Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.
For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.
Blood pressure (BP) and electrocardiography (ECG) are basic parameters for cardiovascular physiology and medicine. Experimental animal models have been widely applied in biomedical research for various cardiovascular diseases such as hypertensive heart failure1 and procedures for ECG recording and BP measurement can be performed in experimental rats.
There are three methods for BP measurement in rats: intra-arterial cannulation (invasive)2, tail cuff plethysmography (noninvasive)3, and radio telemetry (invasive). The reliability of BP measurement by tail cuff plethysmography can be affected by animal handling during the recording. For example, the tail cuff underestimates the core BP changes that occur simultaneously during the restraint and measurement phases4. Radio telemetry is considered the best "gold standard" technique for monitoring BP and heart rate in awake and freely moving animals5. However, since radio telemetry hardware and software are costly, intra-arterial cannulation is also widely used as an economical alternative.
Intra-arterial cannulation requires considerable microsurgical skill but yields the real waveforms of arterial pressure. BP can be recorded through a saline-filled catheter inserted in the radial, femoral, or brachial artery. This method of direct invasive BP measurement requires pre-surgical animal preparation, anesthesia, immobilization of laboratory animals, surgical skill in tissue dissection and arterial cannulation, and proper calibration before acquiring the measurement.
Rodent surface ECG is similar to human ECG. A rat ECG has sequences of P waves, QRS complexes, T waves, and QT intervals6. The P wave, PR interval, QRS complex, and T waves reflect atrial depolarization, impulse conduction from the atrial to the AV node, ventricular depolarization, and repolarization, respectively. The QT interval is defined as the period from the initiation of the Q wave to the end-point of the T wave where it returns to the iso-electrical baseline1.
The ECG indicates the cardiac systole and diastole phases; therefore, the simultaneous recording of the surface ECG correlates with the invasive BP measurement. By using a combination of methodologies, it is possible to elucidate pathophysiological changes in a disease model or the pharmacological effects of a drug or therapy in cardiovascular medicine.
A spontaneous hypertensive rat (SHR) strain had been obtained by inbreeding of Wistar rats with high BP in Japan. The BP rises from 5 to 10 weeks of age and becomes stationary from 30 to 35 weeks of age7. Wistar-Kyoto rats (WKY) have systolic BP about 130 mmHg7 and are commonly used as normo-tensive control. We used SHR and WKY to demonstrate the result of intraarterial cannulation BP and ECG recording.
All the animal experiments described were approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University.
1. Animal Care
2. Experimental Preparation
3. Pressure Transducer Calibration
4. Mini-surgery for Cannulation of the Femoral Artery
5. Recording of Blood Pressure
6. Surface ECG
7. Animal Euthanasia After Completing of the Experiment
We purchased SHR and normotensive Wistar-Kyoto WKY rats from the National Laboratory Animal Center (Taipei, Taiwan). All animals were housed in a temperature-controlled facility (20−22 °C) with free access to water and standard chow on a 12 h light/dark cycle.
We used six 47-week-old rats and they were weighed before the BP and ECG measurement. The representative tracings from simultaneous recording of ECG and BP in SHR and WKY are shown in Figure 6. Table 1 shows parameters for BP and heart rate. Statistical (t) tests revealed significantly higher systolic BP and mean BP in SHR (124.5 mmHg ± 15.1 mmHg and 84.3 mmHg ± 5.0 mmHg) than in WKY (90.0 mmHg ± 7.5 mmHg and 67.5 mmHg ± 5.0 mmHg) (P < 0.05) (Table 1).
The parameters of P wave, PR intervals, QRS width, and QT intervals can be measured from the ECG recordings (Table 2). Choice of right vs. left femoral cannulation in relation to ECG electrode placement (at the right hind limb) did not affect the BP or ECG signals.
Figure 1: Materials. (A) Forceps with teeth, (B) surgical scissors, (C) tissue forceps, (D) tissue forceps with fine tips, (E) angle tip forceps, (F) bulldog vascular clamp, (G) silk string, (H) micro scissors, (I) polyethylene catheter connecting with a 26 G x 1/2'' needle and a three-way stopcock (J), (K) pressure transducer connecting with a three-way stopcock, (L) mercury sphygmomanometer with a stopcock (green arrow) and inflation bulb with an air-leak valve (white arrow). Please click here to view a larger version of this figure.
Figure 2: Surface landmark for femoral artery dissection. (A) Supine rat with left inguinal crease highlighted with a dashed line. (B) Groin skin picked up by forceps with teeth to be cut off by surgical scissors. (C) The surgical zone for femoral artery dissection. Please click here to view a larger version of this figure.
Figure 3: Tissue dissection and cannulation of the femoral artery. (A) Exposed femoral nerve (yellow arrow), femoral vein (blue arrow), and femoral artery (red arrow) after tissue dissection. (B) Femoral vein and artery after the nerve cut-off. (C) Application of a bulldog vascular clamp over the cranial terminal of the dissected femoral artery. (D) Femoral artery with a bulldog clamp and two silk strings with loose ties. (E) A small hole on the ventral side of femoral artery, created using micro scissors. (F) Insertion of the polyethylene catheter into the femoral artery. Please click here to view a larger version of this figure.
Figure 4: Femoral artery cannulation for pressure recording. The intra-arterial cannula (highlighted by a dashed green line) is connected with two three-way stopcocks (blue arrows) with syringes filled with heparinized saline. The pressure transducer (red arrow) is linked to the cannulation through the three-way stopcocks. Please click here to view a larger version of this figure.
Figure 5: The whole experimental setup for invasive femoral arterial pressure and electrocardiography. Three electrocardiographic leads (red arrows) with platinum needle electrodes subcutaneously inserted over bilateral fore legs and right leg (yellow arrows). The exposed right femoral artery with cannulation (blue arrow). Please click here to view a larger version of this figure.
Figure 6: Representative tracings. Simultaneous recordings of ECG and BP in WKY (left) and SHR (right) for the ECG (top), and arterial pressure waves (bottom). Please click here to view a larger version of this figure.
WKY | SHR | P value | |
(n = 3) | (n = 3) | ||
Weight, g | 407.7 ± 6.4 | 353.7 ± 10.3 | <0.01 |
Heart rate, /min | 241.3 ± 15.0 | 262.0 ± 15.7 | 0.18 |
Systolic BP, mmHg | 90.0 ± 7.5 | 124.5 ± 15.1 | <0.05 |
Diastolic BP, mmHg | 56.3 ± 4.0 | 64.2 ± 0.6 | 0.08 |
Mean BP, mmHg | 67.5 ± 5.0 | 84.3 ± 5.0 | <0.05 |
Pulse pressure, mmHg | 33.6 ± 4.4 | 60.4 ± 15.1 | 0.08 |
Table 1: Blood pressure levels measured by invasive femoral artery cannulation. Data are presented as mean ± standard deviation; SHR = spontaneously hypertensive rats; WKY = Wistar-Kyoto rats; BP = blood pressure.
WKY | SHR | P value | |
(n = 3) | (n = 3) | ||
Heart rate, /min | 241.3 ± 15.0 | 262.0 ± 15.7 | 0.18 |
P wave, msec | 38.7 ± 9.3 | 43.0 ± 4.6 | 0.52 |
PR interval, msec | 59.0 ± 7.8 | 61.3 ± 4.7 | 0.69 |
QRS width, msec | 43.0 ± 11.3 | 50.0 ± 2.6 | 0.4 |
QT interval, msec | 112.0 ± 12.8 | 116.7 ± 9.5 | 0.15 |
Table 2: Measurements of electrocardiographic parameters. Data are presented as mean ± standard deviation; SHR = spontaneously hypertensive rats; WKY = Wistar-Kyoto rats.
Invasive arterial cannulation allows highly accurate measurement of BP. It can be done with a PE tube without requiring an expensive catheter. Invasive BP measurement can also be performed simultaneously with a recording of the surface ECG.
The major learning curve for this method is the experimental skill required to cannulate small blood vessels. In experienced hands, the successful rate for femoral artery cannulation can approach 100%. Practice is recommended before performing real experiments. Some noteworthy points during the procedure: (1) make sure animals are completely anesthetized before starting the surgery; (2) use a bulldog clamp at the high position of the exposed artery before cutting the arterial wall to prevent extensive bleeding; (3) make the incision only over the ventral wall of the artery and avoid damaging the dorsal wall; and (4) steady placement of the cannulation system and electrocardiographic leads helps avoid motion artifacts during the recording.
The potential complications with this procedure include hemorrhage from the surgical dissection. The hemorrhage in a successful and smooth cannulation of an artery is very trivial. Major hemorrhage can be caused by accidental damage to the femoral artery or vein or the major branches during the tissue dissection. The hemorrhage can be stopped by focal compression with a clear cotton ball. Another cause of major hemorrhage is dislocation of the femoral cannulation. In this case, the blood loss is usually large enough to result in a significant drop in systemic BP. At this point, BP recording can be terminated early.
Telemeter implantation also is an established technique for invasive BP recording8. The advantage of telemetry is the ability to obtain a high-fidelity continuous recording over relatively long periods of time in conscious, freely moving animals without the limitations of restraint or anesthesia. Direct BP recordings can be done with successful subcutaneous implantation of radio-transmitters and carotid artery cannulation9. However, it can be challenging to implant the telemeter successfully and the hardware and software for radio telemetry are costly.
There are some limitations for invasive BP measurement by femoral artery cannulation. First, it cannot be performed in unrestrained and conscious rats. Second, BP in the femoral artery may be higher or lower than BP in the central aorta. Third, arterial cannulation can be difficult for small rodents, where femoral arteries may be too small to insert the PE tube. Fourth, the cannulated artery should be ligated after completing the BP recording and ligation of femoral artery will result in hind leg ischemia. Due to this limitation, the animals are usually euthanized following invasive BP recording.
Invasive femoral artery cannulation requires restraining and anesthetizing the animals, which potentially introduces stress and influences both BP and electrocardiography data10. The stress can be alleviated by appropriate anesthesia. Anesthesia includes injection or inhalation protocols. In our past experience, high elevated BP in I.P. anesthetized WKY rats suggests that the animals were still under stress or in pain. Normal BP in conscious WKY rats is 130/80 mmHg; this dropped to below 100 mmHg in our inhalation-anesthetized WKY rats (Table 1). Inhalational anesthesia (isoflurane) has several advantages over injectable agents: quick onset, minimal animal handling, ease of control, no controlled drugs, and a quick recovery. The disadvantages are the cost of the equipment, the hazard of human exposure in case with substantial gas leak into the working environment, and a significant suppressive effect on BP. Isoflurane-induced BP reduction should be considered when choosing the anesthesia regime.
Further applications are possible for invasive BP measurement. According to the fluid dynamics principles for pulse wave velocity (PWV), the stiffer artery propagates the pulse wave faster11. When applying double cannulations over the carotid artery and femoral artery, the aortic PWV can be determined. The length of aortic pulse propagation can be measured as the distance between the tips of the cannula at the carotid (proximal) and distal (femoral) arteries. The PWV is the ratio between the aortic length and the difference between the time at the minimal values of the proximal and distal arterial pulses12.
The setup described above for simultaneous electrocardiography and intra-arterial BP recording in experimental rats is an inexpensive and highly accessible technique for physiological or pharmacological studies investigating pathogenic or therapeutic mechanisms in cardiovascular medicine.
The authors have nothing to disclose.
This study was supported by Taiwan Ministry of Science and Technology grants MOST 104-2314-B-037-080-MY3 and MOST 107-2314-B-037-110 to HCL and Taiwan National Health Research Institutes grant NHRI-EX107-10724SC.
Polyethylene tube | BECTON DICKINSON | 427401 | internal diameter of 0.5 mm, outer diameter of 0.9 mm |
26G x 1/2"" needle | TERUMO | 160426D | |
Adson Forceps | TOP Line | 12-540 | 12 cm (4.75") Long, Straight, 1 x 2 Teeth |
Bulldog vascular clamp | Teleflex | 357581 | 8 mm |
Computer | AUSUS | X453M | |
Exernal analog signal recording device | iWorx | T5141538 | This allows the recording of up to three channels of ECG, EMG or EEG as well as GSR (skin conductance) from a single iWire input on the recording Module. |
Graefe Forceps | AESCULAP Surgical Instruments | BD312R | MICRO DRESSING FORCEPS, CURVED, SERRATED, 105 mm, 4 1/8 |
Mecury sphygmomanometer | Spirit | CK-101 | |
Pressure transducer | iWorx | IworxBP100 | |
Semken Forceps | MEDE TECHNIK | 10-104 | 100 mm |
Software | LabScribe3 | ||
Surgical scissors | HEBU | 1714 | 14.5 cm long |
Syringe (1 mL) | TERUMO | 160426D | |
Three-way stopcocks | Cole-Parmer | EW-30600-23 | |
Tipped forceps | World Precision Instruments | 504506 | 11 cm long, 0.1×0.06 mm Tips |
Vannas Scissors | World Precision Instruments | 500086 | 8.5 cm long, Straight, 0.025 x 0.015 mm Tips, 7mm super fine Blades |