Neonatal mice lacking functional Fas death receptors are resistant to hypoxic-ischemic brain injury

Neonatal hypoxia-ischemia (HI) upregulates Fas death receptor expression in the brain, and alterations in expression and activity of Fas signaling intermediates occur in neonatal brain injury. B6.MRL-Tnfrsf6(lpr) mice lacking functional Fas death receptors are protected from HI brain damage in cortex, striatum, and thalamus compared to wild-type mice. Expression of Fas death receptor and active caspases increase in the cortex after HI. In wild-type mice, the hippocampus is most severely injured, and the hippocampus is the only region not protected in the B6.MRL-Tnfrsf6(lpr) mice. The selective vulnerability of the hippocampus to injury correlates with (1) lower basal expression of [Fas-associated death-domain-like IL-1beta-converting enzyme]-inhibitory protein (FLIP), (2) increased degradation of spectrin to its 145 or 150 kDa breakdown product, and (3) a higher percentage of non-apoptotic cell death following neonatal HI. We conclude that Fas signaling via both extrinsic and intrinsic caspase cascades causes brain injury following neonatal HI in a region-dependent manner. Basal levels of endogenous decoy proteins may modulate the response to Fas death receptor signaling and provide a novel approach to understanding mechanisms of neonatal brain injury.     

Extended role of necrotic cell death after hypoxia-ischemia-induced neurodegeneration in the neonatal rat

The relative contribution of apoptosis and necrosis after neonatal hypoxia-ischemia (HI) is still a matter of debate. Here we determined the time course of necrotic cell death after neonatal HI and its relationship to caspase-3 activation and apoptotic cell death. Necrosis was evaluated by intracerebroventricular injection of propidium iodide (PI) before sacrificing the animal and processing brain sections for caspase-3 immunohistochemistry and TUNEL assay. PI-positive cells were found starting from 30 min after HI and increased rapidly in different brain areas. PI co-localized with the neuronal-specific nuclear marker NeuN but not with GFAP indicating that the dye label neurons with damaged plasma membrane but not reactive astrocytes. In the cerebral cortex 24 h after HI, the superficial layers showed cells with strong caspase-3 and TUNEL staining and with nuclei having apoptotic morphology whereas the deep layers of the cortex and the hippocampus showed cells with necrotic features. At later times, cells of the superficial layers were positive to PI, caspase-3, TUNEL and cathepsin-B. These data indicate that necrosis has an extended role in the progression of brain injury after neonatal HI and that a different spectrum of suicidal programs can be activated in the same cell. The extended period of caspase-3 activation in PI-positive necrotic cells supports the possibility that the apoptotic-to-necrotic continuum may ensue as the result of an incomplete execution of the apoptotic program.     

Spinal cord injury induces upregulation of Beclin 1 and promotes autophagic cell death

Autophagy is a degradation of the cytoplasm and it induces autophagic cell death in several neurodegenerative conditions. Beclin 1, a Bcl-2-interacting protein, is known to be a promoter of autophagy. We investigated the alterations in the Beclin 1 protein expression and the involvement of autophagy and autophagic cell death after spinal cord injury using a spinal cord hemisection model in mice. In the present study, the Beclin 1 expression dramatically increased at the lesion site after hemisection. The increased expression of Beclin 1 started from 4 h, peaked at 3 d, and lasted for at least 21 d after hemisection. The Beclin 1 expression was observed in neurons, astrocytes, and oligodendrocytes. The nuclei in the Beclin 1 expressing cells were round, which should normally be observed in autophagic cell death, and they were not either shrunken or fragmented as is observed in apoptotic nuclei. The results of the present study suggested that autophagy is activated in the injured spinal cord. Furthermore, autophagic cell death is considered to clearly contribute to neural tissue damage after spinal cord injury.     

Molecular mediators of hypoxic-ischemic injury and implications for epilepsy in the developing brain

Perinatal hypoxia-ischemia (HI) is the most common cause of cerebral palsy, and an important consequence of perinatal HI is epilepsy. Epilepsy is a disorder in which the balance between cerebral excitability and inhibition is tipped toward uncontrolled excitability. Selected neuronal circuits as well as certain populations of glial cells die from the excitotoxicity triggered by HI. Excitotoxicity, a term referring to cell death caused by overstimulation of the excitatory glutamate neurotransmitter receptors, plays a critical role in brain injury caused by perinatal HI. Ample evidence suggests distinct differences between the immature and mature brain with respect to the pathology and consequences of hypoxic-ischemic brain injury. Thus, the intrinsic vulnerability of specific cell types and systems in the developing brain is particularly important in determining the final pattern of damage and functional disability caused by perinatal HI. These patterns of neuronal vulnerability are associated with clinical syndromes of neurologic disorders such as cerebral palsy, epilepsy, and seizures. Recent studies have uncovered important molecular and cellular aspects of hypoxic-ischemic brain injury. The cascade of biochemical and histopathological events initiated by HI can extend for days to weeks after the insult is triggered, which may provide a "therapeutic window" for intervening in the pathogenesis in the developing brain. Activation of apoptotic programs accounts for the majority of HI-induced pathophysiology in neonatal brain disorders. New experimental approaches to protecting brain tissue from the effects of neonatal HI include administration of neuronal growth factors and effective inhibition of the death effector pathways, such as caspase cascade, and their downstream targets, which execute apoptosis and/or induction of their regulatory cellular proteins. Our recent findings that a novel neuronal protein, neuronal pentraxin 1 (NP1), is induced following HI in neonatal brain and that NP1 gene silencing is neuroprotective suggest that NP1 could be a new molecular target in the central neurons for preventing HI injury in developing brain. Most importantly, the specific interactions between NP1 and the excitatory glutamate receptors and their colocalization further implicate a role for this novel neuronal protein in the excitotoxic cascade. Recent experimental work suggests that these approaches may be effective during a longer therapeutic window after the insult, as they are acting on events that are relatively delayed, creating the potential for therapeutic interventions for these lifelong neurological disabilities.     

IGF-1 protects oligodendrocyte progenitor cells and improves neurological functions following cerebral hypoxia-ischemia in the neonatal rat

To investigate if insulin-like growth factor-1 (IGF-1) provides neuroprotection to oligodendrocyte progenitor cells (OPCs) following cerebral hypoxia-ischemia, a previously developed neonatal rat model of white matter damage was used in this study. Postnatal day 4 (P4) SD rat pups were subjected to bilateral common carotid artery ligation, followed by exposure to 8% oxygen for 10 min. IGF-1 (0.5 microg) or vehicle was injected into the left ventricle after artery ligation and before the hypoxic exposure. Cerebral hypoxia-ischemia caused death of O4+ late OPCs in the P5 rat brain and impaired myelination in the P9 and P21 rat brain. Caspase-3 activation was involved in the death of OPCs. Moreover, cerebral hypoxia-ischemia impaired neurobehavioral performance in juvenile rats. IGF-1 treatment attenuated damages to OPCs and improved neurological functions after cerebral hypoxia-ischemia. It reduced death of O4+ OPCs by 39% on P5 and enhanced myelination on P9 and P21. Bromodeoxyuridine uptake assay showed that cerebral hypoxia-ischemia inhibited proliferation of stem/progenitor cells in the subventricular zone and NG2+ early OPCs in the white matter area. IGF-1 treatment increased cell proliferation in the subventricular zone by 31% 1 day following hypoxic-ischemic insult. Proliferation of early and late OPCs in the IGF-1-treated group was 1.5- and 2.4-fold of that in the vehicle-treated group, respectively. In conclusion, IGF-1 provided potent neuroprotection to OPCs and improved neurological functions following cerebral hypoxia-ischemia in the neonatal rat. The neuroprotection of IGF-1 was associated with its antiapoptotic and mitogenic effects.     

Hypoxic-ischemic injury in neonatal brain: involvement of a novel neuronal molecule in neuronal cell death and potential target for neuroprotection

Perinatal hypoxia-ischemia (HI) is the most common cause of various neurological disabilities in children with high societal cost. Hypoxic-ischemic brain damage is an evolving process and ample evidence suggests distinct difference between the immature and mature brain in the pathology and consequences of brain injury. Therefore, it is of utmost importance to better understand the mechanisms underlying the hypoxic-ischemic injury in neonatal brain to devise effective therapeutic strategies. Nonetheless, the mechanism(s) involved in this pathology in the developing brain remain inadequately understood. Effective neuroprotective strategies will include either inhibition of the death effector pathways or induction of their regulatory and survival promoting cellular proteins. Neuronal pentraxins (NPs) define a family of novel proteins "long pentraxins" that are exclusively expressed in the central neurons, and are homologous to the C-reactive and acute-phase proteins in the immune system. NPs have been shown to be involved in the excitatory synaptic remodeling. We found that the neuronal protein 'neuronal pentraxin 1' (NP1) is induced in neonatal rat brain following HI, and NP1 induction preceded the time of actual tissue loss in brain. In demonstrating this we also found that NP1 gene silencing is neuroprotective against hypoxia-induced neuronal death. This is the first evidence for a pathophysiological function of NP1 in central neurons. Our results suggest that NP1 is part of a death program triggered by HI. Most importantly, our findings of specific interactions of NP1 with the excitatory glutamate receptors AMPA GluR1 subunit and their co-localization suggest a role for this novel neuronal protein NP1 in the excitotoxic cascade. Blockade of AMPA-induced neuronal death following inhibition of NP1 expression further implicates a regulatory interaction between NP1 and AMPA glutamate receptors. Subsequent experiments using NP1 loss-of-function strategies, we have demonstrated specific requirements of NP1 induction in HI-induced neuronal death. Together our findings clearly identify a novel role for NP1 in the coupling between HI and cerebral cell death. Thus, NP1 could be a new molecular target in the central neurons for preventing hypoxic-ischemic neuronal death in developing brain. These very novel results could lead to more effective neuroprotective strategies against hypoxic-ischemic brain injury in neonates.     

Excessive Autophagy Contributes to Neuron Death in Cerebral Ischemia

Aims: To determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia. Methods: We performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo. Results: In both primary cortical neurons and SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD)for 6 h and reperfusion (RP) for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3‐II to LC3‐I and Beclin‐1 (BECN1) expression. Using Fluoro‐Jade C and monodansylcadaverine double‐staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3‐methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3–7 days postinjury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and BECN1 expression. Conclusion: Excessive activation of autophagy contributes to neuronal death in cerebral ischemia.     

Transgenic expression of human FGF-1 protects against hypoxic-ischemic injury in perinatal brain by intervening at caspase-XIAP signaling cascades

Perinatal hypoxia-ischemia (HI) is a major cause of neurological disability and mortality in infant and children. In the present study, we explored the neuroprotective efficacy of FGF-1 in a rat model of perinatal HI. Carotid ligation combined with hypoxia caused marked infarctions in the ipsilateral cerebral hemisphere with significant loss of ipsilateral striatal, cortical and hippocampal volumes. Morphological analyses revealed both apoptotic and necrotic form of neuronal death determined by Nissl histology, dark-field microscopy and TUNEL staining. HI induced a marked increase in activated caspase-9, caspase-3 and PARP cleavage at 12 h to 7 days after HI in brain areas displaying TUNEL (+) cells. In addition, expression of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) was decreased under similar conditions of HI. Expression of human FGF-1 in brain significantly reduced the extent of both apoptotic and necrotic injury caused by HI. FGF-1 attenuated the HI-induced increase in activated caspase-3, caspase-9 and cleaved PARP protein levels and markedly blocked the HI-induced decrease in XIAP expression under the conditions at which FGF-1 showed significant neuroprotection. These findings demonstrate that FGF-1 prevents the onset of both apoptotic and necrotic death in neurons otherwise "destined to die" following hypoxic-ischemic injury by intervening at the level of caspase-signaling cascades and by restoring prosurvival protein XIAP expression in central neurons.     

Intranasal administration of IGF-1 attenuates hypoxic-ischemic brain injury in neonatal rats

To determine whether intranasal administration (iN) of recombinant human insulin-like growth factor-1 (rhIGF-1) provides neuroprotection to the neonatal rat brain following cerebral hypoxia-ischemia (HI), two doses of rhIGF-1 (50 μg at a 1 h interval) were infused into the right naris of postnatal day 7 (P7) rat pups with or without a prior HI insult (right common carotid artery ligation, followed by an exposure to 8% oxygen for 2 h). Our result showed that rhIGF-1 administered via iN was successfully delivered into the brain 30 min after the second dose. In the following studies rhIGF-1 was administered to P7 rat pups at 0, 1 or 2 h after HI at the dose described above. Pups in the control group received cerebral HI and vehicle treatment. Pups that underwent sham operation and vehicle treatment served as the sham group. Brain pathological changes were evaluated 2 and 15 days after HI. Our results showed that rhIGF-1 treatment up to 1 h after cerebral HI effectively reduced brain injury as compared to that in the vehicle-treated rats. Moreover, rhIGF-1 treatment improved neurobehavioral performance (tested on P5–P21) in juvenile rats subjected to HI. Our results further showed that rhIGF-1 inhibited apoptotic cell death, possibly through activating the Akt signal transduction pathway. rhIGF-1 enhanced proliferation of neuronal and oligodendroglial progenitors after cerebral HI as well. These data suggest that iN administration of IGF-1 has the potential to be used for clinical treatment.     

Effects of human umbilical cord blood CD34+ cell transplantation in neonatal hypoxic-ischemia rat model

Perinatal brain injury can cause death in the neonatal period and lifelong neurodevelopmental deficits. Stem cell transplantation had been proved to be effective approach to ameliorate neurological deficits after brain damage. In this study we examine the effect of human umbilical cord blood CD34⁺ cells on model of neonatal rat hypoxic–ischemic brain damage and compared the neuroprotection of transplantation of CD34⁺ cells to mononuclear cells from which CD34⁺ cells isolated on neonatal hypoxic-ischemia rat model. Seven-day-old Sprague-Dawley rats were subjected to hypoxic-ischemic (HI) injury, CD34⁺ cells (1.5?×?10⁴?cells) or mononuclear cells (1.0?×?10⁶?cells) were transplanted into mice by tail vein on the 7?day after HI. The transplantation of CD34⁺ cells significantly improved motor function of rat, and reduced cerebral atrophy, inhibited the expression of glial fibrillary acidic protein (GFAP) and apoptosis-related genes: TNF-α, TNFR1, TNFR2, CD40, Fas, and decreased the activation of Nuclear factor kappa B (NF-κB) in damaged brain. CD34⁺ cells treatment increased the expression of DCX and lectin in ipsilateral brain. Moreover, the transplantation of CD34⁺ cells and MNCs which were obtained from the same amount of human umbilical cord blood had similar effects on HI. Our data demonstrated that transplantation of human umbilical cord blood CD34+ cells can ameliorate the neural functional defect and reduce apoptosis and promote nerve and vascular regeneration in rat brain after HI injury and the effects of transplantation of CD34+ cells were comparable to that of MNCs in neonatal hypoxic-ischemia rat model.     

Erythropoietin prevents long-term sensorimotor deficits and brain injury following neonatal hypoxia-ischemia in rats

Perinatal asphyxia accounts for behavioral dysfunctions that often manifest as sensorimotor, learning or memory disabilities throughout development and into maturity. Erythropoietin (Epo) has been shown to exert neuroprotective effects in different models of brain injury including experimental models of perinatal asphyxia. However, the effect of Epo on functional abilities following cerebral hypoxia-ischemia (HI) in neonatal rats is not known. The aim of the present study is to investigate the effect of Epo on sensorimotor deficits and brain injury induced by hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia. Epo was administered as a single dose immediately after the hypoxic insult (2000 U/kg). The neuroprotective effect of Epo was evaluated at postnatal day 42 by using a battery of behavioral tests and histological analysis. The results of the present study suggest that Epo treatment immediately after HI insult significantly facilitated recovery of sensorimotor function. Consistently, histopathological evaluation demonstrated that Epo significantly attenuated brain injury and preserved the integrity of cerebral cortex. These findings indicate that long-term neuroprotective effect of Epo on neonatal HI-induced brain injury might be associated with the preservation of sensorimotor functions.     

Doxycycline treatment in a neonatal rat model of hypoxia-ischemia reduces cerebral tissue and white matter injury: a longitudinal magnetic resonance imaging study

Doxycycline may potentially be a neuroprotective treatment for neonatal hypoxic-ischemic brain injury through its anti-inflammatory effects. The aim of this study was to examine any long-term neuroprotection by doxycycline treatment on cerebral gray and white matter. Hypoxic-ischemic brain injury was induced in 7-day-old rats. Pups were treated with either doxycycline (HI+doxy) or saline (HI+vehicle) by intraperitoneal injection at 1 h after hypoxia-ischemia (HI). At 6 h after HI, MnCl(2) was injected intraperitoneally for later manganese-enhanced magnetic resonance imaging (MRI). MRI was performed with diffusion-weighted imaging on day 1 and T(1) -weighted imaging and diffusion tensor imaging at 7, 21 and 42 days after HI. Animals were killed after MRI on day 42 and histological examinations of the brains were performed. There was a tendency towards lower lesion volumes on diffusion maps among HI+doxy than HI+vehicle rats at 1 day after HI. Volumetric MRI showed increasing differences between groups with time after HI, with less cyst formation and less cerebral tissue loss among HI+doxy than HI+vehicle pups. HI+doxy pups had less manganese enhancement on day 7 after HI, indicating reduced inflammation. HI+doxy pups had higher fractional anisotropy on diffusion tensor imaging in major white matter tracts in the injured hemisphere than HI+vehicle pups, indicating less injury to white matter and better myelination. Histological examinations supported the MRI results. Lesion size on early MRI was highly correlated with final injury measures. In conclusion, a single dose of doxycycline reduced long-term cerebral tissue loss and white matter injury after neonatal HI, with an increasing effect of treatment with time after injury.     

Prolonged hypothermia protects neonatal rat brain against hypoxic-ischemia by reducing both apoptosis and necrosis

Although hypothermia is an effective treatment for perinatal cerebral hypoxic-ischemic (HI) injury, it remains unclear how long and how deep we need to maintain hypothermia to obtain maximum neuroprotection. We examined effects of prolonged hypothermia on HI immature rat brain and its protective mechanisms using the Rice-Vannucci model. Immediately after the end of hypoxic exposure, the pups divided into a hypothermia group (30 degrees C) and a normothermia one (37 degrees C). Rectal temperature was maintained until they were sacrificed at each time point before 72h post HI. Prolonged hypothermia significantly reduced macroscopic brain injury compared with normothermia group. Quantitative analysis of cell death using H&E-stained sections revealed the number of both apoptotic and necrotic cells was significantly reduced by hypothermia after 24h post HI. Hypothermia seemed to decrease the number of TUNEL-positive cells. Immunohistochemistry and Western blot showed that prolonged hypothermia suppressed cytochrome c release from mitochondria to cytosol and activation of both caspase-3 and calpain in cortex, hippocampus, thalamus and striatum throughout the experiment. These results showed that prolonged hypothermia significantly reduced neonatal brain injury even when it was started after HI insult. Our results suggest that prolonged hypothermia protects neonatal brain after HI by reducing both apoptosis and necrosis.     

Protection of neonatal rat brain from hypoxic-ischemic injury by LY379268, a Group II metabotropic glutamate receptor agonist

Neuroprotective effects of a Group II metabotropic glutamate receptor agonist, LY379268, were examined in a neonatal rat model of hypoxia-ischemia (unilateral common carotid artery ligation followed by hypoxic exposure for 1.5h in 7-day-old rat pups). LY379268 administered 5 min after hypoxic exposure (2, 5, or 10 mg/kg, i.p.) significantly reduced brain injury as measured by reductions in the ipsilateral brain weight and in CA1 hippocampal neuron density. The significant neuroprotective effects were also observed when this compound (5 mg/kg) was administered 30 min, but not 60 min, after hypoxic exposure. The neonatal hypoxia-ischemia (HI) procedure significantly increased caspase-3 activity and induced DNA fragmentation in the ipsilateral cortex compared with that in the contralateral cortex 24 and 72h after the insult, respectively. LY379268 did not prevent this increase in caspase-3 activity and DNA fragmentation in the ipsilateral cortex. These results suggest that activation of Group II metabotropic glutamate receptors may provide neuroprotection against HI brain injury. However, blockade of caspase-3 activation and the apoptotic pathway appears not to be involved in the neuroprotective effects of LY379268 observed in the neonatal rat model of HI.     

Genetic and pharmacologic manipulation of oxidative stress after neonatal hypoxia-ischemia

Oxidative stress is a critical component of the injury response to hypoxia-ischemia (HI) in the neonatal brain, and this response is unique and at times paradoxical to that seen in the mature brain. Previously, we showed that copper-zinc superoxide-dismutase (SOD1) over-expression is not beneficial to the neonatal mouse brain with HI injury, unlike the adult brain with ischemic injury. However, glutathione peroxidase 1 (GPx1) over-expression is protective to the neonatal mouse brain with HI injury. To further test the hypothesis that an adequate supply of GPx is critical to protection from HI injury, we crossed SOD1 over-expressing mice (hSOD-tg) with GPx1 over-expressing mice (hGPx-tg). Resulting litters contained wild-type (wt), hGPx-tg, hSOD-tg and hybrid hGPx-tg/hSOD-tg pups, which were subjected to HI at P7. Confirming previous results, the hGPx-tg mice had reduced injury compared to both Wt and hSOD-tg littermates. Neonatal mice over-expressing both GPx1 and SOD1 also had less injury compared to wt or hSOD-tg alone. A result of oxidative stress after neonatal HI is a decrease in the concentration of reduced (i.e. antioxidant-active) glutathione (GSH). In this study, we tested the effect of systemic administration of alpha-lipoic acid on levels of GSH in the cortex after HI. Although GSH levels were restored by 24h after HI, injury was not reduced compared to vehicle-treated mice. We also tested two other pharmacological approaches to reducing oxidative stress in hSOD-tg and wild-type littermates. Both the specific inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7NI), and the spin-trapping agent alpha-phenyl-tert-butyl-nitrone (PBN) did not reduce HI injury, however. Taken together, these results imply that H2O2 is a critical component of neonatal HI injury, and GPx1 plays an important role in the defense against this H2O2 and is thereby neuroprotective.     

Mice deficient in interleukin-1 converting enzyme are resistant to neonatal hypoxic-ischemic brain damage.

Interleukin-1 (IL-1) converting enzyme (ICE) is a cysteine protease that cleaves inactive pro-IL-1beta to active IL-1beta. The pro-inflammatory cytokine IL-1beta is implicated as a mediator of hypoxic-ischemic (HI) brain injury, both in experimental models and in humans. ICE is a member of a family of ICE-like proteases (caspases) that mediate apoptotic cell death in diverse tissues. The authors hypothesized that in neonatal mice with a homozygous deletion of ICE (ICE-KO) the severity of brain injury elicited by a focal cerebral HI insult would be reduced, relative to wild-type mice. Paired litters of 9- to 10-day-old ICE-KO and wild-type mice underwent right carotid ligation, followed by 70 or 120 minutes of exposure to 10% O2. In this neonatal model of transient focal cerebral ischemia followed by reperfusion, the duration of hypoxia exposure determines the duration of cerebral ischemia and the severity of tissue damage. Outcome was evaluated 5 or 21 days after lesioning; severity of injury was quantified by morphometric estimation of bilateral cortical, striatal, and dorsal hippocampal volumes. In animals that underwent the moderate HI insult (70-minute hypoxia), damage was attenuated in ICE-KO mice, when evaluated at 5 or 21 days post-lesioning. In contrast, in mice that underwent the more severe HI insult (120-minute hypoxia), injury severity was the same in both groups. Reductions in intra-HI CBF, measured by laser Doppler flow-metry, and intra- and post-HI temperatures did not differ between groups. These results show that ICE activity contributes to the progression of neonatal HI brain injury in this model. Whether these deleterious effects are mediated by pro-inflammatory actions of IL-1beta and/or by pro-apoptotic mechanisms is an important question for future studies.     

Transgenerational effects of neonatal hypoxia-ischemia in progeny

Neonatal hypoxia-ischemia (HI) affects 60% of low birth weight infants and up to 40% of preterm births. Cell death and brain injury after HI have been shown to cause long-lasting behavioral deficits. By using a battery of behavioral tests on second generation 3-week-old rodents, we found that neonatal HI is associated with behavioral outcomes in the progeny of HI-affected parents. Our results suggest an epigenetic transfer mechanism of some of the neurological symptoms associated with neonatal HI. Elucidating the transfer of brain injury to the next generation after HI calls attention to the risks associated with HI injury and the need for proper treatment to reverse these effects. Assessing the devastating extent of HI's reach serves as a cautionary tale to the risks associated with neonatal HI, and provides an incentive to create improved therapeutic measures to treat HI.     

HIF-1α/Beclin1-Mediated Autophagy Is Involved in Neuroprotection Induced by Hypoxic Preconditioning

Hypoxic preconditioning (HPC) exerts a protective effect against hypoxic/ischemic brain injury, and one mechanism explaining this effect may involve the upregulation of hypoxia-inducible factor-1 (HIF-1). Autophagy, an endogenous protective mechanism against hypoxic/ischemic injury, is correlated with the activation of the HIF-1α/Beclin1 signaling pathway. Based on previous studies, we hypothesize that the protective role of HPC may involve autophagy occurring via activation of the HIF-1α/Beclin1 signaling pathway. To test this hypothesis, we evaluated the effects of HPC on oxygen-glucose deprivation/reperfusion (OGD/R)-induced apoptosis and autophagy in SH-SY5Y cells. HPC significantly attenuated OGD/R-induced apoptosis, and this effect was suppressed by the autophagy inhibitor 3-methyladenine and mimicked by the autophagy agonist rapamycin. In control SH-SY5Y cells, HPC upregulated the expression of HIF-1α and downstream molecules such as BNIP3 and Beclin1. Additionally, HPC increased the LC3-II/LC3-I ratio and decreased p62 levels. The increase in the LC3-II/LC3-I ratio was inhibited by the HIF-1α inhibitor YC-1 or by Beclin1-short hairpin RNA (shRNA). In OGD/R-treated SH-SY5Y cells, HPC also upregulated the expression levels of HIF-1α, BNIP3, and Beclin1, as well as the LC3-II/LC3-I ratio. Furthermore, YC-1 or Beclin1-shRNA attenuated the HPC-mediated cell viability in OGD/R-treated cells. Taken together, our results demonstrate that HPC protects SH-SY5Y cells against OGD/R via HIF-1α/Beclin1-regulated autophagy.     

Role of apoptosis and autophagy in the control of cell proliferation in dentate gyrus after hippocampal lesion

研究背景：很多病理情况下都可以引起神经发生（新的神经元产生）,新生细胞的命运和这些疾病的预后有着直接的关系。实验证明脑损伤往往导致海马齿状回神经发生区细胞增殖的增加,新生细胞的数量随着时间的推移逐渐减少,但是新生细胞减少的原因尚不知。阐明这些新生细胞的命运将增加对脑损伤病理过程和治疗手段的理解。 目的：本文研究凋亡和自噬这两种程序性细胞死亡在海马损伤诱导的海马齿状回细胞增殖后新生细胞减少中的作用。 设计、时间和设定：海马齿状回细胞增殖的动物实验于2008年2月至2009年3月在苏州大学医学部神经生物学系进行。 材料：成年雄性SD大鼠通过微量注射海人酸(购自美国Sigma公司)建立海马损伤模型。 方法：5-溴脱氧嘧啶核苷（BrdU）用于标记增殖细胞,海马损伤后齿状回的不同时间点BrdU阳性细胞用免疫组织化学方法鉴定,阳性细胞的数量采用无偏差体视学(Stereology)方法进行定量。凋亡相关蛋白caspase-3、Bcl-2、p53和自噬相关蛋白LC3、Beclin1通过免疫荧光和蛋白质印迹(Western blot）的方法进行检测。 主要结果检测：凋亡和自噬的检测,是在海马损伤后不同时间点,在激光共聚焦显微镜下观察BrdU阳性细胞共表达BrdU/caspase-3、BrdU/LC3和BrdU/Beclin 1。同时, 还用Western blot的方法检测齿状回凋亡相关蛋白和自噬相关蛋白的改变。 结果：定量分析显示海马损伤组在连续3天注射BrdU后的第1天有最多的BrdU阳性细胞,BrdU阳性细胞随着时间的推移逐渐减少。海马颗粒细胞层和门区发现BrdU/cleaved caspase-3双标细胞,海马损伤后的各个时间点未发现BrdU/LC3、BrdU/Beclin 1双标细胞。但Western blot结果显示海马损伤后LC3Ⅱ、Beclin 1、p53的表达上调,pro-caspase-3和Bcl-2的表达下调。 结论：成年大鼠海马损伤后能导致齿状回显著的细胞增殖,这些增殖细胞随着时间的推移逐渐减少,凋亡和自噬可能参与海马损伤后增殖细胞的减少。     

Neonatal hypoxic-ischemic injury increases forebrain subventricular zone neurogenesis in the mouse

Neurogenesis persists throughout life in the rodent subventricular zone (SVZ)-olfactory bulb pathway and increases in the adult after brain insults. The influence of neonatal injury on SVZ neural precursors is unknown. We examined the effects of hypoxia-ischemia (HI) on neonatal mouse SVZ cell proliferation and neurogenesis. Postnatal day 10 (P10) mice underwent right carotid artery ligation followed by 10% O2 exposure for 45 min. The SVZ area and hemispheric injury were quantified morphometrically 1-3 weeks later. Bromodeoxyuridine (BrdU) was used to label proliferating cells, and cell phenotypes of the progeny were identified by immunohistochemistry. HI significantly enlarged the ipsilateral SVZ at P18, P24, and P31, and increases in the SVZ area correlated directly with the degree of hemispheric damage. HI also stimulated cell proliferation and neurogenesis in the SVZ and peri-infarct striatum. Some newborn cells expressed a neuronal phenotype at P24, but not at P31, indicating that neurogenesis was short-lived. These results suggest that augmenting SVZ neuroblast recruitment and survival may improve neural repair after neonatal brain injury.