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

Our research scope is congenital heart disease and heart regeneration. We are trying to understand how volume or pressure overload affects the postnatal heart and lung development, including cardiomyopathy, proliferation, and maturation. We have developed various types of postnatal mouse or rat models of congenital heart disease, for example, ventricular overload, pressure overload, and reduced pulmonary blood flow.

These models are fascinating and would advance our understanding of pediatric congenital heart disease. For decades, the AVF model has been used to induce chronic heart VO in adult animals. However, little is known about the model in postnatal mice.

This study generated a VO postnatal mouse model by creating an abdominal AVF. The morphological and hemodynamic changes of the right ventricle are demonstrated. Begin by testing for pinch reflexes on the anesthetized mouse pup's tail to confirm the depth of anesthesia.

Next, disinfect the skin with three alternating rounds of Betadine and 70%ethanol using sterile cotton swabs. Cut the abdominal wall and peritoneum from the lower abdomen to the subxiphoid to fully expose the peritoneal cavity. Use cotton swabs to gently pull away the gastrointestinal tract and the bladder to visualize the vertical abdominal aorta, or AA, and the inferior vena cava, or IVC, under the retroperitoneum.

After rotating the operating table 90 degrees counterclockwise, adjust the microscope magnification to view the two horizontal vessels clearly. Using an 11-0 suture needle having a 0.7 millimeter diameter, puncture the fistula obliquely from the AA into the IVC. Verify the successful puncture by observing the swelling and mixing of the venous and arterial blood in the IVC.

Press the bleeding point with dry cotton swabs for 15 seconds to compress it rapidly. Immediately place the stomach, intestines and bladder back into the abdominal cavity to promote hemostatic compression. Make a blanket stitch on the abdominal wall and peritoneum using the 9-0 suture thread.

Then place the mouse on a warm pad. To confirm the fistula by ultrasound, begin by taping down the anesthetized mouse in a supine position on the warm platform. Then connect the mouse to an ECG monitor with pre-warmed ultrasound gel.

Use hair removal cream to depilate the chest and abdominal skin. Place a 24 megahertz transducer on the mid-abdominal line and rotate the transducer marker to the mouse head. Now, move the platform down to the left or right side of the mouse, and use B-mode and color doppler mode to visualize the long axis view of the vessels and blood signals.

Use the pulsed wave doppler mode to measure the blood flow velocity of the AA, IVC and fistula to confirm arteriovenous fistula patency. To confirm ventricular volume overload, move the tail end of the platform downward, then place the transducer on the chest, rotating the marker to the right shoulder of the mouse. Use the B-mode and the color doppler modes to visualize the modified parasternal long axis view of the pulmonary artery, or PA.Now, using the pulsed wave doppler mode, measure the blood flow signals in the PA, including the velocity time integral, PA valve diameter, pulmonary arterial acceleration, and right ventricular ejection time.

Use the mean of three consecutive measurements in each case. The postnatal VO mice showed an acceptable perioperative survival rate in the early postoperative period. The AVF patency rates were 70%at one and two weeks, 46.7%at one month, and 40%at two months postoperatively.

Both the PA valve diameter and the right ventricular stroke volume increased within two months. The PA velocity time integral was relatively higher in the VO group within two weeks postoperatively, but declined thereafter. The PA flow patterns changed with decreasing PA acceleration.

The right ventricular systolic pressure significantly increased with pulmonary regurgitation two months after surgery. Microscopic analysis showed a significantly enlarged right ventricle of the VO group after AVF, while histological staining showed a thickened RV free wall and enlarged RV cavity.