Marianne S. Wright

Absence of synapsin I and II is accompanied by decreases in vesicular transport of specific neurotransmitters.

Studies of synapsin‐deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild‐type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double‐labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.

Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation

To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post‐mitotic NT2‐N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP‐ribose)‐polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3‐(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 ± 117% of neutral medium, p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 ± 25%, p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2‐N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detrimental during reoxygenation.

Resuscitation of newborn piglets. short-term influence of FiO2 on matrix metalloproteinases, caspase-3 and BDNF.

Background Perinatal hypoxia-ischemia is a major cause of mortality and cerebral morbidity, and using oxygen during newborn resuscitation may further harm the brain. The aim was to examine how supplementary oxygen used for newborn resuscitation would influence early brain tissue injury, cell death and repair processes and the regulation of genes related to apoptosis, neurodegeneration and neuroprotection. Methods and Findings Anesthetized newborn piglets were subjected to global hypoxia and then randomly assigned to resuscitation with 21%, 40% or 100% O2 for 30 min and followed for 9 h. An additional group received 100% O2 for 30 min without preceding hypoxia. The left hemisphere was used for histopathology and immunohistochemistry and the right hemisphere was used for in situ zymography in the corpus striatum; gene expression and the activity of various relevant biofactors were measured in the frontal cortex. There was an increase in the net matrix metalloproteinase gelatinolytic activity in the corpus striatum from piglets resuscitated with 100% oxygen vs. 21%. Hematoxylin-eosin (HE) staining revealed no significant changes. Nine hours after oxygen-assisted resuscitation, caspase-3 expression and activity was increased by 30–40% in the 100% O2 group (n = 9/10) vs. the 21% O2 group (n = 10; p<0.04), whereas brain-derived neurotrophic factor (BDNF) activity was decreased by 65% p<0.03. Conclusions The use of 100% oxygen for resuscitation resulted in increased potentially harmful proteolytic activities and attenuated BDNF activity when compared with 21%. Although there were no significant changes in short term cell loss, hyperoxia seems to cause an early imbalance between neuroprotective and neurotoxic mechanisms that might compromise the final pathological outcome.

Nicotine affects the expression of brain-derived neurotrophic factor mRNA and protein in the hippocampus of hypoxic newborn piglets

Abstract Brain-derived neurotrophic factor (BDNF) is highly expressed in the developing brain. It has anti-apoptotic abilities, and protects the neonatal brain. In experimental settings in adult animals, pre-treatment with nicotine has shown increased BDNF levels, indicating a possible contribution to nicotines anti-apoptotic effect. Apoptosis contributes to the development of brain damage in perinatal asphyxia. We examined the effects of nicotine on apoptosis-inducing factor (AIF), caspase-3 and BDNF in the hippocampus of a neonatal piglet model of global hypoxia. Forty-one anesthetized newborn piglets were randomized to one of four groups receiving different infusions after hypoxia (1) nicotine 130 μg/kg/h, 2) 260 μg/kg/h, 3) adrenaline, and 4) saline, all 2.6 mL/kg/h. Four hours after hypoxia they were euthanized. The left hemisphere/hippocampus was examined by histopathology and immunohistochemistry; the right hippocampus was analyzed using real time PCR. There was a significantly higher expression of BDNF mRNA and protein in the animals treated with nicotine 130 μg/kg/h vs. the saline treated group (mRNA P=0.038; protein P=0.009). There were no differences regarding AIF or caspase-3. We conclude that nicotine (130 μg/kg/h), infused over 1 h after global hypoxia in neonatal piglets, increases levels of both BDNF mRNA and protein in the hippocampus. This might imply neuroprotective effects of nicotine in asphyxiated neonates.

Transcriptome profiling of the newborn mouse brain after hypoxia-reoxygenation: hyperoxic reoxygenation induces inflammatory and energy failure responsive genes

Background:Supplemental oxygen used during resuscitation can be detrimental to the newborn brain. The aim was to determine how different oxygen therapies affect gene transcription in a hypoxia–reoxygenation model.Methods:C57BL/6 mice (n = 56), postnatal day 7, were randomized either to 120 min of hypoxia 8% O2 followed by 30 min of reoxygenation with 21, 40, 60, or 100% O2, or to normoxia followed by 30 min of 21 or 100% O2. Affymetrix 750k expression array was applied with RT-PCR used for validation. Histopathology and immunohistochemistry 3 d after hypoxia–reoxygenation compared groups reoxygenated with 21 or 100% O2 with normoxic controls (n = 22).Results:In total, ~81% of the gene expression changes were altered in response to reoxygenation with 60 or 100% O2 and constituted many inflammatory-responsive genes (i.e., C5ar2, Stat3, and Ccl12). Oxidative phosphorylation was downregulated after 60 or 100% O2. Iba1+ cells were significantly increased in the striatum and hippocampal CA1 after both 21 and 100% O2.Conclusion:In the present model, hypoxia–reoxygenation induces microglial accumulation in subregions of the brain. The transcriptional changes dominating after applying hyperoxic reoxygenation regimes include upregulating genes related to inflammatory responses and suppressing the oxidative phosphorylation pathway.

Transcriptome profiling of the newborn mouse lung after hypoxia and reoxygenation: hyperoxic reoxygenation affects mTOR signaling pathway, DNA repair, and JNK-pathway regulation

Background:The use of oxygen in acute treatment of asphyxiated term newborns is associated with increased mortality. It is unclear how hyperoxic reoxygenation after hypoxia affects transcriptional changes in the newborn lung.Methods:On postnatal day 7, C57BL/6 mice (n = 62) were randomized to 120-min hypoxia (fraction of inspired oxygen (FiO2) 0.08) or normoxia. The hypoxia group was further randomized to reoxygenation for 30 min with FiO2 0.21, 0.40, 0.60, or 1.00, and the normoxia group to FiO2 0.21 or 1.00. Transcriptome profiling was performed on homogenized lung tissue using the Affymetrix 750k expression array, and validation was carried out by real-time polymerase chain reaction and enzyme-linked immunosorbent assay.Results:The hypoxia–reoxygenation model induced hypoxia-inducible factor 1 (HIF-1) targets like Vegfc, Adm, and Aqp1. In total, ~70% of the significantly differentially expressed genes were detected in the two high hyperoxic groups (FiO2 0.60 and 1.00). Reoxygenation with 100% oxygen after hypoxia uniquely upregulated Gadd45g, Dusp1, Peg3, and Tgm2. Pathway analysis identified mammalian target of rapamycin (mTOR) signaling pathway, DNA repair, c-jun N-terminal kinase (JNK)-pathway regulation, and cell cycle after hyperoxic reoxygenation was applied.Conclusion:Acute hypoxia induces HIF-1 targets independent of the reoxygenation regime applied. Hyperoxic reoxygenation affects pathways regulating cell growth and survival. DNA-damage–responsive genes are restricted to reoxygenation with 100% oxygen.

Inflammatory receptors and pathways in human NT2-N neurons during hypoxia and reoxygenation. Impact of acidosis.

Cytokines are released in response to brain injury and inflammation. By binding to receptors, they can cause, exacerbate or inhibit cellular injury and repair. We studied RNA expression of cytokine receptors and members of inflammatory pathways in human NT2-N neurons during 3 h of hypoxia and glucose deprivation followed by 21 h of reoxygenation, and the impact of acidosis. Right after acidotic hypoxia, RNA of IL-10RA and CXCR4 were significantly increased relative to acidotic control, while Bcl-2 and Bcl-xL were significantly decreased. After 21 h of neutral reoxygenation after neutral hypoxia, there was a significant increase in RNA of CXCR1 (relative quantification (RQ)=4.1, p<0.05), CXCR2 (3.6, p<0.05), CCR2 (3.8, p<0.05), Hsp70 (2.4, p<0.05), HIF-1alpha (1.5, p<0.001), TRAF6 (1.3, p<0.05) and TNFR1 (1.6, p<0.05). After 21 h of acidotic reoxygenation after acidotic hypoxia, we found a significant increase in RNA of IL-1R1, IL-10RA, CXCR4 and Hsp70 compared to control, and a significant decrease in FAS and TRAF6. There was a significant increase in Bax expression and a significant decrease in Bcl-2 and Bcl-xL expression in three out of four pH groups after 21 h of reoxygenation. Acidotic, relative to neutral, hypoxia and reoxygenation also influenced the expression of various genes. We conclude that inflammatory receptors and pathways are activated during hypoxia and reoxygenation in NT2-N neurons, and that this activation is pH dependent. This supports the concept that inflammatory pathways play a role in cerebral hypoxic-ischemic damage, and that they may represent important pharmacological targets.

Resuscitation of Hypoxic Newborn Piglets With Supplementary Oxygen Induces Dose-Dependent Increase in Matrix Metalloproteinase-Activity and Down-Regulates Vital Genes

The optimal oxygen concentration for newborn resuscitation is still discussed. Oxygen administration during reoxygenation may induce short- and long-term pathologic changes via oxidative stress and has been associated to later childhood cancer. The aim was to study changes in oxidative stress-associated markers in liver and lung tissue of newborn pigs after acute hypoxia followed by reoxygenation for 30 min with 21, 40, or 100% oxygen compared with room air or to ventilation with 100% oxygen without preceding hypoxia. Nine hours after resuscitation, we found a dose-dependent increase in the matrix metalloproteinase gelatinase activity in liver tissue related to percentage oxygen supply by resuscitation (100% versus 21%; p = 0.002) pointing at more extensive tissue damage. Receiving 100% oxygen for 30 min without preceding hypoxia decreased the expression of VEGFR2 and TGFBR3 mRNA in liver tissue, but not in lung tissue. MMP-, VEGF-, and TGFβ-superfamily are vital for the development, growth, and functional integrity of most tissues and our data rise concern about both short- and long-term consequences of even a brief hyperoxic exposure.

Differential development of vesicular glutamate transporters in brain: An in vitro study of cerebellar granule cells

The cerebellar granule cells have been extensively used for studies on metabolism, neurotransmission and neurotoxicology, since they can easily be grown in cultures. However, knowledge about the development of different proteins essential for synaptic transmission in these cells is lacking. This study has characterized the developmental profiles of the vesicular glutamate transporters (VGLUTs) and the synaptic vesicle proteins synapsins and synaptophysin in cerebellar granule cells and in co-cultures containing both granule cells and astrocytes. The protein levels of VGLUT2 decreased by approximately 70% from days 2 to 7 in vitro, whereas the levels of VGLUT1 increased by approximately 95%. Protein levels of synapsin I, synapsin IIIa and synaptophysin showed a developmental pattern similar to VGLUT1 while synapsin II and VGLUT3 were absent. The mRNA expressions of VGLUT1 and VGLUT2 were in accordance with the protein levels. The results indicate both that cerebellar granule cells are mature at approximately 7 days in vitro, and that the up-regulation of VGLUT1 and down-regulation of VGLUT2 in cerebellar granule cells are both independent of surrounding astrocytes and neuronal input. The results of this study are discussed in relation to general developmental profiles of VGLUTs in other brain regions.

Hypoxia-reoxygenation affects whole-genome expression in the newborn eye.

PURPOSE Resuscitation of newborns is one of the most frequent procedures in neonatal medicine. The use of supplementary oxygen during resuscitation of the asphyxiated newborn has been shown to be detrimental to vulnerable tissues. We wanted to assess transcriptional changes in ocular tissue after the acute use of oxygen in the delivery room in a hypoxia-reoxygenation model of the newborn mouse. METHODS C57BL/6 mice (n = 57), postnatal day 7, were randomized to receive either 120 minutes of hypoxia, at 8% O2, followed by 30 minutes of reoxygenation with 21, 40, 60, or 100% O2 or to normoxia followed by 30 minutes of 21% or 100% O2. Whole ocular homogenates were analyzed by Affymetrix 750k expression array, and RT-PCR was performed for validation. Bayesian analysis of variance for microarray data (BAMarray) was used to identify single significant genes, and Gene Set Enrichment Analysis (GSEA) was applied to reveal significant pathway systems. RESULTS In total, ∼ 92% of the gene expression changes were altered in response to reoxygenation with 60% or 100% O2 compared to expression at the lower percentages of 21% and 40%. After 100% O2 treatment, genes involved in inflammation (Ccl12), angiogenesis (Igfr1, Stat3), and metabolism (Hk2) were upregulated. Pathway analyses after hypoxia-reoxygenation revealed significant alterations of six pathways which included apoptosis, TGF-beta signaling, oxidative phosphorylation, voltage-gated calcium channel complex, mitochondrion, and regulation of RAS protein signal transduction. CONCLUSIONS Hypoxia-reoxygenation can induce immediate transcriptional responses in ocular tissue involving inflammation, angiogenesis, energy failure, and Ras signaling.