Animal Experiments

All mice were male C57Bl/6 mice, aging from 7 weeks old, 15 weeks old, 30 weeks old, to 22 months old; purchased from Harlan Laboratories (Frederick, MD). Mice were placed on standard feed ad libitum. All mouse experimentation was performed following institutional animal welfare protocols at The Johns Hopkins University School of Medicine (protocol # MO16M433).

Radiopharmaceuticals were administered to anesthetized mice via retro-orbital sinus (n=5 per experiment group). Mice dosed with [²²³Ra]RaCl₂ 3.7 kBq (100 nCi) were sacrificed at 1 h, 2 h, and 24 h post administration for prompt dissection, where indicated. Mice administered [^99mTc]Tc-MDP 3700 kBq (100 μCi) were sacrificed at 2 h post administration and those with [¹⁸F]NaF 370 kBq (10 μCi) were sacrificed at 1 h post administration; for prompt dissection. Whole-body distribution was evaluated from organs including blood, heart, lungs, liver, spleen, kidney, stomach, cecum, small intestines, muscle, epididymal fat, and tibia (including fibula). Organ radioactivity was assessed by gamma counting (see below). One leg from each mouse was cryo-embedded (OCT, Sakura Fintec, Torrance, CA) without decalcification procedures to prevent bone radiolabel-loss and sectioned (below).

For expanded skeletal dissection and biodistribution, mice (n=5) were euthanized by CO₂ asphyxiation at 24h post-injection of 3.7 kBq (100 nCi) [²²³Ra]RaCl₂, and immediately dissected to collect bones. Skin, organs and soft tissues were trimmed, long bones were separated at the joints and the vertebral column was separated into cervical, thoracic, lumbar, sacral and tail after the pelvic girdle was removed. The ribcage and sternum were removed from the spinal column. The limbs were separated into long bone segments and paw. The mandible was removed from the skull, which was divided into two portions - caudal half including the calvarium and rostral half including maxilla and the nasal cavity.