Abstract
This study addressed the hypothesis that the delayed impairment in cerebral energy metabolism that develops 10-24 h after transient hypoxia-ischemia in the developing brain is mediated by induction of increased nitric oxide synthesis. Four groups of 14-d-old Wistar rat pups were studied. Group 1 was subjected to unilateral carotid artery ligation and hypoxia followed immediately by treatment with the nitric oxide synthase (NOS) inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg). Group 2 underwent hypoxia-ischemia but received saline vehicle. Group 3 received L-NAME without hypoxia-ischemia, and group 4, saline vehicle alone. At defined times after insult, the expression of neuronal and inducible NOS were determined and calcium-dependent and -independent NOS activities measured. Cerebral energy metabolism was observed using 31P magnetic resonance spectroscopy. At 48 h after insult, the expression of inducible NOS increased, whereas neuronal NOS at 24 h decreased on the infarcted side. Calcium-dependent NOS activity was higher than calcium-independent NOS activity, but did not increase within 36 h after insult, and was significantly inhibited by the administration of L-NAME. However, L-NAME did not prevent delayed impairment of cerebral energy metabolism or ameliorate infarct size. These results suggest that the delayed decline in cerebral energy metabolism after hypoxia-ischemia in the 14-d-old rat brain is not mediated by increased nitric oxide synthesis.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- HI:
-
hypoxia-ischemia
- NO:
-
nitric oxide
- NOS:
-
nitric oxide synthase
- nNOS:
-
neuronal nitric oxide synthase
- eNOS:
-
endothelial nitric oxide synthase
- iNOS:
-
inducible nitric oxide synthase
- L-NAME:
-
Nω-nitro-L-arginine methyl ester
- pHi:
-
intracellular pH
- MRS:
-
magnetic resonance spectroscopy
- NTP:
-
nucleotide triphosphates
- PCr:
-
phosphocreatine
- Pi:
-
inorganic phosphate
- EPP:
-
total exchangeable phosphate pool
- TCF:
-
tissue clearing fluid
References
Hope PL, Costello AM, Cady EB, Delpy DT, Tofts PS, Chu A, Hamilton PA, Reynolds EOR, Wilkie DR 1984 Cerebral energy metabolism studied with phosphorus NMR spectroscopy in normal and birth-asphyxiated infants. Lancet 2: 366–370
Yager JY, Brucklacher RM, Vannucci RC 1991 Cerebral oxidative metabolism and redox state during hypoxia-ischemia and early recovery in immature rats. Am J Physiol 261:H1102–H1108
Lorek A, Takei Y, Cady EB, Wyatt JS, Penrice J, Edwards AD, Peebles D, Wylezinska M, Owen-Reece H, Kirkbride V, Cooper C, Aldridge RF, Roth SC, Brown G, Delpy DT, Reynolds EOR 1994 Delayed ("secondary") cerebral energy failure after acute hypoxia-ischemia in the newborn piglet: continuous 48-hour studies by phosphorus magnetic resonance spectroscopy. Pediatr Res 36: 699–706
Blumberg RM, Cady EB, Wigglesworth JS, McKenzie JE, Edwards AD 1997 Relation between delayed impairment of cerebral energy metabolism and infarction following transient focal hypoxia-ischaemia in the developing brain. Exp Brain Res 113: 130–137
Marks KA, Mallard EC, Roberts I, Williams CE, Sirimanne ES, Johnston B, Gluckman PD, Edwards AD 1996 Delayed vasodilation and altered oxygenation after cerebral ischemia in fetal sheep. Pediatr Res 39: 48–54
Azzopardi D, Wyatt JS, Cady EB, Delpy DT, Baudin J, Stewart AL, Hope PL, Hamilton PA, Reynolds EOR 1989 Prognosis of newborn infants with hypoxic-ischemic brain injury assessed by phosphorus magnetic resonance spectroscopy. Pediatr Res 25: 445–451
Williams CE, Gunn A, Gluckman PD 1991 Time course of intracellular edema and epileptiform activity following prenatal cerebral ischemia in sheep. Stroke 22: 516–521
Yue X, Mehmet H, Penrice J, Cooper C, Cady E, Wyatt JS, Reynolds EO, Edwards AD, Squier MV 1997 Apoptosis and necrosis in the newborn piglet brain following transient cerebral hypoxia-ischaemia. Neuropathol Appl Neurobiol 23: 16–25
Nowicki JP, Duval D, Poignet H, Scatton B 1991 Nitric oxide mediates neuronal death after focal cerebral ischemia in the mouse. Eur J Pharmacol 204: 339–340
Dawson VL, Dawson TM, London ED, Bredt DS, Snyder SH 1991 Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci USA 88: 6368–6371
Dawson VL, Dawson TM, Bartley DA, Uhl GR, Snyder SH 1993 Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. J Neurosci 13: 2651–2661
Dawson VL, Dawson TM 1996 Nitric oxide neurotoxicity. J Chem Neuroanat 10: 179–190
Tan WK, Williams CE, During MJ, Mallard CE, Gunning MI, Gunn AJ, Gluckman PD 1996 Accumulation of cytotoxins during the development of seizures and edema after hypoxic-ischemic injury in late gestation fetal sheep. Pediatr Res 39: 791–797
Thoresen M, Satas S, Puka-Sandvall M, Whitelaw A, Hallstrom A, Loberg E-M, Ungerstedt U, Steen PA, Hagberg H 1997 Post-hypoxic hypothermia reduces cerebrocortical release of NO and excitotoxins. Neuroreport 8: 3359–3362
Brown GC, Cooper CE 1994 Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett 356: 259–298
Iadecola C, Xu X, Zhang F, et al Fakahany EE, Ross ME 1995 Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia. J Cereb Blood Flow Metab 15: 52–59
Iadecola C, Zhang F, Xu S, Casey R, Ross ME 1995 Inducible nitric oxide synthase gene expression in brain following cerebral ischemia. J Cereb Blood Flow Metab 15: 378–384
Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA 1994 Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265: 1883–1885
Ferriero DM, Holtzman DM, Black SM, Sheldon RA 1996 Neonatal mice lacking neuronal nitric oxide synthase are less vulnerable to hypoxic-ischemic injury. Neurobiol Dis 3: 64–71
Panahian Y, Yoshida T, Huang PL, Hedley Whyte ET, Dalkara T, Fishman MC, Moskowitz MA 1996 Attenuated hippocampal damage after global cerebral ischemia in mice mutant in neuronal nitric oxide synthase. Neuroscience 72: 343–354
Samdani AF, Dawson TM, Dawson VL 1997 Nitric oxide synthase in models of focal ischemia. Stroke 28: 1283–1288
Forstermann U, Schmidt HHHW, Pollock JS, Sheng H, Mitchell JA, Warner TD, Nakane M, Murad F 1991 isoforms of nitric oxide synthase-characterization and purification from different cell types. Biochem Pharmacol 42: 1849–1857
Romijn HJ, Hofman MA, Gramsbergen A 1991 At what age is the developing cerebral cortex of the rat comparable to that of the full-term newborn human baby?. Early Hum Dev 26: 61–67
Lizasoain I, Weiner CP, Knowles RG, Moncada S 1996 The ontogeny of cerebral and cerebellar nitric oxide synthase in the guinea pig and rat. Pediatr Res 39: 779–783
Chao CC, Hu S, Molitor TW, Shaskan EG, Peterson PK 1992 Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J Immunol 149: 2736–2741
Sirimanne ES, Guan J, Williams CE, Gluckman PD 1994 Two models for determining the mechanisms of damage and repair after hypoxic-ischaemic injury in the developing rat brain. ethods 55: 7–14
Sherwood NM, Timiras PS 1970 A Stereotaxic Atlas of the Developing Rat Brain. University of California Press, Berkley, CA, pp 23–48
Salter M, Knowles RG, Moncada S 1991 Widespread tissue distribution, species distribution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases. FEBS Lett 291: 145–149
Petroff OA, Prichard JW, Behar KL, Alger JR, den Hollander JA, Shulman RG 1985 Cerebral intracellular pH by 31P nuclear magnetic resonance spectroscopy. Neurology 35: 781–788
Robertson NJ, Cox IJ, Counsell S, Cowan F, Azzopardi D, Edwards AD 1998 Persistent lactate following perinatal hypoxic-ischaemic encephalopathy and its relationship to energy failure studied by magnetic resonance spectroscopy. Early Hum Dev 5: 73
Matsumoto T, Pollock JS, Nakane M, Forstermann U 1993 Developmental changes of cytosolic and particulate nitric oxide synthase in rat brain. Brain Res Dev Brain Res 73: 199–203
Keilhoff G, Seidel B, Noack H, Tischmeyer W, Stanek D, Wolf G 1996 Patterns of nitric oxide synthase at the messenger RNA and protein levels during early rat brain development. Neuroscience 75: 1193–1201
Higuchi Y, Hattori H, Hattori R, Furusho K 1996 Increased neurons containing neuronal nitric oxide synthase in the brain of a hypoxic-ischemic neonatal rat model. Brain Dev 18: 369–375
Shibata M, Araki N, Hamada J, Sasaki T, Shimazu K, Fukuuchi Y 1996 Brain nitrite production during global ischemia and reperfusion: an in vivo microdialysis study. Brain Res 734: 86–90
Bolanos JP, Almeida A, Stewart V, Peuchen S, Land JM, Clark JB, Heales SJ 1997 Nitric oxide-mediated mitochondrial damage in the brain: mechanisms and implications for neurodegenerative diseases. J Neurochem 68: 2227–2240
Buttery LD, Evans TJ, Springall DR, Carpenter A, Cohen J, Polak JM 1994 Immunochemical localization of inducible nitric oxide synthase in endotoxin-treated rats. lab Invest 71: 755–764
Springall DR, Riveros-Moreno V, Buttery L, Suburo A, Bishop AE, Merrett M, Moncada S, Polak JM 1992 Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms. Histochemistry 98: 259–266
Yager JY, Brucklacher RM, Vannucci RC 1992 Cerebral Energy metabolism during hypoxia-ischemia and elderly recovery in immature rats. Am J Physiol 262:H672–H677
Williams CE, Gunn AJ, Mallard C, Gluckman PD 1992 Outcome after ischemia in the developing sheep brain: an electroencephalographic and histological study. Ann Neurol 31: 14–21
Ashwal S, Cole DJ, Osborne S, Osborne TN, Pearce WJ 1995 L-NAME reduces infarct volume in a filament model of transient middle cerebral artery occlusion in the rat pup. Pediatr Res 38: 652–656
Anderson RE, Meyer FB 1996 Nitric oxide synthase inhibition by L-NAME during repetitive focal cerebral ischemia in rabbits. Am J Physiol 271:H588–H594
Dalkara T, Yoshida T, Irikura K, Moskowitz MA 1994 Dual role of nitric oxide in focal cerebral ischemia. Neuropharmacology 33: 1447–1452
Marks KA, Mallard CE, Roberts I, Williams CE, Gluckman PD, Edwards AD 1996 Nitric oxide synthase inhibition attenuates delayed vasodilation and increases injury after cerebral ischemia in fetal sheep. Pediatr Red 40: 185–191
Takei Y, Edwards AD, Lorek A, Peebles DM, Belai A, Cope M, Delpy DT, Reynolds EOR 1993 Effects of Nω-nitro-L-arginine methyl ester on the cerebral circulation of newborn piglets quantified in vivo by near-infrared spectroscopy. Pediatr Res 34: 354–359
Patel K, Pryds O, Roberts I, Harris D, Edwards D 1996 Limited role for nitric oxide in mediating cerebrovascular control of newborn piglets. Arch Dis Child Fetal Neonatal Ed 75:F82–F86
Wei HM, Chi OZ, Liu X, Sinha AK, Weiss HR 1994 Nitric oxide synthase inhibition alters cerebral blood flow and oxygen balance in focal cerebral ischemia in rats. Stroke 25: 445–449
Tasker RC, Sahota SK, Williams SR 1996 Bioenergetic recovery following ischemia in brain slices studied by 31P NMR spectroscopy: differential age effect of depolarization mediated by endogenous nitric oxide. J Cereb Blood Flow Metab 16: 125–133
Acknowledgements
The authors wish to thank Dr. T. Evans and Prof. J. Polak (ICSM, Hammersmith Hospital, London) for generously supplying the antibodies used in this study.
Author information
Authors and Affiliations
Additional information
Supported by the Wellcome Trust (grant No. 038919).
Rights and permissions
About this article
Cite this article
Blumberg, R., Taylor, D., Yue, X. et al. Increased Nitric Oxide Synthesis Is Not Involved in Delayed Cerebral Energy Failure following Focal Hypoxic-Ischemic Injury to the Developing Brain. Pediatr Res 46, 224–231 (1999). https://doi.org/10.1203/00006450-199908000-00016
Received:
Accepted:
Issue date:
DOI: https://doi.org/10.1203/00006450-199908000-00016
