The mechanism of sevoflurane post-treatment alleviating hypoxic-ischemic encephalopathy by affecting histone methyltransferase G9a in rats.

Hypoxic-ischemic encephalopathy (HIE) is recognized as the main cause of neonatal death, and efficient treatment strategies remain limited. This study aims to investigate the mechanism of sevoflurane (SF) post-treatment in alleviating HIE in rats. The HIE rat model and oxygen-glucose deprivation (OGD) cell model were established, and adeno-associated virus (AAV)-histone-lysine N-methyltransferase EHMT2 (G9a) was transfected after SF treatment. The learning and memory ability and the levels of nerve growth factor (NGF)/brain-derived neurotrophic factor (BDNF) were evaluated and determined. The levels of G9a/histone H3 lysine 9 dimethylation (H3K9me2) and the enrichment level of H3K9me2 in the promoter region of BDNF gene were analyzed. After SF post-treatment, the neurons in cerebral cortex, the learning and memory skills and the contents of NGF/BDNF were increased, while the apoptosis and G9a/H3K9me2 levels were reduced. After overexpression of G9a in vitro/vivo, the enrichment levels of H3K9me2 in the promoter region of BDNF were increased, the levels of BDNF were decreased, the neurons were damaged and the learning and memory abilities of HIE rats were impaired. The conclusion is that SF post-treatment can promote the expression of BDNF by inhibiting H3K9me2 on the BDNF gene promoter and inhibiting G9a, thus alleviating HIE in rats.

Pharmacokinetics and short-term safety of the selective NOS inhibitor 2-iminobiotin in asphyxiated neonates treated with therapeutic hypothermia.

BACKGROUND: Neonatal encephalopathy following perinatal asphyxia is a leading cause for neonatal death and disability, despite treatment with therapeutic hypothermia. 2-Iminobiotin is a promising neuroprotective agent additional to therapeutic hypothermia to improve the outcome of these neonates. METHODS: In an open-label study, pharmacokinetics and short-term safety of 2-iminobiotin were investigated in neonates treated with therapeutic hypothermia. Group A (n = 6) received four doses of 0.16 mg/kg intravenously q6h. Blood sampling for pharmacokinetic analysis and monitoring of vital signs for short-term safety analysis were performed. Data from group A was used to determine the dose for group B, aiming at an AUC0-48 h of 4800 ng*h/mL. RESULTS: Exposure in group A was higher than targeted (median AUC0-48 h 9522 ng*h/mL); subsequently, group B (n = 6) received eight doses of 0.08 mg/kg q6h (median AUC0-48 h 4465 ng*h/mL). No changes in vital signs were observed and no adverse events related to 2-iminobiotin occurred. CONCLUSION: This study indicates that 2-iminobiotin is well tolerated and not associated with any adverse events in neonates treated with therapeutic hypothermia after perinatal asphyxia. Target exposure was achieved with eight doses of 0.08 mg/kg q6h. Optimal duration of therapy for clinical efficacy needs to be determined in future clinical trials.

Inhibition of Calcium/Calmodulin-Dependent Protein Kinase Kinase ß Is Detrimental in Hypoxia⁻Ischemia Neonatal Brain Injury.

Neonatal hypoxia-ischemia (HI) is a major cause of death and disability in neonates. HI leads to a dramatic rise in intracellular calcium levels, which was originally thought to be detrimental to the brain. However, it has been increasingly recognized that this calcium signaling may also play an important protective role after injury by triggering endogenous neuroprotective pathways. Calcium/calmodulin-dependent protein kinase kinase ß (CaMKK ß) is a major kinase activated by elevated levels of intracellular calcium. Here we evaluated the functional role of CaMKK ß in neonatal mice after HI in both acute and chronic survival experiments. Postnatal day ten wild-type (WT) and CaMKK ß knockout (KO) mouse male pups were subjected to unilateral carotid artery ligation, followed by 40 min of hypoxia (10% O2 in N2). STO-609, a CaMKK inhibitor, was administered intraperitoneally to WT mice at 5 minutes after HI. TTC (2,3,5-triphenyltetrazolium chloride monohydrate) staining was used to assess infarct volume 24 h after HI. CaMKK ß KO mice had larger infarct volume than WT mice and STO-609 increased the infarct volume in WT mice after HI. In chronic survival experiments, WT mice treated with STO-609 showed increased tissue loss in the ipsilateral hemisphere three weeks after HI. Furthermore, when compared with vehicle-treated mice, they showed poorer functional recovery during the three week survival period, as measured by the wire hang test and corner test. Loss of blood-brain barrier proteins, a reduction in survival protein (Bcl-2), and an increase in pro-apoptotic protein Bax were also seen after HI with CaMKK ß inhibition. In conclusion, inhibition of CaMKK ß exacerbated neonatal hypoxia-ischemia injury in mice. Our data suggests that enhancing CaMKK signaling could be a potential target for the treatment of hypoxic-ischemic brain injury.

The activation of group II metabotropic glutamate receptors protects neonatal rat brains from oxidative stress injury after hypoxia-ischemia.

Birth asphyxia resulting in brain hypoxia-ischemia (H-I) can cause neonatal death or lead to persistent brain damage. Recent investigations have shown that group II metabotropic glutamate receptor (mGluR2/3) activation can provide neuroprotection against H-I but the mechanism of this effect is not clear. The aim of this study was to investigate whether mGluR2/3 agonists applied a short time after H-I reduce brain damage in an experimental model of birth asphyxia, and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I in 7-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intra peritoneally with mGluR2 (LY 379268) or mGluR3 (NAAG) agonists 1 h or 6 h after H-I (5 mg/kg). The weight deficit of the ischemic brain hemisphere, radical oxygen species (ROS) content levels, antioxidant enzymes activity and the concentrations of reduced glutathione (GSH) were measured. Both agonists reduced weight loss in the ischemic hemisphere and mitigated neuronal degeneration in the CA1 hippocampal region and cerebral cortex. Both agonists reduced the elevated levels of ROS in the ipsilateral hemisphere observed after H-I and prevented an increase in antioxidant enzymes activity in the injured hemisphere restoring them to control levels. A decrease in GSH level was also restored after agonists application. The results show that the activation of mGluR2 and mGluR3 a short time after H-I triggers neuroprotective mechanisms that act through the inhibition of oxidative stress and ROS production. The prevention of ROS production by the inhibition of glutamate release and decrease in its extracellular concentration is likely the main mechanism involved in the observed neuroprotection.

Benefits of starting hypothermia treatment within 6 h vs. 6-12 h in newborns with moderate neonatal hypoxic-ischemic encephalopathy.

BACKGROUND: It has been suggested that mild hypothermia treatment of hypoxia-ischemic encephalopathy (HIE) should start within 6 h after HIE, but many children are admitted to the hospital > 6 h, particularly in developing areas. We aimed to determine whether hypothermia treatment could remain effective within 12 h after birth. METHODS: According to their admission, 152 newborns were enrolled in the < 6 h and 6-12 h after HIE groups. All newborns received conventional treatment combined with mild head hypothermia therapy, according to our routine clinical practice. Some newborns only received conventional treatment (lacking informed consent). All newborns received amplitude-integrated electroencephalography (aEEG) monitoring for 4 h and neuron-specific enolase (NSE) measurement before and after 3 days of therapy. RESULTS: Compared to the conventional treatment, hypothermia significantly improved the aEEG scores and NSE values in all newborns of the < 6-h group. In the 6-12-h group, the aEEG scores (F = 5.67, P < 0.05) and NSE values (F = 4.98, P < 0.05) were only improved in newborns with moderate HIE. Hypothermia treatment seems to have no effect in newborns with severe HIE after 6 h (P > 0.05). Hypothermia improved the rates of neonatal death and 18-month disability (all P < 0.01). CONCLUSIONS: In newborns with moderate HIE, starting hypothermia therapy < 6 h and 6-12 h after HIE showed curative effects. In those with severe HIE, only starting hypothermia therapy within 6 h showed curative effects.

Changes in lactate dehydrogenase are associated with central gray matter lesions in newborns with hypoxic-ischemic encephalopathy.

PURPOSE: Biomarkers may predict neurological prognosis in infants with hypoxic-ischemic encephalopathy (HIE). We evaluated the relationship between serum lactate dehydrogenase (LDH) and brain magnetic resonance imaging (MRI), which predicts neurodevelopmental outcomes, in order to assess whether LDH levels are similarly predictive. MATERIALS AND METHODS: Medical records were reviewed for infants with HIE and LDH levels were assessed on the first (LDH1) and third (LDH3) days following birth. Receiver operating characteristic curves were obtained in relation to central gray matter hypoxic-ischemic lesions. RESULTS: Of 92 patients, 52 (56.5%) had hypoxic-ischemic lesions on brain MRI, and 21 of these infants (40.4%) had central gray matter lesions. LDH1 and LDH3 did not differ; however, the percentage change (ΔLDH%) was significantly higher in infants with central gray matter lesions (36.9% versus 6.6%, p = 0.006). With cutoffs of 187 (IU/L, ΔLDH) and 19.4 (%, ΔLDH%), the sensitivity, specificity, positive predictive value and negative predictive value were 71.4, 69.0, 40.5 and 89.1%, respectively. The relative risk was 5.57 (p = 0.001). CONCLUSION: Changes in serum LDH may be a useful biomarker for predicting future neurodevelopmental prognosis in infants with HIE.

Administration of Huperzia quadrifariata Extract, a Cholinesterase Inhibitory Alkaloid Mixture, has Neuroprotective Effects in a Rat Model of Cerebral Hypoxia-Ischemia.

Neonatal hypoxia-ischemia (HI) is an etiologic component of several neurologic pathologies associated to cognitive impairment. The mechanisms involved in HI-induced tissue damage start immediately after HI and extend for days. Acetylcholine is an important neurotransmitter in the central nervous system and exerts a protector effect on tissue damage by modulating inflammation, and cholinesterase inhibitors have shown neuroprotective properties and their action are often attributed to inhibition of the immune response. The administration of Huperzia quadrifariata alkaloid extract (HqAE), with potent and selective cholinesterase inhibitor properties, will reduce the HI induced behavioral deficits and tissue damage. A total of 84 newborn Wistar rat pups at post natal day 7 (PND7) were subjected to right carotid occlusion followed by 1 h of hypoxia (8% of O2) and i.p. injections of saline, vehicle or HqAE (10 mg/kg). Morris Water Maze and inhibitory avoidance tests were used to assess the cognitive function. Flow cytometry was performed at PND11. Histological analysis was performed at PND45. HqAE treatment was able to prevent the HI induced cognitive deficits in both tests and, at PND45, histological analysis showed that HqAE treatment reduced hippocampus tissue damage. Flow cytometry of the injured hippocampus revealed that the treatment was able to reduce cellular death and the number of infiltrating T cells. Altogether, these results show the therapeutic potential of the Huperzia quadrifariata alkaloid extract to prevent cognitive deficits and histological damage caused by neonatal hypoxia-ischemia, probably by reducing cellular death and T cell mobilization.

Effects of activin A and its downstream ERK1/2 in oxygen and glucose deprivation after isoflurane-induced postconditioning.

BACKGROUND: Isoflurane postconditioning (ISPOC) plays a neuroprotection role in the brain. Previous studies confirmed that isoflurane postconditioning can provide better protection than preconditioning in acute hypoxic-ischemic brain damage, such as acute craniocerebral trauma and ischemic stroke. Numerous studies have reported that activin A can protect rat's brain from cell injury. However, whether activin A and its downstream ERK1/2 were involved in isoflurane postconditioning-induced neuroprotection is unknown. METHODS: A total of 80 healthy Sprague-Dawley rats weighing 50-70g were randomly divided into 10 groups of 8: normal control, oxygen and glucose deprivation (OGD), 1.5% ISPOC, 3.0% ISPOC, 4.5% ISPOC, blocker of activin A (SB431542), blocker of ERK1/2 (U0126), 3.0% ISPOC+SB431542, 3.0% ISPOC+U0126, and vehicle (dimethyl sulfoxide(DMSO)) group. Blockers (SB431542 and U0126) were used in each concentration of isoflurane before OGD. Hematoxylin-eosin staining, 2,3,5-triphenyl tetrazolium chloride staining, and propidium iodide (PI) staining were conducted to assess the reliability in the brain slices. Immunofluorescence, Western blot, and quantitative real-time PCR(Q-PCR) were performed to validate the protein expression levels of activin A, Smad2/3, P-Smad2/3, ERK1/2, and phosphorylation ERK1/2 (P-ERK1/2). RESULTS: The number of damaged neurons and mean fluorescence intensity(MFI) of PI staining increased, but formazan generation, expression levels of activin A and P-ERK1/2 protein, and mRNA synthesis level of activin A decreased in the OGD group compared with the normal control group (p<0.05). The number of damaged neurons and MFI of PI staining decreased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A increased significantly in the 1.5% ISPOC and 3.0% ISPOC groups (p<0.05) compared with the OGD group. The result in the 4.5% ISPOC group, was completely opposite to the 1.5% ISPOC and 3.0% ISPOC groups. The number of damage neuron and MFI of PI staining increased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A decreased in the 4.5% ISPOC group. However, the expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A in the 4.5% ISPOC group were higher than the OGD group (p<0.05). The other results were compared between the SB431542 group/the U0126 group and 3.0% ISPOC group. The MFI of PI staining increased, but the expression levels of activin A, P-Smad2/3, and P-ERK1/2 decreased (p<0.05). The expression level of ERK1/2 protein in all groups exhibited no change (p>0.05). CONCLUSION: Results of this study showed that 3.0% concentration of isoflurane postconditioning provided better neuroprotection than 1.5% and 4.5% concentrations of isoflurane. Activin A/Smad 2/3 and activin A/ERK1/2 signaling pathway may be involved in ISPOC-induced neuroprotection.

Interrelationship Between Broadband NIRS Measurements of Cerebral Cytochrome C Oxidase and Systemic Changes Indicates Injury Severity in Neonatal Encephalopathy.

Perinatal hypoxic ischaemic encephalopathy (HIE) is associated with severe neurodevelopmental problems and mortality. There is a clinical need for techniques to provide cotside assessment of the injury extent. This study aims to use non-invasive cerebral broadband near-infrared spectroscopy (NIRS) in combination with systemic physiology to assess the severity of HIE injury. Broadband NIRS is used to measure the changes in haemodynamics, oxygenation and the oxidation state of cytochrome c oxidase (oxCCO). We used canonical correlation analysis (CCA), a multivariate statistical technique, to measure the relationship between cerebral broadband NIRS measurements and systemic physiology. A strong relationship between the metabolic marker, oxCCO, and systemic changes indicated severe brain injury; if more than 60 % of the oxCCO signal could be explained by the systemic variations, then the neurodevelopmental outcome was poor. This boundary has high sensitivity and specificity (100 and 83 %, respectively). Broadband NIRS measured concentration changes of the oxidation state of cytochrome c oxidase has the potential to become a useful cotside tool for assessment of injury severity following hypoxic ischaemic brain injury.

Caveolin-1 is a checkpoint regulator in hypoxia-induced astrocyte apoptosis via Ras/Raf/ERK pathway.

Astrocytes, the most numerous cells in the human brain, play a central role in the metabolic homeostasis following hypoxic injury. Caveolin-1 (Cav-1), a transmembrane scaffolding protein, has been shown to converge prosurvival signaling in the central nerve system. The present study aimed to investigate the role of Cav-1 in the hypoxia-induced astrocyte injury. We also examined how Cav-1 alleviates apoptotic astrocyte death. To this end, primary astrocytes were exposed to oxygen-glucose deprivation (OGD) for 6 h and a subsequent 24-h reoxygenation to mimic hypoxic injury. OGD significantly reduced Cav-1 expression. Downregulation of Cav-1 using Cav-1 small interfering RNA dramatically worsened astrocyte cell damage and impaired cellular glutamate uptake after OGD, whereas overexpression of Cav-1 with Cav-1 scaffolding domain peptide attenuated OGD-induced cell apoptosis. Mechanistically, the expressions of Ras-GTP, phospho-Raf, and phospho-ERK were sequestered in Cav-1 small interfering RNA-treated astrocytes, yet were stimulated after supplementation with caveolin peptide. MEK/ERK inhibitor U0126 remarkably blocked the Cav-1-induced counteraction against apoptosis following hypoxia, indicating Ras/Raf/ERK pathway is required for the Cav-1's prosurvival role. Together, these findings support Cav-1 as a checkpoint for the in hypoxia-induced astrocyte apoptosis and warrant further studies targeting Cav-1 to treat hypoxic-ischemic brain injury.

Helium preconditioning protects against neonatal hypoxia-ischemia via nitric oxide mediated up-regulation of antioxidases in a rat model.

This study aimed to investigate the role of nitric oxide (NO) in the neuroprotective effects of helium preconditioning (He-PC) in a neonatal hypoxia/ischemia (HI) rat model. Seven-day old rat pups were divided into normal control group, He-PC group, HI group, He-PC+HI group, L-NAME+HI group and L-NAME+He-PC+HI group. HI was induced by exposure to 80% oxygen for 90 min. He-PC was conducted with 70% helium-30% oxygen for three 5-min periods. Three hours after He-PC, animals in control group and He-PC group were sacrificed, and the brain was collected for the detection of NO content. At 24h after HI, animals in control group, HI group, He-PC+HI group, and L-NAME+He-PC+HI group were sacrificed, and the brain was collected for detection of infarct ratio, antioxidases (SOD, HO-1 and Nrf2), DNA binding activity of Nrf2 and TUNEL staining. Three weeks later, the neurological function and brain atrophy were determined. Results showed pretreatment with L-NAME alone failed to exert protective effect on HI. He-PC significantly increased NO content, reduced the brain infarct area, increased anti-oxidases expression and DNA binding activity of Nrf2, decreased the apoptotic cells, and improved the neurological function and brain atrophy. In addition, this protection was markedly inhibited by L-NAME (a non-selective NOS inhibitor). These findings suggest that the He-PC may induce NO production to activate Nrf2, exerting neuroprotective effect on neonatal HI.

Folic Acid Can Contribute to Memory Deficit and Na+, K+- ATPase Failure in the Hippocampus of Adolescent Rats Submitted to Hypoxia- Ischemia.

Recent findings have demonstrated a dual effect of the folic acid (FA) supplementation on the nervous system of rats. We found that FA treatment prevented memory impairment and Na(+), K(+)- ATPase inhibition in the striatum and cortex in adult rats that suffered neonatal hypoxia-ischemia (HI). However, spatial memory deficit has been associated with FA supplementation. In the present study we investigated the role of FA supplementation on spatial memory and Na(+), K(+)-ATPase activity in the hippocampus, as well as on morphologic alterations in adolescent rats submitted to neonatal HI. Wistar rats of both sexes at postnatal day (PND) 7 were submitted to Levine-Rice HI procedure. Intraperitoneal doses of FA were administered immediately before HI and repeated daily until the maximum PND 40. Hippocampal volume and striatum area were estimated and Na(+), K(+)-ATPase activity in the hippocampus was measured at PND 31. Also, the performance of the animals in the water maze was assessed and Na(+), K(+)-ATPase activity measured again at PND 52. Interestingly, HI and FA resulted in spatial memory deficits in the Morris water maze and the Na(+), K(+)-ATPase activity was impaired at PND 31 in HI rats which received FA. Additionally, Na(+), K(+)-ATPase activity in adulthood showed a decrease after HI and a recovery in supplemented animals. Hippocampal and striatal atrophy were partially reversed by FA. To conclude, the present results support the hypothesis that FA supplementation during development contributes to memory deficits caused by HI and Na(+), K(+)-ATPase failure in adolescent rats, although, in adulthood, FA has been effective in reversing enzymatic activity in the hippocampus.

Astrocyte GGTI-mediated Rac1 prenylation upregulates NF-κB expression and promotes neuronal apoptosis following hypoxia/ischemia.

Stroke is the fifth leading cause of death for Americans, and about 87% of all strokes are ischemic strokes. Astrogliosis plays a crucial role in the pathophysiology of delayed neuronal death (DND) following ischemic stroke. Here we reported that astrocyte geranylgeranyltransferase I (GGTI)-mediated Rac1 activation up-regulated NF-κB expression and promoted the neuronal apoptosis after oxygen-glucose deprivation followed by oxygen-glucose regeneration (OGD/R). We found that GGTIß (a specific subunit of GGTI) and NF-κB-p65 levels as determined by Western blot and/or immunofluorescent analysis were significantly up-regulated in the reactive astrocytes both in rat transient middle cerebral artery occlusion (tMCAO) and in cell OGD/R models. The increased expression of GGTIß and p65 was associated with the DND in the ischemic brain. Inhibiting astrocyte GGTI activity by its specific inhibitor GGTi-2147 treatment reduced the activity of Rac1 (one of substrates for GGTI), down-regulated the expression of p65, and ameliorated the OGD/R-induced neuronal apoptosis. Astrocytes transfected with wild type Rac1, but not the unprenylated Rac1, up-regulated the p65 protein levels and promoted the co-cultured neuronal apoptosis. Furthermore, over-expression of unprenylated Rac1 in astrocytes significantly decreased the neuronal apoptosis. In addition, over-expression of NF-κB-p65 in astrocytes significantly increased the co-cultured neuronal apoptosis under OGD/R condition. Our findings suggest that astrocyte GGTI-mediated Rac1 activation contributed to the DND and that GGTI-Rac1-NF-κB signaling may be a potential target for the therapy of ischemic stroke.

Potential protective role of endogenous glutamate-oxaloacetate transaminase against glutamate excitotoxicity in fetal hypoxic-ischaemic asphyxia.

AIM: Fetal blood contains higher concentrations of glutamate-oxaloacetate transaminase (GOT; a blood enzyme able to metabolize glutamate) than maternal blood. The aim of this study was to determine the relationship between GOT and glutamate levels in arterial blood samples from umbilical cord in control newborn infants and newborn infants with hypoxic-ischaemic insult and/or symptoms of hypoxia-ischemia after delivery. METHOD: A total of 46 newborn infants (28 females, 18 males) were prospectively included in the study. Twenty-three infants (18 females, five males) were included as control participants and 23 (10 females, 13 males) were included as newborn infants at risk of adverse neurological outcome (defined as umbilical blood with pH <7.1). RESULTS: Analysis of glutamate concentration and GOT activity in umbilical blood samples showed that newborn infants with pH <7.1 had higher levels of glutamate (142.4 µmol/L [SD 61.4] vs 62.8 µmol/L [SD 25.5]; p<0.001) and GOT (83.1 U/L [SD 60.9] vs 34.9 U/L [SD 18.2]; p<0.001) compared to newborn infants without fetal distress. Analysis of Apgar scores and blood pH values (markers of perinatal distress) showed that conditions of severe distress were associated with higher glutamate and GOT levels. INTERPRETATION: During fetal development, the ability of GOT to metabolize glutamate suggests that this enzyme can act as an endogenous protective mechanism in the control of glutamate homeostasis.

Pyridoxal 5ꞌ-phosphate-responsive epilepsy with novel mutations in the PNPO gene: a case report.

Pyridoxal 5'-phosphate (PLP)-responsive epilepsy is a rare autosomal recessive epileptic disorder caused by deficiency of pyridox(am)-ne 5'-phosphate oxidase (PNPO). Neonatal onset seizures in PLP responsive epilepsy are usually resistant to common anticonvulsants and pyridoxine, but respond to PLP. Various PNPO mutations are associated with this disorder. In this report, we have described a case of a female baby with neonatal onset seizures responding to PLP. Exome sequencing revealed that the patient was compound heterozygous for pathogenic mutations [c.546+1G>A (IVS5+1 G>A) and c.620delG (p.G207VfsX215)] in the PNPO gene. The c.546+1G>A was inherited from the mother while the c.620delG was inherited from the father. Both mutations were absent in 122 unrelated Thai controls. The results of this study indicated the presence of two newly identified mutations in this Thai patient with PLP-responsive epilepsy for the first time, expanding the mutational spectrum of PNPO.

Uridine protects against hypoxic-ischemic brain injury by reducing histone deacetylase activity in neonatal rats.

PURPOSE: A significant cause of neurological disability in newborns is hypoxic-ischemic encephalopathy (HIE), a disorder which involves an enhancement in histone deacetylase (HDAC) activity among underlying pathological mechanisms. We showed recently that exogenous administration of uridine to newborn rats with HIE reduced brain injury in a dose-dependent manner. The present study was performed to investigate whether uridine modulates histone acetylation/deacetylation balance in a neonatal rat model of HIE. METHODS: Newborn rats that were subjected to hypoxic-ischemic (HI) insult on postnatal day 7 (P7) were injected intraperitoneally with either saline or uridine (500 mg/kg) for three consecutive days. One day after completion of treatment, brains of pups were collected for evaluation of brain infarct volume, apoptosis, HDAC activity and acetylated-Histone H3 (Ac-H3) and H4 (Ac-H4) protein levels. RESULTS: Results revealed that uridine administration reduced infarct volume, active Caspase-3 levels and HDAC activity while increasing the expressions of Ac-H3 and Ac-H4 proteins. CONCLUSIONS: We conclude that one mechanism by which uridine provides neuroprotection in neonatal rat HIE model involves reduction in HDAC activity.

ER stress related factor ATF6 and caspase-12 trigger apoptosis in neonatal hypoxic-ischemic encephalopathy.

The specific and available markers proteins of neonatal hypoxic-ischemic encephalopathy (HIE) injury are correlated with disease severity and the disability in childhood. Exploring the mechanism of HIE is very helpful to the targeted therapeutic approach in clinical. This study aims to explore the cell death-related proteins or biomarkers that plays roles in the HIE injury. In this study, 15 patients were included the 487 autopsies patients performed at the Department of Pathology. The lactate dehydrogenase (LDH) assay was used to detect the cell viability of NGF-differentiated PC12 cell. TUNEL assay was employed to examine the apoptotic cells in embedded slides samples. Three ER stress-related protein, including ATF6, p-Perk and IRE-1 were investigated using Western blot assay for the ER stress examination. The apoptosis associated caspase-12 and CHOP protein were detected by Western blot. The results indicated that LDH activity of living cells during hypoxia was significantly enhanced to 45% and 64% after 8 hours and 24 hours. The TUNEL results showed that plenty of the PC12 cells became the positive staining cells when treated with 0.1% O2 hypoxia. ER stress UPR pathway protein, cleaved ATF6, was increased significantly when treated with 0.1% O2 compared with the cells treated with 20% O2. Furthermore, the caspase 12 activation was triggered when the cells treated with the 0.1% O2. In conclusion, apoptosis is served as an important factor that triggers the HIE brain injury through cleaving the ATF6 and caspase-12 ER stress-related protein.

Thioredoxin 1 and glutaredoxin 2 contribute to maintain the phenotype and integrity of neurons following perinatal asphyxia.

BACKGROUND: Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion. METHODS: We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment. RESULTS AND CONCLUSIONS: We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype. GENERAL SIGNIFICANCE: These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorders.

Neuroinflammation after neonatal hypoxia-ischemia is associated with alterations in the purinergic system: adenosine deaminase 1 isoenzyme is the most predominant after insult.

Hypoxic-ischemic (HI) injury perinatal brain is a major contributor to morbidity and mortality to infants and children. Adenosine may play a role in the pathophysiology of HI, since it modulates the inflammatory process and the release of several neurotransmitters. Thus, the aim of this study was to identify the isoforms of adenosine deaminase (ADA) responsible for the enzymatic activity as well as the adenosine kinase (ADK) and A1 adenosine receptor (A1R) expression in the cerebral cortex eight days after HI. Myeloperoxidase (MPO) and N-acetyl-glucosaminidase (NAG) were assessed as inflammation markers. ADA activity was analyzed, in the presence or absence of a specific ADA1 inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine. The ADA1 activity (92.6%) was significantly higher than ADA2 (7.4%) activity in the cerebral cortex eight days after HI. A1Rs and ADK protein expression showed decreased 8 days after insult. Interestingly, the ADA1, MPO, and NAG activities were correlated positively. In view of this, we conclude that the inhibitor of ADA1, in in vitro conditions, was effective in decreasing the ADA activity, and that mainly ADA1 isoform is responsible for the increase in the ADA activity 8 days after HI insult. Therefore, HI neonatal was able to alter the ADK and A1R expression. Thus, due to the importance of adenosine signaling in the regulation of inflammatory and immune process and the crucial role of ADA in the postischemic homeostase of adenosine as well as during inflammatory process, we suggest that ADA1 inhibitors may play an important role in the regulation of events that follow the HI insult, favoring the increase in the adenosine in the sites of tissue injury. Together, these results highlight a role of the purinergic signaling cascade in the pathophysiology of HI neonatal.

A dual role for AMP-activated protein kinase (AMPK) during neonatal hypoxic-ischaemic brain injury in mice.

Perinatal hypoxic-ischaemic encephalopathy (HIE) occurs in 1-2 in every 1000 term infants and the devastating consequences range from cerebral palsy, epilepsy and neurological deficit to death. Cellular damage post insult occurs after a delay and is mediated by a secondary neural energy failure. AMP-activated protein kinase (AMPK) is a sensor of cellular stress resulting from ATP depletion and/or calcium dysregulation, hallmarks of the neuronal cell death observed after HIE. AMPK activation has been implicated in the models of adult ischaemic injury but, as yet, there have been no studies defining its role in neonatal asphyxia. Here, we find that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is pathological activation of the calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß)-AMPKα1 signalling pathway. Pharmacological inhibition of AMPK during the insult promotes neuronal survival but, conversely, inhibiting AMPK activity prior to the insult sensitizes neurons, exacerbating cell death. Our data have pathological relevance for neonatal HIE as prior sensitization such as exposure to bacterial infection (reported to reduce AMPK activity) produces a significant increase in injury. We show that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is a pathological activation of the CaMKKß-AMPKα1 signalling pathway. Inhibiting AMPK during OGD promotes neuronal survival; conversely, inhibiting AMPK prior to OGD exacerbates cell death. Our data have clinical relevance as prior sensitization (e.g. exposure to bacterial infection reducing AMPK activity) increases injury. AMPK, AMP-activated protein kinase; HI, hypoxia-ischaemia; OGD, oxygen-glucose deprivation.