Biomedical subjects

David W Walker

Publications and source records attributed to David W Walker.

24 records · Page 2Linked to original sources

PubMed2003

Steroid-sensitive GABAA receptors in the fetal sheep brain.

Neuroactive steroids such as allopregnanolone (3 alpha-hydroxy-5 alpha-pregnan-20-one) influence central nervous system (CNS) excitability by increasing GABA (gamma aminobutyric acid) inhibitory activity. Allopregnanolone concentrations are higher in the fetal compared to the adult ovine brain, suggesting that this neurosteroid may have a role in regulating fetal CNS activity during gestation. We examined the localisation of allopregnanolone-sensitive GABA(A) receptors in the fetal brain to determine if their sensitivity to allopregnanolone changed during late gestation. The binding of [(35)S] tert-butylbicyclophosothionate (TBPS) was used to identify the GABA-chloride ion receptor complex in fetal sheep brains at 90-95, 115-120 and 140-145 days gestational age (GA; term approximately 147 days), by autoradiography. Allopregnanolone (200 nM) reduced [(35)S]TBPS binding by 70-100% throughout the brain at all fetal ages examined. The levels of [(35)S]TBPS binding increased with advancing gestation in all regions examined except some areas of the medulla. Functionally related nuclei and brain areas associated with regulating somato/viscerosensory functions displayed high levels of [(35)S]TBPS binding from mid-gestation. These results indicate that allopregnanolone may interact with GABA(A) receptors to inhibit fetal CNS activity from mid-gestation. This inhibition may contribute to maintaining the sleep-like behaviour and low incidence of arousal-type activity typical of fetal life, and may be neuroprotective by limiting excitatory neurotransmission.

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PubMed2002

Prostaglandin D synthase in the prenatal ovine brain and effects of its inhibition with selenium chloride on fetal sleep/wake activity in utero.

It has been proposed that prostaglandin (PG) D(2) induces physiological sleep in mammals by acting on sleep centers located in the anterior hypothalamus. In fetal sheep, definitive rapid-eye-movement and non-rapid-eye-movement sleep states appear at approximately 125 d gestation (term is approximately 147 d). In adult animals, PGD synthase (PGDS) (functionally and structurally homologous to beta-trace protein) is secreted into CSF with a circadian pattern, with the highest concentrations present during sleep. In this study we show that PGDS/beta-trace protein is present in fetal sheep CSF at 125 and 135 d gestation but not at 90 d gestation. SeCl(4), a specific inhibitor of PGDS, was given to unanesthetized fetal sheep (130-140 d gestation) by intracerebroventricular infusion at a dose of 25, 100, 500, or 1000 pmol/min for 4 hr. Artificial CSF was infused in control experiments. Arousal behavior, defined as the presence of nuchal muscle electromyogram activity, electro-ocular activity, and breathing movements during low-amplitude electrocortical activity, increased from 3.8 +/- 1 min/hr to 6.6 +/- 0.5 and 7.0 +/- 0.3 min/hr at doses of 100 and 500 pmol/min, respectively (p < 0.05). SeCl(4) at 25 and 1000 pmol/min had no significant effect on arousal activity. Infusion of PGD(2) at 500 pmol/min intracerebroventricularly for 4 hr decreased the incidence of arousal from 3.8 +/- 0.5 min/hr to 0.7 +/- 0.3 min/hr (p < 0.05). When 500 pmol/min PGD(2) was infused immediately after a 4 hr infusion of SeCl(4) (500 pmol/min), the SeCl(4)-induced increase in arousal behavior was abolished. Together, the presence of PGDS/beta-trace protein in fetal CSF in late gestation and the effects of SeCl(4) in increasing the incidence of arousal-like behavior suggest that PGD(2) has a role in the induction and maintenance of prenatal sleep.

Animals↗

PubMed2002

Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods.

We report here the identification of a previously unknown transcription regulatory element for heat shock (HS) genes in Caenorhabditis elegans. We monitored the expression pattern of 11,917 genes from C. elegans to determine the genes that were up-regulated on HS. Twenty eight genes were observed to be consistently up-regulated in several different repetitions of the experiments. We analyzed the upstream regions of these genes using computational DNA pattern recognition methods. Two potential cis-regulatory motifs were identified in this way. One of these motifs (TTCTAGAA) was the DNA binding motif for the heat shock factor (HSF), whereas the other (GGGTGTC) was previously unreported in the literature. We determined the significance of these motifs for the HS genes using different statistical tests and parameters. Comparative sequence analysis of orthologous HS genes from C. elegans and Caenorhabditis briggsae indicated that the identified DNA regulatory motifs are conserved across related species. The role of the identified DNA sites in regulation of HS genes was tested by in vitro mutagenesis of a green fluorescent protein (GFP) reporter transgene driven by the C. elegans hsp-16-2 promoter. DNA sites corresponding to both motifs are shown to play a significant role in up-regulation of the hsp-16-2 gene on HS. This is one of the rare instances in which a novel regulatory element, identified using computational methods, is shown to be biologically active. The contributions of individual sites toward induction of transcription on HS are nonadditive, which indicates interaction and cross-talk between the sites, possibly through the transcription factors (TFs) binding to these sites.

Animals↗

PubMed2002

Endotoxin increases sleep and brain allopregnanolone concentrations in newborn lambs.

Infection has been identified as a risk factor for sudden infant death syndrome (SIDS). Synthesis of allopregnanolone, a neuroactive steroid with potent sedative properties, is increased in response to stress. In this study, we investigated the effect of endotoxin (lipopolysaccharide, LPS) on brain and plasma allopregnanolone concentrations and behavior in newborn lambs. LPS was given intravenously (0.7 micro g/kg) at 12 and 15 d of age (n = 7), and resulted in a biphasic febrile response (p < 0.001), hypoglycemia, lactic acidemia (p < 0.05), a reduction in the incidence of wakefulness, and increased nonrapid eye movement sleep and drowsiness (p < 0.05) compared with saline-treated lambs (n = 5). Plasma allopregnanolone and cortisol were significantly (p < 0.05) increased after LPS treatment. These responses to LPS lasted 6-8 h, and were similar at 12 and 15 d of age. Each lamb was then given LPS at 20 d of age and killed 3 h posttreatment to obtain samples of the brain. Allopregnanolone concentrations were increased (p < 0.05) in all brain areas except the cerebellum and diencephalon. We suggest that LPS-induced increase of allopregnanolone in the brain may contribute to somnolence in the newborn, and may be responsible for the reduced arousal thought to contribute to the risk of SIDS in human infants.

Animals↗

PubMedDate not supplied

Melatonin provides neuroprotection in the late-gestation fetal sheep brain in response to umbilical cord occlusion.

Oxygen free radicals, including the highly toxic hydroxyl radical (*OH), initiate lipid peroxidation and DNA/RNA fragmentation and damage cells. The pineal hormone melatonin is an antioxidant and powerful scavenger of *OH. We hypothesized that maternally administered melatonin could reduce *OH formation, lipid peroxidation, and DNA/RNA damage in the fetal brain in response to asphyxia. In 15 fetal sheep, extracellular *OH was measured by microdialysis in white and gray matter of the parasagittal cortex. In 10 fetuses, asphyxia was induced by umbilical cord occlusion for 10 min using an inflatable cuff - the ewes of these fetuses received either intravenous melatonin (1 mg bolus, then 1 mg/h for 2 h; n = 5) or vehicle (1% ethanol in saline; n = 5), and results were compared to fetuses with sham cord occlusion and vehicle-infused ewes (n = 5). Hypoxemia, acidemia, hypertension and bradycardia produced by cord occlusion was similar in the melatonin- and vehicle-treated groups. In the vehicle-treated group, cord occlusion resulted in a significant increase in *OH in gray matter at 8-9.5 h after occlusion (p < 0.05); in contrast, there was no *OH change in the melatonin-treated group. After cord occlusion, lipid peroxidation (4-hydroxynonenal immunoreactivity) found throughout the brain of vehicle-infused ewes was significantly less in the melatonin-infused group. Melatonin had no significant effect on the distribution of DNA/RNA fragmentation, as shown by 8-hydroxydeoxyguanosine immunoreactivity. Thus, brief asphyxia results in significant and delayed entry of *OH into the extracellular space of cortical gray matter in the fetal sheep brain, and melatonin given to the mother at the time of the insult abrogates this increase. Melatonin, in reducing O2 free radical production, may be an effective neuroprotective treatment for the fetus.

Animals↗

PubMedDate not supplied

Expression of erythropoietin and its receptor in the brain of late-gestation fetal sheep, and responses to asphyxia caused by umbilical cord occlusion.

Asphyxia and hypoxia are common threats faced by the fetus in utero. In late-gestation fetal sheep, asphyxia produced by umbilical cord occlusion (UCO) results in widespread lipid peroxidation and apoptosis. Adaptive mechanisms that might limit fetal brain damage include induction of the hemopoietic cytokine, erythropoietin (EPO). In unanesthetized fetal sheep, we investigated if 1 or 2 bouts of brief asphyxia (UCO for 10 min) induced EPO and EPO type I receptor (EPO-R) expressions, with the second UCO repeated 48 h after the first. Fetal brains were recovered 48 h after either sham, 1 x or 2 x UCO at 129-133 (term approximately 147) days of gestation and prepared for immunocytochemistry. In age-matched control brain, low levels of EPO and EPO-R proteins were present in oligodendrocytes (OLs), periventricular and cortical white matter (WM), with no EPO and very low EPO-R expression in neurons. After 1 x UCO, EPO and EPO-R expressions were increased in astrocytes (periventricular and cortical WM, striatum, corpus callosum), choroid plexus epithelial cells, scattered neurons in cortical layers IV-VI, hippocampal CA1 neurons, and in the molecular and granule layers of the cerebellum. After 2 x UCO, higher levels of EPO and EPO-R occurred in the periventricular and cortical WM, corpus callosum, hippocampal CA1, and in neurons of all cortical layers. Paradoxically, EPO and EPO-R were now lower in hippocampal CA1 neurons and cerebellar molecular and granule cell layers. Few OLs expressed EPO or EPO-R after 1 x or 2 x UCO. Thus, brief asphyxia induces EPO and EPO-R in fetal astrocytes, but only after repeated asphyxial insult in neurons. Whether this is a response to increased injury, or represents an adaptive response that limits further cell death and brain damage awaits further investigation.

Animals↗