The von Frey monofilament was designed by Maximilian von Frey as an esthesiometer in 1896. These filaments have been widely used in the assessment of mechanical nociception. The process of assessment of punctuating mechanical allodynia and hyperalgesia involves the application of von Frey filaments of forces ranging anywhere from 0.08 mN to 2940 mN and observing the withdrawal thresholds. The assay can be performed manually or by using an electric von Frey.

The most commonly used method is the up-down method, wherein the tips are smoothly applied perpendicularly to the skin of the plantar surface of the subject until they buckle. However, the technique tends to be time-consuming. The electronic von Frey (EvF) is used to overcome the disadvantages of the conventional von Frey assay. The former has many advantages over the manual von Frey assay, primarily, the use of a single filament to apply varying levels of pressure. Further, unlike other tests such as the Randall-Selitto test, the von Frey monofilament assay does not require restraining the subject. This approach minimizes handling and restraining anxiety which could otherwise potentially influence the performance of the subject.

Documentation

Introduction

Apparatus and Equipment

The Electronic von Frey test is performed using a pressure meter that consists of a hand-held force transducer fitted with the rigid monofilament. The choice of the tip is dependent on the tissue being probed. A digital pressure meter allows observation and setting of a cut-off pressure threshold. During the trials, the subjects are housed individually in holding containers that have grid floors. It is important to note that the type of flooring can have a potential influence on the administration of the von Frey test.

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Training Protocol

Clean the apparatus and equipment before and after every trial. The recording and tracking of the behavioral response can be performed using video and tracking system such as the Noldus EthoVision XT or ANY-Maze.

Perform the von Frey assay in a quiet, temperature-controlled room, ensuring that no external stimuli can influence the performances of the subjects during the test. Also, perform the test at the same time of the day for every trial.

Habituation

Place the subjects individually in their acrylic cages, 15 to 30 minutes before beginning the trials. During this period, poke the paws of the subject 2 to 3 times. This process allows the subject to acclimate to the set-up and remain still during the actual trial.

Training Trial

Using a tilted mirror under the wire flooring, apply the probe tip perpendicularly to the plantar surface of the subject. To minimize confounding variables, ensure that the application is performed when the subject is not grooming.  To obtain baseline values, perform the von Frey assessment at least 48 hours before the administration of antinociceptive or other investigatory treatment. Perform 5 trials spaced appropriately; wherein the pressure is gradually increased until nociceptive behavior such as paw retraction, paw licking, or four-paw jumping are observed. Repeat the process after 24 hours.

Following the antinociceptive treatments or other investigatory treatment, perform three trials in the fashion described above to obtain experimental withdrawal threshold.

Evaluation of mechanical sensitivity in a photothrombotic model of stroke

Alamri et al. (2018) evaluated the applicability of the automated von Frey in assessing the forelimb dysfunction in mice following photothrombosis of the sensorimotor cortex, in comparison with the popularly used grid-walking and cylinder test. After baseline assessments, the male CD-1 mice either underwent photothrombosis or served as the sham group. Subjects were evaluated in the following order of tests: grid-walking, cylinder, forepaw grip strength and von Frey, on days 1, 7, 14 and 21 post-stroke induction. The observation from the assessments revealed automated von Frey to be a time-efficient and sensitive method that was capable of detecting substantial and sustained deficits in the withdrawal threshold of the subject on all evaluation days.

Data Analysis

The data collected using the Hargreaves test is straightforward. Withdrawal latencies, the time taken to withdraw paw as a response to heat, and the average reaction time are recorded. It is important to ensure that observed behavior is a reflex behavior and not a voluntary one. In case an ambiguous behavior is observed, repeat the experiment and record the correct withdrawal latency.

Strengths & Limitations

Strength

The Hargreaves test evaluates thermal nociceptive responses of the subject. As opposed to the Hot plate test which subjects the entire plantar surface of the subject to the heat stimulus, the Hargreaves test uses a directed heat stimulus that tests a particular point on the plantar surface. This allows unilateral response assessments. The apparatus is also simple and straightforward to use. The Hargreaves test uses a compartmentalized area that enables testing multiple subjects at once. The use of glass bottom for the compartments minimizes the errors arising from heat sink effects. The Hargreaves test is widely used in investigations of neural injuries and regeneration to evaluate sensitization or recovery of thermal responses.

Limitations

While testing in the Hargreaves apparatus, it is important to ensure the apparatus is cleaned before and after trials, and as required during trials since the presence of urine can affect the temperature of the heat stimulus. Care must also be taken to observe and differentiate voluntary behaviors from reflex behaviors. In case of voluntary behaviors, the experiment should be repeated to obtain accurate results. It is also essential to not exceed the cut-off time to prevent unnecessary damage and harm to the animals used. The test may require longer periods of habituation and acclimation depending on the species and strains as some are known to have an excited exploratory drive. The experiments should also be performed while the subject is awake. It is important to minimize any external stimulus in the test arena to reduce their impact on the subject’s performance. The Hargreaves test also does not allow the direct measure of paw withdrawal temperatures. Handling and consistency of the surgical process can also potentially influence paw withdrawal responses.

Summary and Key Points

• Hargreaves test was first described in the literature by Hargreaves and colleagues in 1988.
• Only a specific part of the plantar surface of the animal is subjected to heat stimulus.
• Withdrawal latencies are observed as the time taken to respond to the heat stimulus.
• Different strains and species showcase a range of exploratory drive thus habituation time may be increased or decreased to acclimate the subject to the apparatus.
• Voluntary movements should be clearly distinguished from heat-stimulus generated reflex movements.