MiRNA inhibitor and mimetic

A locked-nucleic acid (LNA) based in vivo inhibitor for miR-132-3p and a mismatch control (scrambled oligonucleotide, Scr) were purchased from Exiqon (Vedbaek, Denmark). Sequences of the oligonucleotides were as follows: miR-132-3p (5′-3′ ATGGCTGTAGACTGTT) and Scr (5′-3′ ACGTCTATACGCCCA). A miRIDIAN microRNA mimetic for miR-132-3p (C-320363-03-0002) (Dharmacon, GE Healthcare Europe, Freiburg, Germany) was custom-modified with a 3′-cholesterol conjugation on the passenger strand to facilitate its in vivo uptake. Both inhibitor and mimetics were synthesized from ribonucleotides and their functional efficacy was tested in vivo. Both oligonucleotides were purified via HPLC and the lyophilized powder was reconstituted in 1x PBS at pH 7.4 at concentrations of 1 mM and stored in aliquots at −20°C to avoid freeze-thaw cycles.

The inhibitor and mimetic were administered to awake rats via the i.t. catheters. Prior to injection, active or mismatch inhibitors were mixed with (1:5 w/v) i-Fect^™ in vivo transfection reagent (Neuromics, Edina, USA) to final doses of 5, 2 and 1 μg in 10 μl. Bolus injections were performed every 24 h for 3 consecutive days starting on day 10 after SNI or sham surgery. The mimetic was mixed with i-Fect^™ (1:5 w/v) to final doses of 8, 5, 3 and 1 μg and the injection sequence to naïve animals followed the same pattern as that for the antagonist. Each drug was mixed with sterile saline at pH 7.4 to achieve the desired dilution. Each injection was followed by a 10 μl saline flush.