We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.
Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50–60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.
Small animal models are the most preferred tools for the study of chronic changes in the development of cardiac hypertrophy and its progression to heart failure. Heart failure models by surgical methods were originally developed in rats as these animals are most suitable for open chest surgical procedures, echocardiography and evaluation of hemodynamic parameters. They also provide adequate post mortem samples for tissue, cellular and molecular level studies1. Constriction of the ascending aorta is one of the most common and successful surgical models for creating pressure overload heart failure2,3. This model is well suited to study the cellular and sub cellular remodeling events and contractile properties of the heart during the transition from hypertrophy to heart failure4.
A major advantage of this model is the gradual onset of pressure overload on the heart5,6. This model is clinically relevant because of its slow but steady progression from compensated cardiac hypertrophy to decompensated phase and finally to the heart failure stage. The duration for progression of cardiac hypertrophy to heart failure and the associated physiological changes are mainly influenced by the degree of stenosis produced7. Ascending aortic constriction can be performed in two ways, either by using sutures or by application of metallic clips8,9. The main advantage of using a metallic clip for aortic constriction is that the surgical procedures are less complicated. In this method one has to locate the supra valvular ascending aorta and insert the clip around the aorta to obtain desired level of constriction. A chest retractor is not necessary in this procedure. Aortic banding by suturing requires more surgical manipulations for placing the gauge needle beside the ascending aorta and suturing around it to produce the desired level of aortic constriction. The latter technique is more time consuming when compared with the use of metallic clips for aortic constriction. The method described in this video demonstrates the surgical procedure for ascending aortic constriction in rats that allows creation of pressure overloaded left ventricular hypertrophy model.
Animal experiments were performed after obtaining approval from the Institutional Animal Ethics Committee. Wistar rats were housed under a 12 hr dark to light cycle, constant room temperature (24 ± 2 °C) with food and water ad libitum (as per the CPCSEA guidelines).
1) Pre-operative Care and Anesthesia
2) Preparation of the Surgical Site
3) Tracheal Intubation in Rat
4) Constriction of Ascending Aorta
5) Post-operative Care
6) Confirmation of Successful Constriction of Ascending Aorta
In our experiments we are able to achieve more than 80% survival rates. The effectiveness of aortic constriction was confirmed by performing Doppler echocardiography. Rats (n=6) with a pressure gradient of about 60 mm of Hg at the aortic constricted site were observed for 8 weeks and sacrificed to analyze for the development of cardiac hypertrophy (Figure 1). After 8 weeks post-surgery echocardiographic evaluation revealed a significant increase in left ventricular parameters such as inter ventricular septal thickness, left ventricular posterior wall thickness and decrease in left ventricular internal diameter at diastolic state, in animals which underwent aortic constriction, compared to sham operated controls (Table 1, Figure 2). A significant increase in heart weight/body weight ratios and left ventricular weight/body weight ratios were observed in rats with aortic constriction (Table 2, Figure 3). These data suggest that 8 weeks post-surgery rats with aortic constriction develop a significant level of cardiac hypertrophy, compared to sham operated controls.
Figure 1. Echocardiogram in rats representing the pressure gradient across the ascending aorta. A.a) The representative echocardiogram from a rat with constriction of aorta and having a pressure gradient of 63.0 mm of Hg and A.b represents color Doppler echo of the site across the aorta where pressure gradient was measured. B.a) The representative echocardiogram from a sham operated control rat having a pressure gradient of 2.86 mm of Hg and B.b represents color Doppler echo of the site across the aorta where the pressure gradient was measured. This increase in pressure gradient indicates successful banding of the aorta in rats which underwent partial ascending aortic constriction.
Figure 2. M- mode echocardiogram of the left ventricle in rats with aortic constriction (A) and control rats (B) depicting left ventricular dimensions at the time of sacrifice 8 weeks post-surgery. IVST= inter ventricular septal thickness; LVPWT= left ventricular posterior wall thickness; LVID= left ventricular internal diameter in diastole. In rats with aortic constriction a significant increase in inter ventricular septal thickness, left ventricular posterior wall thickness and decrease in left ventricular internal diameter at diastolic state can be observed.
Figure 3. Images of the whole hearts of a rat which underwent aortic constriction (A) and a sham operated control rat (B) 8 weeks post-surgery. The rat with aortic constriction has significant cardiac hypertrophy compared to sham operated control.
Table 1. M- mode echocardiographic parameters of cardiac left ventricle at the time of sacrifice, 8 weeks post-surgery. IVST= inter ventricular septal thickness; LVPWT= left ventricular posterior wall thickness; LVID= left ventricular internal diameter at diastolic state; AAC= ascending aortic constriction.
Table 2. Heart weight: Body weight ratio and left ventricular weight: body weight ratio in aortic constricted and sham control rats sacrificed 8 weeks post-surgery. Hw= Heart weight; Bw= Body weight; LV+S= Left ventricle+ inter ventricular septum; AAC= ascending aortic constriction.
There are several surgical models for the induction of pressure overload as well as cardiac hypertrophy and heart failure. In small animals, Lorell and colleagues developed the model we have described here and they reported the occurrence of compensated cardiac hypertrophy 8 weeks post aortic banding5. Cardiac changes gradually progress to a decompensated state and then result in heart failure. The main advantage of this model is the gradual onset of increase in left ventricular pressure and thus similar to clinical situations in chronic hypertension. It is also a suitable animal model for studying left heart failure associated pulmonary hypertension10.
Other animal models for pressure overload heart failure, such as Dahl sensitive rats, spontaneously hypertensive rats and rats with constriction of abdominal aorta, also develop chronic pressure overload. They, however, take extended time periods for progression from cardiac hypertrophy to heart failure. The maintenance cost of experimental rats is therefore also higher1. deAlmeida and colleagues employed transverse aortic constriction for inducing left ventricular hypertrophy in mice11. The technique involves median sternotomy and retraction of sternum and hence is more challenging.
Ensure a swift change of anesthesia from nose cone to tracheal intubation before thoracotomy and from tracheal intubation to nose cone after the operative procedures. An alternate method for tracheal intubation in rats is by using polyethylene tube for endotracheal intubation instead of tracheotomy. However, this technique requires adequate skill. Care should be taken to avoid any injury to the lungs while making incision between the ribs. Application of titanium clips sufficient to produce critical aortic constriction for left ventricular stress, but not severe enough to produce acute left ventricular failure and pulmonary oedema. Use of prolene sutures for the approximation of ribs is critical for successful recovery of the animal. During the approximation of ribs, pause the ventilator for about 2-4 sec in order to re-inflate the lungs.
We had only 20% operative mortality in the animals and there were no post-operative complications. The technique we described here is thus an efficient and suitable tool for studying chronic patho-physiological changes in cardiac hypertrophy and progressive heart failure.
The authors have nothing to disclose.
The authors acknowledge Department of Biotechnology, Government of India for financial support of this project. Ajith Kumar G S was supported with senior research fellowship from Council of Scientific and Industrial Research, Government of India and Binil Raj with senior research fellowship from Indian Council of Medical Research, Government of India.
Wistar rats | Maintained at ARF, RGCB | ||
Inhalation anaesthesia systems | VetEquip | AB19276 | |
Rodent ventilator | CWE Inc | SAR- 830/AP | |
Rat tracheal cannula | CWE Inc | 13-21032 | |
Ultrasound system | Philips | HD7 | |
Ultrasound transducer | Philips | S 12 | |
Titanium clip (small) | Horizon | 1204 | |
Clip applicator | Weck | 137081 | |
Electric shaver | Vinverth | VBT0817 | |
BP blade (size:11) | Surgeon | REF10111 | Preparation of surgical site |
Tramadol | Orchid health care | OCH043 | |
Amoxycillin Hydrochloride | Ranbaxy | ||
Isoflurane | Piramal health care | A23M10A | |
Syringes | BD | 2015-09 | |
4-0 braided silk | Diamond | ||
3-0 prolene | Johnson&Johnson | NW018 | |
Surgical tape | Romsons | SH 6301 | |
Povidone iodine | Win Medicare | ||
Temp. controlled heating pad | Flamingo | HC1003 |