Different roles of neuronal and endothelial nitric oxide synthases on ischemic nitric oxide production in gerbil striatum

Neuroscience Letters 288 (2000) 151±154

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Different roles of neuronal and endothelial nitric oxide synthases on ischemic nitric oxide production in gerbil striatum Naoto Adachi*, Baiping Lei, Masao Soutani, Tatsuru Arai Department of Anesthesiology and Resuscitology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan Received 13 April 2000; received in revised form 19 May 2000; accepted 29 May 2000

Abstract The production of nitric oxide (NO) in gerbil striatum during ischemia and reperfusion was monitored by measuring total NO metabolites in dialysates, and the effects of 7-nitroindazole (7-NI), a selective inhibitor of neuronal NO synthase, and N G-nitro-l-arginine methyl ester (l-NAME), a non-selective inhibitor of NO synthase, were examined. The effects of these agents on ischemic neuronal damage were histologically evaluated 7 days after transient ischemia for 5 or 10 min. 7-NI and l-NAME decreased the NO production to similar extents in non-ischemic gerbils. 7-NI inhibited the increased NO production after 5 min of ischemia, and partly attenuated the increase in NO production after 10 min of ischemia, but had no effect on the increase after 15 min of ischemia. l-NAME completely abolished the increased NO production after different durations of ischemia. The extent of ischemic neuronal damage by 5-min ischemia was aggravated by either 7NI or l-NAME, while damage by 10-min ischemia was marked in all groups. These results indicate that neuronal and endothelial NO synthases make different contributions to the post-ischemic NO production and the histological outcomes in gerbil striatum. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral ischemia; Gerbil; Microdialysis; Nitric oxide; Nitric oxide synthase; Striatum

Nitric oxide (NO) is synthesized from l-arginine by the action of one of the three isoforms of NO synthase (NOS); neuronal NOS (nNOS), endothelial NOS (eNOS), or inducible NOS (iNOS) [2,12]. Since post-ischemic NO production is thought to be related to ischemic neuronal damage [4], we examined NO production in gerbil striatum by determining the total metabolites of NO (NOx2) using brain microdialysis. Then, we evaluated the contributions of nNOS and eNOS to NO production by assessing the effects of 7-nitroindazole (7-NI), a speci®c inhibitor of nNOS in vivo, and N G-nitro-l-arginine methyl ester (l-NAME), a non-speci®c inhibitor of NOS. Further, the effects of these agents on the histological outcomes were examined. All experiments were approved by the Committee on Animal Experimentation at Ehime University School of Medicine, Ehime, Japan. Male Mongolian gerbils (60±80 g) (Seiwa Experimental Animals, Fukuoka, Japan) were anesthetized and maintained with 3% halothane in balanced 50% oxygen and 50% nitrous oxide via a face mask. An intraperitoneal catheter was inserted into the abdominal space for drug administration. Then, both common carotid * Corresponding author. Tel.: 181-89-960-5383; fax: 181-89960-5386.

arteries were exposed, and silk threads (4±0) were looped around them. After the animal was placed in a stereotaxic apparatus, two small burr holes were drilled to insert probes for brain temperature measurement and for microdialysis. Brain and rectal temperatures were maintained at 37.0± 37.58C with heating lamps during the experimental period. A 1-mm microdialysis probe (A-I-8-01, o.d. 0.22 mm; Eicom, Kyoto, Japan) was implanted into the left striatum (0.8 mm anterior and 2.8 mm lateral to the bregma and 3.6 mm below the brain surface), and the probe was perfused with Ringer's solution at 2 ml/min. The administration of nitrous oxide was then discontinued, and the anesthesia was maintained with 2% halothane in oxygen. After a stabilization period of 90 min, dialysates were collected every 15 min and stored at 2808C. The amount of NOx2 (NO22 plus NO32) in the dialysate was determined by the 2,3-diaminonaphthalene method as described previously [8]. Transient forebrain ischemia for various durations was achieved by pulling the threads with 8-g weights. In the ®rst experiment, effects of 7-NI and l-NAME (Sigma; St. Louis, MO, USA) on NO production were evaluated in non-ischemic animals. Twenty-three gerbils were divided into four groups: peanut oil, 7-NI (80 mg/kg), saline, and l-NAME (60 mg/kg) groups. l-NAME was

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N. Adachi et al. / Neuroscience Letters 288 (2000) 151±154

dissolved in saline (10 mg/ml), and 7-NI was suspended in peanut oil (40 mg/ml) with sonication. Each agent or vehicle was injected via an intraperitoneal catheter. The effects of 7-

NI and l-NAME on NO production in ischemic animals were also examined. Sixty gerbils were divided into three ischemic groups: 5, 10, and 15 min of ischemia. Animals in

Fig. 1. Effects of 7-nitroindazole (7-NI) and N G-nitro-l-arginine methyl ester (l-NAME) on striatal NOx2 levels in non-ischemic (A), 5-min ischemic (B), 10-min ischemic (C), and 15-min ischemic (D) gerbils. Each value represents the mean ^ SD (%) of the basal level. B designates the basal level by averaging two consecutive dialysate samples. 7-NI (80 mg/kg) or l-NAME (60 mg/kg) was administered intraperitoneally as shown by the arrow. Values at 0 min represent NOx2 levels in dialysates collected during occlusion (B±D). *P , 0:05, **P , 0:01 compared with each corresponding sham vehicle group; 1P , 0:05, 11P , 0:01 compared with each corresponding ischemic 7-NI or l-NAME group; #P , 0:05, ##P , 0:01 compared with each corresponding ischemic vehicle group, respectively.

N. Adachi et al. / Neuroscience Letters 288 (2000) 151±154

each group were further divided into four groups: peanut oil, 7-NI, saline, and l-NAME groups. In the 5-min ischemic groups, ischemia was induced 30 min after the intraperitoneal injection. In the 10-min and 15-min ischemic groups, 7NI or l-NAME was injected immediately after the onset of ischemia, because the gerbils treated with such a dose of lNAME prior to ischemia died during ischemia. In the second experiment, the histological outcomes were examined. Fifty-four gerbils were divided into nine groups: sham-operated group; 5-min ischemic oil or 7-NI groups; 5min ischemic saline or l-NAME groups; 10-min ischemic oil or 7-NI groups; and 10-min ischemic saline or l-NAME groups. Each drug was administered and ischemia was induced by the method described above. Seven days after surgery, neuronal damage in the striatum was assessed using a scoring system described by Zaidan and Sims [14]: 0, ,3% affected neurons; 1, 3±25% affected neurons; 2, 26± 50% affected neurons; 3, .50% affected neurons. In another set of 16 animals, the effects of these agents on mean arterial blood pressure were assessed. After anesthesia, a catheter was inserted into the femoral artery. After a 30-min stabilization period at normothermia, the mean arterial blood pressure was recorded, and each drug or vehicle was given via the intraperitoneal catheter. The results of microdialysis are expressed as percentages of the basal values (the mean of two values before ischemia or drug administration). Statistical signi®cance was determined by analysis of variance, followed by Fisher's protected least signi®cant difference test. The data from the histological experiment were assessed with the Mann±Whitney U test. The results of mean arterial blood pressure were analyzed by Student's t-test. The basal level of NOx2 in the striatum was 0.33 ^ 0.10 mM (n ˆ 35). The striatal NOx2 levels in non-ischemic animals were decreased to ~50% of the control level by the 7-NI treatment (Fig. 1A). Likewise, l-NAME decreased the levels of NOx2 to ~60%. Fig. 1B±D shows the effects of 7-NI and l-NAME on NOx2 levels after various durations of ischemia. The levels of NOx2 were decreased during all durations of ischemia, and the levels of NOx2 were increased after reperfusion. 7-NI completely prevented the postischemic increase in the NOx2 levels after 5 min of ischemia, although the levels of NOx2 were not decreased below the sham control level (Fig. 1B). On the other hand, l-NAME decreased the levels to ,50% of the sham control level. In the 7-NI group subjected to 10 min of ischemia, the levels of NOx2 from 45 to 105 min after reperfusion were lower than those in the ischemic oil group, but still higher than those in the sham oil group (Fig. 1C). l-NAME completely abolished the post-ischemic increase after 10 min of ischemia. In animals subjected to 15 min of ischemia, the increase in the NOx2 levels was observed transiently in the oil group (Fig. 1D). 7-NI did not affect the increase, whereas lNAME abolished it. The extent of neuronal damage by 5-min ischemia was small in both the oil- and saline-treated animals (Fig. 2). The

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Fig. 2. The extent of ischemic neuronal damage in gerbil striatum and the effects of 7-NI or l-NAME. Forebrain ischemia for 5 or 10 min was induced, and neuronal damage was evaluated after 7 days using the scoring system. Values obtained from individual animals are shown. *P , 0:05, **P , 0:01 compared with each corresponding vehicle group; #P , 0:01 compared with the sham-operated group, respectively.

damage score was signi®cantly greater in the 7-NI- and lNAME-treated animals than in the corresponding vehicletreated animals. Neuronal damage by 10-min ischemia was severe in all groups, and there were no signi®cant differences among groups. Mean arterial blood pressure in the lNAME group was increased 15 and 30 min after the administration (Table 1). The ®ndings from the microdialysis experiments suggest that (1) the striatal NO level in non-ischemic states originates from nNOS, (2) the increased NO production after a short duration of ischemia is derived from mainly nNOS and partly eNOS, and (3) NO production after a long duration of ischemia is induced by eNOS. Dissimilar to the present results, 7-NI did not change the basal NO level in the hippocampal CA1 ®eld in our previous study, although l-NAME decreased it [9]. This implies that the basal NO production in the CA1 region is dependent on the action of eNOS. In a study on immunohistochemistry, intense immunoreactivity of nNOS was found in cell bodies and processes of mediumlarge aspiny neurons in the striatum, while eNOS immunoreactivity was selectively concentrated in the CA1 region [3]. The difference in the distributions of nNOS and eNOS may

Table 1 The effects of 7-NI and l-NAME on mean arterial blood pressure (mmHg) a

Oil 7-NI Saline l-NAME a

Before

15 min

30 min

80 ^ 9 80 ^ 7 85 ^ 8 79 ^ 7

76 ^ 9 81 ^ 8 81 ^ 4 93 ^ 6* ,**

77 ^ 10 75 ^ 9 78 ^ 7 90 ^ 6* ,²

Values are means ^ SD of four animals. *P , 0.01 compared with each corresponding value before injection.**P , 0.01, ² P , 0.05, compared with each corresponding vehicle group, respectively.

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N. Adachi et al. / Neuroscience Letters 288 (2000) 151±154

have resulted in the difference in the basal NO production between the two regions. With respect to NO production in the post-ischemic period, a marked increase in the NO level was found in the striatum after 5 min of ischemia, although there was no increase in the CA1 ®eld [9]. Following ischemia, the cytosolic Ca 21 concentration increases by the Ca 21 in¯ux, which leads to the activation of NOS [4]. The sustained increase in the striatal NO production in the present study may have been caused by a persistent activation of NOS. On the other hand, the increased NO production after a long duration of ischemia was not inhibited by 7-NI in the striatum, but by l-NAME, while NO production in the CA1 ®eld was abolished by either 7-NI or l-NAME [9]. These ®ndings indicate that predominant NO production after a long ischemic period is dependent on nNOS in the CA1 ®eld, and on eNOS in the striatum. However, the present pharmacological approach cannot determine the cellular sources of NO. In the present study, ischemia for 5 min did not produce signi®cant neuronal damage in the striatum, and either 7-NI or l-NAME aggravated the damage. Therefore, it is unlikely that the increased NO production in the striatum in the 5min ischemic gerbils is pathological. NO has been demonstrated to reduce neurotoxicity by downregulating the NMDA receptors [10]. It also contributes to vasodilation and anti-aggregation of platelets. The inhibition of NO production by 7-NI or l-NAME observed in this study may be detrimental by altering post-ischemic blood ¯ow and the NMDA receptor activation. In contrast, NO interacts with superoxide to produce the highly toxic peroxynitrite anion, which is regarded as a predominant mechanism for the aggravation [1]. However, the time course of superoxide production is different from that of NO with each ischemic duration. While the increased superoxide production after 5min ischemia is transient, 10-min ischemia produces a lasting increase [7]. Therefore, peroxynitrite toxicity seems to be marked after a prolonged duration of ischemia. Although 7-NI showed no effect on eNOS in the brain in several animal species [11,13,15], there are no available data about its speci®city for nNOS in gerbils. Dissimilar to l-NAME, 7-NI did not increase arterial blood pressure. This may show the absence of the inhibition of eNOS activity in gerbils. In ischemia, iNOS is also responsible for NO production [2,12]. Because l-NAME inhibits all three isoforms and 7-NI inhibits nNOS and iNOS, the observed inhibitory effects might result from the inhibition of iNOS. However, since the enzyme activity of iNOS develops more than 12 to 24 h after ischemia [5,6], the inhibition of iNOS by l-NAME or 7-NI is unlikely. In summary, nNOS and eNOS make different contribu-

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