T. Genetta

Erythropoietin after focal cerebral ischemia activates the Janus kinase-signal transducer and activator of transcription signaling pathway and improves brain injury in postnatal day 7 rats.

Erythropoietin (Epo) plays a central role in erythropoiesis but also has neuroprotective properties. Recently, Epo-related neuroprotective studies used a hypoxic-ischemic neonatal model, which is different from focal stroke, a frequent cause of neonatal brain injury. We report on the effects of Epo treatment given after focal stroke and its potential neuroprotective mechanisms in postnatal day 7 rats with focal cerebral ischemia (FCI) achieved by occlusion of the middle cerebral artery. The experimental groups included sham operation, FCI plus vehicle, and FCI plus Epo. In the Epo-treated group, pups received a single intraperitoneal injection of 1000 U/kg 15 min after FCI or three injections of 100, 1000, or 5000 U/kg, starting at 15 min and repeated at 1 and 2 d after FCI. Epo treatment produced significant reductions in the mean infarct area and volume at 1 and 3 d after FCI, demonstrated by 2,3,5-triphenyltetrazolium chloride staining. Terminal deoxynucleotidyltransferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining showed a markedly reduced number of TUNEL-positive cells in the Epo-treated group when compared with the vehicle control 3 d after FCI (p < 0.01). The most effective dose after FCI was 1000 U/kg for 3 d. Immunoanalyses showed that Epo induced a significant increase in phosphorylated Janus kinase 2 and signal transducer and activator of transcription-5 expressions at 1 and 3 d and up-regulated Bcl-xL expression by 24 h after FCI but did not affect Epo receptor or NF-κB expression. In conclusion, Epo given after FCI in neonatal rats provides significant neuroprotection, mediated possibly by activation of the Janus kinase–signal transducer and activator of transcription–Bcl-xL signaling pathways.

Gender differences in long-term beneficial effects of erythropoietin given after neonatal stroke in postnatal day-7 rats

Recently, we reported that erythropoietin attenuates neonatal brain injury caused by focal cerebral ischemia. The long-term effects of erythropoietin on focal cerebral ischemia-induced injury to the developing brain and the potential gender differences in these long-term effects have not been studied in detail. The current study demonstrated a similarity in the mean infarct volume in both the vehicle-treated male and female rats at 6 and 12 weeks after focal cerebral ischemia. On the other hand, erythropoietin treatment (1000 U/kg x three doses after focal cerebral ischemia) caused a significant reduction in the mean infarct volume in both males and females at 6 weeks after focal cerebral ischemia when compared with the corresponding vehicle-treated animals (males: 141.4+/-48.2 mm3 vs. 194.0+/-59.2 mm3, P<0.05; females: 85.4+/-31.6 mm3 vs. 183.4+/-46.3 mm3, P<0.05). Interestingly, the reduction in the mean infarct volume in the erythropoietin-treated males was significantly less than that in the erythropoietin-treated females at 6 weeks after focal cerebral ischemia (141.4+/-48.2 mm3 vs. 85.4+/-31.6 mm3, P<0.05). At 12 weeks after focal cerebral ischemia, the mean infarct volume in the erythropoietin-treated males significantly increased to 181.0+/-50.4 mm3 (P<0.05). In contrast, the mean infarct volume in the erythropoietin-treated females remained stable (87.0+/-41.7 mm3). Additionally, erythropoietin treatment significantly improved sensorimotor function recovery with a misstep number similar to the sham-operation group at 6 and 12 weeks after focal cerebral ischemia. Moreover, the mean number of missteps in the erythropoietin-treated females was less than that in males at 6 (13.5+/-2.0 vs. 24.5+/-2.5, P<0.05) and 12 (12.5+/-2.0 vs. 20.0+/-2.0, P<0.05) weeks after focal cerebral ischemia. These results indicate that erythropoietin administration after focal cerebral ischemia produces a significant long-term neuroprotective benefit on the developing brain, and that this effect is more beneficial in the female rats.

Permanent focal cerebral ischemia activates erythropoietin receptor in the neonatal rat brain

Erythropoietin (Epo) has been shown to act as a neurotrophic and neuroprotective factor via binding to its receptor (EpoR) which is activated in adult brains following hypoxia and ischemia. However, no evidence suggests that cerebral ischemia can activate EpoR in the neonatal brain. In the present study, the changes in EpoR expression were investigated using a modified model of permanent focal cerebral ischemia (FCI) in 7-day-old rat pups. Western blot analysis with an anti-rabbit EpoR antibody revealed a significant increase in the EpoR protein in the ischemic areas, starting from 6 to 12 h after FCI. Moreover, many EpoR-positive cells were detected in the ischemic areas from 12 h after FCI, and the positive cells were identified as neurons and microglia/macrophage but not astrocytes 24 h after FCI. Additionally, double staining with a red in situ apoptosis detection kit and the EpoR antibody indicated that EpoR-positive cells were in apoptotic cell death in the ischemic area. Therefore, these results suggest that EpoR is activated in the ischemic areas of neonatal rats and plays an important role in brain injury during development.

ZEB1 links p63 and p73 in a novel neuronal survival pathway rapidly induced in response to cortical ischemia.

Background Acute hypoxic/ischemic insults to the forebrain, often resulting in significant cellular loss of the cortical parenchyma, are a major cause of debilitating injury in the industrialized world. A clearer understanding of the pro-death/pro-survival signaling pathways and their downstream targets is critical to the development of therapeutic interventions to mitigate permanent neurological damage. Methodology/Principal Findings We demonstrate here that the transcriptional repressor ZEB1, thought to be involved in regulating the timing and spatial boundaries of basic-Helix-Loop-Helix transactivator-mediated neurogenic determination/differentiation programs, functions to link a pro-survival transcriptional cascade rapidly induced in cortical neurons in response to experimentally induced ischemia. Employing histological, tissue culture, and molecular biological read-outs, we show that this novel pro-survival response, initiated through the rapid induction of p63, is mediated ultimately by the transcriptional repression of a pro-apoptotic isoform of p73 by ZEB1. We show further that this phylogenetically conserved pathway is induced as well in the human cortex subjected to episodes of clinically relevant stroke. Conclusions/Significance The data presented here provide the first evidence that ZEB1 induction is part of a protective response by neurons to ischemia. The stroke-induced increase in ZEB1 mRNA and protein levels in cortical neurons is both developmentally and phylogenetically conserved and may therefore be part of a fundamental cellular response to this insult. Beyond the context of stroke, the finding that ZEB1 is regulated by a member of the p53 family has implications for cell survival in other tissue and cellular environments subjected to ischemia, such as the myocardium and, in particular, tumor masses.

The regulatory role of NF-κB in autophagy-like cell death after focal cerebral ischemia in mice

Autophagy may contribute to ischemia-induced cell death in the brain, but the regulation of autophagic cell death is largely unknown. Nuclear factor kappa B (NF-κB) is a regulator of apoptosis in cerebral ischemia. We examined the hypothesis that autophagy-like cell death could contribute to ischemia-induced brain damage and the process was regulated by NF-κB. In adult wild-type (WT) and NF-κB p50 knockout (p50(-/-)) mice, focal ischemia in the barrel cortex was induced by ligation of distal branches of the middle cerebral artery. Twelve to 24h later, autophagic activity increased as indicated by enhanced expression of Beclin-1 and LC3 in the ischemic core and/or penumbra regions. This increased autophagy contributed to cell injury, evidenced by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) co-staining and a protective effect achieved by the autophagy inhibitor 3-methyladenine. The number of Beclin-1/TUNEL-positive cells was significantly more in p50(-/-) mice than in WT mice. Neuronal and vascular cell death, as determined by TUNEL-positive cells co-staining with NeuN or Collagen IV, was more abundant in p50(-/-) mice. Immunostaining of the endothelial cell tight junction marker occludin revealed more damage to the blood-brain barrier in p50(-/-) mice. Western blotting of the peri-infarct tissue showed a reduction of Akt-the mammalian target of rapamycin (mTOR) signaling in p50(-/-) mice after ischemia. These findings provide the first evidence that cerebral ischemia induced autophagy-like injury is regulated by the NF-κB pathway, which may suggest potential treatments for ischemic stroke.

Cardiotrophin-1 protects cortical neuronal cells against free radical-induced injuries in vitro.

Cardiotrophin-1 (CT-1) was initially defined as a mediator of cardiomyocyte hypertrophy. Additional studies have showed that CT-1 enhanced survival of differentiated cardiac muscle cells and inhibited cardiac myocyte apoptosis after serum deprivation or cytokine stimulation. Moreover, CT-1 has recently been shown to act as a neuroregulatory cytokine in the peripheral nervous system. However, its effects in the central nervous system have not been determined. In the present study, we evaluated whether CT-1 protects cultured cortical neurons against oxidative injuries caused by the hydroxyl radical-producing agent FeSO4 and by the peroxynitrite-producing agent 3-morpholinosydnonimine (SIN-1). CT-1 reduced neuronal cell death caused by FeSO4 and also attenuated the neurotoxic effect of SIN-1 in a dose-dependent manner. These results indicate that CT-1 is neuroprotective in an in vitro model of cerebral ischemia. This study indicates that further evaluation of CT-1 in acute brain injury should be investigated in vivo.

116 THE TRANSCRIPTIONAL REPRESSOR ZINC-FINGER E-BOX BINDING PROTEIN TARGETED BY HYPOXIA-INDUCIBLE FACTOR-1a IN RESPONSE TO STROKE REPRESSES A NUMBER OF PROAPOPTOTIC GENES.

We have recently discovered that a transcriptional repressor protein called ZEB (zinc-finger E-box binding protein), thought to be involved in regulating the timing and spatial boundaries of neurogenic determination/differentiation programs, can function to promote neuronal cell survival against a number of proapoptotic insults, including hypoxia-ischemia (H-I). ZEB protein levels increase dramatically in pyramidal cells of the rat cerebral cortex in response to experimentally administered unilateral focal cerebral ischemia [FCI: 15-fold (postnatal day 7) and 30-fold (8 week adult), compared to the contralateral control]. We show that not only is ZEB a HIF-1a transcriptional target, but in response to H-I, it represses several classes of genes involved in either mediating neuronal apoptosis or in inhibiting HIF-1a-induced survival pathways. Further, we present evidence that, for a subset of these proapoptotic genes, notably PUMA, BMF, etc, ZEB is acting through TAp73 and its truncated isoform, DNp73. Lastly, we show that immunostained paraffin sections derived from archived samples of cortical brain taken at autopsy from human perinatal stroke patients give nearly identical results, with respect to intensity and staining pattern, of ZEB protein induction as seen in our experimental model of permanent FCI. Funded through the Emory Goddard Scholar Award (A.S.) and the United Cerebral Palsy Research Foundation (T.G.).

220 EXPOSURE TO HYPEROXIA CAUSES OXIDATIVE STRESS AND INCREASES APOPTOTIC-LIKE CELL DEATH IN THE MURINE DEVELOPING BRAIN.

Background Exposure to hyperoxia has been associated with significant perinatal morbidity such as retinopathy of prematurity and bronchopulmonary dysplasia but the effect of hyperoxia on the developing brain is less known. Studies have shown that hyperoxia results in apoptosis in the immature murine brain in several animal models. However, little is known about the effect of hyperoxia on neural progenitor/stem cells in the developing brain. Objectives To determine the effects of exposure to high (FiO2 of 1.0) versus room air oxygen concentrations (FiO2 of 0.21) on the neural progenitor/stem cells in newborn mice. Design/Methods Five-day-old transgenic mouse pups that express green fluorescent protein (GFP) under control of the nestin gene were randomized into 2 groups. Animals were subjected to either normoxic conditions (FiO2 of 0.21) (n = 4) or hyperoxic conditions (FiO2 of 1.0) (n = 4) for 90 min. Twenty-four hours after the experiment, pups were euthanized and their brains harvested, fixed, and cryosectioned. Apoptotic-like changes were assessed by terminal transferase-mediated dUTP nick end labeling (TUNEL).Oxidative stress was assessed by immunohistochemical methods using a monoclonal antibody against 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product. Positive cells were counted in 20 different fields in 4 different slices per animal, including cortical region and ventricular zone. Results Neural cells showed weak and patchy HNE immunoreactivity in cortex and ventricular/subventricular zone (VZ) in control pups exposed to.normoxic conditions. Pups exposed to hyperoxic conditions showed marked increase in the intensity of immunoreactivity as well as the number of HNE+ cells, both in cortical (mean 6 SD: 568 6 164 vs 55.3 6 24; p = .0001) and VZ regions (311 6 103 vs 78 6 49; p = .01) On preliminary visual analysis the number of TUNEL+ cells appears to be higher after exposure to hyperoxia, with a five-fold increase compared to the normoxic pups. Furthermore, the number of nestin+ cells undergoing apoptotic-like changes also appears to be greater in the hyperoxia-exposed pups compared to the control group pups. Conclusions These preliminary results suggest that exposure to high oxygen concentration causes an increase in oxidative stress markers. High oxygen concentration exposure also appears to increase apoptosis in nestin+ cells, indicating the possible injurious effects of hyperoxia on neural progenitor/stem cells. We are continuing to investigate the behavior of neural stem cells exposed to hyperoxia in the developing brain.

318 LONG-TERM BENEFICIAL EFFECTS OF ERYTHROPOIETIN GIVEN AFTER NEONATAL STROKE IN POSTNATAL DAY-7 RATS

Background We and others have shown that erythropoietin (Epo) attenuates neonatal brain injury caused by hypoxia-ischemia and neonatal stroke; however, little is known about the long-term effects of Epo on injury to the developing brain. Objective To investigate the long-term effects of Epo on brain injury caused by focal cerebral ischemia (FCI) in postnatal day-7 (P7) rat pups. Methods FCI was induced by using a modified intraluminal catheter technique causing middle cerebral artery occlusion in P7 rats, as previously described. The experimental groups included sham-operated (n = 15), FCI plus vehicle (n = 17) and FCI plus recombinant human Epo (n = 18). In Epo-treated group, pups received a single intraperitoneal injection of 1000 U/kg at 15 minutes after FCI, which was repeated at 1 and 2 days after FCI. At 6 and 12 weeks after surgery, animals were sacrificed, and body weight and brain weight were determined, and then their brains were cut into 2 mm coronal slices. The infarct area and volume were measured using Windows Image J. Statistical comparisons were conducted by using the two-tailed Mann-Whitney U-test. Results In the vehicle-treated FCI group, focal cerebral insult caused a marked reduction in brain weight (6 weeks: 1.23 ± 0.1 g; 12 weeks: 1.50 ± 0.1 g; p = .0017), but there was no significant difference in body weight (6 weeks: 104.4 ± 13.7 g; 12 weeks: 352.0 ± 77.5 g), compared to the sham-operated group. Mean brain weight in the Epo-treated FCI group was significantly increased at 6 weeks (1.39 ± 0.1 g; p = .0015) and at 12 weeks (1.75 ± 0.14 g; p = .001) after FCI compared to the vehicle-treated group. Moreover, Epo treatment after FCI produced significant reductions in the mean infarct area and volume in comparison to vehicle-treated group at 6 weeks (infarct area: 38.2 ± 33.6 mm< vs 94.3 ± 23.4 mm<, p = .002; infarct volume: 67.8 ± 53.1 mm> vs 188.5 ± 46.9 mm>, p = .002) and at 12 weeks (infarct area: 67.4 ± 45.0 mm< vs 127.2 ± 21.9 mm<; infarct volume: 134.9 ± 89.9 mm> vs 253.3 ± 44.2 mm>). Conclusion Epo administration after focal cerebral insult produces a significant neuroprotective benefit over a prolonged period in the developing rat brain with neonatal stroke. These findings indicate that Epo may indeed be of clinical use as a potential neuroprotective agent in neonatal ischemic injury.

319 A NOVEL NEURONAL SURVIVAL PATHWAY IN THE DEVELOPING CORTEX

Background Hypoxic insult to the developing brain results in neuronal and glial cellular loss, in large part through apoptosis. A clearer understanding of the signaling pathways and downstream targets that these cells trigger to survive such insult is critical to the eventual development of therapeutic interventions to mitigate permanent neurological damage. We have discovered that a transcriptional repressor of the pro-apoptotic p73 gene, called ZEB (Zinc finger, E-box-binding factor), is itself up-regulated in response to hypoxic insult in the developing brain. Objective To determine the mechanistic basis for the up-regulation of ZEB, as well as the functional consequences of this effect with regard to neuronal survival. Methods Dominant negative versions of ZEB (that activate rather than repress) were constructed via standard recombinant DNA methodologies and transfected into primary cortical neurons isolated from E16 rat embryos. Additionally, P7 rat pups were subjected to unilateral focal cerebral ischemia via permanent occlusion of the mid cerebral artery (the contralateral side serving as control). At specific time points, brains were processed for and immunostained using antibodies against ZEB and the neuronal-specific antigen, NeuN. TUNEL staining was carried out using a standard kit protocol. Expression vectors encoding cDNAs for HIF-1 and dominant negative HIF-1 were obtained from the ATCC. Results Dominant negative versions of ZEB causes primary cortical neurons to apoptose. In response to focal ischemic insult, ZEB protein was up-regulated in the nuclei of cortical neurons on the ischemic side. This increased expression is co-localized with an up-regulation of the EPO receptor on these neurons. At 12 and 24 Hr Post-FCI, ZEB-positive cells are mutually exclusive from TUNEL-positive cells (an end point indicator of cell death). Overexpressed HIF-1 alpha in normoxia up-regulates ZEB about 3-fold via ZEB-promoter-driven reporter analysis; overexpressing a dominant negative version of HIF-1 in primary neurons fails to up-regulate ZEB (via cytostaining). Conclusions This is the first step in the elucidation of what may be a novel pro-survival pathway in the developing brain, one that is utilized by neurons to defend against proapoptotic insults, such as hypoxia. One well-characterized target of ZEB transcriptional repression, the p73 gene, encodes a protein that, in its full-length form, is known to play a role in neuronal cell death. Taken together, the above data suggest a potential link between pro-survival HIF-1 signaling and repression of the pro-death p73 gene and offer a potential mechanistic explanation for how a particular neuron might survive an ischemic insult.