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

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. Overall design: 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.

Toll样受体4（TLR4）/核因子κB（NF-κB）信号通路在肌肉缺血再灌注损伤（IRI）的危险信号应答过程中发挥核心介导作用。本研究旨在解析缺血再灌注损伤后骨骼肌中受TLR4/NF-κB调控的微小RNA（microRNA, miRNA）表达谱。本研究以C57BL/6、Tlr4基因敲除（Tlr4–/–）及NF-κB基因敲除（NF-κB–/–）小鼠的离体股动脉灌注大腿骨骼肌为模型，先实施2小时缺血处理，随后分别于再灌注指定时间点（0 h、4 h、1 d、7 d）取材，通过miRNA芯片分析检测受TLR4/NF-κB调控的差异表达miRNA。

实验整体设计：选取10~12周龄、体重22~35 g的雄性C57BL/6小鼠，购自中国台湾BioLasco公司；Tlr4基因敲除（Tlr4–/–，C57BL/10ScNJ背景）及NF-κB基因敲除（NF-κB–/–，B6.Cg-Nfkb1tm1Bal/J背景）小鼠购自美国Jackson实验室（美国缅因州巴尔港）。所有饲养环境搭建、手术操作、镇痛处理及实验评估均在经国际动物护理与使用评估认证（AAALAC）认可的无特定病原体（SPF）级动物设施中开展，严格遵循国家及机构相关实验指南。本研究的动物实验方案已获长庚纪念医院实验动物管理与使用委员会（IACUC）批准。小鼠采用腹腔注射麻醉合剂进行麻醉，合剂配方为每克体重给予0.1 mg氯胺酮与0.01 mg甲苯噻嗪，注射体积为每克体重0.01 ml。麻醉诱导完成后，将小鼠仰卧固定于加热垫上，维持其体温在37℃。小心分离小鼠股动脉灌注区的股四头肌，使其与股骨及深层收肌群分离。缺血组小鼠通过在血管蒂近端放置微血管夹阻断血流2小时，随后移除微血管夹，在放大直视下确认血管蒂血流恢复通畅。假手术对照组仅分离肌肉，不施加微血管夹阻断血流。手术切口采用4-0尼龙线间断缝合，待小鼠苏醒后继续完成剩余再灌注周期。获取的骨骼肌标本置于液氮预冷的异戊烷中速冻，随后保存于-80℃冰箱中。本研究的miRNA芯片实验共设置3次生物学重复，取材分组如下：C57BL/6小鼠缺血2小时后分别于再灌注0 h、4 h、1 d、7 d取材；Tlr4–/–及NF-κB–/–小鼠缺血2小时后于再灌注1 d取材。