td>
		</tr>
		<tr>
			<td>Needle-25 G</td>
			<td>BD</td>
			<td>305124</td>
			<td>for making a samll hole in a vessel</td>
		</tr>
		<tr>
			<td>Oxygen controller ProOx Oxygen Sensor</td>
			<td>BioSpherix</td>
			<td>E702</td>
			<td>for oxygen concentration monitoring</td>
		</tr>
		<tr>
			<td>PCU-2000 Pressure Control Unit</td>
			<td>Millar</td>
			<td></td>
			<td>for connecting Millar Mikro-Tip catheter to PowerLab 4/35</td>
		</tr>
		<tr>
			<td>PowerLab 4/35</td>
			<td>ADInstruments</td>
			<td></td>
			<td>for Data Acquisition. 
			Investigator needs to connect the PowerLab 4/35 to a personal laptop containing LabChart 8 software for operation.</td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td>for statistics and scientific graphing</td>
		</tr>
		<tr>
			<td>Semisealable hypoxia chamber</td>
			<td>BioSpherix</td>
			<td></td>
			<td>an artificial environment that simulates high-altitude conditions for animals</td>
		</tr>
		<tr>
			<td>Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15021-15</td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Tweezer Style 4</td>
			<td>Electron Microscopy Sciences</td>
			<td>0302-4-PO</td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>VasoRx compound 7C1/let-7 miRNA</td>
			<td>VasoRx, Inc.</td>
			<td>Lot# B2-L-16Apr</td>
			<td>IV injection compound</td>
		</tr>
		<tr>
			<td>VIP 3000 Veterinary Vaporizer</td>
			<td>COLONIAL MEDICAL SUPPLY CO., INC.</td>
			<td></td>
			<td>for accurate anesthesia delivery</td>
		</tr>
	</tbody>
</table>

multiple intravenous bolus dosing and invasive hemodynamic assessment in a hypoxia-induced mouse pulmonary artery hypertension model

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

Anesthesia often reduces blood pressure. Therefore, a minimum dose of anesthesia was used to abolish the movements in response to a noxious stimulus. Successful right ventricular chamber access can be visualized as the hemodynamic waveform changes in different regions of venous systems (Figure 8).
In this study, mice were randomly assigned to the normoxic (21% O2) group (n = 10), hypoxia (10% O2) group (n = 10), or hypoxia + 7C1/let-7 treatme...

Watch this Scientific Journal Video about Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model at JoVE.com

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...

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