Changes in hyaluronan deposition in the rat myenteric plexus after experimentally-induced colitis

Abstract

Myenteric plexus alterations hamper gastrointestinal motor function during intestinal inflammation. Hyaluronan (HA), an extracellular matrix glycosaminoglycan involved in inflammatory responses, may play a role in this process. In the colon of control rats, HA-binding protein (HABP), was detected in myenteric neuron soma, perineuronal space and ganglia surfaces. Prominent hyaluronan synthase 2 (HAS2) staining was found in myenteric neuron cytoplasm, suggesting that myenteric neurons produce HA. In the myenteric plexus of rats with 2, 4-dinitrobenzene sulfonic (DNBS)-induced colitis HABP staining was altered in the perineuronal space, while both HABP staining and HA levels increased in the muscularis propria. HAS2 immunopositive myenteric neurons and HAS2 mRNA and protein levels also increased. Overall, these observations suggest that inflammation alters HA distribution and levels in the gut neuromuscular compartment. Such changes may contribute to alterations in the myenteric plexus.

Figure 1

HA staining and levels in the rat colon after DNBS treatment. The immuno-localization of HA was performed on paraffin-embedded tissue sections, colonic whole-mount preparations and primary cultures of myenteric ganglia, using a biotin-labeled HA-binding protein (HABP), which is able to localize HA in tissues by streptavidin conjugation with an appropriate fluorophore. (A) HABP staining in rat colon cross-sections obtained from control (vehicle-treated animals) and DNBS-treated animals and relative negative control, bar 100 µm. M, mucosa; SM, submucosa; Mus, muscolaris propria (B) HABP staining in rat colon LMMP whole-mount preparations from control and DNBS-treated animals and relative negative control, bar 50 µm. LM, longitudinal muscle; MP, myenteric plexus. Arrowhead indicates interconnecting fiber strands in the myenteric plexus. (C) Rat small intestine myenteric ganglion in culture double-stained with HABP and and for the pan-neuronal marker HuC/D. Asterisk indicates neurons displaying HA staining. Bar 50 μm. (D) HA levels measured in colonic submucosal and muscularis propria layers from control (empty bars) and DNBS-treated animals (solid bars). Quantitative analysis of HA was performed by HPLC with fluorimetric detection of derivatized disaccharides obtained after isolation and successive degradation of HA from the submucosal and muscularis propria layers. Values are expressed as mean ± S.E.M. of 4 experiments. *P < 0.05 and ***P < 0.001 vs control animals by Student’s t test.

Figure 2

Co-localization of HA with HUC/D in the colonic myenteric plexus of control and DNBS-treated rats. Confocal images showing co-localization of HABP with HuC/D in control (A–C) and DNBS-treated (G–I) colonic LMMP whole-mounts. In control preparations, HABP faintly stained the soma of myenteric neurons (arrow) and more intensely the perineuronal space contributing (insets, panels D–F). After DNBS-treatment, HABP immunofluorescence was still found in the soma of myenteric neurons (arrow) and the well organized perineuronal staining was significantly altered (insets, J–L). Bar 50 μm.

Figure 3

Absence of co-localization between HABP and the glial marker S100β in the rat colon. (A–C) Confocal images of a colonic LMMP whole-mount preparation double-stained with HABP and S100β. (D–F) Confocal images of rat small intestine myenteric ganglia primary cultures double-stained with HABP and S100β. Bar 50 μm.

Figure 4

HAS2 and HuC/D co-localization in the rat colon myenteric plexus of control and DNBS-treated animals. (A–H) Confocal images showing co-localization of HAS2 with HuC/D in LMMP whole-mount preparations obtained from in control (B–D; arrows indicate myenteric neurons co-staining for HAS2 and HuC/D) and after DNBS-treatment (F–H). (I–N) HAS2 and HABP co-localization in the rat colon myenteric plexus of control and DNBS-treated animals. Co-localization of HAS2 with HABP in LMMP whole-mount preparations of control (I–K) and DNBS-treated animals (L–N). Panels A and E show negative controls for HAS2 staining in control and DNBS-treated preparations, respectively. Bar 50 μm.

Figure 5

HAS2 expression in myenteric ganglia. (A) Percentage of myenteric neurons per mm² co-staining for HuC/D and HAS2, in rat colon obtained from control (vehicle-treated, empty bars) and from DNBS-treated animals (solid bars). Values are expressed as mean ± S.E.M. of 3 experiments. (B) RT-PCR quantification of HAS2 transcripts in control (empty bars) and in DNBS-treated animals (solid bars). Values are mean ± S.E.M. of 8 experiments of the percentage variation of relative gene expression with respect to values obtained in control animals. The relative gene expression was determined by comparing 2^−ΔΔCt values normalized to β-actin. (C) HAS2 protein expression analyzed in lysates fractions of LMMPs obtained from control (empty bars) and DNBS-treated animals (solid bars). Blots representative of immunoreactive bands for HAS2 and β-actin in the different experimental conditions are reported on top of each panel. Samples (100 μg) were electrophoresed in SDS-10% polyacrylamide gels. Numbers at the margins of the blots indicate relative molecular weights of the respective protein in kDa. Values are expressed as mean ± S.E.M. of 4 experiments of the percentage variation of the normalized optical density (O.D.) obtained from DNBS-treated preparations with respect to values obtained in control samples. **P < 0.01 and ***P < 0.001 vs control animals by Student’s t test.