Molecular and cellular identification of the immune response in peripheral ganglia following nerve injury

Abstract

Background: Neuroinflammation accompanies neural trauma and most neurological diseases. Axotomy in the peripheral nervous system (PNS) leads to dramatic changes in the injured neuron: the cell body expresses a distinct set of genes known as regeneration-associated genes, the distal axonal segment degenerates and its debris is cleared, and the axons in the proximal segment form growth cones and extend neurites. These processes are orchestrated in part by immune and other non-neuronal cells. Macrophages in ganglia play an integral role in supporting regeneration. Here, we explore further the molecular and cellular components of the injury-induced immune response within peripheral ganglia.

Methods: Adult male wild-type (WT) and Ccr2 ^-/- mice were subjected to a unilateral transection of the sciatic nerve and axotomy of the superior cervical ganglion (SCG). Antibody arrays were used to determine the expression of chemokines and cytokines in the dorsal root ganglion (DRG) and SCG. Flow cytometry and immunohistochemistry were utilized to identify the cellular composition of the injury-induced immune response within ganglia.

Results: Chemokine expression in the ganglia differed 48 h after nerve injury with a large increase in macrophage inflammatory protein-1γ in the SCG but not in the DRG, while C-C class chemokine ligand 2 was highly expressed in both ganglia. Differences between WT and Ccr2 ^-/- mice were also observed with increased C-C class chemokine ligand 6/C10 expression in the WT DRG compared to C-C class chemokine receptor 2 (CCR2)^-/- DRG and increased CXCL5 expression in CCR2^-/- SCG compared to WT. Diminished macrophage accumulation in the DRG and SCG of Ccr2 ^-/- mice was found compared to WT ganglia 7 days after nerve injury. Interestingly, neutrophils were found in the SCG but not in the DRG. Cytokine expression, measured 7 days after injury, differed between ganglion type and genotype. Macrophage activation was assayed by colabeling ganglia with the anti-inflammatory marker CD206 and the macrophage marker CD68, and an almost complete colocalization of the two markers was found in both ganglia.

Conclusions: This study demonstrates both molecular and cellular differences in the nerve injury-induced immune response between DRG and SCG and between WT and Ccr2 ^-/- mice.

Keywords: Axotomy; Dorsal root ganglion (DRG); Macrophage; Neuroinflammation; Neutrophil; Regeneration; Superior cervical ganglion (SCG).

Fig. 1

The axonal injury-induced marker ATF3 is highly upregulated in L4, L5, and L6 DRG and in the SCG 7 days after axotomy. A diagram illustrating the sciatic nerve transection injury and the relative innervation of the sciatic nerve by the individual lumbar DRG (a). WT mice underwent a unilateral transection of the right sciatic nerve and lumbar DRG were removed and stained for the injury marker ATF3 and the neuronal marker HuC/D. The percentage of ATF3⁺HuC/D⁺ neurons were quantified 7 days after injury (b). Representative images of sham-operated (c–f) and axotomized (g–j) DRG for L3 (c, g), L4 (d, h), L5 (e, i), and L6 (f, j) DRG are shown. A diagram illustrating the SCG axotomy and the relative innervation of the external and internal carotid nerves (k). In the SCG diagram, blue represents the preganglionic fibers, the green cell bodies are neurons that project into the ECN, and the purple cell bodies project into the ICN. Mice underwent a unilateral transection of the ECN and ICN of the right SCG. SCG were removed 7 days post injury and stained with ATF3 and HuC/D. The percentage of ATF3⁺ HuC/D⁺ SCG neurons were quantified (l). Representative images of sham-operated (m) and axotomized (n) SCG are shown. The data are presented as the mean ± SEM. DRG images were taken at × 10 magnification. SCG images were taken at × 25 magnification. Scale bar = 50 μm. n = 5 per group. *p < 0.05. **p < 0.001 between groups. ^#p < 0.05. ^##p < 0.001 between sham and axotomy within groups

Fig. 2

Chemokines are differentially expressed in the DRG and SCG 48 h after axotomy. Mice underwent a unilateral transection of the right sciatic nerve and the right ICN and ECN of the SCG. Forty-eight hours after axotomy, the injured and contralateral sham-operated L4/L5 DRG and SCG were collected and flash frozen. Protein was isolated from sham and injured ganglia pooled from five mice and used for measurement of chemokines using a Proteome Profiler Mouse Array kit (g). Representative arrays for WT [85] and Ccr2^−/− (a’–d’) mice for DRG (a, a’, b, b’) and SCG (c, c’, d, d’) are shown. Array images were analyzed by densitometry for integral optical density using ImageJ. The optical density for each cytokine was then normalized to the internal control for each blot. The data are represented as the fold increase in the expression of the injured condition compared to the sham for each genotype for DRG (e) and SCG (f). The spots corresponding to the cytokines CCL2, CXCL5, CCL8/MCP-2, MIP-1γ, and CCL6/C10 are highlighted in the representative arrays based on the template indicating the location of controls and various chemokine capture antibodies (g). N = 3 arrays per group. The data are presented as mean ± SEM. *p < 0.05. **p < 0.001 represents significance of expression relative to sham within genotype. ^#p < 0.05. ^##p < 0.001 represents significance between genotypes

Fig. 3

Macrophage accumulation is significantly diminished in the axotomized DRG of Ccr2^−/− mice compared to WT mice, while satellite glial cells and neutrophils are comparable between genotypes. Three and seven days after sciatic nerve transection, L4, L5 and L6 DRG were dissected and examined using flow cytometry. Both sham and axotomized nerves were analyzed. Gray boxes in a indicate CD11b⁺F4/80⁺ cells. In b, black boxes indicate CD11b⁺Ly6G⁻ cells and gray boxes indicate CD11b⁺Ly6G⁺ cells. Flow cytometric analysis of macrophage populations in the injured DRG (a, b) shows similar percentages of CD11b⁺F4/80⁺ and CD11b⁺Ly6G⁻ cells between genotypes at 3 days (c) and significant attenuation in Ccr2^−/− mice compared to WT mice at 7 days (d) post injury. A significant increase in the percentage of satellite glial cells after axotomy was seen in 3 days in Ccr2^−/− DRG alone, with a trend towards a significant increase over injured WT DRG (e). No differences were observed for CD11b⁺Ly6G⁺ neutrophils after injury in WT mice, while a trend towards a significant increase was observed 3 days after injury in Ccr2^−/− mice (f). Mean ± SEM, two-way ANOVA, Tukey’s post hoc test corrected (within individual time points). *p < 0.05. **p < 0.001. n = 3 mice per genotype per time point

Fig. 4

Macrophage accumulation is attenuated in the axotomized SCG of Ccr2^−/− mice compared to WT mice, while Ccr2^−/− SCG show a larger increase in neutrophil accumulation 3 days after injury, compared to the WT SCG. Three and seven days after axotomy, SCG were dissected and examined using flow cytometry. Both sham and axotomized nerves were analyzed. Gray boxes in a indicate CD11b⁺F4/80⁺ cells. In b, black boxes indicate CD11b⁺Ly6G⁻ cells and gray boxes indicate CD11b⁺Ly6G⁺ cells. Flow cytometric analysis of macrophage populations in the injured SCG (a, b) indicates increased percentages of CD11b⁺F4/80⁺ cells 3 days after axotomy in WT mice alone and a significant increase in CD11b⁺Ly6G⁻ cells in injured WT mice compared to Ccr2^−/− mice (c). Axotomy induced significant increases in macrophages in both genotypes 7 days post injury, with WT mice displaying a larger increase in CD11b⁺Ly6G⁻ cells over Ccr2^−/− mice (d). No changes were observed between genotypes in the satellite glial cell population at either time point (e). Significant increases in CD11b⁺Ly6G⁺ neutrophils were seen in WT and Ccr2^−/− SCG 3 days post injury (f). Neutrophils were more prevalent in the Ccr2^−/− SCG 3 days after axotomy compared to the WT SCG. Mean ± SEM, two-way ANOVA, Tukey’s post hoc test corrected (within individual time points). *p < 0.05. **p < 0.001. n = 3 mice per genotype per time point

Fig. 5

Myeloperoxidase staining of neutrophils in the axotomized SCG 1, 3, and 7 days after axotomy. Immunohistochemical staining of neutrophils with an antibody against MPO showed comparable neutrophil cell counts between WT and Ccr2^−/− SCG 1 and 3 days after injury and a significantly higher number of cells in WT ganglia compared to mutants 7 days after injury. Cell counts were performed using ImageJ at 1, 3, and 7 days post injury (a). The counts represent the sum of cells from three images per section. Representative images of IHC staining in the SCG at 1, 3, and 7 days post axotomy are shown for WT Ax (b, d, f) and Ccr2^−/− Ax (c, e, g). All images were taken at × 25 magnification. Scale bar = 20 μm. n = 5 per group. *p < 0.05. **p < 0.001

Fig. 6

Injury-induced cytokine profiles in the DRG and SCG 7 days after axotomy. Mice underwent a unilateral transection of the right sciatic nerve and the right ICN and ECN of the SCG. Seven days after axotomy, the injured and contralateral sham-operated L4/L5 DRG and SCG were collected and flash frozen. Protein was isolated from sham and injured ganglia pooled from five mice and used for measurement of cytokines using a Proteome Profiler Mouse Array kit (g). Representative arrays for WT [85] and Ccr2^−/− (a’–d’) mice for DRG (a, a’, b, b’) and SCG (c, c’, d, d’) are shown. Array images were analyzed by densitometry for integral optical density using ImageJ. The optical density for each cytokine was then normalized to the internal control for each blot. The data are represented as the fold increase in the expression of the injured condition compared to the sham for each genotype for DRG (e) and SCG (f). The spots corresponding to the cytokines TIMP-1, sICAM-1, C5/C5a, IL-1ra, and IL-16 are highlighted in the representative arrays based on the template indicating the location of controls and various chemokine capture antibodies (g). N = 3 arrays per group. The data are presented as the mean ± SEM. *p < 0.05. **p < 0.001 represents significance of expression relative to sham within genotype. ^#p < 0.05. ^##p < 0.001 represents significance between genotypes

Fig. 7

The anti-inflammatory marker, CD206, is highly expressed in CD68⁺ macrophages in the WT DRG 3 and 7 days after sciatic nerve transection. Immunohistochemical labeling of DRG sections with antibodies against a macrophage marker, CD68, and an anti-inflammatory marker, CD206, shows that a majority of macrophages in the WT DRG at 7 days post injury express CD206 (k). The number of CD68⁺ and CD206⁺ cells was significantly diminished in Ccr2^−/− mice at 3 days (f) and 7 days (k) post injury. Cell counts were performed using ImageJ at 1, 3, and 7 days post injury for CD68⁺-, CD206⁺-, and CD68⁺CD206⁺-colocalized cells (a, f, k). The counts represent the sum of cells from three images per section. Representative images of IHC staining in the L5 DRG at 1, 3, and 7 days post axotomy are shown for WT sham (b, g, l), WT Ax (c, h, m), Ccr2^−/− sham (d, i, n), and Ccr2^−/− Ax (e, j, o). All images were taken at × 25 magnification. Scale bar = 50 μm. n = 5 per group. *p < 0.05. **p < 0.001

Fig. 8

The anti-inflammatory marker, CD206, is highly expressed in CD68⁺ macrophages in the WT SCG at 1, 3, and 7 days after axotomy. Immunohistochemical labeling of SCG sections with antibodies against a macrophage marker, CD68, and an anti-inflammatory marker, CD206, shows that a majority of macrophages in the WT SCG at 3 and 7 days post injury express CD206 (f, k). The number of CD68⁺ and CD206⁺ cells was significantly diminished in Ccr2^−/− mice at 3 and 7 days post injury (f, k). Cell counts were performed using ImageJ at 1, 3, and 7 days post injury for CD68⁺-, CD206⁺-, and CD68⁺CD206⁺-colocalized cells (a, f, k). The counts represent the sum of cells from three images per section. Representative images of IHC staining in the SCG at 1, 3, and 7 days post axotomy are shown for WT sham (b, g, l), WT Ax (c, h, m), Ccr2^−/− sham (d, i, n), and Ccr2^−/− Ax (e, j, o). All images were taken at × 25 magnification. Scale bar = 50 μm. n = 5 per group. *p < 0.05. **p < 0.001