Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks
Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks
Mice were sacrificed by Shedule1 cervical dislocation and femoral head cartilage (hip) and whole intervertebral disc tissues (IVDs) were harvested for RNAseq. All animal studies were performed in accordance with the 1986 UK Home Office Animal Procedures Act. Approval was provided by the local ethics committee. Mice were maintained at 20-22°C, with standard rodent chow available ad libitum and under 12:12 hr light dark schedule (light on at 7 am; light off at 7 pm). The PER2::Luc mice carry the firefly luciferase gene fused in-frame with the 3' end of the Per2 gene, creating a fusion protein reporter (PMID: 14963227). Treadmill exercise. Mice were exercised on TSE Treadmill System setup with eight individual running lanes. Treadmill running was performed 2 hours after lights on according to the following protocol:Day 1: Mice were placed in the treadmill without the motor turned on for acclimatisation for about 5 min, following which the power was turned on and the mice allowed to acclimate to the mechanical noise for another 5 min. Next, mice walked at a 10-degree incline at 5 m/min for 10 minutes. Control group was moved to the same room but remained in their cages.Day 2: 2 min warm up at start 8m/min, then speed was increased to 10 m/min for 15 minutes.Day 3: 2 min warm up at start 8m/min followed by 10 m/min gradually increasing to 15 m/min over 20 minutes Day 4: 2 min warm up at start 8m/min, then 15 m/min for 30 minutes.Day 5: 2 min warm up at start 8m/min, then 15 m/min for 40 Day 6,7: Rest and recoveryDay 8-12: 2 min warm up at start 8m/min, then 15 m/min for 45 minutes Mice were sacrificed by Shedule1 cervical dislocation after treadmill running on day 12 and femoral head cartilage (hip) and whole intervertebral disc tissues (IVDs) were harvested for RNAseq. Tissues were pulverized in liquid nitrogen cooled dismembrator. mRNA was extracted using TRIzol/chloroform method. Quality and integrity of the RNA samples were assessed using a 2200 TapeStation (Agilent Technologies).  Illumina mRNA library