Pressure Fixation of Isolated Murine Pulmonary Valve: A Procedure to Isolate and Chemically Fix the Mouse Pulmonary Valve in Closed Conformation

Protocolo

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

1. Pulmonary valve excision

1. Autoclave the necessary tools needed for the mouse dissection. This includes fine scissors, micro forceps, micro vascular clamps, clamp applying forceps, microneedle holders, spring scissors, and retractors.
2. Acclimate all mice for a minimum of 2 weeks before the operation. Remove C57BL/6 mice, approximately 1 year of age, from their cages and weigh, then euthanize with a ketamine/xylazine cocktail (3:1 ketamine:xylazine, 0.01 mL per g) overdose.
3. Place the mouse in a dorsal recumbence position on a tray and secure its limbs with tape. Once secured, perform the thoracotomy.
   1. Expose the heart by removing any excess adipose tissue and fascia.
   2. Remove the right atrium and perfuse through the left ventricle with room temperature saline solution (0.9% NaCl). The perfusion should take approximately 20 mL over 30 s. This results in the exsanguination of the mouse.
4. Remove the entire heart by severing the superior vena cava, inferior vena cava, pulmonary artery, and aorta. Special care should be taken for the pulmonary artery. Cut approximately 2 mm above the ventriculo-arterial junction, as this will serve as the conduit for pressurization.
5. Remove the left and right ventricles to expose chambers to atmospheric pressure. Ensure that the structure of the pulmonary trunk should be unaffected by the removal of the ventricles.

2. Pressure fixation of pulmonary valve

1. Anastomose pressurization tubing with pulmonary artery, leaving approximately 1 mm distance between the sino-tubular junction and the end of the tubing to accommodate for large movements of the leaflets and pulmonary trunk.
2. Elevate the reservoir to an analogous physiological pressure and fill it with the saline solution. Test the flow-through system to ensure there are no blockages or air bubbles.
3. Attach a stopcock to the anastomosed pulmonary valve and ensure adequate flow through the tubing (i.e., no air bubbles) by switching the outflow tract. Once the flow is adequate, switch the outflow to the anastomosed pulmonary valve and ensure pressurization of the pulmonary trunk. This is identified by pulmonary trunk distention.
4. Once the pressurization of the pulmonary trunk is confirmed, gradually incorporate primary fixative solution (1.25% glutaraldehyde, 1.0% paraformaldehyde in 0.15 M cacodylate) until the saline solution is purged. This is done by removing a portion, approximately 25% of the reservoir capacity, of the saline solution and replacing it with the primary fixative.
   CAUTION: The fixatives used (paraformaldehyde and glutaraldehyde) are highly toxic and appropriate personal protective equipment (PPE) should be worn to ensure safety.
5. Place a fixative-soaked gauze over the tissue sample to prevent drying.
6. Perfuse the fixative for 3 h, refilling the reservoir as needed to maintain a constant pressure. Throughout fixation, it is not uncommon for the pulmonary valve to shrink because of the chemical fixation. If this is the case, constantly replenish the reservoir with primary fixative to maintain physiological pressure.
7. Store the heart valve in the fixative solution at 4 °C until use. Samples were stored for up to 1 week without any discernable difference.

Materiales

Name	Company	Catalog Number	Comments
25% glutaraldehyde (aq)	EMS	16210	Primary fixative component
0.9% sodium chloride injection	Hospira Inc.	NDC 0409-4888-10
1 mL syringe	BD	309659
10 mL syringe	BD	309604
22G needle	BD	305156
3-way stopcock	Smiths Medical ASD, Inc	MX5311L
4% paraformaldehyde (aq)	EMS	157-4-100	Primary fixative component
C57BL/6 mice	Jackson Laboratories	664	Approximately 1 yo
Clamp applying forcep	FST	00072-14
Dumont #5 forcep	FST	11251-20
Dumont #5/45 forceps	FST	11251-35
Dumont #7 fine forcep	FST	11274-20
Fine scissor	FST	14028-10
Saline solution (sterile 0.9% sodium chloride)	Hospira Inc	NDC 0409-0138-22
Surgical microscope	Leica	M80
Tish needle holder/forcep	Micrins	MI1540
Trimmer	Wahl	9854-500
Black polyamide monofilament suture, 10-0	AROSurgical instruments Corporation	TI38402
Black polyamide monofilament suture, 6-0	AROSurgical instruments Corporation	SN-1956
Ketamine hydrochloride injection	Hospira Inc	NDC 0409-2053
L-aspartic acid	Sigma-Aldrich	56-84-8	Staining component
Lead nitrate	EMS	17900	Staining component
Sodium cacodylate trihydrate	Sigma-Aldrich	6131-99-3	Buffer
Non-woven songes	McKesson Corp.	94442000
Micro-adson forcep	FST	11018-12
Millex-GP filter, 0.22 um, PES 33mm, non-sterile	EMD Millipore	SLGP033NS