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1.  Medical management with or without interventional therapy for unruptured brain arteriovenous malformations (ARUBA): a multicentre, non-blinded, randomised trial 
Lancet  2013;383(9917):614-621.
Summary
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.
doi:10.1016/S0140-6736(13)62302-8
PMCID: PMC4119885  PMID: 24268105
2.  Untreated brain arteriovenous malformation 
Neurology  2014;83(7):590-597.
Objective:
To identify risk factors for intracranial hemorrhage in the natural history course of brain arteriovenous malformations (AVMs) using individual patient data meta-analysis of 4 existing cohorts.
Methods:
We harmonized data from Kaiser Permanente of Northern California (n = 856), University of California San Francisco (n = 787), Columbia University (n = 672), and the Scottish Intracranial Vascular Malformation Study (n = 210). We censored patients at first treatment, death, last visit, or 10-year follow-up, and performed stratified Cox regression analysis of time-to-hemorrhage after evaluating hemorrhagic presentation, sex, age at diagnosis, deep venous drainage, and AVM size as predictors. Multiple imputation was performed to assess impact of missing data.
Results:
A total of 141 hemorrhage events occurred during 6,074 patient-years of follow-up (annual rate of 2.3%, 95% confidence interval [CI] 2.0%–2.7%), higher for ruptured (4.8%, 3.9%–5.9%) than unruptured (1.3%, 1.0%–1.7%) AVMs at presentation. Hemorrhagic presentation (hazard ratio 3.86, 95% CI 2.42–6.14) and increasing age (1.34 per decade, 1.17–1.53) independently predicted hemorrhage and remained significant predictors in the imputed dataset. Female sex (1.49, 95% CI 0.96–2.30) and exclusively deep venous drainage (1.60, 0.95–2.68, p = 0.02 in imputed dataset) may be additional predictors. AVM size was not associated with intracerebral hemorrhage in multivariable models (p > 0.5).
Conclusion:
This large, individual patient data meta-analysis identified hemorrhagic presentation and increasing age as independent predictors of hemorrhage during follow-up. Additional AVM cohort data may further improve precision of estimates, identify new risk factors, and allow validation of prediction models.
doi:10.1212/WNL.0000000000000688
PMCID: PMC4141996  PMID: 25015366
3.  Distinctive distribution of lymphocytes in unruptured and previously untreated brain arteriovenous malformation 
Aim
To test the hypothesis that lymphocyte infiltration in brain arteriovenous malformation (bAVM) is not associated with iron deposition (indicator of microhemorrhage).
Methods
Sections of unruptured, previously untreated bAVM specimens (n=19) were stained immunohistochemically for T-lymphocytes (CD3+), B-lymphocytes (CD20+), plasma cells (CD138+) and macrophages (CD68+). Iron deposition was assessed by hematoxylin and eosin and Prussian blue stains. Superficial temporal arteries (STA) were used as control.
Results
Both T lymphocytes and macrophages were present in unruptured, previously untreated bAVM specimens, whereas few B cells and plasma cells were detected. Iron deposition was detected in 8 specimens (42%; 95% confidence interval =20–67%). The samples with iron deposition tended to have more macrophages than those without (666±313 vs 478±174 cells/mm2; P=0.11). T-cells were clustered on the luminal side of the endothelial surface, on the vessel-wall, and in the perivascular regions. There was no correlation between T lymphocyte load and iron deposition (P=0.88). No macrophages and lymphocytes were detected in STA controls.
Conclusions
T-lymphocytes were present in bAVM specimens. Unlike macrophages, the load and location of T-lymphocytes were not associated with iron deposition, suggesting the possibility of an independent cell-mediated immunological mechanism in bAVM pathogenesis.
doi:10.4103/2347-8659.143674
PMCID: PMC4283567  PMID: 25568888
B-lymphocyte; human brain arteriovenous malformation; inflammatory cells; microhemorrhage; T-lymphocyte
4.  Induction of Brain Arteriovenous Malformation in the Adult Mouse 
Summary
Brain arteriovenous malformations (bAVM) are tangles of abnormal, dilated vessels that directly shunt blood between the arteries and veins. The pathogenesis of bAVM is currently unknown. Patients with hereditary hemorrhagic telangiectasia (HHT) have a higher prevalence of bAVM than the general population. Animal models are important tools for dissecting the disease-etiopathogenesis and for testing new therapies. Here, we introduce a method that induces the bAVM phenotype through regional deletion of activin-like kinase 1 (Alk1, the causal gene for HHT2) and vascular endothelial growth factor (VEGF) stimulation.
doi:10.1007/978-1-4939-0320-7_25
PMCID: PMC3988785  PMID: 24510874
Arteriovenous malformation; Activin-like kinase 1; Angiogenesis; VEGF; Hereditary hemorrhagic telangiectasia; Mouse model
5.  Endovascular Biopsy: Evaluating the Feasibility of Harvesting Endothelial Cells Using Detachable Coils 
Interventional Neuroradiology  null;19(4):399-408.
Summary
The absence of safe and reliable methods to harvest vascular tissue in situ limits the discovery of the underlying genetic and pathophysiological mechanisms of many vascular disorders such as aneurysms. We investigated the feasibility and comparable efficacy of endothelial cell collection using a spectrum of endovascular coils.
Nine detachable coils ranging in k coefficient (0.15-0.24), diameter (4.0 mm-16.0 mm), and length (8.0 cm-47.0 cm) were tested in pigs. All coils were deployed and retrieved within the iliac artery of pigs (three coils/pig). Collected coils were evaluated under light microscopy. The total and endothelial cells collected by each coil were quantified. The nucleated cells were identified by Wright-Giemsa and DAPI stains. Endothelial and smooth muscle cells were identified by CD31 and α-smooth muscle actin antibody staining.
Coils were deployed and retrieved without technical difficulty. Light microscopy demonstrated sheets of cellular material concentrated within the coil winds. All coils collected cellular material while five of nine (55.6%) coils retrieved endothelial cells. Coils collected mean endothelial cell counts of 89.0±101.6. Regression analysis demonstrated a positive correlation between increasing coil diameter and endothelial cell counts (R2=0.52, p = 0.029).
Conventional detachable coils can be used to harvest endothelial cells. The number of endothelial cells collected by a coil positively correlated with its diameter. Given the widespread use of coils and their well-described safety profile their potential as an endovascular biopsy device would expand the availability of tissue for cellular and molecular analysis.
PMCID: PMC3902737  PMID: 24355142
endothelial; endovascular; aneurysm; brain; stroke; biopsy
6.  Temporal lobe arteriovenous malformations: anatomical subtypes, surgical strategy, and outcomes 
Journal of neurosurgery  2013;119(3):616-628.
Object
Descriptions of temporal lobe arteriovenous malformations (AVMs) are inconsistent. To standardize reporting, the authors blended existing descriptions in the literature into an intuitive classification with 5 anatomical subtypes: lateral, medial, basal, sylvian, and ventricular. The authors’ surgical experience with temporal lobe AVMs was reviewed according to these subtypes.
Methods
Eighty-eight patients with temporal lobe AVMs were treated surgically.
Results
Lateral temporal lobe AVMs were the most common (58 AVMs, 66%). Thirteen AVMs (15%) were medial, 9 (10%) were basal, and 5 (6%) were sylvian. Ventricular AVMs were least common (3 AVMs, 3%). A temporal craniotomy based over the ear was used in 64%. Complete AVM resection was achieved in 82 patients (93%). Four patients (5%) died in the perioperative period (6 in all were lost to follow-up); 71 (87%) of the remaining 82 patients had good outcomes (modified Rankin Scale scores 0–2); and 68 (83%) were unchanged or improved after surgery.
Conclusions
Categorization of temporal AVMs into subtypes can assist with surgical planning and also standardize reporting. Lateral AVMs are the easiest to expose surgically, with circumferential access to feeding arteries and draining veins at the AVM margins. Basal AVMs require a subtemporal approach, often with some transcortical dissection through the inferior temporal gyrus. Medial AVMs are exposed tangentially with an orbitozygomatic craniotomy and transsylvian dissection of anterior choroidal artery and posterior cerebral artery feeders in the medial cisterns. Medial AVMs posterior to the cerebral peduncle require transcortical approaches through the temporooccipi tal gyrus. Sylvian AVMs require a wide sylvian fissure split and differentiation of normal arteries, terminal feeding arteries, and transit arteries. Ventricular AVMs require a transcortical approach through the inferior temporal gyrus that avoids the Meyer loop. Surgical results with temporal lobe AVMs are generally good, and classifying them does not offer any prediction of surgical risk.
doi:10.3171/2013.6.JNS122333
PMCID: PMC4159961  PMID: 23848823
arteriovenous malformation; temporal lobe; anatomical subtype; microsurgical resection; vascular disorders
7.  Brain Vascular Malformation Consortium: Overview, Progress and Future Directions 
Brain vascular malformations are resource-intensive to manage effectively, are associated with serious neurological morbidity, lack specific medical therapies, and have no validated biomarkers for disease severity and progression. Investigators have tended to work in “research silos” with suboptimal cross-communication. We present here a paradigm for interdisciplinary collaboration to facilitate rare disease research. The Brain Vascular Malformation Consortium (BVMC) is a multidisciplinary, inter-institutional group of investigators, one of 17 consortia in the Office of Rare Disease Research Rare Disease Clinical Research Network (RDCRN). The diseases under study are: familial Cerebral Cavernous Malformations type 1, common Hispanic mutation (CCM1-CHM); Sturge-Weber Syndrome (SWS); and brain arteriovenous malformation in hereditary hemorrhagic telangiectasia (HHT). Each project is developing biomarkers for disease progression and severity, and has established scalable, relational databases for observational and longitudinal studies that are stored centrally by the RDCRN Data Management and Coordinating Center. Patient Support Organizations (PSOs) are a key RDCRN component in the recruitment and support of participants. The BVMC PSOs include Angioma Alliance, Sturge Weber Foundation, and HHT Foundation International. Our networks of clinical centers of excellence in SWS and HHT, as well as our PSOs, have enhanced BVMC patient recruitment. The BVMC provides unique and valuable resources to the clinical neurovascular community, and recently reported findings are reviewed. Future planned studies will apply successful approaches and insights across the three projects to leverage the combined resources of the BVMC and RDCRN in advancing new biomarkers and treatment strategies for patients with vascular malformations.
PMCID: PMC4160161  PMID: 25221778
8.  Deep Arteriovenous Malformations in the Basal Ganglia, Thalamus, and Insula: Microsurgical Management, Techniques, and Results 
Neurosurgery  2013;73(3):417-429.
Background
Arteriovenous malformations (AVMs) in the basal ganglia, thalamus, and insula are considered inoperable given their depth, eloquence, and limited surgical exposure. While many neurosurgeons opt for radiosurgery or observation, others have challenged the belief that deep AVMs are inoperable. Further discussion of patient selection, technique, and multimodality management is needed.
Objective
To describe and discuss the technical considerations of microsurgical resection for deep-seated AVMs.
Methods
Patients with deep AVMs who underwent surgery during a 14-year period were reviewed using a prospective AVM registry.
Results
Microsurgery was performed in 48 patients with AVMs in the basal ganglia (n=10), thalamus (n=13), or insula (n=25). The most common Spetzler-Martin grade was III- (68%). Surgical approaches included transsylvian (67%), transcallosal (19%), and transcortical (15%). Complete resection was achieved in 34 patients (71%), and patients with incomplete resection were treated with radiosurgery. Forty-five patients (94%) were improved or unchanged (mean follow-up 1.6 years).
Conclusion
This experience advances the notion that select deep AVMs may be operable lesions. Patients were highly selected for small size, hemorrhagic presentation, young age, and compactness – factors embodied in the Spetzler-Martin and Supplementary grading systems. Overall, 10 different approaches were used, exploiting direct, transcortical corridors created by hemorrhage or maximizing anatomical corridors through subarachnoid spaces and ventricles that minimize brain transgression. The same cautious attitude exercised in selecting patients for surgery was also exercised in deciding extent of resection, opting for incomplete resection and radiosurgery more than with other AVMs to prioritize neurological outcomes.
doi:10.1227/NEU.0000000000000004
PMCID: PMC4123817  PMID: 23728451
Arteriovenous malformation; Basal ganglia; Insula; Microsurgery; Thalamus
9.  Activation of α-7 Nicotinic Acetylcholine Receptor Reduces Ischemic Stroke Injury through Reduction of Pro-Inflammatory Macrophages and Oxidative Stress 
PLoS ONE  2014;9(8):e105711.
Activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) has a neuro-protective effect on ischemic and hemorrhagic stroke. However, the underlying mechanism is not completely understood. We hypothesized that α-7 nAchR agonist protects brain injury after ischemic stroke through reduction of pro-inflammatory macrophages (M1) and oxidative stress. C57BL/6 mice were treated with PHA568487 (PHA, α-7 nAchR agonist), methyllycaconitine (MLA, nAchR antagonist), or saline immediately and 24 hours after permanent occlusion of the distal middle cerebral artery (pMCAO). Behavior test, lesion volume, CD68+, M1 (CD11b+/Iba1+) and M2 (CD206/Iba1+) microglia/macrophages, and phosphorylated p65 component of NF-kB in microglia/macrophages were quantified using histological stained sections. The expression of M1 and M2 marker genes, anti-oxidant genes and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were quantified using real-time RT-PCR. Compared to the saline-treated mice, PHA mice had fewer behavior deficits 3 and 7 days after pMCAO, and smaller lesion volume, fewer CD68+ and M1 macrophages, and more M2 macrophages 3 and 14 days after pMCAO, whereas MLA's effects were mostly the opposite in several analyses. PHA increased anti-oxidant genes and NADPH oxidase expression associated with decreased phosphorylation of NF-kB p65 in microglia/macrophages. Thus, reduction of inflammatory response and oxidative stress play roles in α-7 nAchR neuro-protective effect.
doi:10.1371/journal.pone.0105711
PMCID: PMC4144901  PMID: 25157794
10.  Perioperative Management of Coagulation in Nontraumatic Intracerebral Hemorrhage 
Anesthesiology  2013;119(1):10.1097/ALN.0b013e318297c18a.
doi:10.1097/ALN.0b013e318297c18a
PMCID: PMC3815673  PMID: 23695175
11.  Bone Fracture Exacerbates Murine Ischemic Cerebral Injury 
Anesthesiology  2013;118(6):1362-1372.
Background
Bone fracture increases alarmins and pro-inflammatory cytokines in the blood, and provokes macrophage infiltration and pro-inflammatory cytokine expression in the hippocampus. We recently reported that stroke is an independent risk factor after bone surgery for adverse outcome, the impact of bone fracture on stroke outcome is 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 three days after.
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 peri-infarct 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.
doi:10.1097/ALN.0b013e31828c23f8
PMCID: PMC3987811  PMID: 23438676
12.  Ferumoxytol-enhanced MRI to Image Inflammation within Human Brain Arteriovenous Malformations: A Pilot Investigation 
Translational stroke research  2012;3(Supplement 1):166-173.
Inflammation cell infiltration and cytokine expression are seen in the vascular walls and intervening stroma of resected brain arteriovenous malformation (bAVM) specimens, even in unruptured and previously untreated lesions. Macrophages may play a critical role in bAVM progression to rupture, and could serve as a marker for rupture risk. We assessed feasibility of imaging macrophages within the bAVM nidus using ferumoxytol-enhanced MRI in four patients with already diagnosed bAVMs using iron-sensitive imaging (ISI; T2*-GE-MRI sequence). Patients were imaged at baseline and at either 1 day (n=2) or 5 days (n=2) after infusion of 5mg/kg of ferumoxytol. Residual intravascular ferumoxytol obscured evaluation for uptake in bAVM vascular walls and stroma at the 1-day time point. The two cases imaged at 5 days showed less intravascular tracer but had signal loss in the nidal region consistent with ferumoxytol localization. One case underwent surgical resection; there was prominent vascular wall CD68 staining. Ferumoxytol-enhanced-MRI for assessing bAVM inflammatory cell burden appears feasible and has the potential to be developed as a biomarker to study lesional inflammatory events.
doi:10.1007/s12975-012-0172-y
PMCID: PMC3445332  PMID: 23002401
Arteriovenous malformations; Inflammation; Magnetic resonance imaging; Ferumoxytol; USPIO
13.  Silent Intralesional Microhemorrhage as a Risk Factor for Brain AVM Rupture 
Background and Purpose
We investigated whether brain arteriovenous malformation (bAVM) silent intralesional microhemorrhage (SIM), i.e., asymptomatic bleeding in the nidal compartment, might serve as a marker for increased risk of symptomatic intracranial hemorrhage (ICH). We evaluated two markers to assess the occurrence of SIM: neuroradiological assessment of evidence of old hemorrhage (EOOH)— imaging evidence of bleeding before the outcome events, and hemosiderin positivity in H&E-stained paraffin block sections.
Methods
We included cases from a bAVM database with recorded neuroradiological data or available surgical paraffin blocks. Using two endpoints, index ICH and new ICH after diagnosis (censored at treatment, loss to follow-up, or death), we performed logistic or Cox regression to assess EOOH and hemosiderin positivity, adjusting for age, sex, deep-only venous drainage, maximal bAVM size, deep location, and associated arterial aneurysms.
Results
EOOH was present in 6.5% (n=975) of patients and highly predictive of index ICH (p<0.001; OR=3.97; 95% CI, 2.1-7.5), adjusting for other risk factors. In a multivariable model (n=643), EOOH was an independent predictor of new ICH (HR=3.53; 95% CI=1.35-9.23; p=.010). Hemosiderin positivity was found in 36.2% (29.6% in unruptured; 47.8% in ruptured; p=.04), and associated with index ICH in univariate (OR=2.18; 95%CI 1.03-4.61; p=.042; n=127) and multivariable models (OR=3.64; 95% CI=1.11-12.00; p=.034; n=79).
Conclusions
The prevalence of SIM is high and there is evidence for an association with both index and subsequent ICH. Further development of means to detect SIM during bAVM evaluation may present an opportunity to improve risk-stratification, especially for unruptured bAVMs.
doi:10.1161/STROKEAHA.111.647263
PMCID: PMC3335931  PMID: 22308253
brain arteriovenous malformation; hemorrhage; risk factor; survival; brain microbleed; hemosiderin
14.  Endoglin Deficiency in Bone Marrow Is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse after VEGF 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 (AAV-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
ENG-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.
doi:10.1161/STROKEAHA.112.671974
PMCID: PMC3582755  PMID: 23306322
arteriovenous malformation; adult mouse; brain angiogenesis
15.  Depletion of Bone Marrow-derived Macrophages Perturbs the Innate Immune Response to Surgery and Reduces Postoperative Memory Dysfunction 
Anesthesiology  2013;118(3):527-536.
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. We hypothesize that hippocampal recruitment of bone marrow-derived (BMD) macrophages plays a causal role in these processes, resulting in memory dysfunction.
Methods
Clodrolip injection (liposomal formulation of clodronate) prior to stabilized tibial fracture under general anesthesia was used to deplete BMD macrophages. Systemic and neuroinflammation were studied on postoperative day 1, and memory in a fear-trace conditioning paradigm was assessed on postoperative day 3. CX3CR1GFP/+ CCR2RFP/+ mice were used to identify BMD macrophages.
Results
Clodrolip effectively depleted splenic CCR2+ BMD 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 MCP-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 to 38% vs. 48%, 95% CI: 38 to 58%, n= 20, P = 0.004), but did not affect memory in nonsurgical mice.
Conclusion
Depletion of BMD macrophages prevents hippocampal neuroinflammation and memory dysfunction after experimental tibial fracture. These data suggest that the hippocampal recruitment of BMD 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.
doi:10.1097/ALN.0b013e3182834d94
PMCID: PMC3779063  PMID: 23426204
16.  Cerebellar Arteriovenous Malformations: Anatomical Subtypes, Surgical Results, and Increased Predictive Accuracy of the Supplementary Grading System 
Neurosurgery  2012;71(6):1111-1124.
Background
Anatomical diversity amongst cerebellar AVMs calls for a classification that is intuitive and surgically informative. Selection tools like the Spetzler-Martin grading system are designed to work best with cerebral AVMs, but have shortcomings with cerebellar AVMs.
Objective
To define subtypes of cerebellar AVMs that clarify anatomy and surgical management, determine results according to subtypes, and compare predictive accuracies of Spetzler-Martin and supplementary systems.
Methods
From a consecutive surgical series of 500 patients, 60 had cerebellar AVMs, 39 had brain stem AVMs and were excluded, and 401 had cerebral AVMs.
Results
Cerebellar AVM subtypes were: 18 vermian, 13 suboccipital, 12 tentorial, 12 petrosal, and 5 tonsillar. Patients with tonsillar and tentorial AVMs fared best. Cerebellar AVMs presented with hemorrhage more than cerebral AVMs (p<0.001). Cerebellar AVMs were more likely to drain deep (p=0.036) and less likely eloquent (p<0.001). The predictive accuracy of supplementary grade was better than that of Spetzler-Martin grade with cerebellar AVMs (areas under the ROC curve 0.74 and 0.59, respectively). The predictive accuracy of the supplementary system was consistent for cerebral and cerebellar AVMs, whereas that of the Spetzler-Martin system was greater with cerebral AVMs.
Conclusion
Patients with cerebellar AVMs present with hemorrhage more than patients with cerebral AVMs, justifying an aggressive treatment posture. The supplementary system is better than the Spetzler-Martin system at predicting outcomes after cerebellar AVM resection. Key components of the Spetzler-Martin system, like venous drainage and eloquence, are distorted by cerebellar anatomy in ways that components of the supplementary system are not.
doi:10.1227/NEU.0b013e318271c081
PMCID: PMC3923506  PMID: 22986595
arteriovenous malformation; cerebellum; microsurgical resection; Spetzler Martin grading scale; supplementary grading scale
17.  Validation of In Vivo MRI Blood-Brain Barrier Permeability Measurements by Comparison with Gold Standard Histology 
Background and Purpose
To validate the blood-brain barrier permeability measurements extracted from perfusion-weighted MRI through a relatively simple and frequently applied model, the Patlak model, by comparison with gold standard histology in a rat model of ischemic stroke.
Methods
Eleven spontaneously hypertensive rats and eleven Wistar rats with unilateral, 2h filament occlusion of the right MCA underwent imaging during occlusion, at 4h and 24h post reperfusion. BBB permeability was imaged by gradient echo imaging after the first pass of the contrast agent bolus and quantified by a Patlak analysis. BBB permeability was shown on histology by the extravasation of Evans blue on fluorescence microscopy sections matching location and orientation of MR images. Cresyl-violet staining was used to detect and characterize hemorrhage. Landmark-based elastic image registration allowed a region-by-region comparison of permeability imaging at 24 hours with Evans blue extravasation and hemorrhage as detected on histological slides obtained immediately after the 24-hour image set.
Results
Permeability values in the non-ischemic tissue (marginal mean±SE: 0.15±0.019ml/min·100ml) were significantly lower compared to all permeability values in regions of Evans blue extravasation or hemorrhage. Permeability values in regions of weak Evans blue extravasation (0.23±0.016ml/min·100ml) were significantly lower compared to permeability values of in regions of strong Evans blue extravasation (0.29±0.020ml/min·100ml) and macroscopic hemorrhage (0.35±0.049ml/min·100ml). Permeability values in regions of microscopic hemorrhage (0.26±0.024ml/min·100ml) only differed significantly from values in regions of non-ischemic tissue (0.15±0.019 ml/min·100ml).
Conclusion
Areas of increased permeability measured in-vivo by imaging coincide with BBB disruption and hemorrhage observed on gold standard histology.
doi:10.1161/STROKEAHA.110.597997
PMCID: PMC3921072  PMID: 21636816
ischemic stroke; hemorrhagic transformation; blood-brain barrier permeability; MRI; validation study
18.  Novel Brain Arteriovenous Malformation Mouse Models for Type 1 Hereditary Hemorrhagic Telangiectasia 
PLoS ONE  2014;9(2):e88511.
Endoglin (ENG) is a causative gene of type 1 hereditary hemorrhagic telangiectasia (HHT1). HHT1 patients have a higher prevalence of brain arteriovenous malformation (AVM) than the general population and patients with other HHT subtypes. The pathogenesis of brain AVM in HHT1 patients is currently unknown and no specific medical therapy is available to treat patients. Proper animal models are crucial for identifying the underlying mechanisms for brain AVM development and for testing new therapies. However, creating HHT1 brain AVM models has been quite challenging because of difficulties related to deleting Eng-floxed sequence in Eng2fl/2fl mice. To create an HHT1 brain AVM mouse model, we used several Cre transgenic mouse lines to delete Eng in different cell-types in Eng2fl/2fl mice: R26CreER (all cell types after tamoxifen treatment), SM22α-Cre (smooth muscle and endothelial cell) and LysM-Cre (lysozyme M-positive macrophage). An adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) was injected into the brain to induce focal angiogenesis. We found that SM22α-Cre-mediated Eng deletion in the embryo caused AVMs in the postnatal brain, spinal cord, and intestines. Induction of Eng deletion in adult mice using R26CreER plus local VEGF stimulation induced the brain AVM phenotype. In both models, Eng-null endothelial cells were detected in the brain AVM lesions, and formed mosaicism with wildtype endothelial cells. However, LysM-Cre-mediated Eng deletion in the embryo did not cause AVM in the postnatal brain even after VEGF stimulation. In this study, we report two novel HHT1 brain AVM models that mimic many phenotypes of human brain AVM and can thus be used for studying brain AVM pathogenesis and testing new therapies. Further, our data indicate that macrophage Eng deletion is insufficient and that endothelial Eng homozygous deletion is required for HHT1 brain AVM development.
doi:10.1371/journal.pone.0088511
PMCID: PMC3919779  PMID: 24520391
19.  Gene expression profiling of blood in brain arteriovenous malformation patients 
Translational stroke research  2011;2(4):575-587.
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. Gene expression profiling of blood has led to the identification of stroke biomarkers, and may help identify BAVM biomarkers and illuminate BAVM pathogenesis. It is unknown whether blood gene expression profiles differ between 1) BAVM patients and healthy controls, or 2) unruptured and ruptured BAVM patients at presentation. We characterized blood transcriptional profiles in 60 subjects (20 unruptured BAVM, 20 ruptured BAVM, and 20 healthy controls) using Affymetrix whole genome expression arrays. Expression differences between groups were tested by ANOVA, adjusting for potential confounders. Genes with absolute fold change ≥ 1.2 (false discovery rate corrected p ≤ 0.1) were selected as differentially expressed and evaluated for over-representation in KEGG biological pathways (p ≤ 0.05). Twenty-nine genes were differentially expressed between unruptured BAVM patients and controls, including 13 which may be predictive of BAVM. Patients with ruptured BAVM compared to unruptured BAVM differed in expression of 1490 genes, with over-representation of genes in 8 pathways including MAPK, VEGF, Wnt signaling and several inflammatory pathways. These results suggest clues to the pathogenesis of BAVM and/or BAVM rupture and point to potential biomarkers or new treatment targets.
doi:10.1007/s12975-011-0103-3
PMCID: PMC3241209  PMID: 22184505
arteriovenous malformation; blood; gene expression; intracranial hemorrhage; microarray analysis
20.  Reduced mural cell coverage and impaired vessel integrity after angiogenic stimulation in the Alk1-deficient brain 
Objective
Vessels in brain arteriovenous malformations (bAVM) are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 (HHT2) patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of bAVM than the general population. We tested the hypothesis that vascular endothelial growth factor (VEGF) impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell-coverage.
Methods and Results
Adult Alk11f/2f mice (loxP sites flanking exons 4-6) and wild-type (WT) mice were injected with 2×107 PFU Ad-Cre and 2×109 genome copies of AAV-VEGF to induce focal homozygous Alk1 deletion (in Alk11f/2f mice) and angiogenesis. Brain vessels were analyzed eight weeks later. Compared to WT mice, the Alk1-deficient brain had more fibrin (99±30×103 pixels/mm2 vs. 40±13×103, P=0.001), iron deposition (508±506 pixels/mm2 vs. 6 ±49, P=0.04), and Iba1+ microglia/macrophage infiltration (888±420 Iba1+ cells/mm2 vs. 240±104 Iba1+, P=0.001) after VEGF stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-SMA- vessels (52±9% vs. 12±7%, P<0.001), fewer vascular associated pericytes (503±179/mm2 vs. 931±115, P<0.001), and reduced PDGFR-β expression (26±9%, P<0.001).
Conclusion
Reduction of mural cell coverage in response to VEGF stimulation is a potential mechanism for the impairment of vessel wall integrity in HHT2-associated bAVM.
doi:10.1161/ATVBAHA.112.300485
PMCID: PMC3569037  PMID: 23241407
brain arteriovenous malformation; activin receptor-like kinase 1; pericyte; iron deposition; PDGFR-β
21.  Intravenous delivery of AAV9 vector 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 vector (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, by injecting an AAV vector expressing vascular endothelial growth factor (AAV-VEGF) into the basal ganglia. ssAAV vectors that have 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 one hour after MCAO or four weeks after the induction of angiogenesis. LacZ gene expression was analyzed in the brain and other organs five days post 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 AAV9-H9LacZ-injected mice.
Conclusion
ssAAV9 vector could be a useful tool to deliver therapeutic genes to the ischemic stroke lesion or brain angiogenic foci.
doi:10.1161/STROKEAHA.112.662965
PMCID: PMC3531817  PMID: 23250995
peri-infarct region; AAV serotype 9; mouse; brain; angiogenesis; intravenous delivery
22.  G Protein-Coupled Receptor 124 (GPR124) Gene Polymorphisms and Risk of Brain Arteriovenous Malformation 
Translational stroke research  2012;3(4):418-427.
Abnormal endothelial proliferation and angiogenesis may contribute to brain arteriovenous malformation (BAVM) formation. G protein-coupled receptor 124 (GPR124) mediates embryonic CNS angiogenesis; thus we investigated the association of single nucleotide polymorphisms (SNPs) and haplotypes in GPR124 with risk of BAVM. Ten tagging SNPs spanning 39 kb of GPR124 were genotyped in 195 Caucasian BAVM patients and 243 Caucasian controls. SNP and haplotype association with risk of BAVM was screened using χ2 analysis. Associated variants were further evaluated using multivariable logistic regression, adjusting for age and sex. The minor alleles of 3 GPR124 SNPs adjacent to exon 2 and localized to a 16 kb region of high linkage disequilibrium were associated with reduced risk of BAVM (rs7015566 A, P=0.001; rs7823249 T, P=0.014; rs12676965 C, P=0.007). SNP rs7015566 (intron 1) remained associated after permutation testing (additive model P=0.033). Haplotype analysis revealed a significant overall association (χ2=12.55, 4 df, P=0.014); 2 haplotypes (ATCC, P=0.006 and GGCT, P=0.008) were associated with risk of BAVM. We genotyped a known synonymous SNP (rs16887051) in exon 2, however genotype frequency did not differ between cases and controls. Sequencing of conserved GPR124 regions revealed a novel indel polymorphism in intron 2. Immunohistochemistry confirmed GPR124 expression in the endothelium with no qualitative difference in expression between BAVM cases and controls. SNP rs7015566 mapping to intron 1 of GPR124 was associated with BAVM susceptibility among Caucasians. Future work is focused on investigating this gene region.
doi:10.1007/s12975-012-0202-9
PMCID: PMC3544200  PMID: 23329986
Angiogenesis; Genetics; Intracerebral hemorrhage; Risk factor; Vascular malformation
23.  Early Change in Ferumoxytol-Enhanced Magnetic Resonance Imaging Signal Suggests Unstable Human Cerebral Aneurysm. A Pilot Study 
Background and Purpose
The clinical significance of early (i.e. within the first 24 hours) uptake of ferumoxytol by macrophages in the wall of human cerebral aneurysms is not clear. The purpose of this study was to determine whether early uptake of ferumoxytol suggests unstable cerebral aneurysm.
Methods
Thirty unruptured aneurysms in 22 patients were imaged with MRI 24 hours after infusion of ferumoxytol. Eighteen aneurysms were also imaged 72 hours after infusion of ferumoxytol. Aneurysm dome tissue was collected from four patients with early MRI signal changes, five patients with late signal changes, and five other patients with ruptured aneurysms. The tissue was immunostained for expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), microsomal-prostaglandin-E2 synthase-1 (mPGES-1) and macrophages.
Results
In 23% (7/30) of aneurysms, there was pronounced early uptake of ferumoxytol. Four aneurysms were clipped. The remaining three aneurysms were managed conservatively; all three ruptured within six months. In 53% (16/30) of aneurysms, there was pronounced uptake of ferumoxytol at 72 hours. Eight aneurysms were surgically clipped and eight were managed conservatively; none ruptured or increased in size after six months. Expression of COX-2, mPGES-1, and macrophages was similar in unruptured aneurysms with early uptake of ferumoxytol and ruptured aneurysms. Expression of these inflammatory molecules was significantly higher in aneurysms with early uptake of ferumoxytol versus aneurysms with late uptake.
Conclusions
Uptake of ferumoxytol in aneurysm walls within the first 24 hours strongly suggests aneurysm instability and probability of rupture within six months, and may warrant urgent intervention.
doi:10.1161/STROKEAHA.112.673400
PMCID: PMC3508354  PMID: 23138441
Aneurysm; MRI; Ferumoxytol; Rupture. Macrophages
24.  Brain Arteriovenous Malformations associated with Hereditary Hemorrhagic Telangiectasia: Gene-Phenotype Correlations 
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disease with a wide spectrum of vascular malformations involving multiple organs. Nine-16% of patients with HHT harbor brain arteriovenous malformations (AVMs), which can cause intracranial hemorrhage. Our objective was to study clinical manifestations of brain AVM in patients with HHT and correlate these with the specific gene mutated. We reviewed records of 171 patients with HHT and brain AVMs. A history of intracranial hemorrhage was found in 27% (41/152) patients, with a mean (range) age of 26 +/− 18 (0–68) years. All patients with intracranial hemorrhage were neurologically asymptomatic prior to intracranial hemorrhage. Multiple brain AVMs were found in 23% (170/39) of patients on initial examination. Genetic test results were available in 109 (64%) patients. Mutations in ENG, ACVRL1, and SMAD4 were present in 75 (69%), 18 (17%) and 2 (2%), respectively. A history of intracranial hemorrhage was reported in 24% of patients with an ENG mutation and 27% of patients with an ACVRL1 mutation, with a mean (range) age of 26 +/− 16 (2–50) and 18 +/− 21 (0–48) years, respectively. No statistically significant differences in age at first brain AVM diagnosis, prevalence of intracranial hemorrhage history, age at intracranial hemorrhage, or other manifestations of brain AVMs were observed among gene groups. In conclusion, no evidence for differences in brain AVM characteristics was observed among HHT gene groups, although we cannot exclude clinically important differences. Larger studies are needed to further guide brain AVM screening decisions in patients with HHT.
doi:10.1002/ajmg.a.35622
PMCID: PMC3610331  PMID: 22991266
Hereditary Hemorrhagic Telangiectasia; Brain Arteriovenous Malformation; Genotype; Intracranial Hemorrhage
25.  Angiopoietin-Like 4 (ANGPTL4) Gene Polymorphisms and Risk of Brain Arteriovenous Malformations 
Background
Brain arteriovenous malformations (BAVM) are high-flow vascular lesions prone to intracranial hemorrhage (ICH). Abnormal angiogenesis is a key characteristic of BAVM tissue. Angiopoietin-like 4 (ANGPTL4), a secreted glycoprotein, is thought to be involved in angiogenesis and required for proper postnatal blood vessel partitioning. We investigated whether common single nucleotide polymorphisms (SNPs) in ANGPTL4 were associated with risk of BAVM or ICH.
Methods and Results
We conducted a case-control study of 216 Caucasian BAVM cases and 246 healthy controls, and a secondary case-only analysis, comparing 83 ruptured (ICH) with 133 unruptured BAVM cases at presentation. Four tagSNPs in ANGPTL4 captured variation over a 10-kb region (rs2278236, rs1044250, rs11672433, and rs1808536) and were tested for association with BAVM or ICH. The minor allele (A) of rs11672433 (exon 6, Pro389Pro) was associated with an increased risk of BAVM (p = 0.006), which persisted after adjusting for multiple comparisons (p = 0.03). After adjustments for age and sex, carriers of the minor allele (A) remained at higher risk for BAVM compared to noncarriers (odds ratio, OR = 1.56; 95% confidence interval, CI = 1.01–2.41; p = 0.046) and risk of BAVM was increased with increasing copy of the minor A allele (OR = 1.49, 95% CI = 1.03–2.15; ptrend = 0.03). Five common haplotypes (frequency >1%) were inferred; overall haplotype distribution differed between BAVM cases and controls (χ2 = 12.2, d.f. = 4, p = 0.02). Neither SNPs (p > 0.05) nor haplotype distribution (χ2 = 1.1, d.f. = 4, p = 0.89) were associated with risk of ICH among BAVM cases.
Conclusion
A synonymous SNP in ANGPTL4 and haplotypes carrying it are associated with risk of BAVM but not with ICH presentation in BAVM cases.
doi:10.1159/000322601
PMCID: PMC3030504  PMID: 21212665
Arteriovenous malformations; Cerebrovascular disorders; Epidemiology; Genetics; Intracranial hemorrhage

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