Abstract
Our studies examined the hypothesis that the distribution of cerebral injury after a focal ischemic insult in the immature rat pup is associated with the regional distribution of nitric oxide synthase (NOS) activity and that differences in the vulnerability to ischemia between pup and adult might be related to differences in cofactor availability. We measured NOS activity in well-defined regions prone to become either core or penumbra in controls and at different times (end of occlusion, 0.5 h, and 24 h reperfusion) after middle cerebral artery occlusion (MCAO) from the right and left hemispheres in a 14- to 18-day-old rat pup filament model. Three groups of corresponding isoflurane sham controls were also included. “Core” NOS activity for combined right and left hemispheres ranged from 113% to 217% more than “penumbral” regions in control and sham groups. In the three MCAO groups, marked decreases in ischemic core and penumbral NOS activity were seen; however, core NOS remained higher than penumbral regions bilaterally. The effects of cofactor addition (10 μM tetrahydrobiopterin, 3 μM flavin adenine dinucleotide, and 3 μM flavin mononucleotide) on NOS activity were similar in “core” and “penumbral” regions in control and sham groups. However, after 24 h MCAO, cofactor addition preferentially increased NOS activity in the ischemic hemisphere. Cofactor addition in the pup also had a greater effect on enhancing NOS activity in all regions compared with the adult. Greater NOS activity in core regions in the rat pup, as in the adult, could in part, explain the increased vulnerability of that region to ischemia. NOS activity also can be influenced by the availability of cofactors and this effect may be greater in the immature animal.
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
- BH4:
-
tetrahydrobiopterin
- CaM:
-
calmodulin
- FAD:
-
flavin adenine dinucleotide
- FMN:
-
flavin mononucleotide
- MCAO:
-
middle cerebral artery occlusion
- NO:
-
nitric oxide
- NOS:
-
NO synthase
- SHR:
-
spontaneously hypertensive rat
- TTC:
-
2, 3, 5-triphenyltetrazolium chloride
References
Dorrepaal CA, Shadid M, Steendijk P, Van der Velde ET, Van de Bor M, Baan J, Van Bel F 1997 Effect of post-hypoxic-ischemic inhibition of nitric oxide synthesis on cerebral blood flow, metabolism and electrocortical brain activity in newborn lambs. Biol Neonate 72: 216–226.
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.
Ashwal S, Cole DJ, Osborne TN, Osborne S, 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.
Matsumoto T, Pollock JS, Nakane M, Förstermann U 1993 Developmental changes of cytosolic and particulate nitric oxide synthase in rat brain. Dev Brain Res 73: 199–203.
Pearce WJ, Tone B, Ashwal S 1997 Maturation alters cerebral NOS kinetics in the spontaneously hypertensive rat. Am J Physiol 42: R1–R7
Kuppusamy P, Ohnishi ST, Numagami Y, Ohnishi T, Zweier JL 1995 Three-dimensional imaging of nitric oxide production in the rat brain subjected to ischemia-hypoxia. J Cereb Blood Flow Metab 15: 904–913.
Salter M, Duffy C, Garthwaite J, Strubos PJLM 1995 Substantial regional and hemispheric differences in brain nitric oxide synthase (NOS) inhibition following intracerebroventricular administration of Nω-nitro-L-arginine (L-NA) and its methyl ester (L-NAME). Neuropharmacology 34: 639–649.
Ashwal S, Tone B, Tian HR, Cole DJ, Pearce WJ 1998 Core and penumbral nitric oxide synthase activity during cerebral ischemia and reperfusion. Stroke 29: 1037–1047.
Bredt DS, Snyder SH 1990 Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 87: 682–685.
Tobin JR, Martin LD, Breslow MJ, Traystman RJ 1994 Selective anesthetic inhibition of brain nitric oxide synthase. Anesthesiology 81: 1264–1269.
Ashwal S, Cole DJ, Osborne S, Osborne TN, Pearce WJ 1995 A new model of neonatal stroke: Reversible middle cerebral artery occlusion in the rat pup. Pediatr Neurol 12: 191–196.
Tamura A, Graham DI, McCulloch J, Teasdale GM 1981 Focal cerebral ischaemia in the rat: 1. J Cereb Blood Flow Metab 1: 53–60.
Memezawa H, Minamisawa H, Smith ML, Siesjö BK 1992 Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat. Exp Brain Res 89: 67–78.
Takagi K, Ginsberg MD, Globus MYT, Dietrich D, Martinez, Kraydieh S, Busto R 1993 Changes in amino acid neurotransmitters and cerebral blood flow in the ischemic penumbra region following middle cerebral artery occlusion in the rat: : correlation with histopathology. J Cereb Blood Flow Metab 13: 575–585.
Müller TB, Haraldseth O, Unsgård G 1994 Characterization of the microcirculation during ischemia and reperfusion in the penumbra of a rat model of temporary middle cerebral artery occlusion: : a laser doppler flowmetry study. Int J Microcirc 14: 289–295.
Zhang F, Iadecola C 1994 Reduction of focal cerebral ischemic damage by delayed treatment with nitric oxide donors. J Cereb Blood Flow Metab 14: 574–580.
Hatfield RH, Mendelow AD, Perry RH, Alvarez LM, Modha P 1991 Triphenyltetrazolium chloride (TTC) as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion. Neuropath Appl Neurobiol 17: 61–67.
Bartus RT, Dean RI, Cavanaugh K, Eveleth D, Carriero DL, Lynch G 1995 Time-related neuronal changes following middle cerebral artery occlusion: : implications for therapeutic intervention and the role of calpain. J Cereb Blood Flow Metab 15: 969–979.
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR 1990 A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10: 290–293.
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.
Higuchi Y, Hattori H, Kume T, Tsuji M, Akaike A, Furusho K 1998 Increase in nitric oxide in the hypoxic-ischemic neonatal rat brain and suppression by 7-nitroindazole and aminoguanidine. Eur J Pharmacol 342: 47–49.
Bredt DS, Snyder SH 1994 Transient nitric oxide synthase neurons in embryonic cerebral cortical plate, sensory ganglia, and olfactory epithelium. Neuron 13: 301–313.
Yan XX, Garey LJ, Jen LS 1994 Development of NADPH-diaphorase activity in the rat neocortex. Dev Brain Res 79: 29–38.
Samama B, Chateau D, Boehm N 1995 Expression of NADPH-diaphorase in the rat forebrain during development. Neurosci Lett 184: 204–207.
Johnston MV 1997 Hypoxic and ischemic disorders of infants and children. Brain Dev 19: 235–239.
Beasley TC, Bari F, Thore C, Thrikawala N, Louis T, Busija D 1998 Cerebral ischemia/reperfusion increases endothelial nitric oxide synthase levels by an indomethacin-sensitive mechanism. J Cereb Blood Flow Metab 18: 88–96.
Roohey T, Raju TN, Moustogiannis AN 1997 Animal models for the study of perinatal hypoxic-ischemic encephalopathy: : a critical analysis. Early Hum Dev 47: 115–146.
Vannucci RC, Vannucci SJ 1997 A model of perinatal hypoxic-ischemic brain damage. Ann N Y Acad Sci 835: 234–249.
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.
Hagberg H, Bona E, Gilland E, Puka-Sundvall M 1997 Hypoxia-ischaemia model in the 7-day-old rat: possibilities and shortcomings. Acta Paediatr Suppl 422: 85–88.
Ikonomidou C, Mosinger JL, Salles KS, Labruyere J, Olney JW 1989 Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methyl-aspartate neurotoxicity. J Neurosci 9: 2809–2818.
McDonald JW, Johnston MV 1990 Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res Rev 15: 41–70.
Andersen DL, Tannenberg AE, Burke CJ, Dodd PR 1998 Regional development of glutamate-N-methyl-D-aspartate receptor sites in asphyxiated newborn infants. J Child Neurol 13: 149–157.
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.
Yan XX, Garey LJ, Jen LS 1996 Prenatal development of NADPH-diaphorase-reactive neurons in human frontal cortex. Cereb Cortex 6: 737–745.
Gonzalez-Hernandez T, Gonzalez-Gonzalez B, Mantolan-Sarmiento B, Mendez-Medina R, Ferres-Torres R, Meyer G 1994 Transient NADPH-diaphorase activity in motor nuclei of the foetal human brain stem. Neuroreport 5: 758–760.
Bredt DS, Snyder SH 1994 Transient nitric oxide synthase neurons in embryonic cerebral cortical plate, sensory ganglia, and olfactory epithelium. Neuron 13: 301–313.
Santacana M, Uttenthal LO, Bentura ML, Fernandez AP, Serrano J, Martinez de Velasco J, Alonso D, Martinez-Murillo R, Rodrigo J 1998 Expression of neuronal nitric oxide synthase during embryonic development of the rat cerebral cortex. Brain Res Dev Brain Res 111: 205–222.
Luth HJ, Hedlich A, Hilbig H, Winkelmann E, Mayer B 1995 Postnatal development of NADPH-diaphorase/nitric oxide synthase positive nerve cells in the visual cortex of the rat. J Hirnforsch 36: 313–328.
Tenorio F, Giraldi-Guimaraes A, Mendez-Otero R 1995 Developmental changes of nitric oxide synthase in the rat superior colliculus. J Neurosci Res 42: 633–637.
Black SM, Bedolli MA, Martinez S, Bristow JD, Ferriero DM, Soifer SJ 1995 Expression of neuronal nitric oxide synthase corresponds to regions of selective vulnerability to hypoxia-ischaemia in the developing rat brain. Neurobiol Dis 2: 145–155.
van Eden CG, Steinbusch HW, Rinkens A, de Vente J 1996 Developmental pattern of NADPH-diaphorase activity and nitric oxide-stimulated cGMP immunoreactivity in the frontal rat cortex and its role in functional recovery from aspiration lesions. J Chem Neuroanat 10: 279–286.
Tomic D, Zobundzija M, Meaugorac M 1994 Postnatal development of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) positive neurons in rat prefrontal cortex. Neurosci Lett 170: 217–220.
Ohyu J, Takashima S 1998 Developmental characteristics of neuronal nitric oxide synthase (nNOS) immunoreactive neurons in fetal to adolescent human brains. Brain Res Dev Brain Res 110: 193–202.
Hamada Y, Hayakawa T, Hattori H, Mikawa H 1994 Inhibitors of nitric oxide synthesis reduces hypoxic-ischemic brain damage in the neonatal rat. Pediatr Res 35: 10–14.
Brand MP, Heales SJ, Land JM, Clark JB 1995 Tetrahydrobiopterin deficiency and brain nitric oxide synthase in the hph1 mouse. J Inherit Metab Dis 18: 33–39.
Author information
Authors and Affiliations
Additional information
Supported by the Pediatric Research Fund from the Department of Pediatrics, Loma Linda University School of Medicine and by the Department of Anesthesiology.
Rights and permissions
About this article
Cite this article
Ashwal, S., Tone, B., Tian, H. et al. Core and Penumbral Nitric Oxide Synthase Activity during Cerebral Ischemia and Reperfusion in the Rat Pup. Pediatr Res 46, 390 (1999). https://doi.org/10.1203/00006450-199910000-00006
Received:
Accepted:
Issue date:
DOI: https://doi.org/10.1203/00006450-199910000-00006
This article is cited by
-
Enhanced autophagy interacting proteins negatively correlated with the activation of apoptosis-related caspase family proteins after focal ischemic stroke of young rats
BMC Neuroscience (2022)
-
Neuronal chemokine-like-factor 1 (CKLF1) up-regulation promotes M1 polarization of microglia in rat brain after stroke
Acta Pharmacologica Sinica (2022)
-
Morin Attenuated Cerebral Ischemia/Reperfusion Injury Through Promoting Angiogenesis Mediated by Angiopoietin-1-Tie-2 Axis and Wnt/β-Catenin Pathway
Neurotoxicity Research (2022)
-
Neferine Protects Against Brain Damage in Permanent Cerebral Ischemic Rat Associated with Autophagy Suppression and AMPK/mTOR Regulation
Molecular Neurobiology (2021)
-
Effects of Paired Associative Stimulation on Metabolites in Ischemia Stroke Rats Model as Studied by Nuclear Magnetic Resonance Spectrum
Neurochemical Research (2021)
