Neuroprotective effects of ghrelin in cuprizone-induced rat model of multiple sclerosis

Abstract

Multiple sclerosis (MS) is an inflammatory central nervous system disease characterized by demyelination and axonal loss and is the main cause of non-traumatic neurological disability in young adults. Although there are several treatment approaches to manage the disease, there is no definitive cure for multiple sclerosis. Inflammation and oxidative stress are known to play important roles in the pathophysiology of MS. Ghrelin, a peptide secreted by the stomach, is reported to have neuroprotective properties through several pathways, including attenuating oxidative stress and inflammation. In the present study cuprizone (CPZ)-induced model of MS was used in Wistar albino rats to study the possible anti-inflammatory, antioxidant and neuroprotective effects of ghrelin. Rats were randomly divided into six groups: Control groups (Control₃₅ and Control-S₄₂), demyelination group, remyelination group, remyelination + ghrelin (20 µg/kg) group and remyelination + ghrelin (40 µg/kg) group. Y maze test was performed on the rats on their last day of the experiment. Oxidative stress and inflammatory parameters were investigated in brain using commercial kits by enzyme-linked immunosorbent assay (ELISA). Luxol fast blue (LFB) and hematoxylen&eosin (H&E) staining were performed in brain tissues. CPZ leads to a significant decrease in glutathione peroxidase (GSH-Px) levels and myelin content and a significant increase in malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-ɑ), interleukin- 6 (IL- 6) levels, the number of lymphatic cells and inflammatory cells. A significant increase in the antioxidant parameter levels and a significant decrease in MDA levels were found in the ghrelin treated groups (p < 0.05). CPZ leads to irregular, fragmented, demyelinating nerve fibers. A more significant remyelination was observed in the ghrelin treated groups compared to the other groups (p < 0.05). In conclusion, ghrelin treatment showed neuroprotective and antioxidant properties and reduced demyelination in the CPZ-induced rat model of multiple sclerosis.

Keywords: Demyelination; Ghrelin; Inflammation; Neuroprotection; Oxidative stress; Remyelination.

Fig. 1

Experimental procedure. (Standard diet: Bayramoglu Feed and Flour Industry Trade Inc., Turkey)

Fig. 2

Alterations in inflammatory and oxidative stress parameters in control (Control₃₅ and Control-Saline₄₂) and treatment groups (demyelination, remyelination, remyelination + ghrelin 20 and remyelination + ghrelin 40) in a cuprizone-induced multiple sclerosis rat model (n = 8). A) SOD, B) GSH-Px, C) CAT, (D) MDA, (E) IL- 6, (F) TNF-α. *p ≤ 0.05 is significant according to the other group (Mean ± SEM). (DM: demyelination, RM: remyelination, G: ghrelin, S: saline, SOD: superoxide dismutase, GSH-Px: glutathione peroxidase, CAT: catalase, MDA: malondialdehyde, IL- 6: interleukin- 6, TNF-α: tumor necrosis factor-alpha)

Fig. 3

Percentages (%) of change ​​in biochemical parameters in ghrelin treated groups (RM-G20 and RM-G40), according to RM (control) and Control-S₄₂ groups (n = 8). (RM: remyelination, G: ghrelin, S: saline, SOD: superoxide dismutase, GSH-Px: glutathione peroxidase, CAT: catalase, MDA: malondialdehyde, IL- 6: interleukin- 6, TNF-α: tumor necrosis factor-alpha)

Fig. 4

Histological alterations in control (Control₃₅ and Control-Saline₄₂) and treatment groups (demyelination, remyelination, remyelination + ghrelin 20 and remyelination + ghrelin 40) in a cuprizone-induced multiple sclerosis rat model (n = 8). (A) General histological organization of brain cortical areas. Black arrows indicate lymphatic infiltration (Scale bar: 200 μm and 100 μm). (B) Bar graph of inflammation score. (C) Arrangement of nerve bundles in the corpus callosum. Black arrowheads indicate disorganized and fragmented demyelinated nerve fibers, and asterisks indicate areas of vacuolization (Scale bar: 100 μm and 50 μm). (D) Bar graph of demyelination score. (p ≤ 0.05 *; p ≤ 0.01 **; p ≤ 0.001 ***; p ≤ 0.0001 ****) (DM: demyelination, RM: remyelination, G: ghrelin)

Fig. 5

Alterations in myelin content in control (Control₃₅ and Control-Saline₄₂) and treatment groups (demyelination, remyelination, remyelination + ghrelin 20 and remyelination + ghrelin 40) in a cuprizone-induced multiple sclerosis rat model (n = 8). (A) Myelin content (blue) of corpus callosum was visualized using LFB staining. (Scale bar: 100 μm and 50 μm). (B) Bar graph of myelin staining intensity in the corpus callosum (p ≤ 0.05 *; p ≤ 0.01 **; p ≤ 0.001 ***; p ≤ 0.0001 ****) (DM: demyelination, RM: remyelination, G: ghrelin)