- Email: [email protected]
Consecutive evaluation of nitric oxide production after transient cerebral ischemia in the rat hippocampus using in vivo brain microdialysis
Neuroscience Letters 240 (1998) 53–57
Consecutive evaluation of nitric oxide production after transient cerebral ischemia in the rat hippocampus using in vivo brain microdialysis Hiroko Togashi a ,*, Kiyoshi Mori a, Ken-ichi Ueno a, Machiko Matsumoto a, Noriyuki Suda b, Hideya Saito b, Mitsuhiro Yoshioka a a
First Department of Pharmacology, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo 060, Japan b Department of Clinical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-02, Japan Received 24 June 1997; received in revised form 27 November 1997; accepted 28 November 1997
Abstract The time-course effects of transient cerebral ischemia on nitric oxide (NO) formation in the rat hippocampus were evaluated by the consecutive determination of oxidative NO metabolites (NO−2 and NO−3), using brain microdialysis under the freely moving condition. Bilateral carotid artery occlusion (CAO; 2-vessel occlusion, 2VO; 10 and 20 min) and combined vertebral artery occlusion (4VO; 10 min) produced a transient increase in hippocampal NO−2 and NO−3 levels, according to the duration and degree of ischemic insults. In addition, 4VO produced a gradual increase in hippocampal NO−2 and NO−3 levels over a 24 h period after reperfusion, which was abolished by an inducible NO synthase inhibitor, aminoguanidine (10 mg/kg, intraperitoneally). These findings suggest that the dynamic changes in oxidative NO metabolite levels reflect NO production following transient cerebral ischemia, which is possibly mediated in part by an inducible NO synthase, in the rat hippocampus. 1998 Elsevier Science Ireland Ltd.
Keywords: Nitric oxide; Nitrite; Nitrate; Cerebral ischemia; 2-Vessel occlusion; 4-Vessel occlusion; Hippocampus; In vivo brain microdialysis
Evidence has accumulated that transient ischemic insults to the brain trigger diverse biochemical perturbations that result in neuronal cell injury and death. Significant roles of nitric oxide (NO) are postulated in the pathophysiological processes following cerebral ischemia [5]. The widespread localization of NO synthase (NOS) in the brain suggests that NO is synthesized in neuronal and grial cells in various cerebral regions, where it may act as an interneuronal messenger and/or neurotoxin [2]. However, it is not fully understood whether the diverse biological changes that are attributed to transient ischemia are consistent with NO production and if so which NOS isoform is involved. More direct evidence for NO involvement in the diverse biologi-
* Corresponding author. Tel.: +81 11 7065059; fax: +81 11 7067872; e-mail: [email protected]
cal changes is needed before we can assess its specific pathophysiological roles in ischemia-reperfusion injury. Especially, it is necessary to evaluate the dynamics of NO production associated with transient cerebral ischemia, since the effects of NO on the ischemic brain are thought to be dependent on the stage of evolution of the ischemic process [5]. NO is an unstable molecule that is easily degraded into nitrite (NO−2) and nitrate (NO−3) ions [15]. Recently an in vivo assay system, combined with a microdialysis technique and an on-line high performance liquid chromatography (HPLC) system equipped with a spectrophotometer, has been developed for consecutive measurement of the cerebral concentrations of oxidative NO metabolites, NO−2 and NO−3 [9,14]. These stable NO metabolite levels have been reported to reflect the levels of regional NO production/ release in the rat striatum and cerebellum [11,16].
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00918- X
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H. Togashi et al. / Neuroscience Letters 240 (1998) 53–57
The present study was designed to elucidate the dynamic changes in the levels of hippocampal NO production/release following transient ischemic insults to the brain in the conscious rat. To clarify the time-course effects of transient ischemia, we measured NO−2 and NO−3 levels, as indices of NO formation. Two different types of cerebral ischemia model were used; the 2-vessel occlusion (2VO) model with an incomplete cerebral ischemia produced by bilateral common carotid artery occlusion (CAO) [4] and the 4-vessel occlusion (4VO) model with a complete cerebral ischemia produced by CAO and combined vertebral artery occlusion as described by Pulsinell and Brierley [13]. In order to assess whether these oxidative NO metabolite levels measured by the in vivo assay system, would reflect the regional NO production, the effect of N-methyl-D-aspartate (NMDA), which is known to stimulate NO production, on hippocampal NO−2 and NO−3 levels was also examined. Male, Wistar rats (12–15 weeks old; Shizuoka Laboratory Animal Center, Hamamatsu, Japan) were used. The animals were allowed free access to food and water in a room with a 12:12 h light-dark alternation cycle. All chemicals used were of analytical grade and were obtained from commercially available sources: NMDA, AP5 and aminoguanidine hemisulfate were purchased from Sigma Chemical, St. Louis, MO, USA, and ketamine hydrochloride from Sanyo, Tokyo, Japan. An 8 mm guide cannula for brain microdialysis was stereotaxically implanted into the hippocampus of a rat anesthetized with ketamine (100 mg/kg, i.p.) and was attached to the skull with dental cement. The coordinates for the guide cannula were as follows [12]: rostral-caudal, 5.8 mm; lateral, 3.0 mm; ventral, 3.3 mm; 30° from ventral axis. After a 2 day recovery period, a concentric microdialysis probe with a 3 mm tip (Eicom, Kyoto, Japan) was inserted into the guide cannula and the hippocampus was perfused with Ringer’s solution at a flow rate of 1 ml/min. After a 2 h equilibration period, the dialysate was collected every 10 min from the freely moving rat for up to 2–12 h
following an ischemic insult and automatically injected into the on-line HPLC system. NO−2 and NO−3 levels were also determined 24 h after reperfusion. NMDA (1–10 mM) was administered through the microdialysis probe for a 20 min period. A NMDA receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid (AP5; 1 mM), was added to the perfusion solution and was infused for 60 min from 40 min prior to the onset of the NMDA (1 mM) perfusion. Rats were subjected to electrical permanent occlusion of bilateral vertebral arteries (for 4VO model) [13] or to sham operation (for 2VO model) under halothane anesthesia. Seven days after, bilateral common carotid arteries were occluded for 10 or 20 min by the ligature technique reported by Himori et al. [4]. An inducible NOS (iNOS) inhibitor, aminoguanidine hemisulfate (10 mg/kg), was intraperitoneally injected into the rat 30 min prior to CAO. At the end of the experiment, brains were removed to assess the correct insertion of the dialysis probe. NO−2 and NO−3 levels in the hippocampal dialysate were measured using the HPLC-NO detector system (ENO-10; Eicom, Kyoto, Japan), as reported previously [9,14]. In brief, NO−2 and NO−3 were separated on a reverse phase column (NO-PAK, 4.6 × 50 mm; Eicom, Kyoto, Japan) and NO−3 was reduced to NO−2 by passage through a reduction column (NO-RED; Eicom, Kyoto, Japan). NO−2 was determined as the azo dye compound formed by the Griess reaction [3] using a spectrophotometer. These oxidative NO products were also evaluated as NOx− (NO−2 plus NO−3). To assess the dialysis efficiency, in vitro recovery of dialysis probes was estimated before each assay. NO−2 and NO−3 levels in the dialysate are expressed as percentages of the respective amounts of NO−2 and NO−3 in the 10 min dialysate obtained before NMDA perfusion or CAO was carried out. Data are expressed as the mean ± SEM. Statistical differences between group means were determined using analysis of variance. The differences between group means were assessed using Student’s t-test for two groups and Dunnett’s or Duncan’s multiple comparison test for three or more
Fig. 1. Effects of NMDA on oxidative NO metabolite levels in the rat hippocampus. Chromatograms showing 100 pmol of the standard nitrite (NO−2) and nitrate (NO−3) solution (A), NO−2 and NO−3 levels in the dialysate before (B) and after (C) 1 mM of NMDA perfusion (1 ml/min, 20 min) through the microdialysis probe into the rat hippocampus. The time course changes in NOx (NO−2 plus NO−3) levels after 1 and 10 mM NMDA perfusion (D) and the blockade of a NMDA receptor antagonist, AP5, on the maximum responses induced by 1 mM NMDA perfusion (E). AP5 (1 mM) was perfused before NMDA (40 min) and co-perfused with NMDA (20 min). Values were expressed as a percentage of the basal value obtained before NMDA perfusion. Bars represent SEM. The number of rats used is given in parentheses. *P , 0.05 and **P , 0.01.
H. Togashi et al. / Neuroscience Letters 240 (1998) 53–57
groups. P-Values less than 0.05 were considered as the level of significance. Extracellular levels of NO−2 and NO−3 in the rat hippocampus were 5.72 ± 0.50 and 35.87 ± 2.38 pmol/10 ml of the 10 min dialysate (n = 54), respectively. The in vitro recovery of the dialysis probe was 40.2 ± 2.4% for NO−2 and 39.3 ± 2.2% for NO−3 (n = 11). As shown in Fig. 1, NMDA, when introduced into the perfusion solution, increased both NO−2 and NO−3 levels in the hippocampus in a concentration (1–10 mM)-dependent manner. NOx− as well as NO−2 and NO−3 levels increased to ~150 and ~300% of the basal levels as the result of 1 and 10 mM NMDA perfusion, respectively (Fig. 1D). The maximum responses were significantly different from those in the control rats. A NMDA receptor antagonist, AP5 (1 mM), which was added to the perfusion medium 40 min prior to the onset of the NMDA (1 mM) perfusion, significantly inhibited the NMDA-induced increase in hippocampal NOx levels (Fig. 1E). The basal levels of NO−2 and NO−3 were not affected by AP5 perfusion (data not shown). As shown in Fig. 2, 2VO produced a temporal increase in hippocampal NO−2 and NO−3 levels in an occlusion time (10 and 20 min)-dependent manner. Oxidative NO metabolite levels increased to ~120 and ~150% of the basal levels following 10 and 20 min occlusions, respectively (Fig. 2A). The maximum response, which was observed 20–40 min after re-circulation, was significantly different from that in the sham-operated rat (Fig. 2B). A 10 min 4VO produced a biphasic increase in hippocampal NO−2 and NO−3 levels: a temporal increase as observed in 2VO rats and a gradual increase over a period of 24 h after reperfusion (Fig. 3A). The maximum response obtained 20–40 min after recirculation in 4VO rats was 130.17 ± 13.01, 141.47 ± 14.34 and 137.82 ± 13.02% of the basal NO−2, NO−3 and NOx levels, respectively (n = 7). The response in NOx was significantly different from that in the sham-operated rats (98.28 ± 3.82%, n = 11) and slightly
Fig. 2. Effects of 2-vessel occlusion (2VO) on oxidative NO metabolite levels in the rat hippocampus. The time course changes (A) and the maximum responses (B) in NOx (NO−2 plus NO−3) levels after bilateral common CAO for 10 and 20 min. Values were expressed as a percentage of the basal value obtained before CAO or sham operation. Bars represent SEM. The number of rats used is given in parentheses. *P , 0.05 and **P , 0.01.
55
Fig. 3. Effects of 4-vessel occlusion (4VO) on oxidative NO metabolite levels in the rat hippocampus. Specimen records showing a gradual increase in hippocampal NO−2 and NO−3 levels over 12 h after 4VO. Data are expressed as NO−2 and NO−3 levels in the 60 min dialysate (60 ml). (B) Changes in NOx levels 24 h after 4VO (10 min) and its blockade of an inducible NO synthase inhibitor, aminoguanidine. Aminoguanidine (10 mg/kg) was intraperitoneally injected 30 min before occlusion. Values were expressed as a percentage of the basal value obtained before occlusion or sham operation. Bars represent SEM. The number of rats used is given in parentheses. **P , 0.01 versus sham-operated controls, #P , 0.05 versus 4VO.
higher than that in the 2VO (10 min) rats (125.96 ± 14.42%, n = 7). The NOx- level as well as NO−2 and NO−3 levels in the 4VO rats, measured 24 h after reperfusion, was significantly higher than that in the sham-operated and 2VO rats. As shown in Fig. 3B, intraperitoneal administration of aminoguanidine (10 mg/kg) 30 min prior to CAO significantly abolished the post-ischemic response of the hippocampal NOx-level of 4VO rats which was observed 24 h after reperfusion. The response observed at earlier times (,1 h) was not affected by aminoguanidine, but significantly higher than that in the sham operated control (120.58 ± 3.14%, n = 7, P , 0.05). The NOx−, NO−2 and NO−3 levels, measured 24 h after reperfusion, were not significantly different from those in the sham-operated and 2VO rats (Fig. 3B). Dynamic evaluation of NO production is thought to be difficult since NO is an unstable molecule with a very short physiological half-life of 3–5 s [7]. In the present study, in vivo brain microdialysis, coupled with on-line HPLC and the colorimetric assay system [9,14] based on the Griess reaction [3], was used for continuous measurement of extracellular NO−2 and NO−3 levels for evaluation of NO production/release. NMDA, which is well known to stimulate NO synthesis from an amino acid precursor L-arginine via a NMDA type-glutamate receptor-mediated mechanism [2], produced a concentration-dependent increase in extracellular NO−2 and NO−3 levels in the perfused region, the hippocampus. The increase was abolished by concurrent perfusion of a NMDA receptor antagonist, AP5. These results indicate that hippocampal NO−2 and NO−3 levels may reflect regional NO production/release. The present data demonstrated that transient cerebral ischemia resulted in an increase in the hippocampal levels
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H. Togashi et al. / Neuroscience Letters 240 (1998) 53–57
of NO−2 and NO−3 in the conscious rat. The time-course response in these oxidative NO metabolite levels was dependent upon the degree and duration of ischemic insults to the brain. Namely, 2VO (10 and 20 min) and 4VO (10 min) resulted in a temporal increase in hippocampal NO−2 and NO−3 levels. In addition, 4VO produced a gradual increase in these NO metabolites over a 24 h period after reperfusion. These time-course changes in hippocampal NO−2 and NO−3 levels may reflect those in the regional NO production/release following transient cerebral ischemia. Thus, consecutive in vivo quantification of NO metabolites is useful to evaluate the dynamics of cerebral NO production and to delineate the role of NO in the pathophysiological processes associated with ischemic insults to the brain. Cerebral ischemia is reported to produce an increase in protein expression and enzymatic activity of endothelial/ neuronal constitutive NOS and iNOS [5]. However, the time-course of iNOS expression is at variance with that of constitutive NOS expression. In the pathogenesis of cerebral ischemia, the effects of NO on the ischemic brain are dependent upon the stage of evolution of the ischemic process [5]. From these points of view, it is important to determine the time-course of NO production in the post-ischemic brain. In our present study, 4VO produced a biphasic increase in oxidative NO metabolites. The precise mechanism underlying our observations is still unknown. However, we have recently reported a crucial role of interleukin (IL)-1b, a proinflammatory citokine, which stimulates iNOS expression in the ischemic brain [1]; IL-1b receptor antagonist ameliorated the 4VO-induced deficiency of long-term potentiation (LTP) [10]. In the rat treated with IL-1b antagonist, the hippocampal NO metabolite levels measured 24 h after 4VO did not increase. In addition, the present study demonstrated that the iNOS inhibitor aminoguanidine abolished the 4VO-induced response at later times (24 h), without a significant effect on the response at earlier times (,1 h). Thus, iNOS-mediated mechanism is likely to contribute to NO production following transient cerebral ischemia at later times. Indeed, iNOS is known to be expressed more than 6–12 h after cerebral ischemia and resulting in large amounts of NO production and in the progression of the post-ischemic tissue damage [5,6]. These findings might support a detrimental role of iNOS in the post-ischemic brain. On the other hand, we also found that both 2VO and 4VO produced a temporal increase (,1 h) in the levels of hippocampal NO metabolites according to the degree or duration of ischemic insults. It remains to be determined which NOS isoform is involved in this response. It is of note that the time-course pattern of NO metabolite changes in the post-ischemic hippocampus was similar to that after regional NMDA perfusion and that of nNOS activity in focal cerebral ischemia [8]. NMDA receptor-nNOS pathway might be involved in the temporal increase of NO production at earlier time, which resulted in the postischemic brain damage [5]. This explains why 2VO produced a functional deficiency, namely attenuated hippocam-
pal LTP [10], even though 2VO did not exert any significant effects on NO metabolite levels at later times as in 4VO. Thus, transient cerebral ischemia possibly induces hippocampal NO production via different mechanisms, although more direct evidence is needed to clarify whether the increase in NO metabolites observed after transient cerebral ischemia arise from the stimulation of NMDA receptor or not. NO−2 is thought to be a stable NO metabolite, however, it is also reported to be readily oxidized to NO−3, particularly by oxyhaemoglobin [15]. This might be true in the rat hippocampus, since we noted that the basal level of NO−3 is markedly higher than that of NO−2. On the other hand, Yamada et al. [16] reported that the cerebellum stimulation, NMDA receptor stimulation or potassium depolarization, did not always elicit parallel changes in extracellular NO−2 and NO−3 levels in the conscious rat. However, in our study, the changes in NO−2 and NO−3 levels basically paralleled each other, although a slight difference in the time-courses or the amplitudes of the responses to ischemic insults was noted. Therefore, both NO−2 and NO−3 levels in the rat hippocampus appear to be related to the regional NO formation associated with transient ischemia. In summary, the results of the present study demonstrated that hippocampal NO−2 and NO−3 levels measured by in vivo microdialysis and an on-line HPLC system coupled with the Griess reaction may reflect the regional NO production/ release. The consecutive measurement of hippocampal NO−2 and NO−3 levels showed that transient cerebral ischemia resulted in an increase in these NO metabolites according to the degree and duration of the ischemic insults. These dynamic changes in oxidative NO metabolites might provide evidence for a crucial role of NO in the pathophysiological process in the post-ischemic brain. The authors are grateful to Mr. Katsunori Miura, Mr. Takuya Fukuda and Miss. Mikako Yamada for their technical assistance. This work was partly supported by a Grant-in-Aid for General Scientific Research (No. 08670097) from the Ministry of Education, Science, Sports and Culture of Japan. [1] Chao, C.C., Hu, S., Molitor, T.W., Shaskan, E.G. and Peterson, P.K., Activated microglia mediate neuronal cell injury via a nitric oxide mechanism, J. Immunol., 149 (1992) 2736–2741. [2] Garthwaite, J., Glutamate, nitric oxide and cell-cell signaling in the nervous system, Trends Neurosci., 14 (1991) 60–67. [3] Griess, J.P., On a new series of bodies in which nitrogen is substituted for hydrogen, Phil. Trans. R. Soc. Lond., 154 (1864) 667–731. [4] Himori, N., Watanabe, H., Akaike, N., Kurasawa, M., Itoh, J. and Tanaka, Y., Cerebral ischemia model with conscious mice, J. Pharmacol. Methods, 23 (1990) 311–327. [5] Iadecola, C., Bright and dark sides of nitric oxide in ischemic brain injury, Trends Neurosci., 20 (1997) 132–139. [6] Iadecola, C., Zhang, F., Casey, R., Clark, H.B. and Ross, E., Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia, Stroke, 27 (1996) 1373–1380.
H. Togashi et al. / Neuroscience Letters 240 (1998) 53–57 [7] Ignarro, L.J., Buga, G.M., Wood, K.S. and Byrns, R.E., Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide, Proc. Natl. Acad. Sci. USA, 84 (1987) 9265–9269. [8] Kader, A., Frazzini, V.I., Solomon, R.A. and Trifiletti, R.R., Nitric oxide production during focal cerebral ischemia in rats, Stroke, 24 (1993) 1709–1716. [9] Luo, D., Knezevich, S. and Vincent, S.R., N-Methyl-D-aspartate-induced nitric oxide release: an in vivo microdialysis study (Letter), Neuroscience, 57 (1993) 897–900. [10] Mori, K., Togashi, H., Itoh, Y., Matsumoto, M., Ueno, K. and Yoshioka, M., Effects of transient cerebral ischemia on nitric oxide metabolites and long-term potentiation in vivo: its blockade by IL-1b analog. In S. Moncada, N. Toda, H. Maeda and E. Higgs (Eds.), The Biology of Nitric Oxide, Part 6, Portland Press, London, 1997, in press. [11] Ohta, K., Araki, N., Shibata, M., Hamada, J., Komatsumoto, S., Shimazu, K. and Fukuuchi, Y., A novel in vivo assay system for consecutive measurement of brain nitric oxide production com-
[12] [13] [14]
[15]
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bined with the microdialysis technique, Neurosci. Lett., 176 (1994) 165–168. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1986. Pulsinell, W.A. and Brierley, J.B., A new model of bilateral hemisphemia in the unanesthetized rat, Stroke, 10 (1979) 267–272. Shintani, F., Kanba, S., Nakaki, T., Sato, K., Yagi, G., Kato, R. and Asai, M., Measurement by in vivo brain microdialysis of nitric oxide release in the rat cerebellum, J. Psychiatr. Neurosci., 19 (1994) 217–221. Spagnuolo, C., Rinelli, P,. Coletta, M., Chiancone, E. and Ascoli, F., Oxidation reaction of human oxyhemoglobin with nitrite: a reexamination, Biochem. Biophys. Acta, 911 (1987) 59–65. Yamada, K. and Nabeshima, T., Simultaneous measurement of nitrite and nitrate levels as indices of nitric oxide release in the cerebellum of the conscious rats, J. Neurochem., 68 (1997) 1234–1243.
Consecutive evaluation of nitric oxide production after transient cerebral ischemia in the rat hippocampus using in vivo brain microdialysis Hiroko Togashi a ,*, Kiyoshi Mori a, Ken-ichi Ueno a, Machiko Matsumoto a, Noriyuki Suda b, Hideya Saito b, Mitsuhiro Yoshioka a a
First Department of Pharmacology, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo 060, Japan b Department of Clinical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-02, Japan Received 24 June 1997; received in revised form 27 November 1997; accepted 28 November 1997
Abstract The time-course effects of transient cerebral ischemia on nitric oxide (NO) formation in the rat hippocampus were evaluated by the consecutive determination of oxidative NO metabolites (NO−2 and NO−3), using brain microdialysis under the freely moving condition. Bilateral carotid artery occlusion (CAO; 2-vessel occlusion, 2VO; 10 and 20 min) and combined vertebral artery occlusion (4VO; 10 min) produced a transient increase in hippocampal NO−2 and NO−3 levels, according to the duration and degree of ischemic insults. In addition, 4VO produced a gradual increase in hippocampal NO−2 and NO−3 levels over a 24 h period after reperfusion, which was abolished by an inducible NO synthase inhibitor, aminoguanidine (10 mg/kg, intraperitoneally). These findings suggest that the dynamic changes in oxidative NO metabolite levels reflect NO production following transient cerebral ischemia, which is possibly mediated in part by an inducible NO synthase, in the rat hippocampus. 1998 Elsevier Science Ireland Ltd.
Keywords: Nitric oxide; Nitrite; Nitrate; Cerebral ischemia; 2-Vessel occlusion; 4-Vessel occlusion; Hippocampus; In vivo brain microdialysis
Evidence has accumulated that transient ischemic insults to the brain trigger diverse biochemical perturbations that result in neuronal cell injury and death. Significant roles of nitric oxide (NO) are postulated in the pathophysiological processes following cerebral ischemia [5]. The widespread localization of NO synthase (NOS) in the brain suggests that NO is synthesized in neuronal and grial cells in various cerebral regions, where it may act as an interneuronal messenger and/or neurotoxin [2]. However, it is not fully understood whether the diverse biological changes that are attributed to transient ischemia are consistent with NO production and if so which NOS isoform is involved. More direct evidence for NO involvement in the diverse biologi-
* Corresponding author. Tel.: +81 11 7065059; fax: +81 11 7067872; e-mail: [email protected]
cal changes is needed before we can assess its specific pathophysiological roles in ischemia-reperfusion injury. Especially, it is necessary to evaluate the dynamics of NO production associated with transient cerebral ischemia, since the effects of NO on the ischemic brain are thought to be dependent on the stage of evolution of the ischemic process [5]. NO is an unstable molecule that is easily degraded into nitrite (NO−2) and nitrate (NO−3) ions [15]. Recently an in vivo assay system, combined with a microdialysis technique and an on-line high performance liquid chromatography (HPLC) system equipped with a spectrophotometer, has been developed for consecutive measurement of the cerebral concentrations of oxidative NO metabolites, NO−2 and NO−3 [9,14]. These stable NO metabolite levels have been reported to reflect the levels of regional NO production/ release in the rat striatum and cerebellum [11,16].
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00918- X
54
H. Togashi et al. / Neuroscience Letters 240 (1998) 53–57
The present study was designed to elucidate the dynamic changes in the levels of hippocampal NO production/release following transient ischemic insults to the brain in the conscious rat. To clarify the time-course effects of transient ischemia, we measured NO−2 and NO−3 levels, as indices of NO formation. Two different types of cerebral ischemia model were used; the 2-vessel occlusion (2VO) model with an incomplete cerebral ischemia produced by bilateral common carotid artery occlusion (CAO) [4] and the 4-vessel occlusion (4VO) model with a complete cerebral ischemia produced by CAO and combined vertebral artery occlusion as described by Pulsinell and Brierley [13]. In order to assess whether these oxidative NO metabolite levels measured by the in vivo assay system, would reflect the regional NO production, the effect of N-methyl-D-aspartate (NMDA), which is known to stimulate NO production, on hippocampal NO−2 and NO−3 levels was also examined. Male, Wistar rats (12–15 weeks old; Shizuoka Laboratory Animal Center, Hamamatsu, Japan) were used. The animals were allowed free access to food and water in a room with a 12:12 h light-dark alternation cycle. All chemicals used were of analytical grade and were obtained from commercially available sources: NMDA, AP5 and aminoguanidine hemisulfate were purchased from Sigma Chemical, St. Louis, MO, USA, and ketamine hydrochloride from Sanyo, Tokyo, Japan. An 8 mm guide cannula for brain microdialysis was stereotaxically implanted into the hippocampus of a rat anesthetized with ketamine (100 mg/kg, i.p.) and was attached to the skull with dental cement. The coordinates for the guide cannula were as follows [12]: rostral-caudal, 5.8 mm; lateral, 3.0 mm; ventral, 3.3 mm; 30° from ventral axis. After a 2 day recovery period, a concentric microdialysis probe with a 3 mm tip (Eicom, Kyoto, Japan) was inserted into the guide cannula and the hippocampus was perfused with Ringer’s solution at a flow rate of 1 ml/min. After a 2 h equilibration period, the dialysate was collected every 10 min from the freely moving rat for up to 2–12 h
following an ischemic insult and automatically injected into the on-line HPLC system. NO−2 and NO−3 levels were also determined 24 h after reperfusion. NMDA (1–10 mM) was administered through the microdialysis probe for a 20 min period. A NMDA receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid (AP5; 1 mM), was added to the perfusion solution and was infused for 60 min from 40 min prior to the onset of the NMDA (1 mM) perfusion. Rats were subjected to electrical permanent occlusion of bilateral vertebral arteries (for 4VO model) [13] or to sham operation (for 2VO model) under halothane anesthesia. Seven days after, bilateral common carotid arteries were occluded for 10 or 20 min by the ligature technique reported by Himori et al. [4]. An inducible NOS (iNOS) inhibitor, aminoguanidine hemisulfate (10 mg/kg), was intraperitoneally injected into the rat 30 min prior to CAO. At the end of the experiment, brains were removed to assess the correct insertion of the dialysis probe. NO−2 and NO−3 levels in the hippocampal dialysate were measured using the HPLC-NO detector system (ENO-10; Eicom, Kyoto, Japan), as reported previously [9,14]. In brief, NO−2 and NO−3 were separated on a reverse phase column (NO-PAK, 4.6 × 50 mm; Eicom, Kyoto, Japan) and NO−3 was reduced to NO−2 by passage through a reduction column (NO-RED; Eicom, Kyoto, Japan). NO−2 was determined as the azo dye compound formed by the Griess reaction [3] using a spectrophotometer. These oxidative NO products were also evaluated as NOx− (NO−2 plus NO−3). To assess the dialysis efficiency, in vitro recovery of dialysis probes was estimated before each assay. NO−2 and NO−3 levels in the dialysate are expressed as percentages of the respective amounts of NO−2 and NO−3 in the 10 min dialysate obtained before NMDA perfusion or CAO was carried out. Data are expressed as the mean ± SEM. Statistical differences between group means were determined using analysis of variance. The differences between group means were assessed using Student’s t-test for two groups and Dunnett’s or Duncan’s multiple comparison test for three or more
Fig. 1. Effects of NMDA on oxidative NO metabolite levels in the rat hippocampus. Chromatograms showing 100 pmol of the standard nitrite (NO−2) and nitrate (NO−3) solution (A), NO−2 and NO−3 levels in the dialysate before (B) and after (C) 1 mM of NMDA perfusion (1 ml/min, 20 min) through the microdialysis probe into the rat hippocampus. The time course changes in NOx (NO−2 plus NO−3) levels after 1 and 10 mM NMDA perfusion (D) and the blockade of a NMDA receptor antagonist, AP5, on the maximum responses induced by 1 mM NMDA perfusion (E). AP5 (1 mM) was perfused before NMDA (40 min) and co-perfused with NMDA (20 min). Values were expressed as a percentage of the basal value obtained before NMDA perfusion. Bars represent SEM. The number of rats used is given in parentheses. *P , 0.05 and **P , 0.01.
H. Togashi et al. / Neuroscience Letters 240 (1998) 53–57
groups. P-Values less than 0.05 were considered as the level of significance. Extracellular levels of NO−2 and NO−3 in the rat hippocampus were 5.72 ± 0.50 and 35.87 ± 2.38 pmol/10 ml of the 10 min dialysate (n = 54), respectively. The in vitro recovery of the dialysis probe was 40.2 ± 2.4% for NO−2 and 39.3 ± 2.2% for NO−3 (n = 11). As shown in Fig. 1, NMDA, when introduced into the perfusion solution, increased both NO−2 and NO−3 levels in the hippocampus in a concentration (1–10 mM)-dependent manner. NOx− as well as NO−2 and NO−3 levels increased to ~150 and ~300% of the basal levels as the result of 1 and 10 mM NMDA perfusion, respectively (Fig. 1D). The maximum responses were significantly different from those in the control rats. A NMDA receptor antagonist, AP5 (1 mM), which was added to the perfusion medium 40 min prior to the onset of the NMDA (1 mM) perfusion, significantly inhibited the NMDA-induced increase in hippocampal NOx levels (Fig. 1E). The basal levels of NO−2 and NO−3 were not affected by AP5 perfusion (data not shown). As shown in Fig. 2, 2VO produced a temporal increase in hippocampal NO−2 and NO−3 levels in an occlusion time (10 and 20 min)-dependent manner. Oxidative NO metabolite levels increased to ~120 and ~150% of the basal levels following 10 and 20 min occlusions, respectively (Fig. 2A). The maximum response, which was observed 20–40 min after re-circulation, was significantly different from that in the sham-operated rat (Fig. 2B). A 10 min 4VO produced a biphasic increase in hippocampal NO−2 and NO−3 levels: a temporal increase as observed in 2VO rats and a gradual increase over a period of 24 h after reperfusion (Fig. 3A). The maximum response obtained 20–40 min after recirculation in 4VO rats was 130.17 ± 13.01, 141.47 ± 14.34 and 137.82 ± 13.02% of the basal NO−2, NO−3 and NOx levels, respectively (n = 7). The response in NOx was significantly different from that in the sham-operated rats (98.28 ± 3.82%, n = 11) and slightly
Fig. 2. Effects of 2-vessel occlusion (2VO) on oxidative NO metabolite levels in the rat hippocampus. The time course changes (A) and the maximum responses (B) in NOx (NO−2 plus NO−3) levels after bilateral common CAO for 10 and 20 min. Values were expressed as a percentage of the basal value obtained before CAO or sham operation. Bars represent SEM. The number of rats used is given in parentheses. *P , 0.05 and **P , 0.01.
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Fig. 3. Effects of 4-vessel occlusion (4VO) on oxidative NO metabolite levels in the rat hippocampus. Specimen records showing a gradual increase in hippocampal NO−2 and NO−3 levels over 12 h after 4VO. Data are expressed as NO−2 and NO−3 levels in the 60 min dialysate (60 ml). (B) Changes in NOx levels 24 h after 4VO (10 min) and its blockade of an inducible NO synthase inhibitor, aminoguanidine. Aminoguanidine (10 mg/kg) was intraperitoneally injected 30 min before occlusion. Values were expressed as a percentage of the basal value obtained before occlusion or sham operation. Bars represent SEM. The number of rats used is given in parentheses. **P , 0.01 versus sham-operated controls, #P , 0.05 versus 4VO.
higher than that in the 2VO (10 min) rats (125.96 ± 14.42%, n = 7). The NOx- level as well as NO−2 and NO−3 levels in the 4VO rats, measured 24 h after reperfusion, was significantly higher than that in the sham-operated and 2VO rats. As shown in Fig. 3B, intraperitoneal administration of aminoguanidine (10 mg/kg) 30 min prior to CAO significantly abolished the post-ischemic response of the hippocampal NOx-level of 4VO rats which was observed 24 h after reperfusion. The response observed at earlier times (,1 h) was not affected by aminoguanidine, but significantly higher than that in the sham operated control (120.58 ± 3.14%, n = 7, P , 0.05). The NOx−, NO−2 and NO−3 levels, measured 24 h after reperfusion, were not significantly different from those in the sham-operated and 2VO rats (Fig. 3B). Dynamic evaluation of NO production is thought to be difficult since NO is an unstable molecule with a very short physiological half-life of 3–5 s [7]. In the present study, in vivo brain microdialysis, coupled with on-line HPLC and the colorimetric assay system [9,14] based on the Griess reaction [3], was used for continuous measurement of extracellular NO−2 and NO−3 levels for evaluation of NO production/release. NMDA, which is well known to stimulate NO synthesis from an amino acid precursor L-arginine via a NMDA type-glutamate receptor-mediated mechanism [2], produced a concentration-dependent increase in extracellular NO−2 and NO−3 levels in the perfused region, the hippocampus. The increase was abolished by concurrent perfusion of a NMDA receptor antagonist, AP5. These results indicate that hippocampal NO−2 and NO−3 levels may reflect regional NO production/release. The present data demonstrated that transient cerebral ischemia resulted in an increase in the hippocampal levels
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of NO−2 and NO−3 in the conscious rat. The time-course response in these oxidative NO metabolite levels was dependent upon the degree and duration of ischemic insults to the brain. Namely, 2VO (10 and 20 min) and 4VO (10 min) resulted in a temporal increase in hippocampal NO−2 and NO−3 levels. In addition, 4VO produced a gradual increase in these NO metabolites over a 24 h period after reperfusion. These time-course changes in hippocampal NO−2 and NO−3 levels may reflect those in the regional NO production/release following transient cerebral ischemia. Thus, consecutive in vivo quantification of NO metabolites is useful to evaluate the dynamics of cerebral NO production and to delineate the role of NO in the pathophysiological processes associated with ischemic insults to the brain. Cerebral ischemia is reported to produce an increase in protein expression and enzymatic activity of endothelial/ neuronal constitutive NOS and iNOS [5]. However, the time-course of iNOS expression is at variance with that of constitutive NOS expression. In the pathogenesis of cerebral ischemia, the effects of NO on the ischemic brain are dependent upon the stage of evolution of the ischemic process [5]. From these points of view, it is important to determine the time-course of NO production in the post-ischemic brain. In our present study, 4VO produced a biphasic increase in oxidative NO metabolites. The precise mechanism underlying our observations is still unknown. However, we have recently reported a crucial role of interleukin (IL)-1b, a proinflammatory citokine, which stimulates iNOS expression in the ischemic brain [1]; IL-1b receptor antagonist ameliorated the 4VO-induced deficiency of long-term potentiation (LTP) [10]. In the rat treated with IL-1b antagonist, the hippocampal NO metabolite levels measured 24 h after 4VO did not increase. In addition, the present study demonstrated that the iNOS inhibitor aminoguanidine abolished the 4VO-induced response at later times (24 h), without a significant effect on the response at earlier times (,1 h). Thus, iNOS-mediated mechanism is likely to contribute to NO production following transient cerebral ischemia at later times. Indeed, iNOS is known to be expressed more than 6–12 h after cerebral ischemia and resulting in large amounts of NO production and in the progression of the post-ischemic tissue damage [5,6]. These findings might support a detrimental role of iNOS in the post-ischemic brain. On the other hand, we also found that both 2VO and 4VO produced a temporal increase (,1 h) in the levels of hippocampal NO metabolites according to the degree or duration of ischemic insults. It remains to be determined which NOS isoform is involved in this response. It is of note that the time-course pattern of NO metabolite changes in the post-ischemic hippocampus was similar to that after regional NMDA perfusion and that of nNOS activity in focal cerebral ischemia [8]. NMDA receptor-nNOS pathway might be involved in the temporal increase of NO production at earlier time, which resulted in the postischemic brain damage [5]. This explains why 2VO produced a functional deficiency, namely attenuated hippocam-
pal LTP [10], even though 2VO did not exert any significant effects on NO metabolite levels at later times as in 4VO. Thus, transient cerebral ischemia possibly induces hippocampal NO production via different mechanisms, although more direct evidence is needed to clarify whether the increase in NO metabolites observed after transient cerebral ischemia arise from the stimulation of NMDA receptor or not. NO−2 is thought to be a stable NO metabolite, however, it is also reported to be readily oxidized to NO−3, particularly by oxyhaemoglobin [15]. This might be true in the rat hippocampus, since we noted that the basal level of NO−3 is markedly higher than that of NO−2. On the other hand, Yamada et al. [16] reported that the cerebellum stimulation, NMDA receptor stimulation or potassium depolarization, did not always elicit parallel changes in extracellular NO−2 and NO−3 levels in the conscious rat. However, in our study, the changes in NO−2 and NO−3 levels basically paralleled each other, although a slight difference in the time-courses or the amplitudes of the responses to ischemic insults was noted. Therefore, both NO−2 and NO−3 levels in the rat hippocampus appear to be related to the regional NO formation associated with transient ischemia. In summary, the results of the present study demonstrated that hippocampal NO−2 and NO−3 levels measured by in vivo microdialysis and an on-line HPLC system coupled with the Griess reaction may reflect the regional NO production/ release. The consecutive measurement of hippocampal NO−2 and NO−3 levels showed that transient cerebral ischemia resulted in an increase in these NO metabolites according to the degree and duration of the ischemic insults. These dynamic changes in oxidative NO metabolites might provide evidence for a crucial role of NO in the pathophysiological process in the post-ischemic brain. The authors are grateful to Mr. Katsunori Miura, Mr. Takuya Fukuda and Miss. Mikako Yamada for their technical assistance. This work was partly supported by a Grant-in-Aid for General Scientific Research (No. 08670097) from the Ministry of Education, Science, Sports and Culture of Japan. [1] Chao, C.C., Hu, S., Molitor, T.W., Shaskan, E.G. and Peterson, P.K., Activated microglia mediate neuronal cell injury via a nitric oxide mechanism, J. Immunol., 149 (1992) 2736–2741. [2] Garthwaite, J., Glutamate, nitric oxide and cell-cell signaling in the nervous system, Trends Neurosci., 14 (1991) 60–67. [3] Griess, J.P., On a new series of bodies in which nitrogen is substituted for hydrogen, Phil. Trans. R. Soc. Lond., 154 (1864) 667–731. [4] Himori, N., Watanabe, H., Akaike, N., Kurasawa, M., Itoh, J. and Tanaka, Y., Cerebral ischemia model with conscious mice, J. Pharmacol. Methods, 23 (1990) 311–327. [5] Iadecola, C., Bright and dark sides of nitric oxide in ischemic brain injury, Trends Neurosci., 20 (1997) 132–139. [6] Iadecola, C., Zhang, F., Casey, R., Clark, H.B. and Ross, E., Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia, Stroke, 27 (1996) 1373–1380.
H. Togashi et al. / Neuroscience Letters 240 (1998) 53–57 [7] Ignarro, L.J., Buga, G.M., Wood, K.S. and Byrns, R.E., Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide, Proc. Natl. Acad. Sci. USA, 84 (1987) 9265–9269. [8] Kader, A., Frazzini, V.I., Solomon, R.A. and Trifiletti, R.R., Nitric oxide production during focal cerebral ischemia in rats, Stroke, 24 (1993) 1709–1716. [9] Luo, D., Knezevich, S. and Vincent, S.R., N-Methyl-D-aspartate-induced nitric oxide release: an in vivo microdialysis study (Letter), Neuroscience, 57 (1993) 897–900. [10] Mori, K., Togashi, H., Itoh, Y., Matsumoto, M., Ueno, K. and Yoshioka, M., Effects of transient cerebral ischemia on nitric oxide metabolites and long-term potentiation in vivo: its blockade by IL-1b analog. In S. Moncada, N. Toda, H. Maeda and E. Higgs (Eds.), The Biology of Nitric Oxide, Part 6, Portland Press, London, 1997, in press. [11] Ohta, K., Araki, N., Shibata, M., Hamada, J., Komatsumoto, S., Shimazu, K. and Fukuuchi, Y., A novel in vivo assay system for consecutive measurement of brain nitric oxide production com-
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