VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF165 (rhVEGF165) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF165 to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF165 to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF165 to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.
In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non-neurogenic regions of the ischemic brain of adult rodents. The present study demonstrated that post-insult treatment with an HDAC inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo-2′-deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid-neural cell adhesion molecule (PSA-NCAM), nestin, GFAP, phospho-CREB, and brain-derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co-localization of BrdU and PSA-NCAM was observed in multiple regions after ischemia, and SB treatment upregulated protein levels of BDNF, phospho-CREB and GFAP. Intraventricular injection of K252a, a TrkB receptor antagonist, markedly reduced SB-induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG and other brain regions, blocked SB-induced nestin expression and CREB activation, and attenuated the long-lasting behavioral benefits of SB. Together, these results suggest that HDAC inhibitor-induced cell proliferation, migration and differentiation require BDNF-TrkB signaling and may contribute to SB’s long-term beneficial effects after ischemic injury.
HDAC inhibitors; cerebral ischemia; neurogenesis; BrdU; BDNF; K252a
Increasing evidence has shown the potential of neuronal plasticity in adult brain after injury. Neural proliferation can be triggered by a focal sublethal ischemic preconditioning event; whether mild global ischemia could cause neurogenesis has been not clear. The present study investigated stimulating effects of sublethal transient global ischemia (TGI) on endogenous neurogenesis and neuroblast migration in the subventricular zone (SVZ), dentate gyrus, and peri-infarct areas of the adult cortex. Adult mice of 129S2/Sv strain were subjected to 8-min bilateral common carotid artery ligation followed by 5-bromo-2′-deoxyuridine (BrdU; 50 mg/kg, intraperitoneal) administration every day until being sacrificed at 1–21 days after reperfusion. The mild TGI did not induce neuronal cell death for up to 7 days after TGI, as evidenced by negative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining among NeuN-positive cells in the hippocampus and neocortex. In TGI animals, BrdU staining revealed enhanced proliferation of neuroblasts and their migration track from the SVZ into the striatum and neocortex. In the corpus callosum, there were more BrdU-positive cells in the TGI group in the first 2 days. Increasing numbers of BrdU-positive cells were seen 7–21 days later in the striatum and cortex of TGI mice. The cortex of TGI animals showed increased expression of erythropoietin, erythropoietin receptor, fibroblast growth factor 2, vascular endothelial growth factor, and phosphorylated Jun N-terminal kinase; the expression was peaked 2 to 3 days after reperfusion. BrdU and NeuN double staining in the dentate gyrus, striatum, and cortex implied increased neurogenesis induced by the TGI preconditioning. Doublecortin (DCX)-positive cells increased in the cortex of TGI mice, localized to cortical layers II, III, and V, and many stained positive for the mature neuronal markers NeuN, neurofilament, N-methyl-d-aspartic acid receptor subunit gene NR1, or the gamma-aminobutyric-acid-synthesizing enzyme glutamic acid decarboxylase (GAD67). The atypical localization of DCX-positive cells and the colabeling with mature neuronal markers suggested that, in addition to indentifying migrating neuroblasts, DCX might also be a stress marker in the cortex. It is suggested that the sublethal TGI-induced regenerative responses may contribute to the beneficial effects of ischemic preconditioning.
Transient global ischemia; Neuroblasts; Neurogenesis; Cell migration; Doublecortin (DCX); Subventricular zone (SVZ)
The observed age-related decline in neurogenesis may result from reduced proliferation or increased death rate of neuronal precursor cells (NPCs). We found that caspase-3, but not caspase-6, -7, or -9, was activated in NPCs in neurogenic regions of young, young-adult, middle-aged and aged rat brains. The number of capase-3-immunoreactive cells was highest in young and lowest in aged rats. Surprisingly, intraventricular administration of a caspase-3 inhibitor failed to restore the number of BrdU-positive cells in the aged dentate gyrus, suggesting that the age-related decline in neurogenesis may be attributable primarily to reduced proliferation. Additionally, we also found that NPCs in the subventricular zone of young-adult and aged rat brain were increased after focal cerebral ischemia, suggesting that the increase in neurogenesis induced by ischemia may result from an increase in the rate of NPC proliferation, but not from a decrease in NPC death. Thus, our results suggest that age-related and injury-induced changes in the rate of neurogenesis are controlled at the level of NPC proliferation. Furthermore, our results may imply that the mechanisms that maintain a stable population of NPCs in the normal adult and in the ischemic brain, which account for the observed age-dependent reduction or injury-induced increases in neurogenesis, impinge on the regulation of cell division at the NPC level.
Neurogenesis; aging; cell death; ischemia; dentate gyrus; subventricular zone
The induction of angiogenesis will stimulate endogenous recovery mechanisms, which are involved in the long-term repair and restoration process of the brain after an ischemic event. Here, we tested whether exercise influences the pro-angiogenic factors and outcomes after cerebral infarction in rats. Wistar rats were exposed to two hours of middle-cerebral artery occlusion and reperfusion. Different durations of treadmill training were performed on the rats. The expression of matrix metalloproteinase 2 (MMP2) and vascular endothelial growth factor (VEGF)-related genes and proteins were higher over time post-ischemia, and exercise enhanced their expression. Sixteen days post-ischemia, the regional cerebral blood flow in the ischemic striatum was significantly increased in the running group over the sedentary. Although no difference was seen in infarct size between the running and sedentary groups, running evidently improved the neurobehavioral score. The effects of running on MMP2 expression, regional cerebral blood flow and outcome were abolished when animals were treated with bevacizumab (BEV), a VEGF-targeting antibody. Exercise therapy improves long-term stroke outcome by MMP2-VEGF-dependent mechanisms related to improved cerebral blood flow.
treadmill training; stroke; vascular endothelial growth factor; matrix metalloproteinase 2; bevacizumab
Vascular endothelial growth factor (VEGF) is thought to contribute to both neuroprotection and angiogenesis after stroke. While increased expression of VEGF has been demonstrated in animal models after experimental ischemia, these studies have focused almost exclusively on the infarct and peri-infarct regions. The present study investigated the association of VEGF to neurons in remote cortical areas at three days after an infarct in primary motor cortex (M1). Although these remote areas are outside of the direct influence of the ischemic injury, remote plasticity has been implicated in recovery of function. For this study, intracortical microstimulation techniques identified primary and premotor cortical areas in a non-human primate. A focal ischemic infarct was induced in the M1 hand representation, and neurons and VEGF protein were identified using immunohistochemical procedures. Stereological techniques quantitatively assessed neuronal-VEGF association in the infarct and peri-infarct regions, M1 hindlimb, M1 orofacial, and ventral premotor hand representations, as well as non-motor control regions. The results indicate that VEGF protein significantly increased association to neurons in specific remote cortical areas outside of the infarct and peri-infarct regions. The increased association of VEGF to neurons was restricted to cortical areas that are functionally and/or behaviorally related to the area of infarct. There was no significant increase in M1 orofacial region or in non-motor control regions. We hypothesize that enhancement of neuronal VEGF in these functionally related remote cortical areas may be involved in recovery of function after stroke, through either neuroprotection or the induction of remote angiogenesis.
VEGF (vascular endothelial growth factor); neuron; stroke; focal cerebral ischemia; stereology; neuroprotection
Following focal cerebral ischemia, blood vessels in the ischemic border, or penumbra, launch an angiogenic response. In light of the critical role for fibronectin in angiogenesis, and the observation that fibronectin and its integrin receptors are strongly upregulated on angiogenic vessels in the hypoxic CNS, the aim of this study was to establish whether angiogenic vessels in the ischemic CNS also show this response. Focal cerebral ischemia was established in C57/Bl6 mice by middle cerebral artery occlusion (MCA:O), and brain tissue analyzed seven days following re-perfusion, a time at which angiogenesis is ongoing. Within the ischemic core, immunofluorescent (IF) studies demonstrated vascular expression of MECA-32, a marker of leaky cerebral vessels, and vascular breakdown, defined by loss of staining for the endothelial marker, CD31, and the vascular adhesion molecules, laminin, dystroglycan and α6 integrin. Within the ischemic penumbra, dual-IF with CD31 and Ki67 revealed the presence of proliferating endothelial cells, indicating ongoing angiogenesis. Significantly, vessels in the ischemic penumbra showed strong upregulation of fibronectin and the fibronectin receptors, α5β1 and αvβ3 integrins. Taken together with our recent finding that the α5β1 integrin plays an important role in promoting cerebral angiogenesis in response to hypoxia, these results suggest that stimulation of the fibronectin-α5β1 integrin signalling pathway may provide a novel approach to amplifying the intrinsic angiogenic response to cerebral ischemia.
cerebral ischemia; angiogenesis; endothelial cells; integrin; fibronectin; laminin; astrocyte
Neural stem cells persist in the adult mammalian forebrain and are a potential source of neurons for repair after brain injury. The two main areas of persistent neurogenesis, the subventricular zone (SVZ)-olfactory bulb pathway and hippocampal dentate gyrus, are stimulated by brain insults such as stroke or trauma. Here we focus on the effects of focal cerebral ischemia on SVZ neural progenitor cells in experimental stroke, and the influence of mechanical injury on adult hippocampal neurogenesis in models of traumatic brain injury (TBI). Stroke potently stimulates forebrain SVZ cell proliferation and neurogenesis. SVZ neuroblasts are induced to migrate to the injured striatum, and to a lesser extent to the peri-infarct cortex. Controversy exists as to the types of neurons that are generated in the injured striatum, and whether adult-born neurons contribute to functional restoration remains uncertain. Advances in understanding the regulation of SVZ neurogenesis in general, and stroke-induced neurogenesis in particular, may lead to improved integration and survival of adult-born neurons at sites of injury. Dentate gyrus cell proliferation and neurogenesis similarly increase after experimental TBI. However, pre-existing neuroblasts in the dentate gyrus are vulnerable to traumatic insults, which appear to stimulate neural stem cells in the SGZ to proliferate and replace them, leading to increased numbers of new granule cells. Interventions that stimulate hippocampal neurogenesis appear to improve cognitive recovery after experimental TBI. Transgenic methods to conditionally label or ablate neural stem cells are beginning to further address critical questions regarding underlying mechanisms and function significance of neurogenesis after stroke or TBI. Future therapies should be aimed at directing appropriate neuronal replacement after ischemic or traumatic injury while suppressing aberrant integration that may contribute to co-morbidities such as epilepsy or cognitive impairment.
It is well known that focal ischemia increases neurogenesis in the adult dentate gyrus of the hippocampal formation but the cellular mechanisms underlying this proliferative response are only poorly understood. We here investigated whether precursor cells which constitutively proliferate before the ischemic infarct contribute to post-ischemic neurogenesis. To this purpose, transgenic mice expressing green fluorescent protein (GFP) under the control of the nestin promoter received repetitive injections of the proliferation marker bromodeoxyuridine (BrdU) prior to induction of cortical infarcts. We then immunocytochemically analyzed the fate of these BrdU-positive precursor cell subtypes from day 4 to day 28 after the lesion.
Quantification of BrdU-expressing precursor cell populations revealed no alteration in number of radial glia-like type 1 cells but a sequential increase of later precursor cell subtypes in lesioned animals (type 2a cells at day 7, type 3 cells/immature neurons at day 14). These alterations result in an enhanced survival of mature neurons 4 weeks postinfarct.
Focal cortical infarcts recruit dentate precursor cells generated already before the infarct and significantly contribute to an enhanced neurogenesis. Our findings thereby increase our understanding of the complex cellular mechanisms of postlesional neurogenesis.
The effect of neurotrophic factors in enhancing stroke-induced neurogenesis in the adult subventricular zone (SVZ) is limited by their poor blood-brain barrier (BBB) permeability.
Intranasal administration is a noninvasive and valid method for delivery of neuropeptides into the brain, to bypass the BBB. We investigated the effect of treatment with intranasal transforming growth factor-β1 (TGF-β1) on neurogenesis in the adult mouse SVZ following focal ischemia. The modified Neurological Severity Scores (NSS) test was used to evaluate neurological function, and infarct volumes were determined from hematoxylin-stained sections. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling was performed at 7 days after middle cerebral artery occlusion (MCAO). Immunohistochemistry was used to detect bromodeoxyuridine (BrdU) and neuron- or glia-specific markers for identifying neurogenesis in the SVZ at 7, 14, 21, 28 days after MCAO.
Intranasal treatment of TGF-β1 shows significant improvement in neurological function and reduction of infarct volume compared with control animals. TGF-β1 treated mice had significantly less TUNEL-positive cells in the ipsilateral striatum than that in control groups. The number of BrdU-incorporated cells in the SVZ and striatum was significantly increased in the TGF-β1 treated group compared with control animals at each time point. In addition, numbers of BrdU- labeled cells coexpressed with the migrating neuroblast marker doublecortin (DCX) and the mature neuronal marker neuronal nuclei (NeuN) were significantly increased after intranasal delivery of TGF-β1, while only a few BrdU labeled cells co-stained with glial fibrillary acidic protein (GFAP).
Intranasal administration of TGF-β1 reduces infarct volume, improves functional recovery and enhances neurogenesis in mice after stroke. Intranasal TGF-β1 may have therapeutic potential for cerebrovascular disorders.
Cell therapy with endothelial progenitor cells (EPCs) has emerged as a promising strategy to regenerate the brain after stroke. Here, we aimed to investigate if treatment with EPCs or their secreted factors could potentiate angiogenesis and neurogenesis after permanent focal cerebral ischemia in a mouse model of ischemic stroke. BALB/C male mice were subjected to distal occlusion of the middle cerebral artery, and EPCs, cell-free conditioned media (CM) obtained from EPCs, or vehicle media were administered one day after ischemia. Magnetic resonance imaging (MRI) was performed at baseline to confirm that the lesions were similar between groups. Immunohistochemical and histological evaluation of the brain was performed to evaluate angio-neurogenesis and neurological outcome at two weeks. CM contained growth factors, such as VEGF, FGF-b and PDGF-bb. A significant increase in capillary density was noted in the peri-infarct areas of EPC- and CM-treated animals. Bielschowsky’s staining revealed a significant increase in axonal rewiring in EPC-treated animals compared with shams, but not in CM-treated mice, in close proximity with DCX-positive migrating neuroblasts. At the functional level, post-ischemia forelimb strength was significantly improved in animals receiving EPCs or CM, but not in those receiving vehicle media. In conclusion, we demonstrate for the first time that the administration of EPC-secreted factors could become a safe and effective cell-free option to be considered in future therapeutic strategies for stroke.
Activation of endogenous stem cells has been proposed as a novel form of therapy in a variety of neurologic disorders including traumatic brain injury (TBI). Vascular endothelial growth factor (VEGF) is expressed in the brain after TBI and serves as a potent activator of angiogenesis and neurogenesis. In this study, we infused exogenous VEGF into the lateral ventricles of mice for 7 days after TBI using mini-osmotic pumps to evaluate the effects on recovery and functional outcome. The results of our study show that VEGF significantly increases the number of proliferating cells in the subventricular zone and in the perilesion cortex. Fate analysis showed that most newborn cells differentiated into astrocytes and oligodendroglia and only a few cells differentiated into neurons. Functional outcome was significantly better in mice treated with VEGF compared with vehicle-treated animals after TBI. Injury size was significantly smaller at 90 days after TBI in VEGF-treated animals, suggesting additional neuroprotective effects of VEGF. In conclusion, VEGF significantly augments neurogenesis and angiogenesis and reduces lesion volumes after TBI. These changes are associated with significant improvement in recovery rates and functional outcome.
angiogenesis; neurogenesis; neuroprotection; traumatic brain injury; vascular endothelial growth factor
Cerebral ischemia stimulates neurogenesis in proliferative zones of the rodent forebrain. To identify the signaling factors involved, cerebral cortical cultures prepared from embryonic mouse brains were deprived of oxygen. Hypoxia increased bromodeoxyuridine (BrdU) incorporation into cells that expressed proliferation markers and immature neuronal markers and that lacked evidence of DNA damage or caspase-3 activation. Hypoxia-conditioned medium and stem cell factor (SCF), which was present in hypoxia-conditioned medium at increased levels, also stimulated BrdU incorporation into normoxic cultures. The SCF receptor, c-kit, was expressed in neuronal cultures and in neuroproliferative zones of the adult rat brain, and in vivo administration of SCF increased BrdU labeling of immature neurons in these regions. Cerebral hypoxia and ischemia may stimulate neurogenesis through trophic factors, including SCF.
Cerebrolysin is a peptide preparation mimicking the action of neurotrophic factors and has beneficial effects on neurodegenerative diseases and stroke. The present study investigated the effect of Cerebrolysin on neurogenesis in a rat model of embolic middle cerebral artery occlusion (MCAo). Treatment with Cerebrolysin at doses of 2.5 and 5 ml/kg significantly increased the number of bromodeoxyuridine positive (BrdU+) subventricular zone (SVZ) neural progenitor cells and doublecortin (DCX) immunoreactivity (migrating neuroblasts) in the ipsilateral SVZ and striatal ischemic boundary 28 days after stroke when the treatment was initiated 24h after stroke. The treatment also reduced TUNEL+ cells by ~50% in the ischemic boundary. However, treatment with Cerebrolysin at a dose of 2.5 ml/kg initiated at 24 and 48h did not significantly reduce infarct volume, but substantially improved neurological outcomes measured by an array of behavioral tests 21 and 28 days after stroke. Incubation of SVZ neural progenitor cells from ischemic rats with Cerebrolysin dose dependently augmented BrdU+ cells and increased the number of Tuj1+ cells (a marker of immature neurons). Blockage of the PI3K/Akt pathway abolished Cerebrolysin-increased BrdU+ cells. Moreover, Cerebrolysin treatment promoted neural progenitor cell migration. Collectively, these data indicate that Cerebrolysin treatment when initiated 24 and 48h after stroke enhances neurogenesis in the ischemic brain and improves functional outcome and that Cerebrolysin-augmented proliferation, differentiation, and migration of adult SVZ neural progenitor cells contribute to Cerebrolysin-induced neurogenesis, which may be related to improvement of neurological outcome. The PI3K/Akt pathway mediates Cerebrolysin-induced progenitor cell proliferation.
Cerebrolysin; neurogenesis; MCAO; rats
The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear.
Methods and Results
We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B−/−) or overexpressing VEGF-B167. After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B167 overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B167 overexpression failed to enhance vascular growth in the skin or ischemic limb.
VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities.
VEGF-B; angiogenesis; arteriogenesis; collateral growth; cardiac ischemia; limb ischemia
Peripheral stimulation and physical therapy can promote neurovascular plasticity and functional recovery after CNS disorders such as ischemic stroke. Using a rodent model of whisker-barrel cortex stroke, we have previously demonstrated that whisker activity promotes angiogenesis in the penumbra of the ischemic barrel cortex. This study explored the potential of increased peripheral activity to promote neurogenesis and neural progenitor migration toward the ischemic barrel cortex.
Three days after focal barrel cortex ischemia in adult mice, whiskers were manually stimulated (15 min × 3 times/day) to enhance afferent signals to the ischemic barrel cortex. 5-bromo-2′-deoxyuridine (BrdU, i.p.) was administered once daily to label newborn cells. At 14 days after stroke, whisker stimulation significantly increased vascular endothelial growth factor (VEGF) and stromal derived factor-1 (SDF-1) expression in the penumbra. The whisker stimulation animals showed increased doublecortin (DCX) positive and DCX/BrdU-positive cells in the ipsilateral corpus of the white matter but no increase in BrdU-positive cells in the subventricular zone, suggesting a selective effect on neuroblast migration. Neurogenesis indicated by neuronal nuclear protein (NeuN) and BrdU double staining was also enhanced by whisker stimulation in the penumbra at 30 days after stroke. Local cerebral blood flow was better recovered in mice that received whisker stimulation. It is suggested that the enriched microenvironment created by specific peripheral stimulation increases regenerative responses in the post-ischemic brain and may benefit long-term functional recovery from ischemic stroke.
Neurogenesis; Ischemic stroke; Barrel cortex; Whisker stimulation; Cell migration
Vascular endothelial growth factor (VEGF) stimulating angiogenesis was shown to be a potential novel therapeutic approach for the treatment of ischemic vascular diseases.
The goal of the present study was to examine whether transfection of VEGF before occurrence of major stroke (part I) and cerebral vasospasm after experimental subarachnoid hemorrhage (SAH; part II) develops neuroprotective qualities. A total of 25 (part I) and 26 (part II) brains were analyzed, respectively. In part one, a significant reduction of infarct volume in the VEGF-treated stroke animals (43% reduction, P < 0.05) could be detected. In part two, significant vasospasm was induced in all hemorrhage groups (P < 0.02). Analyzing microperfusion, a significant higher amount of perfused vessels could be detected (P < 0.01), whereas no significant effect could be detected towards macroperfusion. Histologically, no infarctions were observed in the VEGF-treated SAH group and the sham-operated group. Minor infarction in terms of vasospasm-induced small lesions could be detected in the control vector transduced group (P = 0.05) and saline-treated group (P = 0.09). The present study demonstrates the preconditioning impact of systemic intramuscular VEGF injection in animals after major stroke and induced severe vasospasm after SAH.
Vascular endothelial growth factor (VEGF)-induced neovasculature is immature and leaky. We tested if coexpression of angiopoietin-1 (ANG1) with VEGF improves blood–brain barrier (BBB) integrity and VEGF neuroprotective and neurorestorative effects using a permanent distal middle cerebral artery occlusion (pMCAO) model. Adult CD-1 mice were injected with 2 × 109 virus genomes of adeno-associated viral vectors expressing VEGF (AAV-VEGF) or ANG1 (AAV-ANG1) individually or together in a 1:1 ratio into the ischemic penumbra 1 hour after pMCAO. AAV-LacZ was used as vector control. Samples were collected 3 weeks later. Compared with AAV-LacZ, coinjection of AAV-VEGF and AAV-ANG1 reduced atrophy volume (46%, P=0.004); injection of AAV-VEGF or AAV-ANG1 individually reduced atrophy volume slightly (36%, P=0.08 and 33%, P=0.09, respectively). Overexpression of VEGF reduced tight junction protein expression and increased Evans blue extravasation. Compared with VEGF expression alone, coexpression of ANG1 with VEGF resulted in upregulation of tight junction protein expression and reduction of Evans blue leakage (AAV-ANG1/AAV-VEGF: 1.4±0.3 versus AAV-VEGF: 2.8±0.7, P=0.001). Coinjection of AAV-VEGF and AAV-ANG1 induced a similar degree of angiogenesis as injection of AAV-VEGF alone (P=0.85). Thus, coexpression of ANG1 with VEGF improved BBB integrity and resulted in better neuroprotection compared with VEGF expression alone.
adeno-associated viral vector; angiopoietin-1; middle cerebral artery occlusion; tight junction protein; vascular integrity; VEGF
Stroke in the neonatal brain is an important cause of neurologic morbidity. To characterize the dynamics of neural progenitor cell proliferation and maturation after survival delays in the neonatal brain following ischemia, we utilized unilateral carotid ligation alone to produce infarcts in postnatal day 12 CD1 mice. We investigated the neurogenesis derived from the sub-ventricular zone and the sub-granular zone of the dentate gyrus subsequent to injury. Newly produced cells were labeled by bromodeoxyuridine at ~1week (P18-20) after the insult by 5 I.P. injections (each 50mg/kg). Subsequent migration and differentiation of the newborn cells was investigated at postnatal day 40 by immunohistochemistry for molecular neuronal and glial cell-lineage markers and BrdU incorporation. Cresyl violet stain demonstrated massive loss of neurons in the ipsilateral septal hippocampus in the CA3 and CA1 regions associated with atrophy. Total counts of new cells were significantly lowered not only in the ipsilateral injured but also the contralateral uninjured hippocampi and correlated with the lesion induced atrophy. Bilateral percent neuronal commitments in the dentate gyri however, were not significantly different from control. New cell densities in the neocortex and striatum increased bilaterally after neonatal stroke. The predominantly non-neuronal commitment of the SVZ derived new cells was similar to the percentage of non-neuronal commitment in controls. In conclusion, neurogenesis occurring at 1 week after neonatal ischemia in the model maintained cell-lineage commitment patterns similar to sham controls. However, the total number of hippocampal SGZ-derived new neurons was reduced bilaterally; in contrast, the SVZ-derived neurogenesis was amplified.
Neurogenesis; Bromodeoxyuridine; Neuronal commitment; Hippocampus; Neocortex; Striatum
Stroke in the neonatal brain is an understudied cause of neurologic morbidity. Recently we have characterized a new immature mouse model of stroke utilizing unilateral carotid ligation alone to produce infarcts and acute seizures in postnatal day 12 (P12) CD-1 mice. In this study, the amount of poststroke neural progenitor proliferation was examined in the subgranular (SGZ) of the dentate gyrus and the subventricular zone (SVZ) 7, 14, and 21 days after ischemia (DAI). A single IP injection (50 mg/kg) of bromodeoxyuridine (BrdU) given 2 hr before perfusion fixation labeled newborn cells. Early cell pheno-types were quantified by colabeling with GFAP, nestin, and DCX. Control mice revealed an age-dependent decrease in neural proliferation, with an ~50% drop in BrdU-labeled cell counts at P33 compared with P19 both in the SGZ and in the SVZ. Significant reduction in the amount of neural proliferation in the ipsilateral injured SGZ of ligated mice correlated with both the severity of the stroke-injury and the acute seizure scores. Similar correlations were not detected contralaterally. Contralateral SGZ neural proliferation was initially lowered at 7 DAI but normalized by 21 DAI. In both injured and control brains, ~90% of newborn SGZ cells colabeled with nestin, ~30% colabeled with GFAP, and a few colabeled with DCX. In contrast, poststroke SVZ cell proliferation was enhanced ipsi- more than contralaterally at 7 DAI. In the SVZ, the enhanced neural proliferation normalized to control levels by P33. In conclusion, the neural cell proliferation was differentially altered in the SGZ vs. SVZ after neonatal stroke.
neural proliferation; bromodeoxyuridine; neuroblast; hippocampus; subventricular zone
Neuroprotective strategies in ischemic stroke are an important challenge in clinical and experimental research as an adjunct to reperfusion therapy that may reduce neurologic injury and improve outcome. The neuroprotective properties of levosimendan in traumatic brain injury in vitro, transient global brain ischemia and focal spinal cord ischemia suggest the potential for similar effects in transient brain ischemia.
Transient brain ischemia was induced for 60 min by intraluminal occlusion of the middle cerebral artery in 40 male Wistar rats under general anesthesia with s-ketamine and xylazine and with continuous monitoring of their blood pressure and cerebral perfusion. Five minutes before inducing reperfusion, a levosimendan bolus (24 μg kg -1) was administered over a 20 minute period. Infarct size, brain swelling, neurological function and the expression of inflammatory markers were quantified 24 hours after reperfusion.
Although levosimendan limited the infarct size and brain swelling by 40% and 53%, respectively, no effect on neurological outcome or mortality could be demonstrated. Upregulation of tumor necrosis factor α and intercellular adhesion molecule 1 was significantly impeded. Cerebral blood flow during reperfusion was significantly reduced as a consequence of sustained autoregulation.
Levosimendan demonstrated significant neuroprotective properties in a rat model of transient brain ischemia by reducing reperfusion injury.
Experimental stroke; Postconditioning; Levosimendan; Cerebral reperfusion injury
Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. However, Molecular mechanisms that regulate neuronal cell proliferation in the dentate gyrus (DG) following ischemic stroke insult are poorly understood. This study was designed to investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated cell proliferation in the adult DG and its underlying mechanism.
Src kinase activated continuously in the DG 24 h and 72 h after transient global ischemia, while SU6656, the Src kinase inhibitor significantly decreased the number of bromodeoxyuridine (BrdU) labeling-positive cells of rats 7 days after cerebral ischemia in the DG, as well as down-regulated Raf phosphorylation at Tyr(340/341) site, and its down-stream signaling molecules ERK and CREB expression followed by 24 h and 72 h of reperfusion, suggesting a role of Src kinase as an enhancer on neuronal cell proliferation in the DG via modifying the Raf/ERK/CREB cascade. This hypothesis is supported by further findings that U0126, the ERK inhibitor, induced a reduction of adult hippocampal progenitor cells in DG after cerebral ischemia and down-regulated phospho-ERK and phospho-CREB expression, but no effect was detected on the activities of Src and Raf.
Src kinase increase numbers of newborn neuronal cells in the DG via the activation of Raf/ERK/CREB signaling cascade after cerebral ischemia.
Ongoing neurogenesis in the adult mammalian dentate gyrus and olfactory bulb is generally accepted, but its existence in other adult brain regions is highly controversial. We labeled newly born cells in adult rats with the S-phase marker bromodeoxyuridine (BrdU) and used neuronal markers to characterize new cells at different time points after cell division. In the neocortex and striatum, we found BrdU-labeled cells that expressed each of the eight neuronal markers. Their size as well as staining for γ-aminobutyric acid (GABA), glutamic acid decarboxylase 67, calretinin and/or calbindin, suggest that new neurons in both regions are GABAergic interneurons. BrdU and doublecortin-immunoreactive (BrdU+/DCX+) cells were seen within the striatum, suggesting migration of immature neurons from the subventricular zone. Surprisingly, no DCX+ cells were found within the neocortex. NG2 immunoreactivity in some new neocortical neurons suggested that they may instead be generated from the NG2+ precursors that reside within the cortex itself.
Here, we investigate the effects of endothelial nitric oxide synthase (eNOS) on angiogenesis, neurogenesis, neurotrophic factor expression, and neurological functional outcome after stroke. Wild-type and eNOS knock-out (eNOS−/−) mice were subjected to permanent occlusion of the right middle cerebral artery. eNOS−/− mice exhibited more severe neurological functional deficit after stroke than wild-type mice. Decreased subventricular zone (SVZ) progenitor cell proliferation and migration, measured using bromodeoxyuridine, Ki-67, nestin, and doublecortin immunostaining in the ischemic brain, and decreased angiogenesis, as demonstrated by reduced endothelial cell proliferation, vessel perimeter, and vascular density in the ischemic border, were evident in eNOS−/− mice compared with wild-type mice. eNOS-deficient mice also exhibited a reduced response to vascular endothelial growth factor (VEGF)-induced angiogenesis in a corneal assay. ELISAs showed that eNOS−/− mice have decreased brain-derived neurotrophic factor (BDNF) expression but not VEGF and basic fibroblast growth factor in the ischemic brain compared with wild-type mice. In addition, cultured SVZ neurosphere formation, proliferation, telomerase activity, and neurite outgrowth but not cell viability from eNOS−/− mice were significantly reduced compared with wild-type mice. BDNF treatment of SVZ cells derived from eNOS−/− mice restored the decreased neurosphere formation, proliferation, neurite outgrowth, and telomerase activity in cultured eNOS−/− SVZ neurospheres. SVZ explant cell migration also was significantly decreased in eNOS−/− mice compared with wild-type mice. These data indicate that eNOS is not only a downstream mediator for VEGF and angiogenesis but also regulates BDNF expression in the ischemic brain and influences progenitor cell proliferation, neuronal migration, and neurite outgrowth and affects functional recovery after stroke.
eNOS; angiogenesis; neurogenesis; BDNF; neural progenitor cells; focal cerebral ischemia
Angiogenesis and neurogenesis are coupled processes. Using a coculture system, we tested the hypothesis that cerebral endothelial cells activated by ischemia enhance neural progenitor cell proliferation and differentiation, while neural progenitor cells isolated from the ischemic subventricular zone promote angiogenesis. Coculture of neural progenitor cells isolated from the subventricular zone of the adult normal rat with cerebral endothelial cells isolated from the stroke boundary substantially increased neural progenitor cell proliferation and neuronal differentiation and reduced astrocytic differentiation. Conditioned medium harvested from the stroke neural progenitor cells promoted capillary tube formation of normal cerebral endothelial cells. Blockage of vascular endothelial growth factor receptor 2 suppressed the effect of the endothelial cells activated by stroke on neurogenesis as well as the effect of the supernatant obtained from stroke neural progenitor cells on angiogenesis. These data suggest that angiogenesis couples to neurogenesis after stroke and vascular endothelial growth factor likely mediates this coupling.
angiogenesis; neurogenesis; stroke; rat; VEGF