Short small-interfering RNAs produce interferon-α-mediated analgesia P.H. Tan, Y.J. Gao, T. Berta, Z.Z. Xu, R.R. Ji  British Journal of Anaesthesia  Volume 108, Issue 4, Pages 662-669 (April 2012) DOI: 10.1093/bja/aer492 Copyright © 2012 The Author(s) Terms and Conditions

Fig 1 NT siRNAs inhibit CFA-induced inflammatory pain via INF-α. (a) The mechanism of RNA interference. The long double-stranded (ds) RNA or short hairpin (sh) RNA is processed to siRNA (21–23 nucleotides duplexes) by the enzyme Dicer. The siRNAs are incorporated into the RISC and the duplex is unwound. The RISC guided with antisense oligonucleotide of the siRNA binds to target mRNA and degrades it. (b) Sequences of three NT siRNAs. (c) Intrathecal injection of NT-siRNA increased PWL before CFA injection and inhibited heat hyperalgesia after CFA injection. *P<0.05, n=8; BL, baseline. (d) NT-siRNA-induced inhibition of mechanical allodynia in the inflamed rats was reversed by INF-α neutralizing antibody. Both groups of rats were pretreated with NT-siRNA, followed by INF-α antibody (Ab) or control serum injection. *P<0.05, n=5. British Journal of Anaesthesia 2012 108, 662-669DOI: (10.1093/bja/aer492) Copyright © 2012 The Author(s) Terms and Conditions

Fig 2 IFN-α elicits anti-nociceptive effects. (a) Intraplantar injection of CFA produced marked mechanical allodynia on day 1, as indicated by a reduction in paw withdrawal threshold. *P<0.05, compared with baseline (BL), n=12. (b) Intrathecal injection of IFN-α, on CFA day 1, reversed mechanical allodynia in a dose-dependent manner. *P<0.05, compared with corresponding saline control, n=6. (c) Intrathecal IFN-α raised PWL in naïve rats (*P<0.05, n=6). (d) INF-α-induced inhibition of mechanical allodynia in the CFA-inflamed rats was reversed by the INF-α neutralizing antibody (30 ng intrathecally). Both groups of rats were pretreated with INF-α (100 ng intrathecally), followed by INF-α antibody (Ab) or control serum injection. *P<0.05, n=6. British Journal of Anaesthesia 2012 108, 662-669DOI: (10.1093/bja/aer492) Copyright © 2012 The Author(s) Terms and Conditions

Fig 3 Reversal of NT-siRNA and IFN-α-induced anti-allodynia effect by intrathecal naloxone. Two groups of rats were pretreated with intrathecal INF-α (100 ng) followed by intrathecal naloxone (20 nmol) in one group and vehicle in another group. Two groups of rats were pretreated with intrathecal NT-siRNA (10 μg), followed by intrathecal naloxone (20 nmol) in one group and vehicle in another group (*P<0.05, compared with IFN-α; +P<0.05, ++P<0.01, compared with NT-siRNA; n=6). British Journal of Anaesthesia 2012 108, 662-669DOI: (10.1093/bja/aer492) Copyright © 2012 The Author(s) Terms and Conditions

Fig 4 IFN-α is up-regulated in rat spinal cords by NT siRNA. (a) Western blotting revealed that NT-siRNA (10 μg, intrathecally) elevated the IFN-α level in rat spinal cords 24 h after treatment. *P<0.05, compared with the corresponding PEI vehicle (n=6 in each group). (b) Immunohistochemistry showed more IFN-α-positive cells in the rat dorsal horn 24 h after NT-siRNA treatment than after PEI treatment. Arrows indicate IFN-α-positive cells. Scale bar, 25 μm. Absence of immunostaining either after omission of the primary antibody (c) or after preabsorption of IFN-α antibody with the blocking IFN-α peptide (10 000 U≈100 ng, e). IFN-α staining still remains after preabsorption with 1000 U (≈10 ng) of IFN-α peptide (d). Scale bar, 50 μm (c–e). British Journal of Anaesthesia 2012 108, 662-669DOI: (10.1093/bja/aer492) Copyright © 2012 The Author(s) Terms and Conditions