Effect of nitric oxide synthase inhibitor NG-nitro-L-arginine on the content of amino acid in ischemic brain tissues of rats

Jianxin Zhang; Huixin Zhang; Lanfang Li; Qinzeng Zhang; Yonghui Li

摘要：BACKGROUND: Nitric oxide synthase (NOS) inhibrtors have been widely used to investigate the role of NO on cerebral ischemic injury, but the results are controversial. Moreover, it has been considered to aggravate the ischemic neuronal damage with the release of excessively excitatory amino acids (EAA) during cerebral ischemia. On the other hand, some inhibitory amino acid is suggested to be important for the neuronal protection against ischemic brain damage. Our study has recently showed that treatment with the NOS inhibitor NG-nitro-L-arginine (L-NA) reduced focal cerebral ischemic damage. The effect of L-NA on the contents of excitatory and inhibitory amino acid in the rat brain following cerebral ischemia is still unclear.OBJECTIVE: By evaluating the effect of NOS inhibitor, L-NA on the contents of aspartate, glutamate, glycine and γ-aminobutyric acid (GABA) in striatum, hippocampus and cortex in the rat brain following cerebral ischemia respectively, to investigate the beneficial effect of L-NA on cerebral ischemic injury and the possible mechanism.DESIGN: A randomized and controlled experiment.SETTING: Department of Pharmacology, Hebei Academy of Medical Sciences.MATERIALS: A total of 42 male healthy SD rats (grade Ⅱ, weighting 250-300 g) were provided by the Experimental Animal Center of Hebei Province (Certification: 04036). Aspartate, glutamate, glycine, GABA, L-NA and 2,3,5-triphenyltetrazolium chloride (TTC) were obtained from Sigma Chemicals Co, St Louis, MO, USA.HPLC-ultraviolet detector system consisted of Agilent 1100 HPLC.METHODS: The experiment was carried out in Department of Pharmrcology, Hebei Academy of Medical Sciences from June 2005 to June 2006. Rats were randomly divided into three groups: sham-operated group (n=6), ischemic group (n = 18), L-NA group (n = 18). The model of focal cerebral ischemia in rat was prepared with intraluminal line occlusion methods. In sham-operated rats, the external carotid artery was surgically prepared,but the filament was not inserted. Each group was further divided into 3 subgroups (n = 6 for each): drugs were administrated at 2, 6 and 12 hours after the middle cerebral artery occlusion (MCAO) respectively.L-NA (20 mg/kg, ip) was administrated, twice a day, for 3 consecutive days. Same volume of normal saline was administrated in ischemic and sham operation groups. The changes of infarcted volume and the contents of amino acids were respectively assayed. Image analysis software was used for the measurement of the infarcted area. The results were expressed as a percentage of the infarcted volume of cerebral/volume of whole brain (Ⅳ%) in order to control for edema formation. The contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in the rat brain following cerebral ischemia were respectively measured by HPLC method. All data were analyzed with one-way ANOVA and Dunnett's test.MAIN OUTCOME MEASURES: ① The volume of cerebral infarction; ② The contents of aspartate, glutamate, glycine and GABA in brain tissue after cerebral ischemia.RESULTS: All 42 rats were involved in the final analysis. ① Infarcted volume: Volume was 0 in sham-operated group. When L-NA was administrated at 2 and 6 hours after MCAO, the infarcted volume was (20.13±3.59)%and (23.12±5.84)% in L-NA group, which was not similar to that in ischemic group [(22.10±3.98)%, (25.38±5.37)%, P＞ 0.05]. However, the infarcted volume was markedly decreased compared with that of ischemic group when L-NA was administrated at 12 hours after MCAO [(26.11±3.55)％ and (37.15±3.58)％, P ＜ 0.01]. ② Changes of amino acid content: At 2 and 6 hours after ischemia, the contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in ischemic group were significantly increased compared with those in sham-operated group ( P＜ 0.05-0.01). However, contents in L-NA group were similar to those in ischemic group (P ＞ 0.05). At 12 hours after ischemia, the contents of aspartate [(0.21 ±0.06), (0.36±0.05),(0.29±0.12) mg/g] and glutamate [(0.55±0.06), (0.78±0.10), (0.52±0.10) mg/g] in striatum, hippocampus and cortex in L-NA group were significantly decreased compared with those in ischemic group [(0.49±0.17), (0.63±0.03), (0.51±0.15) mg/g; (0.98±0.30), (1.15±0.15), (0.93±0.15) mg/g, P＜ 0.05-0.01]. Glycine in hippocampus was (0.40±0.07) mg/g, which was higher than that in ischemic group [(0.21±0.07) mg/g, P ＜ 0.05]. GABA in striatum, hippocampus and cortex was (0.93±0.10), (0.62±0.12) and (0.81±0.10) mg/g, respectively, which was higher than that in ischemic group [(0.60±0.08), (0.37±0.17), (0.59±0.10) mg/g, P ＜ 0.05-0.01].CONCLUSION: It may be concluded that L-NA have beneficial effect on ischemic cerebral injury in ischemic later stage in rats. The possible mechanism is that L-NA can decrease the contents of aspartate and glutamate,increase the contents of glycine and GABA.