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 品系大鼠繁育种鼠购自Harlan Industries（印第安纳波利斯，美国印第安纳州），饲养于繁育种群中，采用12:12小时可逆光暗周期（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：妊娠雌鼠每日饮用含糖精（saccharin, Sac）或5%乙醇（PAE造模组）的糖精水溶液，每日给药时长4小时，贯穿整个妊娠周期。仔鼠于出生后24日龄断奶，并以配对笼养方式饲养。所有实验均采用6~7月龄的雌性仔鼠，包括产前酒精暴露组（PAE组）及年龄匹配的糖精对照组（Sac对照组）仔鼠。成年Sac对照组及PAE组大鼠分别接受轻度坐骨神经慢性压迫损伤（chronic constriction injury, CCI）手术或假手术处理。轻度神经损伤后，仅PAE组大鼠出现慢性异常性疼痛，而所有假手术对照组及神经损伤对照组大鼠均未表现出异常性疼痛。CCI术后第28日完成行为学测试后，采集组织样本以检测环状RNA（circular RNA, circRNA）的表达水平。血液样本采集后立即采用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）。所有样本均置于-80℃冻存，总RNA提取采用miRNeasy RNA提取试剂盒（Qiagen，德国）。随后采用Rat Array Star环状RNA表达谱分析服务检测circRNA表达水平，每组包含3个样本。脊髓组织的circRNA表达谱分析涵盖以下四组：(1) 糖精对照+假手术组；(2) 产前酒精暴露+假手术组；(3) 糖精对照+CCI手术组；(4) 产前酒精暴露+CCI手术组。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。以假手术组大鼠的组织作为对照，用于比较PAE所调控的circRNA表达变化。