Determination of cerebrospinal fluid concentrations of arginine and dimethylarginines in patients with subarachnoid haemorrhage

Elevated cerebrospinal fluid (CSF) concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), are assumed to be related to delayed vasospasm after subarachnoid haemorrhage (SAH). However, data on CSF concentrations of L-arginine, ADMA and its structural isomer symmetric dimethylarginine (SDMA) are very sparse in humans. We here present a new hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS-MS) method for the precise determination of these substances in CSF. The method requires only minimal sample preparation and features isotope labeled internal standards. First data of patients with SAH showed that on the day of admission CSF concentration values of L-arginine and ADMA were not significantly different from controls, but increased markedly during the course of the hospital stay. The decrease of the L-arginine to ADMA ratio points to a progressive impairment of the NO production rate in the brain after SAH which is confirmed by a simultaneous decrease in nitrate and nitrite concentrations in CSF.     

Homocysteine increases the production of asymmetric dimethylarginine in cultured neurons

Increased circulating concentrations of homocysteine may be a risk factor for Alzheimer's disease and cognitive dysfunction in normal aging. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide synthase (NOS). ADMA is metabolized in neurons by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Nitric oxide plays an important role in synaptic events involved in learning and memory. We determined the effect of L-homocysteine on ADMA accumulation and nitric oxide production in cultured rat neuronal granule cells. The incubation of neuronal granule cells with L-homocysteine for 24 hr caused a dose-dependent accumulation of ADMA and a dose-dependent decrease in nitric oxide production. The addition of the sulfhydryl antioxidant pyrrolidine dithiocarbamate (PDTC) attenuated the effect of homocysteine on ADMA accumulation and nitric oxide production. DDAH activity had a decreasing dose-response relationship with increasing L-homocysteine concentrations. The addition of PDTC caused a dose-dependent increase in DDAH activity. The addition of the N-methyl-D-aspartate receptor antagonists (+/-)-2-amino-5-phosphopentanoic acid and 7-chlorokynurenate had no effect on the inhibition of DDAH by homocysteine. It is concluded that L-homocysteine inhibits DDAH activity, thereby causing ADMA accumulation and decreasing nitric oxide production in cultured neurons. The protective effect of PDTC suggests that L-homocysteine inactivates DDAH in neurons by reacting with the cysteine residue in its active site. The preservation of DDAH activity and the reduction of ADMA accumulation in neurons may be a new strategy for the treatment of Alzheimer's disease and cognitive impairment in normal aging.     

Regulation by DDAH/ADMA pathway of lipopolysaccharide-induced tissue factor expression in endothelial cells

Previous studies have shown the regulatory effect of nitric oxide (NO) on endotoxin-induced tissue factor (TF) in endothelial cells. Asymmetric dimethylarginine (ADMA), a major endogenous NO synthase (NOS) inhibitor, could inhibit NO production in vivo and in vitro. ADMA and its major hydrolase dimethylarginine dimethylaminohydrolase (DDAH) have recently been thought of as a novel regulatory system of endogenous NO production. The aim of the present study was to determine whether the DDAH/ADMA pathway is involved in the effect of lipopolysaccharide (LPS) on TF expression in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with LPS (1 microg/ml) to induce TF expression. Exogenous ADMA significantly enhanced the increase in both TF mRNA level and activity induced by LPS, whereas L-arginine, the NOS substrate, markedly attenuated the LPS-induced TF increment. LPS markedly increased the level of ADMA in cultured medium and decreased DDAH activity in endothelial cells, and over-expression of DDAH2 could significantly suppress LPS-induced TF increment in endothelial cells. LPS could increase intracellular reactive oxygen species (ROS) production and activate nuclear factor-kappaB, which were enhanced by exogenous ADMA and attenuated by either L-arginine or overexpression of DDAH2. Therefore, our present results for the first time suggest that the DDAH/ADMA pathway can regulate LPS-induced TF expression via ROS-nuclear factor-kappaB-dependent pathway in endothelial cells.     

Endothelial nitric oxide synthase gene polymorphisms predict susceptibility to aneurysmal subarachnoid hemorrhage and cerebral vasospasm.

Rupture of an intracranial aneurysm (subarachnoid hemorrhage) is a potentially devastating condition frequently complicated by delayed cerebral ischemia from sustained contraction of intracranial arteries (cerebral vasospasm). There is mounting evidence linking the formation of intracranial aneurysms and the pathogenesis of post-subarachnoid hemorrhage vasospasm to aberrant bioavailability and action of the vasodilator molecule nitric oxide generated by isoforms of nitric oxide synthase. In humans, the gene encoding the endothelial isoform of nitric oxide synthase (eNOS) is known to be polymorphic, with certain polymorphisms associated with increased cardiovascular disease susceptibility. In this prospective clinical study involving 141 participants, we used gene microarray technology to demonstrate that the eNOS gene intron-4 27-base pair variable number tandem repeat polymorphism (eNOS 27 VNTR) predicts susceptibility to intracranial aneurysm rupture, while the eNOS gene promoter T-786C single nucleotide polymorphism (eNOS T-786C SNP) predicts susceptibility to post-subarachnoid hemorrhage vasospasm. We believe that genetic information such as this, which can be obtained expeditiously at the time of diagnosis, may be used as a helpful adjunct to other clinical information aimed at predicting and favorably modifying the clinical course of persons with intracranial aneurysms.     

血管内一氧化氮合酶基因转染预防脑血管痉挛的实验研究

目的采用血管内基因转染的方法,将重组内皮型一氧化氮合酶(eNOS)基因转染入蛛网膜下腔出血(SAH)后迟发性脑血管痉挛大鼠脑动脉,探讨防治SAH后迟发性脑血管痉挛的新方法。方法首先构建携带eNOS基因的重组腺病毒。采用小脑延髓池二次注血法建立大鼠SAH后迟发性脑血管痉挛模型。通过颈动脉微泵持续滴注方法进行基因转染,并设置对照组。结果第7天采用免疫组化证实重组eNOS基因表达,重组eNOS主要表达于内皮层。第7天显微镜下测定血管内eNOS转染组脑动脉环平均直径较单纯SAH组增大,电镜观察血管痉挛较单纯SAH组减轻。结论通过本研究证实采用颈动脉微泵持续滴注方法可在大鼠脑动脉表达重组eNOS,重组基因主要表达于动脉内皮细胞,可达到缓解SAH后迟发性脑血管痉挛的目的。     

Genetic elimination of eNOS reduces secondary complications of experimental subarachnoid hemorrhage

Delayed complications of subarachnoid hemorrhage (SAH) such as angiographic vasospasm, cortical spreading ischemia, microcirculatory dysfunction, and microthrombosis are reported in both patients and animal models of SAH. We demonstrated previously that SAH is associated with increased oxidative stress in the brain parenchyma, and that this correlates with dysfunction of endothelial nitric oxide synthase (eNOS) (homodimeric uncoupling). Uncoupling of eNOS exacerbated oxidative stress and enhanced nitric oxide (NO) depletion, and was associated with multiple secondary complications such as microthrombosis, neuronal apoptosis, and release of reactive oxygen species. Thus, we hypothesized that genetic abbrogation of eNOS would confer a beneficial effect on the brain after SAH. Using a prechiasmatic injection model of SAH, we show here that eNOS knockout (KO) significantly alleviates vasospasm of the middle cerebral artery and reduces superoxide production. Endothelial nitric oxide synthase KO also affected other nitric oxide synthase isoforms. It significantly increases neuron nitric oxide synthase expression but has no effect on inducible nitric oxide synthase. Endothelial nitric oxide synthase KO decreases Zn²⁺ release after SAH, reduces microthrombi formation, and prevent neuronal degeneration. This work is consistent with our findings where, after SAH, increased oxidative stress can uncouple eNOS via Zn²⁺ thiolate oxidation, or theoretically by depletion or oxidation of tetrahydrobiopterin, resulting in a paradoxical release of superoxide anion radical, further exacerbating oxidative stress and microvascular damage.     

Dimethylarginine dimethylaminohydrolase 1 regulates nerve growth factor-promoted differentiation of PC12 cells in a nitric oxide-dependent but asymmetric dimethylargenine-independent manner

There are significant morphological and biochemical alterations during nerve growth factor (NGF)-promoted neuronal differentiation, and the process is regulated by molecules, including nitric oxide (NO). Dimethylarginine dimethylaminohydrolase (DDAH) is thought to play a critical role in regulating NO production via hydrolyzing the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Thus, we tested the role of DDAH in NGF-promoted differentiation of PC12 (pheochromocytoma) cells. The present results show that both mRNA and protein levels of DDAH1 were increased, whereas those of DDAH2 were decreased, during NGF-promoted cell differentiation. Both the DDAH activity and the ADMA level in cultured medium were unchanged in this process. NGF promoted neurite formation and induced the expression of microtubule-associated protein 2 (MAP2), a neuronal marker, which were both significantly repressed by DDAH1 silence with small interfering RNA but not by DDAH2 silence. The expressions of three isoforms of NOS were markedly upregulated after NGF stimulation with a time course similar to that of DDAH1, which were attenuated by DDAH1 silence. Conversely, overexpression of DDAH1 accelerated neurite formation in PC12 cells, concomitantly with upregulating the expression of three NOS isoforms. In summary, our data reveal the critical regulatory effect of DDAH1 on NGF-promoted differentiation of PC12 cells in an NOS/NO-dependent but ADMA-independent manner.     

High glucose-mediated imbalance of nitric oxide synthase and dimethylarginine dimethylaminohydrolase expression in endothelial cells

The mechanisms involved in endothelial dysfunction are multifactorial. A correlation between oxidative stress and derangements of nitric oxide synthase (NOS) pathways in altered endothelial homeostasis has been most studied and demonstrated in different pathophysiological conditions. NOS activities are regulated by endogenous inhibitors such as asymmetric dimethyl-L-arginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Since recent data demonstrated that some endothelial dysfunction may be related to reduced expression and/or activity of DDAH, the aim of the present research was to investigate the expression of DDAH-2 and NOS isoforms in high glucose-mediated oxidative stress. Endothelial cells were incubated with normal (7 mM) and high concentrations (33 mM) of D-glucose for 5 days; mannose (26 mM) plus D-glucose (7 mM) was used as osmotic control. Data obtained in the present study show that the exposure for 5 days to high glucose increases oxidative stress, reduces DDAH-2 and eNOS expression and increases iNOS expression. These results indicate that DDAH-2 and iNOS/eNOS dysregulation may play a key role in high glucose-mediated oxidative stress, suggesting that selective modulation of DDAH isoforms may result in selective inhibition/activation of NOS isoforms, thereby providing a novel strategy of approach in vascular complications of several pathologies.     

All‐trans retinoic acid inhibits cobalt chloride‐induced apoptosis in PC12 cells: Role of the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine pathway

Previous studies have shown that the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and its specific hydrolase dimethylarginine dimethylaminohydrolase (DDAH) are involved in the regulation of apoptosis in different cell types. In the present study, we investigated the role of the DDAH/ADMA pathway in cobalt chloride (CoCl₂)–induced apoptosis and the antiapoptotic effect of all‐trans retinoic acid (atRA) in undifferentiated pheochromocytoma (PC12) cells. Treatment of CoCl₂ (125 μM) for 48 hr significantly induced the apoptosis of PC12 cells, concomitantly with increased intracellular reactive oxygen species (ROS) production and caspase‐3 activity. CoCl₂ treatment also decreased the activity of DDAH and the expression of DDAH2 (mRNA and protein), resulting in an increased level of ADMA. All these alterations induced by CoCl₂ were attenuated by atRA (0.1, 1, or 10 μM). Interestingly, the antiapoptotic effects of atRA were inhibited by DDAH2 small RNA interference. In contrast, DDAH2 overexpression inhibited the proapoptotic effects of CoCl₂. We also found that treatment of exogenous ADMA (3, 10, or 30 μM) induced the apoptosis of PC12 cells in a concentration‐ and time‐dependent manner, which was inhibited by the antioxidant or the caspase‐3 inhibitor. These findings suggest that the modulation of the DDAH/ADMA/ROS pathway plays an important role in CoCl₂‐induced apoptosis and the antiapoptotic effects of atRA in undifferentiated PC12 cells. © 2009 Wiley‐Liss, Inc.     

Alzheimer's disease is not associated with altered concentrations of the nitric oxide synthase inhibitor asymmetric dimethylarginine in cerebrospinal fluid

Summary. Nitric oxide (NO) may play a role in the pathophysiology of Alzheimer's disease (AD). Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, is involved in regulation of NO production. Recently it has been reported that dimethylarginine dimethylaminohydrolase, an enzyme that hydrolyses ADMA into citrulline and dimethylamine, is specifically elevated in neurons displaying cytoskeletal abnormalities and oxidative stress in AD. We hypothesized that this could lead to altered CSF concentrations of ADMA in AD. Measurement of ADMA and dimethylamine in CSF revealed no significant differences between AD patients (n = 20) and age-matched control subjects (n = 20). Our results suggest that in early stages of AD overall regulation of NO production by ADMA is not aberrant. Received March 14, 2002; accepted May 21, 2002 Published online July 26, 2002     

The interaction between high ammonia diet and bile duct ligation in developing rats: assessment by spatial memory and asymmetric dimethylarginine

Bile duct ligation (BDL) in developing rats causes cholestasis, impaired liver function and cognition. Because both nitric oxide (NO) and ammonia are implicated in hepatic encephalopathy (HE), we hypothesized that asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor, and ammonia affect cognition in young rats with BDL. Four groups of young male Sprague–Dawley rats ages 17 days were used: rat underwent laparotomy (SC group), rat underwent laparotomy plus a 30% ammonium acetate diet (SC + HA group), rat underwent BDL (BDL group), rats underwent BDL plus high ammonia diet (BDL + HA group). Spatial memory was assessed by Morris water maze task. Plasma was collected for biochemical and ADMA analyses. Liver and brain cortex were collected for determination of protein arginine methyltransferase-1 (PRMT1, ADMA-synthesizing enzyme) and dimethylarginine dimethylaminohydrolase (DDAH, ADMA-metabolizing enzyme). We found BDL group had significantly higher plasma direct/total bilirubin, aspartate aminotransferase, alanine aminotransferase, ADMA, liver p22^phox, and worse spatial performance as compared with SC group. High ammonia diet increased plasma ammonia and ADMA concentration, and aggravated spatial deficit in the presence of BDL-induced cholestasis. We conclude plasma ADMA plays a role in BDL-induced spatial deficit. High ammonia aggravated the spatial deficits encountered in cholestatic young rats.     

Uncoupling of endothelial nitric oxide synthase after experimental subarachnoid hemorrhage

We studied whether endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled in large cerebral arteries after subarachnoid hemorrhage (SAH) and also whether this causes cerebral vasospasm in a mouse model of anterior circulation SAH. Control animals underwent injection of saline instead of blood (n=16 SAH and n=16 controls). There was significant vasospasm of the middle cerebral artery 2 days after SAH (lumen radius/wall thickness ratio 4.3±1.3 for SAH, 23.2±2.1 for saline, P<0.001). Subarachnoid hemorrhage was associated with terminal deoxynucleotidyl transferase dUTP nick-end labeling, cleaved caspase-3, and Fluoro-Jade-positive neurons in the cortex and with CA1 and dentate regions in the hippocampus. There were multiple fibrinogen-positive microthromboemboli in the cortex and hippocampus after SAH. Transgenic mice expressing lacZ under control of the eNOS promoter had increased X-gal staining in large arteries after SAH, and this was confirmed by the increased eNOS protein on western blotting. Evidence that eNOS was uncoupled was found in that nitric oxide availability was decreased, and superoxide and peroxynitrite concentrations were increased in the brains of mice with SAH. This study suggests that artery constriction by SAH upregulates eNOS but that it is uncoupled and produces peroxynitrite that may generate microemboli that travel distally and contribute to brain injury.     

盐酸法舒地尔对蛛网膜下腔出血后血管痉挛的实验研究

目的通过建立大鼠蛛网膜下腔出血（SAH）模型,探讨盐酸法舒地尔对蛛网膜下腔出血后血管痉挛的缓解作用和神经保护作用,并与尼莫地平对比,观察疗效。方法通过枕大池二次注血法建立大鼠SAH模型,观察基底动脉和海马神经元形态变化,测量基底动脉管径和管壁厚度,计算海马CA1区神经元密度,检测基底动脉内皮型一氧化氮合成酶（eNOS）的表达。结果各组模型大鼠的基底动脉均出现血管痉挛,海马CA1区正常神经元数目明显减少,多数神经元发生变性,基底动脉的eNOS表达明显减弱。但注射法舒地尔组与其他模型组相比能较大程度的缓解以上变化,具有统计学差异（P〈0.05）,且优于尼莫地平。结论法舒地尔可以有效缓解SAH后的迟发性脑血管痉挛,具有神经保护作用,其缓解迟发性脑血管痉挛作用和神经保护作用与动脉壁产生的一氧化氮（NO）有关。     

Asymmetrical Dimethylarginine Antagonizes Glutamate-Induced Apoptosis in PC12 Cells

Overproduction of nitric oxide (NO) plays an important role in glutamate-induced excitotoxicity. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor. The aim of this study is to explore whether ADMA antagonizes the excitotoxicity of glutamate to neuronal cells and the underlying molecular mechanisms. In this work, we investigated the effects of ADMA on glutamate-induced toxicity in neuronal cells by studying PC12 cells, a clonal rat pheochromocytoma cell line. We show that ADMA obviously protects PC12 cells against glutamate-induced cytotoxicity and apoptosis. We also found that ADMA treatment results in prevention of glutamate-induced mitochondrial membrane potential loss and caspase-3 activation. Moreover, ADMA prevents glutamate-caused down-regulation of bcl-2 protein expression. These results indicate that ADMA protects against glutamate-induced apoptosis and excitotoxicity and the underlying mechanism may be involved in preservation of mitochondrial function by up-regulating the expression of bcl-2. Our study suggests a promising future of ADMA-based therapies for neuropathologies associated with an excess of NO.     

Intrathecal and systemic alterations of L-arginine metabolism in patients after intracerebral hemorrhage

Alterations in the concentration of nitric oxide (NO) and L-arginine metabolites have been associated with the pathophysiology of different vascular diseases. Here, we describe striking changes in L-arginine metabolism after hemorrhagic stroke. Blood and cerebrospinal fluid (CSF) samples of patients with intracerebral hemorrhage (ICH) and/or intraventricular hemorrhage were collected over a ten-day period. Liquid chromatography-tandem mass spectrometry was used to quantify key substrates and products of L-arginine metabolizing enzymes as well as asymmetric (ADMA) and symmetric dimethylarginine (SDMA). Changes in the plasma were limited to early reductions in L-ornithine, L-lysine, and L-citrulline concentrations. Intrathecally, we observed signs of early NO synthase (NOS) upregulation followed by a decrease back to baseline accompanied by a rise in the level of its endogenous NOS-inhibitor ADMA. SDMA demonstrated increased levels throughout the observation period. For arginase, a pattern of persistently elevated activity was measured and arginine:glycine amidinotransferase (AGAT) appeared to be reduced in its activity at later time points. An early reduction in CSF L-arginine concentration was an independent risk factor for poor outcome. Together, these findings further elucidate pathophysiological mechanisms after ICH potentially involved in secondary brain injury and may reveal novel therapeutic targets.     

Functional deterioration of endothelial nitric oxide synthase after focal cerebral ischemia

Endothelial nitric oxide synthase (eNOS) dysfunction is related to secondary injury and lesion expansion after cerebral ischemia. To date, there are few reports about postischemic alterations in the eNOS regulatory system. The purpose of the present study was to clarify eNOS expression, Ser1177 phosphorylation, and monomer formation after cerebral ischemia. Male Wistar rats were subjected to transient focal cerebral ischemia. Endothelial nitric oxide synthase messenger RNA (mRNA) and protein expression increased ∼8-fold in the ischemic lesion. In the middle cerebral artery core, eNOS-Ser1177 phosphorylation increased 6 hours after ischemia; however, there was an approximately 90% decrease in eNOS-Ser1177 phosphorylation observed 24 hours after ischemia that continued until at least 7 days after ischemia. Endothelial nitric oxide synthase monomer formation also increased 24 and 48 hours after ischemia (P<0.05), and protein nitration progressed in parallel with monomerization. To assess the effect of a neuroprotective agent on eNOS dysfunction, we evaluated the effect of fasudil, a Rho-kinase inhibitor, on eNOS phosphorylation and dimerization. Postischemic treatment with fasudil suppressed lesion expansion and dephosphorylation and monomer formation of eNOS. In conclusion, functional deterioration of eNOS progressed after cerebral ischemia. Rho-kinase inhibitors can reduce ischemic lesion expansion as well as eNOS dysfunction in the ischemic brain.     

High asymmetric dimethylarginine,symmetric dimethylarginine and L-arginine levels in migraine patients

Experimental and clinical data strongly suggests that nitric oxide (NO) plays a pivotal role in migraine. This is also supported by studies of migraine induced by substances that release NO. NO is synthesized from L-arginine by endothelial NO synthase (NOS). Asymmetric dimethylarginine (ADMA) is the major endogenous competitive inhibitor of NOS. Symmetric dimethylarginine (SDMA) is an inactive stereoisomer of ADMA. It may reduce NO production by competing with arginine for cellular uptake. The aim of this study was to measure the levels of ADMA, SDMA and L-arginine in migraine patients during the interictal period. One hundred migraine patients and 100 healthy volunteers were recruited. The patients were in the interictal period and classified into two groups as having migraine with aura and migraine without aura. Their serum ADMA, SDMA and L-arginine levels were measured by high-performance liquid chromotography (HPLC) method. ADMA, SDMA and L-arginine levels were significantly higher in migraine patients compared to the control group. But there was no difference between the patients with and without aura. These results suggest that NOS inhibitors and L-arginine/NO pathway plays an important role in migraine pathopysiology.     

Increased cerebrospinal fluid concentrations of asymmetric dimethylarginine correlate with adverse clinical outcome in subarachnoid hemorrhage patients

Elevated cerebrospinal fluid (CSF) concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, have been found in patients with subarachnoid hemorrhage (SAH). In addition, CSF levels of ADMA are associated with the severity of vasospasm. However, the relation between CSF ADMA levels and the clinical outcome of SAH patients is still unclear. We hypothesized that elevated ADMA levels in CSF might be related to the clinical outcome of SAH patients. CSF ADMA levels were measured in 20 SAH patients at days 3–5, days 7–9 and days 12–14 after SAH onset using high-performance liquid chromatography. Cerebral vasospasm was assessed by transcranial Doppler ultra sonography. Clinical outcome at 2 year follow-up was evaluated using the Karnofsky Performance Status scale (KPS). CSF ADMA concentrations in all SAH patients were significantly increased at days 3–5 (p = 0.002) after SAH, peaked on days 7–9 (p < 0.001) and remained elevated until days 12–14 (p < 0.001). In subgroup analysis, significant increases of CSF ADMA levels were found in patients both with and without vasospasm. The KPS scores significantly correlated with CSF levels of ADMA at days 7–9 (correlation coefficient = −0.55, p = 0.012; 95% confidence interval −0.80 to −0.14). Binary logistic regression analysis indicated that higher ADMA level at days 7–9 predicted a poor clinical outcome at 2 year follow-up after SAH (odds ratio = 1.722, p = 0.039, 95% confidence interval 1.029 to 2.882). ADMA may be directly involved in the pathological process and future adverse prognosis of SAH.     

Adenovirus-mediated gene transfer to human cerebral arteries.

Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1,000 microm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant beta-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.     

蛛网膜下腔出血腰池持续引流前后脑脊液中NO浓度的变化

目的 探讨腰池持续引流防治脑血管痉挛的效果及其对脑脊液中一氧化氮（NO）浓度的影响。方法 50例破裂动脉瘤蛛网膜下腔出血患，随机分为引流组25例。对照组25例。引流组在电解可脱弹簧圈（GDC）栓塞术后立即实施腰池持续引流，对照组行间断腰椎穿刺。脑脊液NO浓度采用镉粒子还原法测定。结果 引流组中发生症状性脑血管痉挛2例。对照组7例。血管痉挛患的NO浓度明显降低，引流组脑脊液中NO浓度在出血后第5d起明显高于对照组。结论 腰池持续引流可有效清除蛛网膜下腔积血，提高脑脊液中NO的浓度。