Chronic oral or intraarticular administration of docosahexaenoic acid reduces nociception and knee edema and improves functional outcomes in a mouse model of Complete Freund’s Adjuvant–induced knee arthritis

Experiments were performed on a total of 83 male adult ICR mice (Harlan Laboratories, Mexico City, Mexico) weighing 18 to 25 g at the beginning of the experiments. All animals were housed in our facilities at a constant temperature (22°C to 24°C) and had free access to food and drinking water before the experiments. This study was approved by the Comité de Ética Institucional de la Unidad Académica Multidisciplinaria Reynosa Aztlán de la Universidad Autónoma de Tamaulipas (CEI-UAMRA-2012-10). All experiments were conducted in accordance with the Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals[37]. At the end of the experiments, all animals were killed in a CO₂ chamber.

CFA (catalog no. F5881), DHA (catalog no. D2534), olive oil (vehicle; catalog no. W530191) and naloxone (NLX; catalog no. N7758) were purchased from Sigma-Aldrich (Toluca, Mexico). DHA was dissolved in olive oil and administered by oral gavage or intraarticular injection.

A version of a previously validated model of arthritic inflammation of the knee joint[38‐40] was produced by performing four intraarticular injections of CFA (10 μl) at days 0, 7, 14 and 21 unilaterally into the right knee joint. Briefly, mice were anesthetized using a mixture of ketamine/xylazine (100 mg/kg and 5 mg/kg by intraperitoneal injection, respectively), followed by an intraarticular injection of CFA using a 30-gauge, 0.5-inch needle that was fitted with cannulation tubing such that only 2 to 3 mm of the needle was allowed to puncture the joint. CFA was injected through the patellar ligament into the articular space using the femoral condyles as a guide.

Behavioral measures of arthritic joint pain included flinching of the affected limb (spontaneous pain), number of total vertical rearings requiring the use of both hind limbs and horizontal exploratory activity (used as functional outcomes of the affected limb). Briefly, mice were placed in open cylindrical Plexiglas observation chambers (height 30 cm and inside diameter 20 cm) for 20 minutes to allow the mice to acclimate to their surroundings. Mirrors were placed behind the chambers to enable unhindered observation. The number of spontaneous flinches was recorded during a 5-minute observation period as previously described[41]. Flinches were defined as the number of times the animal raised its hind paw, which was used as a measure of spontaneous arthritic pain. Although spontaneous flinching is not present in patients with RA, this behavior may reflect the spontaneous pain that arthritic patients experience without movement (that is, pain at rest) of the affected extremity[21, 41, 42].

The number of total vertical rearings was recorded during a 5-minute observation period while the animals were in the open Plexiglas observation chamber. Total vertical rearings was defined as the number of times the animals stood on both hind limbs while supporting their entire body weight[43]. A reduction in vertical rearing in mice is consistent with the reduction in movement or joint-loading observed in RA patients[21, 42].

Once flinching behavior and vertical rearing had been evaluated, horizontal exploratory activity was quantified using a modified open-field paradigm. Mice were placed in the center of a rectangular acrylic chamber (18 × 27 × 15 cm). The chamber was divided into a grid of equally sized rectangles (nine in total measuring 9 × 6 cm each) using lines drawn on the chamber floor for visual quantification. The total number of times each line was crossed in a 2-minute period was quantified by the experimenter[44, 45].

A two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni post hoc correction was used to compare the behavioral measures between the experimental groups at different time points. The magnitude of the anti-inflammatory effect of DHA was determined using two-way ANOVA followed by the Bonferroni post hoc correction. The statistical significance level was set at P < 0.05. In all possible cases, the investigator responsible for behavioral testing, measuring and counting was blinded to the experimental conditions of each animal.

Mice that received only an intraarticular injection of saline or DHA showed minimal spontaneous nociceptive behavior or impairment of functional outcomes (data not shown). In contrast, mice that received CFA injections exhibited a greater number of flinches (considered spontaneous pain-related behavior) than observed in sham-treated mice from day 4 through day 25 after the initial CFA injection (data not shown). Additionally, the number of line crosses in the open field test (pain-related functional disability) and vertical rearings (pain-related functional disability) were significantly reduced in mice that received CFA injections than sham-treated mice as time progressed (data not shown).

To determine the effect of chronic oral administration of DHA on spontaneous nociceptive behavior and on pain-related functional disabilities, DHA was administered daily from day 15 until day 25 after the initial CFA injection (Figure 1A). Mice with CFA-induced arthritis and treated with DHA at three different doses (10, 30 and 100 mg/kg by oral administration) showed significantly fewer flinches than mice with CFA-induced arthritis and treated with vehicle (Figure 1B). The antinociceptive effect of DHA was maintained until day 25. Additionally, chronic oral administration of DHA (at 30 and 100 mg/kg) significantly reversed the CFA-induced impairment of horizontal exploratory activity. This beneficial effect was also maintained until day 25 after the initial CFA injection (Figure 1C). The antinociceptive effect of DHA on these two pain-related behaviors reached a maximum effect between the doses of 30 and 100 mg/kg. DHA administration did not significantly change the CFA-induced reduction in vertical rearing (Figure 1D).

Repeated injections of CFA significantly increased knee edema in the ipsilateral knee joint as compared to the contralateral knee joint or in the knee joints of mice injected with saline solution (data not shown). In all mice that received CFA injections, knee edema was restricted to the ipsilateral joint with no visual evidence of knee edema in the contralateral hind limb, and this edema was maintained through day 25 after the initial CFA injection. At day 15 after the initial CFA injection, oral administration of DHA or vehicle was initiated and maintained until day 25. The anti-inflammatory effect of chronic oral administration of DHA at doses of 10, 30 and 100 mg/kg was determined at days 18 and 25 after the initial CFA injection (Figure 2A). The diameter of CFA-induced ipsilateral knee edema was reduced by DHA in a dose-dependent manner. This effect was statistically significant at doses of 10, 30 and 100 mg/kg (Figure 2B).

To determine the effect of DHA in the CFA-induced arthritis model following a single administration, we performed behavioral analysis at day 15 after CFA injection. Behavioral analysis revealed that a single oral administration of DHA (30 mg/kg) did not significantly reduce the spontaneous nociceptive behaviors (Figure 3A) or restore the CFA-induced vertical rearing impairment at hours 1, 2 and 24 after DHA administration (Figure 3C). In contrast, acute DHA administration significantly reversed the CFA-induced horizontal exploratory activity impairment (Figure 3B) and reduced the knee edema of the ipsilateral knee joint at 2 and 24 hours as compared to CFA-injected mice treated with vehicle (Figure 3D).

To determine whether intraarticular administration of DHA would reduce pain-related behaviors and knee edema in the CFA-induce arthritis model, administration of DHA into the knee joint (25 and 50 μg/joint) was performed at days 15, 19 and 22 after initial CFA injection (Figure 4A). Both doses of DHA (25 and 50 μg/joint) decreased spontaneous pain behavior (Figure 4B). The CFA-induced impairment of horizontal exploratory activity was significantly restored by intraarticular DHA administration at both doses on day 25 (Figure 4C). The intraarticular DHA did not significantly change the CFA-induced reduction in vertical rearing (Figure 4D). The anti-inflammatory effect of chronic intraarticular administration of DHA at doses of 25 and 50 μg/joint was determined at days 18 and 25 after the initial CFA injection (Figure 5A). DHA treatment at doses of 25 and 50 μg/joint significantly decreased the diameter of the ipsilateral knee joint on the days evaluated (Figure 5B).

To evaluate a possible mechanism of action for the antinociceptive effect of DHA, we examined the effect of NLX, an opioid receptor antagonist, on DHA antinociceptive action in mice that received CFA injections and were treated with oral DHA. NLX did not reverse the antinociceptive effect of DHA on mice with CFA-induced arthritis (Figure 6). Additionally, NLX by itself did not increase or reduce CFA-induced spontaneous flinches or pain-related functional disabilities (data not shown).

Although we observed that acute administration of DHA was able to significantly reduce knee edema and restore CFA-induced horizontal exploratory activity deficits, this treatment did not significantly reduce spontaneous flinching. Although we do not yet know the exact mechanisms underlying this finding, we suggest that CFA-induced arthritic pain might not be driven only by central and peripheral sensitization triggered by chronic inflammation of the knee[22]. In this context, several studies have demonstrated that osteoclast-mediated acidosis and bone loss are also involved in the generation and maintenance of arthritic pain[24, 51, 52]. Researchers in previous studies have shown that treatment with DHA decreases osteoclast activation[17, 18] and that long-term intake of a mixture of ω-PUFAs (containing DHA) reduces urine excretion of markers of bone and cartilage resorption in patients with RA[53]. In light of these findings, we suggest that the greater beneficial effect of chronic DHA as compared to acute DHA on spontaneous pain and functional outcomes may be due to DHA’s ability to reduce arthritis-mediated bone loss and erosion, thereby preserving mechanical bone strength.

Although RA is one of the most common musculoskeletal disorders in the world[1, 4], current therapies used to alleviate joint pain have limited efficacy and/or are associated with significant side effects[9, 11, 54]. For example, owing to the chronic nature of the disease, systemic long-term treatment with nonsteroidal anti-inflammatory drugs and cyclooxygenase 2 (COX-2) inhibitors in patients with arthritic pain may induce negative side effects in the cardiovascular and gastrointestinal systems[8‐11]. A therapeutic option frequently used to minimize systemic side effects is to deliver the drug directly into the affected joint (for example, by intraarticular administration)[28‐31]. Although intraarticular administration has been widely used to deliver corticosteroids and other disease-modifying antirheumatic drugs[55, 56], this method is not frequently used to deliver analgesics in arthritic conditions. In the present study, we evaluated the chronic effect of intraarticular administration of DHA on spontaneous nociception, knee edema and pain-related functional disabilities. Our results show that intraarticular injection of DHA was as effective as oral administration. However, it should be noted that for intraarticular administration, we used doses 80 times lower than the systemic concentration. Given that intraarticular injections may be associated with discomfort, pain and possible risk of infection[31, 57], the number of intraarticular injections should be kept to a minimum. In this study, DHA intraarticular injections were performed twice per week versus on a daily basis for oral administration; however an analgesic and anti-inflammatory effect of DHA was reached. Thus, the results of this study suggest that local delivery of DHA into the affected joints may offer another alternative treatment for arthritic pain and inflammation without the possible development of side effects associated with systemic administration.

Investigators in previous studies have demonstrated that pretreatment with NLX prevented the antinociceptive effect of DHA after oral administration in acute pain models including formalin-induced nociception and acetic acid writhing tests in the mouse[32]. Additionally, oral administration of DHA increases plasma levels of β-endorphins in mice[58]. These data, taken together, suggest that DHA exerts an antinociceptive effect via activation of an endogenous opioidergic mechanism. In contrast, our experiments demonstrated that administration of NLX (using a dosage and scheme of administration in mice previously validated by others and in our laboratory)[49, 50] did not reverse the beneficial effect of DHA on CFA-induced flinching and horizontal activity deficit. The reasons for these findings are unclear; however, it is possible that mechanisms underlying DHA antinociception in acute pain models may be different from those involved in chronic pain states and/or there may be a decrease in the levels of endorphins in chronic arthritic conditions. In support of this theory, investigators in a clinical study demonstrated that there is an inverse correlation between serum levels of β-endorphins and rheumatoid disease activity score and the duration of RA[59].

Although researchers in several studies have demonstrated anti-inflammatory and antinociceptive effects of DHA following acute administration[32, 34, 36, 60], less is known about the molecular mechanisms underlying these effects. Our possible hypothesis, gleaned from basic scientific literature, is that both direct and indirect effects may explain the biological actions of DHA. First, direct effects may include competition of DHA with arachidonic acid as a substrate for cyclo- and lipoxygenase, thus reducing the production of inflammatory eicosanoids[26, 61, 62]. Moreover, researchers recently demonstrated that the anti-inflammatory actions of DHA may partially be explained by inhibition of lipopolysaccharide-mediated COX-2 induction and activity via direct activation of the free fatty acid receptor 4[63]. However, indirect effects may include the participation of D-resolvins and protectin D1, which are lipid mediators enzymatically synthesized in vivo from DHA and promote the resolution of inflammation with greater potency than their parent precursors[35, 64].

The mechanisms underlying the antinociceptive actions of DHA in arthritic pain conditions remain to be explored. However, the results of a previous study suggest that the anti-inflammatory actions of DHA may also contribute to amelioration of arthritic pain[26]. It also has been shown that DHA may directly modulate the excitability of sensory neurons through inhibition of the voltage-dependent sodium currents in dorsal root ganglion neurons[65]. However, an indirect analgesic effect of DHA on arthritic conditions cannot be ruled out. It was recently shown that DHA affects disease progression in that it reduces knee synovial inflammation, pannus formation, cartilage damage and bone damage as evaluated by histopathological analysis in a collagen-induced arthritis model in the mouse[26]. Both direct actions on sensory neurons and effects on disease progression, in addition to as yet unknown mechanisms, presumably participate in the analgesic actions of DHA in arthritic conditions.

The present study has some limitations. First, we suggest that the beneficial effect of DHA on pain-related functional disabilities may partially be a result of DHA’s inhibiting osteoclast-mediated bone destruction and preserving bone mechanical strength. Whether DHA affects bone mass and microarchitecture in arthritic conditions is unknown. Second, it is possible that intraarticular delivery of olive oil by itself could further increase pain, discomfort and synovial inflammation. However, our results show that treatment-naïve animals that received multiple intraarticular olive oil injections displayed minimal spontaneous flinching and that levels of locomotor activity and vertical rearing were similar in magnitude as those of treatment-naïve mice. The mechanisms driving anti-inflammatory and antinociceptive actions in arthritic conditions by DHA remain to be elucidated. Although our present study suggests that an endogenous opioid pathway does not seem to be involved in the actions of DHA, future studies need to be performed to explore the molecular targets of DHA in painful arthritic conditions.