Neuroglobin is a hypoxia-inducible O2-binding protein with neuroprotective effects in cell and animal models of stroke and Alzheimer’s disease. The mechanism underlying neuroglobin’s cytoprotective action is unknown, although several possibilities have been proposed, including antioxidative and antiapoptotic effects. We used affinity purification-mass spectrometry methods to identify neuroglobin-interacting proteins in normoxic and hypoxic murine neuronal (HN33) cell lysates, and to compare these interactions with those of a structurally and functionally related protein, myoglobin. We report that the protein interactomes of neuroglobin and myoglobin overlap substantially and are modified by hypoxia. In addition, neuroglobin-interacting proteins include partners consistent with both antioxidative and antiapoptotic functions, as well as with a relationship to several neurodegenerative diseases.
hypoxia; ischemia; myoglobin; neuroglobin; proteome
In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.
Vascular endothelial growth factor (VEGF) and neuroglobin (Ngb) participate in neuronal responses to hypoxia and ischemia, but the relationship between their effects, if any, is unknown. To address this issue, we measured Ngb levels in VEGF-treated mouse cerebrocortical cultures and VEGF levels in cerebrocortical cultures from Ngb-overexpressing transgenic mice. VEGF stimulated Ngb expression in a VEGFR2/Flk1 receptor-dependent manner, whereas Ngb overexpression suppressed expression of VEGF. These findings provide further insight into hypoxia-stimulated neuronal signaling pathways.
Vascular endothelial growth factor; neuroglobin; hypoxia
PTEN plays an important role not only in tumorigenesis but also in the normal development of central nervous system. PTEN loss in neural progenitor cells during embryogenesis disrupts migration and proper formation of the brain laminar structure. We generated a conditional PTEN knockout mouse by crossing mice that express Cre recombinase driven by the human GFAP promoter to a floxed PTEN gene to investigate the role of astroglial PTEN signaling pathway in neuronal patterning and lamination. We found PTEN loss not only in astroglial cells, but also in radial glia-derived neurons in hGFAP-Cre+/−/PTENloxp/loxp transgenic mice. Homozygous hGFAP-Cre+/−/PTENloxp/loxp transgenic mice showed progressive brain enlargement with cellular disorganization that occurred predominantly in hippocampus and cerebellum and died by postnatal day 20. Confocal images show that nestin-positive radial glial cells were observed in the hippocampus, cortex, and cerebellum at postnatal day 0 in homozygous hGFAP-Cre+/−/PTENloxp/loxp, but not in heterozygous hGFAP-Cre+/−/PTENloxp/− and hGFAP-Cre−/−/PTENloxp/loxp mice. Homozygous hGFAP-Cre+/−/PTENloxp/loxp transgenic mouse eyes, which lack radial glial lineage, were able to develop normal architectonics after birth. In addition, we also found that neuronal progenitor migration was defected at postnatal day 0 in homozygous hGFAP-Cre+/−/PTENloxp/loxp mice. These results suggest that PTEN has a critical role in regulating radial glial differentiation, proliferation, maturation, and eventually neuronal patterning in central nervous system in a spatio-temporal dependent manner.
PTEN; radial glia; neurogenesis; neuronal lamination
Clinical studies suggest a correlation between changes in activity of the contralesional cerebral cortex and spontaneous recovery from stroke, but whether this is a causal relationship is uncertain.
Young adult Sprague-Dawley male rats underwent unilateral or bilateral permanent distal middle cerebral artery occlusion (dMCAO). Infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 hr after dMCAO, and functional outcome was assessed 1–28 days after dMCAO using the ladder rung walking and limb placing tests.
Infarct volume was unchanged, but functional neurological deficits were reduced 1 day after bilateral compared to unilateral dMCAO.
Activity in the contralesional cerebral cortex may inhibit functional motor recovery acutely after experimental stroke.
Stroke; ischemia; recovery; rat
Vascular endothelial growth factor-B (VEGF-B) protects against experimental stroke, but the effect of stroke on VEGF-B expression is uncertain.
We examined VEGF-B expression by immunohistochemistry in the ischemic border zone 1–7 days after middle cerebral artery occlusion in rats.
VEGF-B immunoreactivity in the border zone was increased after middle cerebral artery occlusion and was associated with neurons and macrophages/microglia, but not astrocytes or endothelial cells.
These findings provide additional evidence for a role of VEGF-B in the endogenous response to cerebral ischemia.
Vascular endothelial growth factor-B (VEGF-B); Stroke; Ischemia
Neuroglobin (Ngb) is a hypoxia-inducible protein with cytoprotective effects in animal models of stroke, Alzheimer's disease, and related disorders, but the molecular mechanisms involved in its induction are unknown. We tested the hypothesis that hypoxia-inducible factor-1 (HIF-1) regulates Ngb levels, using shRNA-mediated knockdown and lentiviral vector-mediated overexpression of the HIF-1α subunit, in cultured neural (HN33) cells. HIF-1α knockdown decreased and HIF-1α overexpression increased Ngb levels, consistent with a connection between HIF-1 and Ngb induction. These findings may have implications for understanding the hypoxia-response repertoire of neural cells and devising therapeutic strategies for neurologic disorders.
neuroglobin; hypoxia; hypoxia-inducible factor-1; stroke
Depletion of neurogenesis worsens functional outcome in young-adult mice after focal cerebral ischemia, but whether a similar effect occurs in older mice is unknown. Using middle-aged (12-month-old) transgenic (DCX-TK(+)) mice that express herpes simplex virus thymidine kinase (HSV-TK) under control of the doublecortin (DCX) promoter, we conditionally depleted DCX-positive cells in the subventricular zone (SVZ) and hippocampus by treatment with ganciclovir (GCV) for 14 days. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery (MCAO) or occlusion of the distal segment of middle cerebral artery (dMCAO) on day 14 of vehicle or GCV treatment and mice were killed 24 hr or 12 weeks later. Increased infarct volume or brain atrophy was found in GCV- compared to vehicle-treated middle-aged DCX-TK(+) mice, both 24 hr after MCAO and 12 weeks after dMCAO. More severe motor deficits were also observed in GCV-treated, middle-aged DCX-TK(+) transgenic mice at both time points. Our results indicate that ischemia-induced newborn neurons contribute to anatomical and functional outcome after experimental stroke in middle-aged mice.
Background and Purpose
Interhemispheric inhibition via the corpus callosum has been proposed as an exacerbating factor in outcome from stroke.
We measured infarct volume and behavioral outcome following middle cerebral artery occlusion in callosotomized rats and acallosal mice.
Neither callosotomy in rats nor callosal agenesis in mice improved infarct volume or behavioral outcome after middle cerebral artery occlusion.
These findings argue against a role for transcallosal projections in exacerbating focal cerebral ischemia.
corpus callosum; stroke; ischemia; callosotomy; callosal agenesis
Supported by the International Society for Translational Medicine (ISTM), Wenzhou Medical College and the First Affiliated Hospital of Wenzhou Medical College, the International Conference on Translational Medicine (ICTM) was held on October 22–23, 2011 in Wenzhou, China. Nearly 800 registrants attended the meeting, primarily representing institutes and hospitals in Europe, The United States of America, And Asia, and China. The meeting was chaired and organized by Dr. Xiangdong Wang, Xiaoming Chen, Richard Coico, Jeffrey M. Drazen, Richard Horton, Francesco M. Marincola, Laurentiu M. Popescu, Jia Qu and Aamir Shahzad.
The meeting focused on the communication of the need to foster translational medicine (TM) by building and broadening bridges between basic research and clinical studies at the international level. The meeting included distinguished TM experts from academia, the pharmaceutical and diagnostics industries, government agencies, regulators, and clinicians and provided the opportunity to identify shared interests and efforts for collaborative approaches utilizing cutting edge technologies, innovative approaches and novel therapeutic interventions. The meeting defined the concept of TM in its two-way operational scheme and emphasized the need for bed to bench efforts based directly on clinical observation.
It was the meeting participants’ realization that the shared main goals of TM include breaking the separation between clinic practice and basic research, establishing positive feedback by understanding the basis of expected and unexpected clinical outcomes and accelerating basic research relevant to human suffering. The primary objectives of the meeting were two-fold: to accelerate the two-way translation by informing the participants representing the different disciplines about the state of art activities around TM approaches; and to identify areas that need to be supported by redirecting limited resources as well as identifying new sources of funding. This report summarizes key concepts presented during the meeting representing the state-of-art translational research and salient aspects of the ensuing discussions.
Translational Medicine (TM); International Society for Translational Medicine (ISTM); the International Conference on Translational Medicine (ICTM); Biomarkers; Biobank globalization and networking
Animal studies indicate that adult renal stem/progenitor cells can undergo rapid proliferation in response to renal injury, but whether the same is true in humans is largely unknown. To examine the profile of renal stem/progenitor cells responsible for acute tubular necrosis in human kidney, double- and triple-immunostaining was performed using proliferative marker and stem/progenitor protein markers on sections from ten kidneys with acute tubular necrosis and four normal adult kidneys. The immunopositive cells were recorded using two-photon confocal laser scanning microscopy. We found that dividing cells were present in the tubules of the cortex and medulla, as well as the glomerulus in normal human kidney. Proliferative cells in the parietal layer of Bowman’s capsule expressed CD133, and dividing cells in the tubules expressed immature cell protein markers paired box gene 2, vimentin and nestin. After acute tubular necrosis, Ki67-positive cells in the cortex tubules significantly increased compared to normal adult kidney. These Ki67-positive cells expressed CD133 and paired box gene 2, but not the cell death marker, activated caspase-3. In addition, the number of dividing cells increased significantly in patients with acute tubular necrosis, who subsequently recovered, compared to patients with acute tubular necrosis, who consequently developed protracted acute tubular necrosis or died. Our data suggest that renal stem/progenitor cells may reside not only in the parietal layer of Bowman’s capsule, but also in the cortex and medulla in normal human kidney, and the proliferative capacity of renal stem/progenitor cells after acute tubular necrosis may be an important determinant of a patient’s outcome.
ATN; kidney; progenitor cells; stem cells; outcome; proliferation
We reported previously that ablation of doublecortin (DCX)-immunopositive newborn neurons in mice worsens anatomical and functional outcome measured 1 day after experimental stroke, but whether this effect persists is unknown. We generated transgenic mice that express herpes simplex virus thymidine kinase under control of the DCX promoter (DCX-TK transgenic mice). DCX-expressing and recently divided cells in the rostral subventricular zone (SVZ) and hippocampus of DCX-TK transgenic mice, but not wild-type mice, were specifically depleted after ganciclovir (GCV) treatment for 14 days. Focal cerebral ischemia was induced by permanent distal middle cerebral artery occlusion (MCAO) on day 14 of vehicle or GCV treatment, and mice were killed 12 weeks after MCAO. Infarct volume was significantly increased and neurologic deficits were more severe in GCV- compared to vehicle-treated DCX-TK transgenic mice at first 8 weeks, after depletion of DCX- and bromodeoxyuridine-immunoreactive cells in the SVZ and dentate gyrus following focal ischemia. Our results indicate that endogenous neurogenesis in a critical period following experimental stroke influences the course of long-term recovery.
Little is known about the relationship between neuronal cell transplantation and endogenous neurogenesis after experimental stroke. We found previously that transplantation of neuronal precursors derived from BG01 human embryonic stem cells reduced infarct volume and improved behavioral outcome after distal middle cerebral artery occlusion (MCAO) in rats. In this study, transplantation was performed 14 d after distal MCAO and doublecortin (Dcx)-expressing cells in the subventricular zone (SVZ) and subgranular zone of dentate gyrus (SGZ) were counted 60 d post-transplant. Transplantation increased neurogenesis (Dcx expression) in ipsilateral SVZ, but not in contralateral SVZ or either SGZ, in both young adult (3 mo-old) and aged (24-mo-old) rats. These findings suggest that cell-based therapy for stroke may be associated with changes in endogenous adaptive processes, including neurogenesis.
Ischemia; stroke; transplantation; neurogenesis; subventricular zone
Neuroglobin (Ngb) is an intracellular, oxygen-binding neuronal protein with protective effects against ischemia and related pathological processes. To identify small molecules capable of inducing Ngb protein expression, which might have therapeutic benefit, we examined Ngb expression by Western blot in cultured HN33 (mouse hippocampal neuron × N18TG2 neuroblastoma) cells. In addition to deferoxamine, which was shown previously to enhance Ngb levels, Ngb expression was increased by the short-chain fatty acids cinnamic acid and valproic acid (≥100 μmol/l), but not by other short-chain fatty acids, histone deacetylase inhibitors, or anticonvulsants. Drugs that stimulate the expression of neuroprotective proteins like Ngb may have therapeutic potential in the treatment of stroke and other neurological disorders.
Neuroglobin; Cinnamic acid; Valproic acid; Ischemia
Surgery, radiotherapy and chemotherapy are universally recognized as the most effective anti-cancer therapies. Despite significant advances directed towards elucidating molecular mechanisms and developing clinical trials, cancer still remains a major public health issue. Recent studies have showed that cancer stem cells (CSCs), a small subpopulation of tumor cells, can generate bulk populations of nontumorigenic cancer cell progeny through the self-renewal and differentiation processes. As CSCs are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors, development of CSC-targeted therapeutic strategies holds new hope for improving survival and quality of life in patients with cancer. Therapeutic innovations will emerge from a better understanding of the biology and environment of CSCs, which, however, are largely unexplored. This review summarizes the characteristics, evidences and development of CSCs, as well as implications and challenges for cancer treatment.
cancer stem cells; differentiation; progenitor; signaling pathway; tumorigenesis
We reported previously that Notch signaling is activated in human arteriovenous malformations (AVMs) and that intracerebral hemorrhage (ICH) in humans is accompanied by increased neurogenesis. The former phenomenon may be involved in AVM pathogenesis and the latter in the brain’s response to ICH-induced injury. Here we describe increased expression of the hypoxia-inducible neuroprotective protein, neuroglobin (Ngb), in neurons surrounding unruptured AVMs and in the perihematomal region adjacent to ICH. In these disorders, as in other clinical settings, such as ischemic stroke, AVM- and ICHinduced overexpression of Ngb may be stimulated by ischemic hypoxia and may help to constrain brain injury.
Neuroglobin; Arteriovenous malformation; Intracerebral hemorrhage; Hypoxia; Ischemia
Neural precursor cell (NPC) transplantation may have a role in restoring brain function after stroke, but how aging might affect the brain’s receptivity to such transplants is unknown. We reported previously that transplantation of human embryonic stem cell (hESC)-derived NPCs together with biomaterial (Matrigel) scaffolding into the brains of young adult Sprague-Dawley rats 3 wks after distal middle cerebral artery occlusion (MCAO) reduced infarct volume, and improved neurobehavioral performance. In this study we compared the effect of NPC and Matrigel transplants in young adult (3-mo-old) and aged (24-mo-old) Fisher 344 rats from the National Institute on Aging’s aged rodent colony. Distal MCAO was induced by electrocoagulation and hESC-derived NPCs were transplanted into the infarct cavity 3 wks later. Aged rats developed larger infarcts, but infarct volume and performance on the cylinder and elevated body swing tests, measured 6–8 wks post-transplant, were improved by transplantation. We conclude that advanced age does not preclude a beneficial response to NPC and Matrigel transplantation following experimental stroke.
transplant; neural precursor; ischemia; stroke; brain
Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viral-mediated transduction of tracer genes in ESC or in ESC-derived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping β-actin promoter The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759–769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a high-expressing EGFP-mESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFP-mESC(G11) line may constitute a useful tool in ESC-based cell and tissue replacement studies.
The subventricular zone (SVZ) is a principal site of adult neurogenesis and appears to participate in the brain’s response to injury. Thus, measures that enhance SVZ neurogenesis may have a role in treatment of neurological disease. To better characterize SVZ cells and identify potential targets for therapeutic intervention, we studied electrophysiological properties of SVZ cells in adult mouse brain slices using patch-clamp techniques. Electrophysiology was correlated with immunohistochemical phenotype by injecting cells with lucifer yellow and by studying transgenic mice carrying green fluorescent protein under control of the doublecortin (DCX) or glial fibrillary acidic protein (GFAP) promoter. We identified five types of cells in the adult mouse SVZ: type 1 cells, with 4-aminopyridine (4-AP)/tetraethylammonium (TEA)-sensitive and CdCl2-sensitive inward currents; type 2 cells, with Ca2+-sensitive K+ and both 4-AP/TEA-sensitive and -insensitive currents; type 3 cells, with 4-AP/TEA-sensitive and -insensitive and small Na+ currents; type 4 cells, with slowly activating, large linear outward current and sustained outward current without fast-inactivating component; and type 5 cells, with a large outward rectifying current with a fast inactivating component. Type 2 and 3 cells expressed DCX, types 4 and 5 cells expressed GFAP, and type 1 cells expressed neither. We propose that SVZ neurogenesis involves a progression of electrophysiological cell phenotypes from types 4 and 5 cells (astrocytes) to type 1 cells (neuronal progenitors) to types 2 and 3 cells (nascent neurons), and that drugs acting on. ion channels expressed during neurogenesis might promote therapeutic neurogenesis in the injured brain.
doublecortin; potassium channels; sodium channels; neurogenesis; stroke
Transplantation of neural cells is a potential approach for stroke treatment, but disruption of tissue architecture may limit transplant efficacy. One strategy for enhancing the ability of transplants to restore brain structure and function is to administer cells together with biomaterial scaffolding. We electrocoagulated the distal middle cerebral artery in adult rats and, 3 weeks later, injected one of the following into the infarct cavity: artificial cerebrospinal fluid, Matrigel scaffolding, human embryonic stem cell-derived neuronal precursor cells, scaffolding plus cells, or cells cultured in and administered together with scaffolding. Five weeks after transplantation, the latter two groups showed ∼50% and ∼60% reductions, respectively, in infarct cavity volume. Rats given cells cultured in and administered together with scaffolding also showed (1) survival and neuronal differentiation of transplanted cells shown by immunostaining for neuronal marker proteins and cleaved caspase-3, and by patch-clamp recording, 8 weeks after transplantation and (2) improved outcome on tests of sensorimotor and cognitive functions, 4 to 9 weeks after transplantation. These results indicate that transplantation of human neural cells together with biomaterial scaffolding has the potential to improve the outcome from stroke, even when treatment is delayed for several weeks after the ischemic event.
ischemia; neurogenesis; neuronal progenitor cell; stem cells; stroke; transplantation
Background and Purpose
We investigated whether neuroglobin (Ngb), a neuronal protein that protects neurons from hypoxic-ischemic injury, is upregulated in ischemic stroke.
Ngb immunoreactivity was measured in brain tissue from control subjects and patients with ischemic stroke.
Ngb was detected in several brain areas and its expression was increased in the cortical peri-infarct region following stroke.
Ischemic stroke increases expression of the neuroprotective protein Ngb, suggesting Ngb may represent a novel target for stroke therapy.
neuroglobin; ischemia; neuroprotection
Despite recent advances in molecular biology and genetics, the mysteries that control human lifespan are yet to be unraveled. Many theories, which fall into two main categories: programmed and error theories, have been proposed to explain the process of aging, but neither of them appears to be fully satisfactory. These theories may interact with each other in a complex way. By understanding and testing the existing and new aging theories, it may be possible to promote successful aging.
Aging; Biological; Theory; Programmed; lifespan
A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch-1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch-1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch-1 ligands Jagged-1 and Delta-like-4 and the downstream Notch-1 target Hes-1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformations.
Notch-1; AVM; human; brain; signalling; angiogenesis
To explore the expression of Notch1 signaling pathway in nasopharyngeal carcinoma (NPC).
We performed immunocytochemistry on surgically resected NPC using antibodies against embryonic stem (ES) cell proteins and against Notch1 signaling components.
We found that ES cell protein markers SOX2 and OCT4 were expressed in a subpopulation of cells for all three subtypes of NPC but barely in the normal control. Double immunostaining shows that SOX2- and OCT4-positive cells coexpressed proliferative markers, suggesting that human NPC may contain cancer stem–like cells. In addition, we found that Notch1 signaling was activated in NPC. Confocal images show that the Notch1 signaling activated form and Hes1, a downstream target of Notch1 signaling, was predominantly found in SOX2- and OCT4-positive cells.
Our findings suggest that the Notch1 signaling pathway might be a regulator of cancer stem–like cells in NPC.
cancer stem cells; nasopharyngeal carcinoma; Notch1 signaling; OCT4; proliferation; SOX2
Adult neuronal stem cells (NSCs) hold great promise for brain repair because of their unique location within the central nervous system, their potential to proliferate and to differentiate into all major neural lineages, and their ability to functionally incorporate into existing neuronal circuitry after stroke. Nevertheless, the ability to exploit these cells for therapeutic purposes is hampered by the lack of knowledge about the signals that control the generation of a functional neuron from adult NSCs after stroke, particularly in the aged brain. Therefore, to further define the regulatory mechanisms that underlie neurogenesis after stroke, it is critically important to develop future NSC-based repair strategies. Notch signaling defines a fundamental pathway controlling cell fate acquisition. Studies have shown that Notch signaling pathways play critical roles during the maintenance, proliferation, and differentiation of NSCs in the developing brain. Recent evidence shows that Notch1 signaling is conserved in the regulation of adult neurogenesis. Here we summarize current knowledge about the role of Notch signaling in the regulation of neurogenesis in the normal and stroke brain.
Neurogenesis; adult; Notch signaling; SVZ; Stroke