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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  Neurosurgery 
Western Journal of Medicine  1993;158(1):64-65.
The Council on Scientific Affairs of the California Medical Association presents the following inventory of items of progress in neurosurgery. Each item, in the judgement of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome, and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, researchers, and scholars to stay abreast of these items of progress in neurosurgery that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another.
The items of progress listed below were selected by the Advisory Panel to the Section on Neurosurgery of the California Medical Association, and the summaries were prepared under its direction.
PMCID: PMC1021942  PMID: 8470387
14.  Defective sphingosine-1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation 
Nature immunology  2013;14(11):1166-1172.
Sphingosine-1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. Sphingosine phosphate receptor 1 (S1P1) agonist, FTY-720 (Gilenya™) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring a S1pr1 gene encoding phosphorylation-deficient receptors [S1P1(S5A)] developed severe experimental autoimmune encephalomyelitis (EAE) due to T helper (TH) 17-mediated autoimmunity in the peripheral immune and nervous system. S1P1 directly activated Janus-like kinase–signal transducer and activator of transcription 3 (JAK-STAT3) pathway via interleukin 6 (IL-6). Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.
doi:10.1038/ni.2730
PMCID: PMC4014310  PMID: 24076635
15.  Intraarterial transplantation of human umbilical cord blood mononuclear cells is more efficacious and safer compared with umbilical cord mesenchymal stromal cells in a rodent stroke model 
Introduction
Stroke is the second leading cause of death worldwide, claims six lives every 60 seconds, and is a leading cause of adult disability across the globe. Tissue plasminogen activator, the only United States Food and Drug Administration (FDA)-approved drug currently available, has a narrow therapeutic time window of less than 5 hours. In the past decade, cells derived from the human umbilical cord (HUC) have emerged as a potential therapeutic alternative for stroke; however, the most effective HUC-derived cell population remains unknown.
Methods
We compared three cell populations derived from the human umbilical cord: cord blood mononuclear cells (cbMNCs); cord blood mesenchymal stromal cells (cbMSCs), a subpopulation of cbMNCs; and cord matrix MSCs (cmMSCs). We characterized these cells in vitro with flow cytometry and assessed the cells’ in vivo efficacy in a 2-hour transient middle cerebral artery occlusion (MCAo) rat model of stroke. cbMNCs, cbMSCs, and cmMSCs were each transplanted intraarterially at 24 hours after stroke.
Results
A reduction in neurologic deficit and infarct area was observed in all three cell groups; however, this reduction was significantly enhanced in the cbMNC group compared with the cmMSC group. At 2 weeks after stroke, human nuclei-positive cells were present in the ischemic hemispheres of immunocompetent stroke rats in all three cell groups. Significantly decreased expression of rat brain-derived neurotrophic factor mRNA was observed in the ischemic hemispheres of all three cell-treated and phosphate-buffered saline (PBS) group animals compared with sham animals, although the decrease was least in cbMNC-treated animals. Significantly decreased expression of rat interleukin (IL)-2 mRNA and IL-6 mRNA was seen only in the cbMSC group. Notably, more severe complications (death, eye inflammation) were observed in the cmMSC group compared with the cbMNC and cbMSC groups.
Conclusions
All three tested cell types promoted recovery after stroke, but cbMNCs showed enhanced recovery and fewer complications compared with cmMSCs.
doi:10.1186/scrt434
PMCID: PMC4055161  PMID: 24690461
16.  Who’s in Favor of Translational Cell Therapy for Stroke: STEPS Forward Please? 
Cell transplantation  2009;18(7):691-693.
A consortium of translational stem cell and stroke experts from multiple academic institutes and biotechnology companies, under the guidance of the government (FDA/NIH), is missing. Here, we build a case for the establishment of this consortium if cell therapy for stroke is to advance from the laboratory to the clinic.
doi:10.3727/096368909X470883
PMCID: PMC3962837  PMID: 19796499
Stem cell transplantation; Tissue regeneration; Cellular therapy; Clinical translation
17.  δPKC mediates microcerebrovascular dysfunction in acute ischemia and in chronic hypertensive stress in vivo 
Brain research  2007;1144:146-155.
Maintaining cerebrovascular function is a priority for reducing damage following acute ischemic events such as stroke, and under chronic stress in diseases such as hypertension. Ischemic episodes lead to endothelial cell damage, deleterious inflammatory responses, and altered neuronal and astrocyte regulation of vascular function. These, in turn, can lead to impaired cerebral blood flow and compromised blood–brain barrier function, promoting microvascular collapse, edema, hemorrhagic transformation, and worsened neurological recovery. Multiple studies demonstrate that protein kinase C (PKC), a widely expressed serine/threonine kinase, is involved in mediating arterial tone and microvascular function. However, there is no clear understanding about the role of individual PKC isozymes. We show that intraperitoneal injection of δV1-1–TAT47–57 (0.2 mg/kg in 1 mL), an isozymespecific peptide inhibitor of δPKC, improved microvascular pathology, increased the number of patent microvessels by 92% compared to control-treated animals, and increased cerebral blood flow by 26% following acute focal ischemia induced by middle cerebral artery occlusion in normotensive rats. In addition, acute delivery of δV1-1–TAT47–57 in hypertensive Dahl rats increased cerebral blood flow by 12%, and sustained delivery δV1-1–TAT47–57 (5 uL/h, 1 mM), reduced infarct size by 25% following an acute stroke induced by MCA occlusion for 90 min. Together, these findings demonstrate that δPKC is an important therapeutic target for protection of microvascular structure and function under both acute and chronic conditions of cerebrovascular stress.
doi:10.1016/j.brainres.2007.01.113
PMCID: PMC3742377  PMID: 17350602
Cerebral blood flow; Hypertension; Microvasculature; Protein kinase C; Stroke; Vasculature
18.  Distinctive effects of T cell subsets in neuronal injury induced by co-cultured splenocytes in vitro and by in vivo stroke in mice 
Background and purpose
T cells and their subsets modulate ischemic brain injury. We studied the effects of the absence of T cell subsets on brain infarction after in vivo stroke and then used an in vitro co-culture system of splenocytes and neurons to further identify the roles of T cell subsets in neuronal death.
Methods
Stroke was induced by MCA suture occlusion in mice and infarct sizes were measured 2 days post-stroke.
Splenocytes were co-cultured with neurons, and neuronal survival was measured 3 days later.
Results
A deficiency of both T and B cells (SCID) and the paucity of CD4 or CD8 T cells equally resulted in smaller infarct sizes as measured 2 days post-stroke. Although a functional deficiency of regulatory T cells had no effect, impaired Th1 immunity reduced infarction and impaired Th2 immunity aggravated brain injury, which may be due to an inhibited and enhanced inflammatory response in mice deficient in Th1 and Th2 immunity, respectively. In the in vitro co-culture system, WT splenocytes resulted in dose-dependent neuronal death. The neurotoxicity of splenocytes from the above immunodeficient mice was consistent with their effects on stroke in vivo , except for the mice with the paucity of CD4 or CD8 T cells, which did not alter the ratio of neuronal death.
Conclusion
T cell subsets play critical roles in brain injury induced by stroke. The detrimental versus beneficial effects of Th1 cells and Th2 cells both in vivo and in vitro reveal differential therapeutic target strategies for stroke treatment.
doi:10.1161/STROKEAHA.112.656611
PMCID: PMC3506376  PMID: 22678086
cerebral ischemia; stroke; T cells; Th1; Th2
19.  CGS 19755 (Selfotel): A Novel Neuroprotective Agent Against CNS Injury 
CNS drug reviews  2006;2(3):257-268.
The hypothesis that excitoxicity is a mechanism of damage following different types of cerebral injury including global and focal ischemia (34), and head and spinal cord trauma (6,7,9,25) has been supported by numerous findings. During ischemia for example, glutamate neurotoxicity is mediated in part through N-methyl-D-aspartate (NMDA) receptors, since selective antagonists to this receptor protect against hypoxic-ischemic injury (10,35,41). In the last few years, different NMDA antagonists have been developed and tested; they can be divided into competitive and noncompetitive antagonists. Noncompetitive NMDA antagonists are extremely lipophilic and reach high levels in the brain after systemic administration. Various studies have demonstrated that these agents provide neuroprotection against hypoxic-ischemic injury (for review see ref. 29).
Many competitive NMDA antagonists are hydrophilic and require direct cerebral administration to obtain high brain levels. Newer competitive NMDA blockers, such as cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755, selfotel), provide neuroprotection against global ischemia, focal ischemia, and trauma when given systemically (2,3,32,33). Selfotel is currently being studied in multicenter safety and efficacy trials for stroke (17) and head trauma (6).
doi:10.1111/j.1527-3458.1996.tb00301.x
PMCID: PMC3678965  PMID: 23766625
Cerebral ischemia; Excitotoxicity; Glutamate; NMDA; Trauma; Anoxia; Neurotoxicity; Neuroprotection; CGS 19755; Selfotel
20.  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
21.  Derivation of Injury-Responsive Dendritic Cells for Acute Brain Targeting and Therapeutic Protein Delivery in the Stroke-Injured Rat 
PLoS ONE  2013;8(4):e61789.
Research with experimental stroke models has identified a wide range of therapeutic proteins that can prevent the brain damage caused by this form of acute neurological injury. Despite this, we do not yet have safe and effective ways to deliver therapeutic proteins to the injured brain, and this remains a major obstacle for clinical translation. Current targeted strategies typically involve invasive neurosurgery, whereas systemic approaches produce the undesirable outcome of non-specific protein delivery to the entire brain, rather than solely to the injury site. As a potential way to address this, we developed a protein delivery system modeled after the endogenous immune cell response to brain injury. Using ex-vivo-engineered dendritic cells (DCs), we find that these cells can transiently home to brain injury in a rat model of stroke with both temporal and spatial selectivity. We present a standardized method to derive injury-responsive DCs from bone marrow and show that injury targeting is dependent on culture conditions that maintain an immature DC phenotype. Further, we find evidence that when loaded with therapeutic cargo, cultured DCs can suppress initial neuron death caused by an ischemic injury. These results demonstrate a non-invasive method to target ischemic brain injury and may ultimately provide a way to selectively deliver therapeutic compounds to the injured brain.
doi:10.1371/journal.pone.0061789
PMCID: PMC3627911  PMID: 23613937
22.  Immune response profiling identifies autoantibodies specific to Moyamoya patients 
Background
Moyamoya Disease is a rare, devastating cerebrovascular disorder characterized by stenosis/occlusion of supraclinoid internal carotid arteries and development of fragile collateral vessels. Moyamoya Disease is typically diagnosed by angiography after clinical presentation of cerebral hemorrhage or ischemia. Despite unclear etiology, previous reports suggest there may be an immunological component.
Methods
To explore the role of autoimmunity in moyamoya disease, we used high-density protein arrays to profile IgG autoantibodies from the sera of angiographically-diagnosed Moyamoya Disease patients and compared these to healthy controls. Protein array data analysis followed by bioinformatics analysis yielded a number of auto-antibodies which were further validated by ELISA for an independent group of MMD patients (n = 59) and control patients with other cerebrovascular diseases including carotid occlusion, carotid stenosis and arteriovenous malformation.
Results
We identified 165 significantly (p < 0.05) elevated autoantibodies in Moyamoya Disease, including those against CAMK2A, CD79A and EFNA3. Pathway analysis associated these autoantibodies with post-translational modification, neurological disease, inflammatory response, and DNA damage repair and maintenance. Using the novel functional interpolating single-nucleotide polymorphisms bioinformatics approach, we identified 6 Moyamoya Disease-associated autoantibodies against APP, GPS1, STRA13, CTNNB1, ROR1 and EDIL3. The expression of these 6 autoantibodies was validated by custom-designed reverse ELISAs for an independent group of Moyamoya Disease patients compared to patients with other cerebrovascular diseases.
Conclusions
We report the first high-throughput analysis of autoantibodies in Moyamoya Disease, the results of which may provide valuable insight into the immune-related pathology of Moyamoya Disease and may potentially advance diagnostic clinical tools.
doi:10.1186/1750-1172-8-45
PMCID: PMC3648437  PMID: 23518061
Autoantibodies; Cerebrovascular disease; Moyamoya; Protein microarray
23.  Long-term behavioral assessment of function in an experimental model for ischemic stroke 
Journal of neuroscience methods  2011;196(2):247-257.
Middle cerebral artery occlusion (MCAO) in rats is a well-studied experimental model for ischemic stroke leading to brain infarction and functional deficits. Many preclinical studies have focused on a small time window after the ischemic episode to evaluate functional outcome for screening therapeutic candidates. Short evaluation periods following injury have led to significant setbacks due to lack of information on the delayed effects of treatments, as well as short-lived and reversible neuroprotection, so called false-positive results. In this report, we evaluated long-term functional deficit for 90 days after MCAO in two rat strains with two durations of ischemic insult, in order to identify the best experimental paradigm to assess injury and subsequent recovery. Behavioral outcomes were measured pre-MCAO followed by weekly assessment post-stroke. Behavioral tests included the 18-point composite neurological score, 28-point neuroscore, rearing test, vibrissae-evoked forelimb placing test, foot fault test and the CatWalk. Brain lesions were assessed to correlate injury to behavior outcomes at the end of study. Our results indicate that infarction volume in Sprague-Dawley rats was dependent on occlusion duration. In contrast, the infarction volume in Wistar rats did not correlate with the duration of ischemic episode. Functional outcomes were not dependent on occlusion time in either strain; however, measureable deficits were detectable long-term in limb asymmetry, 18- and 28-point neuroscores, forelimb placing, paw swing speed, and gait coordination. In conclusion, these behavioral assays, in combination with an extended long-term assessment period, can be used for evaluating therapeutic candidates in preclinical models of ischemic stroke.
doi:10.1016/j.jneumeth.2011.01.010
PMCID: PMC3539723  PMID: 21256866
Ischemic stroke; CatWalk; long-term functional recovery; middle cerebral artery occlusion; rat strain
24.  Gene expression profiling of blood in brain arteriovenous malformation patients 
Translational stroke research  2011;2(4):575-587.
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. Gene expression profiling of blood has led to the identification of stroke biomarkers, and may help identify BAVM biomarkers and illuminate BAVM pathogenesis. It is unknown whether blood gene expression profiles differ between 1) BAVM patients and healthy controls, or 2) unruptured and ruptured BAVM patients at presentation. We characterized blood transcriptional profiles in 60 subjects (20 unruptured BAVM, 20 ruptured BAVM, and 20 healthy controls) using Affymetrix whole genome expression arrays. Expression differences between groups were tested by ANOVA, adjusting for potential confounders. Genes with absolute fold change ≥ 1.2 (false discovery rate corrected p ≤ 0.1) were selected as differentially expressed and evaluated for over-representation in KEGG biological pathways (p ≤ 0.05). Twenty-nine genes were differentially expressed between unruptured BAVM patients and controls, including 13 which may be predictive of BAVM. Patients with ruptured BAVM compared to unruptured BAVM differed in expression of 1490 genes, with over-representation of genes in 8 pathways including MAPK, VEGF, Wnt signaling and several inflammatory pathways. These results suggest clues to the pathogenesis of BAVM and/or BAVM rupture and point to potential biomarkers or new treatment targets.
doi:10.1007/s12975-011-0103-3
PMCID: PMC3241209  PMID: 22184505
arteriovenous malformation; blood; gene expression; intracranial hemorrhage; microarray analysis
25.  Arterial Spin-Labeling MRI Can Identify the Presence and Intensity of Collateral Perfusion in Patients With Moyamoya Disease 
Background and Purpose
Determining the presence and adequacy of collateral blood flow is important in cerebrovascular disease. Therefore, we explored whether a noninvasive imaging modality, arterial spin labeling (ASL) MRI, could be used to detect the presence and intensity of collateral flow using digital subtraction angiography (DSA) and stable xenon CT cerebral blood flow as gold standards for collaterals and cerebral blood flow, respectively.
Methods
ASL and DSA were obtained within 4 days of each other in 18 patients with Moyamoya disease. Two neurointerventionalists scored DSA images using a collateral grading scale in regions of interest corresponding to ASPECTS methodology. Two neuroradiologists similarly scored ASL images based on the presence of arterial transit artifact. Agreement of ASL and DSA consensus scores was determined, including kappa statistics. In 15 patients, additional quantitative xenon CT cerebral blood flow measurements were performed and compared with collateral grades.
Results
The agreement between ASL and DSA consensus readings was moderate to strong, with a weighted kappa value of 0.58 (95% confidence interval, 0.52–0.64), but there was better agreement between readers for ASL compared with DSA. Sensitivity and specificity for identifying collaterals with ASL were 0.83 (95% confidence interval, 0.77–0.88) and 0.82 (95% confidence interval, 0.76–0.87), respectively. Xenon CT cerebral blood flow increased with increasing DSA and ASL collateral grade (P<0.05).
Conclusions
ASL can noninvasively predict the presence and intensity of collateral flow in patients with Moyamoya disease using DSA as a gold standard. Further study of other cerebrovascular diseases, including acute ischemic stroke, is warranted.
doi:10.1161/STROKEAHA.111.616466
PMCID: PMC3164217  PMID: 21799169
angiography; arterial spin labeling; cerebral blood flow; cerebral hemodynamics; cerebrovascular disease; collateral flow; neuroradiology; perfusion

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