Loss of nitric oxide synthase immunoreactivity in cerebral vasospasm

✓ To determine the distribution of nitric oxide synthase (NOS) in the primate cerebral artery nervi vasorum and to examine the potential role of NOS in cerebral vasospasm after subarachnoid hemorrhage (SAH) in primates, the distribution of NOS immunoreactivity (NOS-IR) in the major cerebral arteries was examined immunohistochemically in cynomolgus monkeys by the use of whole, mounted preparations of the circle of Willis. In four normal monkeys, NOS-IR was localized to the endothelial and adventitial layers of the large cerebral arteries. On the abluminal side, NOS-IR staining was densely concentrated in perivascular nerve fibers (nervi vasorum) of the anterior circulation. Staining was less prominent in the posterior circulation. In six monkeys with vasospasm on Day 7 after placement of preclotted arterial blood to form an SAH around the right middle cerebral artery (MCA) (42% ± 8.3% decrease of MCA area, mean ± standard deviation), NOS-IR was virtually absent in nerve fibers around the spastic right MCA but was normal on the contralateral side. In five monkeys in which vasospasm resolved by Day 14 after SAH (36% ± 14% decrease of right MCA area on Day 7, and 5% ± 14% decrease on Day 14), NOS-IR was also absent in the right MCA adventitial nerve fibers and remained normal in the left MCA. Adventitial NOS-IR was also normal in cerebral vessels of a sham-operated, nonspastic monkey. These findings provide further evidence that nitric oxide (NO) functions as a neuronal transmitter to mediate vasodilation in primates and indicate a role for adventitial NO in the pathogenesis of cerebral vasospasm after SAH in humans.

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Neuronal Nitric Oxide Synthase Activation by Vasoactive Intestinal Peptide in Bovine Cerebral Arteries

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-199709000-00007 ◽

1997 ◽

Vol 17 (9) ◽

pp. 977-984 ◽

Cited By ~ 45

Author(s):

Carmen González ◽

Carla Barroso ◽

Carmen Martín ◽

Sergio Gulbenkian ◽

Carmen Estrada

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Vasoactive Intestinal Peptide ◽

Cerebral Artery ◽

Cyclic Gmp ◽

Nerve Stimulation ◽

Cerebral Arteries ◽

Neuronal Nitric Oxide Synthase ◽

Nerve Fibers ◽

Oxide Synthase

The participation of nitric oxide and vasoactive intestinal peptide (VIP) in the neurogenic regulation of bovine cerebral arteries was investigated. Nitrergic nerve fibers and ganglion-like groups of neurons were revealed by NADPH-diaphorase staining in the adventitial layer of bovine cerebral arteries. NADPH diaphorase also was present in endothelial cells but not in the smooth muscle layer. Double immunolabeling for neuronal nitric oxide synthase and VIP indicated that both molecules co-localized in the same nerve fibers in these vessels. Transmural nerve stimulation (200 mA, 0.2 milliseconds, 1 to 8 Hz) of endothelium-denuded bovine cerebral artery rings precontracted with prostaglandin F2α, produced tetrodotoxin-sensitive relaxations that were completely suppressed by NG-nitro-l-arginine methyl ester (l-NAME) and by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline (ODQ), but were not affected by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), nor by VIP tachyphylaxis induced by pretreatment with 1 μmol/L VIP. Transmural nerve stimulation also elicited increases in intracellular cyclic GMP concentration, which were prevented by l-NAME, and small decreases in intracellular cyclic AMP concentration. Addition of VIP to bovine cerebral artery rings without endothelium produced a concentration-dependent relaxation that was partially inhibited by l-NAME, ODQ, and SQ 22,536. The effects of l-NAME and SQ 22,536 were additive. VIP induced a transient increase in intracellular cyclic GMP concentration, which was maximal 1 minute after VIP addition, when the highest relaxation rate was observed, and which was blocked by l-NAME. It is concluded that nitric oxide produced by perivascular neurons and nerve fibers fully accounts for the experimental neurogenic relaxation of bovine cerebral arteries and that VIP, which also is present in the same perivascular fibers, acts as a neuromodulator by activating neuronal nitric oxide synthase.

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Attenuation of nitric oxide synthase isoform expression by mild hypothermia after focal cerebral ischemia: variations depending on timing of cooling

Journal of Neurosurgery ◽

10.3171/jns.2003.98.6.1271 ◽

2003 ◽

Vol 98 (6) ◽

pp. 1271-1276 ◽

Cited By ~ 30

Author(s):

Murat Karabiyikoglu ◽

Hyung Soo Han ◽

Midori A. Yenari ◽

Gary K. Steinberg

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Mild Hypothermia ◽

Inos Expression ◽

Western Blots ◽

Neuronal Marker ◽

Content Type ◽

Oxide Synthase ◽

Nos Isoforms ◽

Fine Print

Object. In this study the authors examined the influence of mild hypothermia on early expression of nitric oxide synthase (NOS) isoforms and peroxynitrite generation after experimental stroke. Methods. In 82 male Sprague—Dawley rats, middle cerebral artery occlusion was performed for 2 hours by using the intraluminal suture model. The rats were maintained at their normal body temperature or exposed to 2 hours of intraischemic or postischemic (2-hour delay) mild hypothermia. Brains were collected 2, 6, and 24 hours after onset of ischemia for immunohistochemical and Western blot analysis of neuronal (n)NOS and inducible (i)NOS expression and peroxynitrite generation. Conclusions. Western blots showed significantly increased nNOS and iNOS expression in the ischemic cortex at 2, 6, and 24 hours compared with sham-operated animals. The NOS expression was highest at 24 hours. Postischemic hypothermia attenuated nNOS expression at 6 and 24 hours to a greater extent than intraischemic hypothermia. Intraischemic hypothermia reduced iNOS expression at both 2 and 24 hours, whereas postischemic hypothermia decreased iNOS expression at 24 hours. Results of immunohistochemical studies showed that nNOS colocalized with the neuronal marker MAP-2 at all time points, whereas iNOS was initially localized to vessels, and then localized to activated microglia by 24 hours. Intraischemic but not postischemic hypothermia decreased the number of nitrotyrosine-positive cells in the ischemic cortex at 24 hours. Mild hypothermia significantly but differentially attenuates increases in NOS isoforms, with more robust nNOS suppression when cooling is delayed. This may have important implications for understanding the mechanism of hypothermic neuroprotection and for stroke therapy.

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Association between cerebrospinal fluid levels of asymmetric dimethyl-L-arginine, an endogenous inhibitor of endothelial nitric oxide synthase, and cerebral vasospasm in a primate model of subarachnoid hemorrhage

Journal of Neurosurgery ◽

10.3171/jns.2004.101.5.0836 ◽

2004 ◽

Vol 101 (5) ◽

pp. 836-842 ◽

Cited By ~ 55

Author(s):

Carla S. Jung ◽

Brian A. Iuliano ◽

Judith Harvey-White ◽

Michael G. Espey ◽

Edward H. Oldfield ◽

...

Keyword(s):

Nitric Oxide ◽

Cerebrospinal Fluid ◽

Cerebral Vasospasm ◽

Primate Model ◽

Content Type ◽

Cerebrospinal Fluid Levels ◽

Delayed Cerebral Vasospasm ◽

The Right ◽

Fluid Levels ◽

Fine Print

Object. Decreased availability of nitric oxide (NO) has been proposed to evoke delayed cerebral vasospasm after subarachnoid hemorrhage (SAH). Asymmetric dimethyl-l-arginine (ADMA) inhibits endothelial NO synthase (eNOS) and, therefore, may be responsible for decreased NO availability in cases of cerebral vasospasm. The goal of this study was to determine whether ADMA levels are associated with cerebral vasospasm in a primate model of SAH. Methods. Twenty-two cynomolgus monkeys (six control animals and 16 with SAH) were used in this study. The levels of ADMA, l-arginine, l-citrulline, nitrites, and nitrates in cerebrospinal fluid (CSF) and serum were determined on Days 0, 7, 14, and 21 following onset of SAH. Cerebral arteriography was performed to assess the degree of vasospasm. Western blot analyses of the right and left middle cerebral arteries (MCAs) were performed to assess the expression of eNOS, type I protein—arginine methyl transferase (PRMT1) and dimethylarginine dimethylaminohydrolase (DDAH2). Cerebrospinal fluid levels of ADMA remained unchanged in the control group (six animals) and in animals with SAH that did not have vasospasm (five animals; p = 0.17), but the levels increased in animals with vasospasm (11 animals) on Day 7 post-SAH (p < 0.01) and decreased on Days 14 through 21 (p < 0.05). Cerebrospinal fluid levels of ADMA correlated directly with the degree of vasospasm (correlation coefficient = 0.7, p = 0.0001; 95% confidence interval: 0.43–0.83). Levels of nitrite and nitrate as well as those of l-citrulline in CSF were decreased in animals with vasospasm. Furthermore, DDAH2 expression was attenuated in the right spastic MCA on Day 7 post-SAH, whereas eNOS and PRMT1 expression remained unchanged. Conclusions. Changes in the CSF levels of ADMA are associated with the development and resolution of vasospasm found on arteriograms after SAH. The results indicate that endogenous inhibition of eNOS by ADMA may be involved in the development of delayed cerebral vasospasm. Inhibition of ADMA production may provide a new therapeutic approach for cerebral vasospasm after SAH.

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Selected Contribution: Cerebrovascular NOS and cyclooxygenase are unaffected by estrogen in mice lacking estrogen receptor-α

Journal of Applied Physiology ◽

10.1152/jappl.2001.91.5.2391 ◽

2001 ◽

Vol 91 (5) ◽

pp. 2391-2399 ◽

Cited By ~ 45

Author(s):

Greg G. Geary ◽

Anne Marie McNeill ◽

Jose A. Ospina ◽

Diana N. Krause ◽

Kenneth S. Korach ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Cerebral Artery ◽

Endothelial Nitric Oxide Synthase ◽

Cerebral Arteries ◽

Estrogen Treatment ◽

Wild Type ◽

Cyclooxygenase 1 ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Estrogen alters reactivity of cerebral arteries by modifying production of endothelium-dependent vasodilators. Estrogen receptors (ER) are thought to be involved, but the responsible ER subtype is unknown. ER-α knockout (αERKO) mice were used to test whether estrogen acts via ER-α. Mice were ovariectomized, with or without estrogen replacement, and cerebral blood vessels were isolated 1 mo later. Estrogen increased levels of endothelial nitric oxide synthase and cyclooxygenase-1 in vessels from wild-type mice but was ineffective in αERKO mice. Endothelium-denuded middle cerebral artery segments from all animals constricted when pressurized. In denuded arteries from αERKO but not wild-type mice, estrogen treatment enhanced constriction. In endothelium-intact, pressurized arteries from wild-type estrogen-treated mice, diameters were larger compared with arteries from untreated wild-type mice. In addition, contractile responses to indomethacin were greater in arteries from wild-type estrogen-treated mice compared with arteries from untreated wild-type mice. In contrast, estrogen treatment of αERKO mice had no effect on diameter or indomethacin responses of endothelium-intact arteries. Thus ER-α regulation of endothelial nitric oxide synthase and cyclooxygenase-1 pathways appears to contribute to effects of estrogen on cerebral artery reactivity.

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Bilateral innervation of the cerebral arteries by the superior cervical ganglion in cats

Journal of Neurosurgery ◽

10.3171/jns.1980.53.1.0088 ◽

1980 ◽

Vol 53 (1) ◽

pp. 88-91 ◽

Cited By ~ 22

Author(s):

Jesús Marín ◽

Mercedes Salaices ◽

Fernando Rivilla ◽

Javier Burgos ◽

Emilio J. Marco

Keyword(s):

Cerebral Artery ◽

Superior Cervical Ganglion ◽

Anterior Cerebral Artery ◽

Contractile Response ◽

Cerebral Arteries ◽

The Other ◽

Content Type ◽

Cervical Ganglion ◽

The Right ◽

Fine Print

✓ The effect of removal of the left superior cervical ganglion on the contractile response to norepinephrine (NE) and 5-hydroxytryptamine (5-HT, serotonin) was studied in isolated segments of the middle cerebral artery (MCA) and posterior communicating artery (PCoA) of the cat. Fifteen days after the excision, each dose of NE elicited a potentiated response in both the MCA and the PCoA, whichever side they originated. By contrast, 5-HT induced enhanced vasoconstriction at each dose only in the MCA and PCoA from the left side. When segments of MCA and PCoA from the right side were challenged against 5-HT, a significantly increased response was found only at the first three doses. On the other hand, the NE content of pools made of MCA, PCoA, and anterior cerebral artery from each side was reduced to the same level on both sides after ganglion removal. These results indicate that the excised superior cervical ganglion innervated the MCA and PCoA from both sides of the circle of Willis.

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Effect of intracarotid nitric oxide on primate cerebral vasospasm after subarachnoid hemorrhage

Journal of Neurosurgery ◽

10.3171/jns.1995.83.1.0118 ◽

1995 ◽

Vol 83 (1) ◽

pp. 118-122 ◽

Cited By ~ 113

Author(s):

John K. B. Afshar ◽

Ryszard M. Pluta ◽

Robert J. Boock ◽

B. Gregory Thompson ◽

Edward H. Oldfield

Keyword(s):

Nitric Oxide ◽

Blood Flow ◽

Subarachnoid Hemorrhage ◽

Cerebral Vasospasm ◽

Primate Model ◽

Content Type ◽

Continuous Release ◽

Blood Flow Velocities ◽

The Right ◽

Fine Print

✓ The continuous release of nitric oxide (NO) is required to maintain basal cerebrovascular tone. Oxyhemoglobin, a putative spasmogen, rapidly binds NO, implicating loss of NO in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). If vasospasm is mediated by depletion of NO in the vessel wall, it should be reversible by replacement with NO. To investigate this hypothesis, the authors placed blood clots around the right middle cerebral artery (RMCA) of four cynomolgus monkeys; four unoperated animals served as controls. Arteriography was performed before and 7 days after surgery to assess the presence and degree of vasospasm, which was quantified in the anteroposterior (AP) projection by computerized image analysis. On Day 7, cortical cerebral blood flow (CBF) in the distribution of the right MCA was measured during four to six runs in the right internal carotid artery (ICA) of brief infusions of saline followed by NO solution. Arteriography was performed immediately after completing the final NO infusion in three of the four animals with vasospasm. Right MCA blood flow velocities were obtained using transcranial Doppler before, during, and after NO infusion in two vasospastic animals. After ICA NO infusion, arteriographic vasospasm resolved (mean percent of preoperative AP area, 55.9%); that is, the AP areas of the proximal portion of the right MCA returned to their preoperative values (mean 91.4%; range 88%–96%). Compared to ICA saline, during ICA NO infusion CBF increased 7% in control animals and 19% in vasospastic animals (p < 0.002) without significant changes in other physiological parameters. During NO infusion, peak systolic right MCA CBF velocity decreased (130 to 109 cm/sec and 116 to 76 cm/sec) in two vasospastic animals. The effects of ICA NO on CBF and CBF velocity disappeared shortly after terminating NO infusion. Intracarotid infusion of NO in a primate model of vasospasm 1) increases CBF, 2) decreases cerebral vascular resistance, 3) reverses arteriographic vasospasm, and 4) decreases CBF velocity in the vasospastic artery without producing systemic hypotension. These findings indicate the potential for the development of targeted therapy to reverse cerebral vasospasm after SAH.

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Possible Origins and Distribution of Immunoreactive Nitric Oxide Synthase-Containing Nerve Fibers in Cerebral Arteries

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1993.9 ◽

1993 ◽

Vol 13 (1) ◽

pp. 70-79 ◽

Cited By ~ 298

Author(s):

Kazuhiko Nozaki ◽

Michael A. Moskowitz ◽

Kenneth I. Maynard ◽

Naoki Koketsu ◽

Ted M. Dawson ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Ganglion Cells ◽

Cerebral Arteries ◽

Nerve Fibers ◽

Circle Of Willis ◽

Sphenopalatine Ganglion ◽

Sprague Dawley ◽

Pial Arteries ◽

Oxide Synthase

The distribution of perivascular nerve fibers expressing nitric oxide synthase (NOS)-immunoreactivity was examined in Sprague–Dawley and Long–Evans rats using affinity-purified rabbit antisera raised against NOS from rat cerebellum. NOS immunoreactivity was expressed within the endothelium and adventitial nerve fibers in both rat strains. Labeled axons were abundant and dense in the proximal anterior and middle cerebral arteries, but were less numerous in the caudal circle of Willis and in small pial arteries. The sphenopalatine ganglia were the major source of positive fibers in these vessels. Sectioning postganglionic parasympathetic fibers from both sphenopalatine ganglia reduced the density of NOS-immunoreactive (IR) nerve fibers by >75% in the rostral circle of Willis. Moreover, NOS-IR was present in 70–80% of sphenopalatine ganglion cells. Twenty percent of these neurons also contained vasoactive intestinal polypeptide (VlP)-immunoreactivity. By contrast, the superior cervical ganglia did not contain NOS-IR cells. In the trigeminal ganglion, NO-IR neurons were found chiefly within the ophthalmic division; ∼10–15% of neurons were positively labeled. Colocalization with calcitonin gene-related peptide (CGRP) was not observed. Sectioning the major trigeminal branch innervating the circle of Willis decreased positive fibers by ≤25% in the ipsilateral vessels. In the nodose ganglion, 20–30% of neurons contained NOS-immunoreactivity, whereas less than 1% were in the C2 and C3 dorsal root ganglia. Three human circles of Willis obtained at autopsy showed sparse immunoreactive fibers, chiefly within vessels of the posterior circulation. Postmortem delay accounted for some of the reduced density. Our findings indicate that nerve fibers innervating cerebral arteries may serve as a nonendothelial source of the vasodilator nitric oxide (NO). The coexistence of NOS and VIP within sphenopalatine ganglion cells raises the possibility that two vasodilatory agents, one, a highly diffusable short-lived, low-molecular-weight molecule, and the other, a polar 28 amino acid-containing peptide, may serve as coneuromediators within the cerebral circulation.

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Inducible nitric oxide synthase: a possible key factor in the pathogenesis of chronic vasospasm after experimental subarachnoid hemorrhage

Journal of Neurosurgery ◽

10.3171/jns.1999.90.6.1098 ◽

1999 ◽

Vol 90 (6) ◽

pp. 1098-1104 ◽

Cited By ~ 48

Author(s):

Darius C. Widenka ◽

Ralph J. Medele ◽

Walter Stummer ◽

Karl Bise ◽

Hans J. Steiger

Keyword(s):

Nitric Oxide ◽

Subarachnoid Hemorrhage ◽

Cerebral Vasospasm ◽

Inducible Nitric Oxide Synthase ◽

Immunohistochemical Staining ◽

Content Type ◽

Chronic Vasospasm ◽

Oxide Synthase ◽

Fine Print ◽

Inducible Nitric Oxide

Object. The role of nitric oxide (NO) in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) is not well understood. Nitric oxide is a well-established vasodilatory substance; however, in SAH, NO may become a major source for the production of injurious free-radical species, leading to chronic cerebral vasospasm. Reactive overproduction of NO to counteract vascular narrowing might potentiate the detrimental effects of NO. The focus of the present study is to determine the extent of reactive induction of inducible nitric oxide synthase (iNOS) after experimental SAH.Methods. Chronic vasospasm was induced in male Wistar rats by an injection of autologous blood (100 µl) into the cisterna magna followed by a second injection 24 hours later. A control group of 10 animals was treated with injections of 0.9% sodium chloride solution. Vasospasm was verified by pressure-controlled angiography after retrograde cannulation of the external carotid artery 7 days later. In 11 of 15 animals radiographic evidence of cerebral vasospasm was seen. The animals were perfusion fixed and their brains were removed for immunohistochemical assessment. With the aid of a microscope, staining for iNOS was quantified in 40-µm floating coronal sections.Immunohistochemical staining for iNOS was markedly more intense in animals with significant angiographic evidence of vasospasm. Virtually no staining was observed in control animals. Seven days after the second experimental SAH, labeling of iNOS was found in endothelial cells, in vascular smooth-muscle cells, and, above all, in adventitial cells. Some immunohistochemical staining of iNOS was observed in rod cells (activated microglia), in glial networks, and in neurons.Conclusions. The present study demonstrates induction of iNOS after experimental SAH.

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Estrogen reduces mouse cerebral artery tone through endothelial NOS- and cyclooxygenase-dependent mechanisms

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.2.h511 ◽

2000 ◽

Vol 279 (2) ◽

pp. H511-H519 ◽

Cited By ~ 104

Author(s):

Greg G. Geary ◽

Diana N. Krause ◽

Sue P. Duckles

Keyword(s):

Nitric Oxide ◽

Methyl Ester ◽

Nitric Oxide Synthase ◽

Cerebral Artery ◽

Cerebral Arteries ◽

Estrogen Replacement ◽

Luminal Diameter ◽

Small Resistance ◽

Oxide Synthase

Gender and estrogen status are known to influence the incidence and severity of cerebrovascular disease. The vasoprotective effects of estrogen are thought to include both nitric oxide-dependent and independent mechanisms. Therefore, using small, resistance-sized arteries pressurized in vitro, the present study determined the effect of gender and estrogen status on myogenic reactivity of mouse cerebral arteries. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (OVX), or OVX with estrogen replacement (OVX + EST). The maximal passive diameters of arteries from all four groups were similar. In response to increases in transmural pressure, diameters of arteries from males and OVX females were smaller compared with diameters of arteries from either untreated or OVX + EST females. In the presence of N G-nitro-l-arginine methyl ester, artery diameters decreased in all groups, but diameters remained significantly smaller in arteries from males and OVX females compared with untreated and OVX + EST females. After endothelium removal or when inhibition of nitric oxide synthase and cyclooxygenase were combined, differences in diameters of arteries from OVX and OVX + EST were abolished. These data suggest that chronic estrogen treatment modulates myogenic reactivity of mouse cerebral arteries through both endothelium-derived cyclooxygenase- and nitric oxide synthase-dependent mechanisms.

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