Animals
Six-week-old male Lewis rats (n = 38) were purchased from Janvier (Le Genest Saint Isle, France). The animals were kept under a 12 h-12 h light:dark cycle and allowed free access to food and water. The experimental procedures were approved by the local committee for ethics in animal experimentation (#0411, date 01/18/2011) of Université de Bourgogne (Dijon, France), and complied with the Animal Research Reporting In vivo Experiments (ARRIVE) guidelines. The same operator performed all steps of the experiments (license number 21CAE035).
Selection of animals
All the rats were first handled gently for a few days and familiarized with the treadmill apparatus (Bioseb, Vitrolles, France) in order to reduce stress due to novelty. Then, they were selected according to their ability to walk regularly on a horizontal treadmill with the speed of the treadmill belt fixed at 30 cm/s. Three 30-second running sessions were given twice a day for seven days. Mild intensity electric shocks to the feet were used as negative reinforcement to improve performance. Rats that failed to walk in a regular manner on the treadmill (contact of the forelimbs with the front wall of the treadmill, frequent immobility or galloping) at the end of the selection period were excluded from further experiments. Of the 38 Lewis rats enrolled in the study, 32 were able to walk in a regular manner on the treadmill. These rats were divided into AIA rats (n = 23) and control rats (n = 9).
Induction of arthritis
AIA was induced under volatile anesthesia (halothane) by a single intradermal injection at the base of the tail of 1 mg of heat-killed Mycobacterium butyricum (Difco, Detroit, MI, USA) suspended in 0.1 ml of Freund’s incomplete adjuvant (Difco). A group of non-arthritic age-matched rats received an equal volume of saline and were used as control rats. The incidence of arthritis as assessed from clinical scoring reached 91 % (21/23 immunized rats). Rats (n = 2) that did not develop arthritis were excluded from further analysis.
Assessment of clinical signs of inflammation
The rats were weighed and monitored for clinical signs of arthritis from immunization to week 27 (every two days from day 9 to 80 and every 15 days from day 80 to 195). The clinical scoring system (arthritis score) was employed as follows [17]: one finger scores 0 (no arthritis) or 0.1 (redness or swelling of one finger) and each big joint (ankle or wrist) scores 0 (no arthritis), 0.5 (mild but definite redness and swelling) or 1 (severe redness and intense swelling). The tarsus and ankle was considered the same joint. The arthritis score for a given limb ranged from 0 to 1.5 and the global arthritis score (four limbs) ranged from 0 to 6. The clinical scores were further divided into five grades: grade 0 (arthritis score = 0), grade 1 (arthritis score between 0.1 and 0.9), grade 2 (arthritis score between 1 and 1.9), grade 3 (arthritis score between 2 and 2.9), grade 4 (arthritis score between 3 and 3.9) and grade 5 (arthritis score more than 4). As the arthritis score only provides a subjective quantification of inflammation, it was coupled with the measurement of hind paw diameter using a digital caliper (Fischer Darex, France). The values were expressed as the mean of the two hind paw diameters. When indicated, values for individual hind paw diameters were presented.
Radiographic analysis of hind paws
Radiographs of hind paws were performed at the end of the experiment (week 27 after immunization) with a BMA High Resolution Digital X-rays machine (40 mV, 10 mA) - D3A Medical Systems (Orléans, France). A global score was determined for each hind paw using a modification of the grading scale described by Esser et al. [18]. This score evaluates both joint degradation, which was assessed from joint space narrowing and erosion, and new bone formation, which was assessed from periostitis and heterotopic bone. Joint degradation was scored at the tibiotalar, tarsal and subtalar joints as follows: normal aspect (0), mild degradation (1), moderate degradation (2) and severe degradation (3). New bone formation was scored at the calcaneum, tibia and tarsal bones as follows: normal aspect (0), mild hyperostosis (1), moderate hyperostosis (2) and severe hyperostosis (3). Radiographs were rated by two independent experienced observers. For each hind-paw, the maximal global radiographic score was 18 while the maximal joint degradation and the maximal new bone formation scores were both 9.
Locomotion recordings
Locomotion was recorded at week 25, 26 and 27 after immunization and at corresponding times in controls. Data were collected using the VICON MX-13 optical motion capture system (Vicon, Oxford, Great Britain), which consists of six high-speed digital infrared cameras as previously described in detail by our laboratory [19, 20]. Briefly, after anesthesia (intraperitoneal chloral hydrate, 400 mg/kg) the four limbs and the back were shaved and tattooed in order to locate the bony processes. Twenty-two reflective hemispherical markers (BTS Bioengineering, Cod FMK0005, Milano, Italy) with a diameter of 6 mm were placed over the following anatomical landmarks: the scapula, the upper (shoulder marker) and lower (elbow marker) humerus epiphysis, the metacarpophalangeal (MCP) joint, the iliac crest, the great trochanter, the knee, the external malleolus and the fifth metatarsophalangeal (MTP). Four markers (markers 1, 2, 3 and 4) were also placed on the back from the neck to the tail at regular distances. Three arthritic rats and one control rats died from anesthesia while being tattooed, thus, locomotion was recorded in eighteen AIA rats and eight control rats. Locomotion was recorded with the speed of the treadmill belt fixed at 30 cm/s, a speed that is within the range of the locomotion speed of rats over ground [21] and for a 1-minute session without delivering foot shocks. Soft tissue movement around the knee (skin slippage) is a recognized source of error when estimating joint kinematics of hind limbs in rats from markers placed on the surface of the body overlying joints [22]. To investigate this potential error, we measured the variation coefficient of the distance between the knee marker and the external malleolus marker at toe contact and at toe-off in both control and AIA rats.
Locomotion analysis
The gait cycle (defined as the time between two successive foot contacts of the same limb), was split into two parts, the stance and the swing phase. The stance phase was defined as the part of the cycle that begins when the foot strikes the treadmill belt and terminates when the foot starts its forward movement (i.e., when the vertical velocity of the MTP markers was higher than a threshold fixed at 5 % of its maximal velocity). The swing phase was considered to begin at the onset of forward movement and to end when the foot strikes the treadmill belt. Using a MATLAB custom-program (Math-Works, Natick, MA, USA), we measured the following locomotion-related parameters with a reference frame fixed to the hip marker:
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stance and swing phases duration, and gait cycle duration
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stride length, which was computed as the Euclidian distance (mm) of the more distal markers (MTP for the hind-limbs, MTC for the fore-limbs) throughout the swing phase
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maximal (Max) and minimal (Min) excursion of joint angles (degrees) during the stance and the swing phases
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paw location (mm) of the more distal marker of limbs (MTP or MCP) at toe-off in the frontal plane with respect to the axis passing through the hip for the hind limbs and the shoulder for the fore-limbs. A positive angular value indicates a paw placement further to the side
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maximal paw elevation during the cycle (mm).
These parameters were calculated for each hemibody in both control and AIA rats. For AIA rats, they were calculated from the more and less impaired hemibody (hemibody with the highest and lowest hind paw diameter just before locomotor recording, respectively).
We also calculated the following parameters:
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ROM of lateral roll of the body (degrees). The parameter was assessed from the measurement of the lateral tilt angle between the horizontal plane of the laboratory and the line passing through the two hip markers
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sagittal tilt of the body (degrees). The parameter was assessed from the measurement of the angle between the horizontal plane of the laboratory and the line passing through markers 1 and 4 of the back. A negative angular value indicated elevation of the hindquarter with respect to the head.
Even though all AIA rats were able to walk on the treadmill, periods with irregular locomotor cycles (walking on only three limbs, successive jumps and short periods of immobility followed by increased velocity of walking) were more frequent in AIA than in control rats. In certain AIA rats, the digits of the hind paws were often curled while walking with no contact of the calcaneum with the treadmill belt. From a careful visual inspection of walking AIA rats, we also detected hind-paw eversion. Unlike control rats, AIA rats often used their tail to walk. Finally, the observation of AIA rats in their housing cages revealed that these rats avoided standing on their feet. It is noteworthy that locomotion-related parameters were all calculated from at least four regular and consecutive step cycles during each trial in order to eliminate deviant curves [23].
Data and statistical analysis
Values are presented as the mean ± standard deviation except for the data on recurrence of inflammatory episodes, which were expressed by the median. Comparisons of locomotion-related parameters between the left hemibody of control rats and the two hemibodies of AIA rats were made using the Kruskall Wallis’ test followed by the Mann-Whitney t test and the Bonferroni correction. Differences between the more and less impaired hemibodies in AIA rats were assessed using Wilcoxon’s test for pairwise comparisons. The relationship between two variables was investigated using the Pearson’s correlation coefficient. P <0.05 was considered statistically significant.