Experimental Autoimmune Encephalomyelitis Influences GH-Axis in Female Rats

Abstract

Inflammation, demyelination, and axonal damage to the central nervous system (CNS) are the hallmarks of multiple sclerosis (MS) and its representative animal model, experimental autoimmune encephalomyelitis (EAE). There is scientific evidence for the involvement of growth hormone (GH) in autoimmune regulation. Previous data on the relationship between the GH/insulin like growth factor-1 (IGF-1) axis and MS/EAE are inconclusive; therefore, the aim of our study was to investigate the changes in the GH axis during acute monophasic EAE. The results show that the gene expression of Ghrh and Sst in the hypothalamus does not change, except for Npy and Agrp, while at the pituitary level the Gh, Ghrhr and Ghr genes are upregulated. Interestingly, the cell volume of somatotropic cells in the pituitary gland remains unchanged at the peak of the disease. We found elevated serum GH levels in association with low IGF-1 concentration and downregulated Ghr and Igf1r expression in the liver, indicating a condition resembling GH resistance. This is likely due to inadequate nutrient intake at the peak of the disease when inflammation in the CNS is greatest. Considering that GH secretion is finely regulated by numerous central and peripheral signals, the involvement of the GH/IGF-1 axis in MS/EAE should be thoroughly investigated for possible future therapeutic strategies, especially with a view to improving EAE disease.

Keywords: EAE; IGF-1; growth hormone; pituitary.

Figure 1

Acute EAE—clinical course and weight loss. Acute EAE is induced via subcutaneous immunization with spinal cord homogenate in complete Freund’s adjuvant. Clinical course of disease is scored daily on a scale of 0 to 5 (Y-axis left, red-round shape). Body weight was presented at Y-axis (right, black-square shape). Clinical signs of disease are usually first observed between days 10 and 15, followed by monophasic disease characterized by severe walking disability, with substantial weight loss followed by spontaneous recovery.

Figure 2

EAE affects relative gene expression of Ghrh, Sst, Npy, and Agrp in the hypothalamus. Levels of target genes are expressed relative to the expression of the Gapdh gene. Expression profiles were analyzed via RT-qPCR from RNA isolated from animals at disease onset (Eo, light gray bars), disease peak (Ep, dark gray bars), disease end (Ee, gray bars), and the control animals (Ctrl, red bars). The data shown are mean ± SEM (n ≥ 5 animals/group), derived from a representative experiment. There are no significant changes in the Ghrh and Sst expression. Npy and Agrp expression increase at the peak of disease. The results were analyzed via one-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05.

Figure 3

EAE upregulates relative gene expression of Gh, Ghrhr and Ghr in the pituitary gland at the peak of the disease. Levels of target genes are expressed relative to the expression of the Gapdh gene. Expression profiles were analyzed via RT-qPCR from RNA isolated from animals at Eo (light gray bars), Ep (dark gray bars), Ee (gray bars), and control animal (Ctrl, red bars). The data shown are mean ± SEM (n ≥ 5 animals/group), derived from a representative experiment. There are significant increases in the expression of these genes at the peak of EAE. The results were analyzed via one-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05.

Figure 4

Immunohistochemistry of GH cells in pituitary sections from EAE rats with supporting quantitative data during the course of EAE. Representative micrographs display GH immunoreactivity in the pituitary gland in healthy (A) control rats (Ctrl, red bar) and animals sacrificed at the (B) onset (Eo, light gray bar), (C) peak (Ep, black bar), and (D) end (Ee, dark gray bar) of the disease. Nuclei are counterstained with hematoxylin. The scale bar applicable to all micrographs is 20 µm. Morphometric parameters were analyzed using stereological analysis of GH cells. The ratio of the pituitary volume (white bars) and volume of GH cells (filled bars) were measured from at least six sections, from seven different animals per EAE phase. Results are given as means ± SEM; analyzed using Kruskal-Wallis followed by Dunn’s multiple comparisons test.

Figure 5

EAE affects Ghr and Igf1r gene expression in the liver. Levels of target genes are expressed relative to the expression of the Gapdh gene. Expression profiles were analyzed via RT-qPCR from RNA isolated from animals at Eo (light gray bars), Ep (dark gray bars), Ee (gray bars), and control animals (Ctrl, red bars). The data shown are mean ± SEM (n ≥ 5 animals/group), derived from a representative experiment. Ghr and Igf1r were down-regulated at the peak of EAE. The results were analyzed via one-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05.