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Regional Difference in Nitric Oxide Production during Forebrain Ischemia
MICROCHEMICAL JOURNAL ARTICLE NO.
56, 188–191 (1997)
MJ961452
Regional Difference in Nitric Oxide Production during Forebrain Ischemia1 Mamoru Shibata,2 Nobuo Araki,* Junichi Hamada,† Takahiro Sasaki, Kouichi Ohta, Kunio Shimazu, and Yasuo Fukuuchi Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan; *Department of Internal Medicine, Nippon Koukan Hospital, Kanagawa, Japan; and †Department of Neurology, Saitama National Hospital, Saitama, Japan The hippocampus has been shown to exhibit particular vulnerability to ischemia, suggesting the existence of inherent processes leading to neuronal death. Nitric oxide (NO) has been implicated in neurotoxicity due to ischemia. The relationship between regional distribution of NO synthesis induced by ischemia and the areas vulnerable to ischemia remains elusive. In the present study, we explored regional variation in NO synthesis by determining the concentrations of nitrite (NO02 ), a major metabolite of NO, in the brain in vivo microdialysis samples obtained from the hippocampus and the striatum subjected to ischemia. Sprague–Dawley rats were anesthetized with pentobarbital (40 mg/kg, i.p.). An in vivo microdialysis probe was implanted into the hippocampus or the striatum. Subsequently, the animals were subjected to forebrain ischemia for 20 min by occlusion of both common carotid arteries and induced hypotension. Forebrain ischemia gave rise to a significant change in NO02 level only in the hippocampus, resulting in an increase to 111.2 { 5.4 (mean { SEM) % of the preischemia level. This finding suggests the presence of a regional difference in NO production during ischemia, which may be concerned with the underlying mechanism of ischemic vulnerability. q 1997 Academic Press
Nitric oxide (NO) has been identified as an important mediator of pathological processes including ischemic damage in the brain (2, 3, 11). NO is synthesized from L-arginine by NO synthase (NOS). Several articles have shown that NO synthesis increases during ischemia (7, 9, 10, 16, 19). However, information about the regional difference in NO production within the brain during ischemia is lacking. It has been established that some structures within the brain exhibit selective vulnerability to ischemia (8, 14, 17). Knowledge of regional variation in NO synthesis during ischemia appears to be important to explore the association of NO with the selective vulnerability to ischemia recognized in some brain regions. We have already shown that measurement of nitrite (NO20) levels in the dialysate samples of brain in vivo microdialysis is useful to monitor NO production (12, 16). In the present study, we measured NO20 levels in in vivo microdialysis samples obtained from the hippocampus and the striatum during forebrain ischemia. MATERIALS AND METHODS
in vivo Microdialysis Male Sprague–Dawley rats weighing 250–350 g were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). During the experiment, body temperature was continuously 1
This article is part of the special section devoted to novel microanalytical methods for the detection and quantification of NO and related NOx species. 2 To whom correspondence should be addressed. 188 0026-265X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
ah0b$$1452
05-21-97 00:10:01
mica
AP: MICROCHEM
NO PRODUCTION DURING FOREBRAIN ISCHEMIA
189
monitored and maintained at 37.57C with a thermostatically controlled heating pad connected to a rectal thermistor probe. A venous catheter was inserted into the femoral vein for exsanguination. The tail artery was catheterized for continuous monitoring of blood pressure. Blood pressure was monitored continuously using a multi-pen recorder via a pressure transducer. Then, both common carotid arteries were carefully isolated, and loops made of PE-10 polyethylene catheter were passed around the carotid arteries without interrupting carotid blood flow for later occlusion. Animals were placed in a stereotaxic frame with the skull level. Following exposure of the skull, a small burr hole was drilled at appropriate sites to allow insertion of a microdialysis probe with double lumen into the hippocampus or the striatum. The numbers of animals used for the study of the hippocampus and the striatum were 21 and 16, respectively. The coordinates of the tip of the microdialysis probe were 4.8 mm posterior, 5.3 mm lateral, and 7.5 mm ventral to the bregma for the hippocampus and 1.7 mm anterior, 2.5 mm lateral, 6.5 mm ventral to the bregma for the striatum. Subsequently, the microdialysis probe was perfused with degassed Ringer’s solution at a constant rate of 2 ml/min employing a microsyringe pump. After a 2-h equilibrium period, fractions were collected every 20 min. Induction of Forebrain Ischemia Forebrain ischemia was induced by occlusion of both common carotid arteries using the polyethylene loops in combination with systemic hypotension, with the mean arterial pressure being maintained below 50 mm Hg (18). After 20 min, cerebral reperfusion was achieved by releasing the tied polyethylene loops around the common carotid arteries and by reinfusion of the blood. NO20 Determination NO20 levels in dialysate samples were measured by the Griess reaction. The principle was described elsewhere (12). Briefly, samples were injected into a flow-through system and delivered by a carrier solution containing 1% NH4Cl at 10 ml/min, reaching a mixing tee where the samples reacted with Griess reagent [1 part1% sulfanilamide and 1 part 0.1% N-(naphthyl)-ethylenediamine] to yield an azo dye after incubation at 407C. Subsequently, samples underwent spectrophotometric analysis for the determination of NO20 levels. We have already demonstrated that absorbance at 546 nm is proportional to NO20 levels in our system (12). Statistical Analysis For the statistical analysis, the data for NO20 levels were expressed as a percentage of the baseline level and were given as mean { SEM. A paired-t test was used to compare the mean NO20 levels during ischemia with the baseline levels; p õ 0.05 was considered statistically significant. The experimental protocol met the Animal Experiment Guidelines of the Keio University School of Medicine and was approved by the Experimental Animal Committee of Keio University. RESULTS As depicted in Fig. 1, during forebrain ischemia, the NO20 level significantly (p õ 0.05) increased to 111.2 { 5.4% (mean { SEM) of the preischemia level in the
ah0b$$1452
05-21-97 00:10:01
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SHIBATA ET AL.
FIG. 1. Changes in NO02 level during ischemia. Data are given as mean { SEM%. Paired-t test was used to compare the mean NO02 levels during ischemia with the baseline levels. *p õ 0.05
hippocampus (n Å 21). In the striatum (n Å 16), forebrain ischemia failed to significantly affect NO20 level (102.1 { 5.6%). DISCUSSION
The present study demonstrated that transient forebrain ischemia induced an increase in NO20 level in the hippocampus while it failed to significantly affect NO20 level in the striatum. Since we have already shown that the change in NO20 level detected by our system correlates well with a change in NO production (12, 16), our present finding suggests the presence of regional variation in NO synthesis in response to ischemia. With the advent of new techniques for the determination of NO, many authors have provided information concerning the change in NO production during ischemia (7, 9, 10, 16, 19). However, it remains largely unknown whether there exist regional differences in NO production during ischemia. Our result appears to be important in that it casts light on this problem. Our finding is endorsed by a recent observation by Kuppusamy et al. (9) which demonstrated that cerebral ischemia/hypoxia induced marked enhancement of NO synthesis in the areas such as the cortex, the hippocampus, and the hypothalamus, but not in the striatum. In the ischemia model adopted in the present study, forebrain structures including the striatum and the hippocampus are subjected to severe ischemia to a similar degree (18). Therefore, it seems unlikely that a difference in ischemic severity is responsible for the variation in NO synthesis. Higher NOS catalytic activity has been identified in the hippocampus than in the striatum (1, 5). Therefore, it seems plausible that our finding would be accounted for by the regional distribution of NOS catalytic activity. In our short-term ischemia model, NO formed during ischemia is likely to derive from constitutive NOS (cNOS) activity. We have already confirmed that the increase in NO synthesis during forebrain ischemia in our model is ascribed to the activation of the neuronal isoform of cNOS
ah0b$$1452
05-21-97 00:10:01
mica
AP: MICROCHEM
NO PRODUCTION DURING FOREBRAIN ISCHEMIA
191
(4). Huang et al. (5) succeeded in producing knock-out mice deficient in the neuronal isoform of cNOS, in which small residual activity of NOS was found throughout the brain structures with the maximum NOS catalytic activity remaining in the hippocampus. As pointed out by the authors, this observation was suggestive of the existence of novel subtypes of neuronal NOS (5). There is a possibility that one subtype of NOS would be more potently activated than others in response to ischemia. Therefore, the regional difference in the composition of NOS subtypes may also have had an effect on our result. The hippocampus is particularly vulnerable to an ischemic insult (8, 14, 17). Several lines of evidence have shown that the neuronal isoform of NOS is implicated in neurotoxicity (6, 15, 20). Our finding suggests a possibility that the enhanced production of NO during ischemia may be associated with the inherent vulnerability to ischemia in the hippocampus. REFERENCES 1. Bredt, D. S.; Glatt, C. E.; Hwang, P. M.; Fotuhi, M.; Dawson, T. M.; Snyder, S. H. Neuron, 1991, 7, 1615–1624. 2. Dawson, T. M.; Snyder, S. H. J. Neurosci., 1994, 14, 5147–5159. 3. Hamada, J.; Greenberg, J. H.; Croul, S.; Dawson, T. M.; Reivich, M. J. Cereb. Blood Flow Metab., 1995, 15, 779–786. 4. Hamada, J.; Fukuuchi, Y.; Shimazu, K.; Araki, N.; Shibata, M., Sasaki, T. Cerebrovasc. Dis., 1996, 6(Suppl. 2), 118. 5. Huang, P. L.; Dawson, T. M.; Bredt, D. S.; Snyder, S. H.; Fishman, M. C. Cell, 1993, 75, 1273–1286. 6. Huang, Z.; Huang, P. L.; Panahian, N.; Dalkara, T.; Fishman, M. C.; Moskowitz, M. A. Science, 1994, 265, 1883–1885. 7. Kader, A.; Frazzini, V. I.; Solomon, R. A.; Trifiletti, R. R. Stroke, 1993, 24, 1709–1716. 8. Kirino, T. Brain Res., 1982, 239, 57–69. 9. Kuppusamy, P.; Ohnishi, S. T.; Numagami, Y.; Ohnishi, T., Zweier, J. L. J. Cereb. Blood Flow Metab., 1995, 15, 899–903. 10. Malinski, T.; Bailey, F.; Zhang, Z. G.; Chopp, M. J. Cereb. Blood Flow Metab., 1993, 13, 355–358. 11. Nowicki, J. P.; Duval, D.; Poignet, H.; Scatton, B. Eur. J. Pharmacol., 1991, 204, 339–340. 12. Ohta, K.; Araki, N.; Shibata, M.; Hamada, J.; Komatsumoto, S.; Shimazu, K.; Fukuuchi, Y. Neurosci. Lett., 1994, 176, 165–168. 13. Paxinos, G.; Watson, C. ‘‘The Rat Brain in Stereotaxic Coordinates,’’ Plate 37. Academic Press, New York, 1986. 14. Pulsinelli, W. A.; Brierley, J. B.; Plum, F. Ann. Neurol., 1982, 11, 491–498. 15. Schulz, J. B.; Matthews, R. T.; Jenkins, B. G.; Ferrante, R. J.; Siwek, D.; Henshaw, D. R.; Cipolloni, P. B.; Mecocci, P.; Kowall, N. W.; Rosen, B. R.; Beal, M. F. J. Neurosci., 1995, 15, 8419–8429. 16. Shibata, M.; Araki, N.; Hamada, J.; Sasaki, T.; Shimazu, K.; Fukuuchi, Y. Brain Res., 1996, 734, 86– 90. 17. Smith, M-L.; Auer, R. N.; Siesjo¨, B. K. Acta Neuropathol. (Berl), 1984, 64, 319–332. 18. Smith, M-L.; Bendek, G.; Dahlgren, N.; Rose´n, I.; Wieloch, T.; Siesjo¨, B. K. Acta Neurol. Scand., 1984, 69, 385–401. 19. Tominaga, T.; Sato, S.; Ohnishi, T.; Ohnishi, T. J. Cereb. Blood Flow Metab., 1994, 14, 715–722. 20. Yoshida, T.; Limmroth, V.; Irikura, K.; Moskowitz, M. A. J. Cereb. Blood Flow Metab., 1994, 14, 924–929.
ah0b$$1452
05-21-97 00:10:01
mica
AP: MICROCHEM
56, 188–191 (1997)
MJ961452
Regional Difference in Nitric Oxide Production during Forebrain Ischemia1 Mamoru Shibata,2 Nobuo Araki,* Junichi Hamada,† Takahiro Sasaki, Kouichi Ohta, Kunio Shimazu, and Yasuo Fukuuchi Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan; *Department of Internal Medicine, Nippon Koukan Hospital, Kanagawa, Japan; and †Department of Neurology, Saitama National Hospital, Saitama, Japan The hippocampus has been shown to exhibit particular vulnerability to ischemia, suggesting the existence of inherent processes leading to neuronal death. Nitric oxide (NO) has been implicated in neurotoxicity due to ischemia. The relationship between regional distribution of NO synthesis induced by ischemia and the areas vulnerable to ischemia remains elusive. In the present study, we explored regional variation in NO synthesis by determining the concentrations of nitrite (NO02 ), a major metabolite of NO, in the brain in vivo microdialysis samples obtained from the hippocampus and the striatum subjected to ischemia. Sprague–Dawley rats were anesthetized with pentobarbital (40 mg/kg, i.p.). An in vivo microdialysis probe was implanted into the hippocampus or the striatum. Subsequently, the animals were subjected to forebrain ischemia for 20 min by occlusion of both common carotid arteries and induced hypotension. Forebrain ischemia gave rise to a significant change in NO02 level only in the hippocampus, resulting in an increase to 111.2 { 5.4 (mean { SEM) % of the preischemia level. This finding suggests the presence of a regional difference in NO production during ischemia, which may be concerned with the underlying mechanism of ischemic vulnerability. q 1997 Academic Press
Nitric oxide (NO) has been identified as an important mediator of pathological processes including ischemic damage in the brain (2, 3, 11). NO is synthesized from L-arginine by NO synthase (NOS). Several articles have shown that NO synthesis increases during ischemia (7, 9, 10, 16, 19). However, information about the regional difference in NO production within the brain during ischemia is lacking. It has been established that some structures within the brain exhibit selective vulnerability to ischemia (8, 14, 17). Knowledge of regional variation in NO synthesis during ischemia appears to be important to explore the association of NO with the selective vulnerability to ischemia recognized in some brain regions. We have already shown that measurement of nitrite (NO20) levels in the dialysate samples of brain in vivo microdialysis is useful to monitor NO production (12, 16). In the present study, we measured NO20 levels in in vivo microdialysis samples obtained from the hippocampus and the striatum during forebrain ischemia. MATERIALS AND METHODS
in vivo Microdialysis Male Sprague–Dawley rats weighing 250–350 g were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). During the experiment, body temperature was continuously 1
This article is part of the special section devoted to novel microanalytical methods for the detection and quantification of NO and related NOx species. 2 To whom correspondence should be addressed. 188 0026-265X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
ah0b$$1452
05-21-97 00:10:01
mica
AP: MICROCHEM
NO PRODUCTION DURING FOREBRAIN ISCHEMIA
189
monitored and maintained at 37.57C with a thermostatically controlled heating pad connected to a rectal thermistor probe. A venous catheter was inserted into the femoral vein for exsanguination. The tail artery was catheterized for continuous monitoring of blood pressure. Blood pressure was monitored continuously using a multi-pen recorder via a pressure transducer. Then, both common carotid arteries were carefully isolated, and loops made of PE-10 polyethylene catheter were passed around the carotid arteries without interrupting carotid blood flow for later occlusion. Animals were placed in a stereotaxic frame with the skull level. Following exposure of the skull, a small burr hole was drilled at appropriate sites to allow insertion of a microdialysis probe with double lumen into the hippocampus or the striatum. The numbers of animals used for the study of the hippocampus and the striatum were 21 and 16, respectively. The coordinates of the tip of the microdialysis probe were 4.8 mm posterior, 5.3 mm lateral, and 7.5 mm ventral to the bregma for the hippocampus and 1.7 mm anterior, 2.5 mm lateral, 6.5 mm ventral to the bregma for the striatum. Subsequently, the microdialysis probe was perfused with degassed Ringer’s solution at a constant rate of 2 ml/min employing a microsyringe pump. After a 2-h equilibrium period, fractions were collected every 20 min. Induction of Forebrain Ischemia Forebrain ischemia was induced by occlusion of both common carotid arteries using the polyethylene loops in combination with systemic hypotension, with the mean arterial pressure being maintained below 50 mm Hg (18). After 20 min, cerebral reperfusion was achieved by releasing the tied polyethylene loops around the common carotid arteries and by reinfusion of the blood. NO20 Determination NO20 levels in dialysate samples were measured by the Griess reaction. The principle was described elsewhere (12). Briefly, samples were injected into a flow-through system and delivered by a carrier solution containing 1% NH4Cl at 10 ml/min, reaching a mixing tee where the samples reacted with Griess reagent [1 part1% sulfanilamide and 1 part 0.1% N-(naphthyl)-ethylenediamine] to yield an azo dye after incubation at 407C. Subsequently, samples underwent spectrophotometric analysis for the determination of NO20 levels. We have already demonstrated that absorbance at 546 nm is proportional to NO20 levels in our system (12). Statistical Analysis For the statistical analysis, the data for NO20 levels were expressed as a percentage of the baseline level and were given as mean { SEM. A paired-t test was used to compare the mean NO20 levels during ischemia with the baseline levels; p õ 0.05 was considered statistically significant. The experimental protocol met the Animal Experiment Guidelines of the Keio University School of Medicine and was approved by the Experimental Animal Committee of Keio University. RESULTS As depicted in Fig. 1, during forebrain ischemia, the NO20 level significantly (p õ 0.05) increased to 111.2 { 5.4% (mean { SEM) of the preischemia level in the
ah0b$$1452
05-21-97 00:10:01
mica
AP: MICROCHEM
190
SHIBATA ET AL.
FIG. 1. Changes in NO02 level during ischemia. Data are given as mean { SEM%. Paired-t test was used to compare the mean NO02 levels during ischemia with the baseline levels. *p õ 0.05
hippocampus (n Å 21). In the striatum (n Å 16), forebrain ischemia failed to significantly affect NO20 level (102.1 { 5.6%). DISCUSSION
The present study demonstrated that transient forebrain ischemia induced an increase in NO20 level in the hippocampus while it failed to significantly affect NO20 level in the striatum. Since we have already shown that the change in NO20 level detected by our system correlates well with a change in NO production (12, 16), our present finding suggests the presence of regional variation in NO synthesis in response to ischemia. With the advent of new techniques for the determination of NO, many authors have provided information concerning the change in NO production during ischemia (7, 9, 10, 16, 19). However, it remains largely unknown whether there exist regional differences in NO production during ischemia. Our result appears to be important in that it casts light on this problem. Our finding is endorsed by a recent observation by Kuppusamy et al. (9) which demonstrated that cerebral ischemia/hypoxia induced marked enhancement of NO synthesis in the areas such as the cortex, the hippocampus, and the hypothalamus, but not in the striatum. In the ischemia model adopted in the present study, forebrain structures including the striatum and the hippocampus are subjected to severe ischemia to a similar degree (18). Therefore, it seems unlikely that a difference in ischemic severity is responsible for the variation in NO synthesis. Higher NOS catalytic activity has been identified in the hippocampus than in the striatum (1, 5). Therefore, it seems plausible that our finding would be accounted for by the regional distribution of NOS catalytic activity. In our short-term ischemia model, NO formed during ischemia is likely to derive from constitutive NOS (cNOS) activity. We have already confirmed that the increase in NO synthesis during forebrain ischemia in our model is ascribed to the activation of the neuronal isoform of cNOS
ah0b$$1452
05-21-97 00:10:01
mica
AP: MICROCHEM
NO PRODUCTION DURING FOREBRAIN ISCHEMIA
191
(4). Huang et al. (5) succeeded in producing knock-out mice deficient in the neuronal isoform of cNOS, in which small residual activity of NOS was found throughout the brain structures with the maximum NOS catalytic activity remaining in the hippocampus. As pointed out by the authors, this observation was suggestive of the existence of novel subtypes of neuronal NOS (5). There is a possibility that one subtype of NOS would be more potently activated than others in response to ischemia. Therefore, the regional difference in the composition of NOS subtypes may also have had an effect on our result. The hippocampus is particularly vulnerable to an ischemic insult (8, 14, 17). Several lines of evidence have shown that the neuronal isoform of NOS is implicated in neurotoxicity (6, 15, 20). Our finding suggests a possibility that the enhanced production of NO during ischemia may be associated with the inherent vulnerability to ischemia in the hippocampus. REFERENCES 1. Bredt, D. S.; Glatt, C. E.; Hwang, P. M.; Fotuhi, M.; Dawson, T. M.; Snyder, S. H. Neuron, 1991, 7, 1615–1624. 2. Dawson, T. M.; Snyder, S. H. J. Neurosci., 1994, 14, 5147–5159. 3. Hamada, J.; Greenberg, J. H.; Croul, S.; Dawson, T. M.; Reivich, M. J. Cereb. Blood Flow Metab., 1995, 15, 779–786. 4. Hamada, J.; Fukuuchi, Y.; Shimazu, K.; Araki, N.; Shibata, M., Sasaki, T. Cerebrovasc. Dis., 1996, 6(Suppl. 2), 118. 5. Huang, P. L.; Dawson, T. M.; Bredt, D. S.; Snyder, S. H.; Fishman, M. C. Cell, 1993, 75, 1273–1286. 6. Huang, Z.; Huang, P. L.; Panahian, N.; Dalkara, T.; Fishman, M. C.; Moskowitz, M. A. Science, 1994, 265, 1883–1885. 7. Kader, A.; Frazzini, V. I.; Solomon, R. A.; Trifiletti, R. R. Stroke, 1993, 24, 1709–1716. 8. Kirino, T. Brain Res., 1982, 239, 57–69. 9. Kuppusamy, P.; Ohnishi, S. T.; Numagami, Y.; Ohnishi, T., Zweier, J. L. J. Cereb. Blood Flow Metab., 1995, 15, 899–903. 10. Malinski, T.; Bailey, F.; Zhang, Z. G.; Chopp, M. J. Cereb. Blood Flow Metab., 1993, 13, 355–358. 11. Nowicki, J. P.; Duval, D.; Poignet, H.; Scatton, B. Eur. J. Pharmacol., 1991, 204, 339–340. 12. Ohta, K.; Araki, N.; Shibata, M.; Hamada, J.; Komatsumoto, S.; Shimazu, K.; Fukuuchi, Y. Neurosci. Lett., 1994, 176, 165–168. 13. Paxinos, G.; Watson, C. ‘‘The Rat Brain in Stereotaxic Coordinates,’’ Plate 37. Academic Press, New York, 1986. 14. Pulsinelli, W. A.; Brierley, J. B.; Plum, F. Ann. Neurol., 1982, 11, 491–498. 15. Schulz, J. B.; Matthews, R. T.; Jenkins, B. G.; Ferrante, R. J.; Siwek, D.; Henshaw, D. R.; Cipolloni, P. B.; Mecocci, P.; Kowall, N. W.; Rosen, B. R.; Beal, M. F. J. Neurosci., 1995, 15, 8419–8429. 16. Shibata, M.; Araki, N.; Hamada, J.; Sasaki, T.; Shimazu, K.; Fukuuchi, Y. Brain Res., 1996, 734, 86– 90. 17. Smith, M-L.; Auer, R. N.; Siesjo¨, B. K. Acta Neuropathol. (Berl), 1984, 64, 319–332. 18. Smith, M-L.; Bendek, G.; Dahlgren, N.; Rose´n, I.; Wieloch, T.; Siesjo¨, B. K. Acta Neurol. Scand., 1984, 69, 385–401. 19. Tominaga, T.; Sato, S.; Ohnishi, T.; Ohnishi, T. J. Cereb. Blood Flow Metab., 1994, 14, 715–722. 20. Yoshida, T.; Limmroth, V.; Irikura, K.; Moskowitz, M. A. J. Cereb. Blood Flow Metab., 1994, 14, 924–929.
ah0b$$1452
05-21-97 00:10:01
mica
AP: MICROCHEM