The impact of optimal dating on the assessment of fetal growth.

BACKGROUND: The impact of using the Intergrowth (IG) dating formulae in comparison to the commonly used Robinson dating on the evaluation of biometrics and estimated fetal weight (EFW) has not been evaluated. METHODS: Nationwide cross-sectional study of routine fetal ultrasound biometry in low-risk pregnant women whose gestational age (GA) had been previously assessed by a first trimester CRL measurement. We compared the CRL-based GA according to the Robinson formula and the IG formula. We evaluated the fetal biometric measurements as well as the EFW taken later in pregnancy depending on the dating formula used. Mean and standard deviation of the Z scores as well as the number and percentage of cases classified as <3rd, < 10th, >90th and > 97th percentile were compared. RESULTS: Three thousand five hundred twenty-two low-risk women with scans carried out after 18 weeks were included. There were differences of zero, one and 2 days in 642 (18.2%), 2700 (76.7%) and 180 (5%) when GA was estimated based on the Robinson or the IG formula, respectively. The biometry Z scores assessed later in pregnancy were all statistically significantly lower when the Intergrowth-based dating formula was used (p < 10- 4). Likewise, the number and percentage of foetuses classified as <3rd, < 10th, >90th and > 97th percentile demonstrated significant differences. As an example, the proportion of SGA foetuses varied from 3.46 to 4.57% (p = 0.02) and that of LGA foetuses from 17.86 to 13.4% (p < 10- 4). CONCLUSION: The dating formula used has a quite significant impact on the subsequent evaluation of biometry and EFW. We suggest that the combined and homogeneous use of a recent dating standard, together with prescriptive growth standards established on the same low-risk pregnancies, allows an optimal assessment of fetal growth.

A rapid chair-side method for the estimation of oral bacterial colonization density.

AIMS: Caries and periodontal disease are associated with inadequate control of oral bacteria. Since conventional microbiological evaluations are impractical in dental clinics or public engagement activities, a rapid test for the quantification of oral bacteria represents a useful tool. We describe the development of a colour change test to rapidly estimate bacterial colonisation density in the mouth. METHODS AND RESULTS: Volunteers rinsed with milk or milkshake. Viability indicators were added and colour changes quantified during incubation. Using milkshake and the resazurin-based solution PrestoBlue (9% v/v), the method distinguished between samples before and after brushing within 5 min. Colour changes were quantified and viable counts were obtained using oral rinses. Measured colour changes strongly correlated with total counts of both anaerobes and streptococci (Spearman's correlation coefficient of 0·782 and 0·769, respectively, P ≤ 0·001) and with perceived changes, as determined by volunteers (n = 10) visually ranking images. CONCLUSIONS: The resazurin milkshake test can rapidly and visually quantify viable bacteria in oral samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The resazurin milkshake test could serve as a sensitive semi-quantitative method for measuring oral bacteria in human oral rinse samples.

Retrospective analysis of technical success rate and procedure-related complications of 867 percutaneous CT-guided needle biopsies of lung lesions.

AIM: To investigate the technical success rate and procedure-related complications of computed tomography (CT)-guided needle biopsy of lung lesions and to identify the factors that are correlated with the occurrence of procedure-related complications. MATERIALS AND METHODS: This was a single- institution retrospective study of 867 consecutive CT-guided needle biopsies of lung lesions performed on 772 patients in a tertiary cancer centre. The technical success rate and complications were correlated with patient, lung lesion, and procedure-related variables. RESULTS: The technical success rate was 87.2% and the mortality rate was 0.12%. Of the 867 total biopsies 25.7% were associated with pneumothorax, and 6.5% required chest tube drainage. The haemothorax rate was 1.8%. There was positive correlation between the development of pneumothorax and smaller lesion diameter (p<0.001), longer transparenchymal distance (p<0.001), and prone position (p=0.027). There was positive correlation between the need for chest tube placement and longer transparenchymal distance (p=0.007) and smaller lesion diameter (p=0.018). Lesions in the left lower lobe had the lowest rates of pneumothorax (p=0.008) and chest tube drainage (p=0.018). Patients whose pneumothoraces were diagnosed on the follow-up chest X-ray, but not on the immediate post-procedural CT scan had significantly higher requirement for chest tube drainage (p=0.039). CONCLUSION: CT-guided lung biopsy has a high rate of technical success and a low rate of major complications. The present study has revealed several variables that can be used to identify high-risk procedures. A post-procedural chest X-ray within hours after the procedure is highly recommended to identify high-risk patients who require chest tube placement.

Modeling non-syndromic autism and the impact of TRPC6 disruption in human neurons.

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.

Biparietal/transverse abdominal diameter ratio ≤ 1: potential marker for open spina bifida at 11-13-week scan.

OBJECTIVE: In the first trimester of pregnancy, a biparietal diameter (BPD) below the 5(th) percentile is a simple marker that enables the prenatal detection of half of all cases of open spina bifida. We hypothesized that relating the BPD measurement to the transverse abdominal diameter (TAD) might be another simple and effective screening method. In this study we assessed the performance of using the BPD/TAD ratio during the first trimester of pregnancy in screening for open spina bifida. METHODS: A total of 20,551 first-trimester ultrasound scans (11-13 weeks' gestation), performed between 2000 and 2013, were analyzed retrospectively; there were 26 cases of open spina bifida and 17,665 unaffected pregnancies with a crown-rump length of 45-84 mm and a record of both BPD and TAD measurements. RESULTS: The mean (± SD) BPD/TAD ratio was 1.00 ± 0.06 for fetuses with spina bifida and 1.13 ± 0.06 for those without (P < 0.0001). A BPD ≤ 5(th) percentile enabled the prenatal detection of 46.2% of spina bifida cases, while a BPD/TAD ratio of ≤ 1.00 detected 69.2%. If we considered cases in which either BPD was ≤ 5(th) percentile or BPD/TAD ratio was ≤ 1, we identified 76.9% of cases. In the latter case, the false-positive rate was 5.1%, while that for using a combination of both BPD ≤ 5th percentile and BPD/TAD ratio ≤ 1 was 0.6%, with a sensitivity of 38.5%. The positive predictive value of using a combination of BPD ≤ 5th percentile and BPD/TAD ratio ≤ 1 for detecting spina bifida was 8.5%. CONCLUSIONS: Between 11 and 13 weeks' gestation, relating BPD to TAD improves considerably the diagnostic performance of using BPD measurement alone in screening for open spina bifida. Screening using this marker is simple and applicable to a large population.

Patterns and outcomes of aortofemoral bypass grafting in the era of endovascular interventions.

OBJECTIVES: The aim of the study is to study contemporary presentation patterns and clinical results in patients undergoing aortofemoral bypass (AFB) surgery. DESIGN: This was a retrospective comparative study. MATERIAL AND METHODS: During a 14-year period, 269 consecutive patients (mean age 65 years) underwent AFB. Indications included occlusive disease with severe intermittent claudication (IC) (n = 86), critical limb ischaemia (CLI, n = 97) and aneurysmo-occlusive disease (n = 86). RESULTS: From 2000-07 on, AFB was performed more frequently for occlusive disease with CLI than for other indications (48% vs. 31% before 2000, P = 0.009) and also in women (51% vs. 32% before 2000, P = 0.003), compared to the period before 2000. Thirty-day mortality was reduced during 2000-2007 to 2.4%, compared with 4.3% during 1993-1999, although this difference was not statistically significant (P = 0.73). Morbidity did not change substantially over the study period. Predictors of 30-day mortality included indication (CLI = 4.1% vs. claudication = 1.2% (P = 0.37)) and chronic kidney disease (CKD, serum creatinine > 1.5 mg dl⁻¹) (11.1% vs. 2.9% in normal renal function, P = 0.07), the latter being the single predictor on multivariate analysis (hazard risk 4.2, P = 0.047). Overall 5 and 10-year assisted primary and secondary patency was 95% and 88%, and 99% and 95%, respectively. Survival at 5 and 10 years was 69% and 48%, respectively. Patient age (hazard risk 1.05, P < 0.001), CKD (hazard risk 1.79, P = 0.018) and diabetes (hazard risk 1.56, P = 0.022) were independent predictors of worse long-term survival. Long-term outcome did not change over the course of the study. CONCLUSIONS: In the contemporary era, AFB is more likely to be performed for CLI and in women than in the past. Despite these changes, perioperative mortality and morbidity remain low and long-term outcome excellent.

[Awareness and knowledge about risks of drinking during pregnancy in pregnant Lebanese women]. / Les conduites d'alcoolisation des femmes enceintes libanaises et leurs connaissances sur les risques et répercussions sur l'enfant à naître.

OBJECTIVE: To assess the awareness and knowledge of pregnant Lebanese women about the risks of drinking during pregnancy and the factors that influence their drinking patterns. MATERIALS AND METHODS: A prospective study was conducted on a sample of 107 women consulting the gynecology outpatient department of Hôtel-Dieu de France in Beirut, Lebanon, who completed the T-ACE screening test included in a 21 multiple choice questionnaire which examine knowledge and beliefs about alcohol use during pregnancy, drinking patterns and awareness of fetal alcohol exposure. RESULTS: The 107 women of our sample were all married, between 20 and 41 years old and had mostly a high educational level (86%). Most of the women (47%) were at their first pregnancy. Of the 20 women who self-reported drinking during pregnancy, 60% obtained a positive score on the T-ACE questionnaire, which indicates that more than 11% of the women engaged with potentially high risk drinking for the baby. There is not a significant difference between the different age categories or educational levels. This proportion is lower than that found in international publications. However, the rate of excessive drinking (4 drinks or more on any one occasion in females) was higher and one woman in five reported excessive drinking in the previous year. There is a high level of knowledge that alcohol use during pregnancy is harmful to the child, and the more consumption the more harmful and likely the effects, but there is confusion about the safety of small amounts of alcohol. Women (37%) think that there is a safe level of drinking during pregnancy; 29% tolerate up to one drink a month, 9% tolerate up to one drink a week and one woman thinks having one drink a day is safe. Women who actually drink during pregnancy are more likely to think that alcohol consumption to a certain level is safe. Women (31%) think that beer and/or wine are safe alcohols to a certain level during pregnancy. When asked about the source of this belief, 22% mention a gynecologist but the majority (61%) says it is a personal belief. Women (65%) in our sample are aware that alcohol use during pregnancy can lead to life-long disabilities in a child, such as delayed development (36%), birth defects/deformities (35%) and mental retardation (32%). However, up to 33% of the respondents report having no information about the effects of alcohol on the fetus and two women believe alcohol is not harmful at all. Women with lower levels of education are somewhat less knowledgeable about the risks of alcohol use during pregnancy than those with higher levels of education. There is no association between the drinking patterns of the women with their age, their professional habits and the alcohol consumption of their husbands. The women in our sample seem to be more aware of the necessity to stop smoking rather than stop drinking during pregnancy. CONCLUSION: Lebanese women are not fully aware of the recommendations and risks related to drinking during pregnancy. This is the reason why action must be taken to ensure better diffusion of these recommendations and better assessment of alcohol intake during prenatal visits.

Basilic vein transposition: what is the optimal technique?

OBJECTIVES: To compare the outcome of the one-stage basilic vein transposition (BVT) fistula with a modified, two-stage technique. DESIGN: Retrospective case-controlled study, performed in an academic centre. MATERIAL: A total of 173 candidates for BVT fistula (87 males, mean age 61 years). METHODS: In one-stage BVT, the basilic vein is mobilised through a single incision, placed inside an anterolateral arm tunnel and anastomosed with the brachial artery. In two-stage procedures, the fistula-arterial anastomosis is created first, followed by the second stage, after fistula maturation several weeks later, when the basilic vein is mobilised through two skip incisions, transected near the anastomosis, placed inside an anterolateral arm tunnel and reanastomosed. Morbidity and fistula maturation rate were the main outcome measures. RESULTS: In one-stage BVT (n=76), the incidence of venous hypertension, wound haematomas and all complications (17%, 13% and 43%, respectively) was significantly higher than in two-stage procedures (n=98) (4%, p=0.004, 3%, p=0.012 and 11%, p<0.001, respectively). Time (68 days) to fistula use was significantly decreased in one-stage BVT than in two-stage procedures (132 days, p<0.001) but failure to mature rate was equivalent (15% vs. 18%, p=0.49). CONCLUSIONS: Our results indicate that the two-stage BVT fistula through two skip-arm incisions is superior to the established one-stage procedure in terms of less morbidity but at the cost of a second operation and longer time to access use. Further research comparing these two techniques is necessary. Until this issue is resolved, an individualised approach is suggested.

Mediterranean Sea surface radiocarbon reservoir age changes since the last glacial maximum.

Sea surface reservoir ages must be known to establish a common chronological framework for marine, continental, and cryospheric paleoproxies, and are crucial for understanding ocean-continent climatic relationships and the paleoventilation of the ocean. Radiocarbon dates of planktonic foraminifera and tephra contemporaneously deposited over Mediterranean marine and terrestrial regions reveal that the reservoir ages were similar to the modern one (approximately 400 years) during most of the past 18,000 carbon-14 years. However, reservoir ages increased by a factor of 2 at the beginning of the last deglaciation. This is attributed to changes of the North Atlantic thermohaline circulation during the massive ice discharge event Heinrich 1.

Association between palmoplantar pustulosis and cigarette smoking in Brazil: a case-control study.

BACKGROUND: Palmoplantar pustulosis (PPP) discloses some differences compared to vulgar psoriasis (PV) in terms of age of onset, female predominance and low occurrence of psoriasis lesions elsewhere. Cigarette smoking has been associated to PPP in international studies; nevertheless, these studies were never performed among Brazilian. OBJECTIVES: To compare prevalence of smoking among PPP, PV and other dermatologic patients (NPD). METHODS: Case-control study involving 25 PPP patients from a reference psoriasis centre. Two control groups were matched according to gender and age: 50 patients with PV and 50 NPD. Confounders were adjusted by conditional multiple logistic regression. RESULTS: Among cases, 84.0% were female and PPP age of disease onset (41.4 years) was greater than PV (34.5 years). Prevalence of ever smoking was higher among cases (92.0%) than PV (52.0%) and NPD (30.0%). Adjusted odds ratio of PPP ever smoking compared to PV and NPD was 9.5 and 36.2, respectively. All smokers reported the onset of their habit before the development of PPP. CONCLUSIONS: There was significant association between PPP and smoking. However, the impact of giving it up in the clinical course of the disease remains to be established.

[An independent observatory in Vendée on ST-elevated acute myocardial infarction (the OVISCA Register). A Departmental evaluation of demographic trends, mortality and delays before treatment]. / Observatoire Vendéen indépendant des syndromes Coronarien aigus avec sus-décalage du segment ST (Registre OVISCA). Évaluation départementale, évolution démographique, mortalité, délais de prise en charge.

AIM: Mortality from acute myocardial infarction has been falling during the past 30 years. The aim of the study was to evaluate the temporal trends of demographics, mortality rates, and time to treatment in patients admitted for acute ST elevation myocardial infarction (STEMI) in Vendée. PATIENTS AND METHODS: From 2008 to 2016, 1994 patients hospitalised in CHD Vendée for STEMI <48hours were included. Two groups were compared, 838 patients admitted between 2008 and 2011 (group 1), and 1156 admitted between 2013 and 2016 (group 2). RESULTS: Between the 2 periods, mean age was comparable (63.8 vs. 64.4 years), the gender ratio decreased (from 3.15 to 2.79 ; P=0.25). The mean duration of hospital stay was 0.8 day shorter (P=0.008). Treatment at discharge was optimum in 97.5% patients versus 92% (P<0.001). Left ventricular ejection fraction was comparable (50.6% vs. 50.2%). There was a non-significant trend to a decrease in hospital mortality (from 6.3% to 4.4%; p=0.12), and 6-month mortality (from 6.9% to 5.9%; P=0.51). There was a reduction in the use of emergency call-outs (74.9% to 68.9%; P<0.01), but an increase in direct presentations from 44% to 48.7% (P<0.05). The time before calling was comparable (2.5hours vs. 2.3hours; P=04.7). The "door-to-balloon" time decreased (0.71 vs. 0.55hour; P<0.001). The mean time between pain and angioplasty increased (5.7 vs. 6.8hours; P<0.05). CONCLUSIONS: In vendee, from 2011 to 2016, hospital and 6-month mortality of STEMI trend to decrease non-significantly. The door to balloon time decreased, although emergency call-out rates and delays did not. Considerable efforts are still required with respect to patient information and education. Our registry offers an excellent tool to improve practices, the aim being to ensure its integration in the CRAC-France PCI registry.

The sodium-activated potassium channel Slack is modulated by hypercapnia and acidosis.

Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.

Secondary patency of thrombosed prosthetic vascular access grafts with aggressive surveillance, monitoring and endovascular management.

BACKGROUND: To study the long-term patency of thrombosed prosthetic vascular access grafts treated with percutaneous mechanical thrombectomy (PMT) followed by aggressive surveillance and monitoring and repeated endovascular interventions. STUDY DESIGN: Two hundred seven vascular access grafts presented with first-time thrombosis were treated with PMT using the AngioJet device (n=185) or the Arrow-Trerotola percutaneous thrombolytic device (n=22) followed by angioplasty (+/- stenting) of the anatomical lesion responsible for the thrombotic event. Clinical success was considered at least one successful subsequent hemodialysis session. Graft surveillance/monitoring included clinical and hemodialysis parameters to detect a failing or thrombosed graft. RESULTS: PMT was technically successful in 202 cases (97.6%) and clinically successful in 193 cases (93.2%). During follow-up, 149 got thrombosed and either abandoned (n=33) or underwent at least once repeat thrombectomy (n=116); finally 100 grafts were abandoned (n=90), ligated (n=5) or removed (n=5). Endovascular management (0.54 procedures per 100 graft-days, thrombectomy, n=307 sessions and angioplasty, n=162 sessions) increased significantly functional assisted-primary patency rates from 29% and 14% at 1 and 2 years to a secondary patency of 62% and 47%, respectively. Secondary patency was worse in loop grafts (P=.02) and intermediate graft thrombosis (occurred between 31-182 days after graft placement, P<.001) and better when renal failure was due to hypertension or diabetes (compared to other or cryptogenic causes, P=.048) or isolated angioplasty for graft dysfunction during follow-up had been performed (P<.001). Multivariate analysis identified intermediate graft thrombosis and isolated angioplasty as independent predictors of secondary patency (P<.001, relative risk 2.77 and P<.001, relative risk 0.28, respectively). CONCLUSIONS: PMT is a highly successful procedure with acceptable long-term secondary patency results, provided that aggressive endovascular management of subsequent thrombotic or dysfunction episode is performed. Further research to identify the causes of intermediate graft thrombosis is justified.

[Outcome of fetuses with malformations discovered before 14 weeks. Where the discovery is revealed by echography during the first trimester, is it responsible for the voluntary termination of the pregnancy? Comparison before and after July 2001]. / Devenir des foetus présentant une ou plusieurs malformations découvertes avant 14 SA. Ou la découverte d'une malformation à l'échographie du premier trimestre est-elle responsable d'interruption volontaire de grossesse? Comparaison avant/après juillet 2001.

OBJECTIVES: The aims of this study were to review detection of fetal malformations during the first trimester and to study pregnancy and infant outcomes. We wanted to check if the lengthening of the legal delay for volontary termination of pregnancy changes the outcome of the pregnancy, in cases outside of the legal requirements. MATERIALS AND METHODS: This study was overseen by the french college of fetal echography (CFEF). All the cases of abnormality detected before 14 weeks' gestational age, excluding the isolated increased nuchal translucency, were extracted from the total population examined, and details were entered into the database of the French College of Fetal Echography. All case records were then analyzed. We compared two populations: before and after July 2001. RESULTS: We observed 336 fetuses with malformation(s), 108 before July 2001 and 208 after that date. One percent (0.5-1.6) of scans performed between 10 and 14 weeks revealed fetal abnormalities apart from isolated increased nuchal translucency. Of the 336 cases retained for investigation, 109 increased nuchal translucency or hygroma associated with other malformation(s), 103 central nervous system anomalies, 85 malformations of the thoracoabdominal wall, 81 limb abnormalities, 41 had renal malformations, 28 spinal abnormalities, 21 had heart malformations, 16 involved biometric abnormalities, 12 involved abnormalities of the appendages, and 11 facial abnormalities. Medical termination of pregnancy was performed in 75% of cases. Death in utero occurred in 9% of cases, 12% of infants were born alive. In 3.9% of cases, an abortion was performed. There were no differences between both populations before and after July 2001. CONCLUSION: Excluding isolated increased nuchal translucency or hygroma, malformation before 14 weeks' gestational age was detected in 1% of fetuses. The most common malformations detected in the first trimester were non-isolated increased nuchal translucency and malformations of the thoracoabdominal wall and the brain. The prognosis for fetuses with malformations detected during the first trimester was very poor as only 12% of these infants were born alive, some of them with severe malformations. In our study, and given its limitations, there were no differences between the number of voluntary terminations performed before and after July 2001.

O(2) deprivation inhibits Ca(2+)-activated K(+) channels via cytosolic factors in mice neocortical neurons.

O(2) deprivation induces membrane depolarization in mammalian central neurons. It is possible that this anoxia-induced depolarization is partly mediated by an inhibition of K(+) channels. We therefore performed experiments using patch-clamp techniques and dissociated neurons from mice neocortex. Three types of K(+) channels were observed in both cell-attached and inside-out configurations, but only one of them was sensitive to lack of O(2). This O(2)-sensitive K(+) channel was identified as a large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), as it exhibited a large conductance of 210 pS under symmetrical K(+) (140 mM) conditions, a strong voltage-dependence of activation, and a marked sensitivity to Ca(2+). A low-O(2) medium (PO(2) = 10-20 mmHg) markedly inhibited this BK(Ca) channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O(2) deprivation on BK(Ca) channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pH(i)), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BK(Ca) channel activity by reducing the sensitivity of this channel to Ca(2+). In contrast, the reducing agents glutathione and DTT increased single BK(Ca) channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BK(Ca) is modulated by O(2) deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca(2+) sensitivity resulting from cytosolic alternations such as in pH(i) and phosphorylation. Because of their large conductance and prevalence in the neocortex, BK(Ca) channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.

Human neocortical excitability is decreased during anoxia via sodium channel modulation.

When the central nervous system in humans is deprived of oxygen, the effects are potentially disastrous. Electroencephalographic activity is lost and higher brain function ceases rapidly. Despite the importance of these effects, the mechanisms underlying the loss of cortical activity are poorly understood. Using intracellular recordings of human neocortical neurons in tissue slices, we show that, whereas anoxia produces a relatively small depolarization and modest alterations in passive properties, it causes a major decrease in excitability. Whole-cell voltage-clamp studies of acutely isolated human neocortical pyramidal neurons demonstrate that anoxia and metabolic inhibition produce a large negative shift in the steady-state inactivation [h infinity (V)] curve for the voltage-dependent sodium current (INa). Inclusion of ATP in the patch pipette decreased the shift of the h infinity (V) curve by two-thirds. Because increased inactivation of INa decreases cellular metabolic demand, we postulate that this promotes neuronal survival during periods of oxygen deprivation. These data show a novel mechanism by which anoxia links metabolism to membrane ionic conductances in human cortical neurons.

Intracellular pH regulation of CA1 neurons in Na(+)/H(+) isoform 1 mutant mice.

To understand the role of Na(+)/H(+) exchanger 1 (NHE1) in intracellular pH (pH(i)) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pH(i), using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO(2)/HCO(3)(-), steady-state pH(i) was higher in WT neurons than in mutant neurons. Using the NH(4)Cl prepulse technique, we also show that H(+) flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na(+) from the extracellular solution or by using 100 microM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO(2)/HCO(3)(-), the difference in H(+) flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 microM/s between them at pH 6.6. H(+) flux in CO(2)/HCO(3)(-) was responsive to diisothiocyanato-stilbene-2, 2'-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pH(i) and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO(3)(-), indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO(3)(-)-dependent and -independent transporters in neurons.

Mutation in pre-mRNA adenosine deaminase markedly attenuates neuronal tolerance to O2 deprivation in Drosophila melanogaster.

O2 deprivation can produce many devastating clinical conditions such as myocardial infarct and stroke. The molecular mechanisms underlying the inherent tissue susceptibility or tolerance to O2 lack are, however, not well defined. Since the fruit fly, Drosophila melanogaster, is extraordinarily tolerant to O2 deprivation, we have performed a genetic screen in the Drosophila to search for loss-of-function mutants that are sensitive to low O2. Here we report on the genetic and molecular characterization of one of the genes identified from this screen, named hypnos-2. This gene encodes a Drosophila pre-mRNA adenosine deaminase (dADAR) and is expressed almost exclusively in the adult central nervous system. Disruption of the dADAR gene results in totally unedited sodium (Para), calcium (Dmca1A), and chloride (DrosGluCl-alpha) channels, a very prolonged recovery from anoxic stupor, a vulnerability to heat shock and increased O2 demands, and neuronal degeneration in aged flies. These data clearly demonstrate that, through the editing of ion channels as targets, dADAR, for which there are mammalian homologues, is essential for adaptation to altered environmental stresses such as O2 deprivation and for the prevention of premature neuronal degeneration.

Prostaglandin transporter expression in mouse brain during development and in response to hypoxia.

Prostaglandins (PGs) are bioactive lipid mediators released following brain hypoxic-ischemic injury. Clearance and re-uptake of these prostaglandins occur via a transmembrane prostaglandin transporter (PGT), which exchanges PG for lactate. We used Western blot analyses to examine the PGT developmental profile and its regional distribution as well as changes in transporter expression during chronic hypoxia in the neonatal mouse brain. Microsomal preparations from four brain regions (cortex, hippocampus, cerebellum and brainstem/diencephalon) showed gradual increases in prostaglandin transporter expression in all brain regions examined from postnatal day 1 till day 30. There was a significant regional heterogeneity in the prostaglandin transporter expression with highest expression in the cortex, followed by cerebellum and hippocampus, and least expressed in the brainstem/diencephalon. To further delineate the pattern of prostaglandin transporter expression, separate astrocytic and neuronal microsomal preparations were also examined. In contrast to neurons, which had a robust expression of prostaglandin transporters, astrocytes had very little PGT expression under basal conditions. In response to chronic hypoxia, there was a significant decline in PGT expression in vivo and in neurons in vitro, whereas cultured astrocytes increased their PGT expression. This is the first report on PGT expression in the CNS and our studies suggest that PGTs have 1) a widespread distribution in the CNS; 2) a gradual increase and a differential expression in various regions during brain development; and 3) striking contrast in expression between glia and neurons, especially in response to hypoxia. Since PGTs play a role as prostaglandin-lactate exchangers, we hypothesize that PGTs are important in the CNS during stress such as hypoxia.