Roscovitine does not affect ERK1/2 activity in SCDH induced by CFA injection.

<p>a. Effects of roscovitine on the heat hyperalgesia compare to the DMSO treatment 2-way ANOVA with repeated measures followed by Bonferroni's multiple-comparison tests. *p<0.05, **p<0.01, n = 6. b. 100 µg roscovitine does not affect rat movement as detected by the inclined plane test before CFA administration, n = 6. c. Effects of roscovitine administration on ERK1/2 activity and p-Cdk5^S159 expression after CFA in the Western blot analysis. d and e. Quantification of the Western blot shows that roscovitine treatment does not affect ERK1/2 activity by CFA, **p<0.01, n = 3. f. Quantification of the Western blot shows that the increase of p-Cdk5^S159 by CFA is attenuated after roscovitine treatment, *p<0.05, **p<0.01, n = 3. g. Roscovitine treatment does not affect total-Cdk5 expression in spinal cord 1 d after CFA injection.</p

Levels of p-ERK and p-Cdk5^S159 increase in SCDH after formalin injection.

<p>a. Time courses of p-ERK1/2 (panel 1) and t-ERK1/2 expression (panel 3) from 5 min to 1 d after formalin injection in rats as compared to naïve condition (lane1) in the Western blot analysis. b. Quantification of ERK1 activity in the dorsal horn. Columns represent means ± SEM. Data were normalized to naïve control and ANOVA followed by Dunnett's Multiple Comparison Test. *P<0.05, **P<0.01, as compared with naive rats. c. Quantification of ERK2 activity in the dorsal horn. d. Time courses of p-Cdk5^S159 (panel 1) and t-Cdk5 (panel 3) expression from 5 min to 1 d after formalin injection in rats as compared to naïve condition (lane1) in the Western blot analysis. e. Quantification of levels in p-Cdk5^S159 in the dorsal horn.</p

CFA injection increases the levels of p-ERK and p-Cdk5^S159 expression in SCDH.

<p>a. Time course of ERK1/2 activity (panel 1) and p-Cdk5^S159expression (panel 2) from 2 h to 1 week after CFA injection in rats as compared to naïve condition (lane1) in the Western blot analysis. b. Quantification of ERK1 activity in the dorsal horn. Columns represent means ± SEM. Data were normalized to naïve control and ANOVA followed by Dunnett's Multiple Comparison Test. *P<0.05, **P<0.01, as compared with naive rats. c. Quantification of ERK2 activity in the dorsal horn. d. Quantification of the levels in p-Cdk5^S159 in the dorsal horn. e. CFA injection does not affect total ERK1/2 and total Cdk5 expression in SCDH compared to naïve control (lane 1) in the Western blot analysis. N = 3.</p

Intrathecal administration of U0126 attenuates CFA-induced heat hyperalgesia.

<p>a. PWLs to thermal stimuli significantly decreases after intraplantar injection of CFA; **p<0.01. b. Compared to the PWL of the control group treated with DMSO, PWL to thermal stimuli significantly increases following intrathecal injection of U0126 30 min before and once per day for 1 week after CFA injection. The symbols represent mean and vertical lines represented SD. Data were analyzed by two-way ANOVA with repeated measures followed by Bonferroni's multiple-comparison tests, *p<0.05, **p<0.01. c. 2 µg U0126 does not affect rat movement as detected by the inclined plane test before CFA administration. N = 6.</p

U0126 attenuates p-Cdk5^S159 increase and Cdk5 activity in SCDH induced by CFA administration.

<p>a. U0126 mitigates CFA-induced p-ERK1/2 and p-Cdk5 increases in spinal cord in the Western blot analysis. b and c. Quantification of the Western blot shows that ERK1/2 activity by CFA is attenuated after U0126 treatment, *p<0.05, **p<0.01. d. Quantification of the Western blot shows that the increase of p-Cdk5^S159 by CFA is attenuated after U0126 treatment, **p<0.01, ***p<0.001. e. U0126 treatment does not affect total-ERK1/2 and total-Cdk5 expression in spinal cord 1 d after CFA injection. f. U0126 mitigates CFA-induced Cdk5 kinase activity in spinal cord detected by an in vitro phosphorylation assay. Histone H1 was used as a substrate. g. Quantification of Cdk5 activity (density of phosphorylated H1 band) in the SCDH. Columns represent means ± SEM for three separate experiments. h. Double-immunofluorescence staining for p-ERK (red), p-Cdk5 (green), Hoechst [a cell nuclear marker (blue)] in ipsilateral spinal enlargement at 1 day after CFA injection. P-Cdk5^S159 colocalizes with p-ERK (white arrow head).</p

CCI surgery decreases NAD and increases NAM in the spinal cord.

<p>CCI surgery decreases (A) NAD and increases (B) NAM as compared with sham surgery. (C) The ratio of NAD/NAM is decreased in CCI mice as compared with that in sham group. (D) NAM and NAD fractions are collected according to HPLC method. The arrows show the retention time of NAM and NAD, which is at around 21 min and 48 min, respectively. The NAM peaks are mixed with those of other impurities. (E) Spectrograms of NAD with MALDI-MS are representative results from a sham-treated (left) and a CCI-treated (right) mouse. The position of NAD and ¹⁸O NAD are indicated. (F) Spectrograms of NAM with ESI-MS are representative results from a sham-treated (left) and a CCI-treated (right) mouse. The position of NAM is indicated. <i>n</i> = 6 at each time point in each group; ^*<i>P</i><0.05, ^**<i>P</i><0.01 <i>vs.</i> sham group at each time point. NAM, nicotinamide; NAD, nicotinamide adenine dinucleotide; CCI, chronic constriction injury; MALDI-MS, matrix-assisted laser desorption ionization-mass spectroscopy; ESI-MS, electrospray ionization-mass spectrometry.</p

EX-527 inhibits the anti-nociceptive effect of resveratrol.

<p>Intrathecal injection of 5 µl EX-527 (1.2 mM) 1 h before resveratrol administration effectively reverses the effect of resveratrol (45 mM) delaying the onset of (A) thermal hyperalgesia and (B) mechanical allodynia produced by CCI surgery. (C) EX-527 inhibits the effect of resveratrol attenuating CCI-induced elevation of H4-K16Ac 1 day after CCI surgery. (D) Quantification of the immunoblots shows that EX-527 inhibits the effects of resveratrol on H4-K16Ac. An arrow indicates a resveratrol injection and a dotted arrow indicates an EX-527 injection. <i>n</i> = 10 in each group for behavior test and <i>n</i> = 6 in each group for H4-K16Ac analysis; ^*<i>P</i><0.05, ^**<i>P</i><0.01 <i>vs.</i> DMSO-DMSO-treated CCI group; ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs.</i> resveratrol-DMSO-treated CCI group. CCI, chronic constriction injury; H4-k16Ac, H4-k16 acetylation; DMSO, dimethyl sulfoxide.</p

CCI surgery increases acetylation of H4-k16.

<p>(A) The levels of H4-k16Ac, but not total H4, are increased 1, 3, 7, 14 and 21 days after CCI surgery as compared with those after sham surgery. (B) Quantification of the immunoblots shows that H4-k16Ac is increased after CCI surgery. <i>n</i> = 6 at each time point in each group; ^*<i>P</i><0.05, ^**<i>P</i><0.01 <i>vs.</i> sham group at each time point. CCI, chronic constriction injury; SIRT1, silent information regulator 1; H4-k16Ac, H4-k16 acetylation.</p

CCI surgery decreases spinal SIRT1 content and this is associated with an increase in pain-related behaviors.

<p>(A) Spinal SIRT1 content is decreased 1, 3, 7, 14 and 21 days after CCI surgery as compared with that after sham surgery. (B) Quantification of the immunoblots shows that SIRT1 levels are decreased after CCI surgery. CCI surgery induces (C) thermal hyperalgesia and (D) mechanical allodynia in ipsolateral hind paw. <i>n</i> = 12 in each group for behavioral test and <i>n</i> = 6 at each time point in each group for SIRT1 analysis; ^*<i>P</i><0.05, ^**<i>P</i><0.01 <i>vs.</i> sham group at each time point. CCI, chronic constriction injury; SIRT1, silent information regulator 1.</p

Resveratrol attenuates the development of neuropathic pain by alleviating the reduction of SIRT1 deacetylase activity.

<p>An intrathecal administration of 5 µl 90 mM resveratrol 1 h before CCI surgery, but not 45 mM suppresses the development of (A) thermal hyperalgesia and (B) mechanical allodynia induced by CCI surgery. (C) 45 mM resveratrol attenuates CCI-induced elevation of H4-K16Ac 2 days after CCI surgery. (D) Quantification of the immunoblots shows that 45 mM resveratrol attenuates CCI-induced increase in H4-K16Ac 2 days after surgery. An arrow indicates an intrathecal injection. <i>n</i> = 10 in each group for behavior test and <i>n</i> = 6 in each group for H4-K16Ac analysis; ^*<i>P</i><0.05, ^**<i>P</i><0.01 <i>vs.</i> DMSO-treated sham group; ^#<i>P</i><0.05, ^##<i>P</i><0.01 <i>vs.</i> DMSO-treated CCI group. Resveratrol1, 45 µM resveratrol; resveratrol2, 90 µM resveratrol; CCI, chronic constriction injury; H4-k16Ac, H4-k16 acetylation.</p