TLR4/NF-κB-responsive microRNAs in a mouse model of skeletal muscle ischemia-reperfusion injury

ABSTRACT: TLR4/NF-κB signaling plays a central mediator in response to danger signals released in the muscle ischemia-reperfusion injury (IRI). This study was designed to profile TLR4/NF-κB-responsive microRNAs (miRNAs) in the skeletal muscles following IRI. Following 2 h of ischemia and subsequent reperfusion for indicated times (0 h, 4 h, 1 d, and 7 d) of the isolated thigh skeletal muscles based on femoral artery perfusion of C57BL/6, Tlr4–/–, and NF-κB–/–mice, the muscle specimens were analyzed with an miRNA array to detect the TLR4/NF-κB-responsive miRNAs. Male C57BL/6 mice (10–12 weeks of age, 22–35 g) were purchased from BioLasco (Taiwan). Tlr4–/– (C57BL/10ScNJ) and NF-κB–/– (B6.Cg-Nfkb1tm1Bal/J) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All housing conditions were established and surgical procedures, analgesia, and assessments were performed in an AAALAC-accredited, SPF facility following national and institutional guidelines. Animal protocols were approved by the IACUC of Chang Gung Memorial Hospital. Mice were anesthetized with an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine, given as an intraperitoneal injection (0.01 ml/g body weight). After the induction of anesthesia, the mice were restrained in a supine position on a heating pad to maintain body temperature at 37°C. The quadriceps muscle perfused based on the femoral artery of the mouse was carefully isolated away from the femoral bone and the underlying adductor muscle group. In the ischemic group, ischemia was induced by placing a microvascular clamp carefully across the proximal site of vascular pedicle for 2 h and then the microvascular clamp was removed. Good vascular inflow and outflow through the pedicle was verified under direct magnified vision. In the sham-operated control group, the muscle was isolated without microvascular clamp being applied. The incision wound was closed with interrupted sutures (4-0 nylon) and the animals were allowed to awaken in the remaining periods of reperfusion. The harvested muscles were frozen in isopentane chilled in liquid nitrogen and stored at -80°C. For the miRNA array experiments, the isolated skeletal muscles of C57BL/6 mice after 2 h of ischemia and 0 h, 4 h, 1 d, and 7 d of reperfusion as well as of Tlr4–/–and NF-κB–/– mice after 2 h of ischemia and 1 d of reperfusion were used in 3 replicate experiments.

ORGANISM(S): Mus musculus

SUBMITTER: Ching-Hua Hsieh 

PROVIDER: E-GEOD-47730 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress