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Increased neurons containing neuronal nitric oxide synthase in the brain of a hypoxic-ischemic neonatal rat model
ELSEVIER
Brain & Development 1996; 18:369-375
Original article
Increased neurons containing neuronal nitric oxide synthase in the brain of a hypoxic-ischemic neonatal rat model Yoshihisa Higuchi a, *, Haruo Hattori a, Ryuichi Hattori h, Kenshi Furusho a a Departmentof Pediatrics, Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Japan Department of Internal Medicine, Faculty of Medicine, Kyoto Uni~'ersity, Kyoto, Japan Received 27 October 1995; accepted 31 January 1996
We evaluated the temporal profile of the number of neurons containing neuronal nitric oxide synthase (nNOS neurons) in the brain of a neonatal hypoxic-ischemic rat model. Hypoxic-ischemic insults were produced in the brains of 7-day-old rat pups using a combination of unilateral carotid artery ligation and hypoxic (8% oxygen) exposure. Sections of brain from rats killed at 0 - 2 4 h after the onset of hypoxia were stained immunohistochemically using a polyclonai anti-nNOS antibody. Histological changes of neuronal injury were evaluated in the adjacent Nissl stained sections. The number of nNOS neurons in the hemisphere ipsilateral to the carotid ligation was significantly increased ( P < 0.05) at 3 h, when the neuronal injury consisted of clusters of degenerating hyperchromic neurons. Neuronal degeneration and an increased number of nNOS neurons were seen only in the ipsilateral hemisphere and the increase was most prominent in the dorsolateral area of the striatum. The increase in the number of nNOS neurons continued at 6 h, when the area of neuronal injury continued to expand. At 24 h, the neuronal injury was diffuse, and the number of nNOS neurons on the ipsilaterai side significantly decreased. The increase of the number of nNOS neurons in the early phase of neonatal neuronal injury suggests its possible involvement in the hypoxicischemic injury. The delineation of its role in neuronal injury may lead to an improvement in managing neonatal hypoxic-ischemic brain injury. Keywords: Nitric oxide synthase; Neonatal rat; Hypoxic-ischemic brain damage
1. INTRODUCTION Nitric oxide (NO) plays an important role in the neuropathological processes of hypoxia-ischemia. Hypoxia-ischemia increases extracellular concentrations of endogenous excitatory amino acids, glutamate and aspartate, in the brain [1-3]. Sustained glutamate stimulation of N-methyl-D-aspartate (NMDA) receptor leads to an influx of Ca 2+ [1,4], and activates intracellular Ca 2 +-dependent enzymatic cascades including nitric oxide synthase (NOS), which generates NO from e-arginine [5,6]. NO freely diffuses to adjacent target neurons where it yields cytotoxic free radicals such as peroxynitrite [5]. In the field of pediatric neurology, hypoxic-ischemic insult in the perinatal period is a common cause of brain injury. The delineation of the
* Corresponding author. [email protected]
Fax: (81) (75) 752-2361; e-mail:
involvement of NO in a model of neonatal brain injury may improve the management of neonatal hypoxic-ischemic injury. The neurotoxicity of NO has been demonstrated in various animal models of ischemic brain injury [7-9], although it is also known that NO can serve as a neuroprotective agent through a vasodilatory effect in adult animals [10-12]. We previously demonstrated the neurotoxicity of NO in the neonatal hypoxicischemic rat model. Prehypoxic administration of N~-nitro-Larginine (NOARG), a competitive inhibitor of NOS, significantly reduced the infarcted volume, whereas posthypoxic NOARG treatment did not produce a neuroprotective effect [13]. There are at least three isoforms of nitric oxide synthase: a constitutive neuronal (nNOS), a constitutive endothelial (eNOS), and an inducible type [14,15]. Some nNOS-containing neurons (nNOS neurons) innervate cerebral arteris [16] and, together with eNOS, show the vasodilatory effect [17]. On the other hand, involvement of nNOS in neuronal injury is suggested by the rapid upregulation of nNOS and its mRNA in the cerebral ischemic lesion caused by occlusion of the middle cerebral artery (MCA) in the
0387-7604/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0387-7604(96)00019-8
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Y. Higuchi et al. / Brain & Deuelopment 1996; 18:369-375
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Fig. 2. The numbers (mean _ S.E.M.) of nNOS neurons in the cortex: the no-insult group (Group N) (13), the ipsilateral (grey columns) and the contralateral side (cross-hatched columns) in the ligation-only group (Group L), and the ipsilateral (l I) and the contralateral (cross-hatched columns) side in the ligation + hypoxia group (Group H). At 0, 3 and 6 h in each group, n = 7 , and at other time points, n = 5 . * P < 0 . 0 5 compared to the contralateral cortex at the same time point. ~ P < 0.05 compared to the ipsilateral cortex in Group L at 0 h.
N
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H 3h
L 3h
H 6h
L 6h
H 12h
L 12h
H 24h
L 24h
Fig. 3. The numbers (mean++_S.E.M.) of nNOS neurons in the striatum: the no-insult group (Group N) ( [] ), the ipsilateral (grey columns) and the contralateral side (cross-hatched columns) in the ligation-only group (Group L), and the ipsilateral ( I ) and the contralateral (cross-hatched columns) side in the ligation + hypoxia group (Group H). At 0, 3, and 6 h in each group, n = 7, and at other time points, n = 5. * P < 0.05 compared to the contralateral striatum at the same time point. ~ P < 0.05 compared to the ipsilateral striatum in Group L at 0 h.
adult rat [18] and by the reduced volume of infarcted tissue after M C A occlusion in mutant mice deficient in nNOS activity [19]. Therefore, we investigated the transient increase of the number of nNOS neurons immunohistochemically in the hypoxicischemic neonatal rat model in an attempt to delineate the role of nNOS in neuronal injury.
hypoxic exposure (group H) or the recovery from the anesthesia (group L). The number of rats in group L was n = 7 at each of 0, 3, and 6 h, and n = 5 at each other time point. That in group H was n = 7 at each of 3 and 6 I~, and n = 5 at each other time point. Pups in the no-insult group (group N, n = 5) were killed without any procedure.
2. MATERIALS AND METHODS
2.2. Antibody
2.1. Animal model
We used rabbit polyclonal antibody against nNOS. Rat cerebellar NOS was purified following the method of Schmidt [21]. Rabbits were immunized with the purified NOS. The IgG fraction was purified on protein A-Sepharose, dialyzed against phosphate buffered saline (PBS). The activity of the purified rat cerebellar NOS was almost completely precipitated by preincubation with this antibody and subsequent centrifugation. In Western blot analysis, the antibody interacted with a single band of relative molecular mass 150,000, corresponding to cerebellar NOS [22], but did not show cross reactivity to rat macrophage NOS (inducible type) [23].
We used the method of Rice et al. to induce hypoxic-ischemic brain damage in neonatal rats [20]. All procedures were in accordance with the Guideline for Animal Experiments of Kyoto University. Under ether anesthesia, the left common carotid artery was exposed through a midline incision, doubly ligated, and severed between the two ligatures. After surgery, the rat pup was allowed to recover from the anesthesia for 2.5 h. Hypoxic exposure was obtained by placing the pup in a 2.0 1 air-tight plastic box submerged in a 37°C water bath and flushing a humidified mixture of 8% oxygen and 92% nitrogen delivered at 1.1 l / m i n for 2.5 h. Sixty 7-day-old Std:Wistar rat pups were randomly assigned to one of three groups. Those in the ligation-hypoxia group (group H) were subjected to the carotid ligation and the hypoxic exposure, and those in the ligation-only group (group L) were subjected to the carotid ligation only. Pups in both groups were killed at various times (0, 3, 6, 12 and 24 h) after the onset of the
2.3. Immunohistochemical staining The rat pups were anesthetized with intraperitoneal chloral hydrate and perfused transcardially with heparinized PBS followed by a perfusion with a fixative containing 3% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB). After post-fixation for 6 - 1 2 h in the same fixative, the brain was
Fig. 1. Immunohistochemically stained sections for nNOS. A: the cortex of rats in the no-insult group (Group N); B: the striatum of rats in the no-insult group (Group N). C-H show nNOS neurons in the dorsolateral area of the striatum in sections from rats in the ligation + hypoxia group (Group H). C,D: at 3 h; E,F: at 6 h; G,H: at 12 h. C,E,G: the ipsilateral side. D,F,H: the contralateral side. The number of nNOS neurons is increased in the ipsilateral side (C,E) as compared with that in the contralateral side (D,F). In G, the nNOS neurons in the ipsilateral side are decreased in number and have lost their dendrites, eC designates the external capsule. Bar, 100 ~m.
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removed and soaked in 15% sucrose in PB overnight followed by 30% sucrose in PB overnight. The brain was frozen in isopentane cooled on dry ice and then cut into coronal sections of 40 txm thickness on a cryostat. The sections were from the level of the caudate-putamen and the anterior commissure, and with longitudinal coordinate of A 5.6 m m according to a stereotaxic atlas of the 10-day-old rat brain [24]. The sections were blocked in 2% goat serum and 0.1% bovine serum albumin (BSA) in PBS, and incubated with the antibody against nNOS at a 1:8000 dilution for 12 h. After being washed in PBS, sections were incubated with biotinylated goat anti-rabbit IgG (Vector Labs, Burlingame, CA) at a 1:200 dilution for 2 h. Endogenous peroxidase activity was inhibited by the incubation
of sections with 3% hydrogen peroxide in PBS for 10 rain. The sections were incubated with avidin-biotin complex (Vectastatin ABC kit, Vector Labs, Burlingame, CA) for 1 h, and were then rinsed in Tris buffer and processed by the nickel-enhanced diaminobenzidine method. The sections were mounted on gelatin-coated glass slides, dehydrated in graded alcohol, and coverslipped. Control sections received identical treatment except use of the antibody against nNOS.
2.4. Nissi stain The sections adjacent to those stained immunohistochemically were washed in PBS and mounted on slide glasses. They were
Fig. 4. Nissl stained sections of the ipsilateral side in rats in Group H. A,C,E, the cortex; B,D,F, the striatum. A,B: at 3 h; C,D: at 6 h; E,F: at 12 h. In A,B: the arrow denotes the area of the neurons with hyperchromia. In C,D: the area of degeneration has expanded, and some neurons have pyknotic or fragmented nuclei. In E,F: most neurons have pyknotic or fragmented nuclei, and the cytoplasm is unrecognizable. Bar, 100 txm.
Y. Higuchi et al./ Brain & Development 1996; 18:369-375
defatted in graded alcohol, and stained with cresyl violet. Then, the sections were dehydrated in graded alcohol and mounted with coverslips.
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h, the area of degeneration had expanded, and some degenerated neurons had fragmented or pyknotic nuclei (Fig. 4C,D). At 12-24 h, diffuse degeneration was observed, and most neurons showed fragmented nuclei and disintegrated cytoplasm (Fig. 4E,F).
3. A N A L Y S I S The light microscopic evaluation was carried out by an examiner without knowing the experimental protocol. All the nNOS neurons in the hemisphere ipsilateral to and contralateral to the carotid ligation in immunohistochemically stained sections were counted. The cortical area as defined herein included the cingulate cortex and the parietal cortex, i.e., above the rhinal fissure, and the striatal area included the caudate-putamen. Only neurons of which the nucleus surrounded by the cytoplasm was visible in the section were counted. The degree of neuronal degeneration was also evaluated microscopically in a Nissl stained section adjacent to the section stained with anti-nNOS antibody. One-way analysis of variance was performed to test the significance of differences among the means at each time point on the two sides both in group L and group H. Then, the means at each time point on the ipsilateral side in group H were compared with that at time 0 on the ipsilateral side in group L using two-tailed t-test with the Bonferroni's correction. The ipsilateral side and the contralateral side were compared by two-tailed, paired t-test. A value of P < 0.05 was considered significant.
4. R E S U L T S Neurons stained by immunohistochemical treatment were distributed both in the cortex and the striatum (Fig. 1A,B). Control sections treated without nNOS antibody showed no specific staining. Figs. 2 and 3 show the numbers of nNOS neurons in the cortex and the striatum, respectively, for the hemisphere ipsilateral to and contralateral to the carotid ligation. At 3 h and 6 h, the numbers of nNOS neurons in the ipsilateral side in group H were significantly increased compared to those in the contralateral side. The numbers of nNOS neurons at 3 h and 6 h in the ipsilateral side in group H were significantly greater than that in the ipsilateral side in group L at 0 h. The nNOS neurons in the striatum were more clustered in the dorsolateral area (Fig. 1C,E) when compared with those in the same area of the contralateral side (Fig. I D,F). No degenerative changes were found at 3 h, although some nNOS neurons in the ipsilateral side showed shrunken dendrites at 6 h as compared with those in group N. At 12 h, the number of nNOS neurons in the ipsilateral side in group H was decreased, although the difference was not significant. These nNOS neurons showed distinct degenerative changes such as loss of dendrites and shrinkage (Fig. 1G), while those in the contralateral side showed no change (Fig. 1H). At 24 h, the number of nNOS neurons, for the cortex and for the striatum, in the ipsilateral side in group H was significantly decreased in comparison to that in the contralateral side at the same time point and compared to that in the ipsilateral side in group L at 0 h. Observation of Nissl stained sections revealed neuronal loss and degeneration were recognized only in the hemisphere ipsilateral to the carotid ligation in group H. At 3 h, patchy or laminar areas of neurons with hyperchromia appeared (Fig. 4A,B). At 6
5. D I S C U S S I O N In this study, neuronal degeneration in the Nissl stained sections was observed only in the hemisphere ipsilateral to the carotid ligation after the hypoxia, i.e., the ipsilateral side in group H. Apparently neither the hypoxia nor ischemia alone caused the neuronal injury in this neonatal rat model. Similarly, nNOS neurons were increased in number only in the hemisphere subjected to both ischemia and hypoxia. Our data represent further evidence of the possible involvement of nNOS in hypoxic-ischemic brain injury in the neonatal rat. The temporal profile of neuronal injury was parallel to the increase of the number of nNOS neurons. The nNOS neurons increased in number as early as 3 h, and the subsequent neuronal degeneration indicates that the intracellular mechanism leading to neuronal death had already commenced in this early period. Consideration of these results in the context of our previous findings that prehypoxic treatment with NOS inhibitor, NOARG, is neuroprotective [13], we consider that NO is neurotoxic and that increased numbers of nNOS neurons are responsible for the production of excessive NO which damages surrounding neurons. Trifiletti also demonstrated the neuroprotective effect of NOARG in the same neonatal model; prehypoxic treatment with NOARG decreased the brain damage as assessed by the disparity in hemispheric brain weight [25]. It has been disputed whether NO is neurotoxic or neuroprotective in the ischemic brain injury in adult animals [26,27]. NO-dependent vasodilation is noted to increase regional cerebral blood flow and reduce infarct volume [7,8,10]. However, we do not know of any report suggesting the neuroprotective effect of NO in a model of neonatal hypoxic-ischemic brain injury. In an ischemic insult, the cerebral circulation of the immature brain is further compromised by such factors as the low systemic blood pressure and the more labile autoregulation than that of the adult [28]. Under these conditions, it is possible that the vasodilatory/neuroprotective effect of NO, if present at all in neonates, is overwhelmed by the neurotoxicity of neuronally produced NO. In adult rats as well, recent data show that a selective nNOS inhibitor decreases the infarct volume and suggest that the effects of eNOS can be discriminated from those of nNOS [29]. Therefore, the neuroprotective effect of endothelial NO production is delineated in contrast to the neurotoxicity of neuronally overproduced NO [26]. Huang et al. also demonstrated the opposing roles of eNOS and nNOS using mutant mice deficient in nNOS activity [19]. They found that the mutant mice had significantly smaller infarcts following MCA occlusion than did the wild-type mice. Conversely, the infarct size in the mutant mice became larger when the activity of eNOS was inhibited by NOARG treatment. Zhang et al. reported an increase in the number of nNOS neurons in the hemisphere ipsilateral to the MCA occlusion and noted that the nNOS mRNA also was increased in the ipsilateral hemisphere of adult rats [18]. The increase was found 2 h after
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occlusion and was present over a longer period (at 2 - 4 8 h after occlusion) than we found in neonatal rats, and it had become bilateral at 4 h. The increase of nNOS neurons during neuronal injury was observed early in the course of injury in adult rats as well as in neonates. Following unilateral injury, bilateral induction of NADPH diaphorase activity in the hippocampus and in the cerebral cortex also is reported in adult rats [30]. NADPH diaphorase has a close association with NOS activity [22,31,32]. However, Ferriero et al., reported that, in the same neonatal rat model as we used, the increased number of NADPH-positive neurons in the cortex was observed only ipsilateral to the carotid ligation [33]. There may be a generalized response that is either humoral or mediated via neural connection against the ischemic insult in adult animals, but which is not present in an immature brain. The NADPH-diaphorase positive or the nNOS neurons have been noted to be resistant to hypoxic-ischemic insult [33] or to other pathological change such as Huntington disease [34,35]. NO modulates the NMDA-receptor and decreases the NMDAmediated increase of intracellular Ca 2+ [36] in a certain redox state [37]. This mechanism does not always prevent neuronal death; we observed the death of nNOS neurons themselves at 12-24 h after the insult. Zhang et al. also found the death of nNOS neurons in spite of their relative resistance to the ischemic insult in adult rats [18]. The increase of the number of nNOS neurons observed in our study may be an initial autoprotective response of the neurons under the insult by modulating their own NMDA-receptors and by producing increase of the regional cerebral blood flow around them. However, overproduced NO kills adjacent neurons, and eventually results in diffuse death of neurons including the nNOS neurons themselves.
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9. Nishikawa T, Kirsch JR, Koehler RC, Miyabe M, Traystman RJ. Nitric oxide synthase inhibition reduces caudate injury following transient focal ischemia in cats. Stroke 1994; 25: 877-85. 10. Morikawa E, Moskowitz MA, Huang Z, Yoshida T, lrikura K, Dalkara T. L-Arginine infusion promotes nitric oxide-dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume. Stroke 1994; 25: 429-35. 11. Kulutz JW, Prado RJ, Dietrich WD, Schleien CL, Watson BD. The effect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats. Stroke 1993: 24: 2023-9. 12. Weissman BA, Kadar T, Brandeis R, Shapira S. NG-nitro-L-arginine enhances neuronal death following transient forebrain ischemia in gerbils. Neurosci Lett 1992; 146: 139-42. 13. Hamada Y, Hayakawa T, Hattori H, Mikawa H. Inhibitor of nitric oxide synthesis reduces hypoxic-ischemic brain damage in the neonatal rat. Pediatr Res 1994: 35: 10-4. 14. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J 1994; 298: 249-58. 15. Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6: 3051-64. 16. Iadecola C, Beitz AJ, Renno W, Xu X, Mayer B, Zhang F. Nitric oxide synthase-containing neural processes on large cerebral arteries and cerebral microvessels. Brain Res 1993; 606: 148-55. 17. Faraci FM, Brian JE. Nitric oxide and the cerebral circulation. Stroke 1994; 25: 692-703. 18. Zhang ZG, Chopp M, Gautam S, et al. Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after focal cerebral ischemia in rat. Brain Res 1994; 654: 85-95. 19. Huang Z, Huang PL, Panahian H, Dalkara T, Fishman MC, Moskowitz MA. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 1994; 265: 1883-5. 20. Rice JE, Vannuci RC, Brierly JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 1981; 9: 131-41. 21. Schmidt HHHW, Pollock JS, Nakane M, Gorsky LD, F~3rstermannU, Murad F. Purification of soluble isoform of guanyl cyclase-activating-factor synthase. Proc Natl Acad Sci USA 1991; 88: 365-9. 22. Hashikawa T, Leggio MG, Hattori R, Yui Y. Nitric oxide synthase immunoreactivity colocalized with NADPH-diaphorase histochemistry in monkey cerebral cortex. Brain Res 1994; 641: 341-9. 23. Yui, Y, Hattori R, Kosuga K, Eizawa H, Hiki K, Kawai, C. Purification of nitric oxide synthase from rat macrophages. J Biol Chem 1991; 266: 12544-7. 24. Sherwood NM, Timiras PS. A Stereotaxic Atlas of the Developing Rat Brain. Berkeley: University of California Press, 1970: pp. 15-75. 25. Trifiletti RR. Neuroprotective effects of NG-nitro-L-arginine in focal stroke in the 7-day old rat. Eur J Pharmacol 1992; 218: 197-8. 26. Dalkara T, Yoshida T, Irikura K, Moskowitz MA. Dual role of nitric oxide in focal cerebral ischemia. Neuropharmacology 1994; 33: 1447-52. 27. ladecola C, Pelligrino DA, Moskowitz MA, Lassen NA. Nitric oxide synthase inhibition and cerebrovascular regulation. J Cereb Blood Flow Metab 1994; 14: 175-92. 28. Volpe JJ. Hypoxic-ischemic encephalopathy: Biochemical and physiological aspects. In: Volpe, JJ, ed. Neurology of the Newborn. 3rd ed. Philadelphia: Saunders, 1994: pp. 211-78. 29. Yoshida T, Limmroth V, Irikura K, Moskowitz MA. The NOS inhibitor, 7-nitroindazole, decreases focal infarct volume but not the response to topical acetylcholine in pial vessels. J Cereb Blood Flow Metab 1994; 14: 924-9. 30. Regidor J, Montesdeoca J, Ramiretz-Gonzalez JA, Hemandez-Urquia CM, Divac I. Bilateral induction of NADPH-diaphorase activity in neocortical and hippocampal neurons by unilateral injury. Brain Res 1993; 631: 171-4.
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Brain & Development 1996; 18:369-375
Original article
Increased neurons containing neuronal nitric oxide synthase in the brain of a hypoxic-ischemic neonatal rat model Yoshihisa Higuchi a, *, Haruo Hattori a, Ryuichi Hattori h, Kenshi Furusho a a Departmentof Pediatrics, Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Japan Department of Internal Medicine, Faculty of Medicine, Kyoto Uni~'ersity, Kyoto, Japan Received 27 October 1995; accepted 31 January 1996
We evaluated the temporal profile of the number of neurons containing neuronal nitric oxide synthase (nNOS neurons) in the brain of a neonatal hypoxic-ischemic rat model. Hypoxic-ischemic insults were produced in the brains of 7-day-old rat pups using a combination of unilateral carotid artery ligation and hypoxic (8% oxygen) exposure. Sections of brain from rats killed at 0 - 2 4 h after the onset of hypoxia were stained immunohistochemically using a polyclonai anti-nNOS antibody. Histological changes of neuronal injury were evaluated in the adjacent Nissl stained sections. The number of nNOS neurons in the hemisphere ipsilateral to the carotid ligation was significantly increased ( P < 0.05) at 3 h, when the neuronal injury consisted of clusters of degenerating hyperchromic neurons. Neuronal degeneration and an increased number of nNOS neurons were seen only in the ipsilateral hemisphere and the increase was most prominent in the dorsolateral area of the striatum. The increase in the number of nNOS neurons continued at 6 h, when the area of neuronal injury continued to expand. At 24 h, the neuronal injury was diffuse, and the number of nNOS neurons on the ipsilaterai side significantly decreased. The increase of the number of nNOS neurons in the early phase of neonatal neuronal injury suggests its possible involvement in the hypoxicischemic injury. The delineation of its role in neuronal injury may lead to an improvement in managing neonatal hypoxic-ischemic brain injury. Keywords: Nitric oxide synthase; Neonatal rat; Hypoxic-ischemic brain damage
1. INTRODUCTION Nitric oxide (NO) plays an important role in the neuropathological processes of hypoxia-ischemia. Hypoxia-ischemia increases extracellular concentrations of endogenous excitatory amino acids, glutamate and aspartate, in the brain [1-3]. Sustained glutamate stimulation of N-methyl-D-aspartate (NMDA) receptor leads to an influx of Ca 2+ [1,4], and activates intracellular Ca 2 +-dependent enzymatic cascades including nitric oxide synthase (NOS), which generates NO from e-arginine [5,6]. NO freely diffuses to adjacent target neurons where it yields cytotoxic free radicals such as peroxynitrite [5]. In the field of pediatric neurology, hypoxic-ischemic insult in the perinatal period is a common cause of brain injury. The delineation of the
* Corresponding author. [email protected]
Fax: (81) (75) 752-2361; e-mail:
involvement of NO in a model of neonatal brain injury may improve the management of neonatal hypoxic-ischemic injury. The neurotoxicity of NO has been demonstrated in various animal models of ischemic brain injury [7-9], although it is also known that NO can serve as a neuroprotective agent through a vasodilatory effect in adult animals [10-12]. We previously demonstrated the neurotoxicity of NO in the neonatal hypoxicischemic rat model. Prehypoxic administration of N~-nitro-Larginine (NOARG), a competitive inhibitor of NOS, significantly reduced the infarcted volume, whereas posthypoxic NOARG treatment did not produce a neuroprotective effect [13]. There are at least three isoforms of nitric oxide synthase: a constitutive neuronal (nNOS), a constitutive endothelial (eNOS), and an inducible type [14,15]. Some nNOS-containing neurons (nNOS neurons) innervate cerebral arteris [16] and, together with eNOS, show the vasodilatory effect [17]. On the other hand, involvement of nNOS in neuronal injury is suggested by the rapid upregulation of nNOS and its mRNA in the cerebral ischemic lesion caused by occlusion of the middle cerebral artery (MCA) in the
0387-7604/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0387-7604(96)00019-8
370
Y. Higuchi et al. / Brain & Deuelopment 1996; 18:369-375
371
Y. Higuchi et a l . / Brain & Development 1996; 18." 369-375 ,
r
160
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300
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7= t00 60 50 40
N
L Oh
H 3h
L 3h
H 6h
L 6h
H 12h
L 12h
H 24h
L 24h
Fig. 2. The numbers (mean _ S.E.M.) of nNOS neurons in the cortex: the no-insult group (Group N) (13), the ipsilateral (grey columns) and the contralateral side (cross-hatched columns) in the ligation-only group (Group L), and the ipsilateral (l I) and the contralateral (cross-hatched columns) side in the ligation + hypoxia group (Group H). At 0, 3 and 6 h in each group, n = 7 , and at other time points, n = 5 . * P < 0 . 0 5 compared to the contralateral cortex at the same time point. ~ P < 0.05 compared to the ipsilateral cortex in Group L at 0 h.
N
L Oh
H 3h
L 3h
H 6h
L 6h
H 12h
L 12h
H 24h
L 24h
Fig. 3. The numbers (mean++_S.E.M.) of nNOS neurons in the striatum: the no-insult group (Group N) ( [] ), the ipsilateral (grey columns) and the contralateral side (cross-hatched columns) in the ligation-only group (Group L), and the ipsilateral ( I ) and the contralateral (cross-hatched columns) side in the ligation + hypoxia group (Group H). At 0, 3, and 6 h in each group, n = 7, and at other time points, n = 5. * P < 0.05 compared to the contralateral striatum at the same time point. ~ P < 0.05 compared to the ipsilateral striatum in Group L at 0 h.
adult rat [18] and by the reduced volume of infarcted tissue after M C A occlusion in mutant mice deficient in nNOS activity [19]. Therefore, we investigated the transient increase of the number of nNOS neurons immunohistochemically in the hypoxicischemic neonatal rat model in an attempt to delineate the role of nNOS in neuronal injury.
hypoxic exposure (group H) or the recovery from the anesthesia (group L). The number of rats in group L was n = 7 at each of 0, 3, and 6 h, and n = 5 at each other time point. That in group H was n = 7 at each of 3 and 6 I~, and n = 5 at each other time point. Pups in the no-insult group (group N, n = 5) were killed without any procedure.
2. MATERIALS AND METHODS
2.2. Antibody
2.1. Animal model
We used rabbit polyclonal antibody against nNOS. Rat cerebellar NOS was purified following the method of Schmidt [21]. Rabbits were immunized with the purified NOS. The IgG fraction was purified on protein A-Sepharose, dialyzed against phosphate buffered saline (PBS). The activity of the purified rat cerebellar NOS was almost completely precipitated by preincubation with this antibody and subsequent centrifugation. In Western blot analysis, the antibody interacted with a single band of relative molecular mass 150,000, corresponding to cerebellar NOS [22], but did not show cross reactivity to rat macrophage NOS (inducible type) [23].
We used the method of Rice et al. to induce hypoxic-ischemic brain damage in neonatal rats [20]. All procedures were in accordance with the Guideline for Animal Experiments of Kyoto University. Under ether anesthesia, the left common carotid artery was exposed through a midline incision, doubly ligated, and severed between the two ligatures. After surgery, the rat pup was allowed to recover from the anesthesia for 2.5 h. Hypoxic exposure was obtained by placing the pup in a 2.0 1 air-tight plastic box submerged in a 37°C water bath and flushing a humidified mixture of 8% oxygen and 92% nitrogen delivered at 1.1 l / m i n for 2.5 h. Sixty 7-day-old Std:Wistar rat pups were randomly assigned to one of three groups. Those in the ligation-hypoxia group (group H) were subjected to the carotid ligation and the hypoxic exposure, and those in the ligation-only group (group L) were subjected to the carotid ligation only. Pups in both groups were killed at various times (0, 3, 6, 12 and 24 h) after the onset of the
2.3. Immunohistochemical staining The rat pups were anesthetized with intraperitoneal chloral hydrate and perfused transcardially with heparinized PBS followed by a perfusion with a fixative containing 3% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB). After post-fixation for 6 - 1 2 h in the same fixative, the brain was
Fig. 1. Immunohistochemically stained sections for nNOS. A: the cortex of rats in the no-insult group (Group N); B: the striatum of rats in the no-insult group (Group N). C-H show nNOS neurons in the dorsolateral area of the striatum in sections from rats in the ligation + hypoxia group (Group H). C,D: at 3 h; E,F: at 6 h; G,H: at 12 h. C,E,G: the ipsilateral side. D,F,H: the contralateral side. The number of nNOS neurons is increased in the ipsilateral side (C,E) as compared with that in the contralateral side (D,F). In G, the nNOS neurons in the ipsilateral side are decreased in number and have lost their dendrites, eC designates the external capsule. Bar, 100 ~m.
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removed and soaked in 15% sucrose in PB overnight followed by 30% sucrose in PB overnight. The brain was frozen in isopentane cooled on dry ice and then cut into coronal sections of 40 txm thickness on a cryostat. The sections were from the level of the caudate-putamen and the anterior commissure, and with longitudinal coordinate of A 5.6 m m according to a stereotaxic atlas of the 10-day-old rat brain [24]. The sections were blocked in 2% goat serum and 0.1% bovine serum albumin (BSA) in PBS, and incubated with the antibody against nNOS at a 1:8000 dilution for 12 h. After being washed in PBS, sections were incubated with biotinylated goat anti-rabbit IgG (Vector Labs, Burlingame, CA) at a 1:200 dilution for 2 h. Endogenous peroxidase activity was inhibited by the incubation
of sections with 3% hydrogen peroxide in PBS for 10 rain. The sections were incubated with avidin-biotin complex (Vectastatin ABC kit, Vector Labs, Burlingame, CA) for 1 h, and were then rinsed in Tris buffer and processed by the nickel-enhanced diaminobenzidine method. The sections were mounted on gelatin-coated glass slides, dehydrated in graded alcohol, and coverslipped. Control sections received identical treatment except use of the antibody against nNOS.
2.4. Nissi stain The sections adjacent to those stained immunohistochemically were washed in PBS and mounted on slide glasses. They were
Fig. 4. Nissl stained sections of the ipsilateral side in rats in Group H. A,C,E, the cortex; B,D,F, the striatum. A,B: at 3 h; C,D: at 6 h; E,F: at 12 h. In A,B: the arrow denotes the area of the neurons with hyperchromia. In C,D: the area of degeneration has expanded, and some neurons have pyknotic or fragmented nuclei. In E,F: most neurons have pyknotic or fragmented nuclei, and the cytoplasm is unrecognizable. Bar, 100 txm.
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defatted in graded alcohol, and stained with cresyl violet. Then, the sections were dehydrated in graded alcohol and mounted with coverslips.
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h, the area of degeneration had expanded, and some degenerated neurons had fragmented or pyknotic nuclei (Fig. 4C,D). At 12-24 h, diffuse degeneration was observed, and most neurons showed fragmented nuclei and disintegrated cytoplasm (Fig. 4E,F).
3. A N A L Y S I S The light microscopic evaluation was carried out by an examiner without knowing the experimental protocol. All the nNOS neurons in the hemisphere ipsilateral to and contralateral to the carotid ligation in immunohistochemically stained sections were counted. The cortical area as defined herein included the cingulate cortex and the parietal cortex, i.e., above the rhinal fissure, and the striatal area included the caudate-putamen. Only neurons of which the nucleus surrounded by the cytoplasm was visible in the section were counted. The degree of neuronal degeneration was also evaluated microscopically in a Nissl stained section adjacent to the section stained with anti-nNOS antibody. One-way analysis of variance was performed to test the significance of differences among the means at each time point on the two sides both in group L and group H. Then, the means at each time point on the ipsilateral side in group H were compared with that at time 0 on the ipsilateral side in group L using two-tailed t-test with the Bonferroni's correction. The ipsilateral side and the contralateral side were compared by two-tailed, paired t-test. A value of P < 0.05 was considered significant.
4. R E S U L T S Neurons stained by immunohistochemical treatment were distributed both in the cortex and the striatum (Fig. 1A,B). Control sections treated without nNOS antibody showed no specific staining. Figs. 2 and 3 show the numbers of nNOS neurons in the cortex and the striatum, respectively, for the hemisphere ipsilateral to and contralateral to the carotid ligation. At 3 h and 6 h, the numbers of nNOS neurons in the ipsilateral side in group H were significantly increased compared to those in the contralateral side. The numbers of nNOS neurons at 3 h and 6 h in the ipsilateral side in group H were significantly greater than that in the ipsilateral side in group L at 0 h. The nNOS neurons in the striatum were more clustered in the dorsolateral area (Fig. 1C,E) when compared with those in the same area of the contralateral side (Fig. I D,F). No degenerative changes were found at 3 h, although some nNOS neurons in the ipsilateral side showed shrunken dendrites at 6 h as compared with those in group N. At 12 h, the number of nNOS neurons in the ipsilateral side in group H was decreased, although the difference was not significant. These nNOS neurons showed distinct degenerative changes such as loss of dendrites and shrinkage (Fig. 1G), while those in the contralateral side showed no change (Fig. 1H). At 24 h, the number of nNOS neurons, for the cortex and for the striatum, in the ipsilateral side in group H was significantly decreased in comparison to that in the contralateral side at the same time point and compared to that in the ipsilateral side in group L at 0 h. Observation of Nissl stained sections revealed neuronal loss and degeneration were recognized only in the hemisphere ipsilateral to the carotid ligation in group H. At 3 h, patchy or laminar areas of neurons with hyperchromia appeared (Fig. 4A,B). At 6
5. D I S C U S S I O N In this study, neuronal degeneration in the Nissl stained sections was observed only in the hemisphere ipsilateral to the carotid ligation after the hypoxia, i.e., the ipsilateral side in group H. Apparently neither the hypoxia nor ischemia alone caused the neuronal injury in this neonatal rat model. Similarly, nNOS neurons were increased in number only in the hemisphere subjected to both ischemia and hypoxia. Our data represent further evidence of the possible involvement of nNOS in hypoxic-ischemic brain injury in the neonatal rat. The temporal profile of neuronal injury was parallel to the increase of the number of nNOS neurons. The nNOS neurons increased in number as early as 3 h, and the subsequent neuronal degeneration indicates that the intracellular mechanism leading to neuronal death had already commenced in this early period. Consideration of these results in the context of our previous findings that prehypoxic treatment with NOS inhibitor, NOARG, is neuroprotective [13], we consider that NO is neurotoxic and that increased numbers of nNOS neurons are responsible for the production of excessive NO which damages surrounding neurons. Trifiletti also demonstrated the neuroprotective effect of NOARG in the same neonatal model; prehypoxic treatment with NOARG decreased the brain damage as assessed by the disparity in hemispheric brain weight [25]. It has been disputed whether NO is neurotoxic or neuroprotective in the ischemic brain injury in adult animals [26,27]. NO-dependent vasodilation is noted to increase regional cerebral blood flow and reduce infarct volume [7,8,10]. However, we do not know of any report suggesting the neuroprotective effect of NO in a model of neonatal hypoxic-ischemic brain injury. In an ischemic insult, the cerebral circulation of the immature brain is further compromised by such factors as the low systemic blood pressure and the more labile autoregulation than that of the adult [28]. Under these conditions, it is possible that the vasodilatory/neuroprotective effect of NO, if present at all in neonates, is overwhelmed by the neurotoxicity of neuronally produced NO. In adult rats as well, recent data show that a selective nNOS inhibitor decreases the infarct volume and suggest that the effects of eNOS can be discriminated from those of nNOS [29]. Therefore, the neuroprotective effect of endothelial NO production is delineated in contrast to the neurotoxicity of neuronally overproduced NO [26]. Huang et al. also demonstrated the opposing roles of eNOS and nNOS using mutant mice deficient in nNOS activity [19]. They found that the mutant mice had significantly smaller infarcts following MCA occlusion than did the wild-type mice. Conversely, the infarct size in the mutant mice became larger when the activity of eNOS was inhibited by NOARG treatment. Zhang et al. reported an increase in the number of nNOS neurons in the hemisphere ipsilateral to the MCA occlusion and noted that the nNOS mRNA also was increased in the ipsilateral hemisphere of adult rats [18]. The increase was found 2 h after
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occlusion and was present over a longer period (at 2 - 4 8 h after occlusion) than we found in neonatal rats, and it had become bilateral at 4 h. The increase of nNOS neurons during neuronal injury was observed early in the course of injury in adult rats as well as in neonates. Following unilateral injury, bilateral induction of NADPH diaphorase activity in the hippocampus and in the cerebral cortex also is reported in adult rats [30]. NADPH diaphorase has a close association with NOS activity [22,31,32]. However, Ferriero et al., reported that, in the same neonatal rat model as we used, the increased number of NADPH-positive neurons in the cortex was observed only ipsilateral to the carotid ligation [33]. There may be a generalized response that is either humoral or mediated via neural connection against the ischemic insult in adult animals, but which is not present in an immature brain. The NADPH-diaphorase positive or the nNOS neurons have been noted to be resistant to hypoxic-ischemic insult [33] or to other pathological change such as Huntington disease [34,35]. NO modulates the NMDA-receptor and decreases the NMDAmediated increase of intracellular Ca 2+ [36] in a certain redox state [37]. This mechanism does not always prevent neuronal death; we observed the death of nNOS neurons themselves at 12-24 h after the insult. Zhang et al. also found the death of nNOS neurons in spite of their relative resistance to the ischemic insult in adult rats [18]. The increase of the number of nNOS neurons observed in our study may be an initial autoprotective response of the neurons under the insult by modulating their own NMDA-receptors and by producing increase of the regional cerebral blood flow around them. However, overproduced NO kills adjacent neurons, and eventually results in diffuse death of neurons including the nNOS neurons themselves.
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