SDF-1/CXCR7 Chemokine Signaling is Induced in the Peri-Infarct Regions in Patients with Ischemic Stroke.

Stromal-derived factor-1 (SDF-1, also known as CXCL12) and its receptors CXCR4 and CXCR7 play important roles in brain repair after ischemic stroke, as SDF-1/ CXCR4/CXCR7 chemokine signaling is critical for recruiting stem cells to sites of ischemic injury. Upregulation of SDF-1/CXCR4/CXCR7 chemokine signaling in the ischemic regions has been well-documented in the animal models of ischemic stroke, but not in human ischemic brain. Here, we found that protein expression of SDF-1 and CXCR7, but not CXCR4, were significantly increased in the cortical peri-infarct regions (penumbra) after ischemic stroke in human, compared with adjacent normal tissues and control subjects. Double-label fluorescence immunohistochemistry shows that SDF-1 and CXCR4 proteins were expressed in neuronal cells and astrocytes in the normal brain tissue and peri-infarct regions. CXCR7 protein was also observed in neuronal cells and astrocytes in the normal cortical regions, but predominantly in astrocytes in the penumbra of ischemic brain. Our data suggest that ischemic stroke in human leads to an increase in the expression of SDF-1 and CXCR7, but not CXCR4, in the peri-infarct cerebral cortex. Our findings suggest that chemokine SFD-1 is expressed not only in animal models of stroke, but also in the human brain after an ischemic injury. In addition, unlike animals, CXCR7 may be the primary receptor of SDF-1 in human stroke brain.

Cerebral ischemia induces angiogenesis in the peri-infarct regions via Notch1 signaling activation.

The Notch1 signaling pathway is considered as one of important regulators of angiogenesis during development, but its role in cerebral ischemia-induced angiogenesis is less well understood. Here, we used human and rodent brains to explore whether Notch1 signaling was involved in the angiogenesis after focal cerebral ischemia. Using immunohistochemistry on surgically resected ischemic stroke brain tissue, we found that the area, volume, and length of the blood vessels in the peri-infarct regions were significantly increased after ischemic stroke in humans, compared with non-ischemic stroke specimens. In addition, the expression of the activated form of Notch1 (Notch intracellular domain; NICD) was increased in endothelial cells of the peri-infarct region. The Notch1 ligand, Jagged1, also increased in abundance in the peri-infarct regions in human. We further confirmed that Notch1 signaling was activated in the peri-infarct regions in a mouse distal middle cerebral artery occlusion (dMCAO) model. Lentivirus-mediated Notch1 knockdown reduced ischemia-induced angiogenesis in the peri-infarct regions of the brain. Our findings suggest that ischemic stroke in human can also induce angiogenesis in the peri-infarct regions as does in animal models of focal ischemia and that Notch1 signaling plays a critical role in mediating this process, which may provide fundamental knowledge regarding the potential mechanisms underlying angiogenesis after ischemic stroke.

Neuroglobin Overexpression Inhibits AMPK Signaling and Promotes Cell Anabolism.

Neuroglobin (Ngb) is a recently discovered globin with preferential localization to neurons. Growing evidence indicates that Ngb has distinct physiological functions separate from the oxygen storage and transport roles of other globins, such as hemoglobin and myoglobin. We found increased ATP production and decreased glycolysis in Ngb-overexpressing immortalized murine hippocampal cell line (HT-22), in parallel with inhibition of AMP-activated protein kinase (AMPK) signaling and activation of acetyl-CoA carboxylase (ACC). In addition, lipid and glycogen content was increased in Ngb-overexpressing HT-22 cells. AMPK signaling was also inhibited in the brain and heart from Ngb-overexpressing transgenic mice. Although Ngb overexpression did not change glycogen content in whole brain, glycogen synthase was activated in cortical neurons of Ngb-overexpressing mouse brain and Ngb overexpression primary neurons. Moreover, lipid and glycogen content was increased in hearts derived from Ngb-overexpressing mice. These findings suggest that Ngb functions as a metabolic regulator and enhances cellular anabolism through the inhibition of AMPK signaling.

Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures.

Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium.

Increasing Proliferation of Intrinsic Tubular Cells after Renal Ischemia-reperfusion Injury in Adult Rat.

The kidney is capable of regeneration following injury. However, whether renal stem/progenitor cells contribute to the repair process after injury, as well as the origin of the cells that repair and replace damaged renal tubule cells remains debated. Therefore, better understanding of the repair process will be critical to developing new strategies for the treatment of acute renal failure. Using an ischemia-reperfusion injury mode and an immunocytochemistry method, we counted the number of BrdU-positive cells in damged regions at different durations of reperfusion. We found that BrdU, a cell proliferative marker, was mainly incorporated in the tubular cells of both medulla and cortex 1 day after reperfusion. The number of BrdU-positive cells reached a peak at 3 days and lasted for two months after injury. BrdU-positive cells were barely found in the renal glomerulus and the parietal layer of Bowman's capsule after injury, and only a few were found in the intersititium. PAX2, an embryonic renal marker, was also increased in renal tubule cells. Confocal images show that BrdU-positive cells co-expressed PAX2, but not the activated form of caspase-3, a cell death marker. Our data suggest that renal stem-like cells or dedifferentiation of surviving renal tubular cells in both the medulla and cortex may predominantly contribute to the repair process after renal ischemia-reperfusion injury in rat.

mTOR signaling inhibition modulates macrophage/microglia-mediated neuroinflammation and secondary injury via regulatory T cells after focal ischemia.

Signaling by the mammalian target of rapamycin (mTOR) plays an important role in the modulation of both innate and adaptive immune responses. However, the role and underlying mechanism of mTOR signaling in poststroke neuroinflammation are largely unexplored. In this study, we injected rapamycin, a mTOR inhibitor, by the intracerebroventricular route 6 h after focal ischemic stroke in rats. We found that rapamycin significantly reduced lesion volume and improved behavioral deficits. Notably, infiltration of γδ T cells and granulocytes, which are detrimental to the ischemic brain, was profoundly reduced after rapamycin treatment, as was the production of proinflammatory cytokines and chemokines by macrophages and microglia. Rapamycin treatment prevented brain macrophage polarization toward the M1 type. In addition, we also found that rapamycin significantly enhanced anti-inflammation activity of regulatory T cells (Tregs), which decreased production of proinflammatory cytokines and chemokines by macrophages and microglia. Depletion of Tregs partially elevated macrophage/microglia-induced neuroinflammation after stroke. Our data suggest that rapamycin can attenuate secondary injury and motor deficits after focal ischemia by enhancing the anti-inflammation activity of Tregs to restrain poststroke neuroinflammation.

LOX-1 expression and oxidized LDL uptake and toxicity in the HN33 neuronal cell line.

Cardiovascular risk factors appear to influence the risk and progression of neurodegenerative disease, but the mechanisms involved are poorly understood. We investigated the possible involvement of oxidized low-density lipoprotein receptor (LOX-1) and oxidized low-density lipoprotein (Ox-LDL) in neurodegeneration by studying the expression of LOX-1 and the effects of Ox-LDL in HN33 cells, a neuronal cell line of central nervous system origin. HN33 cells showed LOX-1 protein expression, hypoxic induction of LOX-1, Ox-LDL uptake and Ox-LDL toxicity. LOX-1/Ox-LDL signaling may contribute to the association between cardiovascular risk factors and neurodegenerative disease.

Flow-induced regulation of brain endothelial cells in vitro.

Endothelial cell (EC) function and susceptibility to vascular disease are regulated by flow; this relationship has been modeled in systemic, but not cerebrovascular, EC culture. We studied the effects of unidirectional flow of medium, produced by orbital rotation of cultures, on morphology and protein expression in bEnd.3 mouse brain ECs. Flow altered the expression of key transcription factors and gasotransmitter-synthesizing enzymes, and increased NO production. Statins and angiotensin receptor blockers reproduced the effect of flow on endothelial nitric oxide synthase expression. Thus, flow modified brain EC properties and function in vitro, with similarities and possible differences compared to previous studies on systemic ECs. Thus, the effect of flow on brain ECs can be modeled in vitro and may assist the investigation of mechanisms of cerebrovascular disease.

Differential activation of mTOR complex 1 signaling in human brain with mild to severe Alzheimer's disease.

Mammalian target of rapamycin (mTOR) signaling has been suggested to be effective in modifying cognitive status in animal models of Alzheimer's disease (AD), but little is known about its role in AD patients. We hereby tested whether mTOR signaling was activated and whether activated mTOR signaling was related to the degree of cognitive deficits in patients with AD. Autopsy brain hippocampal tissues were obtained from controls and patients with AD and Western blots were performed using antibodies against mTOR signaling molecules and RagC, an upstream component of mTOR complex 1 (mTORC1) signaling. We found that expression of mTOR/p-mTOR and its downstream targets S6/p-S6 and Raptor/p-Raptor were expressed in the control and AD hippocampus. The expression levels of these signaling molecules were significantly increased in the hippocampus at the severe stages of AD, compared to controls and other stages of AD. Interestingly, Rictor expression level was unaltered. In addition, RagC was increased in the hippocampus at the early, moderate, and severe stages of AD. Our data indicate that mTORC1, but not mTORC2, was activated in the AD brains and that the level of mTOR signaling activation was correlated with cognitive severity of AD patients.

Notch4 is activated in endothelial and smooth muscle cells in human brain arteriovenous malformations.

Up-regulation of Notch4 was observed in the endothelial cells in the arteriovenous malformations (AVMs) in mice. However, whether Notch4 is also involved in brain AVMs in humans remains unclear. Here, we performed immunohistochemistry on normal brain vascular tissue and surgically resected brain AVMs and found that Notch4 was up-regulated in the subset of abnormal vessels of the brain AVM nidus, compared with control brain vascular tissue. Two-photon confocal images show that Notch4 was expressed not only in the endothelial but also in the smooth muscle cells of the vascular wall in brain AVMs. Western blotting shows that Notch4 was activated in brain AVMs, but not in middle cerebral artery of normal human brain, which was confirmed by immunostaining. Our findings suggest a possible contribution of Notch4 signalling to the development of brain AVMs in human.

Notch1 signaling modulates neuronal progenitor activity in the subventricular zone in response to aging and focal ischemia.

Neurogenesis diminishes with aging and ischemia-induced neurogenesis also occurs, but reduced in aged brain. Currently, the cellular and molecular pathways mediating these effects remain largely unknown. Our previous study has shown that Notch1 signaling regulates neurogenesis in subventricular zone (SVZ) of young adult brain after focal ischemia, but whether a similar effect occurs in aged normal and ischemic animals is unknown. Here, we used normal and ischemic aged rat brains to investigate whether Notch1 signaling was involved in the reduction of neurogenesis in response to aging and modulates neurogenesis in aged brains after focal ischemia. By Western blot, we found that Notch1 and Jagged1 expression in the SVZ of aged brain was significantly reduced compared with young adult brain. Consistently, the activated form of Notch1 (Notch intracellular domain; NICD) expression was also declined. Immunohistochemistry confirmed that expression and activation of Notch1 signaling in the SVZ of aged brain were reduced. Double or triple immunostaining showed that that Notch1 was mainly expressed in doublecortin (DCX)-positive cells, whereas Jagged1 was predominantly expressed in astroglial cells in the SVZ of normal aged rat brain. In addition, disruption or activation of Notch1 signaling altered the number of proliferating cells labeled by bromodeoxyuridine (BrdU) and DCX in the SVZ of aged brain. Moreover, ischemia-induced cell proliferation in the SVZ of aged brain was enhanced by activating the Notch1 pathway and was suppressed by inhibiting the Notch1 signaling. Reduced infarct volume and improved motor deficits were also observed in Notch1 activator-treated aged ischemic rats. Our data suggest that Notch1 signaling modulates the SVZ neurogenesis in aged brain in normal and ischemic conditions.

Neuroglobin protein is upregulated in Alzheimer's disease.

Neuroglobin is a neuronal protein with protective effects in animal models of stroke and Alzheimer's disease, but the relevance of these effects to Alzheimer's disease in humans is unknown. We measured neuroglobin levels by western blot and immunostained hippocampal sections for neuroglobin, cell-type protein markers, and amyloid-ß, in brain tissue obtained at autopsy from patients with Alzheimer's disease. Neuroglobin levels were increased in early and moderately advanced Alzheimer's disease compared to controls, but declined to control levels in severe disease. In patients with Alzheimer's disease, neuroglobin was detected within neurons, as well as at extracellular sites associated with amyloid-ß deposits. We conclude that, as in transgenic mouse models, neuroglobin may influence the course of clinical Alzheimer's disease.

Neuroglobin expression in neurogenesis.

Neuroglobin is a hypoxia-inducible, neuroprotective protein related to myoglobin and hemoglobin, but little is known about its neurodevelopmental expression or function. To begin to explore these issues, we measured neuroglobin protein expression during neuronal differentiation of human embryonic stem cells in vitro and in the neurogenic subventricular zone of adult rats in vivo. Neuroglobin protein expression was barely detectable by western blotting in human embryonic stem cells, but was readily demonstrable in neural stem cells, and was further induced upon differentiation to neurons. In the adult subventricular zone, neuroglobin expression coincided with that of the neuronal lineage marker doublecortin, but not with vimentin or glial fibrillary acidic protein. These findings suggest that neuroglobin is expressed early in the course of neuronal differentiation and may, therefore, have a role in neurodevelopment.

Vascular endothelial growth factor-B expression in postischemic rat brain.

BACKGROUND: Vascular endothelial growth factor-B (VEGF-B) protects against experimental stroke, but the effect of stroke on VEGF-B expression is uncertain. METHODS: We examined VEGF-B expression by immunohistochemistry in the ischemic border zone 1-7 days after middle cerebral artery occlusion in rats. RESULTS: 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. CONCLUSIONS: These findings provide additional evidence for a role of VEGF-B in the endogenous response to cerebral ischemia.

Expression level of vascular endothelial growth factor in hippocampus is associated with cognitive impairment in patients with Alzheimer's disease.

Although the enhanced expression of vascular endothelial growth factor (VEGF) in the brains of patients with Alzheimer's disease (AD) has been reported, the functional significance of VEGF level in the progression of AD is still unclear. We examined the VEGF expression in the hippocampus of patients with AD at different stages of progression by Western blot analysis, and found that the VEGF189 isoform (VEGF(189)) was barely detectable in normal hippocampus, but significantly increased at the early stage of patients with AD. VEGF(189) was decreased with advancing stages of AD. Immunostaining shows that VEGF was significantly increased in the cells in the CA1, CA3, and dentate gyrus regions of hippocampus and layers III and V of entorhinal cortex of patients with AD, compared with normal brain. Confocal images show that VEGF was predominantly expressed in neurons and astrocytes in the hippocampus and entorhinal cortex of patients with AD. Our data suggest that VEGF level is associated with progressive loss of cognitive function in patients with AD.

Neurogenesis in adult human brain after traumatic brain injury.

While much work has been conducted regarding the neurogenesis response to traumatic brain injury (TBI) in rodents, it remains largely unknown whether neurogenesis in adult human brain also responds to TBI in a similar manner. Here, we performed immunocytochemistry on 11 brain specimens from patients with traumatic brain injury, who underwent surgical intervention. We found that expression of neural stem/progenitor cell (NSC) protein markers, including DCX, TUC4, PSA-NCAM, SOX2 and NeuroD, was increased in the perilesional cortex of human brain after TBI compared to that of normal brain. Confocal images showed that these NSC proteins were expressed in one single cell. We also found that proliferative markers were expressed in NSC protein-positive cells after TBI, and the number of proliferative NSCs was significantly increased after TBI. Our data suggest that TBI may also induce neurogenesis in human brain.

Ablation of neurogenesis attenuates recovery of motor function after focal cerebral ischemia in middle-aged mice.

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.

Effect of a contralateral lesion on neurological recovery from stroke in rats.

PURPOSE: 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. METHODS: 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. RESULTS: Infarct volume was unchanged, but functional neurological deficits were reduced 1 day after bilateral compared to unilateral dMCAO. CONCLUSIONS: Activity in the contralesional cerebral cortex may inhibit functional motor recovery after experimental stroke.

Conditional depletion of neurogenesis inhibits long-term recovery after experimental stroke in mice.

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.

Corpus callosum and experimental stroke: studies in callosotomized rats and acallosal mice.

BACKGROUND AND PURPOSE: Interhemispheric inhibition via the corpus callosum has been proposed as an exacerbating factor in outcome from stroke. METHODS: We measured infarct volume and behavioral outcome after middle cerebral artery occlusion in callosotomized rats and acallosal mice. RESULTS: Neither callosotomy in rats nor callosal agenesis in mice improved infarct volume or behavioral outcome after middle cerebral artery occlusion. CONCLUSIONS: These findings argue against a role for transcallosal projections in exacerbating focal cerebral ischemia.