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 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.

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