Rolipram, a Selective Phosphodiesterase 4 Inhibitor, Ameliorates Mechanical Hyperalgesia in a Rat Model of Chemotherapy-Induced Neuropathic Pain through Inhibition of Inflammatory Cytokines in the Dorsal Root Ganglion

Hee Kee Kim, Seon-Hee Hwang, Elizabeth Oh, Salahadin Abdi, Hee Kee Kim, Seon-Hee Hwang, Elizabeth Oh, Salahadin Abdi

Abstract

Chemotherapy-induced neuropathic pain is a significant side effect of chemotherapeutic agents and is the most common reason for stopping chemotherapy. The aim of the present study was to find the major site and mechanisms of action by which rolipram, a selective phosphodiesterase-4 inhibitor, alleviates paclitaxel-induced neuropathic pain. Chemotherapy-induced neuropathic pain was induced in adult male Sprague-Dawley rats by intraperitoneal injection of paclitaxel on four alternate days. Rolipram was administered systemically or locally into the lumbar spinal cord, L5 dorsal root ganglion, sciatic nerve, or skin nerve terminal. The mechanical threshold, the protein level of several inflammatory cytokines, and the cellular locations of phosphodiesterase-4 and interleukin-1β in the dorsal root ganglion were measured by using behavioral testing, Western blotting, and immunohistochemistry, respectively. The local administration (0.03-mg) of rolipram in the L5 dorsal root ganglion ameliorated paclitaxel-induced pain behavior more effectively than did local administration in the other sites. Paclitaxel significantly increased the expression of inflammatory cytokines including tumor necrosis factor-α (2.2 times) and interleukin-1β (2.7 times) in the lumbar dorsal root ganglion, and rolipram significantly decreased it. In addition, phosphodiesterase-4 and interleukin-1β were expressed in the dorsal root ganglion neurons and satellite cells and paclitaxel significantly increased the intensity of interleukin-1β (2 times) and rolipram significantly decreased it. These results suggest that the major site of action of rolipram on paclitaxel-induced neuropathic pain in rats was the dorsal root ganglion. Rolipram decreased the expression of inflammatory cytokines in the dorsal root ganglion. Thus, phosphodiesterase-4 inhibitors may ameliorate chemotherapy-induced neuropathic pain by decreasing expression of inflammatory cytokines in the dorsal root ganglion.

Keywords: DRG; PDE4; chemotherapy; neuropathic pain; paclitaxel; pain behavior; rolipram.

Figures

FIGURE 1
FIGURE 1
Comparison of the analgesic effect of local administration of rolipram (ROL) to the L5 DRG, spinal cord, sciatic nerve, and skin nerve terminal on established paclitaxel-induced neuropathic pain in rats. Paclitaxel (PAC, 2 mg/kg) was injected intraperitoneally in rats on four alternate days (days 0, 2, 4, and 6). Subsequently, the mechanical pain thresholds were significantly reduced. (A) [Analgesic effect of rolipram administered locally at four sites.] After the pain behavior was fully developed (20 days), 0.03 mg of rolipram was locally administered at four different sites. Local administration of rolipram at the L5 DRG significantly increased the mechanical pain threshold at 0.5, 1, and 1.5 h after administration. Vehicle was locally administered in the L5 DRG. Asterisks (∗) indicate significant differences (P < 0.05) from baseline as determined using a one-way ANOVA with one repeated factor followed by Dunnett’s multiple comparison test. (B) [Effect of local administration of rolipram in the L5 DRG] On the 20th post-paclitaxel injection day, the rats were divided into three groups (Vehicle, ROL 0.01, and ROL 0.03). Rats were administered vehicle (3% DMSO in olive oil, 10 μl), 0.01 mg of rolipram, or 0.03 mg of rolipram (indicated by the upward arrowhead), respectively. Administration of 0.03 mg of rolipram significantly increased the mechanical threshold at 0.5, 1, and 1.5 h. Asterisks (∗) indicate significant differences (P < 0.05) from vehicle group as determined using a two-way ANOVA with one repeated factor (time) followed by Sidak’s multiple comparison test. (C) [Effect of local administration of db-cAMP in the L5 DRG] On the 20th post-paclitaxel injection day, the rats were divided into two groups (Saline, db-cAMP). Rats in each group were administered saline or 0.05 mg of dibutyryl cAMP (db-cAMP, indicated by the upward arrowhead), respectively, in the L5 DRG. Administration of 0.05 mg of db-cAMP significantly increased the mechanical threshold at 2, 3, 4, and 5 h. Data are expressed as mean ± SEM. Asterisks (∗) indicate significant differences (P < 0.05) from saline group as determined using a two-way ANOVA with one repeated factor (time) followed by Sidak’s multiple comparison test.
FIGURE 2
FIGURE 2
Paclitaxel increased levels of p-NFκB, TNF-α, and IL-1β in rat DRGs. (A) Western blot showing the expression of p-NFκB, TNF-α, and IL-1β in DRGs after an injection of vehicle (VEH, N = 3) or paclitaxel (PAC, N = 3) on day 20 after the first paclitaxel injection. Rolipram (PAC+ROL, 3 mg/kg, N = 3) was intraperitoneally injected on day 20 after the first injection of paclitaxel, and the L1–L6 DRGs were obtained 1 h after the injection. (B–D) Quantification of p-NFκB, TNF-α, and IL-1β in the DRGs. Paclitaxel increased the levels of p-NFκB, TNF-α, and IL-1β in rat DRGs, and subsequent treatment with rolipram decreased them. The data are expressed as means ± standard deviations for three rats. The asterisks indicate values that are significantly different (P < 0.05) from the values for the vehicle group as determined by the Mann–Whitney U test.
FIGURE 3
FIGURE 3
(Representative immunofluorescent images of PDE4) Co-localization of PDE4, NeuN, and DAPI in the L5 DRGs. (A) PDE4 (green, Alexa Fluor 488) in the L5 DRG of a vehicle (VEH; 4% dimethyl sulfoxide and 4% Tween 80 in saline)-injected rat. (B) PDE4 (green, Alexa Fluor 488) in the L5 DRG of a paclitaxel (PAC; 2 mg/kg/1 ml of vehicle)-injected rat. (C) PDE4 (green, Alexa Fluor 488) in the L5 DRG of a paclitaxel + rolipram (ROL; 3 mg/kg)-injected rat. (D) NeuN (red, Alexa Fluor 568) in the L5 DRG of a paclitaxel-injected rat. (E) DAPI (blue) in the L5 DRG of a paclitaxel-injected rat. (F) PDE4 (green, Alexa Fluor 488), NeuN (red, Alexa Fluor 568), and DAPI (blue) in the L5 DRG of a paclitaxel-injected rat. The L5 DRGs of the VEH (N = 3) and PAC groups (N = 3) were obtained on day 20 after the first paclitaxel injection. For the ROL group, L5 DRGs were obtained 1 h after intraperitoneal injection of rolipram (3 mg/kg) on day 20. PDE4 was expressed in both neurons and satellite cells in the DRG. Stars and arrows indicates satellite cells and neurons, respectively. Scale bars, 50 μm.
FIGURE 4
FIGURE 4
(Representative immunofluorescent images of IL-1 β) Co-localization of IL-1β, NeuN, and GFAP in DRGs. (A) IL-1β (green, Alexa Fluor 488) in the L5 DRG of a vehicle (VEH; 4% dimethyl sulfoxide and 4% Tween 80 in saline)-injected rat. (B) IL-1β (green, Alexa Fluor 488) in the L5 DRG of a paclitaxel (PAC; 2 mg/kg/1 ml of vehicle)-injected rat. (C) IL-1β (green, Alexa Fluor 488) in the L5 DRG of a paclitaxel + rolipram (ROL; 3 mg/kg)-injected rat. (D) IL-1β (green, Alexa Fluor 488) and NeuN (red, Alexa Fluor 568) in the L5 DRG of a paclitaxel-injected rat. (E) IL-1β (green, Alexa Fluor 488) and GFAP (red, Alexa Fluor 568) in the L5 DRG of a paclitaxel-injected rat. (F) Quantification of IL-1β in the DRG. Paclitaxel increased the levels of IL-1β in the DRG, and subsequent treatment with rolipram decreased that. The L5 DRGs of rats in the VEH (N = 3) and PAC groups (N = 3) were obtained on day 20 after the first paclitaxel injection. For rats in the ROL group, L5 DRGs were obtained 1 h after intraperitoneal injection of rolipram (3 mg/kg on day 20). Rolipram decreased the PAC-increased IL-1β intensity in the DRG. Stars and arrows indicates satellite cells and neurons, respectively. Scale bars, 50 μm. The data are expressed as means ± standard error for three rats. The asterisks indicate values that are significantly different (P < 0.05) from the values for the vehicle group or PAC + ROL group as determined by the Mann–Whitney U test.

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