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

Abstract

Background: Spontaneous (non-evoked) pain is a major clinical symptom of neuropathic syndromes, one that is understudied in basic pain research for practical reasons and because of a lack of consensus over precisely which behaviors reflect spontaneous pain in laboratory animals. It is commonly asserted that rodents experiencing pain in a hind limb exhibit hypolocomotion and decreased rearing, engage in both reflexive and organized limb directed behaviors, and avoid supporting their body weight on the affected side. Furthermore, it is assumed that the extent of these positive or negative behaviors can be used as a dependent measure of spontaneous chronic pain severity in such animals. In the present study, we tested these assumptions via blinded, systematic observation of digital video of mice with nerve injuries (chronic constriction or spared nerve injury), and automated assessment of locomotor behavior using photocell detection and dynamic weight bearing (i.e., gait) using the CatWalk system.

Results: We found no deficits in locomotor activity or rearing associated with neuropathic injury. The frequency of asymmetric (ipsilaterally directed) behaviors were too rare to be seriously considered as representing spontaneous pain, and in any case did not statistically exceed what was blindly observed on the contralateral hind paw and in control (sham operated and unoperated) mice. Changes in dynamic weight bearing, on the other hand, were robust and ipsilateral after spared nerve injury (but not chronic constriction injury). However, we observed timing, pharmacological, and genetic dissociation of mechanical allodynia and gait alterations.

Conclusions: We conclude that spontaneous neuropathic pain in mice cannot be assessed using any of these measures, and thus caution is warranted in making such assertions.

Figure 1

Ipsilateral but not contralateral mechanical allodynia in CD-1 mice given CCI (red). No significant changes from baseline von Frey thresholds were seen in either hind paw after sham surgery (blue) or simple repeated testing in unoperated mice (green). Symbols represent mean ± S.E.M. withdrawal threshold (average of two determinations per hind paw) from a nylon filament applying slowly increasing force (Ugo Basile Dynamic Plantar Aesthesiometer) to the plantar surface of the hind paws, measured before surgery (baseline; BL), and 1, 7, 14 and 28 days postoperatively. *p < 0.05 compared to baseline and contralateral side; ***p < 0.001 compared to baseline and contralateral side.

Figure 2

No evidence for reduced locomotor behavior in CCI mice. Symbols represent mean ± S.E.M. (A) walking (total beam breaks minus repetitive breaks of the same beam) and (B) rearing on the hind paws (total breaks of beams located ≈ 10 cm above the floor) in 60 min of mice given CCI surgeries (red), sham surgeries (blue), or unoperated (green), and tested 1, 7, 14 and 28 days post-operatively.

Figure 3

No evidence for asymmetrically directed behaviors in CCI mice. Symbols represent mean ± S.E.M. (A) directed grooming behavior (duration in s), (B) isolated licking behavior (duration in s), (C) hind paw lifting behavior (counts), (D) shaking/flinching behavior (counts), and (E) exaggerated turning behavior (counts) in 60 min of mice given CCI surgeries (red), sham surgeries (blue), or unoperated (green), and tested 1, 7, 14 and 28 days post-operatively. Closed symbols and solid lines (see key in bottom right) refer to the ipsilateral (Ipsi) side; open symbols and dashed lines refer to the contralateral (Contra) side. Definitions of all behaviors can be found in the main text; example video clips are available as Additional files 1, 2, 3, 4 and 5. Full videos are available upon request.

Figure 4

Dissociation of time course of mechanical allodynia and dynamic weight bearing (gait) changes following SNI injury. Data are combined from 22 inbred mouse strains (see text). Symbols represent mean ± S.E.M. 50% withdrawal threshold from von Frey filaments (g; in A), and mean ± S.E.M. CatWalk gait parameters including: (B) mean paw print intensity (arbitrary scale from 0-250), (C) stance phase duration (s), (D) paw print area (mm²), (E) paw placement ratio (ipsilateral: contralateral), and (F) regularity index (arbitrary scale from 0-100). For definitions see main text. In all graphs, data from the ipsilateral hindpaw is shown using closed symbols and solid lines; where relevant, data from the contralateral hindpaw is shown using open symbols and dashed lines. No significant changes in forepaw parameters were seen (data not shown). Note that mechanical allodynia peaks at post-operative day 4 and remains statistically unchanged thereafter, whereas CatWalk gait parameters display maximal changes at post operative day 1 and then partially recover. Individual significance levels are not shown for clarity; owing to the very large sample size (n = 122) all statements above are supported at p < 0.001 by repeated measures ANOVA.

Figure 5

Genetic dissociation between mechanical allodynia and dynamic weight bearing (gait) changes following SNI injury in 22 inbred mouse strains. Bars represent mean ± S.E.M. (A) percentage of maximal allodynia and (B) percentage of maximal weight bearing (mean paw print intensity; see Fig. 4B) change, in each strain separately, over 28 days post-operatively, calculated as areas over the time-effect curve using the trapezoidal rule. Robust strain differences (p < 0.001) were obtained for both measures; the individual pattern of strain responses is the subject of a manuscript in preparation. The non-significant correlation between these two measures is shown in graph C; symbols represent individual strains. Note that the trend towards a negative correlation would argue against genetic variability in allodynia and gait changes being produced by similar gene variants.

Figure 6

Pharmacological dissociation between mechanical allodynia and dynamic weight bearing (gait) changes following SNI injury in CD-1 mice. Symbols in A-C represent mean ± S.E.M. von Frey withdrawal thresholds (g; solid symbols) and CatWalk ipsilateral hind paw print intensity (0-250; open symbols) prior to SNI (baseline; BL) and on post-operative day 14, both prior to (Pre) and subsequent to (time post-injection in min is listed) administration of 10 mg/kg morphine (A), 75 mg/kg gabapentin (B), or topical EMLA (C). *p < 0.05 compared to same-test BL.

Figure 7

Frequency of proposed behaviors indicating chronic spontaneous pain in the neuropathic pain literature. Bars indicate total duration (s) of the behavior (filled) and total observation time (open). All studies used surgical nerve injuries, which are not noted except in studies featuring multiple surgeries. Note that the "scratching" behavior quantified by D'Almeida et al. [16], first described in Kupers et al. [18], is defined as "a rapid vibration of the hind paw," and is probably analogous to what is defined herein as shaking/flinching. Abbreviations: CCI, chronic constriction injury; CST, complete sciatic transection; PSNL, partial sciatic nerve ligation; PST, partial sciatic transection; SNL, spinal nerve ligation. References (from top; only first author noted): D'Almeida, 1999 [16], Paccola, 2008 [61], Guttierez, 2008 [62], Mao, 1993 [63], DeLeo, 1994 [64], Vos, 1994 [65], Deseure, 2004 [66], Kupers, 1992 [67], Kupers, 1998 [18], Kawasaki, 2008 [12], Jin, 2008 [68], Robinson, 2005 [69], Dowdall, 2005 [53], Shimoyama, 2002 [70], Sato, 2001 [71], Decosterd, 1998 [72], Kim, 1997 [52], and Choi, 1994 [73].