Experimental Cancer Cachexia Changes Neuron Numbers and Peptide Levels in the Intestine: Partial Protective Effects after Dietary Supplementation with L-Glutamine

<div><p>Gastrointestinal dysmotility frequently occurs in cancer cachexia and may result from damage to enteric innervation caused by oxidative stress, especially due to glutathione depletion. We assessed the effect of dietary supplementation with 20 g/kg l-glutamine (a glutathione precursor) on the intrinsic innervation of the enteric nervous system in healthy and Walker 256 tumor-bearing Wistar rats during the development of experimental cachexia (14 days), in comparison with non-supplemented rats, by using immunohistochemical methods and Western blotting. The total neural population and cholinergic subpopulation densities in the myenteric plexus, as well as the total population and VIPergic subpopulation in the submucosal plexus of the jejunum and ileum, were reduced in cachectic rats, resulting in adaptive morphometric alterations and an increase in vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) expression, suggesting a neuroplastic response. l-glutamine supplementation prevented decrease in myenteric neuronal density in the ileum, morphometric alterations in the neurons and nerve fibers (in both the plexuses of the jejunum and ileum), and the overexpression of VIP and CGRP. Cancer cachexia severely affected the intrinsic innervation of the jejunum and ileum to various degrees and this injury seems to be associated with adaptive neural plasticity. l-glutamine supplementation presented partial protective effects on the enteric innervation against cancer cachexia, possibly by attenuating oxidative stress.</p></div

Morphometric analysis of CGRP-immunoreactive varicosities areas (μm²) in the submucosal plexus in the jejunum and Ileum from the following groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG).

<p>Morphometric analysis of CGRP-immunoreactive varicosities areas (μm²) in the submucosal plexus in the jejunum and Ileum from the following groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG).</p

Effect of supplementation of L-glutamine and cachexia on proteins (CHAT, VIP and CGRP) expression from jejunal and ileal samples of each experimental group.

<p>Groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG). (a) Jejunum CHAT expression and representative bands (on the top). (b) Ileum CHAT expression and representative bands (on the top). (c) Jejunum VIP expression and representative bands (on the top). (d) Ileum VIP expression and representative bands (on the top). (e) Jejunum CGRP expression and representative bands (on the top). (f) Ileum CGRP expression and representative bands (on the top). Bars represent means ± SEM of samples from four animals. The data are presented as percentage arbitrary units (% of control) after normalization (GAPDH). * indicates significant difference (<i>p</i> < 0.05) versus control group.</p

Neuronal morphometric analysis of submucosal plexus.

<p>Neuronal cell body area (μm²) of jejunum and ileum from groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG).</p

Neuronal density of myenteric neurons (neurons/cm²) in the jejunum and ileum.

<p>Experimental groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG). HuC/D-IR population (neurons/cm²), CHAT-immunoreactive subpopulation (neurons/cm²).</p

Average area of the small intestine and correction factors applied for the of neuronal density correction in the myenteric and submucosal plexuses of the jejunum and ileum.

<p>Experimental groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG). n = 8 rats per group.</p

Physiological parameters assessed in experimental groups.

<p>Final weight body (FW), food intake (FI), tumor mass (TM), weight variation (WV). Experimental groups: control (C), control supplemented with L-glutamine (CG), Walker 256 tumor (TW) and Walker 256 tumor supplemented with L-glutamine (TWG). n = 8 rats per group.</p

Representative images of the myenteric varicosities nerve fibers.

<p>Myenteric VIP-IR and CGRP-IR varicosities of the jejunum: (a-d) VIP-IR nerve fiber and (i-l) CGRP-IR nerve fiber. Ileum: (e-h) VIP-IR nerve fiber and (m-p) CGRP-IR nerve fiber. Experimental groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG). (q-x) representative images of the submucous CGRP-IR varicosities nerve fibers of the jejunum (q-t) and ileum (u-x) from experimental groups (C, CG, TW and TWG). All enlarge images on the top right of each image with white arrows indicate examples of immunoreactive varicosity (a'-x'). Scale Bar 25 μm.</p

Characteristics of the primary and secondary antibodies used in immunoreactions.

<p>Characteristics of the primary and secondary antibodies used in immunoreactions.</p