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Although blood–brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt–β-catenin signaling. Constitutive activation of Wnt–β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

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MicroRNA (miR)-200c increases rapidly in the brain after transient cerebral ischemia but its role in poststroke brain injury is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-200c. We hypothesized that miR-200c contributes to injury from transient cerebral ischemia by targeting reelin.

Brain ischemia causes immediate and delayed cell death that is exacerbated by inflammation. Recent studies show that hypocretin-1orexin-A (Hcrt-1) reduces ischemic brain injury, and Hcrt-positive neurons modulate infection-induced inflammation. Here, we tested the hypothesis that Hcrt plays a protective role against ischemia by modulating inflammation.

MicroRNAs are important in the development, functioning, and pathophysiology of the central nervous system. Here, we show that increasing the levels of microRNA-320 (miR-320) for 3 days markedly increases neurite length, and at 4 days, reduces the total cell number in Neuro-2A cells. In-silico analysis of possible miR-320 targets identified cAMP-regulated phosphoprotein-19 kDa (ARPP-19) and semaphorin 3a as potential targets that could be involved. ARPP-19 was validated by showing reduced mRNA and protein levels when miR-320 was overexpressed, whereas miR-320 had no effect on semaphorin 3a expression. ARPP-19 is known to inhibit protein phosphatase-2A activity, which inhibits mitosis and induces neurite outgrowth, making this the likely mechanism. Thus, increased levels of miR-320 lead to decreased levels of ARPP-19, increased neurite length, and fewer total cells. These data suggest that miR-320 could play a role in neuronal development and might be a target to enhance neuronal regeneration following injury.

Stroke causes brain injury with activation of an inflammatory response that can contribute to injury. We tested the hypothesis that the anti-inflammatory cytokine interleukin-4 (IL-4) reduces injury after stroke using IL-4 knockout (KO) adult male mice.

Impaired locomotor activity and spatial memory are common features in the natural aging process, and aging is an important risk factor for neurodegenerative disease and postoperative cognitive dysfunction. To characterize age-related changes in psychomotor performance, we assessed sensorimotor activity, spatial learning, and memory in C57BL6 mice using the Rotarod, foot fault, and Barnes Maze tests. Old mice exhibit significant deficits in locomotor activity and spatial memory relative to young mice, but improve with training. These tests will be useful to assess outcome in neurodegenerative disease and postoperative cognitive dysfunction models carried out in aged mice.

Blood–brain barrier (BBB) disruption after stroke can worsen ischemic injury by increasing edema and causing hemorrhage. We determined the effect of microglia on the BBB and its primary constituents, endothelial cells (ECs) and astrocytes, after ischemia using in vivo and in vitro models.

Hypoxic/ischemic brain injury in the neonate can activate an inflammatory cascade, which potentiates cellular injury. The role of microglia in this inflammatory response has not been studied extensively. We used an in vitromodel of murine microglia to investigate changes in microglial cytokine release and injury during early development. Isolated microglia were subjected to lipopolysaccharide (LPS) activation or injury by glucose deprivation (GD), serum deprivation (SD), or combined oxygen-glucose deprivation (OGD) for varying durations. The extent and the type of cell death were determined by trypan blue, terminal deoxynucleotidyl end-nick labeling, and annexin staining. Early-culture microglia (2–3 d in purified culture) showed significantly more apoptotic cell death after SD, GD, and OGD compared with microglia maintained in culture for 14–17 d. Measurements of tumor necrosis factor- (TNF-) and IL-1 in culture media demonstrated that OGD induced greater release of both TNF- and IL-1 than LPS activation, with early-culture microglia producing more TNF- compared with late-culture microglia. Microglia that are cultured for a short time are more sensitive to ischemia-like injury in vitrothan those that are cultured for longer durations and may contribute to worsening brain injury by increased release of inflammatory cytokines. Inhibition of microglial activation and decreasing proinflammatory cytokine release may be targets for reduction of neonatal hypoxic/ischemic brain injury.

Hypoxic/ischemic brain injury in the neonate can activate an inflammatory cascade, which potentiates cellular injury. The role of microglia in this inflammatory response has not been studied extensively. We used an in vitro model of murine microglia to investigate changes in microglial cytokine release and injury during early development. Isolated microglia were subjected to lipopolysaccharide (LPS) activation or injury by glucose deprivation (GD), serum deprivation (SD), or combined oxygen-glucose deprivation (OGD) for varying durations. The extent and the type of cell death were determined by trypan blue, terminal deoxynucleotidyl end-nick labeling, and annexin staining. Early-culture microglia (2–3 d in purified culture) showed significantly more apoptotic cell death after SD, GD, and OGD compared with microglia maintained in culture for 14–17 d. Measurements of tumor necrosis factor-a (TNF-a) and IL-1ß in culture media demonstrated that OGD induced greater release of both TNF-a and IL-1ß than LPS activation, with early-culture microglia producing more TNF-a compared with late-culture microglia. Microglia that are cultured for a short time are more sensitive to ischemia-like injury in vitro than those that are cultured for longer durations and may contribute to worsening brain injury by increased release of inflammatory cytokines. Inhibition of microglial activation and decreasing proinflammatory cytokine release may be targets for reduction of neonatal hypoxic/ischemic brain injury.

The 70 kDa heat shock protein (Hsp70) is synthesized in response to a variety of stresses, including ischemia, and is thought to act as a molecular chaperone to prevent protein denaturation and facilitate protein folding. Matrix metalloproteinases (MMPs), a family of serine proteases, are also upregulated by ischemia and are thought to promote cell death and tissue injury. We examined the influence of Hsp70 on expression and activity of MMPs. Astrocyte cultures were prepared from neonatal mice and transfected with retroviral vectors containing hsp70or lacZor mock infected, then exposed to oxygen-glucose deprivation followed by reperfusion. Zymograms and Western blots showed that Hsp70 over-expression suppressed MMP-2 and MMP-9. These findings suggest that Hsp70 may protect by regulating MMPs.

Overexpression of inducible Hsp70 has been shown to provide protection from cerebral ischemia both in animal models of stroke and in cell culture models. New work suggests that there are multiple routes of cell death, including apoptotic and necrotic cell death. Hsp70 is known to protect from both necrotic and apoptotic cell death. In addition to the well-studied role of Hsp70 as a molecular chaperone assisting in correct protein folding, several new mechanisms by which Hsp70 can prevent cell death have been described. Hsp70 is now known to regulate apoptotic cell death both directly by interfering with the function of several proteins that induce apoptotic cell death as well as indirectly by increasing levels of the anti-death protein bcl-2. Despite these new insights into the ways in which Hsp70 functions as an anti-death protein, further surprises are likely as we continue to gain insight into the functioning of this multifaceted protein.

We have previously reported studies of gene therapy using a neurotropic herpes simplex viral (HSV) vector system containing bipromoter vectors to transfer various protective genes to neurons. Using this system in experimental models of stroke, cardiac arrest, and excitotoxicity, we found that it is possible to enhance neuron survival against such cerebral insults by overexpressing genes that target various facets of injury. Among the genes we studied, the anti-apoptotic protein BCL-2 improved neuron survival following various insults, and was protective even when administered after stroke onset. BCL-2 is thought to protect cells from apoptotic death by preventing cytochrome c release from the mitochondria and subsequent caspase activation. We and others have established that cooling the brain by a few degrees markedly reduces ischemic injury and improves neurologic deficits in models of cerebral ischemia and trauma. This hypothermic neuroprotection is also associated with BCL-2 upregulation in some instances. Furthermore, hypothermia suppresses many aspects of apoptotic death including cytochrome c release, caspase activation, and DNA fragmentation. Here we show that two different kinds of protective therapies, BCL-2 overexpression and hypothermia, both inhibit aspects of apoptotic cell death cascades, and that a combination treatment can prolong the temporal therapeutic window for gene therapy.

Bcl-xLis a protein that blocks both apoptotic and necrotic cell death. The authors have previously shown that it is effective in maintaining mitochondrial membrane potential during glucose deprivation in cultured astrocytes. To further investigate the mechanism involved, the authors studied mitochondrial function and cytochrome crelease. Oxygen consumption was monitored to assess oxidative respiration. State III respiration decreased significantly as early as 3 h after removal of glucose. At this time mitochondria hyperpolarize but cytochrome cis not yet released. Damage to the electron transport chain is not responsible for this change because uncoupled respiration was unchanged at this time point. At 5 h of glucose deprivation, when mitochondrial depolarization was observed, state IV respiration increased significantly, cytochrome cbegan to be released, and mitochondrial morphology changed from elongated to punctate. When Bcl-xLwas overexpressed normal state III respiration and mitochondrial morphology were maintained and cytochrome crelease was inhibited in the face of glucose deprivation stress.

Cerebral ischemia and protection is a large field, so for the purposes of this review, which focuses on results published in the last 9 months, we have chosen to discuss a few aspects of ischemia in which our understanding has advanced significantly in this period of time. Recent progress in the clinical use of hypothermia for neurological protection as well as laboratory progress on the role of stress proteins, estrogen and a few other potential adjuncts will be discussed.

We investigated whether HSV gene transfer of HSP72 in vivo and in vitro: (1) protected cornu ammonis 1 region of the hippocampus neurons from global cerebral ischemia; and (2) affected Bcl‐2 expression. HSV vectors expressing HSP72 and β‐galactosidase (reporter) or β‐galactosidase only (control vector) were injected into cornu ammonis 1 region of the hippocampus 15 hours before induction of global cerebral ischemia (n = 10) and sham‐operated rats (n = 8). HSP72 vector–treated rats displayed significantly more surviving transfected neurons (X‐gal‐positive, 31 ± 8) compared with control vector–treated rats (10 ± 4) after global cerebral ischemia. Sham‐operated rats displayed similar numbers of X‐gal–positive neurons (HSP72 vector 18 ± 8 vs control vector 20 ± 7). The percentage of β‐galactosidase and Bcl‐2 coexpressing neurons in HSP72‐treated rats after global cerebral ischemia (84 ± 4%) was greater than that in control vector–treated rats (58 ± 9%). The percentage of β‐galactosidase and Bcl‐2 coexpressing neurons in sham‐operated rats was similar in HSP72 (93 ± 7%) and in control vector–treated rats (88 ± 12%). HSP72 vector transfection led to 12 times as much Bcl‐2 expression as the control vector in uninjured hippocampal neuronal cultures. In injured (oxygen‐glucose deprivation) hippocampal neuron cultures, HSP72 vector transfection led to 2.8 times as much Bcl‐2 expression as control vector. We show that HSP72 overexpression protects cornu ammonis 1 region of the hippocampus neurons from global cerebral ischemia, and that this protection may be mediated in part by increased Bcl‐2 expression.

Recent experimental work has shown that hypothermia with even small decreases in temperature is broadly neuroprotective, but the mechanism of this protection remains unclear. Although reduction of metabolism could explain protection by deep hypothermia, it does not explain the robust protection found with mild hypothermia. Several reports have suggested that ischemic apoptosis is reduced by hypothermia. The authors examined the effects of hypothermia on neuronal apoptosis using serum deprivation, a well-accepted model that induces neuronal apoptosis. Mild hypothermia (33°C) significantly reduced the number of morphologically apoptotic neurons to less than half the number seen in normothermic culture temperatures (37°C) after 48 hours. They examined the effect of hypothermia on several steps in the cascade. Caspase-3, −8, and −9 activity was significantly increased after 24 hours at 37°C, and was significantly lower in cultures deprived of serum at 33°C. Cytochrome ctranslocation was reduced by hypothermia. Western blot analysis failed to detect significant changes in Bax, bcl-2, or hsp-70 at early time points, whereas hypothermia significantly reduced cJun N-terminal kinase activation. The authors conclude that small decreases in temperature inhibit apoptosis very early, possibly at the level of the initiation of apoptosis, as suggested by reduced cJun N-terminal kinase activation and before the translocation of cytochrome c, with subsequent prevention of caspase activation.

The 72-kD inducible heat shock protein (HSP72) can attenuate cerebral ischemic injury when overexpressed before ischemia onset. Whether HSP72 overexpression is protective when applied after ischemia onset is not known, but would have important clinical implications. Fifty-seven rats underwent middle cerebral artery occlusion for 1 hour. Defective herpes simplex viral (HSV) vectors expressing hsp72with lacZas a reporter were delivered 0.5, 2, and 5 hours after ischemia onset into each striatum. Control animals received an identical vector containing only lacZ. Striatal neuron survival at 2 days was improved by 23% and 15% when HSP72 vectors were delayed 0.5 and 2 hours after ischemic onset, respectively (P< 0.05). However, when delayed by 5 hours, HSP72 overexpression was no longer protective. This is the first demonstration that HSP72 gene transfer even after ischemia onset is neuroprotective. Because expression from these HSV vectors begins 4 to 6 hours after injection, this suggests that the temporal therapeutic window for HSP72 is at least 6 hours after ischemia onset. Future strategies aimed at enhancing HSP72 expression after clinical stroke may be worth pursuing. The authors suggest that in the future HSP72 may be an effective treatment for stroke.

We previously showed that overexpressing the 70-kDa inducible heat shock protein in primary astrocyte cultures and in a rodent stroke model using viral vectors resulted in protection from ischemia and ischemia-like injury. However, viral transfection could potentially provoke a stress response itself; therefore, we examined whether transgenic mice constitutively expressing human heat shock protein 70 were protected from ischemic insults. Astrocyte cultures from brains of heat shock protein 70 transgenic mice were resistant to hydrogen peroxide injury in a dose-dependent fashion, but were less resistant to hypoglycemia and oxygen-glucose deprivation. Because hydrogen peroxide exposure and glucose deprivation are partially dependent on glutathione levels, we determined whether heat shock protein 70 transgenic cultures had altered glutathione levels under normal growth conditions. However, there was no significant difference in glutathione levels between heat shock protein 70 transgenic and wildtype astrocytes. Hippocampal, but not cortical neuron cultures from these same transgenic mice were also protected against oxygen-glucose deprivation and glutamate toxicity. In an in vivo model of permanent focal cerebral ischemia, there was no significant difference in infarct size assessed 24 h postinsult. These results suggest that heat shock protein 70 protects against some but not all kinds of central nervous system injury. The protective effects may be related to the nature and severity of the insults, as well as subpopulations of brain cells and dose-dependent effects of HSP70 overexpression.

Different brain regions show differential vulnerability to ischemia in vivo. Despite this, little work has been done to compare vulnerability of brain cells isolated from different brain regions to injury. Relatively pure neuronal and astrocyte cultures were isolated from mouse cortex, hippocampus, and striatum. Astrocyte vulnerability to 6 h oxygen–glucose deprivation was greatest in striatum (81.8 ± 4.6% cell death), intermediate in hippocampus (59.8 ± 4.8%), and least in cortex (37.0 ± 3.5%). In contrast neurons deprived of oxygen and glucose for 3 h showed greater injury to cortical neurons (71.1 ± 5.2%) compared to striatal (39.0 ± 3.1%) or hippocampal (39.0 ± 5.3%) neurons. Astrocyte injury from glucose deprivation or H2O2exposure was significantly greater in cells from cortex than from striatum or hippocampus. Neuronal injury resulting from serum deprivation was greater in cortical neurons than in those from striatum or hippocampus, while excitotoxic neuronal injury was equivalent between regions. Antioxidant status and apoptosis-regulatory genes were measured to assess possible underlying differences. Glutathione was higher in astrocytes and neurons isolated from striatum than in those from hippocampus. Superoxide dismutase activity was significantly higher in striatal astrocytes, while glutathione peroxidase activity and superoxide did not differ by brain region. Bcl-xLwas significantly higher in striatal astrocytes than in astrocytes from other brain regions and higher in striatal and hippocampal neurons than in cortical neurons. Both neurons and astrocytes isolated from different brain regions demonstrate distinct patterns of vulnerability when placed in primary culture. Antioxidant state and levels of expression of bcl-xLcan in part account for the differential injury observed. This suggests that different protective strategies may have different efficacies depending on brain region.

No abstract available.

Pretreatment by a sublethal insult is associated with induction of stress proteins and with protection from subsequent injury. Heat pretreatment protects the brain from subsequent ischemia, and is shown here to protect primary astrocyte cultures from subsequent oxygen-glucose deprivation. To determine whether the expression of a single stress protein, HSP-70, could account for much of this protection, we expressed HSP-70 or β-galactosidase in astrocytes using retroviral vectors. Only 12 of astrocytes expressing HSP-70 died after 7 hours of oxygen-glucose deprivation compared to 65 of astrocytes expressing β-galactosidase and 82 of normal astrocytes. Our data provide direct evidence that selective expression of HSP-70 enhances the survival of astrocytes challenged with heat or oxygen-glucose deprivation.

ACIDOSIS is a well established concomitant of tissue ischemia. Acidosis in the pH range 6.0–7.0 is seen in cerebral ischemia and within solid tumors. Extracellular acidosis of pH 6.0 and 6.4 provided essentially complete protection from & h serum deprivation induced apoptotic death of cultured primary murine neurons. We tested the effect of p53 using transformed mouse embryo fibroblasts of either p53/or p53−/−genotype. Both were markedly protected from serum deprivation by acidity. Hypoxia induced fibroblast injury was also reduced at pH 6.8. Lower pH resulted in a shift from apoptotic to necrotic morphology after 24h hypoxia. Acidosis reduces apoptosis of both normal and transformed cells, irrespective of p53 status.

The in vivo neuroprotective effect and brain levels of cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755), a competitive N-methyl-D-aspartate (NMDA) antagonist, were compared with its in vitro neuroprotective effects. The dose-response for in vitro neuroprotection against both NMDA toxicity and combined oxygen-glucose deprivation (OGD) was determined in murine neocortical cultures. Primary cultures of neocortical cells from fetal mice were injured by exposure to 500 μM NMDA for 10 min or to OGD for 45 min. The effect of CGS 19755 in both injury paradigms was assessed morphologically and quantitated by determination of lactate dehydrogenase release. Near complete neuroprotection was found at high doses of CGS 19755. The ED50for protection against NMDA toxicity was 25.4 μmM, and against OGD the ED50was 15.2 μM. For the in vivo paradigm rabbits underwent 2 h of left internal carotid, anterior cerebral, and middle cerebral artery occlusion followed by 4 h reperfusion; ischemic injury was assessed by magnetic resonance imaging and histopathology. The rabbits were treated with 40 mg/kg i.v. CGS 19755 or saline 10 min after arterial occlusion. CSF and brain levels of CGS 19755 were 12 μM and 5 μM, respectively, at 1 h, 6 μM and 5 μM at 2 h, and 13 μM and 7 μM at 4 h. These levels were neuroprotective in this model, reducing cortical ischemic edema by 48% and ischemic neuronal damage by 76%. These results suggest that a single i.v. dose penetrates the blood-brain barrier, attaining sustained neuroprotective levels that are in the range for in vitro neuroprotection.

We examined the effects of secobarbital and other sedative-hypnotic barbiturates on the neuronal death induced by exposure to excitatory amino acids or deprivation of oxygen or glucose in mouse cortical cell cultures. N-Methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, and kainate toxicities were attenuated in a concentration-dependent fashion by high concentrations of secobarbital or thiopental. Antagonism of NMDA toxicity was not overcome by increasing NMDA concentration and not mimicked by γ-aminobutyrate. Despite these antiexcitotoxic actions, secobarbital exacerbated the neuronal death induced by deprivation of either glucose alone or oxygen and glucose together; death induced by oxygen deprivation alone was little affected. Thiopental and methohexital also increased oxygen-glucose deprivation injury. A possible explanation for this injury potentiation was provided by the observation that secobarbital enhanced the cellular ATP depletion induced by combined oxygen-glucose deprivation. Deleterious effects on ATP production may counterbalance the protective effects of barbiturates under some conditions.