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

Data_Sheet_1.docx

<p>In rodents, the amygdala has been proposed to serve as a key center for the nociceptive perception. Previous studies have shown that extracellular signal-regulated kinase (ERK) signaling cascade in the central nucleus of amygdala (CeA) played a functional role in inflammation-induced peripheral hypersensitivity. Duloxetine (DUL), a serotonin and noradrenaline reuptake inhibitor, produced analgesia on formalin-induced spontaneous pain behaviors. However, it is still unclear whether single DUL pretreatment influences formalin-induced hypersensitivity and what is the underlying mechanism. In the current study, we revealed that systemic pretreatment with DUL not only dose-dependently suppressed the spontaneous pain behaviors, but also relieved mechanical and thermal hypersensitivity induced by formalin hindpaw injection. Consistent with the analgesic effects of DUL on the pain behaviors, the expressions of Fos and pERK that were used to check the neuronal activities in the spinal cord and CeA were also dose-dependently reduced following DUL pretreatment. Meanwhile, no emotional aversive behaviors were observed at 24 h after formalin injection. The concentration of 5-HT in the CeA was correlated with the dose of DUL in a positive manner at 24 h after formalin injection. Direct injecting 5-HT into the CeA suppressed both the spontaneous pain behaviors and hyperalgesia induced by formalin injection. However, DUL did not have protective effects on the formalin-induced edema of hindpaw. In sum, the activation of CeA neurons may account for the transition from acute pain to long-term hyperalgesia after formalin injection. DUL may produce potent analgesic effects on the hyperalgesia and decrease the expressions of p-ERK through increasing the concentration of serotonin in the CeA.</p