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Introduction
Banding of the ascending aorta in rodents is the most commonly used method for generating a small animal model of aortic stenosis (AS), even though the left ventricular hypertrophy (LVH) produced by acute, severe pressure overload is unlike the slow, progressive pressure overload in patients with AS.1)Aortic banding causes LVH and reactive interstitial fibrosis, which have a diffuse distribution within the interstitium, but fibrosis can also be more specifically perivascular, which is observed in patients with AS. Recently, a reproducible mouse model of reverse remodeling by banding and debanding of the ascending aorta was reported.1),2),3),4)In AS patients, longitudinal strain was decreased but circumferential strain (CS) increased (perhaps as a compensatory mechanism) before aortic valve replacement (AVR). These abnormalities were reversed after AVR.5),6),7),8)Two-dimensional speckle-tacking echocardiography (STE) strain has become the leading method for assessing subclinical left ventricular (LV) systolic abnormalities, because it overcomes the limitations of conventional echocardiography in murine models.9)10)This study aimed to evaluate the LV mechanics and associated pathology in a banding and debanding model with a two-dimensional STE.
Methods
Animal study protocol
The ascending aorta banding procedure was performed in 20 rats. Among them, 10 rats were euthanized 5 weeks later. The other 10 rats underwent a debanding operation and were then sacrificed 14 days later. Sham operations were performed in additional 10 rats and were sacrificed 7 weeks later.
Seven-week-old male Sprague Dawley rats were anaesthetized with ketamine (80 mg/kg, intraperitoneally) and xylazine (8 mg/kg, intraperitoneally) and then orotracheal intubations were performed with a 20-gauge IV catheter. Mechanical ventilation was maintained with a Mini-Vent ventilator (Harvard Apparatus, Holliston, MA, USA) containing 2% isoflurane and 98% O2 at a tidal volume of 350 µL and respiratory frequency of 160 min-1.
The ascending aorta banding procedure was performed in 20 rats through upper hemi-sternotomy with careful thymus separation. Then, the ascending aorta was freely dissected. Banding was performed with a Teflon (DuPont Pharmaceuticals, Wilmington, DE, USA) felt supported 5-0 silk ligation around the ascending aorta and a 18-G blunted needle. The Teflon felt that supported aortic banding was helpful for the subsequent debanding operation. The sternum was fixed and the muscle layers and skin were closed with 4-0 silk sutures in three layers. The sham operation was performed as the same thoracotomy procedure without ligation in another 10 rats (Fig. 1). Following surgery, ketoprophen (5 mg/kg, intramuscular, once a day) and gentamycin (5 mg/kg, intramuscular, once a day) were administered over a period of 5 days.
The debanding operation was performed at 5 weeks after aortic banding for 10 rats. The pre- and post-operative procedure was same as the banding operation. Right-sided, muscle-saving thoracotomy for the debanding operation was performed in the second intercostal space. After the banding site was identified, the debanding procedure was performed with cutting the banded silk on the Teflon felt by which aortic rupture was avoided. The thymus was repositioned, and the chest was closed in three layers. For the sham operation group, a similar thoracotomy procedure was performed. The remained 10 rats in the aortic banding model were killed 35 days after the banding operation. Debanding group was sacrificed 14 days after the debanding operation. Sham group was killed 7 weeks after sham operation. The chest was opened, and the hearts were excised.
The study protocol was approved by the Ethics Committee of Daejeon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Daejeon, and Republic of Korea(DMDDJ-AP-2012-020).
Echocardiography
Echocardiography was performed three times in all subjects at baseline, 5 weeks post banding, and then 2 weeks post debanding. Effective banding procedures were confirmed with echocardiographic findings of a maximal peak velocity of the ligation site more than 3 m/sec for the banding group. Relieved constriction of the ascending aorta after debanding procedure was also documented in echo-Doppler findings.
Animals were anesthetized with ketamine (80 mg/kg, intraperitoneally) and xylazine (8 mg/kg, intraperitoneally) during echocardiographic examinations. To obtain a physiologically relevant depth of sedation during echocardiography, the heart rate was maintained at approximately 270-320 beats per minute. Echocardiography was performed using a 15 MHz linear array transducer (Sequoia Acuson system, Mountain View, CA, USA). An acoustic capture B-mode cine clip (120 Hz) (Sequoia Acuson system, Mountain View, CA, USA) with electrocardiographic gating provided 20-30 frames/beat (≥ 110 frames/s) using the smallest possible depth and sector size.11)Off-line analysis was performed with velocity vector imaging (VVI) using Syngo SC2000 workplace (Siemens Medical Solutions Inc., Mountain View, CA, USA). M-mode images from the parasternal long axis view were used to measure conventional echocardiographic parameters [LV end-diastolic dimension, LV end-systolic dimension, interventricular septal dimension, LV mass, fractional shortening (FS), and LV ejection fraction (EF)].12) B-mode clips were acquired in the mid-level region of the parasternal short axis view. The epicardial and endocardial LV borders were manually traced and accurate tracings were verified for more than 3 cycles at end-systole to measure the peak CS and radial strain (RS).11)13) A single investigator who was blinded to the animal groups performed all image acquisitions and offline measurements.
Histopathological analysis
After euthanasia, the hearts were removed, fixed in 4% formaldehyde and embedded in paraffin. Three mid-ventricular sections were stained with hematoxylin-eosin for histological analysis, Periodic acid-Schiff (PAS) strain for determining for size of cardiomyocyte, and Masson's trichrome for determining fibrosis. The histopathologic evaluation of each slides was performed blinded.
Image J version 1.44 (US National Institutes of Health, Bethesda, MD, USA) was used to quantitatively measure fibrosis by determining the blue-stained area in each section. The cardiomyocyte size was measured in each 40X-magnified section area on PAS stained slides.
Statistical analysis
Statistical analysis was performed using commercially available software (SPSS version 18, SPSS Inc., Chicago, IL, USA). The data are presented as the mean value ± standard deviation.
Comparisons among three groups were made using one-way analysis of variance with post-hoc Tukey tests. The relationships between fibrosis and the peak CS were examined using Pearson's correlation coefficient and linear regression analysis. A p value < 0.05 was considered statistically significant.
Results
Compared with the sham operation group, all rats in the banding group had a significantly higher LV mass index after 5 weeks of the aortic banding procedure. The debanding group had significant dilatation of the LV chamber dimension, lower EF and lower FS compared to the banding group (Table 1).
Two-dimensional STE showed no difference in the global peak CS and RS between the sham, banding, and debanding groups (-25.7 ± 6.0 vs. -23.7 ± 5.8 vs. -23.7 ± 5.0, p = 0.661 for CS and 29.8 ± 7.9 vs. 29.0 ± 8.6 vs. 32.6 ± 9.5, p = 281 for RS) (Table 2). In the histological analysis, the size of the myocyte was significantly larger in the banding group than in the sham and debanding groups and was reversed after debanding (Table 2, Fig. 2 and 3). However, myocardial fibrosis was prevalent in the banding and debanding groups compared to the sham group (Table 2, Fig. 3). The 5-weekbanding procedure provoked minimal fibrosis that was lower than approximately 12%. The global peak CS was significantly correlated with the fibrosis severity in the banding group (r = 0.688, p = 0.028) (Fig. 4 and 5). However, there were no correlations between the global peak RS and fibrosis in the banding group (r = -0.618, p = 0.057) (Fig. 4 and 5).
Discussion
In this animal study simulating a severe LV pressure overload state, there was a significant increase in the LV mass index, which was reversible after debanding. Unlike for reversible LVH, myocardial fibrosis did not recover with the debanding procedure 2 weeks later. Quantitative measurement of fibrosis after banding was significantly related to the magnitude of LV mechanics. A mouse model of banding and debanding of the ascending aorta was used to investigate reverse cardiac remodeling. In the pressure overload state, myocytes undergo hypertrophy and proliferated fibroblasts increase extracellular matrix deposition, including collagen accumulation with the activation of bioactive molecules.14)15) Fibrosis induced LV pressure overload states included reactive interstitial fibrosis. Cardiomyocyte hypertrophy and reactive fibrosis are associated with increased ventricular stiffness and deformation that could be determined by myocardial mechanics.8)16)17) STE, such as CS and RS, have been used to better characterize the regional and global myocardial systolic and diastolic function than EF. In the present study, peak CS was correlated with myocardial fibrosis at banding and was not recovered within the 2 weeks after debanding. Peak RS was not significantly correlated with myocardial fibrosis in present study. Also, Peng et al.9) reported that CS efficiently detected the progression of fibrotic changes in mouse model with transverse aortic banding operation. On the other hand, RS showed little concordance with transverse aortic banding induced fibrosis.
Longitudinal strain was decreased in severe AS patients with normal EF. However, CS was increased before AVR and then reversed after AVR according to LV compensation.5),6),7),8)The longitudinal muscle fiber are located in the subendocardium, which is easily affected by increased intracardiac pressures. In addition, due to a larger radius curvature, longitudinal fibers experience greater stress compared to circumferential fibers. Therefore, longitudinal strain analysis could be important in the pressure overload status. However, the longitudinal strain could not be analyzed because of the difficulty of window acquisition in murine model.18) CS was not significantly changed after banding and debanding without a compensation mechanism. An explanation for this discrepant result may be that the severe constriction technique causes acute hemodynamic instability with a reduction in the EF and disturbances in the compensatory increase of CS. Additionally, CS might be a parameter that contributes to the maintenance of LV systolic performance.18)
The debanding procedure had high mortality rate of more than 10% because of aortic rupture during dissection of periaortic adhesions.2)3)In the present study, Teflon-felt supported banding of the ascending aorta could reduce the mortality by avoiding aorta injury during cutting of the banded silk. A decreased pressure gradient after the debanding operation was confirmed by non-invasive Doppler echocardiography.
In our study, reactive interstitial fibrosis was observed after the banding operation. Approximately 5% reactive fibrosis was developed in this study, which was a little higher than in the 4 week banding model, which had less than 2-3% fibrosis. Reactive fibrosis in the aortic banding model is a progressive worsening according to the banding duration until 6-12 months.17)Additionally, interstitial fibrosis precedes irreversible replacement fibrosis and is reversible under agents blocking the renin-angiotensin system.16)19)In our study, fibrosis was not significantly reversible within 2 weeks after debanding, there was no medication and there was a longer banding duration than previous studies.2)3)9)
Several limitations of this study should be considered. The sample size was a small and the frame rate relative to the heart cycle duration was lower than in previous studies performed in humans and animals. Higher frame rates were reported using different instrumentation and algorithms.20)However, a validation study comparing the findings with magnetic resonance imaging showed the feasibility using a VVI and used the same instrument as in the present study.11)Changes in the imaging angle of incidence can result in capturing different fiber layers at different levels and may introduce variability. Echocardiography was performed in rats that were sedated with a ketamine injection that could decrease heart performance. However, the ketamine dosage was kept to a minimum to induced sedation alone. Additionally, all of the groups were treated with identical methods. The velocity of the banding and debanding site was not estimated with invasive carotid catheterization, but we used echocardiography to determine whether the banding and debanding model was appropriate.
In conclusion, left ventricular fibrosis, developed in a pressure overload condition was irreversible within 2 weeks after debanding and was significantly related to deterioration in LV mechanics. Future investigation is needed to evaluate the cutoff point of the quantitatively assessed degree of fibrosis matched with reversible impairment of LV mechanics in a pressure overloaded animal model.
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