Articles in PresS. Am J Physiol Heart Circ Physiol (August 8, 2014). doi:10.1152/ajpheart.00990.2013
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A novel knot method for individually measurable aortic constriction in rats
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Jiaming Liu*, Pengfei Han*, Ying Xiao*, Jiani Liu†, and Y. James Kang*¶
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*Regenerative Medicine Research Center, and †Department of Cardiology, West China Hospital,
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Sichuan University, Chengdu, Sichuan, China 610041, and ¶Department of Pharmacology and
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Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40042, USA
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Short title: A knot method for aortic constriction
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Correspondence address:
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Dr. Y. James Kang
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Regenerative Medicine Research Center
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Sichuan University West China Hospital
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Chengdu, Sichuan 610041, China
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Telephone: (86) 028-8516-4037
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Fax: (86) 028-8516-4037
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E-mail: yjkang01@louisville.edu
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Copyright © 2014 by the American Physiological Society.
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ABSTRACT
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A novel knot method in rats is reported that addresses several drawbacks in the current model of
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aortic constriction-induced heart hypertrophy. Using a rat model, we developed a two-step procedure
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that includes: 1) measurement of individual aorta circumference using a surgical thread; and 2)
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constriction of the aorta using a thread with the desired length pre-defined by a knot at each end for a
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measurable reduction of the aortic circumference as referenced to the measurement in step 1. This
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knot approach produces a manageable gradient of aortic constriction in each rat, reaching a
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consistency among experimental animals, which cannot be achieved by the traditional needle method.
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Notably, the animal model produced by our knot method showed cardiac hypertrophy and
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dysfunction with the severity proportional to the percentage reduction of the aorta circumference (50%
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versus 60%). Additionally, our new procedure produced a lower mortality rate in comparison with
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the traditional needle method. Therefore, we recommend this knot method as an alternative
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procedure for aortic constriction with desired gradient in rats and larger animal models.
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Keywords: aortic circumference; aortic constriction; cardiac hypertrophy; hemodynamic; knot
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method; pressure overload; traditional needle method; rat
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INTRODUCTION
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Pressure overload causes cardiac hypertrophy and dysfunction leading to eventual heart failure.
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Experimental studies using animal models involve constriction of the aorta to reproduce the pressure
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overload-induced heart hypertrophy. The favored rat model employs ascending aortic banding to
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induce pressure overload as first reported in 1983 by Bugaisky et al. In that surgical procedure,
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cardiac hypertrophy was induced by placing a constricting band with an inner diameter (i.d.) of 0.02
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inches (0.51 mm) around the ascending aorta of 25-day rats (3). Subsequently, this model was
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modified by placing a 16-gauge needle, which has an outer diameter (o.d.) of 1.6 mm, alongside the
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ascending aorta. The ascending aorta and needle were tied together with a surgical thread and the
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needle removed rapidly leaving the ascending aorta constricted with the same diameter as the needle
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(14). Several modifications of this fundamental approach have been reported. For example, tantalum
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hemostatic clips or rigid tubes with defined inner or outer diameters have been used to vary the
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gradient, or extent, of aortic constriction (1, 5, 13, 17, 19).
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Although the needle procedure described above has been widely adopted, it is limited by some
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obvious drawbacks. These include: 1) the precise quantifiable constriction of the aorta of individual
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animal is less feasible by using tools with fixed internal or external diameters to all animals; 2) the
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variations among experimental animals are expected to be large due to the difference in the diameter
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of individual aortas; 3) instant occlusion of the aorta leads to unexpected injuries (20). The first two
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concerns are closely related. The fixed diameter of the selected tool provides only a rough-estimate
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of the gradient of constriction. Although the fixed diameter yields uniformity in terms of absolute
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constriction, the exact gradient of aortic constriction is not controlled, or even quantifiable, in any
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individual due to variations in aortic circumference between animals. For instance, a 20-gauge
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needle with an outer diameter of 0.9 mm is estimated to produce approximately 50% reduction of the
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average aorta circumference. However, this is only an average estimate and precise control of the
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gradient of aortic constriction for individual rats cannot be attained. Regarding the third concern, any
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procedure using needles, clips, or tubes to produce constriction involves an instant occlusion of the
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aorta (20). While quickly removing the needle that is tied together with the aorta can reduce the
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ischemic effect, it requires an extensive training of the skill.
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We have encountered each of the unsolved issues noted above in our studies using the
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traditional needle method. In particular, attempts to determine the exact gradient of aortic
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constriction responsible for irreversible cardiac hypertrophy were limited by the needle procedure.
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Therefore, we made an effort to address the stated limitations by developing a new methodology that
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provides precise control over the gradient of aortic constriction in individual rats.
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MATERIALS AND METHODS
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Animals and animal care
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Male Sprague-Dawley rats, 6-8 weeks old and weighing an average 220 g, were obtained from
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Chengdu Da-Shuo experimental animal breeding and research center, a Chinese government
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accredited rodent animal center in Sichuan province, China. The animals were acclimatized to
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laboratory conditions for a period of at least one week in an Association for Assessment and
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Accreditation of Laboratory Animal Care accredited facility. The rats were housed in standard
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laboratory cages with ad libitum access to standard chow and tap water in a temperature-controlled
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room at 22 ± 1°C with a humidity of 50 ± 10% and a 12-hour dark-light cycle (lights on at 8:00 and
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off at 20:00) as approved by the Laboratory Animal Management Committee of Sichuan province.
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To ameliorate pain after surgery, the analgesic dezocine (0.8 mg/kg) was given intramuscularly and
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once daily for the next 2 days. Animal harvest was performed by euthanasia via intravenous injection
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of 10% potassium chloride (2 ml/kg) under anesthesia (10% chloral hydrate 0.35 mg/kg) at the end
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of the experiment. All animal procedures were approved by the Institutional Animal Care and Use
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Committee at Sichuan University West China Hospital, following the guideline of the U.S. National
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Institutes of Health.
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Experimental design
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Three sets of experiments were conducted using a combination of the traditional needle method
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and the new knot method as outlined below. For rats with a body weight of 226 ± 15 g the average
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diameter of the ascending aorta is 1.74 ± 0.23 mm. The aortas subjected to the traditional needle
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method were constricted using a 20-gauge needle (o.d. 0.9 mm) to generate ~50% reduction of the
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circumference of aorta (~50% RC). The aortas of rats subjected to the knot method were individually
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measured and constricted exactly as desired using methods described in the surgical procedure
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section.
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In the first set, rats were divided into the following three groups: sham-operated controls, 50%
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RC, and 60% RC. The groups contained 20, 25, and 30 rats, respectively. Rats were subjected to
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aortic banding using the new knot method. This experiment was designed to evaluate the feasibility
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of producing exactly defined gradients of aortic constriction and the subsequent heart hypertrophy
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using the knot method.
In the second set of experiments, rats were divided into the following three groups:
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sham-operated controls, ~50% RC using the traditional needle method, and 50% RC using the knot
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method. The groups contained 20, 25, and 30 rats, respectively. This experiment was designed to
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compare variations in cardiac hypertrophy and dysfunction developed from aortic constriction
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between the traditional needle method and the new knot method.
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In the third experiment, rats were grouped the same as for the second experiment. The goal of
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this experiment was to compare the mortality rate after the surgical procedure between the two
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methods.
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Surgical procedure for aortic constriction using the knot method
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Prior to the surgical procedure all subjects received an intraperitoneal injection of 10% chloral
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hydrate (0.35 mg/kg) to induce sedation. The hairs covering the left chest were shaved thoroughly
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for operation. Then endotracheal intubation was introduced for ventilation with the tidal volume and
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ventilation rate calculated as previously described (2, 20). A list of tidal volumes and ventilation rates
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for rats of particular body weights is shown in Table 1.
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The ascending aorta of the rat was exposed via the left second intercostal space incision (1-1.5
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cm) on the chest wall. The opening in the thorax was sustained with a retractor. Major vessels are
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located in the upper part of the left atrial appendage. The ascending portion of the aorta was
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dissected from the pulmonary trunk on the right (7, 10, 12, 20).
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The knot method involved two steps. In the first step, the aortic circumference was measured. In
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the second step, the desired gradient of aortic restriction was calculated based upon the measured
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aortic circumference and produced by using a pre-defined length of thread to shorten the
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circumference. The aortic circumference is directly proportional to the diameter of the artery and
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square root of the vessel cross-section area such that a 50% RC decreases the aortic diameter by one
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half and the cross-sectional area by 75%. These two steps are described in details as follows:
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Step 1: Measurement of the aortic circumference. A single piece of 6-0 surgical thread was
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grasped by forceps and placed underneath and then twined around the aorta. A loop was made at
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"end-a" of the thread and a noose knot was made by placing "end-b" through the previously formed
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loop with pulling of "end-a" to tighten the loop (Fig. 1A, 1B). The noose knot was tightened by
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slowly pulling "end-b" until the knot just touched, but did not constrict, the outer wall of the artery.
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Excess thread at "end-b" was trimmed just before the knot site (Fig. 1C). This released the hold
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around the artery while retaining a knot in the thread. The distance from the knot to "end-b" was
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measured providing quantification of the circumference of the measured artery (Fig. 1D).
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Step 2: Aortic constriction. A second piece of 6-0 surgical thread was prepared to constrict the
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aorta to the desired X% RC. First, the final desired length of the constricting surgical thread (Ld) is
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calculated using the following formula: Ld = Lc·(1 - RC); where Lc= measured circumference of the
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aorta from Step 1 and RC for the desired percentage reduction of the aorta circumference. Second, a
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noose knot was prepared and tightend as described in Step 1. Then, a common knot was made at
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"end-b" at the calculated distance Ld (Fig. 1E, 1F).
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The prepared thread was placed around the artery. The "end-b", the end with the common knot,
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was placed through the loop (Fig. 1G), and the "end-a" was pulled to tighten the loop (Fig. 1H). To
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constrict the aorta, the "end-b" was pulled (Fig. 1I) with the gradient of constriction defined by the
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distance between the two knots. Once the two knots meet, further constriction was not possible. The
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constriction was secured by tying another common knot and the excess thread was cut off (Fig. 1J).
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Finally, the chest cavity was closed by bringing together the second and third ribs with 3-0
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nylon sutures and all layers of muscle and skin were closed with 5-0 nylon sutures. With practice, the
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entire procedure including both steps required about 15 minutes.
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Surgical procedure for aortic constriction using a traditional needle method
After the ascending portion of the aorta was dissected from the pulmonary trunk, a single piece
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of 6-0 surgical thread was grasped by forceps and placed underneath the aorta. A loose double knot
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was made. Next, a 20-gauge needle (o.d. 0.9 mm) was delivered through the loose double knot and
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placed directly above and parallel to the aorta. The loop was then tied around the aorta and needle
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and secured with a second knot. The needle was then immediately removed to provide a lumen with
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a stenotic aorta (20). One more knot was made to secure the tie, and the excess thread was cut and
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removed. The chest cavity was closed as described above.
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Echocardiography
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The animals were sedated by intraperitoneal injection of 10% chloral hydrate (0.35 mg/kg) for
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all measurements. At intervals of 2, 4, and 8 weeks after the aortic banding operation, a series of
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echocardiograms were performed using an 11.5-MHz transducer (Vivid 7 Dimension, GE) as
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previously described (4, 15). Interventricular septum depth (IVSD) and left ventricular posterior wall
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depth (LVPWD) were obtained using two-dimensional mode by taking the measurements of
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short-axis cross-sectional areas and left ventricle length (11).
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Measurement of cardiac hemodynamics
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Hemodynamic measurements were acquired and analyzed using Power lab (ML880, AD
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Instrument Inc.) and Lab Chart 7 software (AD Instrument Inc.). LV pressure and aortic pressure
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analyses were conducted using a conductance catheter (1.4 Fr, Millar Instrument Inc.) as previously
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described (18). Briefly, we insert the Millar catheter recessively through the right carotid artery to the
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heart. It means that catheter is situated before the stenosis while inserting meets with resistance; we
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can get the pressure gradient artery end systolic pressure (AESP) before the stenosis. When catheter
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went through the stenosis, the pressure gradient after the stenosis rapidly increases and is equal to
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left ventricle end systolic pressure (LVESP). To define hemodynamic responses to the increased
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cardiac load, isoproterenol was delivered through a femoral vein catheter (0.1 μl/g body weight) with
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a microliter syringe pump (WZ-50C6, Zhejiang Smith Medical Instrument Company). It was
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administered at a constant rate of infusion in varying concentrations of 0.08, 0.16, and 0.32 μgIso/
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(min·g body weight) for a total of 3 min per dose (9, 16). Rats were allowed to recover for 10-15 min
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before administration of each successive dose. Heart performance under the stimulation of
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isoproterenol was analyzed using the Millar instrument (8).
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Histological analysis
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Hearts were harvested after the last hemodynamic measurement, and perfused with 30 ml of
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cold PBS with 0.1 ml of 1% heparin before cutting through the coronal plane and cross-sectional
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plane, and fixed with 4% paraformaldehyde. The heart tissues were embedded in paraffin after being
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fixed for 24 hours and then sectioned. The slides were stained with Masson’s Trichrome (6) by
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automatic staining machine (Shandonvaristain, Thermo SCIENTIFIC) and digitally imaged (eclipse
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80i, Nikon). Collagen staining on the tissue sections was detected by a light microscope and the
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images were digitized. Under 200 magnification, 5 visual fields were randomly observed from each
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slide, defining the average optical density with positive expression (integrated optical density/area)
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for semi-quantitative statistical analysis.
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Statistical analysis
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Data are presented as mean ± standard deviation (SD). One-way ANOVA was used to compare
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intergroup difference followed by LSD test for comparison among different groups. Log-rank was
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used to compare survival difference between groups. SPSS 13.0 for Windows (SPSS, Chicago, IL)
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was applied to perform the statistical processing. For all analyses, p-values < 0.05 were considered
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significant.
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RESULTS
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Effects of percentage reduction of the aorta circumference on cardiac structure and function
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Using the knot method, we made exact 50% or 60% RC of the aorta to determine the effects of
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different percentage reduction on cardiac hypertrophy and dysfunction. At one week after the aortic
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constriction, the pressure across the stenosis site was measured for the sham-control, 50% RC, and
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60% RC groups. As shown in Fig. 2, there were no significant differences in the artery end systolic
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pressure (AESP) before the stenosis site among the three groups. However, the pressure after the
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stenosis site, as reflected by the left ventricle end systolic pressure (LVESP) was significantly
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increased in the 50% and 60% RC groups in comparison to the sham-operated group. The gradient
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across the stenosis site (ESP increase = LVESP - AESP) was significantly higher in the 60% RC
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group than in the 50% RC group. At 8 weeks after the aortic constriction, IVSD and LVPWD
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increased proportionally to the gradient of RC with significantly higher values in the 60% RC than in
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the 50% RC group (Fig. 3). There were also significant differences in LVESP and +dp/dt between the
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50% RC and 60% RC groups. However, no significant differences were observed in LVEDP or
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–dp/dt between the two groups (Fig 4).
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Histopathological analysis revealed more extensive damage in the rats subjected to 60% RC than
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in the rats subjected to 50% RC. Larger heart size, increased heart weight to tibial length ratio, and
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more serious perivascular and interstitial fibrosis were observed in the rats subjected to 60% RC
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relative to the 50% RC group (Fig. 5A, C-H and Table 2). Semi-quantitative analysis of collagen
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staining showed that the level of average optical density (AOD) (the intensity of collagen staining)
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was significantly higher in the rats subjected to 60% RC than those subjected to 50% RC, although
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there was no significant difference in the collagen volume fraction (CVF) (the area of collagen
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staining) between the two groups (Fig. 5I, 5H).
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Comparison in the consistency between the knot and the traditional needle method
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The traditional needle (~50% RC) and the new knot (50% RC) methods produced similar
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cardiac hypertrophic effects (Fig. 5B). However, the traditional needle method produced bigger
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variations than the knot method based on CV values (Table 2). As shown in Fig. 6, at one week after
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the aortic constriction, the pressure across the stenosis site was not significantly different between
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the two groups. However, as observed from the scatter plots, larger variations were observed in the
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group subjected to the traditional needle method, although it was not statistically significant.
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Likewise, no significant differences in IVSD and LVPWD between the two groups were observed,
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but larger variations were seen in the group subjected to the traditional needle method, as judged by
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larger CV values (Fig. 7 and table 3). Hemodynamic examination showed that both methods caused a
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similar increase in LVESP and LVEDP, but the variations of these changes were larger within the
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group subjected to the traditional needle method in comparison to that subjected to the knot method,
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as judged by CV values (Table 4).
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Comparison in the mortality rate between the knot and the traditional needle method
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A significant difference in the mortality rate post operation between groups subjected to the
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knot method versus the traditional needle method was observed. Both methods were designed to
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produce 50% RC. In the group subjected to the traditional needle method, animal death occurred
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immediately following surgery and continued until 32 days post operation. Animal death also
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occurred immediately following surgery in the group subjected to the knot method; however, it
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stopped 15 days post operation. As reflected by the survival curve, there was a significant difference
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in the final survival between the two groups over the course of the study with a significantly larger
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number of surviving animals in the group subjected to the new knot method as compared to the
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traditional needle method group (Fig 8A and Table 5). Interestingly, mortality increased in rats
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subjected to 60% RC using the knot method (Fig. 8B and Table 5) yielding survival rates comparable
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to those subjected to ~50% RC using the traditional needle method.
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DISCUSSION
We have made attempts to quantify the extent of aortic constriction required to induce
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non-reversible cardiac hypertrophy in rats. However, existing methods at that time did not allow such
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analyses to take place because the exact measurement and control of the aortic constriction gradient
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was not possible. Therefore, we developed the knot method described in the current report to
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overcome these limitations in the traditional needle method. A highlight of the new procedure is the
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ability to individually customize the size of the restriction for each animal based upon its own aorta
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circumference. This produces two significant results. First, the exact X% RC can be set for any
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individual animal. As a result, variations in pathological consequences among rats in the same
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sample group are expected to be reduced in comparison to those using the traditional needle method.
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Second, any X% RC can be selected for a sample group. This allows comparison of groups with
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distinct X% RC as shown for 50% versus 60%. We also unexpectedly observed that the post
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operation mortality rate was significantly reduced in this new method relative to the traditional
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needle method.
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In this two-step procedure, the circumference of the aorta is measured first, and the exactly
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desired degree of constriction of the aorta is calculated based on the measured length of the
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circumference. Based upon this calculation, the loop of the surgical thread that is used to constrict
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the aorta is made in the way that the exactly desired degree of constriction is achieved once the loop
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is closed by tightening the two ends of the thread. This procedure does not need any additional tools
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such as needles, clips, or tubes, and with practice, it is completed within 15 min.
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This novel and simple method not only produces measurable degree of constriction of the aorta,
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but it is also expected to reduce the variations in the aortic constriction-induced heart hypertrophy
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and dysfunction among rats, as shown from the scatter plots and analysis tables. This achievement
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would result from the unique feature of the new procedure relative to the traditional procedures using
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needles, clips or tubes. In this new procedure, the measurement of the circumference of each aorta is
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done individually in order to calculate the length of the loop of the thread for the desired percentage
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reduction of the circumference of aorta. In this way, if the desired degree of constriction is 50% RC,
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the aortas of all of the animals subjected to this procedure will be exactly the same. In contrast, the
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needle procedure, as well as other tools, uses only one needle to produce aortic constriction in all
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animals regardless of the size differences in the aorta among animals, such that significant variations
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are inevitable.
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It was unexpected to observe that the new procedure reduces the post-operation mortality rate of
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the animals relative to the traditional needle method. The most noticeable advantage results as the
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new method avoids the instant occlusion of the aorta, which occurs with the needle method when the
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needle and aorta are tightened together, followed by removal of the needle. It was previously
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unknown whether this instant occlusion of aorta affects the post-operation mortality rate. However,
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the newly developed knot method eliminates the instant aortic occlusion and improves the
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post-operation mortality rate. Another potential cause of the reduced post-operation mortality rate is
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the uniformed constriction of the aortas within a sample group. In the process using the traditional
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needle method, the extents of aortic constriction among rats vary due to differences in the size of
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individual aortas. Therefore, it is possible that for any individual in the estimated 50% RC group, the
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actual % RC may be significantly greater or lesser than 50%. Since we observed increased mortality
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rate with increased degree of aortic constriction it is likely that animals subjected to the traditional
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needle procedure would include individuals with more severe aortic constriction than expected and
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an increased risk for post-operation death.
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This new knot method has some limitations in comparison to the traditional needle method. First,
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it takes more time for the precise measurement of aortic circumference and defining the distance
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between the two knots for the desired degree of aortic constriction. Second, it requires more practice
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to become sufficiently skillful for this procedure. Third, this new procedure would not be applicable
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for mice due to their much shorter aortic circumference.
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In conclusion, we developed a knot method of aortic constriction-induced heart hypertrophy and
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dysfunction in rats. This new method not only makes the measurable degree of aortic constriction
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attainable, but it also decreases the post-operation mortality rate. We recommend this method be
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adapted for experimental studies using animals bigger than rats for aortic constriction-induced heart
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hypertrophy and dysfunction.
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ACKNOWLEDGEMENTS
The authors thank Ms. Xiaorong Sun and Mr. Ning Wang for technical assistance and Professor
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Craig A. Grapperhaus (University of Louisville) for assistance with manuscript editing. This work
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was supported by National Science Foundation of China (grant number: 81230004 to Y. J. Kang) and
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Sichuan University West China Hospital. The funding sources had no influence in study design; in
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the collection, analysis and interpretation of data; in the writing of the report; and in the decision to
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submit the article for publication.
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DISCLOSURES
The authors declare that they do not have any conflict of interest.
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AUTHOR CONTRIBUTIONS
JmL, PH and YJK conceived the idea of developing the improved method; JmL developed the
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method and performed the experiments; JnL contributed to echocardiographic measurement; JmL
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and YX contributed to data collection and analysis; JmL and YJK wrote the manuscript. All authors
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read and approved the final version of the manuscript.
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Figure legends
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Fig. 1. Detailed procedure for the knot method of aortic constriction method. (A, B) Place a piece of
372
6-0 surgical thread underneath the aorta. Make a loop at "end-a" of the thread and place "end-b"
373
through the loop to make a noose knot by tightening the loop. (C) Pull "end-b" to tighten the noose
374
until the knot just touches, but does not constrict the outer wall of the artery. Trim excess thread at
375
"end-b" just before the knot site. (D) The noose is released with retaining the knot in the thread.
376
Measure the distance from the knot to "end-b". The distance is equal to the circumference of the
377
artery. (E, F) Make a noose knot as previously in a second piece of 6-0 surgical thread. Tie a
378
common knot at "end-b" at a distance from the noose knot calculated based upon the desired extent
379
of arterial constriction and the length of the circumference of the aorta. (G, H) Place the thread
380
underneath the artery. Place "end-b" through the noose and tighten the noose before the common
381
knot. (I) Pull "end-b" to constrict the aorta. Once the two knots meet, further constriction is not
382
possible. (J) Tie another common knot to secure the constriction and trim excess thread.
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Fig. 2. Pressure changes across the stenosis site one week after aortic constriction operation using the
385
knot method. (A) No difference between the sham-operated control, 50% RC, and 60% RC groups in
386
arterial end systolic pressure (AESP) prior to the stenosis site. (B) The elevation of the left ventricle
387
end systolic pressure (LVESP) in the 50% RC and 60% RC groups in comparison with the
388
sham-operated controls. (C) The gradient across the stenosis site (ESP increase %). Mean ± SD,
389
compared with Sham group, *P < 0.05; compared with sham and 50% RC group, #P < 0.05.
390
391
Fig. 3. Echocardiographic measurements of cardiac structural changes after aortic constriction
392
operation using the knot method. Increases in IVSD (A-C) and LVPWD (D-F) are proportional to
393
the extent of aortic constriction in the knot method groups as measured by echocardiograph at 2, 4, 8
19
394
weeks after the aortic constriction operation. Mean ± SD, compared with sham group, *P < 0.05;
395
compared with sham and 50% RC group, #P < 0.05.
396
397
Fig. 4. Cardiac hemodynamic responses to isoproterenol stimulation after aortic constriction
398
operation using the knot method. All measured parameters changed as a function of increasing
399
concentrations of isoproterenol; a blunted response of systolic function (LVESP and +dp/dt) in rats in
400
the 60% RC group was observed. Compared with sham group, *P < 0.05; compared with sham and
401
50% RC group, #P < 0.05.
402
403
Fig. 5. Postmortem and histological changes of rat hearts. (A) Increases in the heart size subjected to
404
knot method with 50% RC (center) and 60% RC (right) in comparison with sham-operated controls
405
(left). (B) Changes in the heart size between the knot method (center) and traditional needle method
406
(right) groups in comparison with sham-operated controls (left). (C-H) Masson’s trichrome staining
407
of heart tissue slides. Blue, fibrillar collagen; red, myocardium. Perivascular and interstitial fibrosis
408
was more serious in 60% RC group (right) than 50% RC group (center). (I) Average optical density
409
(AOD) value of collagen staining. (J) Quantitative analysis of the collagen volume fraction (CVF) in
410
three groups. Although no difference was observed in CVF, fibrosis was much denser in 60% RC
411
group. Mean ± SD, compared with sham group, *P < 0.05; compared with sham and 50% RC group,
412
#
P < 0.05.
413
20
414
Fig. 6. Pressure changes across the stenosis site one week after cardiac constriction operation. (A)
415
Arterial end systolic pressure (AESP), (B) Left ventricle end systolic pressure (LVESP), (C) The
416
gradient across the stenosis site (ESP increase %). Compared with sham group, *P < 0.05.
417
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Fig. 7. Echocardiographic measurement of cardiac morphological changes 8 weeks after aortic
419
constriction operation. (A) Interventricular septum depth (IVSD), (B) Left ventricular posterior wall
420
depth (LVPWD). Bigger variance was shown in ~50% RC group. Compared with sham group, *P <
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0.05.
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Fig. 8. Survival curve of rats for 8 weeks after aortic constriction operation. (A) Comparison
424
between rats subjected to knot method and those subjected to the traditional needle method at 50%
425
RC of the aorta, P< 0.05. (B) Comparison between rats subjected to 50% versus to 60% RC of the
426
aorta using the knot method, P< 0.05.
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21
Table 1. Tidal volumes and ventilation rates for rats (200–240 g)
mass (g)
tidal volume (ml)
ventilation rate (bpm)
200
1.22
81
210
1.28
80
220
1.34
79
230
1.41
78
240
1.47
78
Tidal volume does not account for system dead space.
Table 2. Postmortem analysis 8 weeks after constriction operation
N
HW
BW
TL
HW/BW
HW/TL
Sham
KM50%
KM60%
12
9
9
TM~50%
9
#
Value (g)
1.758±0.234
2.011±0.215*
2.356±0.240
Variance
0.055
0.046
0.058
2.144±0.300*
0.090
CV (%)
13.311
10.691
10.187
13.993
Value (g)
462±41
439±81
476±65
471±95
Variance
1741
6578
4353
9107
CV (%)
8.874
18.451
13.656
20.170
Value (cm)
4.601±0.135
4.619±0.075
4.488±0.123
4.453±0.104
Variance
0.018
0.006
0.015
0.011
CV (%)
2.934
1.624
2.741
2.336
Value (g/Kg)
3.811±0.450
4.678±0.745*
4.988±0.461*
4.657±0.788*
Variance
0.203
0.555
0.212
0.621
CV (%)
11.808
15.926
9.242
16.921
#
Value (g/dm)
3.822±0.502
4.354±0.460*
5.253±0.563
Variance
0.252
0.212
0.317
4.819±0.694*
0.481
CV (%)
13.134
10.565
10.718
14.401
Values are means ± SD; KM, knot method; TM, traditional needle method; N, number of rats; CV,
coefficient of variation; HW, heart weight; BW, body weight; TL, tibial length. Compared with sham group,
*
P< 0.05; compared with sham and KM50% group, #P < 0.05.
Table 3. Comparison of IVSD and LVPWD after surgical operation at three intervals
Sham
KM50%
KM60%
TM~50%
2W
N
18
20
18
19
IVSD
Value (mm)
0.857±0.148
0.890±0.072
1.181±0.076*
0.866±0.159
Variance
0.022
0.005
0.006
0.025
CV (%)
17.270
8.090
6.435
18.360
Value (mm)
0.831±0.100
0.881±0.061
1.131±0.110*
0.839±0.152
Variance
0.010
0.004
0.012
0.023
CV (%)
12.034
6.924
9.726
18.117
4W
N
18
19
16
14
IVSD
Value (mm)
0.876±0.176
1.231±0.114*
1.301±0.179*
1.229±0.152*
Variance
0.031
0.013
0.032
0.023
CV (%)
20.091
9.261
LVPWD
LVPWD
8W
IVSD
LVPWD
13.759
*
12.368
#
Value (mm)
0.942±0.120
1.188±0.101
1.407±0.143
1.206±0.150*
Variance
0.014
0.010
0.020
0.022
CV (%)
12.739
8.502
10.163
12.438
N
18
19
16
12
#
Value (mm)
1.181±0.058
1.483±0.146*
1.663±0.118
1.567±0.217*
Variance
0.003
0.021
0.014
0.047
CV (%)
4.911
9.845
7.100
13.848
#
Value (mm)
1.181±0.058
1.426±0.120*
1.604±0.116
1.465±0.207*
Variance
0.003
0.014
0.013
0.043
CV (%)
4.911
8.415
7.232
14.130
Values are means ± SD; KM, knot method; TM, traditional needle method; N, number of rats; CV,
coefficient of variation; IVSD, interventricular septum depth; LVPWD, left ventricular posterior wall depth.
Compared with sham group, *P< 0.05; compared with sham and KM50% group, #P < 0.05.
Table 4. Hemodynamics without isoproterenolstimulation8 weeks after constriction operation
HR
AESP
LVESP
LVEDP
Sham
KM50%
KM60%
TM~50%
N
10
8
8
8
Value (b/m)
328±56
365±71
373±63
394±74
Variance
3090
5080
4045
5481
CV (%)
17.1
19.5
16.9
18.8
Value (mmHg)
106.5±9.9
104.7±13.5
98.1±12.0
102.8±17.6
Variance
98.6
183.2
145.1
311.3
CV (%)
9.3
12.9
12.2
17.1
#
Value (mmHg)
104.2±10.2
185.3±46.0*
241.2±30.6
165.9±58.2*
Variance
103.8
2115.6
935.7
3389.7
CV (%)
9.8
24.8
12.7
35.1
Value (mmHg)
9.0±1.1
10.2±1.1
11.0±1.4
10.0±1.2
Variance
1.1
1.2
1.9
1.5
CV (%)
12.2
10.8
12.7
12.0
Values are means ± SD; KM, knot method; TM, traditional needle method; N, number of rats; CV,
coefficient of variation; HR, heart rate; AESP, arterial end systolic pressure; LVESP, left ventricular end
systolic pressure; LVEDP, left ventricular end diastolic pressure. Compared with sham group, *P< 0.05;
compared with sham and KM50% group, #P < 0.05.
Table 5. Mortality in different groups 8 weeks after constriction operation
Animal (NO)
Sham
20
KM50%
Death (NO)
Mortality (%)
2
10
25
6
24*
KM60%
30
14
47
TM~50%
25
13
52
#
#
KM, knot method; TM, traditional needle method. Compared with sham group, *P < 0.05;
compared with sham and KM50% group, #P < 0.05.