Anat Cell Biol.  2010 Sep;43(3):230-240. 10.5115/acb.2010.43.3.230.

Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain

• Mun CH
• Lee WT
• Park KA
• Lee JE

• KMID: 2168878
• DOI: http://doi.org/10.5115/acb.2010.43.3.230

Abstract

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult.

Keyword

Agmatine; endothelial nitric oxide synthase; transient global ischemia; neuroprotection

MeSH Terms

Agmatine
Animals
Arginine
Basement Membrane
Brain
Brain Ischemia
Capillary Permeability
Carboxy-Lyases
Cell Death
Cerebral Cortex
Endothelial Cells
Extracellular Matrix
Glycosaminoglycans
Hippocampus
Ischemia
Matrix Metalloproteinases
Microvessels
Models, Animal
Neurons
Nitric Oxide
Nitric Oxide Synthase Type III
Rats
Reperfusion
Agmatine
Arginine
Carboxy-Lyases
Glycosaminoglycans
Matrix Metalloproteinases
Nitric Oxide
Nitric Oxide Synthase Type III

Figure

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  Fig. 1 Neuronal damages in rat hippocampus (a~f) and cortex (g~i) at 6 (A) and 24 h (B) after transient global ischemia. H-E and TUNEL staining were analyzed for histology, especially in the CA1 region of hippocampus and in the parietal cortex of cerebral cortex. NC, normal control; EC, experimental controls without agmatine treatment; Agm, agmatine treatment groups. Arrows point to necrotic and apoptotic neurons compared with the control. Scale bar=100 µm.

• 

  Fig. 2 Effects of eNOS and iNOS expression by agmatine treatment following transient global ischemia in rat hippocampus. Representative expressional levels of eNOS (A) and iNOS (C) at 6 h after agmatine treatment (100 mg/kg, i.p), and densitometric data (B, D). Data represent means±SD for n=5/NC, n=3/EC and Agm group per each time point. *P<0.05, vs. normal control; †P<0.05, vs. experimental control at same onset of reperfusion. NC, normal control; EC6, at 6 h after reperfusion without agmatine; Agm6, at 6 h after reperfusion with agmatine; EC24, at 24 h after reperfusion without agmatine; Agm24, at 24 h after reperfusion with agmatine.

• 

  Fig. 3 Effects of eNOS and iNOS expression by agmatine treatment following transient global cerebral ischemia in rat cerebral cortex. Representative expressional levels of eNOS (A) and iNOS (C) at 24 h after agmatine treatment (100 mg/kg, i.p), and densitometric data (B, D). Data represent means±SD for n=5/NC, n=3/EC and Agm group per each time point. *P<0.05, vs. normal control; †P<0.05, vs. experimental control at same onset of reperfusion. NC, normal control; EC6, at 6 h after reperfusion without agmatine; Agm6, at 6 h after reperfusion with agmatine; EC24, at 24 h after reperfusion without agmatine; Agm24, at 24 h after reperfusion with agmatine.

• 

  Fig. 4 Photomicrographs of eNOS expression by agmatine treatment (A) and quantification of eNOS positive cells (B, C) in rat brain after global ischemia. Representative expressional levels of eNOS in bregman - 3.8 mm of naïve rats at 6 and 24 h after agmatine treatment (A). The number of eNOS positive cells in the CA 1 sector of hippocampus (B) and in the parietal region of the cerebral cortex (C). Data represent means±SD for n=5/NC, n=3/EC and Agm group per each time point. *P<0.05, vs. normal control; †P<0.05, vs. experimental control at same onset of reperfusion. NC, normal control; EC6, at 6 h after reperfusion without agmatine; Agm6, at 6 h after reperfusion with agmatine; EC24, at 24 h after reperfusion without agmatine; Agm24, at 24 h after reperfusion with agmatine; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; HiF, hippocampal fissure. Higher magnifications of cells indicated by squares show eNOS positive cells in the endothelium of vessels and capillaries. Arrows point to positive cells that showed higher intensity of immunostaining with hematoxylin in rat brain, compared with the control. Scale bar=50 µm (hippocampus), and 100 µm (cortex).

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  Fig. 5 Regulations of MMPs expression by agmatine treatment following transient global ischemia in rat brain. Representative expressional levels of MMP-2 and MMP-9 6 and 24 h after agmatine treatment (100 mg/kg, i.p) in the hippocampus (A), in the cerebral cortex (C), and densitometric data (B, D). Data represent means±SD for n=5/NC, n=3/EC and Agm group per each time point. *P<0.05, vs. normal control. NC, normal control; EC6, at 6 h after reperfusion without agmatine; Agm6, at 6 h after reperfusion with agmatine; EC24, at 24 h after reperfusion without agmatine; Agm24, at 24 h after reperfusion with agmatine.

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