Materials and methods Animals Twenty-five female 6-month-old virg

Materials and methods Animals Twenty-five female 6-month-old virgin Wistar rats (Harlan Laboratories, Torin 1 manufacturer Horst, The Netherlands) were allowed to acclimatize for 7 days before the start of the experiment. The rats were maintained with a cycle of 12 h light and 12 h darkness

and allowed to eat and drink ad libitum. The experiment was approved by the Animals Ethics Committee of the University of Maastricht, The Netherlands. The rats were see more divided into three groups (with equal weight distributions): control (n = 8), ovariectomy (OVX; n = 8), and OVX and PTH treatment (n = 9). All rats were ovariectomized at week 0 and the control MLN2238 solubility dmso group underwent a SHAM ovariectomy. Success of

OVX was confirmed at necropsy by determining atrophy of the uterine horns. Rats were left untreated for 8 weeks to allow for osteopenia to develop. After 8 weeks, rats in the PTH group received daily subcutaneous injections of PTH (60 μg/kg/day) for 6 weeks. This relatively high dose was chosen to maximize the possibility of trabecular tunneling to occur and lies within the dose range investigated in a dose-dependency study [18]. Synthetic human PTH (1–34; Bachem, Bubendorf, Switzerland) was dissolved in a vehicle of acidified saline (0.1 N) and 2% rat serum. Body weight was measured weekly, and the PTH dose adjusted accordingly. Rats were sacrificed at 14 weeks by cervical dislocation under deep

anesthesia after the final CT scan. Micro-CT scanning others Directly after the operation, a 6-mm micro CT-scan (70 kV, 114 μA, 1,000 projections per 180°, 261 ms integration time) with an isotropic resolution of 15 μm was made of the proximal tibia using an in vivo micro-CT scanner (vivaCT 40, Scanco Medical AG, Brütissellen, Switzerland). The CT scanner was calibrated and a beam-hardening correction algorithm was applied to all scans [34]. Another 3.15-mm micro-CT scan of the diaphysis was made with an isotropic resolution of 30 μm (70 kV, 114 μA, 250 projections per 180°, 350 ms integration time). Before this measurement, the most distal and proximal point of the tibia was located in a scout view to ensure that the exact middle of the diaphysis was scanned. Follow-up in vivo CT scans were made after 8, 10, 12, and 14 weeks to monitor bone structure. Every follow-up scan was registered with the first scan by using image registration software that registers two scans based on minimizing the correlation coefficient [35].

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