Korean J Physiol Pharmacol.  2009 Aug;13(4):265-271. 10.4196/kjpp.2009.13.4.265.

Kainic Acid-induced Neuronal Death is Attenuated by Aminoguanidine but Aggravated by L-NAME in Mouse Hippocampus

• Byun JS
• Lee SH
• Jeon SH
• Kwon YS
• Lee HJ
• Kim SS
• Kim YM
• Kim MJ
• Chun W

• KMID: 2071680
• DOI: http://doi.org/10.4196/kjpp.2009.13.4.265

Abstract

Nitric oxide (NO) has both neuroprotective and neurotoxic effects depending on its concentration and the experimental model. We tested the effects of NG-nitro-L-arginine methyl ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, and aminoguanidine, a selective inducible NOS (iNOS) inhibitor, on kainic acid (KA)-induced seizures and hippocampal CA3 neuronal death. L-NAME (50 mg/kg, i.p.) and/or aminoguanidine (200 mg/kg, i.p.) were administered 1 h prior to the intracerebroventricular (i.c.v.) injection of KA. Pretreatment with L-NAME significantly increased KA-induced CA3 neuronal death, iNOS expression, and activation of microglia. However, pretreatment with aminoguanidine significantly suppressed both the KA-induced and L-NAME-aggravated hippocampal CA3 neuronal death with concomitant decreases in iNOS expression and microglial activation. The protective effect of aminoguanidine was maintained for up to 2 weeks. Furthermore, iNOS knockout mice (iNOS-/-) were resistant to KA-induced neuronal death. The present study demonstrates that aminoguanidine attenuates KA-induced neuronal death, whereas L-NAME aggravates neuronal death, in the CA3 region of the hippocampus, suggesting that NOS isoforms play different roles in KA-induced excitotoxicity.

Keyword

Kainic acid; Nitric oxide; L-NAME; Aminoguanidine; iNOS; Neuronal death

MeSH Terms

Animals
Guanidines
Hippocampus
Kainic Acid
Mice
Mice, Knockout
Microglia
Models, Theoretical
Neurons
NG-Nitroarginine Methyl Ester
Nitric Oxide
Nitric Oxide Synthase
Protein Isoforms
Seizures
Guanidines
Kainic Acid
NG-Nitroarginine Methyl Ester
Nitric Oxide
Nitric Oxide Synthase
Protein Isoforms

Figure

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  Fig. 1. Effects of NOS inhibitors on KA-induced neuronal cell death in the CA3 region of mouse hippocampus. Neuronal cell death was determined with cresyl violet staining. KA injection produced a marked loss of pyramidal neurons (IA, E) and L-NAME treatment prior to KA (IB, F) potentiated this KA-induced neuronal loss. Aminoguanidine pretreatment (IC, G) markedly attenuated KA-induced and L-NAME-aggravated (ID, H) neuronal death. Neuronal cell death was not observed in vehicle-, aminoguanidine-only-, or L-NAME-only-treated mice (II). Images in the upper right corner show the entire hippocampus and boxes in these images indicate the enlarged views of the CA3 region. Arrows indicate areas where most neuronal death appears. KA & K stand for kainic acid, L for L-NAME, and A for aminoguanidine. Scale bar: 100 μm.

• 

  Fig. 2. Representative (I) and quantitative (II) neuronal death measured with Terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) assay. KA produces TUNEL-positive neurons within 24 h, and L-NAME (L+KA) treatment potentiates this induction. Aminoguanidine (A+KA) reduces the numbers of TUNEL-positive cells compared with KA-only (KA) and L-NAME-aggravated (A+L+KA) groups. L-NAME and aminoguanidine alone did not affect cell survival (data not shown). Quantitative data represent three independent experiments and are expressed as mean±SEM. ∗p<0.05, ∗∗p<0.01 versus the KA-only treated group. KA & K stand for kainic acid, L for L-NAME, and A for aminoguanidine.

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  Fig. 3. Aminoguanidine produces persistent neuronal survival against KA and L-NAME injury. Pyramidal neurons were measured with cresyl violet staining (Top panels) and NeuN immunoreactivity (Bottom panels) at 2 weeks after KA injection. Arrows indicate areas with highest neuronal death. KA & K stand for kainic acid, L for L-NAME, A for aminoguanidine.

• 

  Fig. 4. Effects of NOS inhibitors on KA-induced microglial activation and subsequent iNOS expression. Expression of iNOS (top panels) and OX-6, a microglial marker, (bottom panels) within the CA3 was determined with immunohistochemical analysis at 24 h after treatment. KA increased iNOS expression (A) and microglial activation (E), and these phenomena were attenuated with aminoguanidine pretreatment (C, G). L-NAME pretreatment (B, F) potentiated the KA effect, but aminoguanidine reduced this potentiation (D, H). L-NAME and aminoguanidine alone did not affect microglial activation and iNOS expression (data not shown). KA & K stand for kainic acid, L for L-NAME, and A for aminoguanidine.

• 

  Fig. 5. Decreased neuronal death after KA in iNOS knockout (iNOS−/–) mice. Neuronal death was measured with cresy violet (I) and TUNEL staining (II&III). KA produced less neuronal death in iNOS−/– mice (I&II-As) than littermates (I&II-Cs). L-NAME pretreatment potentiated neuronal death in both littermates (I&II-Ds) and iNOS−/– mice (I&II-Bs) compared to KA alone, but iNOS−/– mice showed less potentiation (III). Quantitative data represent three independent experiments and are expressed as mean±SEM. ∗∗p<0.01 versus KA-only treated iNOS−/– mice. Images in the upper right corner show the entire hippocampus and boxes in these images indicate the enlarged views of the CA3 region. KA & K stand for kainic acid, L for L-NAME, and A for aminoguanidine. Scale bars: I, 100 μm; II, 50 μm.

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