circular RNA (circRNA) array profiling in adult rats following prenatal alcohol exposure and nerve injury.

Long-Evans rat breeders were purchased from Harlan Industries (Indianapolis, IN) and were maintained in a breeding colony on a 12:12-hour reverse light/dark schedule (lights on from 2100 to 0900 h). We utilized a well-validated PAE paradigm where rat dams were given either saccharin (Sac) or 5% ethanol (PAE) in Sac water, 4 hr/day, throughout pregnancy. Offspring were weaned at 24 days of age and offspring were pair-housed. For all experiments, 6-7-month-old female prenatal alcohol-exposed (PAE) or age-matched Saccharin control (Sac control) rat offspring were used. Adult Sac or PAE rats were exposed to either minor sciatic nerve chronic constriction injury (CCI) or sham surgery. Following minor nerve injury, only PAE rats displayed chronic allodynia, whereas all the sham surgery controls and control rats with nerve injury remained non-allodynic. Following testing on Day 28 post-CCI, tissues were collected to examine circRNA expression. Blood samples were immediately processed to isolate blood circulating peripheral leukocytes (peripheral blood mononuclear cells) utilizing Ficoll density gradient centrifugation (GE Healthcare, IL, USA). All samples were frozen at -80°C and total RNA was isolated using the miRNeasy RNA isolation kit (Qiagen, Germany). Rat array star circRNA profiling service was then used to measure circRNA expression, three samples in each group. circRNA profiling was carried out in the following groups, from the spinal cord: (1) Sac control + Sham surgery, (2) PAE + Sham surgery, (3) Sac control + CCI surgery, (4) PAE + CCI surgery. For peripheral blood circRNA profiling, Sac + Sham surgery and PAE + Sham surgery rats were used. Samples from Sham-injured rats served as control tissue to compare against the circRNA changes modulated by PAE.

Long-Evans大鼠种鼠购自Harlan Industries（印第安纳波利斯，美国印第安纳州），饲养于12:12小时反转光暗周期的繁殖种群中（光照时段为2100至0900时）。本研究采用经过充分验证的产前酒精暴露（prenatal alcohol exposure, PAE）造模方案：妊娠大鼠每日给予4小时的糖精（Sac）水溶液或含5%乙醇的糖精水溶液（PAE组）。仔鼠于出生后24天断奶，并成对饲养。所有实验均使用6~7月龄的产前酒精暴露（PAE）仔鼠或年龄匹配的糖精对照（Sac对照）仔鼠。成年Sac或PAE大鼠分别接受轻度坐骨神经慢性压迫损伤（chronic constriction injury, CCI）或假手术处理。轻度神经损伤后，仅PAE大鼠表现出慢性痛觉异常，而所有假手术对照组及接受神经损伤的对照大鼠均未出现痛觉异常。于CCI术后第28天完成测试后，采集组织以检测环状RNA（circular RNA, circRNA）的表达水平。血液样本立即通过Ficoll密度梯度离心法（GE Healthcare，美国伊利诺伊州）分离外周循环白细胞（外周血单个核细胞，peripheral blood mononuclear cells）。所有样本均冻存于-80℃，并使用miRNeasy RNA提取试剂盒（Qiagen，德国）分离总RNA。随后采用大鼠Array Star环状RNA表达谱分析服务检测circRNA表达水平，每组设置3份样本。circRNA表达谱分析分为以下取材于脊髓的组别：(1) Sac对照+假手术组；(2) PAE+假手术组；(3) Sac对照+CCI手术组；(4) PAE+CCI手术组。针对外周血circRNA表达谱分析，仅纳入Sac+假手术组及PAE+假手术组大鼠。以假手术组大鼠的样本作为对照组织，用于对比由PAE调控的circRNA表达变化。