Antegrade Perfusion Model: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

Overview

This video describes an antegrade perfusion technique of an isolated mouse heart during which a perfusate is injected through the left ventricle, and eventually, it perfuses the entire myocardium. This method can be used to isolate cardiomyocytes from murine models and generate decellularized hearts for further downstream applications.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Antegrade perfusion of the mouse heart

NOTE: The plastic transfer pipette used for sucking the heart should be soft and not be sharply tapered towards the tip. Choose a small vascular clamp with serration. The recommended instruments are listed in the Table of Materials.

1. Excision of the mouse heart and clamping the aorta
   NOTE: The adult mice (>8 weeks old) should be euthanized by an overdose of sodium pentobarbital (>300 mg/kg, intraperitoneal [i.p.] injection) with heparin (8000 unit/kg).
   1. Excise the mouse heart quickly by sucking.
      1. Open the thoracic cavity quickly to expose the heart. Cut the plastic transfer pipette, the tip of which is approximately the same size as, or slightly smaller than, the exposed heart (usually at a site approximately 1 cm from the 0.5-mL mark towards the tip, but it depends on the heart size).
      2. Suck the heart into the pipette, raise the pipette to create enough space to insert scissors, and excise the heart with curved scissors from the dorsal side, avoiding damaging the atria.
      3. Immediately transfer the excised heart to the 30-mL glass beaker containing ice-chilled CIB-EGTA to stop the contraction. This procedure usually takes <1 min.
   2. Cleaning around the aorta
      1. Transfer the heart to a 35-mm culture dish filled with ice-chilled CIB-EGTA and remove the lung and other visible tissues, and then transfer the roughly cleaned heart to the heart stand filled with chilled CIB-EGTA and place it with the apex down.
      2. Under the stereoscopic microscope, remove the fat and connective tissues to clean around the aorta. If the length of the cut aorta is too long including the brachiocephalic artery, the left common carotid artery, or the left subclavian artery, cut off the aorta just under the brachiocephalic artery to shorten it in order to proceed to the next step. This procedure usually takes approximately 4 min
   3. Clamping the aorta and placing the clamped heart on the perfusion plate
      1. Insert the injection needle and start perfusion to discharge blood
         1. Under the microscope, place the heart in the heart stand. The operator should face the anterior surface of the heart, pick up the end of the aorta with tweezers, and clamp the aorta near the atria with a small vascular clamp while gently pushing down on the aorta a bit.
         2. Place the clamped heart on the perfusion plate with the anterior side up, and then cover it with a few drops of CIB-EGTA to keep it from drying out. This procedure usually takes <20 s.
      2. Antegrade perfusion of the heart
         NOTE: First, perfuse the heart with CIB-EGTA to discharge blood and prevent clotting.
         1. Insert the injection needle and start perfusion to discharge blood
            1. Set the 20-mL syringe filled with prewarmed CIB-EGTA connected to the flexible extension tube and a marked injection needle on the infusion pump. Start the pump at a slow rate of 0.5 mL/min to carefully fill the needle and tube with CIB-EGTA and be sure to prevent any air from entering the tube.
            2. Place the injection needle on the perfusion plate with the shorter side of the diagonal shape in front. Slide the needle towards the apex of the heart until it is touching it, and then carefully insert the needle near the apex of the left ventricle into the ventricular chamber without twisting. Do not detach the needle from the plate while performing insertion.
            3. Watch the red mark to estimate the depth of the needle insertion. When the needle insertion is completed, blood flowing from the coronary artery should start to be discharged.
            4. Fix the injection needle on the plate with tape, and increase the pump speed to 1 mL/min. This procedure usually takes approximately 30 s. If the heart is perfused successfully, the flow of the CIB-EGTA in the capillary just under the epicardium can be seen under the microscope.

Materials

Name	Company	Catalog Number	Comments
Extension tube	Top, Japan	X1-50	Connect with syringe and injection needle for antegrade perfusion.
Heater mat	Natsume Seisakusho, Japan	KN-475-3-40	Equipment to warm the perfusion plate.
Infusion pump	TERUMO, Japan	TE-311	Infusion syringe pump for antegrade perfusion.
Injection needle (27 gauge)	TERUMO, Japan	NN-2719S	Needle for insertion into the left ventricle.
Insulin (from bovine pancrease)	Sigma-Aldrich, USA	I5500	Dissolve in 0.1 M HCl.
Plastic syringe (20 mL)	TERUMO, Japan	SS-20ES	Use for infusion of CIB-EGTA.
Vascular clamp	Karl Hammacher GmbH, Germany	HSE 004-35	Small straight vascular clamp used for clamping aorta.
All other reagents	Nacalai Tesque, Japan