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<p> A commercially available cryotherapy system (Erbokryo CS-6-System, Erbe, Tübingen, Germany) was used for cryoablation. This system consists of a casing with a control board and up to 6 vacuum-isolated flexible tubes with a cryoprobe (diameter 3.2 mm) at the end. A cryoprobe creates a cold zone 3 cm long. The Erbokryo is also fitted with computer-controlled temperature sensors (diameter 1.2 mm) witch allow 6 simultaneous measurements. </p> <p> The cryoprobes were introduced via an access hole 3.6 mm in diameter drilled perpendicular to the cortical substance. Temperature was measured inside the cortical substance. Two 15-minute freezing cycles were done with the probe at full power, with a 6-minute thaw in between. Prior to starting the cryoablation, the position of the cryoprobe was checked radiologically and recorded. </p> <p> 24 sheep with a mean weight of 61 kg (range 39–78 kg) were placed under general anaesthesia and, using a single cryoprobe introduced through a lateral access hole, one cryoablation was done in the distal diametaphyseal transitional region of the head of the medial left tibia and the right femur of each sheep. For the tibial head, a medial access hole was drilled and the cryoprobe introduced centrally 1.5 cm below the joint and pushed to the other side of the cortical substance. </p> <p> For the femoral cryoablation, the cryoprobe was introduced through a distal, posterolateral access hole in the area of the linea aspera at the diametaphyseal transition. In addition, 4 temperature sensors were introduced through access holes drilled radially at 1 cm from the bore hole. The cryoprobes were only introduced 1 cm into the bone to avoid freezing the cortical substance. Thus, the necrosis zone (which roughly corresponds to the -10°C isotherm <a></a>[<a href="http://www.biomedcentral.com/1471-2482/5/17#B24">24</a>]) only comprised an area of 2.4 × 2.4 in the outer cortical substance. Control holes and access holes were drilled on the contralateral sides (left femur, head of the right tibia). </p> <p> All operations were done under 600 mg clindamycin i.v. and 12 hours prior to surgery, each animal also received thrombosis prophylaxis (0.3 ml nadroparin calcium [Fraxiparin<sup>®</sup>] s.c.). There was no postop thrombosis prophylaxis since all animals were fully mobile after anaesthesia. The pulmonary-arterial pressure (PAP) and the central venous pressure (CVP) were measured via a pulmonary catheter inserted into the jugular vein. Measurements were done before the first cryoablation (1, Fig. <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F1">1</a>, <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F2">2</a>, <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F3">3</a>), after the first cryoablation on the right femur (2, Fig. <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F1">1</a>, <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F2">2</a>, <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F3">3</a>) and the head of the left tibia (3, Fig. <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F1">1</a>, <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F2">2</a>, <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F3">3</a>), as well as after drilling the control holes immediately after shifting the animals from the right back to the left unilateral recumbent position (4, Fig. <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F1">1</a>, <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F2">2</a>, <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F3">3</a>). Intra- and perioperative monitoring was complemented by measurements of heart rate (Fig. <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F3">3</a>) and oxygen saturation via pulse oxymetry, as well as blood counts (Fig. <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F4">4</a>), deep body temperature (Fig. <a></a><a href="http://www.biomedcentral.com/1471-2482/5/17/figure/F5">5</a>), blood gases and electrolytes after each cryoablation. Postoperative complications were monitored clinically. 8 animals were sacrificed at 8, 16 and 32 weeks postop and tissue samples taken from the lungs and from blood vessels in the areas of cryoablation were examined for signs of embolism. Samples were taken from each lobe and from the femoral vein, fixed in formaline and stained with hematoxylin and eosin (HE). Furthermore, the ablation sites were examined histologically for infection of the soft tissue respectively osteomyelitis. X-rays were taken after the animals were sacrificed. </p>
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