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Background The hornworm Manduca sexta exhibits a defensive strike to noxious assaults, a response that is robust and is easily observed by experimenters. Von Frey filaments and methods typical for studying nociception in other animals were used to assess the strike response in M. sexta. New methods A series of von Frey filaments was applied to the body wall in ascending order and the data generated were used to determine the strike threshold by (i) the up-and-down method, (ii) the first response method, and (iii) the simplified up-and-down order method (SUDO). The effect of a noxious pinch on strike threshold was assessed. Comparison with existing methods To our knowledge none of these methods has been used on M. sexta previously, making the use of the up-and-down and SUDO methods the first in an invertebrate. The use of the first response method has been used in other invertebrates, and the method appears equally suited to M. sexta. Results All three methods were successful in monitoring the threshold sensitivity to touch, which was lowered (sensitized) by tissue damage induced with a pinch. Sensitization lasted 19 h. Conclusions All three methods of assessing nociception were successfully applied to quantify the defensive strike response in M. sexta, although the SUDO method required empirical assessment of which filament to start the test sequence with. The results revealed both short- and long-term sensitization. These methods should prove to be useful for quantifying sensitization in M. sexta.

Here, we reconsider the status quo in testing mechanical sensitivity with von Frey's hairs. The aim is to improve paw withdrawal estimates by integrating current psychometric theory, and to maximise the clinical relevance and statistical power of mechanosensory models. A wealth of research into human tactile stimulus perception may be extended to the quantification of laboratory animal behaviour. We start by reviewing each step of the test, from its design and application through to data analysis. Filament range is assessed as a whole; possible test designs are compared; techniques of filament application to mice and rats are considered; curve fitting software is introduced; possibilities for data pooling and curve fitting are evaluated. A rational update of classical methods in line with recent advances in psychometrics and supported by open source software is expected to improve data homogeneity, and Reduce and Refine animal use in accord with the ‘3R’ principles.

Background: Preclinical studies measure withdrawal responses to evoking thermal and mechanical stimuli instead of the more clinically important spontaneous pain. New method: Therefore, we studied the effect of peripheral inflammation induced by intraplantar administration of complete Freund’s adjuvant (CFA) in mice on the variability of temperature and bioimpedance as an index of pain produced by inflammation. To this end, we initially determined mathematical scores based on changes in temperature and bioimpedance (STB) for animals with an inflamed paw and compared these scores with commonly used measures of inflammatory pain. We then pharmacologically validated the tool using dexamethasone. Results: The STB analysis resembled the response found in the von Frey Hair (vFH) test. The CFA-induced increase in STB and vFH tests were reversed by intraperitoneal administration of dexamethasone. The correlation between the STB and vFH measurements showed a high correlation coefficient (R2=0.911, p < 0.001). Comparison with existing method: Our results also demonstrated that CFA paw injection induced mechanical hyperalgesia in mice and remained virtually unaltered during all time-points tested for 5 days, as measured with vFHs. The administration of CFA into the paw induced a large increase in paw volume that was apparent 1 and 5 days after the injection. The CFA injection resulted in a significant (p

Background Stomatitis induces severe and painful hypersensitivity to pungency and physical contact during meals. Many studies have used anesthetized animals to examine evoked nociception in the oral mucosa, but no reports have used traditional behavioral assays to evaluate nociception in conscious animals. New methods We developed two new methods of applying chemical or mechanical stimulation directly to the oral mucosa of the mandibular vestibule of conscious rats. Nociceptive evaluations were performed by measuring facial grooming time and the head withdrawal threshold to von Frey stimulations. (1) For the intraoral dropping method, rat mucosa was transiently exposed by hand, and a drop of a pungent solution was applied. (2) For the stable intraoral opening method, rat mucosa was long-term exposed following piercing surgery of the mental skin after habitual training for 2–3 weeks. Results In the intraoral dropping method, the application of 100 μM capsaicin or 100 mM allyl isothiocyanate prolonged mouth-rubbing time. Capsaicin-induced mouth-rubbing time was further enhanced following the development of an acetic acid-induced ulcer. The stable intraoral opening method enabled stable measurements of the mechanical withdrawal threshold in the oral mucosa of conscious rats. Ulcer development decreased the mechanical threshold, whereas topical lidocaine treatment increased the threshold. Comparison with existing methods These new methods enable the evaluations of motivational nocifensive behaviors in response to intraoral stimulations without any anesthetic effects. Conclusions The intraoral dropping and stable intraoral opening methods can be used in combination with traditional behavioral assays to evaluate nociception in the oral mucosa of conscious rats.

A von Frey filament (vFF) is a type of aesthesiometer usually made of nylon perpendicularly held in a base. It can be used in paw withdrawal pain threshold assessment, one of the most popular tests for pain evaluation using animal models. For this test, a set of filaments, each able to exert a different force, is applied to the animal paw, from the weakest to the strongest, until the paw is withdrawn.We made 20 low cost vFF using nylon filaments of different lengths and constant diameter glued perpendicularly to the ends of popsicle sticks. They were calibrated using a laboratory balance scale. Building and calibrating took around 4h and confirmed the theoretical prediction that the force exerted is inversely proportional to the length and directly proportional to the width of the filament.The calibration showed that they were precise and accurate. We analyzed the paw withdrawal threshold assessed with the set of home-made vFF and with a high quality commercial set of 5 monofilaments vFF (Stoelting, Wood Dale, USA) in two groups (n=5) of healthy mice.The home-made vFF precisely and accurately measured the hind paw withdrawal threshold (20.3±0.9 g). The commercial vFF have different diameters while our set has the same diameter avoiding the problem of lower sensitivity to larger diameter filaments.Building a set of vFF is easy, cost effective, and depending on the kind of tests, can increase precision and accuracy of animal nociception evaluation.

Von Frey filaments used for testing mechanical thresholds are mechanically unstable and their use is difficult to standardize. We have therefore constructed a hand-held electronic pressure algometer. The pressure algometer is connected to a computerized data collection system, allowing on-line display of the applied force as well as the application rate. Data stored on the computer can be replayed and further analyzed. Using this apparatus, we have measured the pressure-induced withdrawal thresholds in rats with surgically induced neuropathy. The probe, with a circular tip of 1.0 mm diameter, was applied manually with a pressure increasing by approximately 0.05 N/s. Presurgical thresholds were normally distributed with a mean of 0.415 N, showing no significant difference between paws. During 2 weeks after surgery, the thresholds of the operated side were significantly reduced (range, 0.209-0.318 N), while the thresholds of the non-operated side remained at higher values (range, 0.432-0.491 N). Thresholds of control rats without surgery were in the 0.380-0.520 N range, with no significant difference between paws. In an additional experiment it was shown that interobserver reliability was high, both between withdrawal threshold values obtained and between rates of application used. In conclusion, the electronic algometer allows standardization of testing, detailed documentation of each experiment and provides an objective and accurate method for measuring the reactions of test animals to mechanical stimuli.

We describe a device which allows the mechanical sensitivity of trigeminovascular sensory neurons to be monitored over extended time periods. The device can be used to stimulate either the skin or dura mater and consists of a solenoid-driven plunger to which are fixed interchangeable von Frey hairs. The solenoid can be attached to a stereotaxic carrier and mounted on a stereotaxic frame to allow precise positioning over the receptive field. The device is driven from the synchronization signals of a standard stimulator via TTL circuitry and a relay driver, to allow stimulation by a single or multiple stimuli. The advantages of the device over manual stimulation include the reproducibility of the site of stimulation; the ability to apply a known force for a known time; the ability to measure response latencies to millisecond precision and to compare them to latencies to other stimuli and; easy interface with computer-control. We discuss some of the drawbacks of the von Frey system as usually used and illustrate the use of the new device with results from experiments on peripherally induced sensitization.

The effect of testing surface on the rat hind paw withdrawal threshold in the von Frey hair test is investigated in this study. The data indicate that wire mesh, which is typically used to apply von Frey hairs, may have an effect on the paw withdrawal threshold. For example, in control rats tested on the wire mesh, variability in the withdrawal threshold was observed between the left and the right hind paws (51.04+/-12.29 and 64.31+/-9.37 g, respectively) and on different days of testing (35.24+/-9.54 and 45.83+/-12.97 g for the left and right hind paws, respectively, 7 days later). In an attempt to reduce this variability, a customized platform was used to measure the von Frey hair-induced paw withdrawal in the rat. It consists of an opaque, flat-surfaced plastic platform with holes through which von Frey hairs are inserted and applied to the plantar surface of the paw. In control rats tested with von Frey hairs using this customized platform, variability in the paw withdrawal thresholds between the left and right hind paws in single rats over time as well as between different rats was reduced (49.86+/-6.97 and 49.29+/-6.56 g for the left and right hind paws, respectively, on day 0; 48.29+/-5.82 and 53.00+/-4.59 g for the left and right hind paws, respectively, 7 days later). Furthermore, in rats in which a 2 mm polyethylene cuff was used to constrict the left common sciatic nerve, the ipsilateral as well as the contralateral hind paw withdrawal thresholds were decreased (2.45+/-0.65 and 26.09+/-5.86 g, respectively, 7 days later). In similar rats tested on the wire mesh, the ipsilateral but not the contralateral paw withdrawal threshold decreased (12.80+/-2.21 and 65.00+/-10.28 g, respectively, at 7 days). The data suggest that the flat surface and opaque properties of the customized platform enable accurate, reliable and repeatable measurements of ipsilateral and contralateral paw withdrawal threshold using von Frey hairs in normal and nerve-injured rats.

A series of von Frey hairs was subjected to changes in temperature and relative humidity (RH) simulating those encountered on a neonatal intensive care unit. The effect of each environmental change on their calibrated value was monitored. It was found that changes in RH significantly affected their calibration, whereas changes in temperature had a smaller effect. The results of the study indicate that the environmental conditions in which von Frey hairs are used should be taken into account.