Spontaneous Degenerative Aortic Valve Disease in New Zealand Obese Mice.

Background Degenerative aortic valve (AoV) disease and resulting aortic stenosis are major clinical health problems. Murine models of valve disease are rare, resulting in a translational knowledge gap on underlying mechanisms, functional consequences, and potential therapies. Naïve New Zealand obese (NZO) mice were recently found to have a dramatic decline of left ventricular (LV) function at early age. Therefore, we aimed to identify the underlying cause of reduced LV function in NZO mice. Methods and Results Cardiac function and pulmonary hemodynamics of NZO and age-matched C57BL/6J mice were monitored by serial echocardiographic examinations. AoVs in NZO mice demonstrated extensive thickening, asymmetric aortic leaflet formation, and cartilaginous transformation of the valvular stroma. Doppler echocardiography of the aorta revealed increased peak velocity profiles, holodiastolic flow reversal, and dilatation of the ascending aorta, consistent with aortic stenosis and regurgitation. Compensated LV hypertrophy deteriorated to decompensated LV failure and remodeling, as indicated by increased LV mass, interstitial fibrosis, and inflammatory cell infiltration. Elevated LV pressures in NZO mice were associated with lung congestion and cor pulmonale, evident as right ventricular dilatation, decreased right ventricular function, and increased mean right ventricular systolic pressure, indicative for the development of pulmonary hypertension and ultimately right ventricular failure. Conclusions NZO mice demonstrate as a novel murine model to spontaneously develop degenerative AoV disease, aortic stenosis, and the associated end organ damages of both ventricles and the lung. Closely mimicking the clinical scenario of degenerative AoV disease, the model may facilitate a better mechanistic understanding and testing of novel treatment strategies in degenerative AoV disease.

Large Farm Animals Used for Research Purposes: A Survey on Purchase, Housing and Hygiene Management.

BACKGROUND: Farm animals (FAs) are frequently used in biomedical research. Recommendations for the purchase, housing and health monitoring of these animals (sheep, goats, cattle and pigs) are still missing, and many institutes have developed their own strategies and protocols to face the challenges associated with the use of farm animals. This may influence the comparability of research results and increase data variances, thus increasing animal use that contradicts the obligation to apply the 3Rs principle of reduction, refinement and replacement required in Directive 2010/63 EU and the German animal protection law. METHODS: A survey was conducted to define the current state of the art in research institutes working with pigs, and large and small ruminants. RESULTS: The results of the survey clearly show that there are no uniform procedures regarding the purchase, housing and hygiene management of farm animals contrary to small laboratory animals. The facilities make purpose-bound decisions according to their own needs and individual work instructions and implement their own useful protocols to improve and maintain the health of the animals. CONCLUSION: This survey was the first step to filling the gaps and identifying the status quo and practical applied measures regarding the purchase and hygiene monitoring of FAs in order to improve animal welfare and scientific validity.

Breeding and Maintenance of Immunodeficient Mouse Lines under SPF Conditions-A Call for Individualized Severity Analyses and Approval Procedures.

In the EU, the breeding of genetically modified laboratory animals is, by definition, an animal experiment if the offspring may experience pain, suffering, or harm. In order to determine the actual burden of genetically modified mice, established methods are available. However, the breeding of immunodeficient mice is considered an experiment requiring a permit, even if no pain, suffering or harm is observed under scientifically required defined hygienic housing conditions, as determined by established methods of severity assessment. This seems contradictory and leads to uncertainty among scientists. With this commentary, we would like to shed light on this topic from different perspectives and propose a solution in terms of individualized severity assessment and approval procedures.

Sex-Specific Differences of the Inflammatory State in Experimental Autoimmune Myocarditis.

Increasing evidence suggests male sex as a potential risk factor for a higher incidence of cardiac fibrosis, stronger cardiac inflammation, and dilated cardiomyopathy (DCM) in human myocarditis. Chronic activation of the immune response in myocarditis may trigger autoimmunity. The experimental autoimmune myocarditis (EAM) model has been well established for the study of autoimmune myocarditis, however the role of sex in this pathology has not been fully explored. In this study, we investigated sex differences in the inflammatory response in the EAM model. We analyzed the cardiac function, as well as the inflammatory stage and fibrosis formation in the heart of EAM male and female rats. 21 days after induction of EAM, male EAM rats showed a decreased ejection fraction, stroke volume and cardiac output, while females did not. A significantly elevated number of infiltrates was detected in myocardium in both sexes, indicating the activation of macrophages following EAM induction. The level of anti-inflammatory macrophages (CD68+ ArgI+) was only significantly increased in female hearts. The expression of Col3A1 and fibrosis formation were more prominent in males. Furthermore, prominent pro-inflammatory factors were increased only in male rats. These findings indicate sex-specific alterations in the inflammatory stage of EAM, with a pro-inflammatory phenotype appearing in males and an anti-inflammatory phenotype in females, which both significantly affect cardiac function in autoimmune myocarditis.

Measuring myocardial extracellular volume of the right ventricle in patients with congenital heart disease.

The right ventricle´s (RV) characteristics-thin walls and trabeculation-make it challenging to evaluate extracellular volume (ECV). We aimed to assess the feasibility of RV ECV measurements in congenital heart disease (CHD), and to introduce a novel ECV analysis tool. Patients (n = 39) and healthy controls (n = 17) underwent cardiovascular magnetic resonance T1 mapping in midventricular short axis (SAX) and transverse orientation (TRANS). Regions of interest (ROIs) were evaluated with regard to image quality and maximum RV wall thickness per ROI in pixels. ECV from plane ROIs was compared with values obtained with a custom-made tool that derives the mean T1 values from a "line of interest" (LOI) centered in the RV wall. In CHD, average image quality was good (no artifacts in the RV, good contrast between blood/myocardium), and RV wall thickness was 1-2 pixels. RV ECV was not quantifiable in 4/39 patients due to insufficient contrast or wall thickness < 1 pixel. RV myocardium tended to be more clearly delineated in SAX than TRANS. ECV from ROIs and corresponding LOIs correlated strongly in both directions (SAX/TRANS: r = 0.97/0.87, p < 0.001, respectively). In conclusion, RV ECV can be assessed if image quality allows sufficient distinction between myocardium and blood, and RV wall thickness per ROI is ≥ 1 pixel. T1 maps in SAX are recommended for RV ECV analysis. LOI application simplifies RV ECV measurements.

Stress hormones or general well-being are not altered in immune-deficient mice lacking either T- and B- lymphocytes or Interferon gamma signaling if kept under specific pathogen free housing conditions.

It is controversially discussed whether immune-deficient mice experience severity in the absence of infection. Because a comprehensive analysis of the well-being of immune-deficient mice under specific pathogen free conditions is missing, we used a multi-parametric test analyzing, corticosterone, weight, nest building and facial expression over a period of 9 month to determine the well-being of two immune-deficient mouse lines (recombination activating gene 2- and interferon gamma receptor-deficient mice). We do not find evidence for severity when comparing immune-deficient mice to their heterozygous immune-competent littermates. Our data challenge the assumption that immune-deficiency per se regardless of housing conditions causes severity. Based on our study we propose to use objective non-invasive parameters determined by laboratory animal science for decisions concerning severity of immune-deficient mice.

Serelaxin Improves Regional Myocardial Function in Experimental Heart Failure: An In Vivo Cardiac Magnetic Resonance Study.

Background Animal studies demonstrated that serelaxin lessens fibrosis in heart failure. This study assessed its effect on myocardial deformation using cardiac magnetic resonance and elucidated its relationship to gene regulation and histology in a mouse heart failure model. Methods and Results C57BL/6J mice were subjected to SHAM (n=4) or transverse aortic constriction (TAC). At week 10, TAC mice were randomized to receive either serelaxin (0.5 mg/kg per day; n=11) or vehicle (n=13) for 4 weeks. Cardiac magnetic resonance imaging was performed at baseline and repeated at the end of the study (week 14). Cine images were used to calculate left ventricular (LV) global longitudinal, circumferential, and radial strain. Hearts were examined for histology and gene expression. Compared with SHAM, mice 10 weeks after TAC showed increased LV mass with significant decreases in LV deformation parameters, indicating subclinical deterioration of myocardial function. At week 14, TAC mice given serelaxin demonstrated significant improvements in all LV strain parameters and no decrease in LV stroke volume and ejection fraction compared with TAC mice given vehicle. A significant positive correlation between global circumferential strain and the extent of myocardial fibrosis was found, and global circumferential strain correlated significantly with the expression of heart failure genes in serelaxin-treated mice. Conclusions Serelaxin improved cardiac magnetic resonance-derived myocardial deformation parameters as well as histomorphometric and gene expression findings in mice with heart failure. Cardiac magnetic resonance-derived myocardial mechanics correlate with histology and gene expression, stressing its utilization in myocardial remodeling.

Z-score mapping for standardized analysis and reporting of cardiovascular magnetic resonance modified Look-Locker inversion recovery (MOLLI) T1 data: Normal behavior and validation in patients with amyloidosis.

BACKGROUND: T1 mapping using modified Look-Locker inversion recovery (MOLLI) provides quantitative information on myocardial tissue composition. T1 results differ between sites due to variations in hardware and software equipment, limiting the comparability of results. The aim was to test if Z-scores can be used to compare the results of MOLLI T1 mapping from different cardiovascular magnetic resonance (CMR) platforms. METHODS: First, healthy subjects (n = 15) underwent 11 combinations of native short-axis T1 mapping (four CMR systems from two manufacturers at 1.5 T and 3 T, three MOLLI schemes). Mean and standard deviation (SD) of septal myocardial T1 were derived for each combination. T1 maps were transformed into Z-score maps based on mean and SD values using a prototype post-processing module. Second, Z-score mapping was applied to a validation sample of patients with cardiac amyloidosis at 1.5 T (n = 25) or 3 T (n = 13). RESULTS: In conventional T1 analysis, results were confounded by variations in field strength, MOLLI scheme, and manufacturer-specific system characteristics. Z-score-based analysis yielded consistent results without significant differences between any two of the combinations in part 1 of the study. In the validation sample, Z-score mapping differentiated between patients with cardiac amyloidosis and healthy subjects with the same diagnostic accuracy as standard T1 analysis regardless of field strength. CONCLUSIONS: T1 analysis based on Z-score mapping provides consistent results without significant differences due to field strengths, CMR systems, or MOLLI variants, and detects cardiac amyloidosis with the same diagnostic accuracy as conventional T1 analysis. Z-score mapping provides a means to compare native T1 results acquired with MOLLI across different CMR platforms.

Assessment of Bones Deficient in Fibrillin-1 Microfibrils Reveals Pronounced Sex Differences.

Defects in the extracellular matrix protein fibrillin-1 that perturb transforming growth factor beta (TGFß) bioavailability lead to Marfan syndrome (MFS). MFS is an autosomal-dominant disorder, which is associated with connective tissue and skeletal defects, among others. To date, it is unclear how biological sex impacts the structural and functional properties of bone in MFS. The aim of this study was to investigate the effects of sex on bone microarchitecture and mechanical properties in mice with deficient fibrillin-1, a model of human MFS. Bones of 11-week-old male and female Fbn1mgR/mgR mice were investigated. Three-dimensional micro-computed tomography of femora and vertebrae revealed a lower ratio of trabecular bone volume to tissue volume, reduced trabecular number and thickness, and greater trabecular separation in females vs. males. Three-point bending of femora revealed significantly lower post-yield displacement and work-to-fracture in females vs. males. Mechanistically, we found higher Smad2 and ERK1/2 phosphorylation in females vs. males, demonstrating a greater activation of TGFß signaling in females. In summary, the present findings show pronounced sex differences in the matrix and function of bones deficient in fibrillin-1 microfibrils. Consequently, sex-specific analysis of bone characteristics in patients with MFS may prove useful in improving the clinical management and life quality of these patients, through the development of sex-specific therapeutic approaches.

Evaluation of a commercial multi-dimensional echocardiography technique for ventricular volumetry in small animals.

BACKGROUND: The assessment of ventricular volumes using conventional echocardiography methods is limited with regards to the need of geometrical assumptions. In the present study, we aimed to evaluate a novel commercial system for three-dimensional echocardiography (3DE) in preclinical models by direct comparison with conventional 1D- and 2D-echocardiography (1DE; 2DE) and the gold-standard technique magnetic resonance imaging (MRI). Further, we provide a standard operating protocol for image acquisition and analysis with 3DE. METHODS: 3DE was carried out using a 30 MHz center frequency transducer coupled to a Vevo®3100 Imaging System. We evaluated under different experimental conditions: 1) in vitro phantom measurements served as controlled setting in which boundaries were clearly delineated; 2) a validation cohort composed of healthy C57BL/6 J mice and New Zealand Obese (NZO) mice was used in order to validate 3DE against cardiac MRI; 3) a standard mouse model of pressure overload induced-heart failure was investigated to estimate the value of 3DE. RESULTS: First, in vitro volumetry revealed good agreement between 3DE assessed volumes and the MRI-assessed volumes. Second, cardiac volume determination with 3DE showed smaller mean differences compared to cardiac MRI than conventional 1DE and 2DE. Third, 3DE was suitable to detect reduced ejection fractions in heart failure mice. Fourth, inter- and intra-observer variability of 3DE showed good to excellent agreement regarding absolute volumes in healthy mice, whereas agreement rates for the relative metrics ejection fraction and stroke volume demonstrated good to moderate observer variabilities. CONCLUSIONS: 3DE provides a novel method for accurate volumetry in small animals without the need for spatial assumptions, demonstrating a technique for an improved analysis of ventricular function. Further validation work and highly standardized image analyses are required to increase reproducibility of this approach.

Cellular mechanisms of metabolic syndrome-related atrial decompensation in a rat model of HFpEF.

Heart failure (HF) with preserved ejection fraction (HFpEF) is present in about 50% of HF patients. Atrial remodeling is common in HFpEF and associated with increased mortality. We postulate that atrial remodeling is associated with atrial dysfunction in vivo related to alterations in cardiomyocyte Calcium (Ca) signaling and remodeling. We examined atrial function in vivo and Ca transients (CaT) (Fluo4-AM, field stim) in atrial cardiomyocytes of ZSF-1 rats without (Ln; lean hypertensive) and with metabolic syndrome (Ob; obese, hypertensive, diabetic) and HFpEF. RESULTS: At 21weeks Ln showed an increased left ventricular (LV) mass and left ventricular end-diastolic pressure (LVEDP), but unchanged left atrial (LA) size and preserved atrial ejection fraction vs. wild-type (WT). CaT amplitude in atrial cardiomyocytes was increased in Ln (2.9±0.2 vs. 2.3±0.2F/F0 in WT; n=22 cells/group; p<0.05). Studying subcellular Ca release in more detail, we found that local central cytosolic CaT amplitude was increased, while subsarcolemmal CaT amplitudes remained unchanged. Moreover, Sarcoplasmic reticulum (SR) Ca content (caffeine) was preserved while Ca spark frequency and tetracaine-dependent SR Ca leak were significantly increased in Ln. Ob mice developed a HFpEF phenotype in vivo, LA area was significantly increased and atrial in vivo function was impaired, despite increased atrial CaT amplitudes in vitro (2.8±0.2; p<0.05 vs. WT). Ob cells showed alterations of the tubular network possibly contributing to the observed phenotype. CaT kinetics as well as SR Ca in Ob were not significantly different from WT, but SR Ca leak remained increased. Angiotensin II (Ang II) reduced in vitro cytosolic CaT amplitudes and let to active nuclear Ca release in Ob but not in Ln or WT. SUMMARY: In hypertensive ZSF-1 rats, a possibly compensatory increase of cytosolic CaT amplitude and increased SR Ca leak precede atrial remodeling and HFpEF. Atrial remodeling in ZSF-1 HFpEF is associated with an altered tubular network in-vitro and atrial contractile dysfunction in vivo, indicating insufficient compensation. Atrial cardiomyocyte dysfunction in vitro is induced by the addition of angiotensin II.

Cardiovascular magnetic resonance feature tracking in small animals - a preliminary study on reproducibility and sample size calculation.

BACKGROUND: Cardiovascular magnetic resonance feature tracking (CMR-FT) is a novel tissue tracking technique developed for noninvasive assessment of myocardial motion and deformation. This preliminary study aimed to evaluate the observer's reproducibility of CMR-FT in a small animal (mouse) model and define sample size calculation for future trials. METHODS: Six C57BL/6 J mice were selected from the ongoing experimental mouse model onsite and underwent CMR with a 3 Tesla small animal MRI scanner. Myocardial deformation was analyzed using dedicated software (TomTec, Germany) by two observers. Left ventricular (LV) longitudinal, circumferential and radial strain (EllLAX, EccSAX and ErrSAX) were calculated. To assess intra-observer agreement data analysis was repeated after 4 weeks. The sample size required to detect a relative change in strain was calculated. RESULTS: In general, EccSAX and EllLAX demonstrated highest inter-observer reproducibility (ICC 0.79 (0.46-0.91) and 0.73 (0.56-0.83) EccSAX and EllLAX respectively). In contrast, at the intra-observer level EllLAX was more reproducible than EccSAX (ICC 0.83 (0.73-0.90) and 0.74 (0.49-0.87) EllLAX and EccSAX respectively). The reproducibility of ErrSAX was weak at both observer levels. Preliminary sample size calculation showed that a small study sample (e.g. ten animals to detect a relative 10% change in EccSAX) could be sufficient to detect changes if parameter variability is low. CONCLUSIONS: This pilot study demonstrates good to excellent inter- and intra-observer reproducibility of CMR-FT technique in small animal model. The most reproducible measures are global circumferential and global longitudinal strain, whereas reproducibility of radial strain is weak. Furthermore, sample size calculation demonstrates that a small number of animals could be sufficient for future trials.

Myocardial T1 maps reflect histological findings in acute and chronic stages of myocarditis in a rat model.

BACKGROUND: Cardiovascular magnetic resonance offers both diagnostic and prognostic information in myocarditis. Using an established animal model of myocarditis, the aim of this study was to measure myocardial T1 before the onset, in the acute and in the chronic phases of the disease and to compare its course with histological and immunohistochemistry findings. METHODS: Male young Lewis rats were immunized with 0.25 mg porcine myocardial myosin into the rear footpads on day 0. Native and contrast-enhanced ECG-triggered cardiac MRI examinations were performed before immunization on day 0 and on days 14, 21 and 35. Left ventricular function, pre- and post- contrast T1 parameters and LGE images were assessed using Small animal look-locker inversion recovery (SALLI). For each of the indicated time points a minimum of 4 rats were randomly sacrificed for pathological investigations including conventional histology (HE and Sirius-Red staining) and immunohistochemistry (CD 68) investigations. RESULTS: All immunized rats developed myocarditis (morbidity 100%). Histologically we observed increased wall thickness with biventricular macrophage-rich mixed inflammatory infiltrates. All rats with a histologically severe myocarditis showed increased native T1 and decreased post-contrast T1 of the myocardium. CONCLUSIONS: The assessment of native T1 and post-contrast T1 allows accurate differentiation between healthy myocardium and myocardium with inflammation and also between the acute and chronic phases of the disease.

Closed-chest small animal model to study myocardial infarction in an MRI environment in real time.

Current models for real time study of the effects of myocardial ischemia/reperfusion have major limitations and confounders. Confounders include the surgical stresses of a thoracotomy and abnormal physiology of an open chest. The need to reposition the animal interferes with the study of the early changes associated with ischemia. Direct comparison of pre-ischemia and post-ischemia images is then difficult. We developed a novel "closed chest" model of ischemia/reperfusion to overcome these issues. Following thoracotomy, we sutured a balloon occluder to the left coronary artery of male Sprague-Dawley rats. We used both visual inspection and ECG to assess for successful occlusion and reperfusion of the coronary artery at the time of operation by brief inflation and deflation of the balloon. The tubing was then placed under the skin and the incision closed. Following a recovery period (5-10 days), the animals underwent MRI. We performed baseline assessment of left ventricle function, and repeated LV measurement during a 15-min coronary occlusion and again during a 60-min reperfusion period following reopening of the coronary artery. The occluder was successfully placed in 40 of 44 animals. Four developed intraoperative complications; two large myocardial infarction, two terminal bleeding. Six died in the week following surgery, [four sudden deaths (presumed arrhythmic), one anterior infarction, one sepsis]. Cine-MRI demonstrated localised hypokinesia in 31 of the remaining 34 animals. LV ejection fraction (EF) was reduced from 63 ± 7 % at baseline, to 49 ± 9 % during coronary occlusion. LV EF recovered to 61 ± 2 %. The area at risk on staining of the heart was 41.9 ± 15.8 %. This method allows the effects of ischemia/reperfusion to be studied before, during, and after coronary occlusion. Ischemia can be caused while the animal is in the MRI. This new and clinically relevant small animal model is a valuable tool to study the effects of single or repeated coronary occlusion/reperfusion in real-time.

Alterations in creatine metabolism observed in experimental autoimmune myocarditis using ex vivo proton magic angle spinning MRS.

Experimental autoimmune myocarditis (EAM) in rodents is an accepted model of myocarditis and dilated cardiomyopathy (DCM). Altered metabolism is thought to play an important role in the pathogenesis of DCM and heart failure (HF). Study of the metabolism may provide new diagnostic information and insights into the mechanisms of myocarditis and HF. Proton MRS ((1)H-MRS) has not yet been used to study the changes occurring in myocarditis and subsequent HF. We aimed to explore the changes in creatine metabolism using this model and compare them with the findings in healthy animals. Myocardial function of male young Lewis rats with EAM was quantified by performing left ventricular ejection fraction (LVEF) analysis in short-axis cine images throughout the whole heart. Inflammatory cellular infiltrate was assessed by immunohistochemistry. Myocardial tissue was analyzed using ex vivo proton magic angle spinning MRS ((1)H-MAS-MRS). Myocarditis was confirmed histologically by the presence of an inflammatory cellular infiltrate and CD68 positive staining. A significant increase in the metabolic ratio of Tau/tCr (taurine/total creatine) obtained by (1)H-MAS-MRS was observed in myocarditis compared with healthy controls (21 d acute EAM, 4.38 (±0.23); 21 d control, 2.84 (±0.08); 35 d chronic EAM, 4.47 (±0.83); 35 d control, 2.59 (±0.38); P < 0.001). LVEF was reduced in diseased animals (EAM, 55.2% (±11.3%); control, 72.6% (±3.8%); P < 0.01) and correlated with Tau/tCr ratio (R = 0.937, P < 0.001). Metabolic alterations occur acutely with the development of myocarditis. Myocardial Tau/tCr ratio as detected by (1)H-MRS correlates with LVEF and is able to differentiate between healthy myocardium and myocardium from rats with EAM.

T1 mapping in ischaemic heart disease.

A unique feature of cardiac magnetic resonance is its ability to characterize myocardium. Proton relaxation times, T1, T2, and T2* are a reflection of the composition of individual tissues, and change in the presence of disease. Research into T1 mapping has largely been focused in the study of cardiomyopathies, but T1 mapping also shows huge potential in the study of ischaemic heart disease. In fact, the first cardiac T1 maps were used to characterize myocardial infarction. Robust high-resolution myocardial T1 mapping is now available for use as a clinical tool. This quantitative technique is simple to perform and analyse, minimally subjective, and highly reproducible. This review aims to summarize the present state of research on the topic, and to show the clinical potential of this method to aid the diagnosis and treatment of patients with ischaemic heart disease.

Assessment of cardiac function and myocardial morphology using small animal Look-Locker inversion recovery (SALLI) MRI in rats.

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images. The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.