Biomedical subjects

Zinaida S Vexler

Publications and source records attributed to Zinaida S Vexler.

15 recordsLinked to original sources

PubMed2006

Macrophages are comprised of resident brain microglia not infiltrating peripheral monocytes acutely after neonatal stroke.

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post-reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-beta and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell-type specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.

Analysis of Variance↗

PubMed2006

Aminoguanidine inhibits caspase-3 and calpain activation without affecting microglial activation following neonatal transient cerebral ischemia.

Microglial cells, the resident macrophages of the CNS, can be both beneficial and detrimental to the brain. These cells play a central role as mediators of neuroinflammation associated with many neurodegenerative states, including cerebral ischemia. Because microglial cells are both a major source of inducible nitric oxide synthase (iNOS)/nitric oxide (NO) production locally in the injured brain and are activated by NO-mediated injury, we tested whether iNOS inhibition reduces microglial activation and ischemic injury in a neonatal focal ischemia-reperfusion model. Post-natal day 7 rats were subjected to a 2 h transient middle cerebral artery (MCA) occlusion. Pups with confirmed injury on diffusion-weighted magnetic resonance imaging (MRI) during occlusion were administered 300 mg/kg/dose aminoguanidine (AG) or vehicle at 0, 4 and 18 h after reperfusion, and animals were killed at 24 or 72 h post-reperfusion. The effect of AG on microglial activation as judged by the acquisition of ED1 immunoreactivity and proliferation of ED1-positive cells, on activation of cell death pathways and on injury volume, was determined. The study shows that while AG attenuates caspase 3 and calpain activation in the injured tissue, treatment does not affect the rapidly occurring activation and proliferation of microglia following transient MCA occlusion in the immature rat, or reduce injury size.

Animals↗

PubMed2006

Reduction of caspase-8 and -9 cleavage is associated with increased c-FLIP and increased binding of Apaf-1 and Hsp70 after neonatal hypoxic/ischemic injury in mice overexpressing Hsp70.

BACKGROUND AND PURPOSE: Caspase-8 and caspase-9 are essential proteases of the extrinsic and intrinsic apoptotic pathways, respectively. We investigated whether neuroprotection associated with overexpression of heat-shock protein 70 (Hsp70), a natural cellular antiapoptotic protein, is mediated by caspase-8 and caspase-9 signaling in the neonatal mouse brain after hypoxia/ischemia (H/I) injury. METHODS: Postnatal day 7 transgenic mice overexpressing rat Hsp70 (Hsp70 Tg) and their wild-type (Wt) littermates underwent unilateral common carotid artery ligation followed by 30 minutes of exposure to 8% O2. The expression of apoptotic proteins was quantified by Western blot analysis, and the specific interaction between Hsp70 and apoptotic protease activating factor 1 (Apaf-1) was determined by coimmunoprecipitation. RESULTS: Hsp70 overexpression reduced cytosolic translocation of cytochrome c without affecting the levels of Apaf-1 and pro-caspase-9 24 hours after H/I. The expression of these apoptotic proteins in the naïve neonatal brains was also not affected by Hsp70 overexpression. Reduced caspase-9 cleavage occurred in Hsp70 Tg mice compared with Wt littermates 24 hours after H/I and correlated with increased binding of Hsp70 and Apaf-1. Increased cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) expression and decreased caspase-8 cleavage were also observed in Hsp70 Tg compared with Wt mice 24 hours after H/I. CONCLUSIONS: Our results suggest that the extrinsic and intrinsic apoptotic pathways mediate the neuroprotective effects of Hsp70 overexpression in neonatal H/I, specifically by upregulating FLIP and sequestering Apaf-1, leading to reduced cleavage of caspase-8 and caspase-9.

Animals↗

PubMed2006

Translational stroke research in the developing brain.

Preclinical animal models can help guide the development of clinical pediatric and newborn stroke trials. Data obtained using currently available models of hypoxia-ischemia and focal stroke have demonstrated the need for age-appropriate models. There are age-related differences in susceptibility of the immature brain to oxidative stress and inflammation, as well as in the rate and degree of apoptotic neuronal death. These issues need to be carefully addressed in designing future clinical trials.

Adult↗

PubMed2005

Magnetic resonance imaging as a surrogate measure for histological sub-chronic endpoint in a neonatal rat stroke model.

INTRODUCTION: It is becoming increasingly recognized that CNS immaturity at birth affects ischemic injury and recovery, and that the consequences of neonatal stroke need to be studied using age-appropriate focal stroke models. The inclusion of magnetic resonance imaging (MRI) as a surrogate measure of stroke progression has provided useful information in adult models, but the benefit for neonatal stroke studies is yet to be established. METHODS: Postnatal 7-day (P7) rats were subjected to a 3-h transient occlusion of the middle cerebral artery (MCA) which was produced either by inserting a filament via the external carotid artery or via the internal carotid artery. MRI was used to delineate the size and pattern of injury acutely, during MCA occlusion, and 7 days following reperfusion. RESULTS: The size of the diffusion-weighted (DW) MRI-detectable injury during MCA occlusion was similar following both surgical procedures and resulted in histological lesions 7 days later in all animals. The extent of spontaneous recovery in individual animals varied substantially 7 days later within each group, as was depicted by a combination of DW- and T2W-MRI and confirmed by the corresponding histology. CONCLUSIONS: The ability of MRI to provide accurate information on the size of histological outcome at 7 days after neonatal focal transient ischemia suggests that MRI is useful as an intermediate surrogate measure of injury progression in long-term neonatal stroke studies.

Animals↗

PubMed2005

Erythropoietin improves functional and histological outcome in neonatal stroke.

Neonatal stroke is a condition that leads to disability in later life, and as yet there is no effective treatment. Recently, erythropoietin (EPO) has been shown to be cytoprotective following brain injury and may promote neurogenesis. However, the effect of EPO on functional outcome and on morphologic changes in neonatal subventricular zone (SVZ) following experimental neonatal stroke has not been described. We used a transient focal model of neonatal stroke in P10 rat. Injury was documented by diffusion weighted MRI during occlusion. Immediately upon reperfusion, either EPO (5U/gm) or vehicle was administered intraperitoneally and animals were allowed to grow for 2 wk. Sensorimotor function was assessed using the cylinder rearing test and then brains were processed for volumetric analysis of the SVZ. Stroke induced SVZ expansion proportional to hemispheric volume loss. EPO treatment markedly preserved hemispheric volume and decreased the expansion of SVZ unilaterally. Furthermore, EPO treatment significantly improved the asymmetry of forelimb use following neonatal stroke. This functional improvement directly correlated with the amount of preserved hemispheric volume. These results suggest EPO may be a candidate in the treatment of neonatal stroke.

Animals↗

PubMed2005

Hypoxia-inducible factor 1alpha and erythropoietin upregulation with deferoxamine salvage after neonatal stroke.

Treatment with deferoxamine (DFO) is protective against focal ischemia with global hypoxia when given as a preconditioning stimulus in neonatal rodents. DFO acts as an iron chelator and may stabilize HIF1alpha. Therefore, we hypothesized that DFO would protect against pure ischemia-reperfusion injury when given after the insult and that the protection would be associated with expression of hypoxia-inducible factor 1alpha (HIF1alpha) and downstream target genes such as erythropoietin (Epo). To test these hypotheses, we performed middle cerebral artery (MCA) occlusion in postnatal day 10 (P10) rats for 1.5 h followed by treatment with DFO or vehicle upon reperfusion. Preserved brain volumes were measured with cresyl violet staining 1 week after the insult. HIF1alpha and Epo expression were determined by Western blot and immunocytochemical analyses at different time points after injury. We found that DFO treatment preserved brain volumes when compared to vehicle (P < 0.05). In DFO-treated ischemic cortices, HIF1alpha expression peaked early, while Epo expression was seen in two phases and in different cell populations. Epo immunoreactivity colocalized with neuronal markers at 8 h but with astrocytic markers at 1 week. These results suggest that DFO is protective when administered after neonatal ischemic stroke and that this protection may be like that afforded by preconditioning through the upregulation of similar downstream pathways.

Animals↗

PubMed2005

Hsp70 overexpression sequesters AIF and reduces neonatal hypoxic/ischemic brain injury.

Apoptosis is implicated in neonatal hypoxic/ischemic (H/I) brain injury among various forms of cell death. Here we investigate whether overexpression of heat shock protein (Hsp) 70, an antiapoptotic protein, protects the neonatal brain from H/I injury and the pathways involved in the protection. Postnatal day 7 (P7) transgenic mice overexpressing rat Hsp70 (Tg) and their wild-type littermates (Wt) underwent unilateral common carotid artery ligation followed by 30 mins exposure to 8% O(2). Significant neuroprotection was observed in Tg versus Wt mice on both P12 and P21, correlating with a high level of constitutive but not inducible Hsp70 in the Tg. More prominent injury was observed in Wt and Tg mice on P21, suggesting its continuous evolution after P12. Western blot analysis showed that translocation of cytochrome c, but not the second mitochondria-derived activator of caspase (Smac)/DIABLO and apoptosis-inducing factor (AIF), from mitochondria into cytosol was significantly reduced in Tg 24 h after H/I compared with Wt mice. Coimmunoprecipitation detected more Hsp70 bound to AIF in Tg than Wt mice 24 h after H/I, inversely correlating with the amount of nuclear, but not cytosolic, AIF translocation. Our results suggest that interaction between Hsp70 and AIF might have reduced downstream events leading to cell death, including the reduction of nuclear AIF translocation in the neonatal brains of Hsp70 Tg mice after H/I.

Animals↗

PubMed2005

Minocycline confers early but transient protection in the immature brain following focal cerebral ischemia-reperfusion.

The incidence of neonatal stroke is high and currently there are no strategies to protect the neonatal brain from stroke or reduce the sequelae. Agents capable of modifying inflammatory processes hold promise. We set out to determine whether delayed administration of one such agent, minocycline, protects the immature brain in a model of transient middle cerebral artery (MCA) occlusion in 7-day-old rat pups. Injury volume in minocycline (45 mg/kg/dose, beginning at 2 h after MCA occlusion) and vehicle-treated pups was determined 24 h and 7 days after onset of reperfusion. Accumulation of activated microglia/macrophages, phosphorylation of mitogen-activated protein kinase (MAPK) p38 in the brain, and concentrations of inflammatory mediators in plasma and brain were determined at 24 h. Minocycline significantly reduced the volume of injury at 24 h but not 7 days after transient MCA occlusion. The beneficial effect of minocycline acutely after reperfusion was not associated with changed ED1 phenotype, nor was the pattern of MAPK p38 phosphorylation altered. Minocycline reduced accumulation of IL-1beta and CINC-1 in the systemic circulation but failed to affect the increased levels of IL-1beta, IL-18, MCP-1 or CINC-1 in the injured brain tissue. Therefore, minocycline provides early but transient protection, which is largely independent of microglial activation or activation of the MAPK p38 pathway.

Animals↗

PubMed2005

Antiapoptotic and anti-inflammatory mechanisms of heat-shock protein protection.

We and others have previously shown that heat-shock proteins (HSPs) are involved in protecting the brain from a variety of insults including stroke, epilepsy, and other related insults. While the mechanism of this protection has largely been thought to be due to their chaperone functions (i.e., preventing abnormal protein folding or aggregation), recent work has shown that HSPs may also directly interfere with other cell death pathways such as apoptosis and inflammation. Using models of cerebral ischemic and ischemia-like injury, we overexpressed the 70-kDa heat-shock protein (HSP70) using gene transfer or by studying a transgenic mouse model. HSP70 protected neurons and astrocytes from experimental stroke and stroke-like insults. HSP70 transgenic mice also had better neurological scores following experimental stroke compared to their wild-type littermates. Overexpressing HSP70 was associated with less apoptotic cell death and increased expression of the antiapoptotic protein, Bcl-2. Furthermore, HSP70 suppressed microglial/monocyte activation following experimental stroke. HSP70 overexpression also led to the reduction of matrix metalloproteinases. We suggest that HSPs are capable of protecting brain cells from lethal insults through a variety of mechanisms and should be explored as a potential therapy against stroke and other neurodegenerative diseases.

Animals↗

PubMed2004

Manipulation of antioxidant pathways in neonatal murine brain.

To assess the role of brain antioxidant capacity in the pathogenesis of neonatal hypoxic-ischemic brain injury, we measured the activity of glutathione peroxidase (GPX) in both human-superoxide dismutase-1 (hSOD1) and human-GPX1 overexpressing transgenic (Tg) mice after neonatal hypoxia-ischemia (HI). We have previously shown that mice that overexpress the hSOD1 gene are more injured than their wild-type (WT) littermates after HI, and that H(2)O(2) accumulates in HI hSOD1-Tg hippocampus. We hypothesized that lower GPX activity is responsible for the accumulation of H(2)O(2). Therefore, increasing the activity of this enzyme through gene manipulation should be protective. We show that brains of hGPX1-Tg mice, in contrast to those of hSOD-Tg, have less injury after HI than WT littermates: hGPX1-Tg, median injury score = 8 (range, 0-24) versus WT, median injury score = 17 (range, 2-24), p < 0.01. GPX activity in hSOD1-Tg mice, 2 h and 24 h after HI, showed a delayed and bilateral decline in the cortex 24 h after HI (36.0 +/- 1.2 U/mg in naive hSOD1-Tg versus 29.1 +/- 1.7 U/mg in HI cortex and 29.2 +/- 2.0 for hypoxic cortex, p < 0.006). On the other hand, GPX activity in hGPX1-Tg after HI showed a significant increase by 24 h in the cortex ipsilateral to the injury (48.5 +/- 5.2 U/mg, compared with 37.2 +/- 1.5 U/mg in naive hGPX1-Tg cortex, p < 0.008). These findings support the hypothesis that the immature brain has limited GPX activity and is more susceptible to oxidative damage and may explain the paradoxical effect seen in ischemic neonatal brain when SOD1 is overexpressed.

Animals↗

PubMed2004

Differential vulnerability of immature murine neurons to oxygen-glucose deprivation.

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6-8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions-oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.

Animals↗

PubMed2003

Fructose-1,6-bisphosphate preserves intracellular glutathione and protects cortical neurons against oxidative stress.

Fructose-1,6-bisphosphate (FBP), an endogenous intermediate of glycolysis, protects the brain against ischemia-reperfusion injury. The mechanisms of FBP protection after cerebral ischemia are not well understood. The current study was undertaken to determine whether FBP protects primary neurons against hypoxia and oxidative stress by preserving reduced glutathione (GSH). Cultures of pure cortical neurons were subjected to oxygen deprivation, a donor of nitric oxide and superoxide radicals (3-morpholinosydnonimine), an inhibitor of glutathione synthesis (L-buthionine-sulfoximine) or glutathione reductase (1,3-bis(2-chloroethyl)-1-nitrosourea) in the presence or absence of FBP (3.5 mM). Neuronal viability was determined using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. FBP protected neurons against hypoxia-reoxygenation and oxidative stress under conditions of compromised GSH metabolism. The efficacy of FBP depended on duration of hypoxia and was associated with higher intracellular GSH concentration, an effect partly mediated via increased glutathione reductase activity.

Animals↗

PubMed2003

Regulation of hypoxia-inducible factor 1alpha and induction of vascular endothelial growth factor in a rat neonatal stroke model.

Stroke is a devastating condition occurring in at least 1 in 4000 live births in the neonatal period. Since hypoxia-inducible factor (HIF)-1alpha can modulate ischemic injury via induction of target genes that may protect cells against ischemia, and is induced after preconditioning by hypoxia in the neonatal rat brain hypoxia-ischemia model, we evaluated whether HIF-1alpha is induced after focal ischemia-reperfusion, a model for neonatal stroke. We developed an ischemia-reperfusion model in postnatal day 10 (P10) rats by transiently occluding the middle cerebral artery (MCA) for 1.5 h. The MCA territory was reperfused for 0, 4, 8, or 24 h and the expression of HIF-1alpha and its target gene, vascular endothelial growth factor (VEGF), were delineated. HIF-1alpha protein and VEGF protein peaked at 8 h, and declined subsequently at 24 h in injured cortex following 1.5 h of MCA occlusion. Double-immunolabeling indicated that both HIF-1alpha and VEGF are expressed together in neurons with a similar time course of expression. The presence of HIF-1alpha and VEGF after moderate ischemia-reperfusion injury suggests potential avenues to exploit for neuroprotection.

Animals↗

PubMed2002

Rat forebrain neurogenesis and striatal neuron replacement after focal stroke.

The persistence of neurogenesis in the forebrain subventricular zone (SVZ) of adult mammals suggests that the mature brain maintains the potential for neuronal replacement after injury. We examined whether focal ischemic injury in adult rat would increase SVZ neurogenesis and direct migration and neuronal differentiation of endogenous precursors in damaged regions. Focal stroke was induced in adult rats by 90-minute right middle cerebral artery occlusion (tMCAO). Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (BrdU) labeling and immunostaining for cell type-specific markers. Brains examined 10-21 days after stroke showed markedly increased SVZ neurogenesis and chains of neuroblasts extending from the SVZ to the peri-infarct striatum. Many BrdU-labeled cells persisted in the striatum and cortex adjacent to infarcts, but at 35 days after tMCAO only BrdU-labeled cells in the neostriatum expressed neuronal markers. Newly generated cells in the injured neostriatum expressed markers of medium spiny neurons, which characterize most neostriatal neurons lost after tMCAO. These findings indicate that focal ischemic injury increases SVZ neurogenesis and directs neuroblast migration to sites of damage. Moreover, neuroblasts in the injured neostriatum appear to differentiate into a region-appropriate phenotype, which suggests that the mature brain is capable of replacing some neurons lost after ischemic injury.

Animals↗