Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root ganglion

Abstract

Neuronal hyperexcitability in both injured and adjacent uninjured neurons is associated with states of chronic injury and pain and is likely subject to neuroinflammatory processes. Chronic inflammatory responses are largely orchestrated by chemokines. One chemokine, monocyte chemoattractant protein-1 (MCP-1), in the presence of its cognate receptor, the beta chemokine receptor 2 (CCR2), produces neural activity in dissociated neuronal cultures of neonatal dorsal root ganglion (DRG) neurons. Using a neuropathic pain model, chronic compression of the DRG (CCD), we compared anatomically separate populations of noncompressed lumbar DRG (L3/L6) with compressed lumbar DRG (L4/L5) for changes in the gene expression of CCR2. In situ hybridization revealed that CCR2 mRNA was up-regulated in neurons and nonneuronal cells present in both compressed L4/L5 and ipsilateral noncompressed L3/L6 DRGs at postoperative day 5 (POD5). The total percentages of compressed and noncompressed neurons exhibiting CCR2 mRNA transcripts in L3, L5, and L6 DRG were 33 +/- 3.5%, 49 +/- 6.2%, and 41 +/- 5.6%, respectively, and included cell bodies of small, medium, and large size. In addition, the preferred CCR2 ligand, MCP-1, was up-regulated by POD5 in both compressed L4/L5 and noncompressed L3/L6 DRG neurons. Application of MCP-1 to the cell bodies of the intact formerly compressed DRG in vitro produced potent excitatory effects not observed in control ganglia. MCP-1/CCR2 signaling is directly involved with a chronic compression injury and may contribute to associated neuronal hyperexcitability and neuropathic pain.

Fig. 1.

CCR2 mRNA expression in compressed and adjacent noncompressed DRG at POD1, POD3, and POD5. (A) Sense riboprobe hybridization signal in representative compressed L5 DRG at POD5. (B and C) Compressed L5 DRG (B) and adjacent noncompressed L6 DRG (C) do not exhibit CCR2 mRNA expression at POD1. (D) Sham-treated L5 DRG expression levels of CCR2 mRNA at POD5. (E and F) Compressed L5 DRG (E) and adjacent noncompressed L6 (F) exhibit only neuronal CCR2 mRNA expression (white arrows) at POD3. (G–I) High levels of CCR2 mRNA are present in predominantly nonneuronal cells and some neurons (white arrows) of adjacent noncompressed L3 (G) and L6 (I) DRG at POD5. Compressed L5 DRG (H) at POD5 also exhibit high levels of CCR2 mRNA expression in predominantly nonneuronal cells, but many more neurons are positive for CCR2 mRNA transcripts (white arrows). Black asterisks indicate nonlabeled neurons (G–I). (Bar, 50 μm.)

Fig. 2.

MCP-1 immunoreactivity and isolectin B4 binding in compressed lumbar DRG 5 (L5) and adjacent noncompressed L6 DRG at POD5 and in compressed L5 ganglia at POD24. (A, D, and G) MCP-1 immunoreactivity (red arrows) in (A) L5 and (D) L6 DRG at POD5, and (G) L5 DRG at POD24. (B, E, and H) Isolectin B4 binding (green arrows) in compressed L5 (B) and adjacent noncompressed L6 DRG (E) at POD5 and in compressed L5 DRG (H) at POD24. (C, F, and I) Merged images of MCP-1 (red arrows) and IB4 (green arrows) and colocalization (yellow arrows) at POD5 (C and F) and POD24 (I). (Bars, 100 μm.)

Fig. 3.

Responses of DRG neurons to MCP-1 applied to somata on the surface of the formerly compressed ganglion at POD5. The neurons were classified visually by somal size as small (A), medium (B and C), or large (D). Each neuron was further classified by axonal conduction velocity (action potential proceeded by artifact electrically evoked from dorsal root, on the left) and by the presence or absence of a “hump” on the falling phase of the action potential causing an extra deflection in the first derivative (arrows, A and B Left Inset). The hump is typical for nociceptive neurons