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Neonatal hypoxic–ischemic (HI) injury derived from asphyxia during perinatal period, is a serious complication of neonatal asphyxia and the main cause of neonatal acute death and chronic neurological injury. Aberrant autophagy occurs in many nervous system diseases, but its role and underlying mechanism in HI injury is largely unknown. Here, we successfully constructed a newborn rat model of HI brain injury, and the knockout-miR-127-3p (KO-miR-127-3p) rats were structured by using CRISPR/Cas9. Subsequently, the in vitro functional experiments, in vivo zea-longa scores, as well as bioinformatics analyses and biological experiments were applied. The expression of autophagy-related proteins, including ATG12, P62, Beclin-1, LC3II in HI cortex with miR-127-3p knockout was significantly decreased, and autophagic vacuoles were disappeared. Moreover, miR-127-3p has a specific regulatory effect on CISD1 expression, another crucial molecule in autophagy process. Accordingly, the overexpression of CISD1 effectively inhibited the autophagic cell death and physiological dysfunction in the brain of HI injury, whereas si-CISD1 reversed the neuroprotective effects of KO-miR-127-3p. Our findings explained the underlying mechanism for HI injury, and miR-127-3p targeting CISD1 signal could be supposed as a new treatment strategy to prevent and treat HI injury.

BACKGROUND: Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coli LPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model. METHODS: Sixteen piglets were randomized: (i) LPS 2 μg/kg bolus; 1 μg/kg infusion (LPS; n = 5), (ii) Saline with hypoxia (Hypoxia; n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia; n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A, IL6, CXCL8, IL10, TNFA) and brain biomarkers (ENO2, UCHL1, S100B, GFAP, CRP, BDNF, MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h. RESULTS: Within 6 h post-insult, we observed increased IL1A, CXCL8, CCL2 and ENO2 mRNA in LPS+Hypoxia and LPS compared to Hypoxia. IL10 mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated with TNFA (R = 0.69; p

Ischemic strokes cause devastating brain damage and functional deficits with few treatments available. Previous studies have shown that the ischemia-hypoxia rapidly induces clinically similar thrombosis and neuronal loss, but any resulting behavioral changes are largely unknown. The goal of this study was to evaluate motor and cognitive deficits in adult HI mice. Following a previously established procedure, HI mouse models were induced by first ligating the right common carotid artery and followed by hypoxia. Histological data showed significant long-term neuronal losses and reactive glial cells in the ipsilateral striatum and hippocampus of the HI mice. Whereas the open field test and the rotarod test could not reliably distinguish between the sham and HI mice, in the tapered beam and wire-hanging tests, the HI mice showed short-term and long-term deficits, as evidenced by the increased number of foot faults and decreased hanging time respectively. In cognitive tests, the HI mice swam longer distances and needed more time to find the platform in the Morris water maze test and showed shorter freezing time in fear contextual tests after fear training. In conclusion, this study demonstrates that adult HI mice have motor and cognitive deficits and could be useful models for preclinical stroke research.

Defects of vitamin B6 metabolism are responsible for severe neurological disorders, such as pyridoxamine 5′-phosphate oxidase deficiency (PNPOD; OMIM: 610090), an autosomal recessive inborn error of metabolism that usually manifests with neonatal-onset severe seizures and subsequent encephalopathy. At present, 27 pathogenic mutations of the gene encoding human PNPO are known, 13 of which are homozygous missense mutations; however, only 3 of them have been characterised with respect to the molecular and functional properties of the variant enzyme forms. Moreover, studies on wild type and variant human PNPOs have so far largely ignored the regulation properties of this enzyme. Here, we present a detailed characterisation of the inhibition mechanism of PNPO by pyridoxal 5′-phosphate (PLP), the reaction product of the enzyme. Our study reveals that human PNPO has an allosteric PLP binding site that plays a crucial role in the enzyme regulation and therefore in the regulation of vitamin B6 metabolism in humans. Furthermore, we have produced, recombinantly expressed and characterised several PNPO pathogenic variants responsible for PNPOD (G118R, R141C, R225H, R116Q/R225H, and X262Q). Such replacements mainly affect the catalytic activity of PNPO and binding of the enzyme substrate and FMN cofactor, leaving the allosteric properties unaltered.

Birth asphyxia constitutes a major global public health burden for millions of infants with a critical need for real time physiological biomarkers. This proof of concept study targets the translational rigor of such biomarkers and aims to examine whether the variability in the amplitude-integrated EEG (aEEG) outputs impact the determination of neurovascular coupling (NVC) in newborns with encephalopathy. A convenience sample with neonatal asphyxia were monitored for twenty hours in the first day of life with EEG and near infrared spectroscopy (NIRS)-based cerebral tissue oxygen saturation (SctO2). NVC between aEEG and NIRS-SctO2 was assessed using wavelet transform coherence (WTC) analysis, specifically by the wavelet total pixel number of significant coherences within 95% confidence interval. The raw EEG was converted to aEEG using three different methods: Method (M1) derives from the algorithm by Zhang and Ding. Method (M2) uses a Neonatal EEG Analysis Toolbox (WU-NEAT). Method (M3) extracts output directly from a commercial platform with an undisclosed algorithm. Our results demonstrate excellent agreement with Bland Altman comparisons for WTC-based NVC irrespective of the algorithms used, despite significant heterogeneities in the aEEG tracings produced by three algorithms. Our findings confirm the robustness of NVC wavelet analysis in Neonatal Encephalopathy related to HIE.

Preterm brain injury is highly associated with inflammation, which is likely related in part to sterile responses to hypoxia-ischemia. We have recently shown that neuroprotection with inflammatory pre-conditioning in the immature brain is associated with induction of toll-like receptor 7 (TLR7). We therefore tested the hypothesis that central administration of a synthetic TLR7 agonist, gardiquimod (GDQ), after severe hypoxia-ischemia in preterm-equivalent fetal sheep would improve white and gray matter recovery. Fetal sheep at 0.7 of gestation received sham asphyxia or asphyxia induced by umbilical cord occlusion for 25 minutes, followed by a continuous intracerebroventricular infusion of GDQ or vehicle from 1 to 4 hours (total dose 1.8 mg/kg). Sheep were killed 72 hours after asphyxia for histology. GDQ significantly improved survival of immature and mature oligodendrocytes (2′,3′-cyclic-nucleotide 3′-phosphodiesterase, CNPase) and total oligodendrocytes (oligodendrocyte transcription factor 2, Olig-2) within the periventricular and intragyral white matter. There were reduced numbers of cells showing cleaved caspase-3 positive apoptosis and astrogliosis (glial fibrillary acidic protein, GFAP) in both white matter regions. Neuronal survival was increased in the dentate gyrus, caudate and medial thalamic nucleus. Central infusion of GDQ was associated with a robust increase in fetal plasma concentrations of the anti-inflammatory cytokines, interferon-β (IFN-β) and interleukin-10 (IL-10), with no significant change in the concentration of the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α). In conclusion, delayed administration of the TLR7 agonist, GDQ, after severe hypoxia-ischemia in the developing brain markedly ameliorated white and gray matter damage, in association with upregulation of anti-inflammatory cytokines. These data strongly support the hypothesis that modulation of secondary inflammation may be a viable therapeutic target for injury of the preterm brain.

Neonatal hypoxic injury (NHI) is a devastating cause of disease that affects >60% of babies born with a very low birth weight, resulting in significant morbidity and mortality, including life-long neurological consequences such as seizures, cerebral palsy, and intellectual disability. Hypoxic injury results in increased neuronal death, which disrupts normal brain development. Although animal model systems have been useful to study the effects of NHI, they do not fully represent the uniqueness and complexities of the human brain. To better understand the effects of hypoxia on human brain development, we have generated a brain organoid protocol and evaluated these cells over the course of 6 months. As anticipated, the expression of a forebrain marker, FOXG1, increased and then remained expressed over time, while there was a transition in the expression of the deep-layer (TBR1) and upper-layer (SATB2) cortical markers. In addition, ventral genes (Eng1 and Nkx2.1) as well as markers of specialized nonneuronal cells (Olig2 and GFAP) also increased at later time points. We next tested the development of our in vitro cerebral organoid model at different oxygen concentrations and found that hypoxia repressed gene markers for forebrain, oligodendrocytes, glial cells, and cortical layers, as well as genes important for the migration of cortical neurons. In contrast, ventral markers were either unaffected or even increased in expression with hypoxic insult. Interestingly, the negative effect of hypoxia on the dorsal brain genes as well as oligodendrocytes, and neuronal progenitors could be mitigated by the use of minocycline, an FDA-approved small molecule. Taken together, we have generated a unique and relevant in vitro human brain model system to study diseases such as NHI as well as their potential treatments. Using this system, we have shown the efficacy of minocycline for human NHI.

Hypoxic–ischemic encephalopathy (HIE) is a devastating neurological event that contributes to the prolonged neurodevelopmental consequences in infants. Therapeutic strategies focused on attenuating neuronal apoptosis in the penumbra appears to be promising. Given the increasingly recognized neuroprotective roles of adipokines in HIE, we investigated the potential anti-apoptotic roles of a novel member of adipokines, Chemerin, in an experimental model of HIE. In the present study, 10-day-old rat pups underwent right common carotid artery ligation followed by 2.5 h hypoxia. At 1 h post hypoxia, pups were intranasally administered with human recombinant chemerin (rh-chemerin). Here, we showed that rh-chemerin prevented the neuronal apoptosis and degeneration as evidenced by the decreased expression of the pro-apoptotic markers, cleaved caspase 3 and Bax, as well as the numbers of Fluoro-Jade C and TUNEL-positive neurons. Furthermore, rh-Chemerin reversed neurological and morphological impairments induced by hypoxia–ischemia in neonatal rats at 24 h and 4 weeks after HIE. In addition, chemerin-mediated neuronal survival correlated with the elevation of chemerin receptor 23 (chemR23), phosphorylated calmodulin-dependent protein kinase kinase 2 (CAMKK2), as well as phosphorylated adenosine monophosphate-activated protein kinase (AMPK). Specific inhibition of chemR23, CAMKK2, and AMPK abolished the anti-apoptotic effects of rh-chemerin at 24 h after HIE, demonstrating that rh-chemerin ameliorated neuronal apoptosis partially via activating chemR23/CAMKK2/AMPK signaling pathway. Neuronal apoptosis is a well-established contributing factor of pathological changes and the neurological impairment after HIE. These results revealed mechanisms of neuroprotection by rh-chemerin, and indicated that activation of chemR23 might be harnessed to protect from neuronal apoptosis in HIE.

The timing of hypoxia-ischemia (HI) in preterm infants is often uncertain and there are few biomarkers to determine whether infants are in a treatable stage of injury. We evaluated whether epileptiform sharp waves recorded from the parietal cortex could provide early prediction of neuronal loss after HI. Preterm fetal sheep (0.7 gestation) underwent acute HI induced by complete umbilical cord occlusion for 25 minutes (n = 6) or sham occlusion (control, n = 6). Neuronal survival was assessed 7 days after HI by immunohistochemistry. Sharp waves were quantified manually and using a wavelet-type-2-fuzzy-logic-system during the first 4 hours of recovery. HI resulted in significant subcortical neuronal loss. Sharp waves counted by the automated classifier in the first 30 minutes after HI were associated with greater neuronal survival in the caudate nucleus (r = 0.80), whereas sharp waves between 2–4 hours after HI were associated with reduced neuronal survival (r = −0.83). Manual and automated counts were closely correlated. This study suggests that automated quantification of sharp waves may be useful for early assessment of HI injury in preterm infants. However, the pattern of evolution of sharp waves after HI was markedly affected by the severity of neuronal loss, and therefore early, continuous monitoring is essential.

Seizures in neonates, mainly caused by hypoxic-ischemic encephalopathy, are thought to be harmful to the brain. Phenobarbital remains the first line drug therapy for the treatment of suspected neonatal seizures but concerns remain with efficacy and safety. Here we explored the short- and long-term outcomes of phenobarbital treatment in a mouse model of hypoxia-induced neonatal seizures. Seizures were induced in P7 mice by exposure to 5% O2 for 15 minutes. Immediately after hypoxia, pups received a single dose of phenobarbital (25 mg.kg−1) or saline. We observed that after administration of phenobarbital seizure burden and number of seizures were reduced compared to the hypoxic period; however, PhB did not suppress acute histopathology. Behavioural analysis of mice at 5 weeks of age previously subjected to hypoxia-seizures revealed an increase in anxiety-like behaviour and impaired memory function compared to control littermates, and these effects were not normalized by phenobarbital. In a seizure susceptibility test, pups previously exposed to hypoxia, with or without phenobarbital, developed longer and more severe seizures in response to kainic acid injection compared to control mice. Unexpectedly, mice treated with phenobarbital developed less hippocampal damage after kainic acid than untreated counterparts. The present study suggests phenobarbital treatment in immature mice does not improve the long lasting functional deficits induces by hypoxia-induced seizures but, unexpectedly, may reduce neuronal death caused by exposure to a second seizure event in later life.

Therapeutic hypothermia (TH) initiated within 6 h from birth is the most effective therapeutic approach for moderate to severe hypoxic-ischemic encephalopathy (HIE). However, underlying mechanisms and effects on the human metabolism are not yet fully understood. This work aims at studying the evolution of several energy related key metabolites in newborns with HIE undergoing TH employing gas chromatography - mass spectrometry. The method was validated following stringent FDA requirements and applied to 194 samples from a subgroup of newborns with HIE (N = 61) enrolled in a multicenter clinical trial (HYPOTOP) for the determination of lactate, pyruvate, ketone bodies and several Krebs cycle metabolites at different sampling time points. The analysis of plasma samples from newborns with HIE revealed a decrease of lactate, pyruvate and β-hydroxybutyrate concentrations, whereas rising malate concentrations were observed. In healthy control newborns (N = 19) significantly lower levels of pyruvate and lactate were found in comparison to age-matched newborns with HIE undergoing TH, whereas acetoacetate and β-hydroxybutyrate levels were clearly increased. Access to a validated analytical method and a controlled cohort of newborns with HIE undergoing hypothermia treatment for the first time allowed the in-depth study of the evolution of key metabolites of metabolic junctions in this special population., MV acknowledges the EC11-244 grant from the Instituto Carlos III. ANR acknowledges a research contract by the Spanish Maternal, Neonatal and Developmental Network SAMID RETICS funded by the PN 2018–2011 (Spain), ISCIII-Sub-Directorate General for Research Assessment and Promotion and the European Regional Development Fund (FEDER), reference RD12/0026. JK, MCe and ASI are grateful for their personal grants from the Instituto Carlos III (grant numbers CP16/00034, JR16/00038 and FI16/00380, respectively). APL acknowledges her personal grant from the Health Research Institute La Fe (CPR2015/0371)

Although therapeutic hypothermia is known to improve neurologic outcomes after perinatal cerebral hypoxia-ischemia, etiology remains unknown. To decipher the mechanisms whereby hypothermia regulates metabolic dynamics in different brain regions, we used a two-step approach: a metabolomics to target metabolic pathways responding to cooling, and a quantitative imaging mass spectrometry to reveal spatial alterations in targeted metabolites in the brain. Seven-day postnatal rats underwent the permanent ligation of the left common carotid artery followed by exposure to 8% O2 for 2.5 hours. The pups were returned to normoxic conditions at either 38°C or 30°C for 3 hours. The brain metabolic states were rapidly fixed using in situ freezing. The profiling of 107 metabolites showed that hypothermia diminishes the carbon biomass related to acetyl moieties, such as pyruvate and acetyl-CoA; conversely, it increases deacetylated metabolites, such as carnitine and choline. Quantitative imaging mass spectrometry demarcated that hypothermia diminishes the acetylcholine contents specifically in hippocampus and amygdala. Such decreases were associated with an inverse increase in carnitine in the same anatomic regions. These findings imply that hypothermia achieves its neuroprotective effects by mediating the cellular acetylation status through a coordinated suppression of acetyl-CoA, which resides in metabolic junctions of glycolysis, amino-acid catabolism, and ketolysis.

Insults to the developing brain often result in irreparable damage resulting in long-term deficits in motor and cognitive functions. The only treatment today for hypoxic-ischemic encephalopathy (HIE) in newborns is hypothermia, which has limited clinical benefit. We have studied changes to the blood–brain barriers (BBB) as well as regional cerebral blood flow (rCBF) in a neonatal model of HIE to further understand the underlying pathologic mechanisms. Nine-day old mice pups, brain roughly equivalent to the near-term human fetus, were subjected to hypoxia-ischemia. Hypoxia-ischemia increased BBB permeability to small and large molecules within hours after the insult, which normalized in the following days. The opening of the BBB was associated with changes to BBB protein expression whereas gene transcript levels were increased showing direct molecular damage to the BBB but also suggesting compensatory mechanisms. Brain pathology was closely related to reductions in rCBF during the hypoxia as well as the areas with compromised BBB showing that these are intimately linked. The transient opening of the BBB after the insult is likely to contribute to the pathology but at the same time provides an opportunity for therapeutics to better reach the infarcted areas in the brain.

Inflammation plays a critical role in mediating brain injury induced by neonatal hypoxic ischemic encephalopathy (HIE). The mechanisms underlying inflammatory responses to ischemia may be shared by neonatal and adult brains; however, HIE exhibits a unique inflammation phenotype that results from the immaturity of the neonatal immune system. This review will discuss the current knowledge concerning systemic and local inflammatory responses in the acute and subacute stages of HIE. The key components of inflammation, including immune cells, adhesion molecules, cytokines, chemokines and oxidative stress, will be reviewed, and the differences between neonatal and adult inflammatory responses to cerebral ischemic injury will also be discussed.

The prompt and reliable identification of infants at risk of hypoxic-ischemic encephalopathy secondary to perinatal asphyxia in the first critical hours is important for clinical decision-making and yet still remains a challenge. This work strives for the evaluation of a panel of metabolic biomarkers that have been associated with the hypoxic-ischemic insult in the perinatal period. Plasma and urine samples from a consolidated newborn piglet model of hypoxia and withdrawn before and at different time points after a hypoxic insult were analyzed and compared to a control group. Time-dependent metabolic biomarker profiles were studied and observed patterns were similar to those of lactate levels, which are currently considered the gold standard for assessing hypoxia. Class prediction performance could be improved by the use of a combination of the whole panel of determined metabolites in plasma as compared to lactate values. Using a multivariate model including lactate together with the studied metabolic biomarkers allowed to improve the prediction performance of duration of hypoxia time, which correlates with the degree of brain damage. The present study evidences the usefulness of choline and related metabolites for improving the early assessment of the severity of the hypoxic insult.

Investigating and understanding gene-environment interaction (G x E) in a neurodevelopmentally and biologically plausible manner is a major challenge for schizophrenia research. Hypoxia during neurodevelopment is one of several environmental factors related to the risk of schizophrenia, and links between schizophrenia candidate genes and hypoxia regulation or vascular expression have been proposed. Given the availability of a wealth of complex genetic information on schizophrenia in the literature without knowledge on the connections to environmental factors, we now systematically collected genes from candidate studies (using SzGene), genome-wide association studies (GWAS) and copy number variation (CNV) analyses, and then applied four criteria to test for a (theoretical) link to ischemia-hypoxia and/or vascular factors. In all, 55% of the schizophrenia candidate genes (n = 42 genes) met the criteria for a link to ischemia-hypoxia and/or vascular factors. Genes associated with schizophrenia showed a significant, threefold enrichment among genes that were derived from microarray studies of the ischemia-hypoxia response (IHR) in the brain. Thus, the finding of a considerable match between genes associated with the risk of schizophrenia and IHR and/or vascular factors is reproducible. An additional survey of genes identified by GWAS and CNV analyses suggested novel genes that match the criteria. Findings for interactions between specific variants of genes proposed to be IHR and/or vascular factors with obstetric complications in patients with schizophrenia have been reported in the literature. Therefore, the extended gene set defined here may form a reasonable and evidence-based starting point for hypothesis-based testing of G x E interactions in clinical genetic and translational neuroscience studies. Molecular Psychiatry (2012) 17, 1194-1205; doi:10.1038/mp.2011.183; published online 31 January 2012

Therapeutic hypothermia can partially reduce long-term death and disability in neonates after hypoxic-ischemic encephalopathy. The aim of this study was to determine whether prolonging the duration of cooling from 3 days to 5 days could further improve outcomes of cerebral ischemia in near-term fetal sheep. Fetal sheep (0.85 gestation) received 30 minutes bilateral carotid artery occlusion followed by 3 days of normothermia ( n = 8), 3 days of hypothermia ( n = 8), or 5 days of hypothermia ( n = 8) started 3 hours after ischemia. Sham controls received sham ischemia followed by normothermia ( n = 8). Cerebral ischemia was associated with profound loss of electroencephalography power and spectral edge, with greater and more rapid recovery in both hypothermia groups ( P < 0.05). Ischemia was associated with severe loss of neurons in the cortex, hippocampus and thalamus ( P < 0.05), with a significant improvement in both hypothermia groups. However, the ischemia-3-day hypothermia group showed greater neuronal survival in the cortex and dentate gyrus compared with ischemia-5-day hypothermia ( P < 0.05). Ischemia was associated with induction of iba1-positive microglia, which was attenuated in both hypothermia groups ( P < 0.05). Extending the duration of delayed therapeutic hypothermia from 3 to 5 days did not improve outcomes after severe ischemia, and was associated with reduced neuronal survival in some regions.

To examine the temporal relationship of cortical autophagic flux with delayed neuronal cell death after hypoxia-ischemia (HI) in neonatal piglets. HI was produced with 45-min hypoxia and 7-min airway occlusion in 3–5-day-old piglets. Markers of autophagic, lysosomal and cell death signaling were studied via immunohistochemistry, immunoblotting, and histochemistry in piglet brains. In vitro, autophagy was impaired in cultured mouse cortical neurons treated with chloroquine with or without rapamycin for 1 d in the presence of Z-VAD-fmk, cyclosporine A, or vehicle control, and cell viability was assessed with the MTT assay. In vivo, neuronal cell death of sensorimotor cortex was delayed by 1–2 days after HI, whereas LC3-II, Beclin-1, PI3KC3, ATG12-ATG-5, and p-ULK1 increased by 1.5–6 h. Autophagosomes accumulated in cortical neurons by 1 d owing to enhanced autophagy and later to decreased autophagosome clearance, as indicated by LC3, Beclin-1, and p62 accumulation. Autophagy flux impairment was attributable to lysosomal dysfunction, as indicated by low lysosomal-associated membrane protein 2, cathepsin B, and cathepsin D levels at 1 d. Ubiquitin levels increased at 1 d. Autophagosome and p62 accumulated predominantly in neurons at 1 d, with p62 puncta occurring in affected cells. Beclin-1 colocalized with markers of caspase-dependent and caspase-independent apoptosis and necrosis in neurons. In vitro, mouse neonatal cortical neurons treated with rapamycin and chloroquine showed increased autophagosomes, but not autolysosomes, and increased cell death that was attenuated by cyclosporine A. Neonatal HI initially increases autophagy but later impairs autophagosome clearance, coinciding with delayed cortical neuronal death.

Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.

Lithium is used in the treatment of bipolar mood disorder. Reportedly, lithium can be neuroprotective in models of adult brain ischemia. The purpose of this study was to evaluate the effects of lithium in a model of neonatal hypoxic-ischemic brain injury. Nine-day-old male rats were subjected to unilateral hypoxia-ischemia (HI) and 2 mmol/kg lithium chloride was injected i.p. immediately after the insult. Additional lithium injections, 1 mmol/kg, were administered at 24-h intervals. Pups were killed 6, 24 or 72 h after HI. Lithium reduced the infarct volume from 24.7±2.9 to 13.8±3.3 mm(3) (44.1%) and total tissue loss (degeneration + lack of growth) from 67.4±4.4 to 38.4±5.9 mm(3) (43.1%) compared with vehicle at 72 h after HI. Injury was reduced in the cortex, hippocampus, thalamus and striatum. Lithium reduced the ischemia-induced dephosphorylation of glycogen synthase kinase-3β and extracellular signal-regulated kinase, the activation of calpain and caspase-3, the mitochondrial release of cytochrome c and apoptosis-inducing factor, as well as autophagy. We conclude that lithium could mitigate the brain injury after HI by inhibiting neuronal apoptosis. The lithium doses used were in the same range as those used in bipolar patients, suggesting that lithium might be safely used for the avoidance of neonatal brain injury.