Background: Novel preclinical models for prediction of osteoarthritis-like pain are necessary for the elucidation of osteoarthritis (OA) pathology and for assessment of novel analgesics. A widely used behavioral test in rat models of tibiofemoral OA is hind limb weight bearing (WB). However, this method evaluates WB in an unnaturally restricted manner. The aim of this study was therefore to characterize the Tekscan Pressure Measurement System as a means to assess OA-like tibiofemoral pain in rats by determination of plantar pressure distribution in a more natural and unrestricted position, defined as unrestricted WB. Methods: Intra-articular injections of 1 mg monosodium iodoacetate (MIA) or saline were administrated in the left hind knee of 84 male Sprague Dawley rats. Changes in unrestricted WB between ipsilateral and contralateral hindlimbs were determined. Morphine (5 mg/kg administered subcutaneously) and naproxen (60 mg/kg per-oral) were examined for their ability to reverse WB changes. Results: Changes in hind limb unrestricted WB were observed 14 (P < 0.05), 21 (P < 0.001) and 28 (P < 0.001) days post intra-articular injections of MIA compared to control. These alterations were attenuated by morphine 1 hour post administration compared to baseline but were not affected by naproxen. Conclusion: This study indicated that unrestricted WB assessed by the Tekscan system can be utilized as a clinically relevant method to assess aberrations in WB induced by intra-articular MIA injections in rodents. Therefore, this system may be useful for understanding the mechanisms of OA pain in humans and may also assist in the discovery of novel pharmacological agents.
Osteoarthritis (OA) is an age-related disease affecting millions of people worldwide [1,2]. OA symptoms vary considerably and patterns of nociception are distinguished differently by patients varying from dull aches to sharp stabbing pain. Neuropathic descriptors apply to a subset of OA patients indicating a role of sensitization in the underlying pathology [
OA is primarily characterized by damaged articular cartilage and subsequent cartilage loss [
Intra-articular (i.a.) administration of monosodium iodoacetate (MIA) in rats is a validated chemical model used to investigate OA. MIA, when injected through the patellar tendon in the hind limb of rodents, displays multiple components of disease progression and symptoms similar to human OA and induces significant nociceptive behavior [9-11] albeit with a different time course [
The Tekscan Very HR Walkway Floor Mat Pressure Measurement System (Tekscan Inc., USA) is available for the study of plantar pressure in animals by analysis of the force applied to the ground per limb over time. This method seems clinically relevant as it has potential to evaluate WB while the rats are unrestricted in a freely moving position. Moreover, the methodology offers some pre-clinical translatability as the Tekscan device is already used for WB assessment in humans.
This study aimed to characterize the Tekscan system as a means to assess tibiofemoral OA-related nociception by determination of plantar pressure distribution in MIA injected rats in an unrestricted position, defining the method as unrestricted WB. For further validation of unrestricted WB as a relevant, non-evoked behavioral measure or endpoint for pain the effects of two clinically available analgesics on behavior were examined.
All experiments were approved by the Institutional Animal Care and Use Committee at Purdue Pharma L.P. and were in strict accordance with the ethical guidelines laid down by the International Association for the Study of Pain [
For the induction of the unilateral tibiofemoral OA model, 84 animals were briefly anesthetized with isoflurane 2.5% - 4.0% (Baxter Healthcare, USA). Their left hindlimb was shaved and sanitized with Betadine Surgical Scrub (Purdue Pharma, LP, USA) and 72% ethanol. Using a 27-gauge needle through the patellar tendon in the left hind limb, 42 rats received a single i.a. injection of 1 mg MIA (Sigma-Aldrich, USA) (1 mg in 0.02 ml normal saline), while 42 rats (control group) received a single i.a. injection of 0.02 ml normal saline. The rats were allowed to fully recover in a temperature-controlled cage and then returned to their home cages. The dose of MIA used (1 mg/i.a. injection) was selected based on in-house efficacy data as well as from the literature [9,11].
Assessment of bilateral unrestricted WB following unilateral injection of MIA was used as behavioral measurement of nociception in OA-induced rats. Restricted WB was also assessed and used as a reference value. Rats were tested at day 0 (baseline), 7, 14, 21 and 28 postMIA injection.
Changes in weight distribution between the left and right hind limbs and front limbs were assessed by unrestricted WB using the Tekscan WalkwayTM 5101D HR Floor Mat Pressure Measurement System (Tekscan, Inc. South Boston, MA, USA). The Tekscan system is composed of two 5101 very High-Resolution (HR) sensors with a spatial resolution of 15.5 sensels/cm2 embedded into a floor mat. For the purpose of this experiment, the sensors were calibrated to a known weight of 268.5 g according to manufacturer instructions (Tekscan WalkwayTM Software version 7.01, Tekscan Inc. South Boston, MA, USA). Rats were positioned on an open surface inside a 22.4 × 11.2 cm clear Plexiglas chamber, which was placed on top of two sensors without contact between sensels and chamber. In order to acclimate to the equipment and to provoke joint pain, the rats were allowed to walk freely around on the mat and when hind limbs and front limbs were in an equal position (
Changes in restricted weight distribution between the left and right hind limbs were determined using incapacitance testers (Columbus Instruments, USA). Rats were placed in an angled Plexiglas chamber so that each hind limb was positioned on a separate force plate (
Similarly, restricted WB measurements were recorded before (baseline) and after the i.a. procedure (model development). Rats were randomized to treatment group following model induction.
Naproxen sodium (Sigma-Aldrich, USA) was dissolved in 0.5% methylcellulose and administered p.o. at a dose of 60 mg/kg while morphine sulfate (Sigma-Aldrich, USA) was dissolved in 0.9% normal saline and administered at a dose of 5 mg/kg subcutaneously (s.c.). The dose volumes were 5ml/kg and 2 ml/kg, respectively. Control rats were dosed similarly with the appropriate matched vehicle. The doses for both compounds were selected based on in-house assessment as well as efficacy results obtained from several nociceptive models described in the literature [9,11,14-15].
The effect of naproxen on WB was assessed using rats 14 days post-MIA/saline injection. Rats (n = 10/group) were given a single dose of naproxen or vehicle. Changes in hind limb weight distribution were determined by unrestricted and restricted WB as outlined above, before compound administration (pre-drug baseline; day 14) and 1, 3, and 5 hours post-compound administration.
The effect of morphine on WB was assessed in a separate cohort of rats 21 and 28 days post-MIA/saline injection (n = 12/group and n = 20/group, respectively). Rats were given a single dose of morphine or vehicle. Changes in limb weight distribution were determined by unrestricted and restricted WB as outlined above before compound administration (pre-drug baseline; days 21 and 28) and 1 and 3 hours post-compound administration.
For unrestricted and restricted WB measurements, the percent weight (in grams) borne on the left limb was determined using the following formula:
Data were computed in Microsoft® Office Excel® 2007 (Microsoft® Corporation, USA).
The statistical analyses were carried out using GraphPad Prism (version 5.04; GraphPad Software, Inc., USA). The analyses for the time course effect on the hind limb unrestricted and restricted WB following injection of MIA versus saline controls and the effect of administration of analgesic compounds were conducted using a two-way analysis of variance (ANOVA). The analyses of the effect of treatment within groups were performed using a one-way ANOVA. Bonferroni multiple comparison test was performed as a post hoc comparison. Data are presented as mean ± SEM. P < 0.05 was set as the level of statistical significance.
The i.a. administration of either MIA or saline had no effect on the general health of the animals. The animals in the MIAand saline-injected groups gained body weight normally and neither displayed any signs of distress or infection. Following MIA administration a timedependent increase in hind limb joint pain was noted throughout the 28-day study period. The pain was defined by changes in hind limb weight distribution assessed by both unrestricted and restricted WB (Figures 2(a) and
The MIA model resulted in a significant decrease in the mean percentage of weight distribution on the affected hind limb, 41.0% ± 1.8% on day 21 (P < 0.001) and 40.0% ± 2.7% on day 28 (P < 0.001) compared to a between days average of 51.1% ± 1.1% for controls (
Similarly, the MIA model resulted in a significant decrease in the mean percentage of weight on the ipsilateral hind limb, 43.6% ± 0.0% at day 7 (P < 0.0001), 44.00% ± 0.0% at day 14 (P < 0.0001), 42.2% ± 0.0% at day 21 (P < 0.0001), and 40.7% ± 0.0% at day 28 (P < 0.0001) compared to a between days average of 50.0% ± 0.0% for controls (
Experiments designed to determine the analgesic effects of naproxen and morphine on MIA-induced OA-like pain were evaluated by weight distribution assessment between the hind limbs. Behavior was assessed on days 14, 21 and 28 post-MIA injection. In order to ensure that the compounds used above were not impairing the WB assessment on their own, the compounds were administered to saline-injected rats as well. None of the compounds had any significant effect on WB in the control rats.
On day 14 naproxen did not significantly reverse the deficits in hind limb weight distribution noted between MIAand saline-injected animals (P < 0.05) using unrestricted WB assessment (
The mean percentages of weight noted on the ipsilateral hind limb were 50.3% ± 0.0% (P < 0.0001), 48.1% ± 0.0% (P < 0.0001), and 49.6% ± 0.0% (P < 0.0001) at each of the time points, respectively, compared to an average of 45.7% ± 0.0% for saline-injected controls (
Morphine administration on days 21 and 28 post MIA-
injection tended to normalize the changes in hind limb weight distribution assessed by unrestricted WB 1 and 3 hours post-exposure as compared to vehicle treated controls. The mean percentage of weight on the affected hind limb on day 21 increased to 49.6% ± 3.6% and 47.5% ± 0.7% at 1 and 3 hours post-morphine administration, respectively, compared to an average of 42.7% ± 1.1% for vehicle treated animals (
When using the restricted WB methodology, morphine significantly reversed the changes in hind limb weight
distribution 1 and 3 hours post treatment when compared to vehicle treated OA rats both at day 21 and day 28 post-MIA injections.
The mean percentage of weight on the ipsilateral hind limb on day 21 increased to 50.8% ± 0.0% at 1 hour (P < 0.0001) and 47.5% ± 0.0% at 3 hours (P < 0.0001) post-morphine administration compared to an average of 44.7% ± 0.0% for vehicle controls (
The MIA model of OA-like pain produced deficits in unrestricted hind limb WB as evaluated by the Tekscan system. Morphine attenuated this WB deficit within the group of OA rats albeit with a limited effect suggesting
that unrestricted hind limb WB can be assessed by the Tekscan system.
The lack of methods measuring primary hyperalgesia and allodynia has led to the development of several assessment techniques for OA pain measurement in rats (e.g. the validated and frequently used method of restricted hind limb WB published by Bove, et al. [
The Tekscan system has previously demonstrated efficient, reliable, and accurate analysis of behavioral changes that accompany hind limb paresis in rodents during gait [
The observed changes in unrestricted hind limb WB were only statistically significant on days 14 (pharmacology), 21 and 28 post induction of OA, whereas restricted hind limb WB demonstrated statistically significant changes on all assessment days indicating that the restricted WB may be a more sensitive pain indicator. However, while more sensitive, it may be a less relevant measure as rodents are quadrupeds. In the light of this the insignificant unrestricted WB results noted on day 7 post-induction of OA should not be considered a limitation of the Tekscan method. Previous studies [11,14,24] have demonstrated that pharmacological studies using the MIA-model are most clinically relevant when performed at later time points, such as 21+ days post-MIA injection, because of the biphasic nature of MIA injections. Therefore, in the current study the efficacy of naproxen and morphine was assessed 14 - 28 days postinjection.
Previous studies [9,11] have described measurements of hind limb weight distribution as a reliable and practical method to assess nociception associated with experimental OA. The current results are in accordance with those studies, which serve in the validation of the Tekscan system. When comparing unrestricted WB to restricted WB, the values observed were similar indicating that the two methodologies are reliable and that the animals are not stressed. Ferreira-Gomes, et al. [
The Tekscan system offers a comprehensive approach for measuring plantar pressure from front limbs and hind limbs and can thereby reveal possible compensatory patterns in the front limbs and contralateral hind limb as a result of unilateral OA induction in rodents. To our knowledge, only a few studies [18-19] have investigated WB of the front limbs after injury to one of the hind limbs. Boyd, et al. [
Non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclooxygenase 2 inhibitors (COX-2) are some of the most frequently prescribed drugs for the management of OA pain [
Morphine is widely used in animal models investigating OA-related joint pain [11,14-15,27]. The maximum antinociceptive effect of morphine sulfate has been determined to occur between 30 and 90 minutes when dosed at 6 mg/kg [
Pain associated with OA still continues to represent a major unmet medical need. Therefore, novel and effective pharmacological therapeutic options to treat OA pain are still very much warranted. Animal models of OA like MIA continue to be used to provide new information in our evaluation of nociception parameters relevant to the human condition. In this regard, the present study indicates that the Tekscan system can be utilized to assess OA-like pain behavior in rats using a clinically relevant endpoint, unrestricted WB. Although additional pharmacological validation studies are needed to ultimately determine the utility of the Tekscan system as a translational tool, initial results from this study and others are promising [19,22,29].