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Abstract

Vasospasm is a major cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (aSAH). Studies have shown a link between single-nucleotide polymorphisms (SNPs) in the endothelial nitric oxide synthase (eNOS) gene and the incidence of coronary spasm and aneurysms. Alterations in the eNOS T-786 SNP may lead to an increased risk of post-aSAH cerebral vasospasm. In this prospective clinical study, 77 aSAH patients provided genetic material and were followed for the occurrence of vasospasm. In multivariate logistic regression analysis, genotype was the only factor predictive of vasospasm. The odds ratio (OR) for symptomatic vasospasm in patients with one T allele was 3.3 (95% confidence interval (CI): 1.1 to 10.0, P=0.034) and 10.9 for TT. Patients with angiographic spasm were 3.6 times more likely to have a T allele (95% CI: 1.3 to 9.6, P=0.013; for TT: OR 12.6). Patients with severe vasospasm requiring endovascular therapy were more likely to have a T allele (OR 3.5, 95% CI: 1.3 to 9.5, P=0.016; for TT: OR 12.0). Patients with the T allele of the eNOS gene are more likely to have severe vasospasm. Presence of this genotype may allow the identification of individuals at high risk for post-aSAH vasospasm and lead to early treatment and improved outcome.

Keywords

intracranial aneurysm polymorphism endothelial nitric oxide synthase nitric oxide subarachnoid hemorrhage vasospasm

Introduction

The estimated annual rate of aneurysmal subarachnoid hemorrhage (aSAH) among European and North American populations ranges from 6 to 8 per 100,000 population (Broderick et al, 1993; Linn et al, 1996). Approximately 30% of survivors remain dependent, and only 30% to 45% return to previous or comparable jobs (Ropper and Zervas, 1984). Following aSAH, a major cause of morbidity and mortality is delayed ischemic neurologic deterioration secondary to cerebral vasospasm (Bendok et al, 1998). Symptomatic vasospasm occurs in 15% to 65% of patients, is detectable in 30% to 70% of patients by angiography, and results in infarction in 10% to 45% of patients (Kassell et al, 1985; Lanzino et al, 1999; Song et al, 2006; Vajkoczy et al, 2005; Wurm et al, 2004).

In vessel endothelium, nitric oxide (NO) is continuously generated from the endothelial nitric oxide synthase (eNOS) gene to maintain basal vascular tone (Quyyumi et al, 1995). Aneurysmal SAH results in alterations in eNOS gene expression and disruption of the balanced regulation of cerebral vascular tone (Khurana et al, 2002; Weir and MacDonald, 1993). Studies have found abnormal cerebrospinal fluid NO levels in humans after aSAH (Sadamitsu et al, 2001; Woszczyk et al, 2003). Furthermore, in dogs and ex vivo in the arteries of humans, adenovirus gene transfer of eNOS has shown to be protective in SAH (Khurana et al, 2000; Khurana et al, 2002). Nitric oxide is an inhibitor of inflammation and smooth muscle proliferation, which are pathologic changes found in cerebral vasospasm (Dumont et al, 2003; Moncada et al, 1991). Studies have found an association with eNOS polymorphisms and the formation of intracranial aneurysms (Khurana et al, 2004) and coronary spasm (Yoshimura et al, 2000), a process that may be physiologically similar to cerebral vasospasm.

We hypothesize that the eNOS T-786 single-nucleotide polymorphisms (SNPs) are associated with the incidence of post-aSAH cerebral vasospasm. Presence of this allele may allow clinicians to identify high-risk individuals and tailor intensive care management in a patient-specific manner. In this context, the principal aims of our prospective clinical study were to (1) determine if there was an association between the presence of such polymorphisms and the presence of post-aSAH vasospasm; (2) determine if differences in such polymorphisms had an effect on the incidence of cerebral infarction secondary to vasospasm.

Materials and methods

Patient Population

Patients presenting to our institution from January 2004 to December 2006 with nontraumatic, computed-tomography (CT)-confirmed SAH were prospectively approached for enrollment into this IRB-approved study. Patients were eligible if they presented within 2 days of SAH. After obtaining an informed consent, a buccal swab was used to collect cheek cells for DNA analysis. All patients provided samples within 3 days of SAH onset. Patients later found to have non-aneurysmal SAH were excluded from the study.

Management Protocol

A standardized management protocol was used in the management of all aSAH patients including immediate aneurysm repair whenever feasible. Decisions regarding treatment modality were made by combined teams of open-neurovascular and endovascular surgeons. Vasospasm prophylaxis consisted of nimodipine administration and the active maintenance of central venous pressure greater than 5 mm Hg. Postoperatively, patients developing symptomatic vasospasm were treated with induced hypertension and hypervolemia (central pressure ≥8 mm Hg). Elevation of intracranial pressure above 20 mm Hg was treated aggressively with mannitol and/or hypertonic saline.

Study Outcomes

Patient demographic information, medical/surgical history, and clinical course were obtained at the time of enrollment from family members or by review of medical records. Patients were prospectively followed for adverse events until hospital discharge. All outcomes were compiled blinded to the knowledge of a patient's genotype.

Symptomatic vasospasm was defined as any change in mental status of greater than 2 points in the Glasgow Coma Scale or any new neurologic deficit that could not be explained by other etiology (Pickard et al, 1989). Patients suspected of clinical vasospasm underwent physical examination, imaging, and laboratory investigation to rule out other possible causes. Other potential causes of clinical deterioration, such as hydrocephalus, rebleeding, cerebral edema, retraction injury, ventriculitis, metabolic derangements, and seizures were rigorously excluded. Clinical deterioration from symptomatic vasospasm, thought to be consistent with vasospasm, were treated with HHHT (hypertensive, hypervolemic, and hemodilutional therapy) to maintain a hematocrit of approximately 30, systolic blood pressure >160 mm Hg, and central venous pressure of >8. When clinical evidence of vasospasm persisted for more than 2 h despite HHHT, cerebral angiography was used to document vasospasm. Additionally, poor-grade and sedated patients received CT or MR (magnetic resonance) perfusion on day 4 to 8 to assess for vasospasm. Patients with vasospasm on CT or MR perfusion then received angiography. On angiography, patients were considered to have vasospasm if there was arterial narrowing compared with normal vessel diameter (mild<30%, moderate 30% to 60%, severe>60%). Patients with moderate or severe angiographic vasospasm were treated with intraarterial administration of vasodilators and balloon angioplasty when indicated. Cerebral infarction because of vasospasm was defined as any new lucency on CT- or diffusion-weighted-imaging-positive signal on MRI in the appropriate vascular territory after exclusion of other potential causes.

Genetic Analysis

All genetic analyses were performed by laboratory personnel blinded to patient identities. Genomic DNA was extracted from buccal swabs using MasterAmp Extraction kits (Epicentre, Madison, WI, USA) and approximately 50 ng DNA was amplified in 10 μL reactions with primers 5′-GCATGCACTCTGGCCTGAAGT-3′ and 5′-CAGGAAGCTGCCTTCCAGTGC-3′ (500 nmol/L each), 0.25 μL JumpStart RED AccuTaq DNA polymerase (Sigma-Aldrich, St Louis, MO, USA), 1 × AccuTaq reaction buffer, and 500 μmol/L dNTPs. Polymerase chain reaction cycles consisted of denaturation at 94°C for 30 secs, annealing for 30 secs (touchdown from 65°C for first 10 cycles to 57°C for last 20 cycles), and extension at 72°C for 90 secs. After a final 5 mins extension, PCR products were treated with ExoSapIT kits (USB, Cleveland, OH, USA), bidirectional sequencing was performed with the same primers on 20 to 40 ng of PCR products using BigDye Termination v3.1 Cycle Sequencing kits (ABI, Foster City, CA, USA), and sequencing products were analyzed on an ABI 3730xl capillary instrument. Sequence traces were aligned and SNPs determined using SeqMan software (DNAStar, Madison, WI, USA).

Statistical Analysis

Data are presented as mean±s.d. for continuous variables, and as frequency for categorical variables. Statistical analyses were carried out using unpaired Student's t-test, χ²- and Fisher's exact tests, and Mantel—Haenszel test for linear association, as appropriate. Odds ratios (OR) were calculated using univariate logistic regression analysis. Factors predictive of all outcomes of vasospasm in univariate analysis (P<0.10) and age, race, and gender were entered into a multivariable logistic regression. Race, age, gender, stimulant use, smoking, family history, Hunt and Hess and Fischer grade were also controlled for in a comprehensive multivariable logistic regression analysis to assess the ability of the eNOS genotype to predict all outcomes of vasospasm. Conformance with the Hardy—Weinberg equilibrium was tested by χ² goodness of fit. P-values of ≤0.05 were considered statistically significant.

Results

Cohort Characteristics

Seventy-seven patients with SAH and confirmed aneurysms by angiography provided consent and genetic material for this study. The eNOS T-786 SNP was sequenced in all patients. Genotypes were in accordance with the Hardy—Weinberg equilibrium (P=0.46). Minor allele frequencies were found to be similar to those in previously published studies (Akagawa et al, 2005; Khurana et al, 2004; Krex et al, 2006; Krischek et al, 2006; Song et al, 2006). No significant differences were found in baseline characteristics or treatment between patients according to genotype (see Table 1). Eighteen percent of patients had a family history of intracranial aneurysm.

Table 1

Distribution of patient characteristics by genotype

	CC	CT	TT	Significance
	(7)	(37)	(33)	P-value
Mean age (years)	57±12	54±11	54±12	0.832
Female sex	5 (71%)	23 (62%)	21 (64%)	0.897

Medical history
Smokers	4 (57%)	24 (65%)	15 (46%)	0.263
Stimulant use	0	4 (11%)	2 (6%)	0.549
Hypertension	4 (57%)	22 (60%)	21 (64%)	0.915
Diabetes mellitus	1 (14%)	7 (19%)	1 (3%)	0.116
Relative with an aneurysm	1 (14%)	5 (14%)	8 (24%)	0.49

Ethnicity
White	4 (57%)	19 (51%)	13 (39%)	0.483
African-American	0	3 (8%)	2 (6%)
Hispanic	0	9 (24%)	10 (30%)
Others or unknown	3 (43%)	6 (16%)	8 (24%)

Hunt—Hess
1	1 (14%)	10 (27%)	9 (27%)	0.594
2	3 (43%)	13 (35%)	8 (24%)
3	3 (43%)	8 (22%)	9 (27%)
4	0	5 (13%)	3 (9%)
5	0	1 (3%)	4 (12%)

Fisher grade
2	4 (57%)	20 (54%)	11 (33%)	0.313
3	3 (43%)	14 (38%)	16 (49%)
4	0	3 (8%)	6 (18%)

Interventions
Clip	6 (86%)	23 (70%)	25 (83%)	0.371
Coil	1 (14%)	10 (30%)	5 (17%)

Cerebral Vasospasm

In total, 43% of patients had either symptomatic or angiographic vasospasm. Thirty percent of patients had symptomatic vasospasm, 38% had angiographic vasospasm, and 35% had vasospasm necessitating endovascular treatment. Twenty percent of patients had an infarction after aneurysm rupture and 10% had an infarction secondary to vasospasm.

There was a significant increase in symptomatic or angiographic vasospasm in patients with the TT genotype (61%) versus the CT (35%) or CC genotype (0%, P=0.003; Table 2). In univariate analysis, Fisher grade and eNOS genotype were the only factors predictive of vasospasm. In univariate analysis, the OR of developing vasospasm in patients with a T allele was 3.2 (95% confidence interval (CI): 1.4 to 7.3, P=0.004; for TT genotype: OR 10.2). There was a significant increase in angiographic vasospasm and vasospasm necessitating endovascular therapy in the TT (55% and 52%, respectively) versus the CT (30% and 27%) or CC polymorphism (0% and 0%, P=0.003 and P=0.003, respectively). In univariate analysis, patients with a T had 3.6-fold higher odds of having angiographic vasospasm (95% CI: 1.5 to 8.6, P=0.003; for TT genotype: OR 13.0), and an OR of 3.6 for requiring endovascular treatment of vasospasm (95% CI: 1.5 to 8.8, P=0.004; for TT genotype: OR 13.0). Forty-two percent of patients with TT, 27% of patients with CT, and 0% of patients with CC had symptomatic vasospasm (P=0.024). In univariate analysis, the OR for symptomatic vasospasm for those patients with the T allele was 2.7 (95% CI: 1.12 to 6.3, P=0.02; for TT: OR 7.3). In univariate analysis, Fisher grade was the only other variable predictive of vasospasm. Using Fisher grade 2 as a reference point, patients with Fisher grade 3 had a two-fold increased risk of vasospasm (95% CI: 1.0 to 4.0, P=0.044). There was no significant increased risk in Fisher grade 4 patients.

Table 2

Outcome according to genotype

Outcome	CC	CT	TT	P-value^a
Total	7	37	33
Any vasospasm	0	13 (35%)	20 (61%)	0.003
Symptomatic vasospasm	0	10 (27%)	14 (42%)	0.024
Angiographic spasm	0	11 (30%)	18 (55%)	0.003
Endovascularly treated spasm	0	10 (27%)	17 (52%)	0.003
Overall infarction	1 (14%)	5 (14%)	9 (27%)	0.19
Infarction because of vasospasm	0	4 (11%)	4 (12%)	0.52

Significance by the Mantel—Haenszel test for linear association.

Bold values signifies P<0.05.

In multivariate logistic regression analysis, genotype was the only significant predictor of vasospasm (see Table 3). The T allele was a significant predictor of vasospasm (OR 3.4, 95% CI: 1.3 to 9.0, P=0.013; for TT: OR 11.6), symptomatic vasospasm (OR 3.3, 95% CI: 1.1 to 10.0, P=0.034; for TT: OR 10.9), angiographic vasospasm (OR 3.6, 95% CI: 1.3 to 9.6, P=0.013; for TT: OR 12.6), and vasospasm necessitating endovascular treatment (OR 3.5, 95% CI: 1.3 to 9.5, P=0.016; for TT: OR 12.0). When patients were stratified according to race in multivariable analysis, white patients with a T allele had 3.0 times higher odds of having symptomatic spasm (P=0.07), 2.6 times higher odds of angiographic vasospasm (P=0.10), and 2.7 times higher odds of severe vasospasm requiring endovascular treatment (P=0.10). In Hispanic patients with a T allele, the OR of having any vasospasm, angiographic vasospasm, or vasospasm requiring endovascular treatment was significant (12.3, 95% CI: 1.3 to 195, P=0.03; 16, 95% CI: 1.3 to 195, P=0.03; 16, 95% CI: 1.3 to 195, P=0.03, respectively). No alleles were significantly associated with vasospasm when subdivided by other ethnicities (P=0.15).

Table 3

Multivariable logistic regression analysis of genotype as a predictor of vasospasm

Outcome	OR for one T allele ^a	95% CI	P-value
Any vasospasm	3.4	1.29–9.01	0.013
Symptomatic vasospasm	3.3	1.09–9.97	0.034
Angiographic spasm	3.6	1.31–9.57	0.013
Endovascularly treated spasm	3.5	1.26–9.54	0.016

Genotype was the only significant predictor of vasospasm in multivariate logistic regression analysis when controlling for race, age, and gender. Significance was unchanged when controlling for race, age, gender, stimulant use, smoking, family history, and Hunt and Hess and Fisher grade.

Bold values signifies P<0.05.

Infarction

There was no significant difference in infarction in patients according to genotype. Fourteen percent of patients with CC and 14% of patients with CT had infarction versus 27% of TT patients (P=0.19). Zero percent of patients with CC had infarction because of vasospasm versus 11% and 12% of patients with CT and TT, respectively (CC versus CT or TT, P=0.52).

Discussion

Numerous studies have attempted to determine prognostic indicators of vasospasm, as identification of high-risk individuals may allow post-aSAH management to be tailored in a patient-specific manner. History of smoking, preexisting hypertension, and hypovolemia have all been linked with vasospasm (Lasner et al, 1997; Rabinstein, 2006). Although experts have argued over the role of age, the strongest known predictive factor for vasospasm is the amount of blood on CT (Fisher grade), which often correlates with the severity of the vasospasm (Davis et al, 1980; Fox and Ko, 1978). Differences in the incidence of vasospasm and outcomes in patients with similar Fisher grade, and a genetic association with aneurysm formation, have led to the theory that genetic polymorphisms encoding vascular regulatory proteins may result in variable levels of vascular spasticity in response to aSAH.

It is likely that the onset of aSAH is multifactorial in nature and dependent on genetic characteristics and clinical variables associated with aneurysm rupture. The eNOS T-786 SNP has received much attention because of its location within the promoter region of the NO gene (Nakayama et al, 1999). In this study, Fisher grade and genotype were the only significant predictors of vasospasm in univariate analysis. In multivariable analysis, genotype was the only independent predictor of vasospasm when accounting for race, age, gender, stimulant use, smoking, family history of aneurysm, and the Hunt and Hess and Fisher grades. Patients with a T allele had 3.3 times higher odds of developing symptomatic vasospasm and patients with the TT genotype were 10.9 times more likely. Patients with the T allele had an OR of 3.5 of necessitating treatment for vasospasm and patients with the TT genotype had an OR of 12.0; thus, the T allele is not only a predictive marker of vasospasm, but a risk factor for severe vasospasm requiring endovascular therapy.

When stratified according to genotype, there was no significant difference in the incidence of infarction because of vasospasm. Twelve percent of patients with the TT and 11% of patients with the CT genotype had infarction because of vasospasm versus 0% in patients with the CC genotype. Although this difference was not statistically significant, our study was not powered to detect differences in infarction. This may also be because of a large number of clinical and genetic factors that link vasospasm with infarction in a vascular territory. This may also be because we treat vasospasm aggresively with the intent to avoid the development of infarction.

In this study, all polymorphisms were in Hardy—Weinberg equilibrium. Minor allele frequencies were not significantly different from those in previous studies (Akagawa et al, 2005; Khurana et al, 2004; Krex et al, 2006; Krischek et al, 2006; Song et al, 2006), and there was no significant difference in racial groups according to genotype. These results are consistent with more recent studies in larger populations (Rossi et al, 2006). Earlier, smaller studies hypothesized that the CC genotype was because of new mutations as the incidence of CC was 0.9% (n=335) (Nakayama et al, 1999), but recent studies in larger populations (n=1,096) have found the CC allele to be in Hardy—Weinberg equilibrium (19.2%) (Rossi et al, 2006). In multivariate logistic regression analysis, when controlling for clinical variables, the eNOS genotype was the only significant predictor of all forms of vasospasm. There was a trend in white patients with a T allele to have an increased risk of symptomatic vasospasm (OR 3, P=0.07) and severe vasospasm requiring endovascular treatment (OR 2.7, P=0.10). The T allele was not significantly associated with an increased risk of all types of vasospasm in each racial subgroup, but this may have been because of the small numbers of patients in the ethnic subgroup analysis.

Our results contrast to a previous study examining genetic polymorphisms of eNOS T-786 SNP and cerebral vasospasm. This may be because of inadequate power, and/or differences in ethnicity, incidence of alleles, and prevalence of vasospasm. In one study of 28 Fisher grade III aSAH patients, it was found that patients with vasospasm had a nonsignificant increased risk of having the C allele (OR 7.1, 95% CI: 0.88 to 57.5) (Khurana et al, 2004). These results may have been because of underpowering or because of the fact that only one of these patients had the CC genotype. In a study of vasospasm, after aSAH in 133 Korean patients, the eNOS T-786C polymorphism did not confer any increased risk of symptomatic vasospasm, but the CT genotype was associated with worse outcome (Song et al, 2006). These differences may again be accounted for by the variance in racial characteristics; 0% of Korean patients had the CC genotype. They also may be accounted for by the low rate of symptomatic vasospasm (12.8%) seen in this Korean population as compared with previously published studies (20% to 65%) (Kassell et al, 1985; Lanzino et al, 1999; Song et al, 2006; Vajkoczy et al, 2005).

Although NO plays a role in vasospasm after aSAH, the precise pathophysiology remains unclear. Studies have proposed that this process may be because of increased (Ignarro, 1990; Pluta et al, 1996) or decreased NO levels (Dumont et al, 2003; Moncada et al, 1991). Studies have reported decreased levels of NO in animals after experimental aSAH (Kasuya et al, 1995); other studies in humans with aSAH found an initial decrease in NO followed by an increase in NO between 2 and 8 days (Woszczyk et al, 2003).

It is unclear as to how different allelic combinations of the eNOS T-786 SNP lead to pathologic processes. Different allelic combinations have been associated with coronary spasm (CC) (Nakayama et al, 1999), aneurysm size (CT) (Akagawa et al, 2005), and outcomes after coronary spasm (CT) (Nishijima et al, 2007), cerebral vasospasm (CT) (Song et al, 2006), and cardiovascular mortality (TT) (Rossi et al, 2006). The role of eNOS expression in aSAH is controversial (Pluta et al, 1996; Kasuya et al, 1995; Hino et al, 1996; Park et al, 2001; McGirt et al, 2002). Pathogenesis because of alterations in polymorphisms may occur through a variety of mechanisms including decreased promoter activity leading to decreased NO or increased promoter activity leading to increased NO. Increased NO may lead to oxidative stress (Yung et al, 2006), intimal hyperplasia (Hingorani et al, 1999; Kuhlencordt et al, 2001), systemic hypertension (Kuhlencordt et al, 2001), vascular smooth muscle proliferation (Dumont et al, 2003; Moncada et al, 1991), increased platelet aggregation, and proinflammatory adhesion (Dumont et al, 2003; Moncada et al, 1991). These mechanisms of dysfunction may lead to alterations in smooth muscle proliferation, vessel dilation, and inflammation seen in the pathogenesis of vasospasm after aSAH (Dumont et al, 2003; Khurana et al, 2004; Moncada et al, 1991).

In one study, the eNOS promoter was isolated from patients with coronary spasm and after being subjected to hypoxic conditions for 24 h, it was found that genes with the C allele had decreased promoter activity (Nakayama et al, 1999). In this study of Asian patients, only 3 of 335 patients had the CC genotype, which differs significantly from other studies of white patients where the CC genotype comprised 19% of 1,096 patients (Rossi et al, 2006). In this larger study by Rossi et al, patients with the CC genotype had significantly higher levels of nitrotyrosine, a marker of NO production, thus supporting the notion that patients with the TT genotype may produce lower levels of NO. Unlike coronary spasm, most patients with aSAH do not experience cerebral vasospasm until 3 to 14 days after aSAH and the role of polymorphisms, the eNOS promoter, and NO levels have yet to be elucidated over this time period (Pluta et al, 1996; Kasuya et al, 1995; Hino et al, 1996; Kassell et al, 1984; McGirt et al, 2002; Park et al, 2001).

Further studies of eNOS, the promoter, and polymorphisms are needed to develop therapeutic agents that target the NO cascade. Preliminary studies have begun to look at invasive techniques to administer exogenous NO (Afshar et al, 1995; Wolf et al, 1998) or l-arginine (an NO substrate) (Nakaki et al, 1990; Pluta et al, 2000) in the treatment of vasospasm. Adenovirus gene transfer of eNOS to human arteries ex vivo and canine arteries in vivo has been shown to be protective in SAH models (Khurana et al, 2000; Khurana et al, 2002). Statins have recently been found to decrease vasospasm after aSAH. The presumed mechanism is the ability of statins to upregulate eNOS or to decrease reactive oxygen free radicals, platelet aggregation, and inflammation that lead to endothelial damage caused by blood clots (Tseng et al, 2005). Such interventions may decrease the significant morbidity and mortality because of vasospasm, as well as decrease complications because of triple therapy, angiography, and endovascular treatment of vasospasm.

Future studies should focus on the link between vasospasm, eNOS 786 polymorphisms, the eNOS promoter, and the amount of NO produced from the time of aSAH to the end of the vasospasm window (day 14). The role of the eNOS promoter and the corresponding levels of NO after aSAH throughout the window of vasospasm also need to be elucidated. These results should be correlated with long-term outcomes as it is unclear as to whether decreased incidence of vasospasm will result in improved functional outcome. The results of this study should be confirmed in larger racially diverse subgroups.

Conclusion

In conclusion, patients with a T allele have a significantly increased risk of developing severe vasospasm. This may help clinicians predict and identify high-risk patients. Furthermore, genetic analysis may allow clinicians to optimize early treatment for patients at risk for vasospasm and infarction, and improve patient outcomes.

Footnotes

Acknowledgements

We thank Lina Bruno for helping in bringing this project together. RMS was supported in part by an Alpha Omega Alpha Carolyn L Kuckein Student Research Fellowship.

The authors state no conflict of interest. The authors have no personal or institutional financial interest in drugs or materials in relation to this paper.

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Endothelial Nitric Oxide Synthase Gene Single-Nucleotide Polymorphism Predicts Cerebral Vasospasm after Aneurysmal Subarachnoid Hemorrhage

Abstract

Keywords

Introduction

Materials and methods

Patient Population

Management Protocol

Study Outcomes

Genetic Analysis

Statistical Analysis

Results

Cohort Characteristics

Cerebral Vasospasm

Infarction

Discussion

Conclusion

Footnotes

Acknowledgements

References