A simplified up-down method (SUDO) for measuring mechanical nociception in rodents using von Frey filaments

Abstract

Background: The measurement of mechanosensitivity is a key method for the study of pain in animal models. This is often accomplished with the use of von Frey filaments in an up-down testing paradigm. The up-down method described by Chaplan et al. (J Neurosci Methods 53:55-63, 1994) for mechanosensitivity testing in rodents remains one of the most widely used methods for measuring pain in animals. However, this method results in animals receiving a varying number of stimuli, which may lead to animals in different groups receiving different testing experiences that influences their later responses. To standardize the measurement of mechanosensitivity we developed a simplified up-down method (SUDO) for estimating paw withdrawal threshold (PWT) with von Frey filaments that uses a constant number of five stimuli per test. We further refined the PWT calculation to allow the estimation of PWT directly from the behavioral response to the fifth stimulus, omitting the need for look-up tables.

Results: The PWT estimates derived using SUDO strongly correlated (r > 0.96) with the PWT estimates determined with the conventional up-down method of Chaplan et al., and this correlation remained very strong across different levels of tester experience, different experimental conditions, and in tests from both mice and rats. The two testing methods also produced similar PWT estimates in prospective behavioral tests of mice at baseline and after induction of hyperalgesia by intraplantar capsaicin or complete Freund's adjuvant.

Conclusion: SUDO thus offers an accurate, fast and user-friendly replacement for the widely used up-down method of Chaplan et al.

Figures

Figure 1

Mouse mechanosensitivity measured with von Frey filaments. (A) Illustration of the up-down von Frey testing paradigms described by Chaplan et al. [3] and the simplified up-down method (SUDO). (B) Frequency distribution of the paw withdrawal threshold (PWT) estimates calculated using the method of Chaplan et al., [3] for the mouse dataset that was analyzed (n = 1065). (C) Frequency distribution of the mouse PWT divided by experimenter to reveal data heterogeneity (beginner n = 318, intermediate n = 220, expert n = 527). (D) Frequency distribution of the total number of von Frey presentations (trials) per test.

Figure 2

Strong correlation between mouse paw withdrawal threshold (PWT) estimates calculated using the method of Chaplan et al. and SUDO. (A) Correlation between PWT estimates determined using the two methods for the mouse dataset. (B) Correlation between PWT estimates from the two methods divided by experimenter.

Figure 3

Validation of the parameters of SUDO. (A) Strong correlation between mouse paw withdrawal threshold (PWT) estimates calculated using SUDO with 6 von Frey presentations (trials) per test and the method of Chaplan et al. [3]. (B) Correlation between PWT estimates calculated with SUDO using five or six von Frey presentations. (C) Correlation between mouse PWT estimates when calculated as filament bending force using the up-down method of Chaplan et al. and SUDO with 5 von Frey presentations. (D) Effect of changing the adjustment factor for the PWT estimation for SUDO, demonstrating a peak correlation between PWTs calculated using the simplified method and the method of Chaplan et al. [3] with an adjustment factor near 0.5.

Figure 4

Lack of sensitization within single mechanosensitivity tests in mice conducted using up-down methods. (A) Observed frequency of each possible response pattern when 9 von Frey presentations were used in a test and the corresponding difference in the PWT estimates for each pattern as calculated using the method of Chaplan et al. [3] and SUDO. (B) Negative correlation between the magnitude of the difference in PWT estimates calculated using the two methods and the frequency with which each pattern occurred. (C) Probability of a positive nocifensive response by a mouse to the fifth through ninth von Frey filament presentation in all trials. Data in (C) are mean and 95% confidence interval. (D) PWT estimates measured in mice at baseline (n = 58), 3 hours after intraplantar injection of capsaicin (Cap; 5 μl, 0.5% w/v, n = 12), and 3 hours or 3 days after intraplantar injection of complete Freund’s adjuvant (CFA; 10 μl, n = 12) using the method of Chaplan et al. [3] and SUDO. The two testing methods were presented in a randomized crossover manner in each condition. PWT estimates were not significantly different between methods. (E) Same data as in (D) with PWT estimated expressed as force (g). PWT estimates were not significantly different between methods. Data in (D) and (E) are mean ± s.e.m.

Figure 5

SUDO is valid for PWT estimation in rats. (A) Frequency distribution of the rat paw withdrawal threshold (PWT) estimates calculated using the method of Chaplan et al., ([3]; n = 300). (B) Strong correlation between PWT estimates determined using the method of Chaplan et al. [3] and SUDO. (C) Correlation between mouse PWT estimates calculated using six von Frey presentations (trials) and the method of Chaplan et al. [3]. (D) Effect of changing the adjustment factor in the simplified scoring method, demonstrating a peak correlation with an adjustment factor near 0.5. (E) Correlation between mouse PWT estimates calculated with the two methods when calculated as filament bending force. (F) Probability of a positive nocifensive response by a rat to the fifth through ninth von Frey filament presentation in all trials. Data in (F) are mean and 95% confidence interval.

Figure 6

Flowchart summarizing mechanosensitivity testing protocols of Chaplan et al. and SUDO. In the method of Chaplan et al. [3], the threshold is considered to have been reached the first time that the response of the animal to a von Frey filament is different from the previous trial, after which four additional von Frey presentations are conducted in an up-down manner. At the end of the trial the paw withdrawal estimate (PWT) is calculated by taking the value of the final filament used and adding an adjustment factor derived from the response pattern using the calculations of Dixon [5]. In SUDO, only five von Frey filament presentations are done in each test and the PWT is estimated directly from the response to the final filament by incorporating a fixed adjustment factor based on the response to the fifth filament. See text for additional details.