Using the CatWalk method to assess weight-bearing and pain behaviour in walking rats with ankle joint monoarthritis induced by carrageenan: Effects of morphine and rofecoxib

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Abstract

The CatWalk automated quantitative gait analysis technique has been validated as a method to quantify behaviour in rodent models of neuropathic and arthritic pain. Its suitability for pharmacological testing of pain relief has been questioned, however, based on findings using paw soft tissue plantar inflammation as stimulus. In this study, we investigated the effectiveness of morphine and rofecoxib in reducing pain behaviour in monoarthritic rats. The CatWalk was used to assess print area, weight load and duration of stance for each paw, as well as interlimb coordination, before and 3, 5 and 24 h after injection of lambda-carrageenan into one ankle joint. The monoarthritic rat showed a reduced print area, weight load and duration of stance for the injected paw at all times tested, and a significant loss of interlimb coordination at 3 and 5 h after injection. Both morphine (3.75 and 15 μmol/kg s.c.) and rofecoxib (7.5 and 30 μmol/kg p.o.) reduced the effects of carrageenan. In conclusion, behavioural effects interpreted as reflecting movement-related pain in monoarthritic rats and pharmacological treatment of the monoarthritis can objectively and efficiently be quantified in detail by the CatWalk method.

Introduction

Nociception in rodent arthritis models is frequently quantified by evoked responses to thermal or mechanical stimuli but also by measuring behavioural parameters interpreted as signs of ongoing pain, e.g. by measuring weight load exerted onto force-plates by the hind paws (Schött et al., 1994, Bove et al., 2003, Fernihough et al., 2004, Pomonis et al., 2005), or by using a manual rating scale for standing and walking paw pressure scores which is mainly described in terms of visible contact area of the injected limb with the floor such as: “paw is completely on the floor but toes are not spread”, “foot curled with only some parts of the foot lightly touching the floor”, and “foot elevated completely” (Coderre and Wall, 1987, Coderre and Wall, 1988). Measuring behaviours interpreted as signs of pain related to movement in rat models of arthritis has challenged several scientists. Tonussi and Ferreira (1992) compared the time of contact with the floor between the arthritic and the non-arthritic paw, fitted with metal gaiters wrapped around both paws, and the metal mesh floor of a rotarod as a way to grade pain behaviour. López-Muñoz et al. (1993) refined this approach by attaching an electrode to each hind paw, making it possible to measure stance contact time on the rotating cylinder without needing to fit a gaiter each time.

Walker et al. analysed stance duration (Walker et al., 1994) in nerve-injured rats by letting them walk on a runway with a frosted acrylic surface. The rats were recorded on video through the floor and each video frame where the paws were in contact with the floor was counted to compare stance duration of the nerve-injured and the uninjured hind paw. In addition to duration of contact, weight load during walking has been measured in mice (Clarke and Still, 1999, Clarke et al., 2001) and in normal and arthritic rats using load cell platforms, and sometimes also an optical methodology using an internally illuminated glass floor (Clarke, 1995, Clarke et al., 1997). Min et al. (2001) used a walkway equipped with strain gauge weight sensors on the left and right side at four different spots along the path, and Koo et al. (2002) used two electronic balances at the midway point of a walkway, each covering a half-width of the path so that only the paw from one side would step on the balance. By recording and observing chronologically a longer section of the walkway, gait parameters such as stance and swing duration, stride length and velocity have been analysed (Simjee et al., 2004). The CatWalk technique (Hamers et al., 2001) has recently been used to quantify individual paw parameters as well as parameters related to interlimb coordination, after injection of carrageenan into a knee joint in the rat (Gabriel et al., 2007).

Carrageenan, a sulphated mucopolysaccharide derived from the Irish moss Chondrus crispus or from red Scottish seaweed (Smith and Cook, 1953), is known for its remarkable capacity to stimulate local inflammation dominated by intense macrophage aggregation and by fibroblastic proliferation (Williams, 1957, Di Rosa et al., 1971). A single intraarticular injection initiates a localized synovial inflammatory response, which results in a decrease in both the proteoglycan content and in the rate of proteoglycan synthesis in the articular cartilage (Lowther and Gillard, 1976, Santer et al., 1983). One single injection of carrageenan directly into the rat knee joint produces symptoms peaking at 3–4 h and lasting for 1–2 days, and which are sensitive to treatment by most anti-nociceptive drugs (Tonussi and Ferreira, 1992).

In the present study we have characterized the carrageenan-induced rat ankle joint monoarthritis model, using the CatWalk method. Although behavioural effects after injecting carrageenan into the knee joint in rats have been reported (Gabriel et al., 2007), results after injection into the ankle joints are new. The CatWalk was originally developed for studying rats with spinal cord injuries (Hamers et al., 2001), and is a video-based system for automated gait analysis using an enclosed walkway with glass floor where light enters along one long edge. At the placement of a rat paw, the light projected into the glass floor is scattered and produces an illuminated print. Using this method it is possible to measure a number of parameters pertaining to the gait pattern and weight-bearing. The CatWalk has also been used to assess gait changes and weight bearing in the chronic constriction model of neuropathic pain (Vrinten and Hamers, 2003) and, as mentioned above, in rats with arthritis of the knee, but pharmacological treatment was not investigated in these studies. An inflammatory pain model of intraplantar Freund's complete adjuvant also changed CatWalk parameters, but these changes were not reversed by morphine or ibuprofen (Marchand et al., 2007). Using the CatWalk and the carrageenan ankle joint monoarthritis model, this work introduces a new way to assess pharmacological treatment. Here, we have chosen representatives from two classes of analgesics to examine the utility of the model for pharmacological studies. Preliminary results were shown at the 11th World Congress on Pain (Ängeby-Möller et al., 2005).

Section snippets

Animals

This study was approved by the Stockholm Södra Animal Research Ethical Board. Forty-eight Sprague–Dawley male rats, weighing 190–235 g at the start of testing were used. The animals were housed 6 per cage on wood shavings, with free access to food and water and with a 12:12-h artificial light/dark cycle. When using models of monoarthritis, it is of great importance to consider the ethical aspects of inflicting pain in animals (Casey and Dubner, 1989). In the present study, the animals are not

Gait analysis of control animals

The regular pattern of walking rats is seen in Fig. 1A, which shows the prints of a representative animal crossing the walkway. There was no significant difference in print area between front and hind paws or between sides in saline-injected animals (n = 8; Fig. 2A). The weight load of the paws did not change during the test period (Fig. 2B). Stance duration was about 0.3–0.4 s and swing duration about 0.1–0.2 s. The fraction of the stance phase time relative to the total step duration was

Discussion

The major finding of the present study is that the rat monoarthritis model lends itself to rapid, detailed and objective quantification in terms of changes in gait and weight-bearing, using the CatWalk computer-assisted analysis system. The efficacy of morphine and rofecoxib demonstrates that the method is suitable for pharmacological studies. This conclusion is at variance with a previous study in which morphine and ibuprofen were ineffective in reversing behaviour induced by inflammation of

Acknowledgements

We thank Carina Stenfors for her feedback in the preparation of this manuscript, and Kerstin Nilsson for consultative help with statistical analysis.

CatWalk was developed by Dr. Hamers during his tenure as assistant professor at the Department of Pharmacology and Anatomy, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht. CatWalk is now commercialized by Noldus Information Technology B.V., Wageningen, The Netherlands. Both the Department of Pharmacology and Anatomy and

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Present address: Rehabilitation Hospital “De Hoogstraat”, Rudolf Magnus Institute of Neuroscience, Rembrantkade 10, 3583 TM Utrecht, The Netherlands.

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