During the study, care and use of animals were subject to and approved by the Comité d’Éthique de l’Utilisation des Animaux of Université de Montréal (#Rech-1495) and conducted in accordance with principles outlined in the current Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
The present study was conducted on female (n = 63; excluding spares) Sprague-Dawley rats (Charles River Laboratories, Saint-Constant, QC, Canada) ranging from 225–300 g in weight at the beginning of experiment. The animals were housed under regular laboratory conditions and maintained under a light-dark cycle with food and water provided ad libitum. Body weight was obtained weekly. At the end of each experiment, the animals were returned to their housing colony.
Phase 1: reliability of pain assessment methods in normal rats
Phase 1 included a total of 39 normal rats distributed into 8 groups. First, the repeatability of measurements was tested for the influence of environment, including observer, inverted circadian cycle (activity during the day), and exercise (two groups of five animals in crossover). Additionally, repeatability over an extended period was tested again for static weight-bearing (SWB; one group of four animals also tested for exercise effect), and for tactile sensitivity and place escape/avoidance paradigm (PEAP) operant test (one group of five animals). Second, using the most reliable methods only, the influence of four acclimatization protocols (four groups of five animals) was tested to determine the most effective approach to obtain predictable data with low variability. Different pain assessment methods were selected to include reflexive, spontaneous behavior and operant measures.
Influence of environment
First, rats (n = 10) were randomly distributed into two groups of five. Animals were acclimated to the test apparatus on two occasions at day -3 and -1 before starting the experimentation. In a crossover design, the animals were subsequently assessed for three repeated days during light (1000–1400 h) and dark (2000–2400 h) cycles to test the influence of inverted circadian cycles. Both cycles were separated by a 3-day washout period without assessment. Dark cycle evaluations were performed under low-intensity red light. Animals were tested on each of six assessment days by two observers, with the following methods in this order of evaluation: mechanical and tactile sensitivity, SWB, treadmill exercise, mechanical and tactile sensitivity, SWB, PEAP operant test (without nociceptive stimulation) and rotarod acceptance. The mechanical and tactile nociceptive thresholds and SWB evaluation were performed before and after the treadmill exercise to verify the influence of exercise on these three pain assessment methods. Finally, PEAP and rotarod were performed at the end of the evaluation schedule to ensure respectively, that the length of the test, and possible falling from the test device would not impair the other outcomes. To test inter-rater reliability, two female observers were selected for their different levels of experience in laboratory animals (one intermediate, one with advanced expertise).
Second, in complementary studies about test repeatability (after acclimatization on two occasions, at day -3 and -1), SWB was specifically retested over 15 days with two SWB assessments separated by a treadmill session (then testing again the potential effect of exercise on SWB) in a group of four rats. This evaluation was done daily, from days 1 to 5, then on days 8 and 15.
Finally, the repeatability of measurements of tactile sensitivity and PEAP (with nociceptive stimulation) was tested in a group of five rats over 25 days. These evaluations were done daily, from days 1 to 15, and repeated on days 18 and 25.
Influence of acclimatization protocols
In order to determine the most efficient acclimatization protocol associated with the most repeatable data (previously obtained), the next experiment was conducted in a total of 20 animals (four groups of 5 animals). Briefly, over 2 weeks, different acclimatization protocols were tested and included 8 (days -14, -13, -12, -11, -10, -8, -6, and -1), 6 (days -14, -8, -7, -6, -5, and -1), 5 (days -14, -7, -5, -3, and -1) or 4 (days -14, -8, -6, and -1) days of evaluation. The order of assessment was SWB, tactile and mechanical sensitivity, PEAP with nociceptive stimulation, and treadmill. The schedule of pain evaluation methods was determined to obtain nociceptive threshold values before placing the animal on the operant testing device where many paw stimulations were elicited (see subsequent description).
Pain assessment methods
Mechanical sensitivity was assessed by measuring the paw withdrawal threshold (PWT) to an increasing pressure stimulus placed on the dorsal surface of the hind paw using an algorimeter (Randall Selitto test Paw Pressure Meter®, IITC Life Science Inc., Woodland Hills, CA, USA), employing a wedge-shaped probe (1.75 mm2 of surface) and a cutoff value set at 250 g. The animals were placed in a sling apparatus (Lomir Biomedical Inc., Notre-Dame-de-l’Île-Perrot, QC, Canada). The probe was applied once on the dorsal surface at a steadily increasing pressure. The PWT was determined when the animal removed the paw from the apparatus, and the required pressure was recorded. Withdrawal thresholds were measured on the right and left hind paws. The data were expressed as PWT in grams.
First, the animal was placed inside an elevated metal grid cage to allow just enough space for the rat to move while being restricted. After the rat exploration session during the first 2 minutes, tactile sensitivity was assessed using an Electronic von Frey Anesthesiometer® (IITC Life Science Inc., Woodland Hills, CA, USA) applied to the plantar surface of the hind paws and by measuring the PWT to von Frey ascending mechanical stimuli. Gradually increasing pressure was applied with a mechanical von Frey polypropylene probe (0.7 mm2, Rigid Tip®, IITC Life Science Inc., Woodland Hills, CA, USA) fitted to a handheld force transducer. The rigid tip was placed perpendicularly into the mid-plantar surface of the paw. The stimulus was continued until the hind paw was withdrawn or elevated such that the force leveled off. Actions such as vocalization, agitation, jumping, and avoidance were considered indicative of the PWT. Voluntary movements associated with locomotion were not considered to be a withdrawal response. The peak of force in grams was recorded with a cutoff value at 100 g. For each animal, triplicates of each hind paw were taken with a 60-s interval between each stimulus.
Static weight bearing
The weight distribution through the right and left knee was assessed using an Incapacitance Meter® (IITC Life Science Inc., Woodland Hills, CA, USA) to measure SWB distribution in the two hind limbs. The force exerted by each hind limb was measured and analyzed in grams, but reported in percentage of total body weight (%BW) to normalize the data. Rats were allowed to acclimate to the testing apparatus and when stationary, readings were taken over a 3-s period. Triplicates were taken simultaneously for each limb at each time point.
All rats underwent forced training over a 20-minute period at constant treadmill speed (11 m/minute) (IITC Life Science Inc., Woodland Hills, CA, USA). To force the animal to exercise on the treadmill belt, each lane was equipped with an independent shocker grid. The intensity of the shocker grid was kept at the minimum required to keep the animal on the exercise belt. The treadmill number of total crossings (TNTC) was recorded over the whole period, but also reported in blocks of 5 minutes, to potentially detect a within-time change in activity. A total crossing was considered completed when the animal crossed the entire length of the lane. The TNTC was used as an indicator of exercise and/or performance. When the rats were running continuously on the belt, they were exposed to maximal intensity exercise, as they were not pausing, causing them to cross the entire length of the motorized lane.
The PEAP was used as operant testing apparatus [33, 40, 49]. Rats were placed into test cage apparatus that was painted half white and half black. Neither side was illuminated with additional light. With the cage on an elevated metal grid, the observer, located below, determined the preferential location of the rat. The 20-minute observation period began after 2 minutes of acclimatization/exploration to the test environment on each occasion.
Operant testing without nociceptive stimulation
The percentage of time spent on the black or white side of the test apparatus was calculated from observation of the preferential location every 15 s.
Operant testing with nociceptive stimulation
If the rat was on the black side of the test apparatus, the plantar surface of the right (ipsilateral to possible MIA intra-articular injection) hind paw (RHP) was stimulated with a thin wire (60 g) every 15 s, to prompt withdrawal of the limb. When the rat was on the white side of the cage, a similar mechanical stimulation was applied, but to the plantar surface of the left (contralateral) hind paw (LHP). The percentage of time in the black and white side of the test apparatus was calculated from observation of the preferential location every 15 s. The calculations were sequenced by successive blocks (n = 4) of 5-minute periods. Moreover, the total number of crossings from the white to the dark side was noted to detect any decrease in activity. If a rat remained in the crossing tunnel, it would be stimulated to advance and complete its crossing.
Using a Rotamex 4/8® (Columbus Instruments Inc., Columbus, OH, USA) with a previously published protocol , the rats were exposed to an acceleration speed of 5 to 16 rpm, over 60 s, before being maintained at this speed, while the time before falling was monitored with a cutoff time of 3 minutes.
Phase 2: concurrent validity with the MIA model
In the second phase, a pilot study (n = 24 rats) was conducted to test the concurrent validity of different functional and neuropeptide pain assessment methods in the MIA rat OA model. A single intra-articular injection of MIA was performed in the right knee of 16 animals distributed among two groups (n = 8 each). An additional sham group (n = 8) received a single intra-articular injection (50 μL) of 0.9 % NaCl. For the purpose of the study, one of the two MIA groups also received a punctual lidocaine (L) injection (MIA-L group) in the right knee on days 7, 14 and 21. At the end of the 21 days of the experimentation, all animals were euthanized with an overdose of isoflurane and a sacrifice by transection of the cervical spine before spinal cord collection.
Acclimatization period and baseline assessment
The study began with an acclimatization period for the selected optimal outcomes (SWB, tactile sensitivity, PEAP, rotarod, and treadmill), according to the optimal acclimatization protocol of five occurrences (days -14, -7, -5, -3, and -1) obtained in phase 1. Because of pain induction in phase 2, tactile sensitivity could be considered as punctate tactile allodynia evaluation (PTAE). Baseline values were acquired at day -1 in this order of evaluation, following the above-described testing procedures: SWB, PTAE, and PEAP with nociceptive stimulation, rotarod and treadmill, with intra-articular injection of MIA at day 0 in the right knee.
On day 0, fasted (3–6 h) rats from all groups were premedicated with buprenorphine hydrochloride (0.02 mg/kg IM; Buprenex® injectable, Reckitt Benckiser Inc., Mississauga, ON, Canada) and mask-anesthetized with a 2 % isoflurane–O2 mixture. After surgical preparation, a single intra-articular injection of 2 mg MIA (monosodium iodoacetate, BioUltra®, ≥98 %, Sigma-Aldrich Canada Co., no. I9148-5G, Oakville, ON, Canada) dissolved in isotonic saline, or saline 0.9 % (both 50 μL volume) was administered through the infrapatellar ligament of the right knee, using a 26-gauge, 0.5-inch needle mounted on a 0.5-mL syringe. On days 7, 14, and 21 post-MIA injection, 25 minutes before functional assessment, rats from the MIA-L group were again similarly anesthetized with a single intra-articular injection of lidocaine through the infrapatellar ligament of the right knee. Lidocaine Neat® (2 %, Zoetis Canada, Kirkland, QC, Canada) was injected at a volume of 50 μL using a 26-gauge, 0.5-inch needle mounted on a 0.5-mL syringe.
The assessments were performed according to the specific schedules of the different groups on days 3, 7, 14, and 21 post injection, and conducted as described for phase 1. For the MIA-L group on days 7, 14, and 21, the evaluation started 25 minutes after the animals recovered from anesthesia. The evaluation sequence was as follows: SWB (%BW), PTAE (grams), PEAP (percentage of time spent on the dark side), rotarod (seconds) and treadmill (TNTC). The schedule of evaluation was designed to obtain the SWB at rest and the PTAE data before the operant testing evaluation, as this test elicits many PWT stimulations.
Reagents and solutions
Acetic anhydride 99.5 % (Ac2O) and ammonium bicarbonate (NH4HCO3) were obtained from Sigma-Aldrich Inc. (St Louis, MO, USA). SP and CGRP were purchased from Phoenix Pharmaceuticals Inc. (Belmont, CA, USA). Acetonitrile was purchased from Thermo Fisher Scientific Inc. (NJ, USA), and trifluoroacetic acid, formic acid and ammonium hydroxide 28.0–30.0 % (NH4OH) were purchased from J.T. Baker® (Phillipsburg, NJ, USA). Standard solutions were prepared as previously performed .
The tandem mass spectrometry coupled to high-performance liquid chromatography (HPLC-MS/MS) system comprises a Thermo Surveyor autosampler, a Thermo Surveyor MS pump and a Thermo LCQ Advantage Ion Trap Mass Spectrometer (Thermo Fisher Scientific Inc., San Jose, CA, USA). Data were acquired and analyzed with XcaliburTM 1.4 (Thermo Fisher Scientific Inc., San Jose, CA, USA), and regression analysis were performed with PRISM® (version 5.0d) (GraphPad software Inc., La Jolla, CA, USA) using the nonlinear curve fitting module with an estimation of the goodness of fit. The calibration lines were constructed from the peak-area ratios of targeted neuropeptides (SP or CGRP) and the acetylated SP analog internal standard.
Acetylated SP was used as the internal standard. The reaction was performed as previously described  and the analytical method used was also based on a previously published method . The internal standard solution was tested by HPLC-MS/MS in multiple reactions monitoring (MRM) mode and no residual SP were detected.
Spinal cord sample preparation
At the end of the 21 days of experimentation, the entire spinal cord tissue of rats (n = 24) was rapidly collected by a flush of saline within the lumbar spinal canal following deep anesthesia with isoflurane and sacrifice by transection of the cervical spine. Samples were snap-frozen in liquid nitrogen and stored at –80 °C pending analysis. Each spinal cord was weighed accurately and homogenized using a tissue tear or following the addition of phosphate-buffered saline solution (PBS) 0.01 M at a ratio of 1:5 (v/v) and protease inhibitor cocktail (Sigma-Aldrich Inc., Oakville, ON, Canada, number PP8340) at the same ratio. The samples were sonicated and the homogenate was mixed with acetonitrile at a ratio of 1:1 (v/v) to remove larger proteins. The samples were vortexed and centrifuged for 10 minutes (×12,000 g) and the supernatant was transferred into an injection vial then spiked with the internal standard solution at a ratio of 1:1 (v/v). The spinal cords from a naive group (n = 5) in phase 1 were also collected to obtain a baseline value from normal rats to normalize values obtained from the MIA, MIA-L, and sham groups.
All statistical analyses were performed two-sided with an alpha value set at 0.05 (phase 1) or 0.10 (phase 2) using a statistical software program (SAS system for Windows, version 9.2, Cary, NC, USA). The alpha value for phase 2 was set at 0.1 because this phase was an exploratory study. In a pilot study, it is acceptable to set a higher alpha value when the study has the hopes of finding an effect that could lead to a promising scientific discovery  in order to increase the power (consequently decreasing the risk of type II error), but increasing the chances of type I error (i.e., saying there is a difference when there is not). To be consistent with the statistical rules of correction for multiple comparisons, phase 2 results were presented as adjusted p values (adj-P) because the values obtained in the statistical report need to be multiplied by the total number of comparisons. The normality of the outcomes was verified using the Shapiro-Wilk test and the homogeneity of variance was assessed using the absolute values of the residuals of the mixed model, when appropriate.
Phase 1: reliability of pain assessment methods in normal rats
For mechanical nociceptive thresholds and SWB, the effect of the circadian cycle was assessed using the paired t test adapted for a crossover design. Moreover, the effect of covariates of interest, namely observer, exercise, limb (when both left and right limbs were tested), or trials (when replicates were conducted), was assessed using a general linear model. Generalized linear mixed model analyses for repeated measures were conducted to test the effect of groups on TNTC and rotarod (lognormal distribution), and PEAP (Poisson distribution). Models accounted for baseline measurements using the baseline as covariates. This enabled assessment of the effect of the procedure over time using each subject as its own control. For each model, the best structure of the covariance model was assessed using information criteria that measure the relative fit of competing covariance models. When comparing the 5-minute periods, the Bonferroni adjustment was applied (initial alpha value divided by 4).
Outcome repeatability (test-retest reliability) was assessed by computing the intraclass correlation coefficient (ICC). The ICC is a measure of the proportion of variance that is attributable to objects of measurement. Quantifying the test-retest reliability, the closer the ICC is to 1.0, the higher the reliability and the lower the error variance . A ratio of 0.3–0.4 indicates fair agreement, 0.5–0.6 moderate agreement, 0.7–0.8 strong agreement, and >0.8 almost perfect agreement. Moreover, the coefficient of variation (CV), as a normalized measure of dispersion of the distribution, was used to test the effect of the proposed acclimatization protocols. The CV for each variable was calculated at day -14 (initial assessment), and the variation in CV was assessed at the end of each acclimatization protocol as the CV ratio of day -1 (final assessment) to day -14. At the initial assessment (day -14), the CV interpretation was as follows: <10 % indicated almost perfect dispersion, 11–25 % light dispersion, and 26–40 % fair dispersion. The day -1/day -14 CV ratio indicated improvement (decrease in variability) related to the acclimatization protocol if it was <1, and deterioration (increase in variability) if >1.
Phase 2: concurrent validity with the MIA model
The SWB and PTAE data were expressed as the average obtained from the three trials on the RHP. Data were then analyzed using linear mixed models (SWB and PTAE) or generalized linear mixed models for repeated measures. Treatment groups and day were considered as fixed effects and animals in groups as random effects. Models accounted for baseline measurement using the baseline as a covariate. For each model, the best structure of the covariance model was assessed using a graphical method (plots of covariance versus lag in time between pairs of observations compared to different covariance models), and using information criteria that measure the relative fit of competing covariance models. When multiple comparisons were carried out, the Tukey-Kramer adjustment was used to obtain adj-P values. Neuropeptide data were analyzed using the unpaired exact Wilcoxon test with an alpha value set at 0.10 following non-parametric Kruskal-Wallis one-way analysis of variance.