Subjects and housing
This study was conducted at the CNPRC which is the United States Department of Agriculture-registered and the Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. The CNPRC maintains an approval from the Institutional Animal Care and Use Committee of the University of California-Davis and Public Health Services Assurance. All rhesus macaques enrolled in the study were housed in the CNPRC. Echocardiographic examination and blood pressure measurement under sedation were implemented as a part of routine examinations in the CNPRC, and these examinations were performed to all healthy rhesus macaques before assigned to other experiments, allocations, and transportation to other facilities. All rhesus macaques were returned to their cages after the examination once they were fully recovered from sedation. None of animals were euthanized for completion of this study.
All rhesus macaques at this facility are taken care in accordance with the Animal Welfare Act and Guide for the Care and Use of Laboratory Animals [43]. Outdoor rhesus macaques are all housed as groups in rectangular enclosures sized 0.5-acre. Most of rhesus macaques housed indoor are paired in stainless steel cages sized based on the regulation for primary cage-space limitation. Some of indoor rhesus macaques are housed individually in the same indoor condition. All rhesus macaques are managed with environmental enrichment. They were fed primate chow (LabDiet Monkey Diet 5047, Purina Mills International, St Louis, MO) with vegetables and fruits supplement. Water is provided using automatic watering devise to animals without any restriction to access. Room lighting in the indoor room for the indoor rhesus macaques is automatically controlled with alternating 12 h:12 h light and darkness. Complete physical examination and blood tests including complete blood count analysis and serum biochemistry are performed accordingly. Tuberculosis testing and dental prophylaxis are also performed accordingly. When health issues are noted, these rhesus macaques were transferred to a separated colony and excluded from the present study. All rhesus macaques were also monitored for possible viral infections (herpes B virus, simian type D retrovirus, simian immunodeficiency virus, and simian T-lymphotropic virus).
Sedation
Ketamine hydrochloride (10 mg/kg IM; Ketaject, Phoenix Pharmaceutical, St. Joseph, MO) was given within five to 10 min prior to echocardiographic examination and blood pressure measurement for sedation for all animals. If necessary, an additional dose of ketamine (5–10 mg/kg IM) was given during echocardiographic assessment to maintain appropriate sedation.
Echocardiographic measurement
Echocardiographic examination was performed using one of two echocardiographic ultrasound devices (Affiniti 50, Phillips, Best, Netherland, and CX50 Ultrasound System, Phillips, Best, Netherlands) with a 4- to 12-mHz sector-array transducer or a 1- to 5-mHz sector-array transducer. Animals were positioned in right and left lateral recumbency subsequently during the procedure, and 2D and M-mode echocardiography with color and spectral Doppler was performed. In this study, the leading-edge to leading-edge method was employed, and three consecutive measurements were saved for each echocardiographic parameter. All results were analyzed by an author (YU) and reviewed by an ACVIM board-certified cardiologist (JS) using an offline software (Syngo Dynamics, Siemens, Erlangen, Germany) in accordance with the guidelines [44]. After comleting measurement and assessment, rhesus macaques without any significant cardiac abnormalities and changes were selected as control animals from the echocardiographic database developed by the authors (YU, JS).
Complete echocardiographic examination was performed as previously reported as a part of routine screening and for other ongoing projects by a veterinary cardiologist (JS) or a cardiology research fellow (YU) under the direction of a veterinary cardiologist [3]. Briefly, on right parasternal long-axis four-chamber views, left atrial diameter in diastole (LA [la]) and aortic root diameter in diastole (Ao [la]) were acquired (Fig. 1a and b). On right parasternal short-axis views, LA (LA [sa]) and Ao (Ao [sa]) were acquired at the level of the aortic valve (Fig. 1c). The interventricular septal thickness during diastole (IVSd) and left ventricular posterior wall thickness in diastole (LVPWd) were obtained from the two-dimensional (2D) right parasternal long-axis and short-axis 2-chamber views, and the maximal thickness of these parameters were reported as IVSd (2D) and LVPWd in this study (2D) (Fig. 1a and d). The interventricular septal thickness during systole (IVSs) and diastole (IVSd), left ventricular posterior wall thickness in systole (LVPWs) and diastole (LVPWd), left ventricular internal diameter during systole (LVDs) and diastole (LVDd), and mitral valve E-point to septum separation (EPSS) were measured from the right parasternal short-axis M-mode views at the chordae level (Figs. 1d, 2a, and b). From the right parasternal short-axis right ventricular outflow tract view at the level of the aortic valve, peak pulmonary flow velocity (PV Vmax) and its acceleration time (PV AT), and ejection time (PV ET) were obtained. The sample gate using the pulsed-wave spectral Doppler technique was positioned immediately distal to the pulmonic valve (Fig. 2c and d). On the left parasternal apical four-chamber view, passive early filling (E wave) and atrial contraction later filling (A wave) velocities were obtained (Fig. 1e). The sample gate using the continuous spectral Doppler technique was positioned at the tips of the mitral valve leaflets when they were completely open (Fig. 2e). Color-tissue Doppler imaging (TDI) was performed to obtain free-wall (lateral) and septal (medial) mitral annulus motions from the left apical 4-chamber view. Peak velocities were measured in early (E’ [medial] and E’ [lateral]) and late diastole (A’ [lateral] and A’ [lateral]) (Fig. 2f). Aortic flow profile with maximal aortic flow velocity (Ao Vmax) was obtained from left parasternal apical aortic outflow view with parallel alignment to the aorta using the continuous spectral Doppler technique (Fig. 2g and h) [45, 46]. Acceptable parallel alignment of the Doppler gate was possible in all rhesus macaques, no angle corrections were performed. Using the same images, isovolumic relaxation time (LV IVRT) and mitral E deceleration time (MV DT) were also measured. On the left apical four-chamber view optimized for the right cardiac chambers, tricuspid annular plane systolic excursion (TAPSE) was obtained based on the M-mode by qualifying the maximal longitudinal displacement of the lateral tricuspid valve annulus toward the right ventricular apex during systole. During the measurements, the cursor was placed as parallel as possible to the majority of the right ventricular free wall (Fig. 2i). Pulsed-wave TDI velocities of longitudinal myocardial motion at the lateral tricuspid annulus were also obtained to measure peak systolic annular velocity (RV S′ Vmax) (Fig. 2j).
Using the color Doppler technique, the presence and severity of valve regurgitations were determined on all four cardiac valves as previously performed [3]. Briefly, the severity of aortic regurgitation was categorized as mild (ratio of jet height to left ventricular outflow tract width less than 24%), moderate (25 to 64%), or severe (greater than 65%). The severity of mitral valve regurgitation was categorized as mild (jets occupying less than 29 of left atrial area), moderate (30 to 69%), or severe (more than 70%). The severity of tricuspid valve regurgitation was categorized as mild (right ventricular, right atrium, and vena cava all normal size), moderate (normal or dilated right ventricle, right atrium, or vena cava), or severe (all dilated). Pulmonic regurgitation was quantified as mild (thin narrow origin jet with normal right ventricular size), moderate (wide origin jet with normal or mildly dilated right ventricular size), or severe (wide origin jet with severely dilated right ventricle). Left ventricular outflow tract obstruction (LVOTO) was diagnosed by color Doppler evaluation from right parasternal long axis or left parasternal left ventricular outflow view. Rhesus macaques with moderate or severe valve regurgitation and those with LVOTO were excluded from the present study. Rhesus macaques with no or mild valve regurgitation were enrolled in the present study as long as no other structural and/or functional abnormalities were noted on the echocardiographic examination.
Left ventricular fractional shortening (LV FS) and ejection fraction (LV EF) were calculated as previously described and the animals with LV FS less than 25% and/or LV EF less than 50% were excluded from this study [3].
Diastolic dysfunction was diagnosed by pulsed-wave spectral Doppler trans-mitral filling patterns with a ratio of early to late filling velocities ≤0.9, or with the spectral tissue Doppler lateral or medial peak mitral annular velocity during early and late filling phase ≤0.9 [3, 10, 42]. Rhesus macaques < 18 years old were excluded from this study if they were diagnosed with diastolic dysfunction. Rhesus macaques ≥18 years old with diastolic dysfunction were not excluded from this study as long as there was no concurrent structural or function abnormalities noted on echocardiographic examination [3]. However, the reference intervals of several echocardiographic parameters, including A wave, E wave, E/A, LV IVRT, E:LV IVRT, E’ wave medial and lateral, A’ wave medial and lateral, E’/A’ medial and lateral, E/E’ medial and lateral, were obtained by excluding the cohort of geriatric rhesus macaques that had isolated diastolic dysfunction.
Intra-observer measurement variability was determined by having one of the authors (YU) measure blinded echocardiographic parameters twice on different days from saved echocardiographic images from ten randomly selected rhesus macaques with good image quality. Inter-observer measurement variability was calculated by having two of the authors (YU, LD) measure all echocardiographic parameters, while they are blinded to each other’s measurements.
Blood pressure measurement
Under sedation, indirect BP measurement was performed in 181 rhesus macaques using an oscillometric systemic BP measurement device (Cardell 9401, Midmark Corp, Versailles, OH, USA) at the same time of echocardiographic examination. Briefly, animals had a systolic, mean and direct BP measured on the left forelimb while the animal was in right lateral recumbency [47]. BP was measured two to three times ensuring that the displayed heart rate on the BP measurement device was confirmed to match the heart rate obtained on echocardiogram and all obtained values were averaged.
Statistical analysis
This study is a prospective observational study to establish references intervals for echocardiographic parameters on healthy rhesus macaques. 823 rhesus macaques were enrolled in the study when echocardiographic examination was performed as a part of routine examination before assigning to experiments, allocation, and transportation. All data obtained were acquired by the authors (YU, JS) between January 2015 and November 2019 at the CNPRC.
The mean percent coefficient of variation (CV) was calculated for intraobserver and interobserver measurement variabilities using an equation: CV = (SD of the measurement/average of measurement) × 100.
D’Agostino-Pearson test was performed for testing normality of continuous data. Descriptive statistics (mean, SD, and range) was provided as mean with SD or median with interquartile rage for normally distributed parameters and for non-normally distributed parameters, respectively. Outliers were determined and removed by performing post-hoc Tukey test, and double-sided 95% reference intervals were established. Ninety percent confidence interval for each reference limit was determined without the robust method [16].
Pairwise Pearson correlation analysis with animal characteristics (BW, age, sex, HR, and systolic and mean BP) and echocardiographic variables were conducted. The degree of correlation was determined with r as weak correlation with r < 0.3, moderate correlation with 0.3 ≤ r ≤ 0.5, strong correlation with r > 0.5.
Multiple regression analysis was conducted between all animal characteristics and the echocardiographic parameters. A model was performed stepwise using BW (kg), age (in days), heart rate (HR; bpm), and sex. The coefficient of the linear association with each echocardiographic parameter and its associated p-value was obtained. The coefficients represent the mean change in parameters for an increase of one unit of the explanatory variable while other variables are kept constant.
Linear echocardiographic variables were normalized to BW (kg) based on the constants from power equation, Y = aXb or allometric scaling. In this equation, a, b, Y, and X represent proportionality constant, scaling exponent, and linear echocardiographic parameter, and BW, respectively. Linear regression analysis was then conducted on log10(BW) versus log10 (echo parameter) for each echocardiographic parameter. This process produced the equation, log(Y) = log (a) + b x log(x), where b and a represent the slope and antilog Y-intercept, respectively. The constant (ac) based on the formula: ac = log10− 1[log(a) +/− t x Sx,y] determined prediction intervals, where a, t, and Sx,y represent the proportionality constant, desired Student’s t-statistic for n-2 degrees of freedom, and the standard error of the Y estimate, respectively.
Statistical analysis was performed using commercially available softwares (MedCalc version 12.7.4, MedCalc Software, Ostend, Belgium and Stata Corporation v15.1, College Station, TX). A P-value of < 0.05 was considered as significant for all analyses.