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Background: Calcification is a bad prognostic factor in heart valve disease. Calcifying aortic valve disease affects over 35% of patients above 75 years of age. In vivo rat models are increasingly used to investigate the underlying pathophysiology. The purpose of this study was to investigate aortic valve calcification in the healthy, aging rat. For this, we used echocardiographic calibrated integrated backscatter (cIB), compared to ex-vivo micro-CT and histology as reference. Methods: A mixed inbred/outbred population of 32 male Lewis/Wistar rats was studied. Group 1 (n=10) of 3 months old rats, group 2 (n=8) 12 months old, group 3 (n=11) 24 months old, and group 4 (n=3) 30 months old. cIB values and blood parameters (creatinine, total cholesterol, calcium, parathyroid hormone (PTH)) were measured. Results: There was a significant increase of cIB values of the aortic valve with age (group 1: 11.3 ± 0.6 dB; group 2: 11.9 ± 0.4 dB; group 3: 13.7 ± 0.5 dB; group 4: 14.8 ± 1.0 dB; P=0.012). This was confirmed by an increase of the calcified volume on ex-vivo micro-CT (P=0.003) and of the histological calcium score (P=0.009). There was no significant difference in total cholesterol and calcium between groups 2-4. There was a significant increase of creatinine and PTH with age (P?0.001). Conclusions: 1. Aortic valve calcification increases with age in the healthy rat. Aging is also associated with renal failure and secondary hyperparathyroidism. 2. This underlines the importance of taking aging into account when studying aortic valve disease. 3. Additionally, it was confirmed that calibrated integrated backscatter is a promising, non-ionising and non-invasive technique for the quantitative echo assessment of aortic valve calcifications in vivo.

Purpose: calcification associated with heart valve degeneration and atheromatosis is a common finding in the elderly and conveys a bad prognosis. In this pilot study, we sought to develop a rat model of cardiovascular calcification and characterize it by using different imaging modalities. Methods: we studied a total of 15 male Wistar rats receiving 3 subcutaneous injections of cholecalciferol (vitamin D3) 12,500 IU/kg a week (n=8), or the vehicle (control, n=7) for 12 weeks. Echocardiography was performed blindly at baseline, and after 12 weeks, followed by ex-vivo micro-CT and histopathological evaluation. Results: At baseline, no differences between groups were found with echocardiography. After 12 weeks, there was no difference in left ventricular function between both groups (Ejection fraction 72 ± 16 % vs. 75 ± 11 %, P=0.867). The integrated backscatter (IB) of ultrasound reflectivity of the ascending aorta was significantly higher in the vitamin D3 treated animals (-22 ± -5 dB vs. -29.6 ± -2.5 dB,P=0.004) compared to controls, as for the myocardium (-28.5 ± -3 dB vs. -35.1 ± -1.8 dB, P=0.004) and mitral valve annulus (-12,5 ± -6 dB vs. -18,9 ± -3.7 dB, P=0.04). On micro-CT, the thoracic aorta (Pixel value of 30,061 ± 14,920 vs. 13,967 ± 766, P=0.005) and the myocardium (17,720 ± 2,510 vs. 13,869 ± 427, P=0.008) also showed a significantly higher maximal density in vitamin D3 rats. On histopathology, calcifications of the aortic wall, myocardium, valve annuli and cusps were only present in the vitamin D3 treated animals. Conclusion: cardiovascular calcifications in a rat model can be detected in-vivo by echocardiographic IB, as confirmed ex-vivo by micro-CT and histopathology. This offers a unique opportunity to evaluate experimental treatment strategies.

Anesthetic agents have different effects on hemodynamic and cardiac functional parameters. The influence of these changes on valvular function has not been studied in small animals. For this purpose, 48 male Wistar rats were divided into three equal groups. An echocardiogram was performed under inhaled isoflurane 2% gas (group I) or under intraperitoneal pentobarbital 50 mg/kg (group II) or ketamine/xylazine (group III) 40/8 mg/kg. Aortic regurgitation was only found in group III (80%, p