Sinoatrial Node Isolation from Mouse Model: A Procedure to Harvest SA Node from Murine Heart

Protocolo

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

1. Dissecting the sinoatrial node (SAN)

1. Prepare heparinized Complete Tyrode's solution for SAN dissection.
   1. Add 400 µL of heparin (1,000 USP/mL) to 40 mL of Complete Tyrode's solution and warm in a 37 °C water bath.
2. Inject the mouse intraperitoneally with 200-300 μL of heparin (1000 USP/mL) and allow the animal to sit for 10 min.
3. Euthanize the heparinized mouse by isoflurane overdose.
   1. Place the mouse in a small glass chamber that contains isoflurane vapors generated by adding 200-300 μL of liquid isoflurane to a filter paper inside a perforated plastic tube.
      NOTE: Because isoflurane can cause skin irritation and can also be absorbed through the skin, the liquid should not contact the mouse directly. Therefore, the isoflurane-soaked wipe is placed in a perforated tube for administration.
   2. Verify death by cessation of movement and breathing effort and by the absence of a toe pinch reflex. Death usually takes about 1-2 min following placement into the chamber.
      NOTE: Death is usually accompanied by urination.
4. Place the mouse in a supine position on a dissection board with paws outstretched and fix the paws to the board using 1 inch long, 23-gauge syringe needles. Then remove the fur in the vicinity of the bottom of the rib cage by using surgical scissors and cutting the fur at the roots.
   NOTE: For a dissection board, polystyrene cooler lids can be used.
5. While holding the skin with a hemostat, use surgical scissors to make a transverse incision in the skin just beneath the bottom of the rib cage from about the left costal arch to the right costal arch (Figure 1A).
6. Cut open the peritoneum with surgical scissors and carefully separate the liver from the diaphragm, being careful not to nick the liver, which will cause excessive bleeding (Figure 1B). Incise the diaphragm along the thorax to expose the thoracic cavity (Figure 1C-D).
7. Using surgical scissors, cut the lateral walls of the rib cage from the edges of the costal arches up to the clavicles to expose the heart, being careful to avoid damaging the heart (Figure 1D). Then use a 23-gauge syringe needle to pin the rib cage over the shoulder, holding it in place and out of the way of the surgical field.
8. Use a transfer pipette to drip warm (37 °C) heparinized Complete Tyrode's solution onto the heart to keep it moist.
   NOTE: Do not allow the heart to dry out.
9. Remove the lungs by holding them with extra fine Graefe forceps and severing the trachea with surgical scissors (Figure 1E).
10. Hold the apex of the heart with extra fine Graefe forceps and remove it by cutting the aorta and venae cavae with surgical scissors. Transfer the heart to a Petri dish containing cured silicone elastomer (Figure 2A) and use a transfer pipette to bathe the heart with 2-3 mL of warm (37 °C) heparinized Complete Tyrode's solution.
    NOTE: Be careful not to damage the delicate posterior wall of the right atria, which contains the SAN, and the connected right atrial veins. Bathing the heart with Complete Tyrode's solution keeps the heart from drying out but do not fully submerge the heart in solution as it will impair visibility during dissection.
11. Orient the heart with the right atrium on the experimenter's right and the left atrium on the experimenter's left.
    NOTE: Dissection of the SAN tissue should be done quickly in order to prevent ischemia-related injury.
12. Attach the apex of the heart to the dish with a dissection pin. Then, while holding the inferior vena cava with Dumont #2 laminectomy forceps, insert a 22 G syringe needle through the inferior and superior vena cava to locate their position in the right atrium, which also identifies the approximate position of the SAN (located in the patch of tissue between the inferior and superior vena cava (Figure 2B).
13. Using small dissection pins, pin the left and right atrial appendages to the dish.
    1. While holding the left atrial appendage with Dumont #2 laminectomy forceps, put a dissection pin through the left atrial appendage to hold it in place.
    2. While holding the right atrial appendage with Dumont #55 forceps, put a dissection pin through the right atrial appendage to hold it in place.
       NOTE: The same type of forceps can be used to hold the left and right atrial appendages if desired.
14. Remove the syringe needle that spans the venae cavae.
15. To release blood from the heart, use Castroviejo scissors to remove the apex of the heart (i.e., the bottom half) by making a transverse incision across the ventricles (Figure 2C). Then, wash the heart by adding warm (37 °C) heparinized Complete Tyrode's solution with a transfer pipette.
16. Use Castroviejo scissors to cut along the atrioventricular septum keeping the incision closer to the ventricle than the atria. Continue cutting along the atrioventricular septum until the atria are separated from the ventricles.
17. Cut along the interatrial septum to remove the left atrium.
18. Place dissection pins in the periphery of the right atrium to make it lay flat (Figure 2D). Remove any remaining fat, vessels, or tissue from the atrium using the Castroviejo scissors.
19. Locate the SAN in the right atrium, which in this orientation is approximately bordered by the superior vena cava (on the top), inferior vena cava (on the bottom), and cristae terminalis (on the left) (Figure 2D).
    ​NOTE: The crista terminalis appears as a dark muscular ridge between the right atrial appendage and the SAN. Often the SAN artery can also be seen coursing through the SAN (Figure 2D).

Resultados

Figure 1: Removal of the heart. (A) Transverse incision in the skin just beneath the bottom of the rib cage from about the left costal arch to the right costal arch. (B) Peritoneal incision. (C,D) Incision of the diaphragm along the thorax to expose the thoracic cavity. (E) Removal of the heart following ...

Materiais

Name	Company	Catalog Number	Comments
22 gauge syringe needle	Fisher Scientific	14-826-5A	Used for dissection
23 gauge syringe needle	Fisher Scientific	14-826-6C	Used for dissection
60mm Petri Dishes	Genesee Scientific	32-105G
Castroviejo Scissors, 4"	Fine Science Tools	15024-10
Dissection Microscope	Jenco
Dissecting Pins	Fine Science Tools	26002-20
Dumont #2 Laminectomy Forceps	Fine Science Tools	11223-20
Dumont #55 Forceps	Fine Science Tools	11295-51
Extra Fine Graefe Forceps	Fine Science Tools	11152-10
Hemostat	Fine Science Tools	13013-14
Heparin	Aurobindo Pharma Limited IDA, Pashamylaram	NDC 63739-953-25
Sylgruard Elastomer Kit	Dow Corning	184 SIL ELAST KIT 0.5KG
Surgical Scissors	Fine Science Tools	14074-09
Transfer Pipets (3mL graduated)	Samco Scientific	225
Isoflurane	Patterson Veterinary	07-893-1389