Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice

To confirm the adequacy of our CCI surgeries, a separate cohort of mice (n = 8) were given surgeries and tested for mechanical allodynia (automated von Frey test) preoperatively, and at the same time points used for video analysis, 1, 7, 14 and 28 days post-surgery. Results, shown in Fig. 1, reveal significant and robust allodynia of the ipsilateral hindpaw (F _3,21 = 9.2, p < 0.001 by repeated measures ANOVA), reaching a maximum at days 7 and 14 and showing signs of amelioration by day 28. In CD-1 mice, CCI produces no contralateral allodynia (F _3,21 = 1.5, p = 0.25). Mice receiving sham surgeries and unoperated mice displayed no significant changes from baseline in either hind paw (all p's > 0.05). Highly similar data have been obtained in our laboratory previously using the same surgeon (J. S.A.) and strain, but with conventional (i.e., manual) von Frey fibers using the up down method (data not shown).

Walking and rearing behavior were assessed using the automated system. As can be seen in Fig. 2, no significant differences between CCI, sham and unoperated groups were detected overall, or on any post-operative day. For walking--defined as the total number of photocell beam breaks minus repetitive breaks of the same beam--repeated measures ANOVA revealed no main effect of group (F _2,15 = 1.4, p = 0.28), repeated measure (F _3,45 = 2.0, p = 0.14), nor group × repeated measure interaction (F _6,45 = 1.6, p = 0.16). For rearing--defined as total number of breaks of photocell beams located ≈ 10 cm above the floor--no main effect of group (F _2,23 = 1.1, p = 0.34), repeated measure (F _3,69 = 1.8, p = 0.16), nor group × repeated measure interaction (F _6,69 = 0.9, p = 0.50) was observed.

Analysis of 60-min videos made at each post-operative time point in all experimental groups is shown in Fig. 3. Each measure was analyzed via a two-way (surgical condition, side) repeated measures ANOVA. For directed grooming (Fig. 3A), a significant main effect of side was observed (F _1,46 = 5.8, p = 0.02), as was a significant effect of repeated measure (F _3,138 = 8.6, p < 0.001) and a significant repeated measure × side interaction (F _3,138 = 7.0, p < 0.001). This reflects the higher levels of grooming on post operative day 1 on the ipsilateral side in all groups, and can be interpreted simply as reflecting attention being paid to the shaved ipsilateral flank. The fact that unoperated mice groom as much as sham-operated mice suggests that grooming does not represent post-operative pain from the surgery. For isolated licking (Fig. 3B) and lifting (Fig. 3C), no main effects nor interactions were observed. For shaking/flinching (Fig. 3D), a significant main effect of side was observed (F _1,46 = 6.9, p < 0.05), reflecting slightly higher levels of this behavior in the ipsilateral hind paws in all surgical conditions. Finally, for exaggerated turning (Fig. 3E), a significant main effect of side (F_1,46 = 28.9, p < 0.001) and main effect of repeated measure (F _3,138 = 4.5, p < 0.005) were observed. This reflects the generally (but unevenly) higher turning to the ipsilateral side compared to the contralateral side in all conditions.

Importantly, in no case did we observe a significant condition × side interaction in the absence of a significant main effect of repeated measure, or a significant condition × side × repeated measure interaction (nor any trend towards either), as would be demanded by any useful dependent measure of neuropathic pain in this experiment. That is, in no case did we see evidence of these behaviors being more frequent in CCI mice. Also, all behaviors were in fact extraordinarily rare. Grooming and licking behavior were typically observed for a total of 50-100 seconds, representing <3.0% of the total scoring time. Hindpaw lifting and shaking/flinching were observed less than 0.5 times per hour on average, and exaggerated (ipsilateral) turns only 2-4 times per hour.

Mechanical allodynia as measured by conventional von Frey testing was observed in each of the 22 mouse strains. In addition, robust changes in all CatWalk parameters were seen in each strain. However, these changes could be dissociated both in terms of their time courses and their strain-dependence. Fig. 4 shows the time course of von Frey withdrawal thresholds and the five CatWalk parameters in the complete data set, irrespective of strain (n = 123-127/measure). As can be seen, von Frey allodynia was present on post operative day 1, peaked on day 4, and remained statistically equivalent thereafter all the way to day 28 (Fig. 4A; all p's < 0.001). Sham-operated mice displayed no significant changes in either von Frey withdrawal thresholds or CatWalk parameters, even on post-operative day 1 (data not shown). For every CatWalk parameter maximum changes were seen at day 1 (in contrast with the day 4 peak for allodynia), and in every case statistically significant improvement was seen after that point, reaching a plateau at approximately day 21 (Fig. 4B-F; all p's < 0.001). Improvement in all parameters except print area represented approximately half of the initial change on post operative day 1.

There were significant main effects of strain in overall von Frey and CatWalk changes over the 28-day time course, calculated as areas over the time effect curve using the trapezoidal rule (p's < 0.001). Strain means of all CatWalk parameters were highly inter-correlated (r = 0.67-0.97, all p's < 0.01). Correlations of all other CatWalk parameters with mean intensity were all r ≥ 0.76 (p < 0.001), and so we used this parameter--featuring a completely stable baseline and no potentially confounding post surgical alterations whatsoever in any other paw--to represent CatWalk changes by strain. Fig. 5 shows strain-dependent total von Frey allodynia of the ipsilateral hind paw (Fig. 5A), CatWalk total mean intensity decreases in the ipsilateral hind paw (Fig. 5B), and their correlation (Fig. 5C). As can be seen, the correlation between von Frey and CatWalk changes was a non-significant r = -0.28 (p = 0.20), and it should be noted that the trend is in the opposite direction to that which would indicate they measure the same underlying phenomenon. This conclusion is not confounded by body weight or willingness to cross the runway (and thus average walking speed), since although these parameters were highly strain dependent both at baseline and in their changes after neuropathic injury (data not shown), no correlations of these parameters with any CatWalk measures even approached significance. It should be noted that Clarke and Still [23], in their seminal analysis of gait in the mouse, demonstrated that all major gait variables were consistent over a wide range of velocities.

No CatWalk measures were altered significantly from baseline at any post-surgery time point in CD-1 mice (n = 11) given CCI surgeries (data not shown). This is not due to genotype differences, since CD 1 mice displayed highly significant changes in gait as measured by CatWalk after SNI (see below). Why SNI would produce guarding and CCI would not is not immediately obvious, but it should be noted that Piesla and colleagues [24] demonstrated differences in the gait changes produced by various nerve injuries (and inflammatory compounds) in the rat. In their hands, however, CCI in rats clearly produces changes in gait, suggestive of a rather striking species difference.

On day 14 post-surgery, robust ipsilateral mechanical allodynia on the von Frey test and significant CatWalk hind paw print intensity decreases were observed in each drug group (Fig. 6A-C; all p's < 0.005), replicating in CD-1 mice what was previously observed in the 22 inbred strains. Repeated-measures ANOVAs were performed separately on von Frey and CatWalk data for each drug. Morphine, gabapentin and EMLA all produced efficacious anti allodynia, restoring von Frey thresholds to baseline (pre operative) levels, and in the case of morphine, beyond (all p's < 0.05 compared to day 14 pre-injection baseline by repeated measures ANOVA). However, at the same doses and time points, none of the three drugs significantly altered CatWalk mean intensity values (p's = 0.60, 0.10, and 0.10, respectively, compared to day 14 pre injection baseline, with trends towards decreased weight bearing on the ipsilateral hindpaw) or any other CatWalk parameter (data not shown). That is, morphine, gabapentin and EMLA reversed mechanical allodynia produced by the neuropathic injury, but did not affect the gait abnormalities also associated with that injury. Pilot studies (data not shown) confirmed that morphine, gabapentin and EMLA produced no changes in any CatWalk parameters by themselves (i.e., in the absence of injury). Non significant trends towards increases in von Frey withdrawal thresholds of unoperated mice were produced by morphine (baseline: 0.50 ± 0.08 g;

30 min post injection: 0.64 ± 0.21 g) and gabapentin (baseline: 0.43 ± 0.30 g; 120 min post-injection: 1.10 ± 0.27 g), but not EMLA (baseline: 0.70 ± 0.28 g; 20 min post injection: 0.65 ± 0.28 g).

We find no statistical evidence of any hypolocomotion or spontaneously emitted behaviors directed at the presumed site of pain (i.e., the ipsilateral hind paw) in nerve injured mice that exceed levels observed in sham-operated or unoperated mice. Because of the possibility of mirror pain [25], comparison to sham-operated animals may be more appropriate than comparison to the contralateral hindpaw. To remain consistent with standard procedures in the field, we tested all mice in their light cycle. However, a pilot experiment in which neuropathic mice were tested for 6 hours overnight (i.e., during their active phase) under red illumination only also revealed no trend whatsoever towards ipsilateral predominance of spontaneously emitted behaviors (data not shown). We note that although sample sizes in these experiments were not exceptionally large (n = 8-12/measure), they equal or exceed the median sample size of n = 8 used in modern pain research [27]. We note that it remains possible that small subsets of mice might display such behaviors, just as only a minority of humans receiving nerve damage go on to develop chronic pain syndromes (see [28]), but even if this is true as a practical matter these dependent measures would not be valuable in pain research experiments featuring typical sample sizes.

In an attempt to compare the current data to the wider literature, a literature search was performed (PubMed search terms "spontaneous pain" AND [mouse OR rat] AND [chronic OR neuropathic]", plus papers already known to the last author), revealing approximately 50 relevant papers that were studied in further detail. What is striking from these studies is the brevity of the observation period (only two studies exceeding 15 min), the multiple instances where behaviors were asserted but not quantified, the comparisons to baselines, contralateral hind paws, or unoperated controls instead of sham operated animals, and the great variability encountered in the frequency/duration of the behaviors themselves. In an attempt to compare apples-to-apples, Fig. 7 displays data from 17 studies using neuropathic pain models that explicitly quantified the total duration of the behavior. As can be seen, these behaviors are rare, comprising a median percentage of 10% of total observation time. It is possible that the even less-frequent observation of these behaviors in the present study is due to the more conservative definitions that we used. We note that the frequency of some of these same asymmetrically directed behaviors in models of inflammatory and especially cancer pain are sometimes reported to be much higher (e.g., [29‐32]), raising the possibility that neuropathic pain models are fundamentally different in this respect.

In marked contrast to our findings with respect to asymmetric behaviors, we observed robust alterations in gait (i.e., weight bearing during locomotion) produced by SNI (but not CCI). Such changes have been interpreted either as evidence of spontaneous pain and/or guarding; that is, attempts by the animal to avoid the mechanical allodynia that would result from hind paw to-floor contact [33]. In fact, weight bearing changes have been proposed as a confound of mechanical allodynia measurement via von Frey fibres [34]. Direct evidence that gait changes represent pain or guarding (to avoid pain) is somewhat sparse. Using incapacitance testing to measure static (i.e., standing) weight bearing in inflammatory models, a variety of analgesic manipulations appear efficacious [35]. With respect to dynamic weight bearing measured by the CatWalk and its predecessors, effects on arthritic changes have been shown with buprenorphine [36], muscimol [37], morphine [38, 39] and cyclooxygenase inhibitors [38, 39]. However, we are unaware of any prior evaluation of the effects of known analgesics on dynamic weight bearing changes caused by nerve injury. In fact, to our knowledge only one study has directly tested CatWalk changes in a common animal model of neuropathic pain. In CCI rats, Vrinten and Hamers [22] observed that the time course of CatWalk changes paralleled that of von Frey measured mechanical allodynia, and that correlations (in individual rats) between von Frey and various CatWalk measures ranged from r = 0.61 to 0.67. The contrast with present data might be explained by species or pain model differences, but it should also be noted that their sample size was n = 12 whereas ours is n = 122.

In the present strain survey experiment, we demonstrated that there is no genetic correlation whatsoever between von Frey allodynia and any CatWalk measure, suggesting that these are independent phenomena [40]. The independence of allodynia and gait is reinforced by the time course of changes, which although similar in general terms are actually quite different on closer inspection, with von Frey allodynia still clearly developing when gait alterations are at their maximum, and von Frey allodynia remaining constant over the 28 day testing period while gait recovers considerably over the same time span. Finally, the existence of allodynia but not concomitant gait alterations after CCI in the mouse argues for the independence of these phenomena.

It is entirely possible that all these data simply point to the fact that spontaneous and evoked pain are mechanistically distinct (see ref. [50]). However, the fact that three separate analgesic manipulations at doses that were entirely successful at reversing allodynia were wholly unable to affect gait strongly suggests that gait changes may not be related to pain at all. Of interest is a very recent study in the rat by Piesla and colleagues demonstrating that gabapentin and duloxetine reverse mechanical hyperalgesia after nerve injuries but have no effect on gait parameters measured using the DigiGait™system [24]. In their hands, some gait abnormalities produced by carrageenan were normalized by morphine and indomethacin. With respect to knee joint antigen-induced arthritis in rats, one recent study concludes that some gait parameters represent pain but others are indices of mechanical joint deformation [41], and another observed only transient gait changes [42].

Weight bearing and gait may be altered independently of whether the animal is experiencing spontaneous pain after nerve injury. In the SNI and other surgical procedures motor axons that innervate the muscles of the paw and lower leg are damaged [43]. Na and colleagues [44] concluded that the postural abnormalities seen in the SNI model were partially due to this motor deficit. The ventroflexion of the toes, loss of toe spreading, and eversion of the hind paw seen after CCI in rats (but not in mice; unpublished observations) are likewise due to motor axon damage. Other possibilities include tethering of the nerve to the adjacent muscle due to scar tissue formation [45]. Damaged axons are mechanosensitive [46] and it is possible that the animal is trying to avoid the sensations that are produced by stretching the nerve.

Subjects in all experiments but one were naïve, adult (7-12 week old) outbred CD 1^® (ICR:Crl) mice of both sexes [47], bred in our laboratory from mice obtained from Charles River (Boucherville, QC). Mice were weaned at 18-21 days and housed with their same sex littermates. In one experiment, adult mice of both sexes from 22 inbred mouse strains (129P3, A, A/He, AKR, B10.D2-H2/oSn, BALB/c, BALB/cBy, BUB/Bn, C3H/He, C57BL/6, C57BL/10, C58, CBA, DBA/2, FVB/N, LG, LP, MRL/Mp, NZB/BIn, NZW/LaC, RIIIS, and SM; all "J" substrains), were obtained from The Jackson Laboratory (Bar Harbor, ME), and habituated to the vivarium for at least one week before testing. Mice were housed in standard shoebox cages, 2-4 per cage, maintained in a temperature-controlled (20 ± 1°C) environment (14:10 h light cycle; with lights on at 07:00 h), and fed (Harlan Teklad 8604) and watered ad libitum. All procedures were approved by a McGill University animal care and use committee and were consistent with Canadian Council on Animal Care guidelines.

After baseline testing for mechanical sensitivity (in some experiments; see below), all mice received unilateral surgical nerve injuries under isoflurane/oxygen anesthesia using either the chronic constriction injury (CCI) model [48] or the spared nerve injury (SNI) model [49], as adapted for the laboratory mouse [50, 51]. Sham CCI surgeries were conducted identically except that no ligations were placed around the sciatic nerve. Sham SNI surgeries were conducted identically except that no nerves were cut. Unoperated mice were housed, handled and tested identically to other groups (including anesthesia and shaving the flanks), but received no surgeries. Because both Kim et al. [52] and Dowdell et al. [53] suggested that CCI in the rat produces the strongest evidence of spontaneous pain (defined as hind paw lifting) of multiple neuropathic injury models studied [also see [54]], most of the current experiments were conducted using the CCI model. The gait analysis experiment was performed using the SNI model, since data from this experiment were being collected originally for other purposes, and since no evidence of gait changes were observed in mice after CCI surgeries (data not shown).

Mice were placed individually in transparent Plexiglas cubicles (5 cm wide × 8.5 cm long × 6 cm high; each cubicle was separated from the other by an opaque divider to abolish "neighbour effects" [55]) placed upon a perforated metal floor (with 5 mm diameter holes placed 7 mm apart), and habituated for 2 h before behavioral testing began. In one study, nylon monofilaments (Stoelting Touch Test Sensory Evaluator Kit #2 to #9; calibrated weekly using a microbalance; ≈ 0.015, 0.04, 0.07, 0.15, 0.44, 0.55, 1.0 and 1.3 g) were firmly applied to the plantar surface of the hindpaw (alternating the side of the body being tested) until they bowed for 5 s. Only withdrawal responses performed obviously in response to the applied stimulus were scored. The up-down method of Dixon [56] was used to estimate 50% withdrawal thresholds. In other studies, an automated von Frey test (Ugo Basile Dynamic Plantar Aesthesiometer) was employed. In the CCI model, von Frey fibers were applied to the mid-plantar hind paw. In the SNI model, we spared the sural nerve and thus applied von Frey fibers to the lateral aspect of the plantar hind paw to measure mechanical allodynia. Mice were only tested when alert or resting [57]. At each time point, two separate threshold determinations were made on each hind paw, and then averaged.

Mice were singly housed within standard shoebox cages with an aerated Plexiglas cover (to reduce hanging behavior, which confuses the software), habituated for 30 min, and then monitored for 60 min via a fully automated, photocell-based system (Opto Varimex Micro Animal Activity System™; Columbus Instruments; Columbus, OH). Sessions occurred at 1, 7, 14 and 28 days post-surgery, all at 19:30 h (± 1 h). The decision to test animals near the beginning of their dark (active) phase was made to decrease the probability of sleeping, but mice were tested with normal room lighting (30 lux) to better approximate current procedures in the field.

So as not to bias the observation, an undergraduate researcher (A.G.) not familiar with the neuropathic pain literature was instructed simply to observe, unblinded, multiple videos of neuropathic mice (CCI model; n = 8), and identify behaviors seeming potentially significant to her (data not shown). She identified: hindpaw licking (both that occurring as part of the normal, stereotyped grooming sequence [58], and also that isolated from it), hindpaw lifting, hindpaw shaking/flinching, and what she termed "exaggerated turning" to the side of the injury. Definitions are as follows, and video clips illustrating these behaviors are available to interested researchers:

Measured as number of discrete events.

The CatWalk^® system (Noldus Inc.) of automated gait analysis has been described in detail previously [59]. Subjects traverse a walkway (50.5 cm long; 3.3 cm wide) atop a glass floor in a darkened room. Light enters the distal long edge of the glass floor from a fluorescent bulb located at the side, and is internally reflected, scattering only at points where a paw touches the glass, producing bright illumination of the contact area. Internal reflection is incomplete, allowing a faint superimposed image of the animal to be seen as well. A video camera monitors the corridor, and the digitized signal is stored for later analysis of animals crossing the walkway.

A number of "single-paw" and interlimb coordination parameters can be obtained; we found the following to be the most reliable in the mouse:

These parameters were calculated for each paw in each mouse on each testing day. Mice were allowed (and, if necessary, encouraged by prodding) to cross the CatWalk three times on each testing day, with results averaged. Possible confounds of the CatWalk are body weight and motivation to cross the walkway (leading to changes in velocity). Thus, on each testing day, body weight was measured, and the experimenter assigned a score to each mouse indicating its "willingness" to cross (3: mouse moves down runway smoothly with no need of prodding; 2: mouse needs slight prodding but then moves down runway smoothly; 1: mouse needs constant prodding to get it to move; 0: mouse freezes, climbs up side of runway wall, or attempts to turn around or back up).

Mechanical allodynia or CatWalk guarding behavior (measured by mean intensity) over the entire 28-day testing period was calculated for each mouse as the area-over-the curve using the trapezoidal rule. Percentages of maximum possible allodynia and guarding were calculated by comparing observed areas-over-the-curve to the maximum possible for each individual mouse considering its own baseline.

Separate groups of unoperated mice (n = 8/drug) were simply administered the three analgesics and tested for von Frey withdrawal thresholds and on the CatWalk to see if they produced any effects on mechanical sensitivity or dynamic weight bearing per se.

Data were analyzed by ANOVA or Student's t-test, as appropriate. A criterion significance level of α = 0.05 was adopted in all cases. A small number (n = 6) of statistical outliers (>3 S.D.) not developing mechanical allodynia were removed from the analysis in the SNI strain survey data set. In four cases, locomotor activity data was not collected by the software on one testing day.