Nerve growth factor and glutamate increase the density and expression of substance P-containing nerve fibers in healthy human masseter muscles

Abstract

Nocifensive behavior induced by injection of glutamate or nerve growth factor (NGF) into rats masseter muscle is mediated, in part, through the activation of peripheral NMDA receptors. However, information is lacking about the mechanism that contributes to pain and sensitization induced by these substances in humans. Immunohistochemical analysis of microbiopsies obtained from human masseter muscle was used to investigate if injection of glutamate into the NGF-sensitized masseter muscle alters the density or expression of the NMDA receptor subtype 2B (NR2B) or NGF by putative sensory afferent (that express SP) fibers. The relationship between expression and pain characteristics was also examined. NGF and glutamate administration increased the density and expression of NR2B and NGF by muscle putative sensory afferent fibers (P < 0.050). This increase in expression was greater in women than in men (P < 0.050). Expression of NR2B receptors by putative sensory afferent fibers was positively correlated with pain characteristics. Results suggest that increased expression of peripheral NMDA receptors partly contributes to the increased pain and sensitivity induced by intramuscular injection of NGF and glutamate in healthy humans; a model of myofascial temporomandibular disorder (TMD) pain. Whether a similar increase in peripheral NMDA expression occurs in patients with painful TMDs warrants further investigation.

Figure 1

Figure displaying the methodology of the study. (a) Glutamate was injected three days after NGF into the left masseter muscle. One day after injection, a microbiopsy was taken from the injection site. (b) The left photomicrograph shows example image of PGP9.5 staining (green) while the right one shows SP expressing nerve fibers (blue) (1) associated with myocytes and (2) within connective tissue from one female participant on day 4. The white arrows indicate positive PGP 9.5 immunofluorescence signals co-expressed with positive SP signals, while the red arrows indicate positive PGP 9.5 signals without SP co-expression. Green: PGP 9.5, Blue: SP, Scale bar = 25 µm.

Figure 2

The scatter plot illustrates correlations between different pain characteristics and NR2B receptor expression by putative sensory afferent nerve fibers. The correlation between the co-expression of SP with NR2B by nerve fibers and the percentage change (day 3 post glutamate injection/day 0 baseline) in (A) temporal summation pain and (B) chewing-evoked pain. (C) is presenting the correlation of SP with NR2B co-expression with the peak pain intensity induced by injection of glutamate three days after NGF administration. r Pearson, rs Spearman correlation coefficient.