Note
Effective Location of the Plantar Surface of the Hind Paw for the Evaluation of Mechanical Hyperalgesia Using von Frey Filaments in Mice Treated with Paclitaxel
2025 年 48 巻 6 号 p. 835-837
詳細
2025 年 48 巻 6 号 p. 835-837
Mechanical hyperalgesia is commonly evaluated using von Frey filaments (vFFs) in the rodent hind paw plantar. However, it is difficult to select the plantar location to stimulate with the vFFs. In the present study, we investigated the effective location of the plantar surface for the evaluation of mechanical hyperalgesia using vFFs in mice treated with paclitaxel (PTX). PTX or vehicle was injected intraperitoneally once daily, 4 times every other day. On Day 14, mechanical hyperalgesia was evaluated using vFFs. The evaluation was performed by stimulation with vFFs on the skin directly under the 3rd and 4th paw pads (base of the index and middle fingers) from closest to the heel on the thumb side, in the central skin surrounded by the pads, and in the plantar skin one-third of the way down from the heel toward the toe. In comparison to vehicle-treated mice, mechanical hyperalgesia was significantly observed in the skin directly under the 3rd and 4th paw pads counted from closest to the heel, and in the plantar skin one-third of the way down from the heel toward the toe. However, this was not significant in the central skin surrounded by the pads. Interestingly, in vehicle-treated mice, the paw withdrawal threshold varied for each location evaluated. In contrast, in PTX-treated mice, the thresholds were similar across all evaluated locations. These results suggest that the selection of the location on the plantar surface to be stimulated is important for pain evaluation using vFFs.
The evaluation of pain in animals is extremely important for elucidating the mechanisms of pain and developing analgesics. However, it is extremely difficult to obtain information from animals that cannot communicate verbally, and many methods for evaluating pain have been devised,1) including the von Frey test, Randall–Selitto test, tail flick test, hot plate test, Hargreaves test, cold plate test, acetone evaporation test, grimace scale, weight-bearing, and gait analysis. Among these evaluations, the von Frey test using a thin filament makes it difficult to select the stimulation site on the plantar surface. Except for the evaluation of pain in rodent postoperative pain models, there are no reports that clearly define the stimulation site of von Frey filaments (vFFs).2) Therefore, identifying the effective location of stimulation with vFFs in the plantar region, where a clear difference in the response between healthy controls and mouse pain models is observed, is important for elucidating the mechanisms of pain and screening therapeutic drugs.
Paclitaxel (PTX) is an antimicrotubule agent and anticancer drug that is widely used to treat solid neoplasms such as breast, ovarian, and lung cancers.3,4) PTX causes peripheral neuropathy, which is characterized by mechanical allodynia, spontaneous pain and numbness, with a stocking-and-glove distribution in human cancer patients,5) and by mechanical allodynia and hyperalgesia in rodents.6,7) PTX promotes demyelination, which is associated with the induction of pain in the plantar nerve.8–10) Myelinated Aβ- and Aδ-fibers, but not C-fibers, are involved in the mechanical hypersensitivity evaluated with vFFs.11) Therefore, it is suggested that all sensory nerves innervating the plantar skin respond to vFFs in PTX-treated mice. In this study, the effective location of plantar surface for the evaluation of mechanical hyperalgesia using vFFs was investigated in PTX-treated mice.
Male C57BL/6NCr mice (Japan SLC Ltd., Shizuoka, Japan) were used. All animals were 6 weeks old at the start of the experiment. Four mice were housed per cage under a controlled temperature (21–23°C) and humidity (45–65%) conditions. The room was lit between 08 : 00 and 20 : 00. All animal experiments were approved by the Committee for Animal Experiments of Kinjo Gakuin University (No. 247).
MaterialsPTX (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was dissolved in physiological saline containing 10% (v/v) Cremophor® EL (Sigma-Aldrich, St. Louis, MO, U.S.A.) and 10% (v/v) ethanol. PTX (8 mg/kg) or vehicle (VH) was injected intraperitoneally once daily, 4 times, every other day (Days 0, 2, 4, and 6) at a volume of 0.1 mL/10 g body weight.6,12)
Behavioral EvaluationMice were placed individually in a plastic cage (8 × 10 × 18 cm) with a wire mesh bottom for an acclimation period of at least 30 min, and then the pain threshold was evaluated using a fine von Frey filament (vFF; Aesthesio® Precision Tactile Sensory Evaluator, Muromachi Kikai Co., Ltd., Tokyo, Japan). In this study, vFFs with bending forces of 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, and 0.6 g were used. The filaments were tested in descending order from 0.6 to 0.08 g; each filament was applied for 1–3 s until a slight bend was observed. A response was determined to have occurred if the mouse lifted or moved its paw in response to the vFF stimulus. To avoid repeated assessments on the same area within a short time frame, an interval of at least 20 s was maintained between measurements of the left and right paws. The filaments were pressed to various locations (Fig. 1) of the plantar surface of a freely moving mouse. After evaluation using a vFF of a single bending force on a part of both foot pads of all mice, the response in the same location was evaluated using a vFF of another single bending force. This series of evaluations was repeated twice, and the minimum bending force that elicited a response both times was determined as the threshold. After pain evaluations using all vFFs were completed twice in one location, evaluations in another location were performed in the same manner.
Position Nos.①–⑤ show the locations stimulated with von Frey filaments.
All data are presented as means and standard errors of the mean (S.E.M.). Statistical significance was analyzed using the Mann–Whitney rank-sum test. P values of <0.05 were considered to indicate statistical significance. Statistical analyses were performed using the SigmaPlot software program (ver. 14.5; Systat Software Inc., Chicago, IL, U.S.A.).
Figure 1 shows the locations of the mouse hind paws stimulated with vFFs. The evaluation was performed by stimulating the skin with vFFs directly under the 3rd position (Position No. ②) and 4th (Position No. ①) paw pads (base of the index and middle fingers) counted from closest to the heel on the thumb side, in the central skin surrounded by the pads (Position Nos. ③ and ④), and in the plantar skin one-third of the way down from the heel toward the toe (Position No. ⑤). There was no difference in the pain thresholds between the left and right paws at each location in the VH- and PTX-treated mice (Fig. 2). Pain thresholds in the hind paws of PTX-treated mice were similar at all positions, whereas those in VH-treated mice varied depending on the position. In other words, the pain thresholds were greater at Position Nos. ①, ②, and ⑤ than at Position Nos. ③ and ④ (Fig. 2). Compared to the VH-treated mice, the pain thresholds at Position Nos. ①, ②, and ⑤ in the PTX-treated mice were significantly decreased in both hind paws (Fig. 2). The pain threshold at Position No. ④ in the right paws of PTX-treated mice was also significantly decreased (Fig. 2). However, at Position Nos. ③ (both paws) and ④ (left paws), although there was a tendency for the pain threshold to decrease, this was not statistically significant (Fig. 2).
The paw withdrawal threshold was evaluated using von Frey filaments (vFFs) at various locations (see Fig. 1) on the plantar surface of PTX- or VH- treated mice. “Right” and “Left” show the plantar in right hind paw and left hind paw, respectively. Data are presented as the mean ± S.E.M. (n = 4). *p < 0.05 (Mann–Whitney rank-sum test). PTX: paclitaxel; VH: vehicle.
The present study showed the effective location of the plantar region for the evaluation of mechanical hyperalgesia using vFFs in mice treated with PTX. In vehicle-treated mice, the responsiveness to vFF stimulation varied depending on the location of stimulation in the plantar region, but did not change in PTX-treated mice. However, the difference in responsiveness to vFF stimulation between vehicle- and PTX-treated mice was observed in the skin just under the paw pads at the base of the index and middle fingers and in the plantar skin one-third of the way down from the heel toward the toe, but there was no difference in the central area surrounded by the paw pads. Therefore, at least in PTX-treated mice, it is suggested that it would be more effective to evaluate vFF stimulation in either location, such as the skin just under the paw pads at the base of the index and middle fingers or in the plantar skin one-third of the way down from the heel toward the toe.
In healthy mice, the reason for the difference in responsiveness to vFF stimulation in various locations in the plantar skin remains unclear. The skin on the plantar surface is roughly the same thickness, except for the paw pads.13) However, the distance from the skin surface to the metatarsal bone was smaller and the skin was softer in the area surrounded by the paw pad than in other areas, suggesting that the response to vFF stimulation in the area surrounded by the paw pad was more sensitive than in the other areas evaluated.
Merkel cells and Meissner corpuscles are mechanoreceptors.14) Although Merkel cells are widely distributed in the plantar skin, Meissner corpuscles are mainly distributed in the paw pads of the plantar skin.13) Merkel cells respond to sustained indentation in proportion to the indentation depth, and Meissner corpuscles respond to slip between the skin and an object,14) suggesting that Merkel cells may be involved in the response to vFF stimulation. However, the detailed distribution of Merkel cells in plantar skin is not well understood. In addition, the center or entire plantar area of the hind paw of rodents is innervated by the tibial nerve, and the medial and lateral sides of the plantar region are innervated by the saphenous and sural nerves, respectively.15,16) However, the detailed distribution of the nerve terminals in the plantar region remains unclear. Therefore, it is still unknown whether the difference in the response to vFF stimulation in the plantar of healthy mice is related to the distribution of Merkel cells and sensory nerve terminals.
In this study, high sensitivity to vFF stimulation at the same degree was observed at all locations evaluated in PTX-treated mice. Therefore, it is suggested that the difference in sensitivity to vFF stimulation may depend on the local environment rather than the distribution of Merkel cells and sensory nerve terminals.
In conclusion, the present results suggest that to obtain effective and apparent results in comparison to controls, the selection of the location on the plantar surface stimulated using vFFs is important.
This study was supported in part by a Kinjo Gakuin University–Parent Teacher Association Research Grant (Research B).
The authors declare no conflict of interest.
