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Hemodynamics plays a key role in the natural history of intracranial aneurysms (IAs). However, studies exploring the association between aneurysmal hemodynamics and the biological and mechanical characteristics of the IA wall in humans are sparse. In this review, we survey the current body of literature, summarize the studies' methodologies and findings, and assess the degree of consensus among them. We used PubMed to perform a systematic review of studies that explored the association between hemodynamics and human IA wall features using different sources. We identified 28 publications characterizing aneurysmal flow and the IA wall: 4 using resected tissues, 17 using intraoperative images, and 7 using vessel wall magnetic resonance imaging (MRI). Based on correlation to IA tissue, higher flow conditions, such as high wall shear stress (WSS) with complex pattern and elevated pressure, were associated with degenerated walls and collagens with unphysiological orientation and faster synthesis. MRI studies strongly supported that low flow, characterized by low WSS and high blood residence time, was associated with thicker walls and post-contrast enhancement. While significant discrepancies were found among those utilized intraoperative images, they generally supported that thicker walls coexist at regions with prolonged residence time and that thinner regions are mainly exposed to higher pressure with complex WSS patterns. The current body of literature supports a theory of two general hemodynamic-biologic mechanisms for IA development. One, where low flow conditions are associated with thickening and atherosclerotic-like remodeling, and the other where high and impinging flow conditions are related to wall degeneration, thinning, and collagen remodeling., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Intracranial aneurysm (IA) rupture leads to deadly subarachnoid hemorrhages. However, the mechanisms leading to rupture remain poorly understood. Altered gene expression within IA tissue is linked to the pathobiology of aneurysm development and progression. Here, we analyzed expression patterns of control tissue samples and compared them to those of unruptured and ruptured IA tissue samples using data from the Gene Expression Omnibus (GEO)., Methods: FASTQ files for 21 ruptured IAs, 21 unruptured IAs, and 16 control tissue samples were accessed from the GEO database. DESeq2 was used for differential expression analysis in three comparisons: unruptured IA versus control, ruptured IA versus control, and ruptured versus unruptured IA. Genes that were differentially expressed in multiple comparisons were evaluated to find those progressively increasing/decreasing from control to unruptured to ruptured. Significance was tested by either analysis of variance/Gabriel or Brown-Forsythe/Games Howell (p < 0.05 was considered significant). We used additional RNA sequencing and proteomics datasets to evaluate if our differentially expressed genes (DEGs) were present in other studies. Bioinformatics analyses were performed with g:Profiler and Ingenuity Pathway Analysis., Results: In total, we identified 1768 DEGs, of which 318 were found in multiple comparisons. Unruptured versus control reflected vascular remodeling processes, while ruptured versus control reflected inflammatory responses and cell activation/signaling. When comparing ruptured to unruptured IAs, we found massive activation of inflammation, inflammatory responses, and leukocyte responses. Of the 318 genes in multiple comparisons, 127 were found to be significant in the multi-cohort correlation analysis. Those that progressively increased (70 genes) were associated with immune system processes, while those that progressively decreased (38 genes) did not return any gene ontology terms. Many of our DEGs were also found in the other IA tissue sequencing studies., Conclusions: We found unruptured IAs relate more to remodeling processes, while ruptured IAs reflect more inflammatory and immune responses., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The pathogenesis and natural history of intracranial aneurysm (IA) remains poorly understood. To this end, animal models with induced cerebral vessel lesions mimicking human aneurysms have provided the ability to greatly expand our understanding. In this review, we comprehensively searched the published literature to identify studies that endogenously induced IA formation in animals. Studies that constructed aneurysms (i.e., by surgically creating a sac) were excluded. From the eligible studies, we reported information including the animal species, method for aneurysm induction, aneurysm definitions, evaluation methods, aneurysm characteristics, formation rate, rupture rate, and time course. Between 1960 and 2019, 174 articles reported endogenous animal models of IA. The majority used flow modification, hypertension, and vessel wall weakening (i.e., elastase treatment) to induce IAs, primarily in rats and mice. Most studies utilized subjective or qualitative descriptions to define experimental aneurysms and histology to study them. In general, experimental IAs resembled the pathobiology of the human disease in terms of internal elastic lamina loss, medial layer degradation, and inflammatory cell infiltration. After the early 2000s, many endogenous animal models of IA began to incorporate state-of-the-art technology, such as gene expression profiling and 9.4-T magnetic resonance imaging (MRI) in vivo imaging, to quantitatively analyze the biological mechanisms of IA. Future studies aimed at longitudinally assessing IA pathobiology in models that incorporate aneurysm growth will likely have the largest impact on our understanding of the disease. We believe this will be aided by high-resolution, small animal, survival imaging, in situ live-cell imaging, and next-generation omics technology., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Background: The rupture of an intracranial aneurysm (IA) causes devastating subarachnoid hemorrhages, yet most IAs remain undiscovered until they rupture. Recently, we found an IA RNA expression signature of circulating neutrophils, and used transcriptome data to build predictive models for unruptured IAs. In this study, we evaluate the feasibility of using whole blood transcriptomes to predict the presence of unruptured IAs., Methods: We subjected RNA from peripheral whole blood of 67 patients (34 with unruptured IA, 33 without IA) to next-generation RNA sequencing. Model genes were identified using the least absolute shrinkage and selection operator (LASSO) in a random training cohort (n = 47). These genes were used to train a Gaussian Support Vector Machine (gSVM) model to distinguish patients with IA. The model was applied to an independent testing cohort (n = 20) to evaluate performance by receiver operating characteristic (ROC) curve. Gene ontology and pathway analyses investigated the underlying biology of the model genes., Results: We identified 18 genes that could distinguish IA patients in a training cohort with 85% accuracy. This SVM model also had 85% accuracy in the testing cohort, with an area under the ROC curve of 0.91. Bioinformatics reflected activation and recruitment of leukocytes, activation of macrophages, and inflammatory response, suggesting that the biomarker captures important processes in IA pathogenesis., Conclusions: Circulating whole blood transcriptomes can detect the presence of unruptured IAs. Pending additional testing in larger cohorts, this could serve as a foundation to develop a simple blood-based test to facilitate screening and early detection of IAs., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: JNJ—Principal Investigator: NIH Grant R01-AR-060604. KVS—Consulting/teaching: Canon Medical Systems Corporation, Penumbra Inc., Medtronic, Jacobs Institute. Co-Founder: Neurovascular Diagnostics, Inc. EIL—Intratech Medical Ltd. NeXtGen Biologics. Principal investigator: Medtronic US SWIFT PRIME Trials. Honoraria–Medtronic. Consultant–Pulsar Vascular. Advisory Board-Stryker, NeXtGen Biologics, MEDX, Cognition Medical. Other financial support—Abbott Vascular for carotid training sessions. AHS—Financial Interest/Investor/Stock Options/Ownership: Amnis Therapeutics, Apama Medical,BlinkTBI, Inc., Buffalo Technology Partners, Inc., Cardinal Health, Cerebrotech Medical Systems, Inc., Claret Medical, Cognition Medical, Endostream Medical, Ltd., Imperative Care, International Medical Distribution Partners, Rebound Therapeutics Corp., Silk Road Medical, StimMed, Synchron, Three Rivers Medical, Inc., Viseon Spine, Inc. Consultant/Advisory Board: Amnis Therapeutics, Boston Scientific, Canon Medical Systems USA, Inc., Cerebrotech Medical Systems, Inc., Cerenovus, Claret Medical, Corindus, Inc., Endostream Medical, Ltd., Guidepoint 15Global Consulting, Imperative Care, Integra, Medtronic, MicroVention, Northwest University—DSMB Chair for HEAT Trial, Penumbra, Rapid Medical,Rebound Therapeutics Corp., Silk Road Medical, StimMed, Stryker, Three Rivers Medical, Inc.,VasSol, W.L. Gore & Associates. National PI/Steering Committees: Cerenovus LARGE Trial and ARISE II Trial,Medtronic SWIFTPRIME and SWIFT DIRECT Trials, MicroVention FRED Trial & CONFIDENCE Study, MUSC POSITIVE Trial,Penumbra 3D Separator Trial, COMPASS Trial, INVEST Trial. Principal investigator: Cummings Foundation grant. HM—Principal investigator:NIH Grants R01-NS-091075 and R01-NS-064592. Grant support: Canon Medical Systems. Co-founder: Neurovascular Diagnostics, Inc. VMT—Principal investigator: National Science Foundation Award No. 1746694, Brain Aneurysm Foundation grant, and Center for Advanced Technology grant. Co-founder:Neurovascular Diagnostics, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Intracranial aneurysms (IAs) are dangerous because of their potential to rupture. We previously found significant RNA expression differences in circulating neutrophils between patients with and without unruptured IAs and trained machine learning models to predict presence of IA using 40 neutrophil transcriptomes. Here, we aim to develop a predictive model for unruptured IA using neutrophil transcriptomes from a larger population and more robust machine learning methods., Methods: Neutrophil RNA extracted from the blood of 134 patients (55 with IA, 79 IA-free controls) was subjected to next-generation RNA sequencing. In a randomly-selected training cohort (n = 94), the Least Absolute Shrinkage and Selection Operator (LASSO) selected transcripts, from which we constructed prediction models via 4 well-established supervised machine-learning algorithms (K-Nearest Neighbors, Random Forest, and Support Vector Machines with Gaussian and cubic kernels). We tested the models in the remaining samples (n = 40) and assessed model performance by receiver-operating-characteristic (ROC) curves. Real-time quantitative polymerase chain reaction (RT-qPCR) of 9 IA-associated genes was used to verify gene expression in a subset of 49 neutrophil RNA samples. We also examined the potential influence of demographics and comorbidities on model prediction., Results: Feature selection using LASSO in the training cohort identified 37 IA-associated transcripts. Models trained using these transcripts had a maximum accuracy of 90% in the testing cohort. The testing performance across all methods had an average area under ROC curve (AUC) = 0.97, an improvement over our previous models. The Random Forest model performed best across both training and testing cohorts. RT-qPCR confirmed expression differences in 7 of 9 genes tested. Gene ontology and IPA network analyses performed on the 37 model genes reflected dysregulated inflammation, cell signaling, and apoptosis processes. In our data, demographics and comorbidities did not affect model performance., Conclusions: We improved upon our previous IA prediction models based on circulating neutrophil transcriptomes by increasing sample size and by implementing LASSO and more robust machine learning methods. Future studies are needed to validate these models in larger cohorts and further investigate effect of covariates.

Background: Due to the scarcity of longitudinal data, the morphologic development of intracranial aneurysms (IAs) during their natural history remains poorly understood. However, longitudinal information can often be inferred from cross-sectional datasets as demonstrated by anatomists' use of geometric morphometrics to build evolutionary trees, reconstructing species inter-relationships based on morphologic landmarks., Objective: We adopted these tools to analyze cross-sectional image data and infer relationships between IA morphologies., Methods: On 3D reconstructions of 52 middle cerebral arteries (MCA) IAs (9 ruptured) and 10 IAfree MCAs (baseline geometries), 7 semi-automated landmarks were placed at the proximal parent artery and maximum height. From these, 64 additional landmarks were computationally generated to create a 71-landmark point cloud of 213 xyz coordinates. This data was normalized by Procrustes transformation and used in the principal component analysis, hierarchical clustering, and phylogenetic analyses., Results: Principal component analysis showed separation of IA-free MCA geometries and grouping of ruptured IAs from unruptured IAs. Hierarchical clustering delineated a cluster of only unruptured IAs that were significantly smaller and more spherical than clusters that had ruptured IAs. Phylogenetic classification placed ruptured IAs more distally in the tree than unruptured IAs, indicating greater shape derivation. Groups of unruptured IAs were observed, but ruptured IAs were invariably found in mixed lineages with unruptured IAs, suggesting that some pathways of shape change may be benign while others are more associated with rupture., Conclusion: Geometric morphometric analyses of larger datasets may indicate particular pathways of shape change leading toward aneurysm rupture versus stabilization., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Genetics play an important role in intracranial aneurysm (IA) pathophysiology. Genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) that are linked to IA but how they affect disease pathobiology remains poorly understood. We used Encyclopedia of DNA Elements (ENCODE) data to investigate the epigenetic landscapes surrounding genetic risk loci to determine if IA-associated SNPs affect functional elements that regulate gene expression and if those SNPs are most likely to impact a specific type of cells., Methods: We mapped 16 highly significant IA-associated SNPs to linkage disequilibrium (LD) blocks within the human genome. Within these regions, we examined the presence of H3K4me1 and H3K27ac histone marks and CCCTC-binding factor (CTCF) and transcription-factor binding sites using chromatin immunoprecipitation-sequencing (ChIP-Seq) data. This analysis was conducted in several cell types relevant to endothelial (human umbilical vein endothelial cells [HUVECs]) and inflammatory (monocytes, neutrophils, and peripheral blood mononuclear cells [PBMCs]) biology. Gene ontology analysis was performed on genes within extended IA-risk regions to understand which biological processes could be affected by IA-risk SNPs. We also evaluated recently published data that showed differential methylation and differential ribonucleic acid (RNA) expression in IA to investigate the correlation between differentially regulated elements and the IA-risk LD blocks., Results: The IA-associated LD blocks were statistically significantly enriched for H3K4me1 and/or H3K27ac marks (markers of enhancer function) in endothelial cells but not in immune cells. The IA-associated LD blocks also contained more binding sites for CTCF in endothelial cells than monocytes, although not statistically significant. Differentially methylated regions of DNA identified in IA tissue were also present in several IA-risk LD blocks, suggesting SNPs could affect this epigenetic machinery. Gene ontology analysis supports that genes affected by IA-risk SNPs are associated with extracellular matrix reorganization and endopeptidase activity., Conclusion: These findings suggest that known genetic alterations linked to IA risk act on endothelial cell function. These alterations do not correlate with IA-associated gene expression signatures of circulating blood cells, which suggests that such signatures are a secondary response reflecting the presence of IA rather than indicating risk for IA.

Background: Intracranial aneurysms (IAs) are dangerous because of their potential to rupture and cause deadly subarachnoid hemorrhages. Previously, we found significant RNA expression differences in circulating neutrophils between patients with unruptured IAs and aneurysm-free controls. Searching for circulating biomarkers for unruptured IAs, we tested the feasibility of developing classification algorithms that use neutrophil RNA expression levels from blood samples to predict the presence of an IA., Methods: Neutrophil RNA extracted from blood samples from 40 patients (20 with angiography-confirmed unruptured IA, 20 angiography-confirmed IA-free controls) was subjected to next-generation RNA sequencing to obtain neutrophil transcriptomes. In a randomly-selected training cohort of 30 of the 40 samples (15 with IA, 15 controls), we performed differential expression analysis. Significantly differentially expressed transcripts (false discovery rate < 0.05, fold change ≥ 1.5) were used to construct prediction models for IA using four well-known supervised machine-learning approaches (diagonal linear discriminant analysis, cosine nearest neighbors, nearest shrunken centroids, and support vector machines). These models were tested in a testing cohort of the remaining 10 neutrophil samples from the 40 patients (5 with IA, 5 controls), and model performance was assessed by receiver-operating-characteristic (ROC) curves. Real-time quantitative polymerase chain reaction (PCR) was used to corroborate expression differences of a subset of model transcripts in neutrophil samples from a new, separate validation cohort of 10 patients (5 with IA, 5 controls)., Results: The training cohort yielded 26 highly significantly differentially expressed neutrophil transcripts. Models using these transcripts identified IA patients in the testing cohort with accuracy ranging from 0.60 to 0.90. The best performing model was the diagonal linear discriminant analysis classifier (area under the ROC curve = 0.80 and accuracy = 0.90). Six of seven differentially expressed genes we tested were confirmed by quantitative PCR using isolated neutrophils from the separate validation cohort., Conclusions: Our findings demonstrate the potential of machine-learning methods to classify IA cases and create predictive models for unruptured IAs using circulating neutrophil transcriptome data. Future studies are needed to replicate these findings in larger cohorts.

Background: Unruptured intracranial aneurysms (IAs) are typically asymptomatic and undetected except for incidental discovery on imaging. Blood-based diagnostic biomarkers could lead to improvements in IA management. This exploratory study examined circulating neutrophils to determine whether they carry RNA expression signatures of IAs., Methods: Blood samples were collected from patients receiving cerebral angiography. Eleven samples were collected from patients with IAs and 11 from patients without IAs as controls. Samples from the two groups were paired based on demographics and comorbidities. RNA was extracted from isolated neutrophils and subjected to next-generation RNA sequencing to obtain differential expressions for identification of an IA-associated signature. Bioinformatics analyses, including gene set enrichment analysis and Ingenuity Pathway Analysis, were used to investigate the biological function of all differentially expressed transcripts., Results: Transcriptome profiling identified 258 differentially expressed transcripts in patients with and without IAs. Expression differences were consistent with peripheral neutrophil activation. An IA-associated RNA expression signature was identified in 82 transcripts (p<0.05, fold-change ≥2). This signature was able to separate patients with and without IAs on hierarchical clustering. Furthermore, in an independent, unpaired, replication cohort of patients with IAs (n = 5) and controls (n = 5), the 82 transcripts separated 9 of 10 patients into their respective groups., Conclusion: Preliminary findings show that RNA expression from circulating neutrophils carries an IA-associated signature. These findings highlight a potential to use predictive biomarkers from peripheral blood samples to identify patients with IAs.

Background: The neurovasculature dynamically responds to changes in cerebral blood flow by vascular remodeling processes. Serial imaging studies in mouse models could help characterize pathologic and physiologic flow-induced remodeling of the Circle of Willis (CoW)., Method: We induced flow-driven pathologic cerebral vascular remodeling in the CoW of mice (n=3) by ligation of the left Common Carotid Artery (CCA), and the right external carotid and pterygopalatine arteries, increasing blood flow through the basilar and the right internal carotid arteries. One additional mouse was used as a wild-type control. Magnetic Resonance Imaging (MRI) at 9.4 Tesla (T) was used to serially image the mouse CoW over three months, and to obtain threedimensional images for use in Computational Fluid Dynamic (CFD) simulations. Terminal vascular corrosion casting and scanning electron microscope imaging were used to identify regions of macroscopic and microscopic arterial damage., Results: We demonstrated the feasibility of detecting and serially measuring pathologic cerebral vascular changes in the mouse CoW, specifically in the anterior vasculature. These changes were characterized by bulging and increased vessel tortuosity on the anterior cerebral artery and aneurysm- like remodeling at the right olfactory artery origin. The resolution of the 9.4T system further allowed us to perform CFD simulations in the anterior CoW, which showed a correlation between elevated wall shear stress and pathological vascular changes., Conclusion: In the future, serial high-resolution MRI could be useful for characterizing the flow environments corresponding to other pathologic remodeling processes in the mouse CoW, such as aneurysm formation, subarachnoid hemorrhage, and ischemia., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Bilateral common carotid artery (CCA) ligation in rabbits is a model for basilar terminus (BT) aneurysm formation. We asked if this model could be replicated in rats. Fourteen female Sprague Dawley rats underwent bilateral CCA ligation (n=8) or sham surgery (n=6). After 7 days, 5 ligated and 3 sham rats were euthanized for histological evaluation of BT aneurysm formation, while the remaining rats were imaged with magnetic resonance angiography, euthanized, and subjected to corrosion casting of the Circle of Willis (CoW). 3D micro computed tomography images of CoW casts were used for flow simulations at the rat BT, and electron micrographs of the casts were analyzed for aneurysmal and morphological changes. Results from these analyses were compared to rabbit model data (n=10 ligated and n=6 sham). Bilateral CCA ligation did not produce aneurysmal damage at the rat BT. While the surgical manipulation increased rat basilar artery flow, fluid dynamics simulations showed that the initial hemodynamic stress at the rat BT was significantly less than in rabbits. Rats also exhibited fewer morphological and pathological changes (minor changes only occurred in the posterior CoW) than rabbits, which had drastic changes throughout the CoW. A comparison of CoW anatomies demonstrated a greater number of branching arteries at the BT, larger CoW arteries in relation to basilar artery, and a steeper BT bifurcation angle in the rat. These differences could account for the lower hemodynamic stress at the BT and in the cerebrovasculature of the rat. In conclusion, bilateral CCA ligation in rats does not recapitulate the rabbit model of early flow-induced BT aneurysm. We suspect that the different CoW morphology of the rat lessens hemodynamic insults, thereby diminishing flow-induced aneurysmal remodeling.

Increased cerebral blood flow has been shown to induce pathological structural changes in the Circle of Willis (CoW) in experimental models. Previously, we reported flow-induced aneurysm-like remodeling in the CoW secondary to flow redistribution after bilateral common carotid artery (CCA) ligation in rabbits. In the current study, we tested the hypothesis that loading rabbits with biological risk factors for vascular disease would increase flow-induced aneurysmal remodeling in the CoW. In the same series as the previously-reported bilateral CCA-ligation-alone (n  = 6) and sham surgery (n = 3) groups, eight additional female rabbits (the experimental group in this study) were subjected to two risk factors for intracranial aneurysm (hypertension and estrogen deficiency) and then bilateral CCA ligation. Upon euthanasia at 6 months, vascular corrosion casts of the CoW were created and analyzed by scanning electron microscopy for morphological changes and aneurysmal damage. In rabbits with hypertension and estrogen deficiency, arterial caliber increased throughout the CoW, similar to rabbits with CCA ligation alone. However, aneurysmal remodeling (i.e., local bulging) in the CoW was significantly greater than in CCA-ligation-only rabbits and was more widespread, presenting in regions that did not show aneurysmal changes after CCA ligation alone. Furthermore, hypertension and estrogen deficiency caused greater increases in vessel length and tortuosity. These results suggest that hypertension and estrogen deficiency make the CoW more vulnerable to flow-induced aneurysmal remodeling and tortuosity. We propose they do so by lowering the tolerance of vascular tissue to hemodynamic forces caused by CCA ligation, thus lowering the threshold necessary to incite vascular damage., (© 2015 Wiley Periodicals, Inc.)

Background: Hemodynamic insults at arterial bifurcations are believed to play a critical role in initiating intracranial aneurysms. Recent studies in a rabbit model indicate that aneurysmal damage initiates under specific wall shear stress conditions when smooth muscle cells (SMCs) become pro-inflammatory and produce matrix metalloproteinases (MMPs). The mechanisms leading to SMC activation and MMP production during hemodynamic aneurysm initiation are unknown. The goal is to determine if nitric oxide and/or superoxide induce SMC changes, MMP production and aneurysmal remodeling following hemodynamic insult., Methods: Bilateral common carotid artery ligation was performed on rabbits (n = 19, plus 5 sham operations) to induce aneurysmal damage at the basilar terminus. Ligated animals were treated with the nitric oxide synthase (NOS) inhibitor LNAME (n = 7) or the superoxide scavenger TEMPOL (n = 5) and compared to untreated animals (n = 7). Aneurysm development was assessed histologically 5 days after ligation. Changes in NOS isoforms, peroxynitrite, reactive oxygen species (ROS), MMP-2, MMP-9, and smooth muscle α-actin were analyzed by immunohistochemistry., Results: LNAME attenuated ligation-induced IEL loss, media thinning and bulge formation. In untreated animals, immunofluorescence showed increased endothelial NOS (eNOS) after ligation, but no change in inducible or neuronal NOS. Furthermore, during aneurysm initiation ROS increased in the media, but not the intima, and there was no change in peroxynitrite. In LNAME-treated animals, ROS production did not change. Together, this suggests that eNOS is important for aneurysm initiation but not by producing superoxide. TEMPOL treatment reduced aneurysm development, indicating that the increased medial superoxide is also necessary for aneurysm initiation. LNAME and TEMPOL treatment in ligated animals restored α-actin and decreased MMPs, suggesting that eNOS and superoxide both lead to SMC de-differentiation and MMP production., Conclusion: Aneurysm-inducing hemodynamics lead to increased eNOS and superoxide, which both affect SMC phenotype, increasing MMP production and aneurysmal damage.

Carotid occlusions are associated with de novo intracranial aneurysm formation in clinical case reports, but this phenomenon is not widely studied. We performed bilateral carotid ligation (n=9) in rabbits to simulate carotid occlusion, and sham surgery (n=3) for control. Upon euthanasia (n=3 at 5 days, n=6 at 6 months post ligation, and n=3 at 5 days after sham operation), vascular corrosion casts of the circle of Willis (CoW) were created. Using scanning electron microscopy, we quantified gross morphologic, macroscopic, and microscopic changes on the endocasts and compared findings with histologic data. At 5 days, CoW arteries of ligated animals increased caliber. The posterior communicating artery (PCom) increased length and tortuosity, and the ophthalmic artery (OA) origin presented preaneurysmal bulges. At 6 months, calibers were unchanged from 5 days, PComs further increased tortuosity while presenting segmental dilations, and the OA origin and basilar terminus presented preaneurysmal bulges. This exploratory study provides evidence that flow increase after carotid occlusion produces both compensatory arterial augmentation and pathologic remodeling such as tortuosity and saccular/fusiform aneurysm. Our findings may have considerable clinical implications, as these lesser-known consequences should be considered when managing patients with carotid artery disease or choosing carotid ligation as a therapeutic option.

Background: Intracranial aneurysm initiation is poorly understood, although hemodynamic insult is believed to play an important role in triggering the pathology. It has recently been found in a rabbit model that while macrophages are absent during hemodynamic aneurysm initiation, matrix metalloproteinases (MMPs) are elevated and co-localize with smooth muscle cells (SMCs). This study investigates whether SMCs play a mechanistic role in aneurysm initiation triggered by hemodynamics., Methods: Aneurysmal damage was induced at the basilar terminus via bilateral common carotid artery ligation in rabbits (n = 45, plus 7 sham controls). 16 ligated rabbits were treated with doxycycline to inhibit MMPs, 7 received clodronate liposomes to deplete circulating monocytes, and the rest received no drug. Effects of the treatments on aneurysm development were assessed histologically 5 days and 6 months after ligation. MMP production and expression of inflammatory markers by SMCs was monitored by immunohistochemistry and in situ hybridization., Results: Treatment with doxycycline attenuated aneurysmal development examined at 5 days and 6 months, suggesting that MMPs contribute to aneurysm initiation. However, systemic depletion of macrophages did not decrease MMPs or suppress aneurysmal development. Immunofluorescence showed that during aneurysm initiation MMP-2 and MMP-9 were distributed in SMCs, and in situ hybridization indicated that they were transcribed by SMCs. In regions of early aneurysmal lesion, SMCs exhibited decreased expression of smooth muscle actin and increased NF-κB and MCP-1 expressions., Conclusions: During aneurysm initiation triggered by hemodynamics, SMCs rather than macrophages are responsible for MMP production that is critical for aneurysmal lesion development. These SMCs exhibit proinflammatory behavior.

Background: Intracranial aneurysms (IAs) occur more frequently at certain bifurcations than at others. Hemodynamic stress, which promotes aneurysm formation in animal models, also differs among bifurcations, depending on flow and vessel geometry., Objective: To determine whether locations that are more likely to develop IAs experience different hemodynamic stresses that might contribute to higher IA susceptibility., Methods: We characterized the hemodynamic microenvironment at 10 sites in or around the circle of Willis where IAs commonly occur and examined statistical relationships between hemodynamic factors and the tendency for a site to form IAs. The tendency for each site to develop IAs was quantified on the basis of the site distribution from systematic literature analysis of 19 reports including 26418 aneurysms. Hemodynamic parameters for these sites were derived from image-based computational fluid dynamics of 114 cerebral bifurcations from 31 individuals. Wall shear stress and its spatial gradient were calculated in the impact zone surrounding the bifurcation apex. Linear and exponential regression analyses evaluated correlations between the tendency for IA formation and the typical hemodynamics of a site., Results: IA susceptibility significantly correlated with the magnitudes of wall shear stress and positive wall shear stress gradient within the hemodynamic impact zone calculated for each site., Conclusion: IAs occur more frequently at cerebral bifurcations that typically experience higher hemodynamic shear stress and stronger flow acceleration, conditions previously shown to promote aneurysm initiation in animals.

Objective: Computational fluid dynamics (CFD) simulations of intracranial aneurysm hemodynamics usually adopt the simplification of the Newtonian blood rheology model. A study was undertaken to examine whether such a model affects the predicted hemodynamics in realistic intracranial aneurysm geometries., Methods: Pulsatile CFD simulations were carried out using the Newtonian viscosity model and two non-Newtonian models (Casson and Herschel-Bulkley) in three typical internal carotid artery saccular aneurysms (A, sidewall, oblong-shaped with a daughter sac; B, sidewall, quasi-spherical; C, near-spherical bifurcation). For each aneurysm model the surface distributions of shear rate, blood viscosity and wall shear stress (WSS) predicted by the three rheology models were compared., Results: All three rheology models produced similar intra-aneurysmal flow patterns: aneurysm A had a slowly recirculating secondary vortex near the dome whereas aneurysms B and C contained only a large single vortex. All models predicted similar shear rate, blood viscosity and WSS in parent vessels of all aneurysms and in the sacs of B and C. However, large discrepancies in shear rate, viscosity and WSS among predictions by the various rheology models were found in the dome area of A where the flow was relatively stagnant. Here the Newtonian model predicted higher shear rate and WSS values and lower blood viscosity than the two non-Newtonian models., Conclusions: The Newtonian fluid assumption can underestimate viscosity and overestimate shear rate and WSS in regions of stasis or slowly recirculating secondary vortices, typically found at the dome in elongated or complex-shaped saccular aneurysms as well as in aneurysms following endovascular treatment. Because low shear rates and low WSS in such flow conditions indicate a high propensity for thrombus formation and rupture, CFD based on the Newtonian assumption may underestimate the propensity of these events.

Cerebral aneurysms develop near bifurcation apices, where complex hemodynamics occur: Flow impinges on the apex, accelerates into branches, then slows again distally, creating high wall shear stress (WSS) and positive and negative spatial gradients in WSS (WSSG). Endothelial responses to these kinds of high WSS hemodynamic environments are not well characterized. We examined endothelial cells (ECs) under elevated WSS and positive and negative WSSG using a flow chamber with constant-height channels to create regions of uniform WSS and converging and diverging channels to create positive and negative WSSG, respectively. Cultured bovine aortic ECs were subjected to 3.5 and 28.4 Pa with and without WSSG for 24 and 36 h. High WSS inhibited EC alignment to flow, increased EC proliferation assessed by bromodeoxyuridine incorporation, and increased apoptosis determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling. These responses to high WSS were either accentuated or ameliorated by WSSG: Positive WSSG (+980 Pa/m) inhibited alignment and stimulated proliferation and apoptosis, whereas negative WSSG (-1120 Pa/m) promoted alignment and suppressed proliferation and apoptosis. These results demonstrate that ECs discriminate between positive and negative WSSG under high WSS conditions. EC responses to positive WSSG may contribute to pathogenic remodeling that occurs at bifurcations preceding aneurysm formation.

Object: Hemodynamic insult has been speculated to be a key factor in intracranial aneurysm formation; however, it is unclear whether a sustained insult is necessary. The authors examined whether aneurysmal degradation would continue despite the normalization of wall shear stress (WSS) by adaptive outward vascular remodeling., Methods: Twenty-five rabbits underwent either sham operation (5 animals) or bilateral common carotid artery ligation (20 animals) to augment basilar artery (BA) flow. Basilar termini (BTs) were harvested at 5 days and 3, 12, and 27 weeks postoperation. Histological changes at the BTs were quantified using an aneurysm development score (ADS) wherein the luminal length of the vessel wall exhibiting internal elastic lamina (IEL) loss, media thinning (> 30% media loss), and bulging was multiplied by the percentage of media thinning divided by the BA diameter. This score and its component variables were evaluated over the specified time points and compared with the WSS time course obtained from multiple angiography and BA flow velocity measurements., Results: Serial examination of histological sections from the ligation group (17 rabbits survived the procedure) demonstrated localized, progressive, degenerative, and aneurysmal changes at the BTs. Prominent IEL loss was observed in BT specimens from all ligated animals. Media thinning and luminal bulging significantly progressed over the 27-week follow-up. The composite ADS significantly increased over the study period, indicating progressive aneurysm development, although the WSS returned to preligation baseline values within 5 weeks of ligation., Conclusions: Hemodynamic insult can elicit a pathological vascular response leading to a self-sustaining aneurysmal remodeling that does not require persistence of the original inciting factor to continue its pathological progression.

Background/aims: Hemodynamics constitute a critical factor in the formation of intracranial aneurysms. However, little is known about how intracranial arteries respond to hemodynamic insult and how that response contributes to aneurysm formation. We examined early cellular responses at rabbit basilar termini exposed to hemodynamic insult that initiates aneurysmal remodeling., Methods: Flow in the basilar artery was increased by bilateral carotid artery ligation. After 2 and 5 days, basilar terminus tissue was examined by immunohistochemistry and quantitative PCR., Results: Within 2 days of flow increase, internal elastic lamina (IEL) was lost in the periapical region of the bifurcation, which experienced high wall shear stress and positive wall shear stress gradient. Overlying endothelium was still largely present in this region. IEL loss was associated with localized apoptosis and elevated expression of matrix metalloproteinases (MMPs) 2 and 9. A small number of inflammatory cells were sporadically scattered in the bifurcation adventitia and were not concentrated in regions of IEL loss and MMP elevation. Elevated MMP expression colocalized with smooth muscle α-actin in the media., Conclusion: The initial vascular response to aneurysm-initiating hemodynamic insult includes localized matrix degradation and cell apoptosis. Such destructive remodeling arises from intrinsic mural cells, rather than through inflammatory cell infiltration., (Copyright © 2011 S. Karger AG, Basel.)

Background and Purpose: Hemodynamic insult by bilateral common carotid artery ligation has been shown to induce aneurysmal remodeling at the basilar terminus in a rabbit model. To characterize critical hemodynamics that initiate this remodeling, we applied a novel hemodynamics-histology comapping technique., Methods: Eight rabbits received bilateral common carotid artery ligation to increase basilar artery flow. Three underwent sham operations. Hemodynamic insult at the basilar terminus was assessed by computational fluid dynamics. Bifurcation tissue was harvested on day 5; histology was comapped with initial postligation hemodynamic fields of wall shear stress (WSS) and WSS gradient., Results: All bifurcations showed internal elastic lamina loss in periapical regions exposed to accelerating flow with high WSS and positive WSS gradient. Internal elastic lamina damage happened 100% of the time at locations where WSS was >122 Pa and WSS gradient was >530 Pa/mm. The degree of destructive remodeling accounting for internal elastic lamina loss, medial thinning, and luminal bulging correlated with the magnitude of the hemodynamic insult., Conclusions: Aneurysmal remodeling initiates when local hemodynamic forces exceed specific limits at the rabbit basilar terminus. A combination of high WSS and positive WSS gradient represents dangerous hemodynamics likely to induce aneurysmal remodeling.

Objective: Although elevated hemodynamics has been speculated to play a key role in intracranial aneurysm (IA) initiation, little is known about the specific hemodynamic microenvironment that triggers aneurysmal vascular degradation. We previously demonstrated maladaptive remodeling characteristic of IA initiation occurring in hemodynamic regions of combined high wall shear stress (WSS) and high WSS gradient near the apex of an experimentally created carotid bifurcation. This study examines whether this remodeling recapitulates the molecular changes found in IAs and whether molecular changes also correspond to specific hemodynamic environments., Methods: De novo bifurcations were surgically created using both native common carotid arteries in each of 6 dogs. Bifurcations were imaged 2 weeks or 2 months after surgery by high-resolution 3-dimensional angiography, from which flow fields were obtained by computational fluid dynamics simulations. Subsequently, harvested tissues, demonstrating early aneurysmal changes near the apex, were immunostained for interleukin-1beta, endothelial and inducible nitric oxide synthases, nitrotyrosine, and matrix metalloproteinase-2 and -9. Spatial distributions of these molecules were comapped with computational fluid dynamics results., Results: The aneurysmal wall showed decreased endothelial nitric oxide synthase expression compared with surrounding segments, the feeding artery, and native controls, whereas all other markers increased. Anti-CD68 staining indicated the absence of inflammatory cells in the aneurysmal wall. Comapping molecular marker distributions with flow fields revealed confinement of these molecular changes within the hemodynamic region of high WSS and high, positive WSS gradient., Conclusion: Aneurysm-initiating remodeling induced by combined high WSS and high, positive WSS gradient is associated with molecular changes implicated in IAs.

Background and Purpose: Development of hemodynamic modifying devices to treat intracranial aneurysms is an active area of research. The asymmetrical vascular stent (AVS), a stent containing a low-porosity patch, is such device. We evaluate AVS efficacy in an in vivo intracranial aneurysm model., Methods: We created 24 elastase rabbit model aneurysms: 13 treated with the AVS, 5 treated with standard coronary stents, and 6 untreated controls. Four weeks after treatment, aneurysms underwent follow-up angiography, cone-beam micro-CT, histological evaluation, and selective electron microscopy scanning., Results: Four rabbits died early in the study: 3 during AVS treatment and 1 control (secondary to intraprocedural vessel injury and an unrelated tumor, respectively). AVS-treated aneurysms exhibited very weak or no aneurysm flow immediately after treatment and no flow in all aneurysms at follow-up. Standard stent-treated aneurysms showed flow both after treatment (5/5) and at follow-up (3/5). All control aneurysms remained patent during the study. Micro-CT scans showed: 9 of 9 scanned AVS aneurysms were occluded, 6 of 9 AVS were ideally placed, and 3 of 9 low-porosity region partially covered the aneurysm neck; standard stent-treated aneurysms were 1 of 5 occluded, 2 of 5 patent, and 2 of 5 partially patent. Histology results demonstrated: for AVS-treated aneurysms, advanced thrombus organization in the (9/9); for standard stent-treated aneurysms, (1/4) no thrombus, (2/4) partially thrombosed, and (1/4) fully thrombosed; for control aneurysms (4/4), no thrombus., Conclusions: The use of AVS shows promise as a viable new therapeutic in intracranial aneurysm treatment. These data encourage further investigation and provide substantial support to the AVS concept.

Background and Purpose: Hemodynamic insults at arterial bifurcations are hypothesized to play a key role in intracranial aneurysm formation. This study investigates aneurysm-initiating vascular responses at the rabbit basilar terminus subsequent to common carotid artery ligation., Methods: Nine adult female New Zealand white rabbits were subjected to sham, unilateral, or bilateral common carotid artery ligation to produce varying degrees of compensatory basilar artery flow increase. Basilar artery flow velocity and geometry were monitored by transcranial Doppler and rotational angiography, respectively, for 12 weeks after surgery. Bifurcation tissues were harvested at 12 weeks and examined histologically. From the histological sections, we quantified the destructive structural changes at the basilar terminus and correlated them with the basilar artery flow rate increase., Results: Subsequent to common carotid artery ligation, basilar artery flow rate increased by 105% to 900% at the maximum. All common carotid artery-ligated rabbits presented nascent aneurysm formation characterized by a bulge with thinned media and absent internal elastic lamina near the basilar terminus. We defined a nascent aneurysm index based on a multiplicative combination of the local destructive remodeling lengths measured at the nascent aneurysm. The nascent aneurysm index strongly correlated with the increase in basilar artery flow rate with R(2)=0.91., Conclusions: Without other known predisposition, flow increase alone at the basilar bifurcation can lead to a nascent aneurysm. This nascent aneurysm formation is dose-dependent on basilar artery flow increase.

Background and Purpose: Intracranial aneurysm (IA) treatment through hemodynamic modification with novel stent designs is a burgeoning area of research. We present a feasibility study for a new low-porosity patch-containing stent designed to treat intracranial aneurysms. The device is deployed so the patch covers the aneurysm neck ensuring strong flow diversion away from the aneurysm while keeping a low probability of occlusion of perforating vessels., Methods: We created 17 side-wall aneurysms in 6 dogs, 2 per carotid artery if animal size permitted. Twelve proximal aneurysms were treated with AVSs: 5 distal aneurysms were untreated, serving as controls against self-thrombosis; 7 treated aneurysms were fully-covered; and 5 were partially-covered. After 4 weeks, a final angiogram was performed and aneurysms were explanted. Angiograms acquired pre- and posttreatment and at 4-week follow-up were analyzed quantitatively using normalized time-density curves (NTDC). Cone-beam micro-CT and histological specimen analysis were then performed., Results: Posttreatment, NTDC average peaks dropped to 45% of initial values for the partially-covered aneurysms and 78% for the fully-covered aneurysms. Cone-beam micro-CT imaging performed at 4 weeks posttreatment showed partial thrombosis in 4 of 5 partially-covered aneurysms and complete thrombosis in all fully-covered aneurysms. Histology revealed neointimal coverage of all asymmetrical patch regions and thrombus formation in both fully- and partially-covered aneurysms. Four-week follow-up was not done for 1 animal (2 controls, 2 treated) that expired because of groin hemorrhage and for another animal (1 aneurysm) with an occluded carotid., Conclusions: We demonstrate aneurysmal blood flow diversion using a new low-porosity patch-containing asymmetrical vascular stent in a canine side-wall aneurysm model. Overall results are encouraging and support continued AVS development.

Background and Purpose: Arterial bifurcation apices are common sites for cerebral aneurysms, raising the possibility that the unique hemodynamic conditions associated with flow dividers predispose the apical vessel wall to aneurysm formation. This study sought to identify the specific hemodynamic insults that lead to maladaptive vascular remodeling associated with aneurysm development and to identify early remodeling events at the tissue and cellular levels., Methods: We surgically created new branch points in the carotid vasculature of 6 female adult dogs. In vivo angiographic imaging and computational fluid dynamics simulations revealed the detailed hemodynamic microenvironment for each bifurcation, which were then spatially correlated with histologic features showing specific tissue responses., Results: We observed 2 distinct patterns of vessel wall remodeling: (1) hyperplasia that formed an intimal pad at the bifurcation apex and (2) destructive remodeling in the adjacent region of flow acceleration that resembled the initiation of an intracranial aneurysm, characterized by disruption of the internal elastic lamina, loss of medial smooth muscle cells, reduced proliferation of smooth muscle cells, and loss of fibronectin., Conclusions: Strong localization of aneurysm-type remodeling to the region of accelerating flow suggests that a combination of high wall shear stress and a high gradient in wall shear stress represents a "dangerous" hemodynamic condition that predisposes the apical vessel wall to aneurysm formation.