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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.

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.

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.

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.

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.

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 weeks 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-month-old) and aged (24-month-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 weeks later. Aged rats developed larger infarcts, but infarct volume and performance on the cylinder and elevated body swing tests, measured 6-8 weeks post-transplant, were improved by transplantation. We conclude that advanced age does not preclude a beneficial response to NPC transplantation following experimental 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 approximately 50% and approximately 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.

The Notch1 signaling pathway is regarded as one of the main regulators of neural stem cell behavior during development, but its role in the adult brain is less well understood. We found that Notch1 was mainly expressed in doublecortin (DCX)-positive cells corresponding to newborn neurons, whereas the Notch1 ligand, Jagged1, was predominantly expressed in glial fibrillary acidic protein (GFAP)-positive astrocytic cells in the subventricular zone (SVZ) of the normal adult brain. These findings were confirmed by conditional depletion of DCX-positive cells in transgenic mice carrying herpes simplex virus thymidine kinase (HSV-TK) under the control of the DCX promoter. In addition, the activated form of Notch1 (Notch intracellular domain, NICD) and its downstream transcriptional targets, Hes1 and sonic hedgehog (Shh), were also expressed in SVZ cells. Increased activation of Notch1 signaling increased SVZ cell proliferation, whereas inhibiting Notch1 signaling resulted in a reduction of proliferating cells in the SVZ. Levels of NICD, Hes1, and Shh were increased in the SVZ at 4 and 24 h after focal cerebral ischemia. Finally, ischemia-induced cell proliferation in the SVZ was blocked by inhibition of the Notch1 signaling pathway, suggesting that Notch1 signaling may have a key role in normal adult and ischemia-induced neurogenesis.

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.

We used high-throughput Western blotting to identify proteins that are up- or down-regulated by neuronal hypoxia in vitro. Exposure to hypoxia for 24 h produced > or = 1.5-fold increases in the expression of 10/700 proteins (1.4%) and decreases in the expression of 16/700 proteins (2.3%). Up-regulated proteins included Arc, doublecortin/calmodulin kinase-like 1, integrin alpha(v), and fibronectin; down-regulated proteins included nuclear autoantigenic sperm protein, protein kinase C-related kinase 2, and E2F transcription factor 1. The prominence of cytoskeleton-related proteins among those showing altered expression highlights the role of the cytoskeleton in neuronal responses to hypoxia.

A recombinant adeno-associated virus (rAAV) vector was used to overexpress the anti-apoptotic Bcl-2-family protein, BCL-w, in rat brain. Three weeks after injecting the vector into cerebral cortex and striatum on one side, temporary focal ischemia was induced by occlusion of the ipsilateral middle cerebral artery for 90 min, followed by reperfusion for 24 h. BCL-w expression was increased in cerebral cortex and striatum – and in neurons, astroglia and endothelial cells – in the brains of rats that received the rAAV-BCL-w vector, compared to rats given phosphate-buffered saline or a control vector containing the gene for green fluorescent protein. Recipients of the rAAV-BCL-w vector also showed a 30% reduction in infarct size and a 33–40% improvement in neurological function, compared to the control groups. These results provide evidence for a role of BCL-w in regulating histological and functional outcome after focal cerebral ischemia.

Caspase family genes play a critical role in the initiation and execution of programmed cell death. Programmed cell death is an important contributor to neuronal loss following cerebral ischemia. We have performed a series of experiments to investigate the role of a specific caspase, caspase-2, in the development of delayed neuronal death following transient global ischemia in the rat. A rat ischemic brain cDNA library was screened, and two splice-variants of caspase-2 mRNA were identified, caspase-2S and caspase-2L, which were highly homologous with the sequences of human and mouse caspase-2S and caspase-2L genes, respectively. RT-PCR demonstrated an increase in expression of both caspase-2S and caspase-2L mRNA at 8, 24 and 72 h of reperfusion after global ischemia. The ratio of the two PCR fragments did not change significantly throughout the time course of reperfusion. Western blot with monoclonal antibody specific to the pro-apoptotic caspase-2L splice variant revealed an increase in procaspase-2 (51 kDa) protein from 4 to 72 h following ischemia compared with sham-operated controls. Furthermore, an approximately 30-kDa cleavage product appeared at 8 h and increased with increasing duration of reperfusion. Thus, caspase-2L is both translated and activated following transient global ischemia. Finally, intraventricular administration of the caspase-2-like inhibitor (VDVAD-FMK) 30 min before induction of ischemia decreased the number of CA1 neurons staining positively for DNA damage (Klenow-labeling assay) and increased the number of healthy-appearing CA1 neurons (cresyl violet) compared with vehicle-treated controls. Taken together, the data suggest that caspase-2 induction and activation are important mediators of delayed neuronal death following transient global ischemia.

We used cDNA microarray gene expression profiling to characterize the transcriptional response to exposure of cultured mouse cerebral cortical neurons to hypoxia for 24 hr. Of 11,200 genes examined, 1,405 (12.5%) were induced or repressed at least 1.5-fold, whereas 26 known genes were induced and 20 known genes were repressed at least 2.5-fold. The most strongly induced genes included genes coding for endoplasmic reticulum proteins (Ero1L/Giig11, Sac1p, Ddit3/Gadd153), proteins involved in ubiquitination (Arih2, P4hb), proteins induced by hypoxia in non-neuronal systems (Gpi1, Aldo1, Anxa2, Hig1), and proteins that might promote cell death (Gas5, Egr1, Ndr1, Vdac2). These findings reinforce the importance of endoplasmic reticulum-based mechanisms and of protein-ubiquitination pathways in the neuronal response to hypoxia.

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.

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 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-β, 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-β deposits. We conclude that, as in transgenic mouse models, neuroglobin may influence the course of clinical Alzheimer's disease.

Derivation and numerical calculation shows a new solution for surface plasmons excitable with 1.55 µm operating wavelength at Si/SiO2 interface when the electron density of heavily doped silicon equals to 2.35*10^27 m^-3.

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.

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 ICH-induced overexpression of Ngb may be stimulated by ischemic hypoxia and may help to constrain 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.

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 days after distal MCAO and doublecortin (Dcx)-expressing cells in the subventricular zone (SVZ) and subgranular zone of dentate gyrus (SGZ) were counted 60 days post-transplant. Transplantation increased neurogenesis (Dcx expression) in ipsilateral SVZ, but not in contralateral SVZ or either SGZ, in both young adult (3-month-old) and aged (24-month-old) rats. These findings suggest that cell-based therapy for stroke may be associated with changes in endogenous adaptive processes, including neurogenesis.

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.

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.

Tumor stem cells are implicated in tumor initiation and maintenance. Recent studies have shown that a subpopulation of cells isolated from brain tumors can form neurospheres in vitro, and have multiple characteristic properties observed in neural stem cells. In vivo implantation of these cells can induce tumors that phenocopy original tumors, suggesting that tumor stem cells are involved in brain carcinogenesis. We found that a population of cells in human glioblastoma multiforme expressed multiple protein markers of neural stem cells including nestin, TUC-4, doublecortin and beta III-tubulin. In contrast, these markers were not expressed in human capillary hemangioblastoma or meningioma. Double immunolabeling showed that a portion of doublecortin-, beta III-tubulin-, TUC-4- and nestin-positive cells express Ki67 antigen, a cell proliferation marker. To investigate further whether these properties of tumor stem cells are correlated with their biological behavior, immunohistochemistry was performed on brain sections from astrocytomas of different grades using antibodies against neural stem cell markers. The number of cells expressing Ki67 antigen and neural stem cell markers was increased in relation to worsening histological grade of astrocytomas, indicating that the capacity for tumor stem cell proliferation may be clinically relevant. Thus, tumor stem cells in astrocytomas may be involved in carcinogenesis.

Basic fibroblast growth factor (FGF-2) has been reported to protect against ischemic injury in the brains of young adult rodents. However, little is known about whether FGF-2 retains this capability in the aged ischemic brain. Since stroke in human is much more common in older people than among younger adults, to address this question is clinically important. In this study, aged (24-month-old) rats were treated with intracerebroventricular infusion of FGF-2 or vehicle for 3 days, beginning 48 hr before (pre-ischemia), 24 hr after (early post-ischemia), or 96 hr after (late post-ischemia) 60 min of middle cerebral artery occlusion, and were killed 10 days after ischemia. Aged rats given FGF-2 pre-ischemia showed better symmetry of movement and forepaw outstretching, and reduced infarct volumes, compared to rats treated with vehicle, but no significant improvement was found in aged rats given FGF-2 after focal ischemia. In contrast, young adult (3-month-old) rats treated with FGF-2 for 3 days beginning 24 hr post-ischemia showed significant neurobehavioral improvement and better histological outcome. In addition, we also found that newborn neurons in the rostral subventricular zone (SVZ) were increased in aged rats treated with FGF-2 prior to ischemia. However, unlike in young adult ischemic rats, only a few of newly generated cells migrated into the damaged region in aged brain after focal ischemia. These findings point to differences in the response of aged versus young adult rats to FGF-2 in cerebral ischemia, and suggest that such differences need to be considered in the development of neuroprotective agents for stroke.

Caspase family genes play a critical role in the initiation and execution of programmed cell death. Programmed cell death is an important contributor to neuronal loss following cerebral ischemia. We have performed a series of experiments to investigate the role of a specific caspase, caspase-2, in the development of delayed neuronal death following transient global ischemia in the rat. A rat ischemic brain cDNA library was screened, and two splice-variants of caspase-2 mRNA were identified, caspase-2S and caspase-2L, which were highly homologous with the sequences of human and mouse caspase-2S and caspase-2L genes, respectively. RT-PCR demonstrated an increase in expression of both caspase-2S and caspase-2L mRNA at 8, 24 and 72 h of reperfusion after global ischemia. The ratio of the two PCR fragments did not change significantly throughout the time course of reperfusion. Western blot with monoclonal antibody specific to the pro-apoptotic caspase-2L splice variant revealed an increase in procaspase-2 (51 kDa) protein from 4 to 72 h following ischemia compared with sham-operated controls. Furthermore, an approximately 30-kDa cleavage product appeared at 8 h and increased with increasing duration of reperfusion. Thus, caspase-2L is both translated and activated following transient global ischemia. Finally, intraventricular administration of the caspase-2-like inhibitor (VDVAD-FMK) 30 min before induction of ischemia decreased the number of CA1 neurons staining positively for DNA damage (Klenow-labeling assay) and increased the number of healthy-appearing CA1 neurons (cresyl violet) compared with vehicle-treated controls. Taken together, the data suggest that caspase-2 induction and activation are important mediators of delayed neuronal death following transient global ischemia.

Cell death–regulatory genes like caspases and bcl-2 family genes are involved in delayed cell death in the CA1 sector of hippocampus after global cerebral ischemia, but little is known about the mechanisms that trigger their expression. The authors found that expression of Fas and Fas-ligand messenger ribonucleic acid and protein was induced in vulnerable CA1 neurons at 24 and 72 hours after global ischemia. Fas-associating protein with a novel death domain (FADD) also was upregulated and immunoprecipitated and co-localized with Fas. Caspase-10 was activated and interacted with FADD protein to an increasing extent as the duration of ischemia increased. Moreover, caspase-10 co-localized with both FADD and caspase-3. These findings suggest that Fas-mediated death signaling may play an important role in signaling hippocampal neuronal death in CA1 after global cerebral ischemia.

T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.

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.