Inhibition of Microglial Activation in the Amygdala Reverses Stress-Induced Abdominal Pain in the Male Rat

Abstract

Background & aims: Psychological stress is a trigger for the development of irritable bowel syndrome and associated symptoms including abdominal pain. Although irritable bowel syndrome patients show increased activation in the limbic brain, including the amygdala, the underlying molecular and cellular mechanisms regulating visceral nociception in the central nervous system are incompletely understood. In a rodent model of chronic stress, we explored the role of microglia in the central nucleus of the amygdala (CeA) in controlling visceral sensitivity. Microglia are activated by environmental challenges such as stress, and are able to modify neuronal activity via synaptic remodeling and inflammatory cytokine release. Inflammatory gene expression and microglial activity are regulated negatively by nuclear glucocorticoid receptors (GR), which are suppressed by the stress-activated pain mediator p38 mitogen-activated protein kinases (MAPK).

Methods: Fisher-344 male rats were exposed to water avoidance stress (WAS) for 1 hour per day for 7 days. Microglia morphology and the expression of phospho-p38 MAPK and GR were analyzed via immunofluorescence. Microglia-mediated synaptic remodeling was investigated by quantifying the number of postsynaptic density protein 95-positive puncta. Cytokine expression levels in the CeA were assessed via quantitative polymerase chain reaction and a Luminex assay (Bio-Rad, Hercules, CA). Stereotaxic infusion into the CeA of minocycline to inhibit, or fractalkine to activate, microglia was followed by colonic sensitivity measurement via a visceromotor behavioral response to isobaric graded pressures of tonic colorectal distension.

Results: WAS induced microglial deramification in the CeA. Moreover, WAS induced a 3-fold increase in the expression of phospho-p38 and decreased the ratio of nuclear GR in the microglia. The number of microglia-engulfed postsynaptic density protein 95-positive puncta in the CeA was increased 3-fold by WAS, while cytokine levels were unchanged. WAS-induced changes in microglial morphology, microglia-mediated synaptic engulfment in the CeA, and visceral hypersensitivity were reversed by minocycline whereas in stress-naïve rats, fractalkine induced microglial deramification and visceral hypersensitivity.

Conclusions: Our data show that chronic stress induces visceral hypersensitivity in male rats and is associated with microglial p38 MAPK activation, GR dysfunction, and neuronal remodeling in the CeA.

Keywords: Brain–Gut Axis; Chronic Psychological Stress; IBS; Visceral Hypersensitivity.

Graphical abstract

Figure 1

Repeated WAS induced visceral hypersensitivity in rats. (A) The in vivo experimental design. (B) Daily FPO (n = 10 per group, all P < .05). (C) Average FPO over 7 days. (D) Visceral sensitivity was assessed by quantification of abdominal contractions in response to tonic CRD (n = 10 per group). Data are presented as the means ± SD. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

Figure 2

WAS altered microglial morphology in the CeA. (A and B) Iba-1/4′,6-diamidino-2-phenylindole (DAPI) (red/blue) immunofluorescence staining in the CeA of (A) SHAM and (B) WAS animals. (C–F) Enlarged images of representative (C) ramified, (D) primed, (E) reactive, and (F) amoeboid subtypes in panels A and B. Arrows in C and D point to microglial processes. C-F are higher magnification of of C'-F' in panels A and B. (G) Ratio of microglial subtypes in the CeA after exposure to SHAM or WAS. (H) Quantification of microglial cell density in the CeA (n = 7 per group). (I) Ratio of microglial subtypes in the caudate putamen after exposure to SHAM or WAS. (J) Quantification of microglial cell density in the caudate putamen (n = 7 per group). Data are presented as the means ± SD. Scale bars: 20 μm.

Figure 3

WAS induced microglial deramification in the CeA. (A and B) Representative images of microglial from (A) SHAM and (B) WAS showing measurement of soma size (outlined by yellow solid line). (C) Quantification of microglial soma size (n = 7 per group). (D and E) Measurement of microglial process occupied area from (D) SHAM and (E) WAS (green dashed line). (F) Quantification of the microglial process occupied area (n = 7 per group). Data are presented as the means ± SD. Scale bars: 20 μm.

Figure 4

WAS increased microglial phospho-p38 MAPK expression in the CeA. (A–L) Co-labeling of phospho-p38 (green) with Iba-1 (red) in the CeA of (A–F) SHAM and (G–L) WAS animals showing phospho-p38 expression in microglia (arrows) and Iba-1–negative cells (arrowheads). (D–F) Higher magnification of boxed areas in panels A–C. (J–L) Higher magnification of boxed areas in panels G–I. (M) Quantification of phospho-p38 expression (% volume) in Iba-1–positive cells (n = 7 per group). (N–R) Co-labeling of phospho-p38 (green) with Neuronal Nuclei (NeuN) (red) showing phospho-p38 expression in neurons (arrows) and non-neuronal cells (arrowheads). (S) Quantification of phospho-p38 expression (% volume) in neurons. n_SHAM = 6, n_WAS = 7. (T–X) Co-labeling of phospho-p38 (green) with glial fibrillary acidic protein (GFAP) (red) showing phospho-p38 expression in GFAP-negative cells (arrowheads). (Q, R, W, and X) Higher magnification of boxed regions in panels N, P, T, and V, respectively. Data are presented as the means ± SD. Scale bars: 20 μm.

Figure 5

WAS inhibited microglial GR nuclear translocation in the CeA. (A–F) Selected slices from z-stack images show co-labeling of GR (green) with Iba-1 (red) in the CeA of (A–C) SHAM and (D–F) WAS animals. The white dashed line indicates the outline of nuclei (4′,6-diamidino-2-phenylindole [DAPI] staining). (G) Ratio of microglial GR expression in the cytoplasm and nucleus (n = 5 per group). Data are presented as the means ± SD. Scale bars: 10 μm.

Figure 6

WAS exposure increased synaptic engulfment by microglia in the CeA. (A–P) Co-labeling of PSD95 (green) with Iba-1 (red) in the CeA of (A–H) SHAM and (I–P) WAS animals. (A–E and I–M) Selected slices from z-stack images indicate PSD95-positive puncta overlapping (arrows) or not overlapping with (arrowheads) Iba-1. The numerals in the lower right corner in each panel indicate the distance (μm) below (-) or above (+) the center focal plate of the z-stack image. (F–H and N–P) Generated maximum intensity projection (MIP) images. (Q) Quantification of PSD95-positive puncta in Iba-1–positive cells. n_SHAM = 6, n_WAS = 7. (R–T) Quantitative PCR analysis showing the relative mRNA expression level of IL1β (n = 10 per group), TNFα (n = 10 per group), and IL6 (n_SHAM = 4, n_WAS = 6) in the CeA. (U–W) IL1β, TNFα, and IL6 concentration in the CeA (n = 6 per group). Data are presented as the means ± SD. Scale bars: 10 μm.

Figure 7

Infusion of minocycline into CeA reversed stress-induced microglial deramification and visceral hypersensitivity. (A) Experimental design. (B) Illustrated image showing the cannula position above the CeA and delivery of vehicle or minocycline into CeA. (C) VMR to CRD in vehicle- (VEH) and minocycline- (MC) infused animals after WAS. n_WAS+VEH = 6 per group, n_WAS+MC = 10 per group. (D) Ratio of microglial subtypes in the CeA of vehicle- or minocycline-infused animals after exposure to WAS. (E–J) Co-labeling of phospho-p38 (green) and Iba-1 (red) in the CeA showing phospho-p38 expression in Iba-1–positive (arrows) and Iba-1–negative (arrowheads) cells after infusion of (E–G) vehicle- and (H–J) minocycline. (K) Quantification of phospho-p38 expression (% volume) in microglia (n = 5 per group). (L) Ratio of GR expression in the cytoplasm and nucleus after vehicle or minocycline infusion (n = 5 per group). (M) Quantification of PSD95-positive puncta in Iba-1–positive cells in the CeA (n = 5 per group) (N) Representative images of apoptosis assay in the CeA of SHAM and WAS animals (n = 5/group). Data are presented as the means ± SD. Scale bars: 50 μm.

Figure 8

Infusion of fractalkine into CeA-induced microglial deramification and visceral hypersensitivity in stress-naïve animals (n = 6 per group). (A) The experimental design. (B) VMR to CRD of vehicle- or fractalkine- (FNK) infused animals after exposure to SHAM. (C) Ratio of microglial subtypes in the CeA. (D) Quantification of phospho-p38 expression (volume %) in microglia in the CeA. (E–J) Representative immunofluorescence images of phospho-p38 (green) co-labeling with Iba-1 (red) showing phospho-p38 expression in Iba-1–positive (arrows) and Iba-1–negative (arrowheads) cells after infusion of (E–G) vehicle or (H–J) fractalkine. Data are presented as the means ± SD. Scale bars: 50 μm. VEH, vehicle.