Korean J Physiol Pharmacol.  2012 Feb;16(1):43-48. 10.4196/kjpp.2012.16.1.43.

Ghrelin Protects Spinal Cord Motoneurons Against Chronic Glutamate Excitotoxicity by Inhibiting Microglial Activation

Affiliations
  • 1Department of Pharmacology and Medical Research Center for Bioreaction to ROS and Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul 130-701, Korea. sjpark@khu.ac.kr

Abstract

Glutamate excitotoxicity is emerging as a contributor to degeneration of spinal cord motoneurons in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin protects motoneurons against chronic glutamate excitotoxicity through the activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3beta pathways. Previous studies suggest that activated microglia actively participate in the pathogenesis of ALS motoneuron degeneration. However, it is still unknown whether ghrelin exerts its protective effect on motoneurons via inhibition of microglial activation. In this study, we investigate organotypic spinal cord cultures (OSCCs) exposed to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration, to determine if ghrelin prevents microglial activation. Exposure of OSCCs to THA for 3 weeks produced typical motoneuron death, and treatment of ghrelin significantly attenuated THA-induced motoneuron loss, as previously reported. Ghrelin prevented THA-induced microglial activation in the spinal cord and the expression of pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta. Our data indicate that ghrelin may act as a survival factor for motoneurons by functioning as a microglia-deactivating factor and suggest that ghrelin may have therapeutic potential for the treatment of ALS and other neurodegenerative disorders where inflammatory responses play a critical role.

Keyword

Ghrelin; Neuroinflammation; Microglial activation; Excitotoxicity; Motoneuron

MeSH Terms

Amyotrophic Lateral Sclerosis
Cytokines
Ghrelin
Glutamic Acid
Interleukin-1beta
Microglia
Neurodegenerative Diseases
Phosphotransferases
Spinal Cord
Tacrine
Tumor Necrosis Factor-alpha
Cytokines
Ghrelin
Glutamic Acid
Interleukin-1beta
Phosphotransferases
Tacrine
Tumor Necrosis Factor-alpha

Figure

  • Fig. 1 Ghrelin protects spinal cord motoneurons against THA-induced chronic glutamate excitotoxicity. Spinal cord cultures were treated for 3 weeks with 100 µM THA. Either vehicle or ghrelin (100 nM) was added to the culture medium at the same time as THA. Motoneurons were identified by SMI-32 immunostaining and on the basis of their morphology, size and location. Representative micrographs of SMI-32-(+) motoneurons in the ventral horn of the spinal cord are shown. Scale bar represents 100 µm. Values are the mean±SEM of at least 15 sections per treatment. Each experiment was repeated three times. *p<0.05 vs. control cultures; †p<0.05 vs. THA-insulted, vehicle-treated cultures.

  • Fig. 2 Ghrelin suppresses THA-induced microglial activation in organotypic spinal cord cultures. Spinal cord cultures were treated for 1 to 3 weeks with 100 µM THA. Either vehicle or ghrelin (100 nM) was added to the culture medium at the same time as THA. (A) The intensity of Mac-1 immunostaining was measured and presented as percent of control (mean±SEM). (B) Representative photomicrographs of Mac-1 immunostaining in each group are shown. Scale bar represents 100 µm. Each experiment was repeated three times. *p<0.05 vs. control cultures; †p<0.05 vs. THA-insulted, vehicle-treated cultures.

  • Fig. 3 Ghrelin inhibits the production of TNF-α from activated microglia in organotypic spinal cord cultures. Spinal cord cultures were treated for 1 to 3 weeks with 100 µM THA. Either vehicle or ghrelin (100 nM) was added to the culture medium at the same time as THA. (A) Levels of TNF-α were determined in the media by ELISA. Values are the mean±SEM. (B) Representative photomicrographs of double immunofluorescence with anti-Mac-1 and anti-TNF-α after 2-week exposure to THA. Scale bar represents 100 µm. Each experiment was repeated three times. *p<0.05 vs. control cultures; †p<0.05 vs. THA-insulted, vehicle-treated cultures.

  • Fig. 4 Ghrelin inhibits the production of IL-1β from activated microglia in organotypic spinal cord cultures. Spinal cord cultures were treated for 1 to 3 weeks with 100 µM THA. Either vehicle or ghrelin (100 nM) was added to the culture medium at the same time as THA. (A) Levels of IL-1β were determined in the media by ELISA. Values are the mean±SEM. (B) Representative photomicrographs of double immunofluorescence with anti-Mac-1 and anti-IL-1β after 2-week exposure to THA. Scale bar represents 100 µm. Each experiment was repeated three times. *p<0.05 vs. control cultures; †p<0.05 vs. THA-insulted, vehicle-treated cultures.


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