Comprehensive gene expression profiling in the prefrontal cortex links immune activation and neutrophil infiltration to antinociception

Abstract

Functional neuroimaging studies have implicated the prefrontal cortex (PFCTX) in descending modulation of pain and the placebo effect. This study was performed to elucidate comprehensive PFCTX gene expression in an animal model of persistent trigeminal pain. Adult male C57BL/6J mice received facial carrageenan injection and showed sustained increase in nociceptive responses. Microarray analyses of differentially expressed genes in the PFCTX at 3 d after injection showed "immune system process" as the dominant ontology term and increased mRNA expression of S100a8, S100a9, Lcn2, Il2rg, Fcgr1, Fcgr2b, C1qb, Ptprc, Ccl12, and Cd52 were verified by RT-PCR. Upregulation of S100A8, S100A9, and lipocalin 2 (LCN2) were confirmed by Western blots, and cells in the PFCTX were double immunolabeled with MPO, indicating they were neutrophils. Analyses of blood of facial carrageenan-injected mice also showed increased mRNA expression of these markers, suggesting transmigration of activated neutrophils into the brain. Other immune-related genes, Il2rg, Fcgr2b, C1qb, Ptprc, and Ccl12 were upregulated in the PFCTX but not blood. Approximately 70% of S100A9-positive cells in the PFCTX of carrageenan-injected mice were located in capillaries adherent to endothelial cells, whereas 30% were within the brain parenchyma. Carrageenan-injected mice showed significantly reduced nociceptive responses after injection of C terminus of murine S100A9 protein in the lateral ventricles and PFCTX but not somatosensory barrel cortex. Together, these findings demonstrate activation of immune-related genes in the PFCTX during inflammatory pain and highlight an exciting role of neutrophils in linking peripheral inflammation with immune activation of the PFCTX and antinociception.

Figure 1.

Responses to von Frey hair stimulation of the face after facial carrageenan injection. A, Left-sided facial carrageenan injection. B, Right-sided facial carrageenan injection. The y-axis represents number of responses to von Frey hair stimulation of the carrageenan-injected areas of the face. BI, Before injection; 1D, 2D, 3D, 1, 2, and 3 d after facial carrageenan injection; Control, untreated control; Carrageenan, facial carrageenan injection. Analyzed by Student's t test. **p < 0.01; ***p < 0.001.

Figure 2.

Real-time RT-PCR analyses of immune-related genes in (non-perfused) PFCTX of facial carrageenan-injected mice compared with untreated controls. A, Right PFCTX of left facial carrageenan-injected mice. B, Left PFCTX of right facial carrageenan-injected mice. C, Left PFCTX of left facial carrageenan-injected mice. D, Right PFCTX of right facial carrageenan-injected mice. Analyzed by Student's t test. *p <0.05; **p < 0.01; ***p < 0.001.

Figure 3.

Western blot analyses of S100A8, S100A9, and LCN2 in PFCTX homogenates of untreated and 3 d facial post-carrageenan-injected mice. A, The antibody to S100A8 detected a single band at ∼10 kDa in homogenates from facial carrageenan-injected mice but not untreated controls. B, The antibody to S100A9 detected a single band at ∼13 kDa in homogenates from carrageenan-injected mice. C, The antibody to LCN2 detected a single band at ∼ 23 kDa in homogenates from carrageenan-injected mice. No bands were detected in negative controls. D, Quantitation of densitometry readings. Significant increase in the density of detected bands was observed in carrageenan-injected mice compared with untreated controls. Analyzed by Student's t test. *p <0.05; **p < 0.01; ***p < 0.001.

Figure 4.

Immunohistochemical labeling of S100A8, S100A9, or LCN2 in the PFCTX. A–C, S100A8 immunolabeled sections of the PFCTX. Low level of S100A8 labeling is present in the cortex of untreated controls (A), but an increased number of stained cells is present in the PFCTX of facial carrageenan-injected mice (B, arrows). C, Higher-magnification micrograph from a facial carrageenan-injected mouse showed colocalization of S100A8 with MPO, a neutrophil marker (red–green overlay, arrow). D–F, S100A9 immunolabeled sections of the PFCTX. Low level of S100A9 labeling is present in the cortex of untreated controls (D), but an increased number of stained cells is present in the PFCTX of facial carrageenan-injected mice (E, arrows). F, Higher-magnification micrograph from a facial carrageenan-injected mouse showed colocalization of S100A9 with MPO (red–green overlay, arrow). G–I, LCN2 immunolabeled sections of the PFCTX. Low level of LCN2 labeling is present in the cortex of untreated controls (G), but an increased number of stained cells is present in the PFCTX of facial carrageenan-injected mice (H, arrows). I, Higher-magnification micrograph from a facial carrageenan-injected mouse showed colocalization of LCN2 with MPO (red–green overlay, arrow). J, Significantly increased number of S100A8-, S100A9-, and LCN2-expressing cells are detected in the PFCTX of carrageenan-injected mice compared with untreated controls. Analyzed by Student's t test. *p < 0.05; **p < 0.01. The areas examined were 2 × 1.6 mm (3.2 mm²), and the arrows in the figures indicate some of the immunopositive cells. Scale bar: A, B, D, E, G, H, 400 μm; C, F, I, 10 μm.

Figure 5.

Comparison of non-perfused versus perfused PFCTX and results from blood. A, Effects of perfusion on mRNA expression of immune-related genes in the PFCTX after facial carrageenan injection compared with untreated perfused controls. B, Analyses of blood of facial carrageenan-injected versus untreated control mice. Analyzed by one-way ANOVA with Bonferroni's multiple comparison post hoc test. Asterisks indicate significant difference compared with controls: *p <0.05; **p < 0.01; ***p < 0.001.

Figure 6.

Immunohistochemical labeling of S100A9 in the PFCTX of non-perfused versus perfused mice that received facial carrageenan injection 3 d earlier. A, Micrograph of non-perfused PFCTX from facial carrageenan-injected mice showing S100A9 immunolabeled cells (green), localized in capillaries immunostained with vWF (red, arrowhead) or the brain parenchyma (arrow) (B). B, Micrograph of perfused PFCTX from facial carrageenan-injected mice showing S100A9 immunolabeled cells (green), localized in capillaries immunostained with vWF (red, arrowhead) or the brain parenchyma (arrow). C, Number of S100A9-expressing cells in blood vessels or brain parenchyma/unit area (3.2 mm²). No significant difference in number of S100A9-positive cells was found between perfused and non-perfused PFCTX. Scale bar: A, B, 20 μm.

Figure 7.

Responses to von Frey hair stimulation of the face after facial carrageenan injection, followed by bilateral injection of mS100A9p into the lateral ventricles (A), dorsolateral PFCTX (B), or somatosensory cortex (C). The y-axis represents number of responses to von Frey hair stimulation of the carrageenan-injected areas of the face. Mice that received facial carrageenan injection plus intracerebroventricular injection of 2 μg of mS100A9p (per side) in the lateral ventricles or intracortical injection of 1 μg of mS100A9p (per side) in the dorsolateral PFCTX showed significantly reduced nociceptive responses 12 h after injection compared with facial carrageenan plus intracerebroventricular or intracortical saline-injected mice (p < 0.05). In contrast, intracortical injection of 1 μg of mS100A9p (per side) into the somatosensory cortex was ineffective in producing antinociception. U, Untreated mice; C, carrageenan-injected mice; BI, before injection; 1D to 12D, 1–12 d after injection. Analyzed by one-way ANOVA with Bonferroni's multiple comparison post hoc test. *p < 0.05.