This work was supported by the National Science Foundation of China (no. 82200309) and the Innovation Project of Distinguished Medical Team in Ningbo (no. 2022020405)

All of the procedures presented here conformed to the principles outlined in the Declaration of Helsinki and were approved by the Animal Welfare and Human Studies Committee at Shanghai Children&#39;s Medical Center (SCMC-LAWEC-2023-003). C57BL/6 mice pups (P7, males, 3-4 g) were used for the present study. The animals were obtained from a commercial source (see Table of Materials). The mice pups and their nursing mothers (pups:mothers = 6:1 in a single cage) were kept under specific-pathogen-free laboratory conditions under a 12 h light and dark cycle at 22 &#177; 2 &#176;C with free access to water and a nutritional diet. The pups were randomized into two groups: a VO group and a sham-operated (sham) group.
1. Equipment and surgical tool preparation
NOTE: The commercial details of all the materials/equipment are listed in the Table of Materials.
<ol>
	<li>Ensure that the following types of equipment are ready and properly functioning: operating table (foam plastic panel), inhalational anesthesia machine, microscope with vertical illumination and a built-in camera, ultrasound device with a 24 MHz transducer, and thermostatic heating platform.</li>
	<li>Sterilize the surgical instruments (i.e., a micro-needle holder, fine-tip forceps, and round-handled Vannas spring scissors).</li>
	<li>Assemble the following consumables: 11-0 and 9-0 surgical suture needles (taper-point) with thread, tape strips, 5 mL syringe needles, 2-0 silk (surgical fixation), sterile cotton swabs, and ultrasound gel.</li>
	<li>Ensure the following reagents are present: Betadine, 70% ethanol, normal sterile saline, isoflurane, acetaminophen, ophthalmic ointment, and hair removal cream.</li>
</ol>
2. Surgical procedure
NOTE: The fistula surgery procedure was modified according to the previously described method11. Figure 1 shows a schematic diagram of the AVF operation in postnatal mice.
<ol>
	<li>Anesthesia and restraint
	<ol>
		<li>Place the mice pups into an anesthesia-induction box supplied with 2% isoflurane/oxygen for 2 min&#160;with the flow set to 1 L/min. Administer acetaminophen (0.1 ml PO of 80 mg/2.5 ml) using a TB syringe.</li>
		<li>Place the pups in a supine position on the operating table with nasal inhalation of 1.5% isoflurane with a 0.8 L/min flow to maintain anesthesia. Adjust the pup&#39;s position by tying the legs to the fixed syringe needles. Apply ophthalmic ointment to the pups&#39; eyes to prevent corneal desiccation.</li>
		<li>Pinch the anesthetized pup&#39;s tail to check its pain responsiveness; no obvious body movements indicate adequate anesthesia.</li>
	</ol>
	</li>
	<li>Fistula surgery
	<ol>
		<li>Disinfect the skin with three alternating scrubs of betadine and 70% ethanol, and then drape the surgical site. Cut the abdominal wall and peritoneum from the lower abdomen to the subxiphoid to fully expose the peritoneal cavity, taking care not to injure the abdominal organs. Drip normal sterile saline to moisten externalized organs.</li>
		<li>Gently pull the gastrointestinal tract and bladder away from the surgical site using cotton swabs to visualize the vertical abdominal aorta (AA) and inferior vena cava (IVC) under the retroperitoneum. Rotate the operating table 90&#176; counterclockwise and adjust the microscope magnification to visualize the two horizontal vessels clearly.</li>
		<li>Puncture the fistula from the AA into the IVC in an oblique direction 1 cm distal to the renal artery with an 11-0 suture needle (diameter = 0.07 mm). Verify successful fistula creation based on the swelling and mixing of venous and arterial blood in the IVC.</li>
		<li>Next, rapidly compress the bleeding point using an appropriate force applied with dry cotton swabs for 15 s. Replace the stomach, intestines, and bladder in the abdominal cavity as soon as possible to promote hemostatic compression.</li>
		<li>Suture the abdominal wall and peritoneum with a blanket stitch using a 9-0 suture thread. Discontinue anesthesia and provide the pups with 100% oxygen for 1 min.</li>
	</ol>
	</li>
	<li>Anesthesia resuscitation
	<ol>
		<li>Place the pups on a 38 &#176;C heating platform. After a complete awakening with vitality, return the pups to their nursing mother. The entire procedure lasts approximately 15 min. 
		&#8203;NOTE: In the present study, the sham group undergoes the same procedure except for the puncturing step.</li>
	</ol>
	</li>
</ol>
3. Ultrasound confirmation of fistula
NOTE: The general operation of the ultrasound device was identical to previous reports18,19.
<ol>
	<li>Confirmation of fistula by abdominal ultrasound
	<ol>
		<li>After induction of anesthesia (step 2.1.1), fix the mice with tape strips in the supine position on the warm platform. Then, connect the mice to an electrocardiogram (ECG) monitor with ultrasound gel. Maintain anesthesia using 1.5% isoflurane at a 0.8 L/min flow.</li>
		<li>Prepare the chest and abdominal skin using hair removal cream. After a few seconds, remove the cream with a warm water-soaked cotton tip. Place the transducer (24 MHz) on the midabdominal line and rotate the transducer marker to the head of the mice.</li>
		<li>Move the platform down to the left or right side of the mice and use B-mode and color Doppler mode to visualize the long-axis view of the vessels and blood signals18,19. Measure the blood flow velocity of the AA, IVC, and fistula to confirm AVF patency through pulsed wave Doppler mode. 
		NOTE: Successful fistula creation on the ultrasound was indicated by a turbulent flow signal visible between the AA and IVC (Figure 2C). Doppler blood flow velocity at the AVF site was significantly elevated compared with a relatively lower systolic velocity in the AA (Figure 2A,C). Moreover, in contrast with normal flow patterns in the IVC (Figure 2B), the pulsatile waveform of IVC blood flow proximal to the AVF also confirmed the successful creation of the fistula (Figure 2D).</li>
	</ol>
	</li>
	<li>Confirmation of VO by echocardiography
	<ol>
		<li>Move the tail-end part of the platform downward, place the transducer (24 MHz) on the chest, and rotate the transducer marker to the right shoulder of the mice. Visualize the modified parasternal long-axis view of the pulmonary artery (PA) using B-mode and color Doppler mode.</li>
		<li>Using pulsed wave Doppler mode, measure blood flow signals in the PA, including the velocity time integral (PA-VTI), diameter of the PA valve (PAD), pulmonary arterial acceleration time (PAT), and RV ejection time (RVET) (Figure 2E,F and Figure 3A,B).</li>
		<li>Measure the ultrasound parameters from the mean of three consecutive measurements. Calculate the RV stroke volume (RVSV, mL) and RV systolic pressure (RVSP, mmHg) using the following formulas20: 
		RVSV [mL] =1/4&#160;&#215;&#160;&#960;D2 &#215; VTIPA 
		RVSP [mmHg]&#160;= -83.7 &#215; PAT/RVET - index + 63.7 
		NOTE: Considering the ultrasound measurement bias, an increase of &#62;15% in the RVSV or VTIPA in VO mice compared to mice in the sham group was considered VO in the RV (Figure 2E,F).</li>
	</ol>
	</li>
</ol>

Modified Arteriovenous Fistula Surgery to Establish the RV Volume Overload Mouse Model

<ol>
	<li>Sanz, J., Sanchez-Quintana, D., Bossone, E., Bogaard, H. J., Naeije, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy,+function,+and+dysfunction+of+the+right+ventricle:+JACC+state-of-the-art+review.">Anatomy, function, and dysfunction of the right ventricle: JACC state-of-the-art review.</a> Journal of the American College of Cardiology. 73 (12), 1463-1482 (2019).</li><li>Alonso-Gonzalez, R., Dimopoulos, K., Ho, S., Oliver, J. M., Gatzoulis, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+right+heart+and+pulmonary+circulation+(IX).+The+right+heart+in+adults+with+congenital+heart+disease.">The right heart and pulmonary circulation (IX). The right heart in adults with congenital heart disease.</a> Revista Espa&#241;ola de Cardiolog&#237;a. 63 (9), 1070-1086 (2010).</li><li>Kovacs, A., Lakatos, B., Tokodi, M., Merkely, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+ventricular+mechanical+pattern+in+health+and+disease:+beyond+longitudinal+shortening.">Right ventricular mechanical pattern in health and disease: beyond longitudinal shortening.</a> Heart Failure Reviews. 24 (4), 511-520 (2019).</li><li>Ye, 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=Role+of+blood+oxygen+saturation+during+postnatal+human+cardiomyocyte+cell+cycle+activities.">Role of blood oxygen saturation during postnatal human cardiomyocyte cell cycle activities.</a> JACC: Basic to Translational Science. 5 (5), 447-460 (2020).</li><li>Ye, 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=Pressure+overload+greatly+promotes+neonatal+right+ventricular+cardiomyocyte+proliferation:+a+new+model+for+the+study+of+heart+regeneration.">Pressure overload greatly promotes neonatal right ventricular cardiomyocyte proliferation: a new model for the study of heart regeneration.</a> Journal of the American Heart Association. 9 (11), e015574 (2020).</li><li>Geraets, I. M. E., Glatz, J. F. C., Luiken, J., Nabben, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+role+of+membrane+substrate+transporters+on+the+metabolic+alterations+in+the+pressure-overloaded+heart.">Pivotal role of membrane substrate transporters on the metabolic alterations in the pressure-overloaded heart.</a> Cardiovascular Research. 115 (6), 1000-1012 (2019).</li><li>Burns, K. 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=New+mechanistic+and+therapeutic+targets+for+pediatric+heart+failure:+report+from+a+National+Heart,+Lung,+and+Blood+Institute+working+group.">New mechanistic and therapeutic targets for pediatric heart failure: report from a National Heart, Lung, and Blood Institute working group.</a> Circulation. 130 (1), 79-86 (2014).</li><li>Shaddy, R. E., 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=Carvedilol+for+children+and+adolescents+with+heart+failure:+a+randomized+controlled+trial.">Carvedilol for children and adolescents with heart failure: a randomized controlled trial.</a> Journal of the American Medical Association. 298 (10), 1171-1179 (2007).</li><li>Flaim, S. F., Minteer, W. J., Nellis, S. H., Clark, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+arteriovenous+shunt:+evaluation+of+a+model+for+heart+failure+in+rat.">Chronic arteriovenous shunt: evaluation of a model for heart failure in rat.</a> American Journal of Physiology. 236 (5), H698-H704 (1979).</li><li>Liu, Z., Hilbelink, D. R., Crockett, W. B., Gerdes, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+changes+in+hemodynamics+and+cardiac+myocyte+size+in+rats+with+aortocaval+fistulas.+1.+Developing+and+established+hypertrophy.">Regional changes in hemodynamics and cardiac myocyte size in rats with aortocaval fistulas. 1. Developing and established hypertrophy.</a> Circulation Research. 69 (1), 52-58 (1991).</li><li>Scheuermann-Freestone, 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=A+new+model+of+congestive+heart+failure+in+the+mouse+due+to+chronic+volume+overload.">A new model of congestive heart failure in the mouse due to chronic volume overload.</a> European Journal of Heart Failure. 3 (5), 535-543 (2001).</li><li>Du, Y., Plante, E., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+evaluation+of+cardiac+myocyte+hypertrophy+and+hyperplasia+in+male+rats+secondary+to+chronic+volume+overload.">Temporal evaluation of cardiac myocyte hypertrophy and hyperplasia in male rats secondary to chronic volume overload.</a> The American Journal of Pathology. 177 (3), 1155-1163 (2010).</li><li>Wu, J., Luo, X., Huang, Y., He, Y., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+right-ventricle+functional+characteristics+of+a+swine+carotid+artery-jugular+vein+shunt+model+of+pulmonary+arterial+hypertension:+An+18-month+experimental+study.">Hemodynamics and right-ventricle functional characteristics of a swine carotid artery-jugular vein shunt model of pulmonary arterial hypertension: An 18-month experimental study.</a> Experimental Biology and Medicine. 240 (10), 1362-1372 (2015).</li><li>Sun, S., 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=Postnatal+right+ventricular+developmental+track+changed+by+volume+overload.">Postnatal right ventricular developmental track changed by volume overload.</a> Journal of the American Heart Association. 10 (16), e020854 (2021).</li><li>Wang, S., 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=Metabolic+maturation+during+postnatal+right+ventricular+development+switches+to+heart-contraction+regulation+due+to+volume+overload.">Metabolic maturation during postnatal right ventricular development switches to heart-contraction regulation due to volume overload.</a> Journal of Cardiology. 79 (1), 110-120 (2022).</li><li>Zhou, C., 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=Downregulated+developmental+processes+in+the+postnatal+right+ventricle+under+the+influence+of+a+volume+overload.">Downregulated developmental processes in the postnatal right ventricle under the influence of a volume overload.</a> Cell Death Discovery. 7 (1), 208 (2021).</li><li>Cui, Q., 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=Volume+overload+initiates+an+immune+response+in+the+right+ventricle+at+the+neonatal+stage.">Volume overload initiates an immune response in the right ventricle at the neonatal stage.</a> Frontiers in Cardiovascular Medicine. 8, 772336 (2021).</li><li>Cheng, H. W., 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=Assessment+of+right+ventricular+structure+and+function+in+mouse+model+of+pulmonary+artery+constriction+by+transthoracic+echocardiography.">Assessment of right ventricular structure and function in mouse model of pulmonary artery constriction by transthoracic echocardiography.</a> Journal of Visualized Experiments. (84), e51041 (2014).</li><li>Sawada, H., 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=Ultrasound+imaging+of+the+thoracic+and+abdominal+aorta+in+mice+to+determine+aneurysm+dimensions.">Ultrasound imaging of the thoracic and abdominal aorta in mice to determine aneurysm dimensions.</a> Journal of Visualized Experiments. (145), e59013 (2019).</li><li>Thibault, H. B., 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=Noninvasive+assessment+of+murine+pulmonary+arterial+pressure:+validation+and+application+to+models+of+pulmonary+hypertension.">Noninvasive assessment of murine pulmonary arterial pressure: validation and application to models of pulmonary hypertension.</a> Circulation: Cardiovascular Imaging. 3 (2), 157-163 (2010).</li><li>Mori, 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+new+dynamic+three-dimensional+digital+color+doppler+method+for+quantification+of+pulmonary+regurgitation:+validation+study+in+an+animal+model.">A new dynamic three-dimensional digital color doppler method for quantification of pulmonary regurgitation: validation study in an animal model.</a> Journal of the American College of Cardiology. 40 (6), 1179-1185 (2002).</li><li>Bossers, G. P. 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=Volume+load-induced+right+ventricular+dysfunction+in+animal+models:+insights+in+a+translational+gap+in+congenital+heart+disease.">Volume load-induced right ventricular dysfunction in animal models: insights in a translational gap in congenital heart disease.</a> European Journal of Heart Failure. 20 (4), 808-812 (2018).</li><li>Yamamoto, K., 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=The+mouse+aortocaval+fistula+recapitulates+human+arteriovenous+fistula+maturation.">The mouse aortocaval fistula recapitulates human arteriovenous fistula maturation.</a> American Journal of Physiology. Heart and Circulatory Physiology. 305 (12), H1718-H1725 (2013).</li><li>Jouannic, J. 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=The+effect+of+a+systemic+arteriovenous+fistula+on+the+pulmonary+arterial+blood+pressure+in+the+fetal+sheep.">The effect of a systemic arteriovenous fistula on the pulmonary arterial blood pressure in the fetal sheep.</a> Prenatal Diagnosis. 22 (1), 48-51 (2002).</li><li>Jouannic, J. 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=Systemic+arteriovenous+fistula+leads+to+pulmonary+artery+remodeling+and+abnormal+vasoreactivity+in+the+fetal+lamb.">Systemic arteriovenous fistula leads to pulmonary artery remodeling and abnormal vasoreactivity in the fetal lamb.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 285 (3), L701-L709 (2003).</li><li>Patel, M. D., 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=Echocardiographic+assessment+of+right+ventricular+afterload+in+preterm+infants:+maturational+patterns+of+pulmonary+artery+acceleration+time+over+the+first+year+of+age+and+implications+for+pulmonary+hypertension.">Echocardiographic assessment of right ventricular afterload in preterm infants: maturational patterns of pulmonary artery acceleration time over the first year of age and implications for pulmonary hypertension.</a> Journal of the American Society of Echocardiography. 32 (7), 884-894 (2019).</li><li>Habash, S., 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=Normal+values+of+the+pulmonary+artery+acceleration+time+(PAAT)+and+the+right+ventricular+ejection+time+(RVET)+in+children+and+adolescents+and+the+impact+of+the+PAAT/RVET-index+in+the+assessment+of+pulmonary+hypertension.">Normal values of the pulmonary artery acceleration time (PAAT) and the right ventricular ejection time (RVET) in children and adolescents and the impact of the PAAT/RVET-index in the assessment of pulmonary hypertension.</a> The International Journal of Cardiovascular Imaging. 35 (2), 295-306 (2019).</li><li>Arkles, J. S., 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=Shape+of+the+right+ventricular+Doppler+envelope+predicts+hemodynamics+and+right+heart+function+in+pulmonary+hypertension.">Shape of the right ventricular Doppler envelope predicts hemodynamics and right heart function in pulmonary hypertension.</a> American Journal of Respiratory and Critical Care Medicine. 183 (2), 268-276 (2011).</li></ol>

There are no conflicts of interest to declare.

Previously, the classic RV VO model was created using valve regurgitation21; however, compared to AVF, open-heart valve surgery may require more sophisticated techniques and may be associated with significantly higher mortality, particularly in postnatal mice. As animal studies have shown that the same effect of VO has been achieved by AVF22, modified abdominal fistula surgery with less trauma was used in this study.
Certain factors were consider...

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease (CHD), which leads to pathological myocardial remodeling and a poor long-term prognosis1,2,3. An in-depth understanding of RV remodeling and related early targeted interventions is essential for a good outcome in children with CHD. There are several differences in the molecular structures, physiological functions, and responses to stimuli in the hearts of adults and children1,4,5,6. For example, under the influence of pressure overload, cardiomyocyte proliferation is the main response in neonatal hearts, whereas fibrosis occurs in adult hearts5,6. In addition, many effective drugs in treating heart failure in adults have no therapeutic effect on heart failure in children, and may even cause further damage7,8. Therefore, conclusions drawn from adult animals cannot be directly applied to young animals.
The arteriovenous fistula (AVF) model has been used to induce chronic heart VO and corresponding cardiac dysfunction for decades in adult animals of different species9,10,11,12,13. However, little is known about the model in postnatal mice. In our previous studies, a VO postnatal mouse model was successfully generated by the creation of an abdominal AVF. The changed RV developmental track in the postnatal heart was also demonstrated14,15,16,17.
To explore the underlying modified surgical process and characteristics of the present model, a detailed protocol is presented; the model is evaluated for 3 months in this study.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70% Ethanol</td>
			<td>Tiandz,Chia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACETAMINOPHEN Oral Solution</td>
			<td>VistaPharm, Inc. Largo, FL 33771, USA</td>
			<td>NDC 66689-054-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>RWD Life Science,China</td>
			<td>R550IP</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia mask</td>
			<td>RWD Life Science,China</td>
			<td>68680</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Xipu&#8217;er-bikai Experimental Animal Co., Ltd (Shanghai, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Veet, France</td>
			<td>VT-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin Kit&#160;</td>
			<td>Beyotime biotech&#160;</td>
			<td>C0105M&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science,China</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>&#160;Yuyan Instruments, China</td>
			<td>SM-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical suture needles</td>
			<td>NINGBO MEDICAL NEEDLE CO.,LTD, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic heating platform</td>
			<td>Qingdao Juchuang Environmental Protection Group Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound device</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>Vevo 2100</td>
			<td>Image modes includes B-Mode, Color Doppler Mode and Pulsed Wave Doppler Mode</td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Parker Laboratories,United States</td>
			<td>REF 01-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound transducer</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>MS 400</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment and confirmation of a postnatal right ventricular volume overload mouse model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

Survival rate and AVF patency within 3 months 
A total of 30 (75%) mice in the VO group and 19 (95%) mice in the sham group survived the AVF surgery (Figure 4A). In the VO group, eight mice died within 1 day after surgery due to excessive bleeding (n = 5) or cannibalization (n = 3), whereas two mice died of unknown causes at 1 month.
Of the surviving VO mice (n = 30), ultrasound confirmed the successful establishment of fistulas in 21 mice po...

Watch this Scientific Journal Video about Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model at JoVE.com

Author Spotlight: Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV ...

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970 Views.  Shanghai Jiaotong University. This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.

Video: Author Spotlight: Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model  

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...

Chronic Ovine Model of Right Ventricular Failure and Functional Tricuspid Regurgitation

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to suboptimal clinical results. We set out to establish a chronic ovine model of FTR and right heart failure to investigate the mechanisms of FTR. Twenty adult male sheep (6-12 months old, 62 &#177; 7 kg) underwent a left thoracotomy and baseline echocardiography. A pulmonary artery band (PAB) was placed and cinched around the main pulmonary artery (PA) to at least double the systolic pulmonary artery pressure (SPAP), inducing right ventricular (RV) pressure overload and signs of RV dilatation. PAB acutely increased the SPAP from 21 &#177; 2 mmHg to 62 &#177; 2 mmHg. The animals were followed for 8 weeks, symptoms of heart failure were treated with diuretics, and surveillance echocardiography was used to assess for pleural and abdominal fluid collection. Three animals died during the follow-up period due to stroke, hemorrhage, and acute heart failure. After 2 months, a median sternotomy and epicardial echocardiography were performed. Of the surviving 17 animals, 3 developed mild tricuspid regurgitation, 3 developed moderate tricuspid regurgitation, and 11 developed severe tricuspid regurgitation. Eight weeks of pulmonary artery banding resulted in a stable chronic ovine model of right ventricular dysfunction and significant FTR. This large animal platform can be used to further investigate the structural and molecular basis of RV failure and functional tricuspid regurgitation.

Right ventricular failure and functional tricuspid regurgitation are associated with left-sided heart disease and pulmonary hypertension, which contribute significantly to morbidity and mortality in patients. Establishing a chronic ovine model to study right ventricular failure and functional tricuspid regurgitation will help in understanding their mechanisms, progression, and possible treatments.

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to ...

Investigating Physiological Responses to Acute Volume Overload in Porcine Models

Adult and Pediatric Porcine Model of Acute Volume Overload

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, endotracheal intubation, and mechanical ventilation. A central venous catheter and an arterial catheter are placed via surgical cutdown in the external jugular vein and carotid artery, respectively. A pulmonary artery catheter is placed through an introducer sheath of the central venous catheter. PlasmaLyte crystalloid solution is then administered at a rate of 100 mL/min in adult pigs and at 20 mL/kg boluses over 10 min in piglets. Hypervolemia is achieved either at 15% decrease in cardiac output or at 5 L in adult pigs and at 500 mL in piglets. Hemodynamic data, such as heart rate, respiratory rate, end-tidal carbon dioxide, fraction of oxygen-saturated hemoglobin, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, partial arterial oxygen pressure, lactate, pH, base excess, and pulmonary artery fraction of oxygen-saturated hemoglobin, are monitored during experimentation. Preliminary data observed with this model has demonstrated statistically significant changes and strong linear regressions between central hemodynamic parameters and acute volume overload in adult pigs. Only pulmonary capillary wedge pressure demonstrated both a linear regression and a statistical significance to acute volume overload in piglets. These models can aid scientists in the discovery of age-appropriate therapeutic and monitoring strategies to understand and prevent acute volume overload.

Author Spotlight: Exploring Venous Waveforms in Porcine Models to Tackle Volume Overload in Medicine

The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, ...

Creating a Murine Model of RV Hypertrophy and Failure by Pulmonary Trunk Banding

A Murine Model of Pressure Overload-Induced Right Ventricular Hypertrophy and Failure by Pulmonary Trunk Banding

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary diseases. The pathogenesis of RV failure is complex and remains inadequately understood. To identify new therapeutic strategies for the treatment of RV failure, robust and reproducible animal models are essential. Models of pulmonary trunk banding (PTB) have gained popularity, as RV function can be assessed independently of changes in the pulmonary vasculature.
In this paper, we present a murine model of RV pressure overload induced by PTB in 5-week-old mice. The model can be used to induce different degrees of RV pathology, ranging from mild RV hypertrophy to decompensated RV failure. Detailed protocols for intubation, PTB surgery, and phenotyping by echocardiography are included in the paper. Furthermore, instructions for customizing instruments for intubation and PTB surgery are given, enabling fast and inexpensive reproduction of the PTB model.
Titanium ligating clips were used to constrict the pulmonary trunk, ensuring a highly reproducible and operator-independent degree of pulmonary trunk constriction. The severity of PTB was graded by using different inner ligating clip diameters (mild: 450 &#181;m and severe: 250 &#181;m). This resulted in RV pathology ranging from hypertrophy with preserved RV function to decompensated RV failure with reduced cardiac output and extracardiac manifestations. RV function was assessed by echocardiography at 1 week and 3 weeks after surgery. Examples of echocardiographic images and results are presented here. Furthermore, results from right heart catheterization and histological analyses of cardiac tissue are shown.

Author Spotlight: Investigating the Underlying Mechanisms of Right Ventricular Failure in Pulmonary Hypertension

We describe a murine model of right ventricular pressure overload-induced by pulmonary trunk banding. Detailed protocols for intubation, surgery, and phenotyping by echocardiography are included in the paper. Custom-made instruments are used for intubation and surgery, allowing for fast and inexpensive reproduction of the model.

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary ...