Minimally Invasive Transverse Aortic Constriction in Mice

Summary

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

Abstract

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Introduction

The overall purpose of this article is to demonstrate how transverse aortic constriction (TAC) in mice can be modified to produce a simpler and more expedient surgery. TAC was first introduced in 1991¹ and enabled molecular biologists to study the cellular and molecular pathways that lead to hypertrophy in the left ventricle and to heart failure, particularly in small animals with manipulated genomes^2,3,4,5,6. Despite its efficacy at producing the desired pressure overload, the technique is ultimately limited by the difficulty in performing the procedure and by its low survival rate. A surgeon conducting TAC must be able to perform endotracheal intubation, thoracotomy, and use artificial ventilation in order to prevent lung collapse¹.

Oftentimes, a simple and less invasive method is desired. As a consequence, minimally invasive transverse aortic constriction (MTAC) was devised³. Though the outcome (specifically, left ventricular hypertrophy) is the same, the procedure involves significantly fewer apparatuses and less damage to the animal. MTAC proves to be a desirable method that has a greater survival rate, above 90%, and has an operation time of only 15 to 25 min. The infrequent cases of death after successful surgery can be attributable to too-tight constriction of the aortic arch, resulting in tremendous left ventricular overload and fatal reduction of blood supply to critical organs such as the kidneys.

After the procedure is completed, echocardiography can be used to evaluate the success of banding and the cardiac morphology and function. A high-frequency ultrasound system can be utilized to measure the diameter and flow changes in the left and right carotid arteries, to visualize the aortic arch constriction for peak flow velocity measurements, and to determine the left ventricle wall thickness and functional parameters^7,8. After euthanasia, the organs can also be collected for histopathological studies with hematoxylin and eosin staining and Picrosirius collagen staining, the latter being particularly useful for the observation of fibrosis.

Protocol

The protocol follows the guidelines of the Committee on the Use of Live Animals in Teaching and Research (CULATR) at the University of Hong Kong.

1. Operation Site Preparation

1. 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.
2. Use surgical instruments sterilized in a gravity displacement sterilizer at 121 °C for 30 min or by other appropriate methods.
3. 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.
4. 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

1. 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.
2. 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.
3. Apply ophthalmic ointment or gel to the eyes of the animal to prevent the drying out of the cornea.
4. 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.
5. 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.
6. Disinfect the skin alternately with alcohol and povidone-iodine solution three times.
7. 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

1. Open the skin of the mouse at the midline position of the neck and chest with a scalpel.
2. Pull the thyroid gland towards the head by gently separating connective tissues with blunt scissors.
3. Separate the muscle layer on the trachea at the midline towards both sides with the curved needles.
4. Using the blunt scissors, cut the sternum to the second rib (approximately 5 mm). Open the cut with a retractor or curved forceps.
5. 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.
6. 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.
7. Using the curved needle, pull the 6-0 monofilament suture under the aortic arch.
8. Place the spacer in the loop and fix the suture in place with a double-knot. Remove the spacer gently.
9. Confirm a successful constriction with the knot position and cut the ends of the suture.
10. 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

1. Apply povidone-iodine solution to the suture site and place the animal in a pre-warmed cage for recovery and post-operative monitoring.
2. 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.
3. Return the animal to a 12-h light/dark cycle room after full recovery.

5. Echocardiography: Confirmation of Successful Ligation and Measurements

1. After one week, anesthetize the previously banded mouse with 3 - 5% isoflurane, for induction, and 1 - 1.5%, for maintenance, with a 1 L/min oxygen flow rate.
2. Remove the chest hair using depilatory cream. Thoroughly remove the cream with warm water and a cotton applicator.
3. Secure the mouse in the supine position to the animal-handling platform, maintained at 37 °C. Apply electrode gel to the paws and tape them to the pad with electrodes.
4. Apply ultrasound gel to the mouse chest. With a 40-MHz MS550D probe and a high-frequency ultrasound system, perform color Doppler and pulsed-wave Doppler scanning on the left and right carotid arteries, as well as on the aortic arch.
5. Using B-Mode, locate the left ventricle. Using M-Mode, the systolic and diastolic wall thickness can be measured. Use Cine Store and Frame Store to save data and images as required.
6. Using B-Mode, locate the left common carotid artery before the branching point. Use pulsed-wave and color Doppler to evaluate the flow. Repeat with the right carotid artery at a similar point. Save the images in Cine Store and Frame Store modes to analyze later.
7. Tilt the animal-handling platform to the far left so that the mouse is in the left decubitus position. Place the probe on the mouse, 30° offset from a position parallel to the head at the parasternal position, and manipulate X and Y to find the aortic arch.
8. Locate the constriction. Use color Doppler mode to make the site of the constriction more visible, as blood will be flowing through a narrow channel between the point where the two carotid arteries branch out from the aortic arch.
9. Use pulsed-wave and color Doppler to measure the peak flow velocity. Use Cine Store and Frame Store to save data and images as required.
10. Allow the animal to recover on a heating pad in the prone position. Return the animal to the original cage in a 12-h light/dark cycle room after it has fully recovered from anesthesia.

Results

MTAC enables the induction of significant hypertension in the left ventricle in a timely manner.

As seen in Figure 1, the preparation of the primary tools for the procedure is simple and minimal, requiring only 2 - 4 30-gauge needles to be bent and held in place with a polyurethane tube. The surgical procedure itself is also less complicated, as compared to TAC, and can be performed easily. Once the thyroid has ...

Discussion

Rockman and colleagues devised the first method for aortic banding as a method to study pressure overload in the heart. This enabled research to begin to uncover the cellular and molecular consequences of hypertension and heart failure¹. An improved method, minimally invasive transverse aortic constriction (MTAC), provides an amenable, time-dependent method for the induction of left ventricular pressure overload and heart failure, without the drawbacks of the conventional transverse aortic constri...

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
Delapitory 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 Micrioscope	WPI, Sarasota, FL 34240, USA.	PZMIII-BS
GenTeal 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