Background: Collateral formation by adaptive microvascular remodeling during chronic myocardial ischemia may ameliorate the development of heart failure. However, little quantitative information is available about potentially protective vascular changes in chronic ischemia leading to heart failure. In this study, we aimed to quantify adaptive changes in chronically ischemic rabbit hearts using high-resolution 3D imaging of the microvascular network. The rabbit heart is known for its lack of innate collateral vessels, while its intermediate size allows assessment of changes in coronary vasculature and transmural perfusion resulting from gradual coronary occlusion. Methods and Results: New Zealand White rabbits (n=5, male, 2,5-3kg) were anesthetised with a mixture of ketamine (15mg/kg) and dexmedetomidine (0.2mg/kg) with buprenorphine (0.03mg/kg) for analgesia, all i.m.. Anesthesia was maintained by 1.5-3% isoflurane. Regional myocardial ischemia was induced in rabbits by a left thoracotomy and placement of an ameroid constrictor on a side branch of one of the major coronary arteries. Progressive occlusion within 7-10 days produced distal tissue ischemia. A sham-operated animal served as control. Cardiac function was monitored with echocardiography. Post operative, animals received carprofen (4mg/kg i.m.) After 8 weeks, the animals were sacrificed, the heart was excised and the coronary arteries were filled with fluorescent replica material. Each heart was frozen and alternately cut and block-face imaged at 14 µm slice thickness using a custom-developed imaging cryomicrotome (Spaan et al., 2005). The microvascular structure is clearly visible in the unprocessed image stacks (Fig. 1 A) and replica material can be detected in transmural arteries of up to 50 µm diameter. Subsequent image processing of the raw images is performed by application of a Hessian based vesselness enhancing algorithm (Sato et al. 1998). Thereby removing the halo around the large coronary arteries and enhancing the small arteriolar structures with low brightness, figure 1B. From the enhanced structure, a topologically representative vascular tree can be reconstructed following the application of a peeling algorithm (Palagyi et al. 1998) reducing the tubular structures to the centerline pixels. From the analysis of the vascular tree skeleton, vascular diameters and collaterals can be obtained, figure 1C (van den Wijngaard et al. 2010). No signs of myocardial infarction were observed in ischemic hearts, although left ventricular wall thinning was present as compared to the control heart. Computer analysis of the segmented vascular tree identified coronary collateral formation in the ischemic hearts, with collateral connections ranging 50-100 micron between well-perfused territories and the perfusion territory distal to the ameroid constrictor. Few innate collateral anastomoses were detected in the sham operated heart. Conclusion: With use of 3D episcopic cryomicrotome imaging, coronary microvascular adaptation in ischemic rabbit hearts was demonstrated. Future studies employing fluorescent microspheres will serve to assess regional myocardial blood flow across the ventricular wall in relation to collateral development between the normally perfused and ischemic territories.