Analgesic effects and arthritic changes following intra-articular injection of diclofenac etalhyaluronate in a rat knee osteoarthritis model

Abstract

Background: Diclofenac etalhyaluronate (DF-HA) is a recently developed analgesic conjugate of diclofenac and hyaluronic acid that has analgesic and anti-inflammatory effects on acute arthritis. In this study, we investigated its analgesic effect on osteoarthritis, using a rat model of monoiodoacetate (MIA).

Methods: We injected MIA into the right knees of eight 6-weeks-old male Sprague-Dawley rats. Four weeks later, rats were randomly injected with DF-HA or vehicle into the right knee. Seven weeks after the MIA injection, fluorogold (FG) and sterile saline were injected into the right knees of all the rats. We assessed hyperalgesia with weekly von Frey tests for 8 weeks after MIA administration. We took the right knee computed tomography (CT) as radiographical evaluation every 2 weeks. All rats were sacrificed 8 weeks after administration of MIA for histological evaluation of the right knee and immunohistochemical evaluation of the DRG and spinal cord. We also evaluated the number of FG-labeled calcitonin gene-related peptide (CGRP)-immunoreactive(ir) neurons in the dorsal root ganglion (DRG) and ionized calcium-binding adapter molecule 1 (Iba1)-ir microglia in the spinal cord.

Results: Administration of DF-HA significantly improved pain sensitivity and reduced CGRP and Iba1 expression in the DRG and spinal cord, respectively. However, computed tomography and histological evaluation of the right knee showed similar levels of joint deformity, despite DF-HA administration.

Conclusion: DF-HA exerted analgesic effects on osteoarthritic pain, but did not affect joint deformity.

Keywords: Diclofenac etalhyaluronate; Osteoarthritis; Pain; Rat monoiodoacetate model.

Fig. 1

Effect of DF-HA on the pressure withdrawal threshold applied to one posterior ipsilateral limb Changes in hyperalgesia are measured using a von Frey filament and expressed in grams (g) as a 50% paw withdrawal threshold. Behavioral tests are performed before MIA administration (BL). Further, 2 mg of MIA is administered, and the von Frey test is performed weekly for 8 weeks. DF-HA (DF-HA group) or vehicle (vehicle group) is administered at week 4 and FG is administered at week 7 after the test. Lower threshold values indicate enhanced hyperalgesia. Data are expressed as the mean ± standard error of four animals per group

Fig. 2

Fluorescence photomicrographs of the DRG after administration of DF-HA or vehicle for 4 weeks a FG-labeled DRG neurons, b CGRP-immunoreactive (ir) DRG neurons, and c overlaid picture a on b. All photomicrographs are from the same section. White arrows in the photomicrograph of a indicate FG-labeled DRG neurons, and white arrowheads in the photomicrograph of b and c indicate FG-labeled CGRP-ir DRG neurons. The proportions of FG-labeled CGRP-ir neurons in the DF-HA groups are significantly higher than those in vehicle group (n = 4 rats per group)

Fig. 3

Fluorescence photomicrographs of the spinal cord after administration of DF-HA or vehicle for 4 weeks a and b Representative fluorescent photomicrographs of the right dorsal horn of the spinal cord. Scale bars, 100 μm. White arrowheads indicate Iba1-immunoreactive (ir) microglia in a DF-HA and b vehicle groups. The number of Iba1-ir microglia is significantly higher in the vehicle group than in DF-HA group

Fig. 4

Histopathological images of the rat’s knee in the vehicle and DF-HA groups a and b Vehicle group, and c and d DF-HA group. Scale bars, 400 μm. a and c Hematoxylin–eosin staining. b and d Safranin O (SO) staining. a and c White arrows indicate significant cartilage loss in both groups. b and d Little cartilages are stained red with SO. b and d Red-stained areas are irregularly fragmented and distributed deep in the remaining cartilage layer (sections surrounded by a red frame)

Fig. 5

Graphs of OARSI score in each group after 4 weeks of DF-HA or vehicle administration The mean score of the vehicle group is slightly higher than that of DF-HA group (P > 0.05)

Fig. 6

CT images of the right knee after intra-articular injection of MIA every 2 weeks a–d Vehicle and e–h DF-HA groups. The graph shows the transition of the mean Larsen grade of each group. Joint degeneration tends to progress with time (P > 0.05)