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1.  Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse 
Neuron  2015;89(1):37-53.
Summary
The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains, and to maintain these cells in serum-free cultures. We found that human astrocytes have similar abilities to murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to existing observations in mice, we found that mature human astrocytes respond robustly to glutamate. We next performed RNA-sequencing of healthy human astrocytes along with astrocytes from epileptic and tumor foci, and compared these to human neurons, oligodendrocytes, microglia, and endothelial cells. With these profiles, we identified novel human-specific astrocyte genes, and discovered a transcriptome-wide transformation between astrocyte precursor cells and mature post-mitotic astrocytes. These data represent some of the first cell type-specific molecular profiles of the healthy and diseased human brain.
doi:10.1016/j.neuron.2015.11.013
PMCID: PMC4707064  PMID: 26687838
2.  Neurorestoration after stroke 
Neurosurgical focus  2016;40(5):E2.
Recent advancements in stem cell biology and neuromodulation have ushered in a battery of new neurorestorative therapies for ischemic stroke. While the understanding of stroke pathophysiology has matured, the ability to restore patients’ quality of life remains inadequate. New therapeutic approaches, including cell transplantation and neurostimulation, focus on reestablishing the circuits disrupted by ischemia through multidimensional mechanisms to improve neuroplasticity and remodeling. The authors provide a broad overview of stroke pathophysiology and existing therapies to highlight the scientifc and clinical implications of neurorestorative therapies for stroke.
doi:10.3171/2016.2.FOCUS15637
PMCID: PMC4916840  PMID: 27132523
optogenetics; exogenous stem cells; brain-computer interface; neuroplasticity
3.  Personalized Medicine in Cerebrovascular Neurosurgery: Precision Neurosurgical Management of Cerebral Aneurysms and Subarachnoid Hemorrhage 
Frontiers in Surgery  2016;3:34.
Cerebral aneurysms are common vascular lesions. Little is known about the pathogenesis of these lesions and the process by which they destabilize and progress to rupture. Treatment decisions are motivated by a desire to prevent rupture and the devastating morbidity and mortality associated with resulting subarachnoid hemorrhage (SAH). For patients presenting with SAH, urgent intervention is required to stabilize the lesion and prevent re-rupture. Those patients fortunate enough to survive a presenting SAH and subsequent securing of their aneurysm must still face a spectrum of secondary sequelae, which can include cerebral vasospasm, delayed ischemia, seizures, cerebral edema, hydrocephalus, and endocrinologic and catecholamine-induced systemic dysfunction in cardiac, pulmonary, and renal systems. Increased focus on understanding the pathophysiology and molecular characteristics of these secondary processes will enable the development of targeted therapeutics and novel diagnostics for improved patient selection in personalized medicine trials for SAH. In unruptured cerebral aneurysms, treatment decisions are less clear and currently based solely on treating larger lesions, using rigid aneurysm size cutoffs generalized from recent studies that are the subject of ongoing controversy. Further compounding this controversy is the fact that the vast majority of aneurysms that come to clinical attention at the time of a hemorrhagic presentation are of smaller size, suggesting that small aneurysms are indeed not benign lesions. As such, patient-specific biomarkers that better predict which aneurysms represent high-risk lesions that warrant clinical intervention are of vital importance. Recent advancements in genomic and proteomic technologies have enabled the identification of molecular characteristics that may prove useful in tracking aneurysm growth and progression and identifying targets for prophylactic therapeutic interventions. Novel quantitative neuroimaging technologies have also recently emerged, capable of non-invasive characterization of hemodynamic factors, inflammation, and structural changes in aneurysmal walls. The combined use of these quantitative neuroimaging and molecular-based approaches, called Radiogenomics, is a technique that holds great promise in better characterizing individual aneurysms. In the near future, these radiogenomic techniques may help improve quality of life and patient outcomes via patient-specific approaches to the treatment of unruptured cerebral aneurysms and personalized medical management of secondary processes following aneurysmal SAH.
doi:10.3389/fsurg.2016.00034
PMCID: PMC4916172  PMID: 27446925
biomarkers; cerebral aneurysm; neuroimaging; radiogenomics; subarachnoid hemorrhage
4.  High-Resolution Microfluidic Single-Cell Transcriptional Profiling Reveals Clinically Relevant Subtypes among Human Stem Cell Populations Commonly Utilized in Cell-Based Therapies 
Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application.
doi:10.3389/fneur.2016.00041
PMCID: PMC4801858  PMID: 27047447
single-cell analysis; stem cell therapeutics; cellular heterogeneity
5.  Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target 
Brain  2015;139(2):468-480.
While tonic GABA appears to suppress brain repair after stroke, the effects of phasic (synaptic) GABA signalling are unclear. Hiu et al. reveal an increase in phasic GABA signalling during the repair phase that enhances plasticity-related recovery in mice. Increasing phasic signalling with zolpidem improves behavioural recovery, suggesting therapeutic potential.
While tonic GABA appears to suppress brain repair after stroke, the effects of phasic (synaptic) GABA signalling are unclear. Hiu et al. reveal an increase in phasic GABA signalling during the repair phase that enhances plasticity-related recovery in mice. Increasing phasic signalling with zolpidem improves behavioural recovery, suggesting therapeutic potential.
Ischaemic stroke is the leading cause of severe long-term disability yet lacks drug therapies that promote the repair phase of recovery. This repair phase of stroke occurs days to months after stroke onset and involves brain remapping and plasticity within the peri-infarct zone. Elucidating mechanisms that promote this plasticity is critical for the development of new therapeutics with a broad treatment window. Inhibiting tonic (extrasynaptic) GABA signalling during the repair phase was reported to enhance functional recovery in mice suggesting that GABA plays an important function in modulating brain repair. While tonic GABA appears to suppress brain repair after stroke, less is known about the role of phasic (synaptic) GABA during the repair phase. We observed an increase in postsynaptic phasic GABA signalling in mice within the peri-infarct cortex specific to layer 5; we found increased numbers of α1 receptor subunit-containing GABAergic synapses detected using array tomography, and an associated increased efficacy of spontaneous and miniature inhibitory postsynaptic currents in pyramidal neurons. Furthermore, we demonstrate that enhancing phasic GABA signalling using zolpidem, a Food and Drug Administration (FDA)-approved GABA-positive allosteric modulator, during the repair phase improved behavioural recovery. These data identify potentiation of phasic GABA signalling as a novel therapeutic strategy, indicate zolpidem’s potential to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA signalling in stroke recovery.
doi:10.1093/brain/awv360
PMCID: PMC4805083  PMID: 26685158
stroke; phasic GABA; brain repair; zolpidem
6.  Acute Pre-operative Infarcts and Poor Cerebrovascular Reserve are Independent Risk Factors for Severe Ischemic Complications Following Direct Extracranial-Intracranial Bypass for Moyamoya Disease 
Background and Purpose
Severe ischemic changes are a rare but devastating complication following direct superficial temporal artery to middle cerebral artery (STA MCA) bypass in Moyamoya patients. This study was undertaken to determine whether pre-operative MR imaging and/or cerebrovascular reserve (CVR) assessment using reference standard stable xenon enhanced computed tomography (xeCT) could predict such complications.
Materials and Methods
Among all adult patients receiving direct bypass at our institution between 2005 and 2010 who received a clinically interpretable xeCT examination, we identified index cases (patients with >15 ml post-operative infarcts) and control cases (patients without post-operative infarcts and without transient or permanent ischemic symptoms). Differences between groups were evaluated using the Mann Whitney test. Univariate and multivariate generalized linear model regression were employed to test predictors of post-operative infarct.
Results
Six index cases were identified and compared with 25 controls. Infarct size in the index cases was 95±55 ml. Four of six index cases (67%), but no control patients, had pre-operative acute infarcts. Baseline CBF was similar, but CVR was significantly lower in the index cases compared with control cases. For example, in the anterior circulation, median CVR was 0.4% (range: −38.0% to 16.6%) in index vs. 26.3% (range: −8.2% to 60.5%) in control patients (p=0.003). Multivariate analysis demonstrated that the presence of a small pre-operative infarct (regardless of location) and impaired CVR were independent, significant predictors of severe post-operative ischemic injury.
Conclusion
Acute infarcts and impaired CVR on pre operative imaging are independent risk factors for severe ischemic complications following STA MCA bypass in Moyamoya disease.
doi:10.3174/ajnr.A4535
PMCID: PMC4752884  PMID: 26564435
Stroke; Moyamoya; cerebrovascular reserve; perfusion; xenon CT; cerebral blood flow; EC-IC bypass
8.  Novel Stroke Therapeutics: Unraveling Stroke Pathophysiology and Its Impact on Clinical Treatments 
Neuron  2015;87(2):297-309.
Stroke remains a leading cause of death and disability in the world. Over the past few decades our understanding of the pathophysiology of stroke has increased, but greater insight is required to advance the field of stroke recovery. Clinical treatments have improved in the acute time window, but long-term therapeutics remain limited. Complex neural circuits damaged by ischemia make restoration of function after stroke difficult. New therapeutic approaches, including cell transplantation or stimulation, focus on reestablishing these circuits through multiple mechanisms to improve circuit plasticity and remodeling. Other research targets intact networks to compensate for damaged regions. This review highlights several important mechanisms of stroke injury and describes emerging therapies aimed at improving clinical outcomes.
doi:10.1016/j.neuron.2015.05.041
PMCID: PMC4911814  PMID: 26182415
9.  Stem cell therapy for acute cerebral injury: What do we know and what will the future bring? 
Current opinion in neurology  2013;26(6):617-625.
Purpose of review
The central nervous system has limited capacity for regeneration after acute and chronic injury. An attractive approach to stimulate neural plasticity in the brain is to transplant stem cells in order to restore function. Here we discuss potential mechanisms of action, current knowledge and future perspectives of clinical stem cell research for stroke and traumatic brain injury.
Recent findings
Preclinical data using various models suggest stem cell therapy to be a promising therapeutic avenue. Progress has been made in elucidating the mechanism of action of various cell types used, shifting the hypothesis from neural replacement to enhancing endogenous repair processes. Translation of these findings in clinical trials is currently being pursued with emphasis on both safety as well as efficacy.
Summary
Clinical trials are currently recruiting patients in phase I and II trials to gain more insight in the therapeutic potential of stem cells in acute cerebral injury. A close interplay between results of these clinical trials and more extensive basic research is essential for future trial design: Choosing the optimal transplantation strategy and selecting the right patients.
doi:10.1097/WCO.0000000000000023
PMCID: PMC4465754  PMID: 24136128
Neural repair; recovery; stem cell therapy; stroke; traumatic brain injury
10.  Treatment of Nystagmus in Brainstem Cavernous Malformation with Botulinum Toxin 
Cureus  null;8(4):e553.
We report a long-term eye movement study of a 68-year-old female with pontomedullary junction cavernous malformation whose dysconjugate nystagmus was treated with retrobulbar botulinum toxin A injections. Sequential, bilateral retrobulbar injections of botulinum toxin A were performed. Injections immediately decreased oscillopsia and nystagmus, and improved visual acuities. One to three months following injection, three-dimensional infrared oculography measured a significant 39-100% (P = 0.001) decrease in nystagmus amplitudes at multiple dimensions. This improvement diminished by six months in the right eye but sustained for about one year in the left eye. Over two years, botulinum toxin A injections were performed twice in the left eye and five times in the right eye. Our study supported the safe and effective use of repetitive retrobulbar botulinum toxin A injections in symptomatic nystagmus that failed medical therapy.
doi:10.7759/cureus.553
PMCID: PMC4852187  PMID: 27182467
nystagmus; oscillopsia; botulinum toxin; cavernous malformation; vascular malformation
11.  RNF213 Mutations in an Ethnically Diverse Population with Moyamoya Disease 
Background and Purpose
Moyamoya disease (MMD) is a rare, genetically heterogeneous cerebrovascular disease resulting from occlusion of the distal internal carotid arteries. A variant in the Ring Finger 213 gene (RNF213), altering arginine at position 4810 (p.R4810K), is associated with MMD in Asian populations. However, there is a lack of data on the role of RNF213 in MMD patients of additional ethnicities and diasporic Asian populations. We investigate the contribution of RNF213 alterations to MMD in an ethnically diverse population based in the United States (U.S).
Methods
We initially sequenced RNF213 exons 43, 44, 45 (encoding the eponymous RING finger domain), and exon 60 (encoding p.R4810K), in 86 ethnically diverse patients with MMD. Comprehensive exome sequencing data from 24 additional MMD patients was then analyzed to globally identify RNF213 variants. Segregation of variants with MMD and other vascular diseases was assessed in families.
Results
RNF213 p.R4810K was identified in 56% (9/16) of MMD patients of Asian descent, and not in 94 patients of non-Asian descent. 3.6% (4/110) of patients had variants in the exons encoding the RING finger domain. Seven additional variants were identified in 29% (7/24) of MMD patients who underwent exome sequencing. Segregation analysis supported an association with MMD for two variants, and a lack of association with disease for one variant.
Conclusions
These results confirm that alterations in RNF213 predispose patients of diverse ethnicities to MMD, and that the p.R4810K variant predisposes individuals of Asian descent in the U.S. to MMD.
doi:10.1161/STROKEAHA.114.006244
PMCID: PMC4420622  PMID: 25278557
moyamoya disease; stroke; RNF213; genetic; rare genetic variants
12.  Disease Variant Landscape of a Large Multiethnic Population of Moyamoya Patients by Exome Sequencing 
G3: Genes|Genomes|Genetics  2015;6(1):41-49.
Moyamoya disease (MMD) is a rare disorder characterized by cerebrovascular occlusion and development of hemorrhage-prone collateral vessels. Approximately 10–12% of cases are familial, with a presumed low penetrance autosomal dominant pattern of inheritance. Diagnosis commonly occurs only after clinical presentation. The recent identification of the RNF213 founder mutation (p.R4810K) in the Asian population has made a significant contribution, but the etiology of this disease remains unclear. To further develop the variant landscape of MMD, we performed high-depth whole exome sequencing of 125 unrelated, predominantly nonfamilial, ethnically diverse MMD patients in parallel with 125 internally sequenced, matched controls using the same exome and analysis platform. Three subpopulations were established: Asian, Caucasian, and non-RNF213 founder mutation cases. We provided additional support for the previously observed RNF213 founder mutation (p.R4810K) in Asian cases (P = 6.01×10−5) that was enriched among East Asians compared to Southeast Asian and Pacific Islander cases (P = 9.52×10−4) and was absent in all Caucasian cases. The most enriched variant in Caucasian (P = 7.93×10−4) and non-RNF213 founder mutation (P = 1.51×10−3) cases was ZXDC (p.P562L), a gene involved in MHC Class II activation. Collapsing variant methodology ranked OBSCN, a gene involved in myofibrillogenesis, as most enriched in Caucasian (P = 1.07×10−4) and non-RNF213 founder mutation cases (P = 5.31×10−5). These findings further support the East Asian origins of the RNF213 (p.R4810K) variant and more fully describe the genetic landscape of multiethnic MMD, revealing novel, alternative candidate variants and genes that may be important in MMD etiology and diagnosis.
doi:10.1534/g3.115.020321
PMCID: PMC4704723  PMID: 26530418
cerebrovascular disease; hemorrhage; RNF213; ZXDC; OBSCN
13.  Combined Endovascular and Microsurgical Management of Complex Cerebral Aneurysms 
Cerebral aneurysms are associated with a 50% mortality rate after rupture and patients can suffer significant morbidity during subsequent treatment. Neurosurgical management of both ruptured and unruptured aneurysms has evolved over the years. The historical practice of using microsurgical clipping to treat aneurysms has benefited in the last two decades from tremendous improvement in endovascular technology. Microsurgery and endovascular therapies are often viewed as competing treatments but it is important to recognize their individual limitations. Some aneurysms are considered complex, due to several factors such as aneurysm anatomy and a patient’s clinical condition. A complex aneurysm often cannot be completely excluded with a single approach and its successful treatment requires a combination of microsurgical and endovascular techniques. Planning such an approach relies on understanding aneurysm anatomy and thus should routinely include 3D angiographic imaging. In patients with ruptured aneurysms, endovascular coiling is a well-tolerated early treatment and residual aneurysms can be treated with intervals of definitive clipping. Microsurgical clipping also can be used to reconstruct the neck of a complex aneurysm, allowing successful placement of coils across a narrow neck. Endovascular techniques are assisted by balloons, which can be used in coiling and testing parent vessel occlusion before sacrifice. In some cases microsurgical bypasses can provide alternate flow for planned vessel sacrifice. We present current paradigms for combining endovascular and microsurgical approaches to treat complex aneurysms and share our experience in 67 such cases. A dual microsurgical–endovascular approach addresses the challenge of intracranial aneurysms. This combination can be performed safely and produces excellent rates of aneurysm obliteration. Hybrid angiographic operating-room suites can foster seamless and efficient complementary application of these two modalities.
doi:10.3389/fneur.2013.00108
PMCID: PMC3737456  PMID: 23964263
coil embolization; combined therapy; complex aneurysms; microsurgical clipping; revascularization bypass; vessel sacrifice
14.  Phosphorylated MAPK/ERK1/2 may not always represent its kinase activity in a rat model of focal cerebral ischemia with or without ischemic preconditioning 
Neuroscience  2012;209:155-160.
The ERK 1/2 protein require a dual phosphorylation at conserved threonine and tyrosine residues to be fully activated under normal physiological conditions. Thus, ERK1/2 kinase activity is often defined by the quantity of phosphorylated kinase. However, this may not accurately represent its true activity under certain pathological conditions. We investigated whether ERK1/2 kinase activity is proportional to its phosphorylation state in a rat focal ischemia model with and without rapid ischemic preconditioning. We showed that phosphorylated-ERK1/2 protein levels were increased 2.6±0.07 fold, and ERK1/2 kinase activity was increased 10.6±1.9 fold in animals receiving ischemic preconditioning alone without test ischemia compared with sham group (P<0.05, n=6/group), suggesting that phosphorylated-ERK1/2 protein levels represent its kinase activity under these conditions. However, preconditioning plus test ischemia robustly blocked ERK1/2 kinase activity, while it increased phosphorylated-ERK1/2 protein levels beyond those receiving test ischemia alone, suggesting that phosphorylated-ERK1/2 protein levels were not representative of actual kinase activity in this pathological condition. In conclusion, protein phosphorylation levels of ERK1/2 do not always correspond to kinase activity, thus, measuring the true kinase activity is essential.
doi:10.1016/j.neuroscience.2012.02.005
PMCID: PMC3322316  PMID: 22366512
ischemic preconditioning; kinase activity; MAPK; ERK1/2; focal ischemia; stroke
15.  Transplanted stem cell-secreted VEGF effects post-stroke recovery, inflammation, and vascular repair 
Stem cells (Dayton, Ohio)  2011;29(2):10.1002/stem.584.
Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after sub-acute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human VEGF (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair, and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier (BBB) integrity and suppression of inflammation was followed by a delayed spatio-temporal regulated increase in neovascularization. These events coincided with a bi-modal pattern of functional recovery: an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multi-modal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.
doi:10.1002/stem.584
PMCID: PMC3524414  PMID: 21732485
angiogenesis; blood brain barrier; dystroglycan; inflammation; Avastin
16.  Glioblastoma Multiforme: Exploratory Radiogenomic Analysis by Using Quantitative Image Features 
Radiology  2014;273(1):168-174.
We have demonstrated the potential of combining quantitative image features with advanced bioinformatics analysis to create radiogenomic maps providing molecular correlates of morphologic characteristics of glioblastoma multiforme.
Purpose
To derive quantitative image features from magnetic resonance (MR) images that characterize the radiographic phenotype of glioblastoma multiforme (GBM) lesions and to create radiogenomic maps associating these features with various molecular data.
Materials and Methods
Clinical, molecular, and MR imaging data for GBMs in 55 patients were obtained from the Cancer Genome Atlas and the Cancer Imaging Archive after local ethics committee and institutional review board approval. Regions of interest (ROIs) corresponding to enhancing necrotic portions of tumor and peritumoral edema were drawn, and quantitative image features were derived from these ROIs. Robust quantitative image features were defined on the basis of an intraclass correlation coefficient of 0.6 for a digital algorithmic modification and a test-retest analysis. The robust features were visualized by using hierarchic clustering and were correlated with survival by using Cox proportional hazards modeling. Next, these robust image features were correlated with manual radiologist annotations from the Visually Accessible Rembrandt Images (VASARI) feature set and GBM molecular subgroups by using nonparametric statistical tests. A bioinformatic algorithm was used to create gene expression modules, defined as a set of coexpressed genes together with a multivariate model of cancer driver genes predictive of the module’s expression pattern. Modules were correlated with robust image features by using the Spearman correlation test to create radiogenomic maps and to link robust image features with molecular pathways.
Results
Eighteen image features passed the robustness analysis and were further analyzed for the three types of ROIs, for a total of 54 image features. Three enhancement features were significantly correlated with survival, 77 significant correlations were found between robust quantitative features and the VASARI feature set, and seven image features were correlated with molecular subgroups (P < .05 for all). A radiogenomics map was created to link image features with gene expression modules and allowed linkage of 56% (30 of 54) of the image features with biologic processes.
Conclusion
Radiogenomic approaches in GBM have the potential to predict clinical and molecular characteristics of tumors noninvasively.
© RSNA, 2014
Online supplemental material is available for this article.
doi:10.1148/radiol.14131731
PMCID: PMC4263772  PMID: 24827998
17.  Stem Cells and Stroke: Opportunities, Challenges, and Strategies 
INTRODUCTION
Stroke remains the leading cause of disability in the Western world. Despite decades of work, no clinically effective therapies exist to facilitate recovery from stroke. Stem cells may have the potential to minimize injury and promote recovery after stroke.
AREAS COVERED
Transplanted stem cells have been shown in animal models to migrate to the injured region, secrete neurotrophic compounds, promote revascularization, enhance plasticity and regulate the inflammatory response, thereby minimizing injury. Endogenous neural stem cells also have a remarkable propensity to respond to injury. Under select conditions, subventricular zone progenitors may be mobilized to replace lost neurons. In response to focal infarcts, neuroblasts play important trophic roles to minimize neural injury. Importantly, these endogenous repair mechanisms may be experimentally augmented, leading to robust improvements in function. Ongoing clinical studies are now assessing the safety and feasibility of cell-based therapies for stroke.
EXPERT OPINION
We outline the unique challenges and potential pitfalls in the clinical translation of stem cell research for stroke. We then detail what we believe to be the specific basic science and clinical strategies needed to overcome these challenges, fill remaining gaps in knowledge and facilitate development of clinically viable stem cell-based therapies for stroke.
doi:10.1517/14712598.2011.552883
PMCID: PMC3087443  PMID: 21323594
stem cell; neurogenesis; neuroregeneration; stroke; ischemic brain injury; neural progenitor cell; neuroblast; neuroprotection; clinical trial; translational research; plasticity; subventricular zone; migration; differentiation
18.  Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain 
Brain  2011;134(6):1777-1789.
Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.
doi:10.1093/brain/awr094
PMCID: PMC3102243  PMID: 21616972
stroke; transplantation; brain rewiring; APP; dendrites
19.  Stepwise Recruitment of Transcellular and Paracellular Pathways Underlies Blood-Brain Barrier Breakdown in Stroke 
Neuron  2014;82(3):603-617.
SUMMARY
Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a novel transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 h post-stroke, TJs display profound structural defects only after two days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 h post-stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.
doi:10.1016/j.neuron.2014.03.003
PMCID: PMC4016169  PMID: 24746419
20.  Manufacturing neurons from human embryonic stem cells: biological and regulatory aspects to develop a safe cellular product for stroke cell therapy 
Regenerative medicine  2009;4(2):251-263.
Demographic trends, particularly those related to longer life expectancy, suggest that the demand for tissue and organ transplants will further increase since many disorders result from degeneration, injury or organ failure. The most urgent problem in transplantation medicine is the shortage or lack of suitable donor organs and tissue, leading to ethical and societal problems such as organ trafficking. The discovery of stem cells in the inner cell mass of developing embryos and in adult tissue has revolutionized the medical field by introducing new therapeutic dimensions to consider for previously untreatable diseases and injuries. The unlimited self-renewal ability and pluripotent capacity to become any cell type of the organism make human embryonic stem cells (hESCs) a compelling source of cells to study tissue histogenesis and to apply in a wide array of tissue engineering, cell transplantation therapy and drug discovery applications. In this article, we will focus on hESCs and address the derivation of therapeutic neural stem cell lines from hESCs, as well as the biological and regulatory aspects to developing a safe cellular product for stroke cell therapy.
doi:10.2217/17460751.4.2.251
PMCID: PMC4337782  PMID: 19317644
cell therapy; cGMP; good manufacturing practice; human embryonic stem cells; human neural stem cells; investigational new drug application; manufacturing neurons; master cell bank; neuroplasticity; product development; regulation of stem cell products; stroke
21.  Mouse model of focal cerebral ischemia using endothelin-1 
Journal of neuroscience methods  2008;173(2):286-290.
Intracerebral injection of the vasoconstrictor peptide, endothelin-1 (ET-1), has been used as a method to induce focal ischemia in rats. The relative technical simplicity of this model makes it attractive for use in mice. However, the effect of ET-1 on mouse brains has not been firmly established. In this study, we determined the ability of ET-1 to induce focal cerebral ischemia in four different mouse strains (CD1, C57/BL6, NOD/SCID, and FVB). In contrast to rats, intracerebral injection of ET-1 did not produce a lesion in any mouse strain tested. A combination of ET-1 injection with either CCA occlusion or N, G-nitro-L-arginine methyl ester (L-NAME) injection produced only a small infarct and its size was strain-dependent. A triple combination of CCA occlusion with co-injection of ET-1 and L-NAME produced a lesion in all mouse strains tested, and this resulted in a significant motor deficit. However, lesion size was still relatively small and strain-dependent. This study shows that ET-1 has a much less potent effect for producing an infarct in mice than rats.
doi:10.1016/j.jneumeth.2008.06.013
PMCID: PMC2572560  PMID: 18621079
Endothelin-1; focal ischemia; mouse; receptor; endothelial nitric oxide
22.  Viral caspase inhibitor p35, but not crmA, is neuroprotective in the ischemic penumbra following experimental stroke 
Neuroscience  2007;149(4):804-812.
Apoptosis, a predominant cause of neuronal death after stroke, can be executed in a caspase-dependent or apoptosis inducing factor (AIF)-dependent manner. Herpes Simplex Virus (HSV) vectors expressing caspase inhibitors p35 and crmA have been shown to be neuroprotective against various excitotoxic insults. Here we further evaluated the possible neuroprotective role of p35 and crmA in a rat stroke model. Overexpression of p35, but not crmA, significantly increased neuronal survival. Results of double immunofluorescence staining indicate that compared with neurons infected with crmA or control vectors, p35-infected neurons had less active caspase-3 expression, cytosolic cytochrome c and nuclear AIF translocation.
doi:10.1016/j.neuroscience.2007.07.030
PMCID: PMC2144739  PMID: 17945431
cerebral ischemia; apoptosis; caspase inhibitor; crmA; p35; gene therapy; stroke
23.  Moderate Hypothermia Inhibits Brain Inflammation and Attenuates Stroke-induced Immunodepression in Rats 
CNS neuroscience & therapeutics  2013;20(1):10.1111/cns.12160.
Summary
Aims
Stroke causes both brain inflammation and immunodepression. Mild to moderate hypothermia is known to attenuate brain inflammation but its role in stroke-induced immunodepression (SIID) of the peripheral immune system remains unknown. This study investigated the effects in rats of moderate intra-ischemic hypothermia on SIID and brain inflammation.
Methods
Stroke was induced in rats by permanent distal MCA occlusion combined with transient bilateral CCA occlusion while body temperature was reduced to 30°C. Real-time PCR, flow cytometry, in vitro T cell proliferation assays and confocal microscopy were used to study SIID and brain inflammation.
Results
Brief Intra-Ischemic hypothermia helped maintain certain leukocytes in the peripheral blood and spleen, and enhanced T cell proliferation in vitro and delayed-type hypersensitivity in vivo, suggesting that hypothermia reduces SIID. In contrast, in the brain, brief intra-Ischemic hypothermia inhibited mRNA expression of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines INF-γ, TNF-α, IL-2, IL-1β and MIP-2. Brief intra-Ischemic hypothermia also attenuated the infiltration of lymphocytes, neutrophils (MPO+ cells) and macrophages (CD68+ cells) into the ischemic brain, suggesting that hypothermia inhibited brain inflammation.
Conclusions
Brief intra-ischemic hypothermia attenuated SIID and protected against acute brain inflammation.
doi:10.1111/cns.12160
PMCID: PMC3867545  PMID: 23981596
focal cerebral ischemia; hypothermia; inflammation; immunodepression; leukocytes
24.  Human brain arteriovenous malformations express lymphatic-associated genes 
Objective
Brain arteriovenous malformations (AVMs) are devastating, hemorrhage-prone, cerebrovascular lesions characterized by well-defined feeding arteries, draining vein(s) and the absence of a capillary bed. The endothelial cells (ECs) that comprise AVMs exhibit a loss of arterial and venous specification. Given the role of the transcription factor COUP-TFII in vascular development, EC specification, and pathological angiogenesis, we examined human AVM tissue to determine if COUP-FTII may have a role in AVM disease biology.
Methods
We examined 40 human brain AVMs by immunohistochemistry (IHC) and qRT-PCR for the expression of COUP-TFII as well as other genes involved in venous and lymphatic development, maintenance, and signaling. We also examined proliferation and EC tube formation with human umbilical ECs (HUVEC) following COUP-TFII overexpression.
Results
We report that AVMs expressed COUP-TFII, SOX18, PROX1, NFATC1, FOXC2, TBX1, LYVE1, Podoplanin, and vascular endothelial growth factor (VEGF)-C, contained Ki67-positive cells and heterogeneously expressed genes involved in Hedgehog, Notch, Wnt, and VEGF signaling pathways. Overexpression of COUP-TFII alone in vitro resulted in increased EC proliferation and dilated tubes in an EC tube formation assay in HUVEC.
Interpretation
This suggests AVM ECs are further losing their arterial/venous specificity and acquiring a partial lymphatic molecular phenotype. There was significant correlation of gene expression with presence of clinical edema and acute hemorrhage. While the precise role of these genes in the formation, stabilization, growth and risk of hemorrhage of AVMs remains unclear, these findings have potentially important implications for patient management and treatment choice, and opens new avenues for future work on AVM disease mechanisms.
doi:10.1002/acn3.142
PMCID: PMC4284124  PMID: 25574473
25.  Revascularization of the Posterior Circulation 
Skull Base  2005;15(1):43-62.
ABSTRACT
The primary objective of revascularization procedures in the posterior circulation is the prevention of vertebrobasilar ischemic stroke. Specific anatomical and neurophysiologic characteristics such as posterior communicating artery size affect the susceptibility to ischemia. Current indications for revascularization include symptomatic vertebrobasilar ischemia refractory to medical therapy and ischemia caused by parent vessel occlusion as treatment for complex aneurysms. Treatment options include endovascular angioplasty and stenting, surgical endarterectomy, arterial reimplantation, extracranial-to-intracranial anastomosis, and indirect bypasses. Pretreatment studies including cerebral blood flow measurements with assessment of hemodynamic reserve can affect treatment decisions. Careful blood pressure regulation, neurophysiologic monitoring, and neuroprotective measures such as mild brain hypothermia can help minimize the risks of intervention. Microscope, microinstruments and intraoperative Doppler are routinely used. The superficial temporal artery, occipital artery, and external carotid artery can be used to augment blood flow to the superior cerebellar artery, posterior cerebral artery, posterior inferior cerebellar artery, or anterior inferior cerebellar artery. Interposition venous or arterial grafts can be used to increase length. Several published series report improvement or relief of symptoms in 60 to 100% of patients with a reduction of risk of future stroke and low complication rates.
doi:10.1055/s-2005-868162
PMCID: PMC1151703  PMID: 16148983
Vertebrobasilar ischemia; angioplasty; parent vessel occlusion; bypass; revascularization

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