Eccentric muscle contraction and stretching evoke mechanical hyperalgesia and modulate CGRP and P2X(3) expression in a functionally relevant manner

Abstract

Non-invasive, movement-based models were used to investigate muscle pain. In rats, the masseter muscle was rapidly stretched or electrically stimulated during forced lengthening to produce eccentric muscle contractions (EC). Both EC and stretching disrupted scattered myofibers and produced intramuscular plasma extravasation. Pro-inflammatory cytokines (IL-1beta, TNF-alpha, IL-6) and vascular endothelial growth factor (VEGF) were elevated in the masseter 24h following EC. At 48h, neutrophils increased and ED1 macrophages infiltrated myofibers while ED2 macrophages were abundant at 4d. Mechanical hyperalgesia was evident in the ipsilateral head 4h-4d after a single bout of EC and for 7d following multiple bouts (1 bout/d for 4d). Calcitonin gene-related peptide (CGRP) mRNA increased in the trigeminal ganglion 24h following EC while immunoreactive CGRP decreased. By 2d, CGRP-muscle afferent numbers equaled naive numbers implying that CGRP is released following EC and replenished within 2d. EC elevated P2X(3) mRNA and increased P2X(3) muscle afferent neuron number for 12d while electrical stimulation without muscle contraction altered neither CGRP nor P2X(3) mRNA levels. Muscle stretching produced hyperalgesia for 2d whereas contraction alone produced no hyperalgesia. Stretching increased CGRP mRNA at 24h but not CGRP-muscle afferent number at 2-12d. In contrast, stretching significantly increased the number of P2X(3) muscle afferent neurons for 12d. The sustained, elevated P2X(3) expression evoked by EC and stretching may enhance nociceptor responsiveness to ATP released during subsequent myofiber damage. Movement-based actions such as EC and muscle stretching produce unique tissue responses and modulate neuropeptide and nociceptive receptor expression in a manner particularly relevant to repeated muscle damage.

Fig. 1

Force and displacement during eccentric contraction of the masseter muscle. Upper trace shows typical torque developed during EC of the masseter muscle. Lower trace shows mandibular displacement during the EC. Note that the muscle is pre-activated (arrow) prior to muscle displacement (i.e. jaw opening).

Fig. 2

Plasma extravasation measured using Evans Blue after eccentric contraction of the masseter muscle. Note that significant plasma extravasation (asterisk, p<0.05) is evoked in the masseter 24h following EC but not in the overlying skin. Naive n=26, 4h n=7, 1d n=13, 2d n=6, 4d n=12, Box plot shows median with edges of box denoting 25th and 75th percentile.

Fig. 3

Masseter muscle following systemic injection of Evans Blue. A: naive, B: 24h following EC, arrows point to myofibers containing Evans Blue indicative of membrane disruption. C: 24h following injection of CFA into masseter muscle. Arrowheads show the localization of Evans Blue in the extracellular space but not within myofibers. Scale bar=50μm

Fig. 4

Eccentric contraction evokes muscle inflammation with selective inflammatory cell infiltration of myofibers. A: H+E staining of the masseter muscle 48h after EC showing typical inflammatory cell infiltration (arrow). B: Higher magnification photomicrograph showing inflammatory cell infiltration into single masseter myofiber (arrow). C: Infiltration of masseter myofibers by ED1 macrophages (arrows) 96h after muscle contraction. D: Higher magnification photomicrograph showing selective infiltration of ED1 macrophages (arrows) into myofibers after EC. Asterisk denotes swollen myofiber. E: ED2 macrophages (arrows) in the masseter muscle 96h following contraction. F: Intramuscular injection of adjuvant produces massive non-specific inflammation with tissue erosion. Large vacuoles (V) are evident, arrowheads identify ED1 macrophages, arrows point to myofibers. Scale bars A,C,D,F=50μm; B=10μm; E=25μm

Fig. 5

Single bout of eccentric muscle contraction evoked ipsilateral mechanical hyperalgesia (asterisks denote significant reduction in head withdrawal threshold). Median values are plotted with the 25th and 75th percentile. Asterisks denote significant reduction in withdrawal threshold from baseline. n=10 animals

Fig. 6

Multiple bouts of eccentric contraction evokes ipsi- and bilateral mechanical hyperalgesia. Asterisks denote significant reduction in head withdrawal thresholds. Medians and 25th and 75th percentiles are plotted for comparison to Fig 5 even though parametric statistics were used for analysis. n=4 animals

Fig. 7

Real-time polymerase chain reaction (RT-PCR) data from the ipsilateral mandibular division (V3) of the trigeminal ganglion 24h following eccentric muscle contraction. Means and SE are plotted, asterisks denote significant fold increases from naive. Note that both EC and rapid stretching increase CGRP mRNA while only EC increases P2X₃ mRNA at 24h.

Fig 8

Percentage of immuno-positive muscle afferent neurons following eccentric muscle contraction and stretching. A. Percentage of CGRP and P2X₃ positive muscle afferent neurons following EC. Note the increase in P2X₃ muscle afferent neurons 2d and 12d following EC. B. Percentage of CGRP and P2X₃ muscle afferent neurons following stretching. Note the increase in P2X₃ muscle afferent neurons 2d and 12d following stretching. Means and SE are plotted, asterisks denote significant differences from naive.