Role of endothelial nitric oxide synthase for early brain injury after subarachnoid hemorrhage in mice

The first few hours and days after subarachnoid hemorrhage (SAH) are characterized by cerebral ischemia, spasms of pial arterioles, and a significant reduction of cerebral microperfusion, however, the mechanisms of this early microcirculatory dysfunction are still unknown. Endothelial nitric oxide production is reduced after SAH and exogenous application of NO reduces post-hemorrhagic microvasospasm. Therefore, we hypothesize that the endothelial NO-synthase (eNOS) may be involved in the formation of microvasospasms, microcirculatory dysfunction, and unfavorable outcome after SAH. SAH was induced in male eNOS deficient (eNOS^–/–) mice by endovascular MCA perforation. Three hours later, the cerebral microcirculation was visualized using in vivo 2-photon-microscopy. eNOS^–/– mice had more severe SAHs, more severe ischemia, three time more rebleedings, and a massively increased mortality (50 vs. 0%) as compared to wild type (WT) littermate controls. Three hours after SAH eNOS^–/– mice had fewer perfused microvessels and 40% more microvasospasms than WT mice. The current study indicates that a proper function of eNOS plays a key role for a favorable outcome after SAH and helps to explain why patients suffering from hypertension or other conditions associated with impaired eNOS function, have a higher risk of unfavorable outcome after SAH.     

The Role of Platelet Activation and Inflammation in Early Brain Injury Following Subarachnoid Hemorrhage

Background

Early brain injury (EBI) following aneurysmal subarachnoid hemorrhage (SAH) is an important predictor of poor functional outcome, yet the underlying mechanism is not well understood. Animal studies suggest that platelet activation and inflammation with subsequent microthrombosis and ischemia may be a mechanism of EBI.

Methods

A prospective, hypothesis-driven study of spontaneous, SAH patients and controls was conducted. Platelet activation [thromboelastography maximum amplitude (MA)] and inflammation [C-reactive protein (CRP)] were measured serially over time during the first 72 h following SAH onset. Platelet activation and inflammatory markers were compared between controls and SAH patients with mild [Hunt–Hess (HH) 1–3] versus severe (HH 4–5) EBI. The association of these biomarkers with 3-month functional outcomes was evaluated.

Results

We enrolled 127 patients (106 SAH; 21 controls). Platelet activation and CRP increased incrementally with worse EBI/HH grade, and both increased over 72 h (all P < 0.01). Both were higher in severe versus mild EBI (MA 68.9 vs. 64.8 mm, P = 0.001; CRP 12.5 vs. 1.5 mg/L, P = 0.003) and compared to controls (both P < 0.003). Patients with delayed cerebral ischemia (DCI) had more platelet activation (66.6 vs. 64.9 in those without DCI, P = 0.02) within 72 h of ictus. At 3 months, death or severe disability was more likely with higher levels of platelet activation (mRS4–6 OR 1.18, 95 % CI 1.05–1.32, P = 0.007) and CRP (mRS4–6 OR 1.02, 95 % CI 1.00–1.03, P = 0.041).

Conclusions

Platelet activation and inflammation occur acutely after SAH and are associated with worse EBI, DCI and poor 3-month functional outcomes. These markers may provide insight into the mechanism of EBI following SAH.

     

Sexual dimorphism in gene expression after aneurysmal subarachnoid hemorrhage

Background and Purpose: Inflammation and compromise in structure and function of cerebral parenchymal microvasculature begins early after subarachnoid hemorrhage (SAH). We recently found greater inflammation and greater vascular compromise in male than in female rats following SAH. In this study, we investigated whether this cross-sexual difference in pathology is reflected in expression levels of genes related to vascular inflammation and structural compromise.

Method: Age-matched male and female rats underwent sham surgery or SAH by endovascular perforation. Early physiology (intracranial pressure (ICP), blood pressure (BP), heart rate, and cerebral blood flow) was monitored. Cerebral RNA was extracted at sacrifice 3 h after surgery and assayed for expression of thrombomodulin (Thbd), endothelial nitric oxide synthase (eNos;Nos3), intracellular adhesion molecule-1 (Icam1), vascular endothelial growth factor (Vegf), interleukin-1beta (Il1β) tumor necrosis factor-alpha (Tnf-α), and arginine vasopressin (Avp).

Results: Increases in ICP and BP at SAH appeared slightly greater in males but the difference did not reach statistical difference, indicating that SAH intensity did not differ significantly between the sexes. Of the seven genes studied two; Tnf-α and Vegf, did not change after injury, while the remainder showed significant responses to SAH. Response of Nos3 and Thbd was markedly different between the sexes, with expression greater in males.

Conclusion: This study finds that sexual dimorphism is present in the response of some but not all genes to SAH. Since products of genes exhibiting sexual dimorphism have anti-inflammatory activities, our results indicate that previously found sex-based differences in vascular pathology are paralleled by sexually dimorphic changes in gene expression following SAH.     

Imaging of hypoxic�Cischemic penumbra with 18F-fluoromisonidazole PET/CT and measurement of related cerebral metabolism in aneurysmal subarachnoid hemorrhage

This study aimed to characterize hypoxic, but salvageable, tissue imaged by ¹⁸F-fluoromisonidazole (¹⁸F-FMISO), combining with perfusion-computed tomography (PCT) for regional cerebral blood flow (rCBF) measurement and metabolism by microdialysis (MD) in aneurysmal subarachnoidal hemorrhage (SAH) patients. ¹⁸F-FMISO positron-emission tomography (PET)/CT was performed within the period of possible vasospasm (day 6.8±3 after SAH) in seven SAH patients. In parallel, rCBF was determined within the MD region of interest (MD-ROI) (n=5). The MD catheter was inserted into the brain parenchyma with highest risk for ischemia; extracellular levels of glutamate and energy metabolites were registered at time of PET and hourly for 10 days. Twelve-month outcome was evaluated. In asymptomatic patients (n=3) no hypoxia was detected and glutamate levels were low (<10 mmol/L), whereas symptomatic patients had higher glutamate concentrations (P<0.001). Increased ¹⁸F-FMISO uptake within the MD-ROI (n=3) was related to higher glutamate levels, while rCBF was above the ischemic range. Hypoxia (increased ¹⁸F-FMISO uptake) was present in symptomatic patients and associated with relevant metabolic derangement of extracellular glutamate levels, whereas energy metabolism and rCBF were preserved. This technique has the potential to improve our understanding of the role of cellular hypoxia in aneurysmal SAH.     

Serum Glucose and Potassium Ratio as Risk Factors for Cerebral Vasospasm after Aneurysmal Subarachnoid Hemorrhage

Objective: Cerebral vasospasm is associated with poor prognosis in patients with aneurysmal subarachnoid hemorrhage (SAH), and biomarkers for predicting poor prognosis have not yet been established. We attempted to clarify the relationship between serum glucose/potassium ratio and cerebral vasospasm in patients with aneurysmal SAH. Methods: We studied 333 of 535 aneurysmal SAH patients treated between 2006 and 2016 (123 males, 210 females; mean age 59.7 years; range 24-93). We retrospectively analyzed the relationship between cerebral vasospasm grade and clinical risk factors, including serum glucose/potassium ratio. Results: Postoperative angiography revealed cerebral vasospasm in 112 patients (33.6%). Significant correlations existed between the ischemic complication due to cerebral vasospasm and glucose/potassium ratio (P < .0001), glucose (P = .016), and potassium (P = .0017). Serum glucose/potassium ratio was elevated in the cerebral vasospasm grade dependent manner (Spearman's r = 0.1207, P = .0279). According to the Glasgow Outcome Scale (GOS) score at discharge, 185 patients (55.5%) had a poor outcome (GOS scores 1-3). Serum glucose/potassium ratio was significantly correlated between poor outcome (GOS scores 1-3) and age (P < .0001), serum glucose/potassium ratio (P < .0001), glucose (P < .0001), potassium (P = .0004), white blood cell count (P = .0012), and cerebral infarction due to cerebral vasospasm (P < .0001). Multivariate logistic regression analyzes showed significant correlations between cerebral infarction due to cerebral vasospasm and serum glucose/potassium ratio (P = .018), glucose (P = .027), and potassium (P = .052). Conclusions: Serum glucose/potassium ratio in cases of aneurysmal SAH was significantly associated with cerebral infarction due to cerebral vasospasm and GOS at discharge. Therefore, this factor was useful to predict prognosis in patients with cerebral vasospasm and aneurysmal SAH.     

Identification of Differentially-Methylated Genes and Pathways in Patients with Delayed Cerebral Ischemia Following Subarachnoid Hemorrhage

ObjectiveWe reported the differentially methylated genes in patients with subarachnoid hemorrhage (SAH) using bioinformatics analyses to explore the biological characteristics of the development of delayed cerebral ischemia (DCI). MethodsDNA methylation profiles obtained from 40 SAH patients from an epigenome-wide association study were analyzed. Functional enrichment analysis, protein-protein interaction (PPI) network, and module analyses were carried out. ResultsA total of 13 patients (32.5%) experienced DCI during the follow-up. In total, we categorized the genes into the two groups of hypermethylation (n=910) and hypomethylation (n=870). The hypermethylated genes referred to biological processes of organic cyclic compound biosynthesis, nucleobase-containing compound biosynthesis, heterocycle biosynthesis, aromatic compound biosynthesis and cellular nitrogen compound biosynthesis. The hypomethylated genes referred to biological processes of carbohydrate metabolism, the regulation of cell size, and the detection of a stimulus, and molecular functions of amylase activity, and hydrolase activity. Based on PPI network and module analysis, three hypermethylation modules were mainly associated with antigen-processing, Golgi-to-ER retrograde transport, and G alpha (i) signaling events, and two hypomethylation modules were associated with post-translational protein phosphorylation and the regulation of natural killer cell chemotaxis. VHL, KIF3A, KIFAP3, RACGAP1, and OPRM1 were identified as hub genes for hypermethylation, and ALB and IL5 as hub genes for hypomethylation. ConclusionThis study provided novel insights into DCI pathogenesis following SAH. Differently methylated hub genes can be useful biomarkers for the accurate DCI diagnosis.     

Transdermal Nicotine Replacement Therapy in Cigarette Smokers with Acute Subarachnoid Hemorrhage

Background

We evaluated the safety of nicotine replacement therapy (NRT) in active smokers with acute (aneurysmal) subarachnoid hemorrhage (SAH).

Methods

A retrospective observational cohort study was conducted in a prospectively collected database including all SAH patients admitted to an 18-bed neuro-ICU between January 1, 2001 and October 1, 2007. Univariate and multivariable models were constructed, employing stepwise logistic regression. The primary endpoint was 3-month mortality. Delayed cerebral ischemia (DCI) due to vasospasm, angiographic and TCD evidence of vasospasm, and delirium were secondary endpoints.

Results

Active cigarette smokers admitted with SAH included 128 that received NRT and 106 that did not. Patients were well-matched for age, admission Hunt-Hess Grade, radiographic findings, and APACHE II scores, but those who received NRT were more likely to be heavy smokers (>10 cigarettes daily), diabetic, heavy alcohol users, and to have cerebral edema on admission. NRT was associated in multivariate analysis with a lower risk of death at 3?months (OR 0.12, 95% CI 0.04?C0.37, P?P?=?0.006) and seizures (9 vs. 2%, P?=?0.024) were more common in patients who received NRT.

Conclusions

Despite vasoactive properties, administration of NRT among active smokers with acute SAH appeared to be safe, with similar rates of vasospasm and DCI, and a slightly higher rate of seizures. The association of NRT with lower mortality could be due to chance, to uncontrolled factors, or to a neuroprotective effect of nicotine in active smokers hospitalized with SAH, and should be tested prospectively.     

Correlating interleukin-10 promoter gene polymorphisms with human cerebral infarction onset

Evidence suggests that interleukin-10 (IL-10) deficiency exacerbates inflammation and worsens the outcome of brain ischemia. In view of the critical role of the single nucleotide polymorphic sites -1082 (A/G) and -819 (C/T) in the promoter region of the IL-10 gene, we hypothesized that they are associated with cerebral infarction morbidity in the Chinese Han population. We genotyped these allelic gene polymorphisms by amplification refractory mutation system-polymerase chain reaction methods in 181 patients with cerebral infarction (cerebral infarction group) and 115 healthy subjects (control group). We identified significant differences in genotype distribution and allele frequency of the IL-10-1082 A/G allele between cerebral infarction and control groups (χ² = 6.643, P = 0.010). The IL-10-1082 A allele frequency was significantly higher in the cerebral infarction group (92.3%) than in the control group (86.1%) (P = 0.015). Moreover, cerebral infarction risk of the AA genotype was 2-fold higher than with the AG genotype (OR = 2.031, 95%CI: 1.134–3.637). In addition, AA genotype together with hypertension was the independent risk factor of cerebral infarction (OR = 2.073, 95%CI: 1.278–3.364). No statistical difference in genotype distribution or allele frequency of IL-10-819 C/T was found between cerebral infarction and control groups (P > 0.05). These findings suggest that the IL-10-1082 A/G gene polymorphism is involved in cerebral infarction, and increased A allele frequency is closely associated with occurrence of cerebral infarction.     

Predictors of 30-Day Readmission After Subarachnoid Hemorrhage

Background

Readmission within 30 days is increasingly evaluated as a measure of quality of care. There are few data on the rates of readmission after subarachnoid hemorrhage (SAH).

Objective

We sought to determine the predictors of 30-day readmission in patients with SAH.

Methods

We prospectively identified 283 patients with SAH admitted between 2006 and 2012. Readmission was determined by means of an automated query with confirmation in the electronic medical record.

Results

Overall, 21 (8 %) patients were readmitted for infection (n = 8), headache (n = 5), hydrocephalus (n = 4), cardiovascular causes (n = 2), medication-related complications (n = 1), and cerebral ischemia (n = 1). Readmission was associated with longer intensive care unit (ICU) length of stay (LOS) (15.4 [13.4–19.3] vs. 12.2 [8.2–18.5] days, P = 0.02), hospital LOS (22.2 [17.4–23.0] vs. 16.8 [12.0–24.1] days, P = 0.01), and placement of an external ventricular drain (EVD, OR 3.9, 95 % CI 1.3–12.0, P = 0.01). Readmission was not associated with admission neurologic grade, NIH Stroke scale at 14 days, modified Rankin scale at 3 months, history of cardiovascular disease, or radiographic cerebral infarction (P > 0.1).

Conclusions

Demographics, severity of neurologic injury, radiographic cerebral infarction, and outcomes were not associated with readmission after SAH. Markers of a more complicated hospital course (ICU and hospital LOS, EVD placement) were associated with 30-day readmission. Most readmissions were for infections acquired after discharge. Readmission within 30 days is difficult to predict, and, since the most common reason was infection acquired after discharge, it may be difficult to prevent without an integrated health system and coordinated care.     

Post-subarachnoid Hemorrhage Vasospasm in Patients with Primary Headache Disorders

Background

Altered cerebral vasomotor reactivity leading to vasospasm can be seen both in patients with primary headache disorders (PHD) and in patients with subarachnoid hemorrhage (SAH). The pathogenesis of vasospasm in post-SAH patients and in headache disorder sufferers may be related. To address this hypothesis, we analyzed a large cohort of SAH patients to determine whether a diagnosis of PHD predisposes to vasospasm, delayed cerebral ischemia, or worsened clinical outcome.

Methods

Prospectively collected data from patients enrolled in the SAH Outcomes Project between 1996 and 2006 were analyzed. Patients were segregated based on whether they had a diagnosis of PHD or not and were subsequently compared for differences in clinical and radiographic outcome.

Results

A total of 921 SAH patients were analyzed, 265 of which had a diagnosis of PHD. In total, symptomatic vasospasm was seen in 17 %, while angiographic vasospasm was seen in 28 %. Vasospasm rates were similar among patients with a PHD and in those without a PHD (p > 0.05). However, on multivariate analysis new ischemic infarcts were more common in patients with a PHD as compared to patients without a PHD (p = 0.015). Functional outcomes at 3 months were similar among PHD and non-PHD patients (p > 0.05).

Conclusion

A history of PHD is associated with an increased rate of ischemic infarcts during admission for SAH. Increased rates of vasospasm within small cerebral blood vessels may be implicated. Further studies are warranted to more closely link the mechanisms of vasospasm in PHD and SAH patients.     

Intracerebral Monitoring of Silent Infarcts After Subarachnoid Hemorrhage

Background

Silent infarction is common in poor-grade subarachnoid hemorrhage (SAH) patients and associated with poor outcome. Invasive neuromonitoring devices may detect changes in cerebral metabolism and oxygenation.

Methods

From a consecutive series of 32 poor-grade SAH patients we identified all CT-scans obtained during multimodal neuromonitoring and analyzed microdialysis parameters and brain tissue oxygen tension (PbtO2) preceding CT-scanning.

Results

Eighteen percent of the reviewed head-CTs (12/67) revealed new infarcts. Of the eight infarcts in the vascular territory of the neuromonitoring, seven were clinically silent. Neuromonitoring changes preceding radiological evidence of infarction included lactate-pyruvate-ratio elevation and brain glucose decreases when compared to those with distant or no ischemia (P ≤ 0.03, respectively). PbtO2 was lower, but this did not reach statistical significance.

Conclusions

These data suggest that there may be distinct changes in brain metabolism and oxygenation associated with the development of silent infarction within the monitored vascular territory in poor-grade SAH patients. Larger prospective studies are needed to determine whether treatment triggered by neuromonitoring data has an impact on outcome.     

Blockade of the MEK/ERK pathway with a raf inhibitor prevents activation of pro-inflammatory mediators in cerebral arteries and reduction in cerebral blood flow after subarachnoid hemorrhage in a rat model

Cerebral ischemia that develops after subarachnoid hemorrhage (SAH) carries high morbidity and mortality. Inflammatory mediators are involved in the development of cerebral ischemia through activation of the mitogen-activated protein kinase pathway. We hypothesized that blockade of the MAPkinase/ERK (MEK)/extracellular signal-regulated kinase (ERK) pathway upstream with a specific raf inhibitor would prevent SAH-induced activation of the cerebrovascular inflammatory response. The raf inhibitor SB-386023-b was injected intracisternally in our rat model at 0, 6, or 12 hours after the SAH. After 48 hours, cerebral arteries were harvested, and iNOS, interleukin (IL)-6, IL-1β, matrix metalloproteinase (MMP)-9, tissue inhibitors of metalloproteinase (TIMP)-1, and phosphorylated ERK1/2 were investigated by immunofluorescence, real-time polymerase chain reaction (PCR), and Western blot analysis. Cerebral blood flow (CBF) was measured using autoradiography. Protein levels of MMP-9, TIMP-1, iNOS, IL-6, and IL-1β were increased after SAH, as were mRNA levels of IL-6, MMP-9, and TIMP-1. After SAH, pERK1/2 was increased, but CBF was reduced. Treatment with SB-386023-b at 0 or 6 hours after SAH normalized CBF and prevented SAH-induced upregulation of MMPs, pro-inflammatory cytokines, and pERK1/2 proteins. These results suggested that inhibition of MEK/ERK signal transduction by a specific raf inhibitor administered up to 6 hours after SAH normalized the expression of pro-inflammatory mediators and extracellular matrix-related genes.