Role of Enteric Glia as Bridging Element between Gut Inflammation and Visceral Pain Consolidation during Acute Colitis in Rats

Abstract

Acute inflammation is particularly relevant in the pathogenesis of visceral hypersensitivity associated with inflammatory bowel diseases. Glia within the enteric nervous system, as well as within the central nervous system, contributes to neuroplasticity during inflammation, but whether enteric glia has the potential to modify visceral sensitivity following colitis is still unknown. This work aimed to investigate the occurrence of changes in the neuron-glial networks controlling visceral perception along the gut-brain axis during colitis, and to assess the effects of peripheral glial manipulation. Enteric glia activity was altered by the poison fluorocitrate (FC; 10 µmol kg^-1 i.p.) before inducing colitis in animals (2,4-dinitrobenzenesulfonic acid, DNBS; 30 mg in 0.25 mL EtOH 50%), and visceral sensitivity, colon damage, and glia activation along the pain pathway were studied. FC injection significantly reduced the visceral hyperalgesia, the histological damage, and the immune activation caused by DNBS. Intestinal inflammation is associated with a parallel overexpression of TRPV1 and S100β along the gut-brain axis (colonic myenteric plexuses, dorsal root ganglion, and periaqueductal grey area). This effect was prevented by FC. Peripheral glia activity modulation emerges as a promising strategy for counteracting visceral pain induced by colitis.

Keywords: S100β; TRPV1 receptors; astrocytes; dorsal root ganglion; enteric glia; inflammatory bowel disease; myenteric plexus; periaqueductal grey matter.

Figure 1

Effect of systemic fluorocitrate administration on the development of visceral pain after DNBS injection. Experimental scheme (A); Abdominal withdrawal reflex (AWR) in response to colorectal distension (B). Each value represents the mean ± SEM of 8 animals per group. ** p < 0.01 vs. vehicle. ^ p < 0.05 and ^^ p < 0.01 vs. DNBS.

Figure 2

Effect of systemic fluorocitrate administration on colon damage induced by DNBS. (A) Colon macroscopic damage score. (B) Representative pictures of haematoxylin–eosin-stained sections of full-thickness colon. Original magnification: 10× and 40×. Each value represents the mean ± SEM of 8 animals per group. ** p < 0.01 vs. vehicle. ^^ p < 0.01 vs. DNBS. Original magnification: 10× and 40×. Scale bars: 50 and 100 μm.

Figure 3

Effect of systemic fluorocitrate administration on submucosal MCs increase induced by DNBS. The panel shows pictures captured from submucosa of MCs stained with antibody to tryptase (A) or with GIEMSA (B). Column graphs display the mean values of MC density per area of colonic wall (cells/field) ± S.E.M. obtained from 6 animals for each group. * p < 0.05 and ** p < 0.01 vs. vehicle. ^ p < 0.05 vs. DNBS. Original magnification: 40×. Scale bar: 50 μm.

Figure 4

Effect of systemic fluorocitrate administration on submucosal eosinophils and activated macrophages increase induced by DNBS. The panel shows pictures captured from submucosa of eosinophils stained with GIEMSA (A) or foamy macrophages naturally stained by their yellow-brown granules (B). Column graphs display the mean values of eosinophil or macrophage density per area of colonic wall (cells/field) ± S.E.M. obtained from 6 animals for each group. ** p < 0.01 vs. vehicle. ^ p < 0.05 vs. DNBS. Original magnification: 40×. Scale bar: 50 μm.

Figure 5

Effect of systemic fluorocitrate on S100β and TRPV1 increased expression within the colonic myenteric plexus of DNBS rats at day 7. (A) Immunofluorescence images show the expression of PLP1 (red), S100β (purple), and TRPV1 (green) in the myenteric plexus of the colon and (B,C) relative immunolabeling quantification at day 7 after colitis induction. Data were analysed by one-way ANOVA and Bonferroni post-hoc. Results are expressed as a mean ± SEM of the percentage of PLP1 immunopositive cells that co-express S100β per area unit (μm²) of n assessments. Results about TRPV1 expression are expressed as average relative fluorescence units (RFUs) ± SEM per area unit of (μm²) of n assessments. **** p < 0.0001 vs. vehicle, ^^^ p < 0.001 and ^^ p < 0.01 vs. DNBS. Original magnification: 20×. Scale bar: 50 μm.

Figure 6

Effect of systemic fluorocitrate on S100β and TRPV1 increased expression in the DRG of DNBS rats at day 7. (A) Representative images show the expression of PLP1 (red), S100β (purple), and TRPV1 (green) in the DRG and (B,C) relative immunolabeling quantification at day 7 after colitis induction. Data were analysed by one-way ANOVA and Bonferroni post-hoc. Results are expressed as a mean ± SEM of the percentage of PLP1 immunopositive cells that express S100β per area unit (μm²) of n assessments. Results about TRPV1 expression are expressed as average relative fluorescence units (RFUs) ± SEM per area unit of (μm²) of n assessments. **** p < 0.0001 vs. vehicle and ^^ p < 0.01 vs. DNBS. Original magnification: 10×. Scale bar: 80 μm.

Figure 7

Effect of systemic fluorocitrate on S100β and TRPV1 increased expression in the periaqueductal grey area of DNBS rats at day 7 (A) Representative images show the expression of PLP1 (red), S100β (purple), and TRPV1 (green) in the periaqueductal grey area and (B,C) relative immunolabeling quantification at day 7 after colitis induction. Data were analysed by one-way ANOVA and Bonferroni post-hoc. Results are expressed as a mean ± SEM of the percentage of PLP1 immunopositive cells that express S100β per area unit (μm²) of n assessments. Results about TRPV1 expression are expressed as average relative fluorescence units (RFUs) ± SEM per area unit of (μm²) of n assessments. *** p < 0.001 vs. vehicle and ^ p < 0.1 vs. DNBS. Original magnification: 4×, 10×, and 40×. Scale bars: 5 and 40 μm.