Mice for this study were 12 weeks old C57/Bl6 males, bred in-house from a colony established in 2007 using 2 breeding pairs obtained from Jackson Laboratories. At weaning, males were distributed randomly and maintained with free access to standard chow and water and with constant light cycles (12 hour light, 12 hours dark). All mice were maintained in the same groups for maturation to 12 weeks and randomly distributed to experimental protocols which were approved by the IACUC of Rush University.
We have previously published on the induction of tendinopathy in murine Achilles tendon by a single injection of TGF-β1 into the body of the tendon, and we noted that the early loss of biomechanical properties and matrix disorganization was largely reversed by extended treadmill running (10 (link)). To generate a model in which structural, biochemical, and cell biological changes become chronic, a more severe injury was generated by two consecutive injections of 100ng active TGF-β1 (human recombinant, Peprotech, Inc), 2 days apart under anesthesia with xylazine (5ug/g body weight) /ketamine (100ug/g body weight). This resulted in persistent histopathological changes up to 14 days with cage activity (CA), and worsening of the pathology with treadmill running (TM) to 14 days and 28 days. All injections were between 2pm and 4pm, and treadmill running was between 9am and 11am daily in a designated room as previously described (10 (link)). Mice were euthanized by CO2 asphyxiation and cervical dislocation between 9am and 10am and tissues collected immediately into RNALater (for RNA isolation) or proteinase K (for DNA isolation).
To control for potential epigenetic effects of anesthesia and needle injuries to the tendon body, we included the following experimental groups: mice (n=12) were anaesthetized as above on days 0 and 2 and sacrificed on day 3; mice (n=12) received anesthesia and needle injury (without TGF-β1) on days 0 and day 2. A summary of experimental groups and outcome measures is provided inTable S1
We have previously published on the induction of tendinopathy in murine Achilles tendon by a single injection of TGF-β1 into the body of the tendon, and we noted that the early loss of biomechanical properties and matrix disorganization was largely reversed by extended treadmill running (10 (link)). To generate a model in which structural, biochemical, and cell biological changes become chronic, a more severe injury was generated by two consecutive injections of 100ng active TGF-β1 (human recombinant, Peprotech, Inc), 2 days apart under anesthesia with xylazine (5ug/g body weight) /ketamine (100ug/g body weight). This resulted in persistent histopathological changes up to 14 days with cage activity (CA), and worsening of the pathology with treadmill running (TM) to 14 days and 28 days. All injections were between 2pm and 4pm, and treadmill running was between 9am and 11am daily in a designated room as previously described (10 (link)). Mice were euthanized by CO2 asphyxiation and cervical dislocation between 9am and 10am and tissues collected immediately into RNALater (for RNA isolation) or proteinase K (for DNA isolation).
To control for potential epigenetic effects of anesthesia and needle injuries to the tendon body, we included the following experimental groups: mice (n=12) were anaesthetized as above on days 0 and 2 and sacrificed on day 3; mice (n=12) received anesthesia and needle injury (without TGF-β1) on days 0 and day 2. A summary of experimental groups and outcome measures is provided in
