Reduction by Tetrahydrobiopterin of H2O2–Induced Endothelial Cell Injury

Abstract: The purpose of this study was to examine the effect of tetrahydrobiopterin, a co–factor of nitric oxide synthase, on H₂O₂–induced endothelial cell injury. Pretreatment with sepiapterin, a precursor of tetrahydrobiopterin biosynthesis, increased tetrahydrobiopterin content of endothelial cells, and reduced H₂O₂–induced endothelial cell injury, which was measured by leakage of lactate dehydrogenase. Both the increase in tetrahydrobiopterin content and the protective effect of sepiapterin were prevented by co–pretreatment with N–acetylserotonin, an inhibitor of sepiapterin reductase. Although Ca²⁺ ionophore ionomycin–induccd nitric oxide synthesis was increased by pretreatment with sepiapterin, the protective effect of sepiapterin was not affected by an inhibitor of nitric oxide synthesis. On the other hand, pretreatment with sepiapterin also reduced H₂O₂–induced rat foetal lung fibroblast cell injury via an increase in tetrahydrobiopterin content, despite rat foetal lung fibroblast cells lacking nitric oxide synthase. Moreover, increase in tetrahydrobiopterin strongly reduced H₂O₂–induced intracellular oxidative stress. These findings indicate that sepiapterin reduces H₂O₂–induced endothelial cell injury via an increase in tetrahydrobiopterin content. Although increase in endothelial tetrahydrobiopterin content stimulated nitric oxide production, the protective effect of tetrahydrobiopterin against H₂O₂–induced endothelial cell injury is unlikely to be related to the stimulation of nitric oxide release from nitric oxide synthase. The protective effect of tetrahydrobiopterin may involve reactive oxygen species–scavenging activity.     

氯沙坦对血管紧张素II致培养的牛脑微血管内皮细胞损伤的保护作用

目的观察氯沙坦对血管紧张素II(Ang II)致牛脑微血管内皮细胞(BCMECs)损伤的保护作用。方法用分光光度计测定培养的BCMECs乳酸脱氢酶(LDH)的漏出量,流式细胞仪测定BCMECs细胞间粘附分子-1(ICAM-1)的表达量,硝酸还原酶法和放射免疫分析法分别测定BCMECs上清液中一氧化氮(NO)和内皮素-1(ET₁)的含量。结果Ang II呈剂量依赖性增加BCMECs LDH漏出、NO和ET₁释放及ICAM-1表达,氯沙坦对此均有明显抑制作用。结论氯沙坦抑制Ang II致体外培养BCMECs的损伤。     

Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-ß

RAW 264.7 macrophages induced with lipopolysaccharide and interferon-γ expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by N^G-nitro-L-arginine and N^G-methyl-L-arginine. Transforming growth factor-ß1 prevented the induction of both enzymes.     

NG-Nitro-L-Arginine-Methyl-Ester Protects against Ischaemia-Induced Biochemical Changes and Hippocampal Neurodegeneration in the Gerbil

To assess the effects of the nitric oxide inhibitor N^G-nitro-l -arginine methyl ester (L-NAME) on behavioural, biochemical and histological changes following global ischaemia, the Mongolian gerbil was used. Ischaemia was induced by bilateral carotid occlusion (BCO) for 5 min. N^G-nitro-l -arginine methyl ester was administered i.p. at 10 mg/kg 30 min, 6, 24, and 48 h after surgery. Results indicated that 5 min BCO caused a large increase in home cage activity. N^G-nitro-l -arginine methyl ester caused some attenuation in this hyperactivity. The activity of nitric oxide synthase (NOS) was increased in the cerebellum, brain stem, striatum, cerebral cortex and hippocampus of 5-min bilateral carotid occluded animals. N^G-nitro-l -arginine methyl ester reversed the increase in nitric oxide synthase activity in all brain regions. Extensive neuronal death was observed in the CA₁ layer of the hippocampus in 5-min BCO animals 96 h after surgery. N^G-nitro-l -arginine methyl ester significantly protected against the neuronal death of cells in the CA₁ layer.     

In vitro toxicity of sodium nitroprusside to human endothelial ECV304 cells

The cytotoxicity of sodium nitroprusside (SNP) to the human endothelial cell line, ECV304, was studied. The cytotoxicity of SNP was primarily related to the liberation of nitric oxide (NO). S-nitroso-N-acetyl-d-penicillamine (SNAP), an NO donor, was highly toxic. Other degradation products of SNP either exerted much less toxicity (i.e. cyanide and nitrite) or were non-toxic (i.e. ferricyanide and ferrocyanide). SNP induced multinucleation, inhibited cell proliferation, lowered the endogenous level of reduced glutathione (GSH), and induced apoptotic cell death. The plasma membrane was not the prime site of toxic action, as leakage of lactic acid dehydrogenase (LDH) occurred only at a relatively high concentration of SNP. Cells treated with non-toxic levels of the glutathione-depleting agents, 1-chloro-2,4-dinitrobenzene (CDNB), dl-buthionine-[S,R]-sulfoximine (BSO), and 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU), were hypersensitive to subsequent exposure to SNP. The GSH status of the cells was, therefore, a key factor in determining the cytotoxicity of SNP.     

Reduction by NG-nitro-L-arginine of H2O2-induced endothelial cell injury.

1. The effects of three analogues of NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, on hydrogen peroxide (H2O2)-induced endothelial cell injury were studied. 2. Endothelial cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH) and 51Cr. 3. Addition of H2O2 (250-1,000 microM) to endothelial cells induced the release of LDH dose-dependently. The release of LDH was reduced by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4)-4 x 10(-3) M), L-NOARG (10(-4)-4 x 10(-3) M) and NG-nitro-L-arginine benzyl ester (L-NABE, 10(-4)-4 x 10(-3) M), inhibitors of NO synthase. 4. L-NOARG analogues also reduced H2O2-induced 51Cr release from endothelial cells, while L-NMMA had no effect. 5. The protective effect of L-NAME was not reversed by addition of L-arginine (L-Arg, 1-10 mM). 6. Both L-NAME and L-NMMA completely inhibited L-Arg metabolism to L-citrulline coupled with NO synthesis. 7. These findings suggest that L-NOARG analogues but not L-NMMA reduced H2O2-induced endothelial cell injury, and that these effects may not be related to inhibition of NO production.     

氯沙坦对小而密低密度脂蛋白致培养的人脐静脉内皮细胞损伤的保护作用

目的：观察氯沙坦对小而密低密度脂蛋白(sLDL)致人哜静脉内皮细胞(HUVECs)损伤的保护作用。方法：分离、鉴定 sLDL；用连续监测法测各组细胞乳酸脱氢酶(LDH)漏出量；用丙二醛试剂盒测定各组细胞上清中丙二醛(MDA)浓度；用硝酸还原酶法测定各组细胞分泌的一氧化氮(NO)量；用细胞-酶联免疫吸附法检测细胞膜上细胞间粘附分子-1(ICAM-1)蛋白表达量；反转录-多聚酶链反应(RT-PCR)法检测 ICAM-1mRNA 水平。结果：sLDL 使 HUVECs LDH 漏出增多、MDA 生成增多、NO 分泌减少、ICAM-1 表达增多,氯沙坦对此均有明显抑制作用,且呈剂量依赖性。结论：氯沙坦抑制 sLDL 致体外培养 HUVEcs 的损伤。     

OXYGEN RADICALS AND NITRIC OXIDE IN RAT MESENTERIC ISCHAEMIA-REPERFUSION: MODULATION BY L-ARGININE AND NG-NITRO-L-ARGININE METHYL ESTER

1. The aims of the present study were to detect changes in superoxide anion (O₂^?), nitric oxide (NO) and other reactive oxygen species (ROS) directly by measurement of chemilumin-escence (CL) and to investigate the role of L-arginine, a nitric oxide synthase (NOS) substrate, and N^G-nitro-L -arginine methyl ester (L-NAME), a NOS inhibitor, together with their molecular enantiomers D-arginine and D-NAME, in a rat mesenteric ischaemia-reperfusion (I/R) model. 2. Seventy-nine female Wistar albino rats were divided into eight groups. The first three groups underwent sham operation; group 1 was the control group, group 2 received L-arginine and group 3 received L-NAME. Ischaemia was produced in the remaining five groups by ligation of the superior mesenteric artery for 30 min followed by 60 min reperfusion. Group 4 rats were control I/R rats and groups 5-8 received either L-arginine, L-NAME, D-arginine or D-NAME, respectively. 3. Both luminol and lucigenin CL was significantly increased in I/R groups compared with sham-operated groups. L-Arginine significantly reduced CL measurements. D-Arginine was also protective, but not as much as L-arginine. Both L - and D-arginine had in vitro O₂^?-scavenging potential, as tested by the xanthine-xanthine oxidase system. N^G-Nitro-L-arginine methyl ester decreased lipid peroxidation values in addition to reducing CL measurements. Nitric oxide concentrations were significantly increased in I/R groups in comparison with sham-operated groups. Peroxynitrite formation was increased by I/R. Treatment with L-NAME was beneficial by reducing NO concentrations in the reperfused ileum. 4. In our I/R model, O₂^?, NO and other ROS were increased. Although NOS inhibitors were effective in reducing oxidative damage, increasing NO concentrations with L-arginine was also beneficial, presumably due to the ability of L-arginine to inhibit phagocyte adherence and its radical scavenging potential. In fact, NO may have different effects in terms of tissue injury or protection depending on the concentration of oxygen and the haemodynamic state of the tissue.     

Down‐Regulation of CD40 Gene Expression and Inhibition of Apoptosis with Danshensu in Endothelial Cells

Abstract: Danshen is commonly used in China for the treatment of atherosclerosis‐related disorders such as cardiovascular and cerebrovascular diseases. Research shows that it also has immunostimulation properties. The present study evaluates the protective effect of danshensu, an active water‐extractable component isolated from danshen, on an endothelial cell line (CRL‐1730) treated with hydrogen peroxide (H₂O₂). Danshensu significantly inhibited endothelial cell viability induced by H₂O₂. The treatment of endothelial cells with danshensu resulted in most cells being arrested in the S and G₂/M phases of the cell cycle. The fraction of cells in G₀/G₁ phase was markedly decreased by danshensu treatment compared to the control groups. The apoptosis was also markedly decreased after danshensu treatment. Additionally, danshensu restrains decreased nitric oxide level, increased the release of lactate dehydrogenase and expression of cluster of differentiation 40 (CD40) significantly. These results suggest that danshensu protects endothelial cells from the damage induced by H₂O₂ through its CD40 anti‐inflammatory approach and cell apoptosis inhibition.     

Blockade of Sensitization to the Toxic Effects of Cocaine in Mice by Nitric Oxide Synthase Inhibitors

Repeated administration of cocaine to animals results in sensitization to several reactions to the drug, including seizures and mortality. These consequences are thought to be related to the pathology that develops in humans abusing cocaine. Previous studies implied the involvement of the N-methyl-D-aspartate (NMDA) type of glutamate receptors in cocaine-induced toxicity, and recent studies documented a role for nitric oxide in NMDA-receptor mediated neurotoxicity. The present study was undertaken to determine whether nitric oxide synthase inhibitors block the development of sensitization to the toxic effects of cocaine in mice. Repeated administration of cocaine (45 mg/kg/day; intraperitoneally) to Swiss Webster mice, for 7 days, resulted in a progressive increase in the duration of the convulsive response to cocaine, an increase in the number of animals that had seizures, and augmentation in lethality rate. Pretreatment with N^G–nitro-L-arginine methyl ester (L-NAME; 100 mg/kg/day; intraperitoneally) or N^G–nitro-L-arginine (NO-Arg; 25 mg/kg/day; intraperitoneally) completely abolished the sensitization to the convulsive and lethal responses to cocaine. Receptor binding assays indicated first, that pretreatment with L-NAME apparently diminished cocaine-induced upregulation of cortical NMDA receptors, and second, that the effects of the nitric oxide synthase inhibitors tested are not mediated via a direct interaction of the drugs with the phencyclidine/NMDA receptor complex. Taken together, the present study suggests an important role for nitric oxide in the development of sensitization to the toxic effects of cocaine, and further supports the relationship between NMDA-receptor mediated neurotoxicity and nitric oxide.     

Inhibitory effects of crocetin on high glucose-induced apoptosis in cultured human umbilical vein endothelial cells and its mechanism

Dysfunction of endothelial cell is considered as a major cause of vascular complications in diabetes. Crocetin has been shown to have strong antioxidant activities. In present study, we tested whether crocetin inhibited high glucose-induced apoptosis in cultured human umbilical vein endothelial cells (HUVEC_s) and to explore its possible mechanism. Exposure to high glucose (33 mM) for 72h induced a pronounced increase in apoptosis compared with normal glucose (5.5 mM), as evaluated by cell chromatin staining with Hoechst 33,258 and cell death detection ELISA. High glucose attenuated activation of Akt and endothelial nitric oxide synthase (eNOS). Crocetin (0.1 μM, 1.0 μM) prevented high glucose-induced apoptosis, which correlates with the increase of activation of p-Akt, following the up-regulation of eNOS and NO production. Pretreatment with phosphatidylinositol 3′ kinase (Pl3K) inhibitor LY294002 or eNOS inhibitor N^G-nitro-arginine methyl ester (LN or L-NAME) inhibited crocetin’ effect on p-Akt or eNOS, respectively. For the first time, results of our study suggest that crocetin inhibits high glucose-induced apoptosis, at least partly, via Pl3K/Akt/eNOS pathway in HUVEC_s and crocetin may exert a beneficial effect in preventing diabetes-associated cardiovascular complications.     

Endothelium-dependent contractions to NG-nitro-L-arginine methyl ester in the porcine isolated splenic artery are sensitive to cyclooxygenase and lipoxygenase inhibitors

Summary The nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), produced large endothelium-dependent contractions in isolated segments of the porcine splenic artery, equivalent to approximately 30% of the maximum responses to 5-hydroxytyptamine (5-HT). These responses were inhibited by 1mM L-arginine, but not by either 1mM D-arginine or the superoxide anion scavenger, superoxide dismutase. However, L-NAME-induced contractions were markedly inhibited by the cyclooxygenase inhibitor, flurbiprofen, and the lipoxygenase inhibitor, 2,3,5-tri-methyl-6-(12-hydroxy-5,10-dodecadiynyl)1,4-benzoquinone (AA-861). The combined cyclooxygenase and lipoxygenase inhibitor 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW 755C) abolished L-NAME-induced contractions. These findings suggest that suppression of endothelial nitric oxide synthase in the porcine isolated splenic artery results in activation of arachidonic metabolism and production of vasoconstrictor eicosanoids.Correspondence to V. G. Wilson at the above address     

Possible role of nitric oxide in 5-hydroxytryptamine-induced increase in vascular permeability in mouse skin

In order to test the hypothesis that a 5-hydroxytryptamine (5-HT)-induced increase in vascular permeability results from a cascade triggered by activation of the synthesis of nitric oxide (NO), the vascular permeability was investigated using the Pontamine sky blue leakage method in male mice. Subcutaneous injection of 5-HT induced a dose-related increase of vascular permeability at the injection site. The vascular permeability induced by 5-HT was inhibited by pretreatment with intraperitoneal injection of ketanserin (5-HT_2A antagonist) and methysergide (5-HT_1/2A antagonist), less efficiently by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine (NAN-190) (5-HT_1A antagonist), but not by granisetron (5-HT₃ antagonist). Increase in vascular permeability induced by 5-HT was inhibited by concurrent intravenous administration of NO synthase inhibitors N^G-nitro-Lrarginine methyl ester (L-NAME) and methylene blue but not by the inactive enantiomer N^G-nitro-D-arginine methyl ester (D-NAME). These results suggest that 5-HT increases vascular permeability by activating the 5-HT receptors and that endogenous NO is involved in this effect of 5-HT. Correspondence to: E. Fujii at the above address     

Effect of N-nitro-l-arginine on shock induced by endotoxin and by platelet activating factor in dogs

When N^G-nitro-l-arginine, a nitric oxide synthase inhibitor, administration was started 5 min prior to shock induction in anesthetized dogs, a partial restoration was observed in endotoxin-induced shock and a complete recovery in platelet activating factor (PAF)-induced shock. When N^G-nitro-l-arginine infusion was started 5 min after shock induction, no significant recovery was observed in endotoxin-induced shock and a complete recovery in PAF-induced shock. These data indicate that enhanced production of nitric oxide by vascular endothelial cells may contribute to endotoxin- or PAF-induced shock and also that some mediators including inducible nitric oxide synthase and/or cellular damage might be involved in endotoxin-induced shock.     

The Role of Nitric Oxide and Sulphydryls in Gastric Mucosal Protection Induced by Sodium Cromoglycate in Rats

The role of endogenous nitric oxide and sulphydryls in gastric protection afforded by sodium cromoglycate against ethanol-induced gastric lesions was studied in rats. Drugs were administered either intraperitoneally (i.p.) or subcutaneously (s.c.) 30, 45 or 60 min before oral administration of ethanol. Administration of cromoglycate before ethanol dose-dependently inhibited ethanol-induced gastric lesions. Pretreatment with N^G-nitro-l -arginine methyl ester (L-NAME), an inhibitor of nitric oxide biosynthesis, dose-dependently aggravated gastric lesions and reduced cromoglycate-induced gastric protection. The attenuating effect of L-NAME on gastric protection elicited by cromoglycate was reversible by pretreatment with l -arginine but not by d -arginine. On the other hand, ethanol-induced gastric lesions were found to be associated with a reduction of nonprotein sulphydryl content of glandular stomachs. Pretreatment with cromoglycate prevented non protein sulphydryl depletion and afforded protection. Pretreatment with N-ethylmaleimide, a sulphydryl blocker, caused dose-dependent enhancement of ethanol-induced gastric lesions and further depletion of non protein-sulphydryl. Treatment with N-ethylmaleimide before cromoglycate reduced the gastric protection that was associated with depletion of nonprotein sulphydryls. Furthermore, combined N-ethylmaleimide and L-NAME pretreatment caused a greater aggravation of ethanol-induced gastric lesions and significantly produced a higher reduction of the protective effects of cromoglycate. However, pretreatment with l -arginine only partially restored the protective effects of cromoglycate. These results suggest that the protective effects of cromoglycate may be dependent on the maintenance of a critical level of both endogenous nitric oxide and nonprotein sulphydryls in the gastric mucosa.     

Relationship between protecitve effect of probucol on endothelial cells and asymmetrical dimethylarginine levels

AIM: To investigate the relationship between protective effect of probucol on endothelial cells and endogenous nitric oxide synthase inhibitor levels. METHODS: Endothelial cells were treated with oxidative-low density lipoprotein (ox-LDL) (100 rag/L) or lysophosphatidyl choline (LPC) (5 mg/L) for 48 h, and the release of lactate dehydrogenase (LDH), levels of nitric oxide (NO),     

Comparative effects of azelnidipine and other Ca2+-channel blockers on the induction of inducible nitric oxide synthase in vascular smooth muscle cells

Overproduction of nitric oxide by inducible nitric oxide synthase contributes to the progression of cardiovascular disease. We investigated the effects of azelnidipine and other Ca2+-channel blockers on nitric oxide production by cultured aortic smooth muscle cells isolated from Wistar rats and human umbilical vein endothelial cells (HUVECs), using the Griess reaction and oxyhemoglobin method. Release of lactic dehydrogenase (LDH) was measured to evaluate cell damage, and immunohistochemistry was performed to examine the expression of inducible nitric oxide synthase and nitrotyrosine protein. Azelnidipine and other Ca2+-channel blockers inhibited the release of nitric oxide induced by lipopolysaccharide plus interferon-gamma. Azelnidipine inhibited it most potently among the Ca2+-channel blockers tested (azelnidipine, amlodipine, nifedipine, diltiazem, verapamil, and nicardipine) at a concentration of 10 microM. Longer stimulation with these agents induced the expression of inducible nitric oxide synthase and nitrotyrosine, with an increase of lactic dehydrogenase release, whereas azelnidipine suppressed these changes. In human umbilical vein endothelial cells, azelnidipine enhanced basal nitric oxide production by endothelial nitric oxide synthase. In conclusion, azelnidipine potently inhibited the induction of inducible nitric oxide synthase and then nitric oxide production in vascular smooth muscle cells, while enhancing constitutive nitric oxide production by endothelial cells. Azelnidipine may inhibit nitrotyrosine expression and cell damage caused by overproduction of nitric oxide, suggesting a mechanism for its cardiovascular protective effect.     

Spontaneous nitric oxide in hepatocyte monolayers and inhibition of compound-induced apoptosis

Primary cultures of hepatocytes are a widely used in vitro model for biochemical research. Following isolation, hepatocytes produce large amounts of nitric oxide (NO), which is known to have both pro- and anti-apoptotic effects in hepatocytes in vivo and in vitro. Previous work has not determined the effect of these increased levels of NO on the response of hepatocytes to apoptotic stimuli. Here we report that levels of nitrites are elevated in hepatocyte monolayers from 24 h onwards. Addition of the inducible nitric oxide synthase (iNOS) inhibitor, Nω-nitro- -arginine methyl ester (L-NAME), to the medium inhibited this increase in nitrites. These results indicate that the increase in nitrite is most likely due to the formation of NO. Elevated nitrite levels had no effect either on basal levels of apoptosis or on ATP and GSH. Apoptosis was induced by transforming growth factor β-1 (TGFβ-1) or glycochenodeoxycholate (GCDC). Both compounds caused moderate hepatocyte apoptosis; however, addition of L-NAME prior to exposure significantly increased the level of apoptosis observed with the two compounds. Both TGFβ-1 and GCDC had no effect on hepatocyte ATP or GSH levels; however, as a consequence of secondary necrosis, TGFβ-1 exposure significantly increased levels of lactate dehydrogenase (LDH) leakage. These findings indicate that the increased levels of NO associated with the culture of hepatocytes have an inhibitory effect on compound-induced apoptosis in the cells.     

Role of nitric oxide on the endothelium-dependent vasodilation in newborn piglet cerebral arteries

1. The present study was undertaken to determine whether endothelial nitric oxide (NO) is involved in the endothelium-dependent vasodilation elicited by bradykinin (BK) in rings of newborn (1–7-day-old) piglet cerebral arteries precontracted with KCl (25 mM).2. In these rings, BK (10⁻¹⁰−10⁻⁶ M) induced concentration-dependent relaxation. The preincubation with the precursor of NO synthesis, l-arginine (10⁻⁴ M), reduced KCl-induced contraction and increased the BK relaxation. However, preincubation with the NO synthase inhibitor, N^G-nitro-l-arginine-methyl ester (L-NAME; 3 × 10⁻⁵ M, increased KCl contraction and basal tone, and inhibited BK relaxation.3. These results suggest that the endothelium of these arteries possesses the ability to produce NO, either basal or stimulated by agents like BK.     

N-Nitro-L-arginine protects against ischaemia-induced increases in nitric oxide and hippocampal neuro-degeneration in the gerbil

To assess the effects of the nitric oxide synthase inhibitor N^G-Nitro-L-arginine on behavioural, biochemical and histological changes following global ischaemia, the Mongolian gerbil was used. Ischaemia was induced by bilateral carotid occlusion for 5 min. N^G-Nitro-L-arginine was administered i.p. at either 1 or 10 mg/kg 30 min, 6, 24, and 48 h after surgery. 5 min bilateral carotid occluded animals were hyperactive 24, 48 and 72 h after surgery. N^G-Nitro-L-arginine caused some attenuation in this hyperactivity. The activity of nitric oxide synthase was increased in the cerebellum, brain stem, striatum, cerebral cortex and hippocampus of 5 min bilateral carotoid occluded animals. N^G-Nitro-L-arginine reversed the increase in nitric oxide synthase activity in all brain regions. Extensive neuronal death was observed in the CA₁ layer of the hippocampus in 5 min bilateral carotid occluded animals 96 h after surgery. N^G-Nitro-L-arginine significantly protected against the neuronal death of cells in the CA₁ layer.