Anti-inflammatory and antinociceptive effects of phonophoresis in animal models: a randomized experimental study

Abstract

The aim of this study was to evaluate the therapeutic effects of ultrasound (US)-mediated phonophoresis alone or in association with diclofenac diethylammonium (DCF) administered topically in animal models of inflammation. A pre-clinical, prospective, and randomized experimental study of quantitative and qualitative nature was carried out. Phonophoresis was performed using a therapeutic ultrasound apparatus in two distinct models of acute inflammation. Edema was induced by an intraplantar injection of carrageenan and measured by plethysmography. The Hargreaves test was used to evaluate the antinociceptive activity and investigate the action of phonophoresis on tumor necrosis factor (TNF)-α production. A histological analysis with hematoxylin-eosin was used to evaluate tissue repair, and the expression of COX-2 was determined by immunohistochemical analysis. At the peak of inflammatory activity (3 h), treatment with US, US+DCF, and DCF significantly reduced edema formation compared to the control group. Treatment with US+DCF was more effective than treatment with US alone at both analyzed times. In the analysis of the antinociceptive activity, the treatments significantly increased the latency time in response to the thermal stimulus. Histopathological analysis revealed a reduction of the inflammatory infiltrates and immunohistochemistry demonstrated that the association was effective in reducing COX-2 expression compared to the control group. The association of DCF with US produced anti-inflammatory and antinociceptive effects in rat models of inflammation, which may be associated with inhibition of COX-2 and TNF-α production.

Figure 1.. Evaluation of acute treatment on the volume of paw edema 3 h after carrageenan administration in rats treated with ultrasound (US), ultrasound associated with diclofenac diethylammonium (US+DCF), and diclofenac diethylammonium alone (DCF). Data are reported as mean±SE. Indomethacin 20 mg/kg (po) was used as reference drug. ^aP<0.001, ^bP<0.05, ^cP<0.001, ^eP<0.01 vs control; ^dP<0.05 vs US (ANOVA).

Figure 2.. Means±SE of the volume of paw edema 3 h after administration of carrageenan in diclofenac (DCF) pre-treated rats with or without ultrasound (US+DCF) and pentoxifylline (PENTOX 1). Indomethacin 20 mg/kg (po) (INDO 20) was used as reference drug. ^aP<0.05, ^bP<0.001 vs control; ^cP<0.05 vs PENTOX 1, PENTOX 1+US, and PENTOX 1+DCF (ANOVA).

Figure 3.. Evaluation of acute treatment with diclofenac (DCF) or ultrasound-associated DCF (US+DCF), and US on thermal nociception in cutaneous hyperalgesia (Hargreaves's test) in the third hour (T3). Data are reported as mean±SE. Indomethacin 20 mg/kg (po) (INDO 20) was used as reference drug. ^aP<0.05; ^bP<0.01; ^cP<0.001 vs control (ANOVA).

Figure 4.. Photomicrographs representative of histopathological analysis (100×, bar=200 μm) of tissues from the right paw of rats submitted to the carrageenan-induced paw edema model, stained with hematoxylin-eosin. The images demonstrate cell infiltrates for the Control and groups treated with 20 mg/kg indomethacin, po (INDO 20), 1 mg pentoxifylline (PENTOX 1), 1 mg pentoxifylline associated with ultrasound (PENTOX 1+US), 1 mg pentoxifylline associated with ultrasound, and 10 mg diclofenac (PENTOX 1+US+DCF), 1 mg pentoxifylline associated with 10 mg diclofenac (PENTOX 1+DCF). Percent of cellular infiltrate analyzed by the ImageJ program is shown in the graph. Data are reported as means.

Figure 5.. Representative photomicrographs of immunohistochemistry for cyclooxygenase-2 (COX-2) (400×, bar=200 μm) of tissues from the right paw of rats submitted to the carrageenan-induced paw edema model. The Control (untreated) group was compared with the groups treated with 20 mg/kg indomethacin, po (INDO), 10 mg/g diclofenac (DCF), ultrasound (US, 1 w/cm², 1 min), and ultrasound associated with diclofenac (DCF+US). The graph shows the data analyzed by the ImageJ program. Data are reported as mean±SE. ^aP<0.001 vs CONTROL; ^bP<0.05 vs DCF; ^cP<0.01, ^dP<0.01 vs DCF+US. ^eP<0.001 vs INDO. (one-way ANOVA and Newman-Keuls with post hoc test).