Simulated whiplash modulates expression of the glutamatergic system in the spinal cord suggesting spinal plasticity is associated with painful dynamic cervical facet loading

Abstract

The cervical facet joint and its capsule have been reported to be injured during whiplash scenarios and are a common source of chronic neck pain from whiplash. Both the metabotropic glutamate receptor 5 (mGluR5) and the excitatory amino acid carrier 1 (EAAC1) have pivotal roles in chronic pain. In this study, spinal mGluR5 and EAAC1 were quantified following painful facet joint distraction in a rat model of facet-mediated painful loading and were evaluated for their correlation with the severity of capsule loading. Rats underwent either a dynamic C6/C7 joint distraction simulating loading experienced during whiplash (distraction; n = 12) or no distraction (sham; n = 6) to serve as control. The severity of capsular loading was quantified using strain metrics, and mechanical allodynia was assessed after surgery. Spinal cord tissue was harvested at day 7 and the expression of mGluR5 and EAAC1 were quantified using Western blot analysis. Mechanical allodynia following distraction was significantly (p < 0.001) higher than sham. Spinal expression of mGluR5 was also significantly (p < 0.05) greater following distraction relative to sham. However, spinal EAAC1 was significantly (p = 0.0003) reduced compared to sham. Further, spinal mGluR5 expression was significantly positively correlated to capsule strain (p < 0.02) and mechanical allodynia (p < 0.02). Spinal EAAC1 expression was significantly negatively related to one of the strain metrics (p < 0.003) and mechanical allodynia at day 7 (p = 0.03). These results suggest that the spinal glutamatergic system may potentiate the persistent behavioral hypersensitivity that is produced following dynamic whiplash-like joint loading; chronic whiplash pain may be alleviated by blocking mGluR5 expression and/or enhancing glutamate transport through the neuronal transporter EAAC1.

Figures

FIG. 1.

Customized facet joint loading device showing the distraction device and set up including the microforceps, motor, LVDT, load cell, and nose cone holder for anesthesia delivery. A surgical microscope was mounted above the device to acquire image data throughout loading while the microforceps coupled to the C6 vertebra was displaced using the motor automated by a LABVIEW program; the microforceps coupled to the C7 vertebra remained stationary.

FIG. 2.

Representative images showing the facet joint and capsule as well as the associated strain field. (A) The C6 and C7 vertebrae were marked by vertebral markers and their initial separation length (xref) defined the joint reference condition. (B) At the peak of capsule stretch (xpeak) C6 was distracted away from C7. Vertebral distraction was calculated as the vector difference between these separations and the maximum tensile strain (not shown) and maximum principal strain in the capsule was calculated using the capsule markers. (C) The maximum principal strain field associated with the images (A, B) is shown.

FIG. 3.

Average forepaw mechanical allodynia as measured by the total number of paw withdrawals on each day for distraction and sham. Higher numbers of withdrawals indicated increased sensitivity. Distraction produced a significantly (p < 0.001) elevated and sustained response compared to sham for each day during the post-operative testing period, for testing with both the 4 and 2 g von Frey filaments. Asterisk (*) indicates significant increase over sham (p < 0.001).

FIG. 4.

(A) Spinal metabotropic glutamate receptor 5 (mGluR5) and (B) excitatory amino acid carrier 1 (EAAC1) expression was significantly modified following facet joint distraction compared to sham. Bands were detected at expected locations based on to their molecular weight. Quantitative analysis using normalization to actin for each specimen showed that mGluR5 was significantly (*p < 0.01) increased in distraction compared to sham. EAAC1 levels were significantly (**p < 0.001) reduced following distraction compared to sham levels. Data are shown as average ± standard deviation.

FIG. 5.

Regression plots showing relationships between the strain metrics (maximum tensile strain and maximum principal strain) in the capsule and each of the behavioral and spinal outcomes. Both strain metrics were positively correlated with total mechanical allodynia (MA) detected using both the (A, B) 4 g and the (C, D) 2 g von Frey filaments. (E, F) Both strain metrics were also positively related to spinal metabotropic glutamate receptor 5 (mGluR5) expression. (G) Maximum tensile strain was significantly negatively correlated to excitatory amino acid carrier 1 (EAAC1), (H) but maximum principal strain was not significantly correlated to spinal EAAC1 expression. The coefficient of determination (R2) for each correlation is displayed on each plot to illustrate the goodness of fit and significant relationships are indicated by asterisks (*p < 0.05; **p < 0.001).

FIG. 6.

Regression plots showing relationships between mechanical allodynia (MA) and each of spinal metabotropic glutamate receptor 5 (mGluR5) and excitatory amino acid carrier 1 (EAAC1) expression at day 7. MA values shown here represent the number of paw withdrawals for each rat on day 7. The level of mGluR5 expression was positively correlated with allodynia measured at day 7 for testing with both (A) the 4 g and (B) the 2 g von Frey filaments. In contrast, spinal EAAC1 expression was negatively correlated with allodynia at day 7 for both (C) the 4 g and (D) the 2 g filament. The coefficient of determination (R2) for each correlation is shown to illustrate the goodness of fit and significant relationships are indicated by asterisks (*p < 0.03; **p = 0.001).