Cancer-induced morphological changes in enteric glial cells in the jejunum of Walker-256 tumor-bearing rats

Abstract

Cancer-induced cachexia is associated with systemic inflammation and gastrointestinal dysfunction. How changes to cells of the enteric nervous system contribute to gut dysfunction in tumor development and cancer cachexia is unknown. Here, we tested the hypothesis that changes to enteric glia, a type of peripheral glia that surround enteric neurons and regulate gut homeostasis, are associated with tumor development and that supplementing with the antioxidant L-glutathione is protective against the changes induced. Immunohistochemistry for neurons, enteric glial cells and immune cells was performed in whole-mount preparations and frozen histological sections of the jejunum from 20 Wistar rats, distributed in 4 groups: control, tumor of Walker-256, control administered with 1 % L-glutathione, and tumor of Walker-256 administered with 1 % L-glutathione. Morphoquantitative analyses were made using Image-Pro® Plus 4.5 and ImageJ® 1.43° software. Tumor development significantly reduced neuronal and glial cell populations in the myenteric and submucosal plexuses and enlarged glial cell body area in the submucosal plexus. In contrast, tumors increased glia in the jejunal mucosa and this effect was accompanied by B-lymphocyte recruitment. GSH-supplemented diet was not sufficient to protect against changes to neurons and glia in the submucosal plexus but was partially protective in the myenteric plexus. L-glutathione had no effect on physiological parameters of cachexia but was sufficient to preserve enteric glial cell density in the myenteric plexus. These results suggest that changes to both enteric neurons and glia likely contribute to the gastrointestinal effects of tumor development and that oxidative stress contributes to these effects in the enteric nervous system.

Keywords: Cachexia; Cancer; Enteric Glial Cells; L-glutathione; Morphology.

Figure 1.

Immunolabeling for analysis of HuC/D-IR neurons and S100-IR enteric glia in the myenteric plexus. Experimental groups: control (C), control administered with 1% GSH (CGT), tumor of Walker-256 (TW), and tumor of Walker-256 administered with 1% GSH (TWGT). Scale bar=50 μm

Figure 2.

Immunolabeling for analysis of HuC/D-IR neurons and S100-IR enteric glia in the submucosal plexus. Experimental groups: control (C), control administered with 1% GSH (CGT), tumor of Walker-256 (TW), and tumor of Walker-256 administered with 1% GSH (TWGT). Scale bar=50 μm

Figure 3.

Quantitative analysis of HuC/D-IR neurons (A, D) and S100-IR enteric glial cells (B, E) expressed in cm² and proportion between enteric glia and neurons (C, F). Experimental groups: control (C), control administered with 1% GSH (CGT), tumor of Walker-256 (TW), and tumor of Walker-256 administered with 1% GSH (TWGT). * indicates a significant difference when compared with C group (p<0.05) and # indicates a significant difference from TW group. n=5 rats per group

Figure 4.

Morphometric analysis of HuC/D-IR neurons (A, C) and S100-IR enteric glia (B, D) expressed in μm². Experimental groups: control (C), control administered with 1% GSH (CGT), tumor of Walker-256 (TW), and tumor of Walker-256 administered with 1% GSH (TWGT). * indicates a significant difference when compared with C group (p<0.05) and # indicates a significant difference from TW group. n=5 rats per group.

Figure 5.

Frequency of distribution of morphometric values of HuC/D-IR neurons (A, C) and S100-IR enteric glia (B, D) expressed in μm². Graphics A and B demonstrate data from the myenteric plexus and graphics C and D show data from the submucosal plexus. Experimental groups: control (C), control administered with 1% GSH (CGT), tumor of Walker-256 (TW), and tumor of Walker-256 administered with 1% GSH (TWGT). n=5 rats per group

Figure 6.

Quantification of area occupied by GFAP-IR intermediate filaments (A) and representative immunostaining for GFAP-IR filaments (B) in the myenteric plexus. Experimental groups: control (C), control administered with 1% GSH (CGT), tumor of Walker-256 (TW), and tumor of Walker-256 administered with 1% GSH (TWGT). Scale bar=20 μm; n=5 rats per group

Figure 7.

Density of GFAP and CD20+ cells in the jejunum mucosa. Results are expressed as mean/villus (A). Representative images of immunolabeling for GFAP in the jejunum mucosa are shown in C. Experimental groups: control (C), control administered with 1% GSH (CGT), tumor of Walker-256 (TW), and tumor of Walker-256 administered with 1% GSH (TWGT). * indicates a significant difference when compared with C group (p < 0.05) and # indicates a significant difference from TW group (p<0.05). n=5 rats per group.