Genome-wide association study of sporadic brain arteriovenous malformations.

BACKGROUND: The pathogenesis of sporadic brain arteriovenous malformations (BAVMs) remains unknown, but studies suggest a genetic component. We estimated the heritability of sporadic BAVM and performed a genome-wide association study (GWAS) to investigate association of common single nucleotide polymorphisms (SNPs) with risk of sporadic BAVM in the international, multicentre Genetics of Arteriovenous Malformation (GEN-AVM) consortium. METHODS: The Caucasian discovery cohort included 515 BAVM cases and 1191 controls genotyped using Affymetrix genome-wide SNP arrays. Genotype data were imputed to 1000 Genomes Project data, and well-imputed SNPs (>0.01 minor allele frequency) were analysed for association with BAVM. 57 top BAVM-associated SNPs (51 SNPs with p<10(-05) or p<10(-04) in candidate pathway genes, and 6 candidate BAVM SNPs) were tested in a replication cohort including 608 BAVM cases and 744 controls. RESULTS: The estimated heritability of BAVM was 17.6% (SE 8.9%, age and sex-adjusted p=0.015). None of the SNPs were significantly associated with BAVM in the replication cohort after correction for multiple testing. 6 SNPs had a nominal p<0.1 in the replication cohort and map to introns in EGFEM1P, SP4 and CDKAL1 or near JAG1 and BNC2. Of the 6 candidate SNPs, 2 in ACVRL1 and MMP3 had a nominal p<0.05 in the replication cohort. CONCLUSIONS: We performed the first GWAS of sporadic BAVM in the largest BAVM cohort assembled to date. No GWAS SNPs were replicated, suggesting that common SNPs do not contribute strongly to BAVM susceptibility. However, heritability estimates suggest a modest but significant genetic contribution.

Morphometric characterization of brain arteriovenous malformations for clinical and radiological studies to identify silent intralesional microhemorrhages.

Brain arteriovenous malformations (bAVMs) are vascular lesions that can cause significant morbidity and mortality, particularly when they bleed, i.e., rupture. Determining the risk of rupture for bAVMs is a crucial task to determine the most appropriate approach to patients with bAVM. Furthermore, patients who present with a hemorrhagic event also have a higher risk of subsequent hemorrhage. Determination of the hemorrhage risk and management strategy for incidentally discovered bAVMs still remains a controversial subject. In recent years, we have identified silent intralesional microhemorrhages (SIMs) as a possible risk factor for subsequent hemorrhage in patients with bAVMs. The principal aim of this study was to determine critical histological features that can be correlated with preoperative radioimaging findings, and allow better identification of patients with greater risk of adverse outcome. Here we provide a detailed descriptive analysis of the morphometric assessment of bAVMs in order to provide reproducible methodology that will aid in correlating preoperative radioimaging findings with histological features that may be significantly associated with increased risk of hemorrhage/rupture.

Hemorrhage rates from brain arteriovenous malformation in patients with hereditary hemorrhagic telangiectasia.

BACKGROUND AND PURPOSE: Hereditary hemorrhagic telangiectasia (HHT) is a systemic disease characterized by mucocutaneous telangiectasias, epistaxis, and arteriovenous malformations (AVMs). Intracranial hemorrhage (ICH) rates in this population are not well described. We report ICH rates and characteristics in HHT patients with brain AVMs (HHT-BAVMs). METHODS: We studied the first 153 HHT-BAVM patients with follow-up data enrolled in the Brain Vascular Malformation Consortium HHT Project. We estimated ICH rates after BAVM diagnosis. RESULTS: The majority of patients were women (58%) and white (98%). The mean age at BAVM diagnosis was 31±19 years (range, 0-70), with 61% of cases diagnosed on asymptomatic screening. Overall, 14% presented with ICH; among symptomatic cases, 37% presented ruptured. During 493 patient-years of follow-up, 5 ICH events occurred yielding a rate of 1.02% per year (95% confidence interval, 0.42-2.44%). ICH-free survival differed significantly by ICH presentation (P=0.003); ruptured cases had a higher ICH rate (10.07%; 95% confidence interval, 3.25-31.21%) than unruptured cases (0.43%; 95% confidence interval, 0.11-1.73%). CONCLUSIONS: Patients with HHT-BAVM who present with hemorrhage are at a higher risk for rehemorrhage compared with patients with BAVM detected presymptomatically.

Medical management with or without interventional therapy for unruptured brain arteriovenous malformations (ARUBA): a multicentre, non-blinded, randomised trial.

BACKGROUND: The clinical benefit of preventive eradication of unruptured brain arteriovenous malformations remains uncertain. A Randomised trial of Unruptured Brain Arteriovenous malformations (ARUBA) aims to compare the risk of death and symptomatic stroke in patients with an unruptured brain arteriovenous malformation who are allocated to either medical management alone or medical management with interventional therapy. METHODS: Adult patients (≥18 years) with an unruptured brain arteriovenous malformation were enrolled into this trial at 39 clinical sites in nine countries. Patients were randomised (by web-based system, in a 1:1 ratio, with random permuted block design [block size 2, 4, or 6], stratified by clinical site) to medical management with interventional therapy (ie, neurosurgery, embolisation, or stereotactic radiotherapy, alone or in combination) or medical management alone (ie, pharmacological therapy for neurological symptoms as needed). Patients, clinicians, and investigators are aware of treatment assignment. The primary outcome is time to the composite endpoint of death or symptomatic stroke; the primary analysis is by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00389181. FINDINGS: Randomisation was started on April 4, 2007, and was stopped on April 15, 2013, when a data and safety monitoring board appointed by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health recommended halting randomisation because of superiority of the medical management group (log-rank Z statistic of 4·10, exceeding the prespecified stopping boundary value of 2·87). At this point, outcome data were available for 223 patients (mean follow-up 33·3 months [SD 19·7]), 114 assigned to interventional therapy and 109 to medical management. The primary endpoint had been reached by 11 (10·1%) patients in the medical management group compared with 35 (30·7%) in the interventional therapy group. The risk of death or stroke was significantly lower in the medical management group than in the interventional therapy group (hazard ratio 0·27, 95% CI 0·14-0·54). No harms were identified, other than a higher number of strokes (45 vs 12, p<0·0001) and neurological deficits unrelated to stroke (14 vs 1, p=0·0008) in patients allocated to interventional therapy compared with medical management. INTERPRETATION: The ARUBA trial showed that medical management alone is superior to medical management with interventional therapy for the prevention of death or stroke in patients with unruptured brain arteriovenous malformations followed up for 33 months. The trial is continuing its observational phase to establish whether the disparities will persist over an additional 5 years of follow-up. FUNDING: National Institutes of Health, National Institute of Neurological Disorders and Stroke.

The ACVRL1 c.314-35A>G polymorphism is associated with organ vascular malformations in hereditary hemorrhagic telangiectasia patients with ENG mutations, but not in patients with ACVRL1 mutations.

Hereditary hemorrhagic telangiectasia (HHT) is characterized by vascular malformations (VMs) and caused by mutations in TGFß/BMP9 pathway genes, most commonly ENG or ACVRL1. Patients with HHT have diverse manifestations related to skin and mucosal telangiectases and organ VMs, including arteriovenous malformations (AVM). The clinical heterogeneity of HHT suggests a role for genetic modifiers. We hypothesized that the ACVRL1 c.314-35A>G and ENG c.207G>A polymorphisms, previously associated with sporadic brain AVM, are associated with organ VM in HHT. We genotyped these variants in 716 patients with HHT and evaluated association of genotype with presence of any organ VM, and specifically with brain VM, liver VM and pulmonary AVM, by multivariate logistic regression analyses stratified by HHT mutation. Among all patients with HHT, neither polymorphism was significantly associated with presence of any organ VM; ACVRL1 c.314-35A>G showed a trend toward association with pulmonary AVM (OR = 1.48, P = 0.062). ACVRL1 c.314-35A>G was significantly associated with any VM among patients with HHT with ENG (OR = 2.66, P = 0.022), but not ACVRL1 (OR = 0.79, P = 0.52) mutations. ACVRL1 c.314-35A>G was also associated with pulmonary AVM and liver VM among ENG mutation heterozygotes. There were no significant associations between ENG c.207G>A and any VM phenotype. These results suggest that common polymorphisms in HHT genes other than the mutated gene modulate phenotype severity of HHT disease, specifically presence of organ VM.

De novo cerebrovascular malformation in the adult mouse after endothelial Alk1 deletion and angiogenic stimulation.

BACKGROUND AND PURPOSE: In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor stimulation induces brain AVMs in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal vascular endothelial growth factor stimulation is sufficient to induce brain AVM in the adult mouse. METHODS: Focal angiogenesis was induced in the brain of 8-week-old Pdgfb-iCreER;Alk1(2f/2f) mice by injection of adeno-associated viral vectors expressing vascular endothelial growth factor. Two weeks later, EC-Alk1 deletion was induced by tamoxifen treatment. Vascular morphology was analyzed, and EC proliferation and dysplasia index (number of vessels with diameter>15 µm per 200 vessels) were quantified 10 days after tamoxifen administration. RESULTS: Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of tamoxifen-treated Pdgfb-iCreER;Alk1(2f/2f) mice. Induced brain AVMs were marked by increased dysplasia index (P<0.001) and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs. CONCLUSIONS: Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo brain AVM.

Endoglin deficiency impairs stroke recovery.

BACKGROUND AND PURPOSE: Endoglin deficiency causes hereditary hemorrhagic telangiectasia-1 and impairs myocardial repair. Pulmonary arteriovenous malformations in patients with hereditary hemorrhagic telangiectasia-1 are associated with a high incidence of paradoxical embolism in the cerebral circulation and ischemic brain injury. We hypothesized that endoglin deficiency impairs stroke recovery. METHODS: Eng heterozygous (Eng+/-) and wild-type mice underwent permanent distal middle cerebral artery occlusion (pMCAO). Pial collateral vessels were quantified before pMCAO. Infarct/atrophic volume, vascular density, and macrophages were quantified in various days after pMCAO, and behavioral function was assessed using corner and adhesive removal tests on days 3, 15, 30, and 60 after pMCAO. The association between ENG 207G>A polymorphism and brain arteriovenous malformation rupture and surgery outcome was analyzed using logistic regression analysis in 256 ruptured and 157 unruptured patients. RESULTS: After pMCAO, Eng+/- mice showed larger infarct/atrophic volumes at all time points (P<0.05) and showed worse behavior performance (P<0.05) at 15, 30, and 60 days when compared with wild-type mice. Eng+/- mice had fewer macrophages on day 3 (P=0.009) and more macrophages on day 60 (P=0.02) in the peri-infarct region. Although Eng+/- and wild-type mice had similar numbers of pial collateral vessels before pMCAO, Eng+/- mice had lower vascular density in the peri-infarct region (P=0.05) on day 60 after pMCAO. In humans, ENG 207A allele has been associated with worse outcomes after arteriovenous malformation rupture or surgery of patients with unruptured arteriovenous malformation. CONCLUSIONS: Endoglin deficiency impairs brain injury recovery. Reduced angiogenesis, impaired macrophage homing, and delayed inflammation resolution could be the underlying mechanism.

Common variants on 9p21.3 are associated with brain arteriovenous malformations with accompanying arterial aneurysms.

OBJECTIVE: To investigate whether previously reported 9p21.3 single nucleotide polymorphisms (SNPs) are associated with risk of brain arteriovenous malformations (BAVM), which often have accompanying arterial aneurysms. Common variants in the 9p21.3 locus have been reported to be associated with multiple cardiovascular phenotypes, including coronary artery disease and intracranial aneurysms (rs10757278 and rs1333040). METHODS: We used data from 338 BAVM cases participating in the University of California, San Francisco (UCSF)-Kaiser Brain AVM Study Project and 504 healthy controls to evaluate genotypes for seven common SNPs (minor allele frequency>0.05) that were imputed using 1000 Genomes Phase 1 European data (R(2)>0.87). Association with BAVM was tested using logistic regression adjusting for age, sex and the top three principal components of ancestry. Subgroup analysis included 205 BAVM cases with aneurysm data: 74 BAVM with aneurysm versus 504 controls and 131 BAVM without aneurysm versus 504 controls. RESULTS: We observed suggestive association with BAVM and rs10757278-G (OR=1.23, 95% CI 0.99 to 1.53, p=0.064) and rs1333040-T (OR=1.27, 95% CI 1.01 to 1.58, p=0.04). For rs10757278-G, the association was stronger in BAVM cases with aneurysm (OR=1.52, 95% CI 1.03 to 2.22, p=0.032) than in BAVM without aneurysm (OR=0.98, 95% CI 0.72 to 1.34, p=0.91). Similar patterns of effects were observed for rs1333040 and for other SNPs in linkage disequilibrium (r(2)>0.8) with rs10757278. CONCLUSIONS: Common 9p21.3 variants showed similar effect sizes for association with BAVM as previously reported for aneurysmal disease. The association with BAVM appears to be explained by known associations with aneurysms, suggesting that BAVM-associated aneurysms share similar vascular pathology mechanisms with other aneurysm types.

Reduced mural cell coverage and impaired vessel integrity after angiogenic stimulation in the Alk1-deficient brain.

OBJECTIVE: Vessels in brain arteriovenous malformations are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of brain arteriovenous malformation than the general population. We tested the hypothesis that vascular endothelial growth factor impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell coverage. METHODS AND RESULTS: Adult Alk1(1f/2f) mice (loxP sites flanking exons 4-6) and wild-type mice were injected with 2×10(7) PFU adenovious-cre recombinase and 2×10(9) genome copies of adeno-associated virus-vascular endothelial growth factor to induce focal homozygous Alk1 deletion (in Alk1(1f/2f) mice) and angiogenesis. Brain vessels were analyzed 8 weeks later. Compared with wild-type mice, the Alk1-deficient brain had more fibrin (99±30×10(3) pixels/mm(2) versus 40±13×10(3); P=0.001), iron deposition (508±506 pixels/mm(2) versus 6±49; P=0.04), and Iba1(+) microglia/macrophage infiltration (888±420 Iba1(+) cells/mm(2) versus 240±104 Iba1(+); P=0.001) after vascular endothelial growth factor stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-smooth muscle actin negative vessels (52±9% versus 12±7%, P<0.001), fewer vascular-associated pericytes (503±179/mm(2) versus 931±115, P<0.001), and reduced platelet-derived growth factor receptor-ß expression. CONCLUSIONS: Reduction of mural cell coverage in response to vascular endothelial growth factor stimulation is a potential mechanism for the impairment of vessel wall integrity in hereditary hemorrhagic telangiectasia type 2-associated brain arteriovenous malformation.

Angiographic features help predict outcome after stereotactic radiosurgery for the treatment of pediatric arteriovenous malformations.

PURPOSE: Arteriovenous malformations (AVMs) are a frequent cause of hemorrhagic stroke in children. Stereotactic radiosurgery (SRS) is an established treatment for these lesions, particularly those that are surgically inaccessible. Because only complete AVM obliteration is believed to protect against the future risk of hemorrhage, identifying lesion characteristics that predict response to therapy is an important objective. The goal of this study is to evaluate the influence of angiographic features of AVMs on the rate of obliteration following treatment with SRS. METHODS: This is a retrospective cohort study of pediatric patients (age ≤18 years) treated with Gamma Knife SRS for cerebral AVMs between 2000 and 2012. Detailed angiographic data at the time of initial angiographic evaluation were prospectively recorded by experienced neurointerventional radiologists. The primary outcome was the rate of obliteration on a 3-year follow-up angiogram. RESULTS: We identified 42 pediatric patients treated with SRS for cerebral AVMs. Twenty-seven patients completed 3-year angiographic follow-ups. Complete obliteration was seen in 30%, partial response in 67%, and no response in 4%. Higher SRS dose was associated with complete obliteration. Larger AVM diameter, presence of multiple draining veins, and presence of multiple draining veins reaching a sinus were associated with partial response. In this small cohort, diffuse AVM borders, presence of aneurysm, and pre-SRS embolization were not associated with obliteration. CONCLUSIONS: Our study identifies AVMs in the pediatric population with a nidus diameter of <2.5 cm and a solitary draining vein as the most likely to undergo complete obliteration after SRS treatment.

The natural history of AVM hemorrhage in the posterior fossa: comparison of hematoma volumes and neurological outcomes in patients with ruptured infra- and supratentorial AVMs.

OBJECT: Patients with posterior fossa arteriovenous malformations (AVMs) are more likely to present with hemorrhage than those with supratentorial AVMs. Observed patients subject to the AVM natural history should be informed of the individualized effects of AVM characteristics on the clinical course following a new, first-time hemorrhage. The authors hypothesize that the debilitating effects of first-time bleeding from an AVM in a previously intact patient with an unruptured AVM are more pronounced when AVMs are located in the posterior fossa. METHODS: The University of California, San Francisco prospective registry of brain AVMs was searched for patients with a ruptured AVM who had a pre-hemorrhage modified Rankin Scale (mRS) score of 0 and a post-hemorrhage mRS score obtained within 2 days of the hemorrhagic event. A total of 154 patients met the inclusion criteria for this study. Immediate post-hemorrhage presentation mRS scores were dichotomized into nonsevere outcome (mRS ≤ 3) and severe outcome (mRS > 3). There were 77 patients in each group. Univariate and multivariate logistic regression analyses using severe outcome as the binary response were run. The authors also performed a logistic regression analysis to measure the effects of hematoma volume and AVM location on severe outcome. RESULTS: Posterior fossa location was a significant predictor of severe outcome (OR 2.60, 95% CI 1.20-5.67, p = 0.016) and the results were strengthened in a multivariate model (OR 4.96, 95% CI 1.73-14.17, p = 0.003). Eloquent location (OR 3.47, 95% CI 1.37-8.80, p = 0.009) and associated arterial aneurysms (OR 2.58, 95% CI 1.09, 6.10; p = 0.031) were also significant predictors of poor outcome. Hematoma volume for patients with a posterior fossa AVM was 10.1 ± 10.1 cm(3) compared with 25.6 ±28.0 cm(3) in supratentorial locations (p = 0.003). A logistic analysis (based on imputed hemorrhage volume values) found that posterior fossa location was a significant predictor of severe outcome (OR 8.03, 95% CI 1.20-53.77, p = 0.033) and logarithmic hematoma volume showed a positive, but not statistically significant, association in the model (p = 0.079). CONCLUSIONS: Patients with posterior fossa AVMs are more likely to have severe outcomes than those with supratentorial AVMs based on this natural history study. Age, sex, and ethnicity were not associated with an increased risk of severe outcome after AVM rupture, but posterior fossa location, associated aneurysms, and eloquent location were associated with poor post-hemorrhage mRS scores. Posterior fossa hematomas are poorly tolerated, with severe outcomes observed even with smaller hematoma volumes. These findings support an aggressive surgical posture with respect to posterior fossa AVMs, both before and after rupture.

Intravenous delivery of adeno-associated viral vector serotype 9 mediates effective gene expression in ischemic stroke lesion and brain angiogenic foci.

BACKGROUND AND PURPOSE: Adeno-associated viral vector (AAV) is a powerful tool for delivering genes to treat brain diseases. Intravenous delivery of a self-complementary but not single-stranded AAV9 (ssAAV9) mediates robust gene expression in the adult brain. We tested if ssAAV9 effectively mediates gene expression in the ischemic stroke lesion and angiogenic foci. METHODS: Focal ischemic stroke was induced by permanent occlusion of the left middle cerebral artery (MCAO) and focal angiogenesis was induced by injecting an AAV expressing vascular endothelial growth factor (AAV-VEGF) into the basal ganglia. ssAAV vectors that have cytomegalovirus (CMV) promoter driving (AAV-CMVLacZ) or hypoxia response elements controlling (AAV-H9LacZ) LacZ expression were packaged in AAV9 or AAV1 capsid and injected into mice through the jugular vein 1 hour after MCAO or 4 weeks after the induction of angiogenesis. LacZ gene expression was analyzed in the brain and other organs 5 days after LacZ vector injection. RESULTS: LacZ expression was detected in the peri-infarct region of AAV9-CMVLacZ and AAV9-H9LacZ-injected MCAO mice and the brain angiogenic foci of AAV9-CMVLacZ-injected mice. Minimum LacZ expression was detected in the brain of AAV1-CMVLacZ-injected mice. Robust LacZ expression was found in the liver and heart of AAV-CMVLacZ-injected mice, but not in AAV9-H9LacZ-injected mice. CONCLUSIONS: ssAAV9 could be a useful tool to deliver therapeutic genes to the ischemic stroke lesion or brain angiogenic foci.

Endoglin deficiency in bone marrow is sufficient to cause cerebrovascular dysplasia in the adult mouse after vascular endothelial growth factor stimulation.

BACKGROUND AND PURPOSE: Bone marrow-derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)-induced brain angiogenic foci, and VEGF induces cerebrovascular dysplasia in adult endoglin heterozygous (Eng(+/-)) mice. We hypothesized that Eng(+/-) BMDCs cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation. METHODS: BM transplantation was performed using adult wild-type (WT) and Eng(+/-) mice as donors/recipients. An adeno-associated viral vector expressing VEGF was injected into the basal ganglia 4 weeks after transplantation. Vascular density, dysplasia index (vessels >15 µm/100 vessels), and BMDCs in the angiogenic foci were analyzed. RESULTS: The dysplasia index of WT/Eng(+/-) BM mice was higher than WT/WT BM mice (P<0.001) and was similar to Eng(+/-)/Eng(+/-) BM mice (P=0.2). Dysplasia in Eng(+/-) mice was partially rescued by WT BM (P<0.001). WT/WT BM and WT/Eng(+/-) BM mice had similar numbers of BMDCs in the angiogenic foci (P=0.4), most of which were CD68(+). Eng(+/-) monocytes/macrophages expressed less matrix metalloproteinase-9 and Notch1. CONCLUSIONS: Endoglin-deficient BMDCs are sufficient for VEGF to induce vascular dysplasia in the adult mouse brain. Our data support a previously unrecognized role of BM in the development of cerebrovascular malformations.

Depletion of bone marrow-derived macrophages perturbs the innate immune response to surgery and reduces postoperative memory dysfunction.

BACKGROUND: According to rodent models of postoperative cognitive decline, activation of the innate immune response following aseptic surgical trauma results in the elaboration of hippocampal proinflammatory cytokines, which are capable of disrupting long-term potentiation, the neurobiologic correlate of memory. The authors hypothesize that hippocampal recruitment of bone marrow-derived macrophages plays a causal role in these processes, resulting in memory dysfunction. METHODS: Clodrolip injection (liposomal formulation of clodronate) before stabilized tibial fracture under general anesthesia was used to deplete bone marrow-derived macrophages. Systemic inflammation and neuroinflammation were studied on postoperative day 1, and memory in a fear-trace conditioning paradigm was assessed on postoperative day 3. CX3CR1 CCR2 mice were used to identify bone marrow-derived macrophages. RESULTS: Clodrolip effectively depleted splenic CCR2 bone marrow-derived macrophages. It also attenuated the surgery-induced increase of interleukin-6 in the serum and the hippocampus, and prevented hippocampal infiltration of CCR2 cells without affecting the number of CX3CR1 microglia. It did not alter the surgery-induced increase in hippocampal monocyte chemoattractant protein-1, the recruitment signal for CCR2 cells. Clodrolip prevented surgery-induced memory dysfunction, as evidenced by a significant increase in freezing time (29% [95% CI, 21-38%] vs. 48% [95% CI, 38-58%], n = 20, P = 0.004), but did not affect memory in nonsurgical mice. CONCLUSION: Depletion of bone marrow-derived macrophages prevents hippocampal neuroinflammation and memory dysfunction after experimental tibial fracture. These data suggest that the hippocampal recruitment of bone marrow-derived macrophages is a necessary mechanism in murine postoperative cognitive dysfunction. Interventions designed to prevent its activation and/or migration into the brain may represent a feasible preemptive strategy.

Bone fracture exacerbates murine ischemic cerebral injury.

BACKGROUND: Bone fracture increases alarmins and proinflammatory cytokines in the blood, and provokes macrophage infiltration and proinflammatory cytokine expression in the hippocampus. We recently reported that stroke is an independent risk factor after bone surgery for adverse outcome; however, the impact of bone fracture on stroke outcome remains unknown. We tested the hypothesis that bone fracture, shortly after ischemic stroke, enhances stroke-related injuries by augmenting the neuroinflammatory response. METHODS: Tibia fracture (bone fracture) was induced in mice one day after permanent occlusion of the distal middle cerebral artery (stroke). High-mobility-group box chromosomal protein-1 (HMGB1) was tested to mimic the bone fracture effects. HMGB1 neutralizing antibody and clodrolip (macrophage depletion) were tested to attenuate the bone fracture effects. Neurobehavioral function (n = 10), infarct volume, neuronal death, and macrophages/microglia infiltration (n = 6-7) were analyzed after 3 days. RESULTS: We found that mice with both stroke and bone fracture had larger infarct volumes (mean percentage of ipsilateral hemisphere ± SD: 30 ± 7% vs.12 ± 3%, n = 6, P < 0.001), more severe neurobehavioral dysfunction, and more macrophages/microglia in the periinfarct region than mice with stroke only. Intraperitoneal injection of HMGB1 mimicked, whereas neutralizing HMGB1 attenuated, the bone fracture effects and the macrophage/microglia infiltration. Depleting macrophages with clodrolip also attenuated the aggravating effects of bone fracture on stroke lesion and behavioral dysfunction. CONCLUSIONS: These novel findings suggest that bone fracture shortly after stroke enhances stroke injury via augmented inflammation through HMGB1 and macrophage/microglia infiltration. Interventions to modulate early macrophage/microglia activation could be therapeutic goals to limit the adverse consequences of bone fracture after stroke.

Macrophage imaging within human cerebral aneurysms wall using ferumoxytol-enhanced MRI: a pilot study.

OBJECTIVE: Macrophages play a critical role in cerebral aneurysm formation and rupture. The purpose of this study is to demonstrate the feasibility and optimal parameters of imaging macrophages within human cerebral aneurysm wall using ferumoxytol-enhanced MRI. METHODS AND RESULTS: Nineteen unruptured aneurysms in 11 patients were imaged using T2*-GE-MRI sequence. Two protocols were used. Protocol A was an infusion of 2.5 mg/kg of ferumoxytol and imaging at day 0 and 1. Protocol B was an infusion of 5 mg/kg of ferumoxytol and imaging at day 0 and 3. All images were reviewed independently by 2 neuroradiologists to assess for ferumoxytol-associated loss of MRI signal intensity within aneurysm wall. Aneurysm tissue was harvested for histological analysis. Fifty percent (5/10) of aneurysms in protocol A showed ferumoxytol-associated signal changes in aneurysm walls compared to 78% (7/9) of aneurysms in protocol B. Aneurysm tissue harvested from patients infused with ferumoxytol stained positive for both CD68+, demonstrating macrophage infiltration, and Prussian blue, demonstrating uptake of iron particles. Tissue harvested from controls stained positive for CD68 but not Prussian blue. CONCLUSIONS: Imaging with T2*-GE-MRI at 72 hours postinfusion of 5 mg/kg of ferumoxytol establishes a valid and useful approximation of optimal dose and timing parameters for macrophages imaging within aneurysm wall. Further studies are needed to correlate these imaging findings with risk of intracranial aneurysm rupture.

Imaging aspirin effect on macrophages in the wall of human cerebral aneurysms using ferumoxytol-enhanced MRI: preliminary results.

BACKGROUND AND PURPOSE: Daily intake of aspirin was shown to decrease human cerebral aneurysm rupture by 60%. The feasibility of imaging macrophages in human cerebral aneurysm walls using ferumoxytol-enhanced MRI has been demonstrated. The goal of the present study is to image aspirin effect on macrophages in the wall of human cerebral aneurysm using ferumoxytol-enhanced MRI. MATERIAL AND METHODS: Five patients with known intracranial aneurysms underwent baseline imaging using T2(*) gradient-echo and T1 MRI sequences using ferumoxytol-enhanced MRI 72-hour post-ferumoxytol infusion. Patients then received 81 mg aspirin per os daily. After 3 months, imaging studies were repeated and analyzed by co-registration using a histogram and subtraction of follow-up images from baseline. RESULTS: In all five patients, after 3 months of treatment with aspirin, the signal intensity corresponding to the uptake of ferumoxytol by macrophages in the aneurysm wall was less intense than in the baseline images. This was confirmed by co-registration of images using histogram and subtraction of follow-up images from baseline. CONCLUSION: These preliminary results suggest the feasibility of imaging aspirin effect on macrophages localized in the wall of human cerebral aneurysm using ferumoxytol-enhanced MRI. The findings provide radiographic evidence of decreased inflammation in human cerebral aneurysms with daily intake of aspirin using macrophages as a surrogate marker for inflammation.

Evaluating performance of the spetzler-martin supplemented model in selecting patients with brain arteriovenous malformation for surgery.

BACKGROUND AND PURPOSE: Our recently proposed point scoring model includes the widely-used Spetzler-Martin (SM)-5 variables, along with age, unruptured presentation, and diffuse border (SM-Supp). Here we evaluate the SM-Supp model performance compared with SM-5, SM-3, and Toronto prediction models using net reclassification index, which quantifies the correct movement in risk reclassification, and validate the model in an independent data set. METHODS: Bad outcome was defined as worsening between preoperative and final postoperative modified Rankin Scale score. Point scores for each model were used as predictors in logistic regression and predictions evaluated using net reclassification index at varying thresholds (10%-30%) and any threshold (continuous net reclassification index >0). Performance was validated in an independent data set (n=117). RESULTS: Net gain in risk reclassification was better using the SM-Supp model over a range of threshold values (net reclassification index=9%-25%) and significantly improved overall predictions for outcomes in the development data set, yielding a continuous net reclassification index of 64% versus SM-5, 67% versus SM-3, and 61% versus Toronto (all P<0.001). In the validation data set, the SM-Supp model again correctly reclassified a greater proportion of patients versus SM-5 (82%), SM-3 (85%), and Toronto models (69%). CONCLUSIONS: The SM-Supp model demonstrated better discrimination and risk reclassification than several existing models and should be considered for clinical practice to estimate surgical risk in patients with brain arteriovenous malformation.

Bevacizumab attenuates VEGF-induced angiogenesis and vascular malformations in the adult mouse brain.

BACKGROUND AND PURPOSE: Vascular endothelial growth factor (VEGF) expression is elevated in human brain arteriovenous malformations (bAVM). We have developed a bAVM model in the adult mouse by focal Alk1 gene deletion and human VEGF stimulation. We hypothesized that once the abnormal vasculature has been established, tonic VEGF stimulation is necessary to maintain the abnormal phenotype, and VEGF antagonism by bevacizumab (Avastin) would reduce vessel density and attenuate the dysplastic vascular phenotype. METHODS: Angiogenesis and bAVM were induced by injection of adeno-associated viral vector expressing human VEGF alone into the brain of wild-type mice or with adenoviral vector expressing Cre recombinase (Ad-Cre) into Alk1(2f/2f) mice. Six weeks later, bevacizumab or trastuzumab (Herceptin, bevacizumab control) was administered. Vessel density, dysplasia index, vascular cell proliferation and apoptosis, and human IgG were assessed (n=6/group). RESULTS: Compared with trastuzumab (15 mg/kg), administration of 5, 10, and 15 mg/kg of bevacizumab to adeno-associated viral vector expressing human VEGF treated wild-type mice reduced focal vessel density (P<0.05); administration of 5 mg/kg bevacizumab decreased proliferating vascular cells (P=0.04) and increased TUNEL-positive vascular cells (P=0.03). More importantly, bevacizumab (5 mg/kg) treatment reduced both vessel density (P=0.01) and dysplasia index (P=0.02) in our bAVM model. Human IgG was detected in the vessel wall and in the parenchyma in the angiogenic foci of bevacizumab-treated mice. CONCLUSIONS: We provide proof-of-principle that, once abnormal AVM vessels have formed, VEGF antagonism may reduce the number of dysplastic vessels and should be evaluated further as a therapeutic strategy for the human disease.

Brain-derived neurotrophic factor Val66Met polymorphism predicts worse functional outcome after surgery in patients with unruptured brain arteriovenous malformation.

BACKGROUND AND PURPOSE: The Val66Met polymorphism of brain-derived neurotrophic factor is associated with decreased brain-derived neurotrophic factor secretion and poor outcome after acute neurological injury. We hypothesized that the Met allele is associated with worsening of functional outcome after brain arteriovenous malformation resection. METHODS: Three hundred forty-one surgically treated patients with brain arteriovenous malformation with outcome data were genotyped for Val66Met. Outcome was change in modified Rankin Scale preoperatively versus postoperatively, dichotomized into poor (change >0) or good outcome (change ≤0). Likelihood ratio tests for interactions and logistic regression analysis were performed. RESULTS: A significant interaction (P=0.03) of Val66Met genotype and hemorrhagic presentation existed; thus, ruptured and unruptured patients were considered separately. The Met allele was associated with increased risk of poor outcome among patients presenting unruptured (OR, 2.15; 95% CI, 1.02-4.55; P=0.045) but not ruptured (OR, 0.54; 95% CI, 0.19-1.53; P=0.25), adjusting for covariates. CONCLUSIONS: The Val66Met polymorphism is associated with worsened surgical outcome in patients with unruptured brain arteriovenous malformation, a group that currently has no good risk predictors. Further studies replicating these findings are needed.