1. Antinociceptive potentiation and attenuation of tolerance by intrathecal β-arrestin 2 small interfering RNA in rats 
C.-H. Yang, H.-W. Huang, K.-H. Chen, Y.-S. Chen, S.-M. Sheen-Chen, C.-R. Lin 
British Journal of Anaesthesia 
Volume 107, Issue 5, Pages (November 2011)
DOI: /bja/aer291
Copyright © 2011 The Author(s) Terms and Conditions

2. Fig 1
(a) Experimental design. (IT, intrathecal.) Male Sprague–Dawley rats received intrathecal injections of i-Fect encapsulated β-arrestin 2 siRNA, non-targeting dsRNA, the transfection reagent (i-Fect), or normal saline once daily, for 3 days. After pretreatment, the rats received continuous intrathecal (osmotic minipump) saline or morphine (2 nmol h−1) infusions for 7 days. Five animals were included in each treatment group. (b) The mechanism of β-arrestin 2 siRNA processing. After cellular uptake of synthetic β-arrestin 2 siRNA, one of the two siRNA strands is cleaved and released, whereas the remaining single strand binds to and activates the RNA-induced silencing complex (RISC). The single-strand siRNA together with the activated RISC binds to its complementary β-arrestin 2 mRNA. The RISC contains an enzyme to cleave β-arrestin 2 mRNA and therefore cause gene knock down.
British Journal of Anaesthesia  , DOI: ( /bja/aer291)
Copyright © 2011 The Author(s) Terms and Conditions

3. Fig 2
Real-time RT–PCR and western blot analysis showing a decrease in β-arrestin 2 but not β-arrestin 1 mRNA and protein level in the spinal cord after intrathecal injection with β-arrestin 2 siRNA. β-arrestin 1 and 2 mRNA and protein level in control and β-arrestin 2 siRNA-injected rats were monitored by qRT–PCR over 9 days after injection. Each kinetic point was performed in triplicate on three to seven pooled rats. For each sample, β-arrestin 1 and 2 transcripts or protein level was normalized against GAPDH. Normalized β-arrestin 1 and 2 expression in β-arrestin 2 siRNA-injected rats was expressed as the proportion of the expression recorded in the control. Average percentages of mRNA depletion are indicated. Data are presented as mean (sd), *P<0.05.
British Journal of Anaesthesia  , DOI: ( /bja/aer291)
Copyright © 2011 The Author(s) Terms and Conditions

4. Fig 3
(a) Thermal TF latencies in rats receiving intrathecal injection with β-arrestin 2 siRNA for 3 days. Significant difference of prolonged latency was noted in rats receiving β-arrestin 2 siRNA (b). Time course of the changes in thermal antinociception and %MPE as measured by the TF test in rats receiving continuous intrathecal infusions. Data are presented as means (sd) (n=10). See the text for details of treatment groups. *Different from morphine 2 nmol h−1 on that day, P< Different from day 2 value within the group, P<0.05.
British Journal of Anaesthesia  , DOI: ( /bja/aer291)
Copyright © 2011 The Author(s) Terms and Conditions

5. Fig 4
Effect of β-arrestin 2 siRNA treatment on the occurrence of withdrawal signs in response to naloxone in (a) saline-infused rats, or (b) morphine-infused rats. a, vocalization in response to light touch with a piece of polyethylene tubing; b, spontaneous vocalization; c, abnormal posture; d, ejaculation; e, ‘wet dog head shakes’; f, escape attempts. Each group contained eight rats. *Different from morphine 2 nmol h−1 on that day, P<0.05.
British Journal of Anaesthesia  , DOI: ( /bja/aer291)
Copyright © 2011 The Author(s) Terms and Conditions

6. Fig 5
Tail withdrawal latency and %MPE to an intrathecal probe morphine bolus dose administered on day 7 after continuous intrathecal drug infusion. Each point represents mean (sd) (n=6). *P<0.05 compared with saline-treated (rats receiving morphine 0 nmol h−1 infusion). +P<0.05 compared with saline-treated (rats receiving morphine 2 nmol h−1 infusion).
British Journal of Anaesthesia  , DOI: ( /bja/aer291)
Copyright © 2011 The Author(s) Terms and Conditions