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Comparative Study
. 2005 Apr 1;564(Pt 1):259-68.
doi: 10.1113/jphysiol.2004.079483. Epub 2005 Jan 27.

Muscular mechanical hyperalgesia revealed by behavioural pain test and c-Fos expression in the spinal dorsal horn after eccentric contraction in rats

Toru Taguchi  1 , Teru Matsuda, Ryoko Tamura, Jun Sato, Kazue Mizumura
Affiliations
Comparative Study

Muscular mechanical hyperalgesia revealed by behavioural pain test and c-Fos expression in the spinal dorsal horn after eccentric contraction in rats

Toru Taguchi et al. J Physiol. .

Abstract

Delayed onset muscle soreness (DOMS) is quite common, but the mechanism for this phenomenon is still not understood; even the existence of muscle tenderness (mechanical hyperalgesia) has not been demonstrated in experimental models. We developed an animal model of DOMS by inducing eccentric contraction (lengthening contraction, ECC) to the extensor digitorum longus muscle (EDL), and investigated the existence of mechanical hyperalgesia in the EDL by means of behavioural pain tests (Randall-Selitto test and von Frey hair test, applied to/through the skin on the EDL muscle) and c-Fos expression in the spinal dorsal horn. We found that the mechanical withdrawal threshold measured with the Randall-Selitto apparatus decreased significantly between 1 and 3 days after ECC, while that measured by von Frey hairs did not. The group that underwent stretching of the muscle only (SHAM group) showed no change in mechanical pain threshold in either test. These results demonstrated that the pain threshold of deep tissues (possibly of the muscle) decreased after ECC. c-Fos immunoreactivity in the dorsal horn (examined 2 days after ECC/SHAM exercise) was not changed by either ECC or compression (1568 mN) to the EDL muscle by itself, but it was significantly increased by applying compression to the EDL muscle 2 days after ECC. This increase was observed in the superficial dorsal horn of the L4 segment of the ipsilateral side, and was clearly suppressed by morphine treatment (10 mg kg(-1), i.p.). These results demonstrated the existence of mechanical hyperalgesia in the muscle subjected to ECC. This model could be used for future study of the neural mechanism of muscle soreness.

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Figures

Figure 1
Figure 1. Schematic drawing of the experimental procedures
A, methods of applying eccentric contraction to the extensor digitorum longus (EDL) muscle. One electrode was inserted transcutaneously near the sciatic nerve (+ pole) and the other near the common peroneal nerve (− pole). Repetitive contractions of the muscle were induced by cyclic electrical stimulation of the nerve. The hindpaw was fixed to a bar that was connected to the linearized motor, and it was pulled (‘lengthened’) synchronously with the muscle contraction so that the EDL muscle was stretched. Current intensity of the electrical stimulation was set at 3 times the threshold for twitch contraction, and frequency was set at 50 Hz to induce tetanic contraction. Contraction was repeated 500 times (1 s contraction followed by 3 s resting period). B, schedule of exercise and compression (upper panel), and the protocol of compression (lower panel) in the c-Fos experiment. Upper panel: on day 0 the animals underwent either eccentric contraction (ECC) or stretching of the muscle (SHAM). On day 2 the animals in the ECC and SHAM groups were each divided into two subgroups, one with and one without muscle compression. Lower panel: exercised muscle (EDL muscle) was compressed with a force of 160 g by Randall-Selitto apparatus for 10 s, and there was an interval of 20 s before the next compression. This session was repeated 60 times.
Figure 2
Figure 2. Decreased pain thresholds after ECC were found with Randall-Selitto test but not with von Frey hair test
A, withdrawal thresholds of the eccentrically exercised muscle measured by Randall-Selitto apparatus (n= 6 for each group). Abscissa: days after exercise; ordinate: withdrawal threshold. Data from the ECC group, •; result of the SHAM group, ○. Randall-Selitto test showed decreased pain threshold after ECC. *P < 0.05, ***P < 0.001 compared with the value immediately before the day of exercise (−1 day). ##P < 0.01, ###P < 0.001 compared with the SHAM group on each day after treatment. B, withdrawal threshold for the skin over the exercised muscle measured by von Frey hairs (n= 7 for each group). The presentation is the same as in A. No significant differences were found between groups on each day, or between −1 day and days after ECC (non-parametric Friedman test followed by Dunn's multiple comparison test). These results suggest that the pain threshold was decreased in the deeper tissue, probably in the muscle.
Figure 3
Figure 3. c-Fos-ir neurones in the ipsilateral dorsal horn of the spinal cord at L4
A–D, camera lucida drawings of representative sections from four groups 2 days after treatment: SHAM group (A), ECC group (B), SHAM + compression group (C), and ECC + compression group (D). Labelled neurones are represented as black dots. Note that the number of labelled neurones increased in the ECC + compression group on the second day. This was especially marked in the superficial dorsal horn. E and F, photographs with different magnification of the same section as in D, showing c-Fos-ir neurones in the ipsilateral dorsal horn of the spinal cord at L4. Calibration bars: 100 μm in E and 50 μm in F.
Figure 4
Figure 4. Segmental distribution of the c-Fos-ir neurones in the dorsal horn of the spinal cord at L2–L6
A, representative camera lucida drawing of the c-Fos-ir neurones in the dorsal horn of the spinal cord at L2–L6 in a rat with ECC + compression. Labelled neurones are represented as black dots. The largest number of labelled neurones was observed in the superficial dorsal horn of segment L4, and smaller numbers were also seen in segments L2 and L3. B, summary of segmental distribution of c-Fos-ir neurones in the spinal dorsal horn (L2–L6). Number of labelled neurones per section in the entire dorsal horn in each segment is shown. The number of animals used was 4 in SHAM, SHAM + compression, and ECC + compression groups, and 5 in ECC group. Note that there is a significant increase in the number of c-Fos-ir neurones at L4 in ECC + compression group compared with the other three groups (**P < 0.01). The number of c-Fos-ir neurones at L2 and L3 in ECC + compression group tended to be greater, but differences among the four groups were not significant.
Figure 5
Figure 5. Number of c-Fos-ir neurones in the different areas of the spinal dorsal horn at L4 (A) and suppression of its increase by morphine (B)
In A, TTL (DH) represents total number of labelled neurones in the entire dorsal horn; SDH, superficial dorsal horn; PN, proprius nucleus; NDH, neck of the dorsal horn. The number of animals was 4 for SHAM, SHAM + compression, and ECC + compression groups, and 5 in ECC group. A significant increase in the number of c-Fos-ir neurones was observed in the superficial dorsal horn compared with each of the other three groups (**P < 0.01, ***P < 0.001). The numbers of c-Fos-ir neurones in the proprius nucleus and the neck of the dorsal horn appeared to be somewhat increased in the ECC + compression group, but the difference was not statistically significant. In B, filled columns show the number of c-Fos-ir neurones per section in the animals that received morphine (10 mg kg−1, i.p.) 20 min before compression of EDL muscle 2 days after ECC, and open columns show those in the control group that received saline instead of morphine. Number of animals was 5 for the morphine group, and 6 for the control group. Note that c-Fos immunoreactivity was clearly suppressed in the morphine-treated group (**P < 0.01, Mann-Whitney test).
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Comment in

  • Muscle tenderness from exercise: mechanisms?
    Proske U. Proske U. J Physiol. 2005 Apr 1;564(Pt 1):1. doi: 10.1113/jphysiol.2005.085514. Epub 2005 Feb 24. J Physiol. 2005. PMID: 15731183 Free PMC article. Review. No abstract available.

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