Overview
In this video, we demonstrate an experimental protocol for minimally invasive transverse aortic constriction in mice to examine pressure overload-induced heart failure.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Operation Site Preparation
- Disinfect the operation site with 75% isopropyl alcohol. Adjust the heating pad temperature to 37 °C to avoid changes in heart and respiratory rates of the subject animal.
- Use surgical instruments sterilized in a gravity displacement sterilizer at 121 °C for 30 min or by other appropriate methods.
- Take 2–4 30-gauge needles and curve them with a needle holder, as seen in Figure 1, to make a tool for ligation and tissue separation and for use as a retractor. Mount the now-curved needle on a cotton applicator for use in surgery.
- Blunt a 27-gauge needle and curve it 90º with the needle holder. Smooth the tip by rubbing a hard surface to make a 0.4 mm spacer in ligation step.
2. Animal Preparation
- Anesthetize a 2-month-old, male, 22–25 g C57BL/6N mouse with an appropriate ketamine and xylazine mixture in relation to body weight: 100 mg/kg + 10 mg/kg i.p. Confirm the anesthetization with the negative tail pinch reflex.
- Remove the hair on the anterior neck and chest with a 2 to 3 min application of depilatory cream. Remove the cream with warm water and a cotton applicator.
- Apply ophthalmic ointment or gel to the eyes of the animal to prevent the drying out of the cornea.
- Place the animal on a heating pad in the supine position and conduct intra-operative monitoring, being sure to keep appropriate documentation at every 15 min interval.
- Use surgical paper tape to secure the limbs to the heating pad in order to keep the animal in the correct position during the surgery.
- Disinfect the skin alternately with alcohol and povidone-iodine solution three times.
- Use a sterile drape with an exposed operation field to prevent contamination. Use new sterile gloves for each mouse and as necessary.
3. Ligation Procedure
- Open the skin of the mouse at the midline position of the neck and chest with a scalpel.
- Pull the thyroid gland towards the head by gently separating connective tissues with blunt scissors.
- Separate the muscle layer on the trachea at the midline towards both sides with the curved needles.
- Using the blunt scissors, cut the sternum to the second rib (approximately 5 mm). Open the cut with a retractor or curved forceps.
- Separate the thymus lobes from one another and the lower chest wall by separating the connective tissue with the curved needle; the transverse aortic arch and two carotid arteries will be clearly visible at this point.
- Place the curved needle under the arch and perforate between the vessel wall and connective tissue on the other side by pushing the curved head out.
- Using the curved needle, pull the 6-0 monofilament suture under the aortic arch.
- Place the spacer in the loop and fix the suture in place with a double-knot. Remove the spacer gently.
- Confirm a successful constriction with the knot position and cut the ends of the suture.
- Close the chest wall using 6/0 silk suture with a simple interrupted suture pattern. Close the skin with a 6/0 monofilament suture in a continuous suture pattern.
4. Post-operative Care
- Apply povidone-iodine solution to the suture site and place the animal in a pre-warmed cage for recovery and post-operative monitoring.
- Inject buprenorphine (0.05–0.1 mg/kg s.c. every 12 h) after the animal has regained consciousness to relieve pain and provide the mouse with appropriate soft food.
- Return the animal to a 12 h light/dark cycle room after full recovery.
Subscription Required. Please recommend JoVE to your librarian.
Representative Results
Figure 1: Process of forming the desired needle hooks by bending a 1-in, 30-gauge needle tip. (A) The original 1-in, 30-gauge needle tip. (B) Grasp the needle tip with the needle holder and bend approximately halfway along its length. Bend the needle into a loop, using the needle holder and the hands, as necessary, to form the loop pictured in the upper-right of the figure. (C) Cover the needle with a polyurethane tube in order to hold the bent segment in place. (D) Using the needle holder, bend the tip of the loop into the desired shape. In this case, a needle tip with two 45–60° bends has been shown for illustrative purposes.
Subscription Required. Please recommend JoVE to your librarian.
Materials
Name | Company | Catalog Number | Comments |
1 inch 30-gauge Needles | BD, Franklin Lakes, NJ 07417, USA | 305128 | Curved as in the procedure and Figure 1 |
27-gauge Needle | BD, Franklin Lakes, NJ 07417, USA | 301629 | Make blunt and smooth for spacer |
6/0 Dafilon | B. Braun, 34212 Melsungen, Hessen, Germany | C0933066 | |
6/0 Silkam | B. Braun, 34212 Melsungen, Hessen, Germany | C0762067 | |
Sterile gloves | A.R. Medicom, Inc (Asia), Hong Kong | ||
Cotton Applicator | Mannings, Quarry Bay, Hong Kong | Local Shopping Center | |
Depilatory Cream | Veet (Hong Kong), Kwun Tong, Kowloon, Hong Kong | Local Shopping Center | |
Lexer-Baby Scissor | FST, British Columbia V7H 0A6, Canada | 14078-10 | |
Curved Iris Forcep | FST, British Columbia V7H 0A6, Canada | 11065-07 | |
Micro Olsen-Hegar Needle Holder | WPI, Sarasota, FL 34240, USA | 501989 | |
Stereo Microscope | WPI, Sarasota, FL 34240, USA | PZMIII-BS | |
GenTeal Eye Gel | Novartis, East Hanover, NJ 07936, USA | Local Pharmacy | |
Heating Pad | Kent Scientific Corporation, Torrington, CT 06790, USA | DCT-20 | |
Surgical tape | Laboratory Animal Unit | ||
Ketamine and Xylazine | Laboratory Animal Unit | ||
Betadine | Laboratory Animal Unit | ||
Buprenorphine | Laboratory Animal Unit |