Effect of i.t. Dex, Ropi, or their combination on CFA-induced astrocytic activation.

<p>s.c. CFA injection induced appreciable astrocytic activation and GFAP up-regulation in the ipsilateral SDH (<b>A</b>). The activated astrocytes presented hypertrophied cell bodies and the thickened processes (<b>A₁</b>), compared with contralateral side (<b>A₂</b>). Scheme showed an overview of detected region (laminae I∼III) of immunohistochemical quantification and western blot (<b>B₁</b> and <b>B₂</b>). CFA-induced aroused GFAP activation from day 3, and reached the peak on day 7 in the ipsilateral SDH (<b>C₁∼C₄</b>). i.t. individual or concomitant medication down-regulated GFAP expression in the ipsilateral SDH on day 7 (<b>D∼G</b>). Fluorescent intensities of GFAP expression at 1, 3, 5 and 7 d after s.c. CFA inejction were presented in <b>H</b>. Western blot of GFAP expression at 3 and 7 d after s.c. CFA injection was shown in <b>I</b>. ** <i>p</i><0.01, *** <i>p</i><0.001, compared with Saline-Veh group; ## <i>p</i><0.01, ### <i>p</i><0.001, compared with CFA-Veh group; +++ <i>p</i><0.001, compared with CFA-Dex&Ropi group. Scale bars = 100 μm in <b>A</b>, 10 μm in <b>A₁</b> and <b>A₂</b>, 50 μm in <b>C₁∼C₄</b> and <b>D∼G</b>.</p

i.t. Dex and Ropi combinations dose-dependently inhibited CFA-induced thermal hyperalgesia of the injected hind paw.

<p>i.t. Dex and Ropi combinations dose-dependently prolonged analgesia duration (<b>A</b>). Iosobologram for combination analgesia was shown in <b>B</b>. The AUCs for different groups were calculated to perform statistical analysis (<b>C</b>). The dose-effect or log (dose)-effect curves for combination analgesic effects were shown in <b>D</b> and <b>E</b>. * <i>p</i><0.05; *** <i>p</i><0.001, compared with CFA-Veh group; arrows indicated s.c. CFA injection and i.t. intervention time point, respectively.</p

Inhibiting Spinal Neuron-Astrocytic Activation Correlates with Synergistic Analgesia of Dexmedetomidine and Ropivacaine

<div><p>Background</p><p>This study aims to identify that intrathecal (i.t.) injection of dexmedetomidine (Dex) and ropivacaine (Ropi) induces synergistic analgesia on chronic inflammatory pain and is accompanied with corresponding “neuron-astrocytic” alterations.</p><p>Methods</p><p>Male, adult Sprague-Dawley rats were randomly divided into sham, control and i.t. medication groups. The analgesia profiles of i.t. Dex, Ropi, and their combination detected by Hargreaves heat test were investigated on the subcutaneous (s.c.) injection of complete Freund adjuvant (CFA) induced chronic pain in rat and their synergistic analgesia was confirmed by using isobolographic analysis. During consecutive daily administration, pain behavior was daily recorded, and immunohistochemical staining was applied to investigate the number of Fos-immunoreactive (Fos-ir) neurons on hour 2 and day 1, 3 and 7, and the expression of glial fibrillary acidic protein (GFAP) within the spinal dorsal horn (SDH) on day 1, 3, 5 and 7 after s.c. injection of CFA, respectively, and then Western blot to examine spinal GFAP and β-actin levels on day 3 and 7.</p><p>Results</p><p>i.t. Dex or Ropi displayed a short-term analgesia in a dose-dependent manner, and consecutive daily administrations of their combination showed synergistic analgesia and remarkably down-regulated neuronal and astrocytic activations indicated by decreases in the number of Fos-ir neurons and the GFAP expression within the SDH, respectively.</p><p>Conclusion</p><p>i.t. co-delivery of Dex and Ropi shows synergistic analgesia on the chronic inflammatory pain, in which spinal “neuron-astrocytic activation” mechanism may play an important role.</p></div

Effect of i.t. Dex, Ropi, or their combination on CFA-induced chronic hyperalgesia.

<p>Analgesia proporties of i.t. delivery of individual and concomitant medications during 7 d after s.c. CFA injection were shown in <b>A</b>. The AUCs for different groups were calculated to perform statistical analysis (<b>B</b>). *** <i>p</i><0.001, compared with CFA+Veh group; arrows indicated s.c. CFA injection time point.</p

Effect of i.t. Dex, Ropi, or their combination on CFA-induced neuronal activation.

<p>s.c. CFA-induced thermal hyperalgesia was accompanied with the increase in the number of Fos-ir neurons to the peak at 2 h after injection, and gradually reduced but sustained to day 7 in the ipsilateral SDH (<b>A₁∼A₄</b>). Both i.t. individual and concomitant medications significantly inhibited neuronal activation indicated by less number of Fos-ir neurons in the ipsilateral SDH at 2 h after s.c. CFA injection (<b>B∼E</b>). Number of Fos-ir neurons was presented in <b>F</b> at 2 h, 1, 3 and 7 d after s.c. CFA injection, respectively. * p<0.05, ** <i>p</i><0.01, *** <i>p</i><0.001, compared with Saline-Veh group; ## <i>p</i><0.01, ### <i>p</i><0.001, compared with CFA-Dex&Ropi group. Scalebars = 10 μm in <b>A₄^’</b>, 50 μm in <b>A₁∼A₄</b> and <b>B∼E</b>.</p

i.t. Dex or Ropi dose-dependently inhibited CFA-induced thermal hyperalgesia of the injected hind paw.

<p>Analgesia duration of different doses of i.t. Dex or Ropi was shown in <b>A</b> and <b>E</b>. The AUCs for different groups were calculated to perform statistical analysis (<b>B</b>) and (<b>F</b>). The dose-effect or log (dose)-effect curves for the analgesic effects in attenuating CFA-induced thermal hyperalgesia after i.t. vehicle and Dex or Ropi were shown in <b>C</b> or <b>G</b> and <b>D</b> or <b>H</b>, respectively.* <i>p</i><0.05; *** <i>p</i><0.001, compared with CFA-Veh group; arrows indicated s.c. CFA injection and i.t. intervention time point, respectively.</p

Experimental protocol.

<p>Time window indicated the days after s.c. CFA or saline injection. I: Intrathecal catheter implantation; B: Behavior test; C: CFA subcutaneous injection; S: Saline subcutaneous injection ; D: Drugs intrathecal injection; V: Vehicle intrathecal injection; E: End of the effect of i.t. medication; K: Killing the rats for further immunofluorescence histochemical staining, western blot, and HE staining.</p

Celecoxib had no effects on the formalin induced USVs.

<p>USVs curves from different groups were shown in A. The AUCs for different groups were calculated to perform statistical analysis (B). The dose-effect or log (dose)-effect curves for celecoxib’s analgesic effects were shown in C and D.</p

Duloxetine (DUL 30), celecoxib (CEL 20) or their combination (DUL + CEL) did not affect animals’ locomotion, anxiety/depression level and motor coordination.

<p>Total distance traveled and center time% in OF (A), OA entries% and OA time% in EPM (B) and latency to fall in rotarod test (C) were used to detect the locomotion, anxiety/depression and motor coordination, respectively.</p

Duloxetine dose-dependently inhibited formalin induced spontaneous flinching of the injected hind paw.

<p>Spontaneous flinchings during 60 min after s.c. formalin injection from different groups were shown in A. The AUCs for different groups were calculated to perform statistical analysis on the first (B) and second (B’) phases. The dose-effect or log (dose)-effect curves for duloxetine’s analgesic effects were shown in C and D (first phase) or C’ and D’ (second phase).</p