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. 2011 Dec;106(6):2838-48.
doi: 10.1152/jn.00183.2011. Epub 2011 Aug 24.

CCL2 has similar excitatory effects to TNF-α in a subgroup of inflamed C-fiber axons

Natalie Richards  1 , Thomas Batty, Andrew Dilley
Affiliations

CCL2 has similar excitatory effects to TNF-α in a subgroup of inflamed C-fiber axons

Natalie Richards et al. J Neurophysiol. 2011 Dec.

Abstract

Peripheral nerve inflammation can cause neuronal excitability changes that have been implicated in the pathogenesis of chronic pain. Although the neuroimmune interactions that lead to such physiological changes are unclear, in vitro studies suggest that the chemokine CCL2 may be involved. This in vivo study examines the effects of CCL2 on untreated and inflamed neurons and compares its effects with those of TNF-α. Extracellular recordings were performed in the anesthetized rat on isolated neurons with C-fiber axons. On untreated neurons, CCL2, as well as TNF-α, had negligible effects. Following neuritis, both cytokines transiently caused the firing of action potentials in 27-30% of neurons, which were either silent or had background (ongoing) activity. The neurons with ongoing activity, which responded to either cytokine, had significantly slower baseline firing rates {median = 3.0 spikes/min [interquartile range (IQR) 3.0]} compared with the nonresponders [median = 24.4 spikes/min (IQR 24.6); P < 0.001]. In an additional group, 26-27% of neurons, which were sensitized due to repeated noxious mechanical stimulation of the periphery, also responded to the effects of both cytokines. Neither cytokine caused axons to become mechanically sensitive. Immunohistochemistry confirmed that the cognate CCL2 receptor, CCR2, is mainly expressed on glia and is therefore not likely to be an axonal target for CCL2 following inflammation. In contrast, the cognate TNF-α receptor (TNFR), TNFR1, was present on untreated and inflamed neurons. In summary, CCL2 can excite inflamed C-fiber neurons with similar effects to TNF-α, although the underlying mechanisms may be different. The modulatory effects of both cytokines are limited to a subgroup of neurons, which may be subtly inflamed.

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Figures

Fig. 1.
Fig. 1.
Schematic diagram of the experimental setup for (A) paradigm 1 and (B) paradigm 2. Each diagram shows the sciatic nerve in the thigh with the trifurcation behind the knee. The recording site at the lumbar (L)5 dorsal root ganglion (DRG) is also shown (Rec). A: for paradigm 1, a well was used to apply treatments to the lesion site or equivalent. To confirm that recorded axons passed through the treatment site, the nerve was stimulated distally (Stim). The 3 terminal branches were crushed to prevent movement artefacts (Crush). B: for paradigm 2, the sciatic nerve was positioned on a plastic platform notched to support the nerve. Treatments were applied using absorbable cotton positioned around the nerve on the platform (gray area). The nerve was electrically stimulated at the L5 DRG (Stim). C: a typical C-fiber axon with axonal mechanical sensitivity recorded from the neuritis group. The short horizontal lines above the trace represent the duration of the mechanical stimulation of the nerve. D: electrical collision of an action potential. Five consecutive traces were triggered by electrical stimulation. In trace 3, an action potential was also elicited by mechanically stimulating the neuron's receptive field (marked *). In this trace, the electrically stimulated action potential (marked **) was delayed, because it fell within the relative refractory period of the previous action potential.
Fig. 2.
Fig. 2.
Percentages of neurons responding to BSA saline, CCL2, and TNF-α application in the untreated and neuritis groups. Higher proportions of neurons responded to the cytokines in the neuritis group vs. BSA saline (*P < 0.05, Fisher's exact test). The total number of neurons sampled and the number that is responsive are shown.
Fig. 3.
Fig. 3.
A–D: typical responses of neuritis-treated C-fiber axons to CCL2 and TNF-α application (A: CCL2 silent; B: CCL2 ongoing; C: TNF-α silent; D: TNF-α ongoing). Horizontal lines above traces represent the exposure period to BSA saline and CCL2/TNF-α. E: interspike-interval plot for a C-fiber axon with baseline ongoing activity that shows the transient response to CCL2 application.
Fig. 4.
Fig. 4.
Percentages of neurons responding to cytokine treatment (CCL2 and TNF-α combined) in the neuritis group based on conduction velocity. As conduction velocity increases, the proportion of responders increases. The total number of neurons sampled and the number that is responsive are shown.
Fig. 5.
Fig. 5.
A: cumulative plot showing the time from start of the experiment, at which neurons with baseline ongoing activity were identified in the untreated group with peripheral field searches (paradigm 2; n = 30 neurons) and the neuritis group (paradigm 1; n = 36 neurons). Time points represent 30-min bins. Dotted lines represent the time from setup, at which 50% of ongoing neurons was identified, which was significantly earlier in the neuritis (50% neurons identified by 118 min) group compared with the untreated group (50% of neurons identified by 151 min); *P < 0.05, Mann-Whitney test. B: percentage of neurons (silent and ongoing) recorded early (<151-min postsetup) and late (>151 min) in the untreated group with peripheral field searches (paradigm 2) that responded to cytokine treatment (CCL2 and TNF-α combined). Significantly more late neurons responded compared with early neurons (*P < 0.05, Fisher's exact test). The total number of neurons sampled and the number that is responsive are shown.
Fig. 6.
Fig. 6.
A–D: CCR2 and TNF-α receptor 1 (TNFR1) immunofluorescent labeling of DRG cell bodies in the untreated and neuritis groups. A: CCR2 untreated; B: CCR2 neuritis; C: TNFR1 untreated; D: TNFR1 neuritis. The nuclei of 4′,6-diamidino-2-phenylindole-positive neuronal cells appear as large, circular, pale-blue structures compared with the smaller, elongated, brighter-blue nuclei of the satellite cells. The CCR2- and TNFR1-positive cells appear bright green (see arrows). Original scale bars = 50 μm. E: summary of CCR2 and TNFR1 expression in DRG cell bodies. There was a significant decrease in CCR2 expression in the neuritis compared with the untreated group (**P < 0.01, χ2 test). Error bars represent SE.
Fig. 7.
Fig. 7.
CCR2 and TNFR1 immunolabeling of the sciatic nerve in the untreated and neuritis group. Sciatic nerve from an untreated (A) and neuritis animal (B), showing CCR2 staining of Schwann cells and associated myelin (arrows in A and B). Note increase in staining following neuritis. Sciatic nerve from an untreated (C and D) and neuritis animal (E), showing TNFR1 labeling of axons (arrows in D and E), endothelial cells, and the perineurium (arrow in C). Original scale bars = 20 μm for A–C and E and 10 μm for D. A–C and E: transverse sections; D: longitudinal section.
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