The Function of the Hypothalamic-Pituitary-Adrenal Axis During Experimental Autoimmune Encephalomyelitis: Involvement of Oxidative Stress Mediators

Abstract

Multiple sclerosis (MS) is an inflammatory, demyelinating disease with an unknown origin. Previous studies showed the involvement of the hypothalamic-pituitary-adrenal (HPA) axis to susceptibility to autoimmune diseases, including MS, and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). During MS/EAE, innate immune cells are activated and release cytokines and other inflammatory mediators, leading to a vicious cycle of inflammation. In response to inflammation, the activated HPA axis modulates immune responses via glucocorticoid activity. Because the mechanisms involving oxidative stress to the HPA axis are relatively unrevealed, in this study, we investigate the inflammatory and oxidative stress status of HPA axis during EAE. Our results reveal an upregulation of Pomc gene expression, followed by POMC and ACTH protein increase at the peak of the EAE in the pituitary. Also, prostaglandins are well-known contributors of HPA axis activation, which increases during EAE at the periphery. The upregulated Tnf expression in the pituitary during the peak of EAE occurred. This leads to the activation of oxidative pathways, followed by upregulation of inducible NO synthase expression. The reactive oxidant/nitrosative species (ROS/RNS), such as superoxide anion and NO, increase their levels at the onset and peak of the disease in the pituitary and adrenal glands, returning to control levels at the end of EAE. The corticotrophs in the pituitary increased in number and volume at the peak of EAE that coincides with high lipid peroxidation levels. The expression of MC2R in the adrenal glands increases at the peak of EAE, where strong induction of superoxide anion and malondialdehyde (MDA), reduced total glutathione (GSH) content, and catalase activity occurred at the peak and end of EAE compared with controls. The results obtained from this study may help in understanding the mechanisms and possible pharmacological modulation in MS and demonstrate an effect of oxidative stress exposure in the HPA activation during the course of EAE.

Keywords: cytokines; experimental autoimmune encephalomyelitis; hypothalamic–pituitary–adrenal axis; multiple sclerosis; oxidative stress.

FIGURE 1

(A) Timeline of the experimental design. Rats were immunized with the homogenate of the spinal cord and CFA. The rats were sacrificed at the onset of the first symptom (Eo), peak of disease (Ep), and end of symptoms (Ee). (B) Time course of experimental autoimmune encephalomyelitis (EAE) symptoms and variation in the weight of the animals. Data have been expressed as mean ± SEM of daily measurements of each animal. (C) Inflammation was observed using Hoechst stain (blue), while demyelination was detected using the MBP antibody (green fluorescence).

FIGURE 2

(A) POMC gene expressions were assessed by qRT-PCR in immunized (Eo, Ep, and Ee) and control animals (C–control), with Gapdh as an internal reference standard in the hypothalamus and pituitary. (B) Protein levels of POMC and adrenocorticotropic hormone (ACTH) relative to β-actin in the pituitary were determined using the Western blot method and results are expressed as mean% of control values ± SEM. *p < 0.05; **p < 0.001 compared with the control.

FIGURE 3

Temporal changes in the immunoreactivity of ACTH cells during EAE. Pituitary glands were obtained from rats afflicted with EAE, (A) control rat, and EAE rats sacrificed at onset (B), peak (C), and end of the disease (D). Scale bar 50 μm.

FIGURE 4

Stereological analysis of the temporal changes of ACTH cells. (A) Changes in the volume of the pituitary during EAE. Variation of ACTH secreting cells in the pituitary in volume density (B), number (C), and volume of the single cell (D) in the course of EAE. Pituitary from control – C and EAE rats, obtained from different time points—Eo (onset of the disease), Ep (peak of the disease), and Ee (end of the disease), and used for determination in morphometric parameters. Results are given as means ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 on a corresponding day.

FIGURE 5

Nitrosative/oxidative status assessment in the pituitary gland during EAE. Total nitrites (NOx; μmol/mg protein) were determined to assess nitrosative status. For oxidative status assessment, O₂^– (μmol/min/mg protein), GSH (μmol/mg protein), and MDA (nmol/mg protein) levels and enzymatic activities of GPx (M NADPH/mg protein), GSR (μmol NADPH/mg protein), catalase (U/mg protein), and SOD2 (U/mg protein) were determined in the pituitary gland at the onset (Eo), peak (Ep), and end (Ee) of EAE rats. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 statistically significant difference compared with the control (C) group.

FIGURE 6

Oxidative and lipid peroxidation status assessment in the adrenal glands of rats during EAE. To assess the oxidative status, O₂^– (μmol/min/mg protein), GSH (μmol/mg protein), and MDA (nmol/mg protein) levels were determined and catalase (U/mg protein) and SOD2 (U/mg protein) activities were assayed in the adrenal gland at the onset (Eo), peak (Ep), and end (Ee) of EAE. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 statistically significant difference compared with the control (C) group.