Start Of Reoxygenation Research Articles

Evaluation of In Vitro Neuronal Protection by Postconditioning with Poloxamer 188 Following Simulated Traumatic Brain Injury.

Traumatic brain injury (TBI) leads to morbidity and mortality worldwide. Reperfusion after ischemia adds detrimental injury to cells. Ischemia/reperfusion (I/R) injures cells in a variety of ways including cell membrane disruption. Hence, methods to improve endogenous membrane resealing capacity are crucial. Poloxamer (P) 188, an amphiphilic triblock copolymer, was found to be effective against I/R and mechanical injury in various experimental settings. The aim of this study was to establish an in vitro mouse neuronal TBI model and, further, to investigate if postconditioning with P188 directly interacts with neurons after compression and simulated I/R injury, when administered at the start of reoxygenation. Cellular function was assessed by cell number/viability, mitochondrial viability, membrane damage by lactated dehydrogenase (LDH) release and FM1-43 incorporation as well as apoptosis-activation by Caspase 3. Five hours hypoxia ± compression with 2 h reoxygenation proved to be a suitable model for TBI. Compared to normoxic cells not exposed to compression, cell number and mitochondrial viability decreased, whereas membrane injury by LDH release/FM1-43 dye incorporation and Caspase 3 activity increased in cells exposed to hypoxic conditions with compression followed by reoxygenation. P188 did not protect neurons from simulated I/R and/or compression injury. Future research is indicated.

Prostaglandin E1 attenuates post‑cardiac arrest myocardial dysfunction through inhibition of mitochondria‑mediated cardiomyocyte apoptosis.

Post-cardiac arrest myocardial dysfunction (PAMD) is a leading cause of death in patients undergoing resuscitation patients following cardiac arrest (CA). Although prostaglandin E1 (PGE1) is a clinical drug used to mitigate ischemia injury, its effect on PAMD remains unknown. In the present study, the protective effects of PGE1 on PAMD were evaluated in a rat model of CA and in a hypoxia-reoxygenation (H/R) in vitro model. Rats were randomly assigned to CA, CA+PGE1 or sham groups. Asphyxia for 8 min followed by cardiopulmonary resuscitation were performed in the CA and CA+PGE1 groups. PGE1 was intravenously administered at the onset of return of spontaneous circulation (ROSC). PGE1 treatment significantly increased the ejection fraction and cardiac output within 4 h following ROSC and improved the survival rate, compared with the CA group. Moreover, PGE1 inactivated GSK3β, prevented mitochondrial permeability transition pore (mPTP) opening, while reducing cytochrome c and cleaved caspase-3 expression, as well as cardiomyocyte apoptosis in the rat model. To examine the underlying mechanism, H/R H9c2 cells were treated with PGE1 at the start of reoxygenation. The changes in GSK3β activity, mPTP opening, cytochrome c and cleaved caspase-3 expression, and apoptosis of H9c2 cells were consistent with those noted in vivo. The results indicated that PGE1 attenuated PAMD by inhibiting mitochondria-mediated cardiomyocyte apoptosis.

Semisynthetic supra plasma expanders: a new class of therapeutics to improve microcircualtion in sickle cell anaemia

Compromised microcirculation and endothelial dysfunction are hallmarks of sickle cell disease (SCD). EAF PEG Haemoglobin (Hb) and EAF PEG Albumin (Alb) represent a novel class of semisynthetic colloidal supra plasma expanders that improve microcirculation. The therapeutic activity of supra plasma expanders to attenuate vaso-occlusion and restore the haemodynamic functions in patients with SCD has been investigated using NY1DD, a transgenic mouse model of mild SCD without anaemia. Vaso-occlusion and perturbation of haemodynamics are amplified in NY1DD by hypoxia-reoxygenation protocol. EAF P5K6 Alb and Alb T12 (Alb conjugated with 12 copies of antioxidant tempo) attenuate vaso-occlusion when infused at the start of reoxygenation. However, only EAF PEG Alb restores haemodynamics close to levels in control C57BL. EAF P5K6 Alb-T12, active plasma expander conjugated with antioxidant, completely clears vaso-occlusion and restores normal haemodynamics. EAF PEG Hb also completely clears vaso-occlusion and restores normal haemodynamics. Pretreating NY1DD with EAF PEG Hb protects it from hypoxia reoxygenation-induced damages. EAF P5K6 Alb T12 attenuates the endothelial dysfunction in S + S Antilles mice as reflected by the vasodilatory response of its arteries and arterioles to vaso-dilators. Active plasma expanders are novel therapeutics to restore normal haemodynamics in SCD patients to improve tissue oxygenation during episodes of painful vaso-occlusive crisis. Abbreviations: 2-IT: 2-immothiolane; Mal-T: 4-Maleimido tempo; Alb: human serum albumin (HSA); Alb-T12: human albumin conjugated with 12 copies of tempo; EAF: extension arm facilitated; EAF PEG Hb: extension arm facilitated PEGylated haemoglobin; EAF PEG Alb: extension arm facilitated PEGylated albumin; EAF P3K6 Hb: extension arm facilitated PEGylated haemoglobin conjugated with 6 copies of PEG3K; EAF P5K6 Alb T12: extension arm facilitated PEGylated albumin conjugated with 6 copies of PEG5K and 12 copies of tempo; Hb: haemoglobin; HAS: human serum albumin (Alb); PEG: polyethylene glycol; MP4: MalPEG Hb, is formulated at 4.2 g/dL in lactated Ringer's solution, a product of Sangart; SCD: sickle cell disease; NO: nitric oxide; SEC: size exclusive chromatography; Vrbc: red cell velocity; Q: volumetric flow rates, Q; SNP: sodium nitroprusside

Poloxamer 188 does not Target Altered Ca2+ Channels in Cardiomyocytes during Hypoxia/Reoxygenation Injury

IntroductionIschemia/reperfusion (I/R) injury is a complex pathological event. Membrane disruptions and altered calcium (Ca2+) transport during ischemia, continue in early reperfusion. With Ca2+ being a major regulator of cardiomyocyte function, increases in intracellular Ca2+ ([Ca2+]i) can have detrimental effects. Ca2+ may not only enter through membrane disruptions, but through altered Ca2+ channels or exchangers. The tri‐block copolymer cell membrane stabilizer, Poloxamer 188 (P188), has been shown to be protective, by targeting membrane disruptions caused by I/R. However, it is not known if P188 also targets altered Ca2+ channels or exchangers. We investigated the potential role of Ca2+ channels and exchangers that may contribute to increased Ca2+ influx during ischemia, and more importantly, reperfusion. Pharmacologic compounds were used to modify Ca2+ influx, and assess the effect of P188 present during reperfusion, on these modifications.HypothesisP188, with its unique hydrophobic/hydrophilic properties, protects cardiomyocytes (CMs) against hypoxia/reoxygenation (H/R; simulated I/R) injury by stabilizing membrane disruptions and blocking altered Ca2+ channels or exchangers.MethodsIsolated CMs were kept in control/normoxic (C/N) conditions or underwent 5 hrs of hypoxia (0.01% O2; serum‐ & glucose‐free medium), then 2 hrs reoxygenation (21% O2; complete medium). The pharmacologic compounds S‐(−)‐BayK8644 (a L‐type Ca2+ channel agonist), R‐(+)‐BayK8644 (a L‐type Ca2+ channel blocker), dobutamine (a β1 receptor agonist/activator of the voltage‐gated Na+ channel), and KB‐R7943 (a blocker of the reverse mode of the Na+/Ca2+ exchanger) were used. Each was used under both C/N and H/R conditions, and added at the start of reoxygenation (or final 2 hr period in C/N conditions) ± 100μM P188. Endpoints were markers of cell number/viability and [Ca2+]i. Statistics: ANOVA and SNK post hoc comparisons, p<0.05 *vs C/N, **vs H/R, Ɨ vs vehicle.ResultsUnder C/N conditions, S‐(−)‐BayK8644 significantly increased [Ca2+]i, but dobutamine did not, over a 2 hr period. P188 had no effect on attenuating the Ca2+ influx caused by S‐(−)‐BayK8644. R‐(+)‐BayK8644 significantly decreased Ca2+ influx. Under H/R conditions, S‐(−)‐BayK8644 and dobutamine significantly increased [Ca2+]i over the 2 hr reoxygenation period. P188 had no effect on attenuating the Ca2+ influx caused by the above compounds; however, P188 did significantly decrease the Ca2+ influx caused by the H/R itself. R‐(+)‐BayK8644 and KB‐R7943 significantly decreased Ca2+ influx that occurred during the 2 hr reoxygenation period; and, when P188 was also present, this decrease in Ca2+ influx was significantly enhanced.ConclusionsIn CMs, P188 does not appear to target Ca2+ channels or exchangers when administered during reoxygenation. The protective effect of P188 most likely comes from its ability to repair membrane disruptions, and thus restore membrane integrity and cellular function.Support or Funding InformationThis work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veteran Affairs Biomedical Laboratory R&D Service.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of transglutaminase 2 in A1 adenosine receptor- and β2-adrenoceptor-mediated pharmacological pre- and post-conditioning against hypoxia-reoxygenation-induced cell death in H9c2 cells

Pharmacologically-induced pre- and post-conditioning represent attractive therapeutic strategies to reduce ischaemia/reperfusion injury during cardiac surgery and following myocardial infarction. We have previously reported that transglutaminase 2 (TG2) activity is modulated by the A1 adenosine receptor and β2-adrenoceptor in H9c2 cardiomyoblasts. The primary aim of this study was to determine the role of TG2 in A1 adenosine receptor and β2-adrenoceptor-induced pharmacological pre- and post-conditioning in the H9c2 cells. H9c2 cells were exposed to 8h hypoxia (1% O2) followed by 18h reoxygenation, after which cell viability was assessed by monitoring mitochondrial reduction of MTT, lactate dehydrogenase release and caspase-3 activation. N6-cyclopentyladenosine (CPA; A1 adenosine receptor agonist), formoterol (β2-adrenoceptor agonist) or isoprenaline (non-selective β-adrenoceptor agonist) were added before hypoxia/reoxygenation (pre-conditioning) or at the start of reoxygenation following hypoxia (post-conditioning). Pharmacological pre- and post-conditioning with CPA and isoprenaline significantly reduced hypoxia/reoxygenation-induced cell death. In contrast, formoterol did not elicit protection. Pre-treatment with pertussis toxin (Gi/o-protein inhibitor), DPCPX (A1 adenosine receptor antagonist) or TG2 inhibitors (Z-DON and R283) attenuated the A1 adenosine receptor-induced pharmacological pre- and post-conditioning. Similarly, pertussis toxin, ICI 118,551 (β2-adrenoceptor antagonist) or TG2 inhibition attenuated the isoprenaline-induced cell survival. Knockdown of TG2 using small interfering RNA (siRNA) attenuated CPA and isoprenaline-induced pharmacological pre- and post-conditioning. Finally, proteomic analysis following isoprenaline treatment identified known (e.g. protein S100-A6) and novel (e.g. adenine phosphoribosyltransferase) protein substrates for TG2. These results have shown that A1 adenosine receptor and β2-adrenoceptor-induced protection against simulated hypoxia/reoxygenation occurs in a TG2 and Gi/o-protein dependent manner in H9c2 cardiomyoblasts.

Moderate hypothermia initiated during oxygen–glucose deprivation preserves HL-1 cardiomyocytes

ObjectivesTherapeutic hypothermia (TH) is an acknowledged strategy for neuroprotection for patients suffering from hypoxic–anoxic brain injury (HAI). Albeit similar pathomechanisms of HAI for both brain and heart, moderate TH (32–34°C) has not been established as a heart-protective measure. Therefore, we investigated the cardioprotective effects of moderate TH on oxygen–glucose deprivation/re-oxygenation (OGD/R)-induced injury in HL-1 cardiomyocytes. MethodsCardiac OGD/R injury was induced by exposing HL-1 cardiomyocytes to 0.2% oxygen in serum/glucose-free medium for 6h. OGD injured cells were subsequently re-oxygenated with 21% oxygen in complete medium. Two hypothermic protocols were investigated: Post-OGD cooling to 33.5°C for 24h initiated at the start of re-oxygenation and intra-OGD cooling to 33.5°C for 24h initiated after 3h of OGD and maintained throughout the re-oxygenation phase. Cell viability was determined by LDH and cTnT releases. Mitochondria dysfunction was evaluated by intracellular ATP content and cellular metabolic activity was accessed by MTT reduction. Activation of caspase 3 was analyzed by Western blot. ResultsOGD/R-induced injury resulted in increased cell death (higher LDH and cTnT releases), mitochondrial impairment (decreased ATP content), and decreased cellular metabolic activity (decreased MTT reduction). Only intra-OGD cooling attenuated both OGD and OGD–R-induced injuries (significantly decreased LDH and cTnT releases and increased ATP contents and MTT reduction). Furthermore, caspase 3 activation was abated by intra-OGD cooling. No protective effects were observed by post-OGD cooling. ConclusionsModerate TH initiated during OGD is a promising intervention for the protection of cardiomyocytes from OGD/R-induced injury. The attenuation of mitochondrial dysfunction and apoptosis by intra-OGD cooling are beneficial effects of hypothermia-induced cardioprotection, resulting in minimized myocardial cell death after OGD and OGD–R-induced injuries.

Role of large-conductance Ca2+-activated K+ channels in adenosine A1 receptor-mediated pharmacological postconditioning in H9c2 cells

Ischaemic postconditioning is a phenomenon whereby short periods of ischaemia applied during the start of reperfusion protect the myocardium from the damaging consequences of reperfusion. As such, pharmacological-induced postconditioning represents an attractive therapeutic strategy for reducing reperfusion injury during cardiac surgery and following myocardial infarction. The primary aim of this study was to determine the role of large-conductance Ca²(+)-activated potassium channels (BK(Ca) channels) in adenosine A₁ receptor-induced pharmacological postconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. H9c2 cells were exposed to 6 h hypoxia (0.5% O₂) followed by 18 h reoxygenation (H/R) after which cell viability was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. The adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA; 100 nmol/L) or the BK(Ca) channel opener NS1619 (10 µmol/L) were added for 30 min at the start of reoxygenation following 6 h hypoxic exposure. Where appropriate, cells were treated (15 min) before pharmacological postconditioning with the BK(Ca) channel blockers paxilline (1 µmol/L) or iberiotoxin (100 nmol/L). Pharmacological postconditioning with CPA or NS1619 significantly reduced H/R-induced LDH release. Treatment with paxilline or iberiotoxin attenuated adenosine A₁ receptor and NS1619-induced pharmacological postconditioning. These results have shown for the first time that BK(Ca) channels are involved in adenosine A₁ receptor-induced pharmacological postconditioning in a cell model system.

Pinocembrin protects rat brain against oxidation and apoptosis induced by ischemia–reperfusion both in vivo and in vitro

Pinocembrin is one of the flavonoids at the highest concentration in propolis. In this study, we investigated the neuroprotective effect of pinocembrin on ischemia/reperfusion and ischemia/reperfusion-like insults. Protection by pinocembrin was studied at the in vivo level using a model of middle cerebral artery occlusion and reperfusion in rats. Pinocembrin was administrated at the start of reperfusion. Pinocembrin markedly increased rat viability, reduced infarct volumes and neurological deficit scores in all treatment groups. Primary cortical neuronal cultures were subjected to oxygen–glucose deprivation/reoxygenation, a model of ischemia/reperfusion-like injury, and treated with pinocembrin at the start of reoxygenation. Neuronal survival rates were increased, LDH release was decreased and both neurite length and apoptosis were alleviated when pinocembrin was present during reoxygenation, and this protection was associated with the reduction of reactive oxygen species, nitric oxide and neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS), and an increase of glutathione. Moreover, DNA laddering was decreased in treatment groups of pinocembrin. Caspase-3 protein was down-regulated and PARP degradation was alleviated after pinocembrin treatments. Our results suggest that pinocembrin may be a novel therapeutic strategy to reduce cerebral ischemia/reperfusion injury, and may act by the anti-oxidative and anti-apoptotic effects.

Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways

A sequence of intermittent interruptions of oxygen supply (i.e., postconditioning, Postcon) at reoxygenation reduces oxidant-induced cardiomyocyte loss. This study tested the hypothesis that prevention of cardiomyocyte apoptosis by Postcon is mediated by mitogen-activated protein kinases pathways. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Relative to hypoxia alone, reoxygenation stimulated expression of JNKs and p38 kinases, corresponding to increased activity of JNKs (phospho-c-Jun) and p38 (phospho-ATF2). The level of TNFalpha in cell lysates, activity of cytosolic caspases-8, -3, expression of Bax and the number of apoptotic cardiomyocytes were increased while expression of Bcl-2 was decreased with reoxygenation. Consistent with an attenuation in generation of superoxide anions detected by lucigenin-enhanced chemiluminescence at early period of reoxygenation, treatment of cardiomyocytes with Postcon further reduced expression and activity of JNKs and p38 kinases, level of TNFalpha, the frequency of apoptotic cells and expression of Bax. However, the inhibitory effects of Postcon on these changes were lost when its application was delayed by 5 min after the start of reoxygenation. Addition of a JNK/p38 stimulator, anisomycin into cardiomyocytes at the beginning of reoxygenation eliminated protection by Postcon. These data suggest that 1) hypoxia/reoxygenation elicits cardiomyocyte apoptosis in conjunction with expression and activation of JNK and p38 kinases, release of TNFalpha, activation of caspases, and an increase in imbalance of pro-/anti-apoptotic proteins; 2) Postcon attenuates cardiomyocyte apoptosis, potentially mediated by inhibiting JNKs/p-38 signaling pathways and reducing TNFalpha release and caspase expression.

Therapeutic time window for the neuroprotective action of MK-801 after decapitation ischemia: hippocampal slice data

Neuroprotective action of MK-801 administrated pre- and postischemically, in vivo or in vitro, respectively, was studied on hippocampal slices using decapitation ischemia model. Recovery of orthodromic population spikes in CA1 region was measured during postischemic incubation of the slices with oxygenated artificial cerebrospinal fluid (ACSF). The ability of postischemically applied MK-801 to restore the electrical activity dramatically depended on the timing of its application during the reoxygenation period. When applied in vitro, together with the start of reoxygenation, MK-801 was as effective as in the case of in vivo administration before the ischemia. The delay in the in vitro administration for only a few minutes led to a dramatic decrease in the drug effectiveness. When applied in 30 min after the start of reoxygenation, MK-801 was totally ineffective. The dose/response relationship between MK-801 concentration and the amplitude of recovered orthodromic population spikes of hippocampal pyramidal neurons is logarithmic. The ED 50 value for the action of “postischemic” MK-801 is ca. 10 −5 M. Preischemic in vivo application of the drug [intraperitoneal (i.p.) injection 15 min prior to decapitation] results in ED 50 ca. 0,2 mg/kg. The slope of both dose/concentration–response curves is similar. The time course of population spike recovery after 90-min ischemia is identical for pre- and postischemic action of MK-801 (estimated for ED 50 in both cases). These data allow to suggest that “preischemic” MK-801 is predominantly active as a neuroprotector only after ischemia, within a short therapeutic window at the start of the reoxygenation period.

Comparison of Pulmonary and Inflammatory Effects of Lipid- and Water-Soluble Components in Meconium in Newborn Piglets

To understand the pathogenesis of meconium aspiration syndrome, we compared the pulmonary and inflammatory effects of the water and lipid extracts of human meconium instilled into the lungs of newborn piglets. The piglets were artificially ventilated, made hypoxemic, and randomized into three groups. At start of reoxygenation, 3 ml/kg of one of the following mixtures was instilled intratracheally: (1) meconium (n = 12); (2) water extract of meconium (n = 12), and (3) lipid extract of meconium (n = 12). During 8 h of reoxygenation, hemodynamics, pulmonary gas exchange, lung mechanics, and interleukin-8 concentrations in tracheobronchial aspirates were monitored. Oxygenation index (p = 0.04) and mean airway pressure (p = 0.04) increased more in the lipid extract group than in the water extract group. Dynamic compliance and mean arterial blood pressure decreased (p < 0.05) in the meconium and lipid extract groups, but not in the water extract group. At 8 h of reoxygenation, the interleukin-8 concentration in the tracheobronchial aspirates was three times higher in the lipid extract group as compared with the water extract group (110 ± 102 vs. 37 ± 27 pg/ml; p = 0.02). In conclusion, pulmonary dysfunction in meconium aspiration syndrome is caused by both the water- and lipid-soluble fractions of meconium, with stronger inflammatory and more detrimental effects promoted by the lipid extract than the water extract.

Heat shock protein 70 or heat shock protein 27 overexpressed in human endothelial cells during posthypoxic reoxygenation can protect from delayed apoptosis.

Overexpression of heat shock protein (Hsp) 70 and Hsp27 in vivo was proclaimed as a potential tool in therapy of ischemia-reperfusion injury. However, it was so far not known whether these Hsps can beneficially act when increased in cells just at the stage of postischemic reperfusion. This issue was examined in a model of ischemia-reperfusion stress when cultures of endothelial cells (EC) from human umbilical vein were infected with virus-based vectors expressing Hsp70 or Hsp27, or Hsp56, or green fluorescent protein (GFP) and exposed to 20 hours of hypoxia followed by reoxygenation. The infection was performed either 10 hours before hypoxia or immediately after hypoxia, or at different time points of reoxygenation. Only low cell death was detected during hypoxia, but later, up to 40% of the treated cells died via caspase-dependent apoptosis between 6 and 12 hours of reoxygenation. The percentage of apoptotic cells was 1.6- to 3-fold greater in Hsp56- and GFP-infected EC than in Hsp70- or Hsp27-infected EC. The last 2 groups exhibited a lesser extent of procaspase-9 and procaspase-3 activation within 6-9 hours of reoxygenation. The cytoprotective effects of overexpressed Hsp70 and Hsp27 were observed not only in the case of infection before hypoxia but also when EC were infected at the start of reoxygenation or 1-2 hours later. An increase in the Hsp70 and Hsp27 levels in infected EC correlated well with their resistance to apoptosis under reoxygenation. These findings suggest that overexpression of Hsp70 or Hsp27, if it occurs in the involved cells at the early stage of postischemic reperfusion, can still be cytoprotective.

Albumin mixed with meconium attenuates pulmonary dysfunction in a newborn piglet model with meconium aspiration.

We hypothesized that lipids and bile acids in meconium may induce pulmonary insufficiency in newborns. Because albumin may bind these components we studied the effect of albumin on meconium-induced lung injury in piglets. We measured concentration of FFA in the meconium (110 mg dry weight/mL) and added albumin to provide a molar FFA to albumin ratio of 1:1. Newborn piglets, 0-2 d of age, artificially ventilated and exposed to hypoxemia by ventilation with 8% O2, were randomized to group A receiving meconium (n = 12) or group B receiving meconium + albumin (n = 12), 3 mL/kg intratracheally. The animals were reoxygenated for 8 h. Reoxygenation was started when mean blood pressure was <20 mm Hg or base excess was <-20 mM. Pulmonary function was assessed in parallel with pulmonary hemodynamics. From the start of reoxygenation and the next 8 h we found a significant difference (by ANOVA) between the two groups in oxygenation index (p = 0.005), with an increase from 1.6 +/- 0.2 to 6.1 +/- 6.8 (p = 0.04) in the meconium group and from 1.8 +/- 0.3 to 3.1 +/- 3.1 (NS) in meconium + albumin group. There were also significant differences (by ANOVA) between the groups in favor of the treatment group concerning need of inspired fraction of O2, mean airway pressure, dynamic compliance of the respiratory system, time constant, ventilation index, and pulmonary vascular resistance. In conclusion, albumin given concurrently with meconium significantly reduced detrimental effects of meconium aspiration in the lungs of newborn piglets.

Newborn piglets with meconium aspiration resuscitated with room air or 100% oxygen.

We investigated whether newborn piglets exposed to hypoxemia and severe meconium aspiration could be reoxygenated with room air as efficiently as with 100% O(2). Twenty-one 2- to 5-d-old piglets were randomly divided into three groups: 1) the room air group: hypoxemia, meconium aspiration, and reoxygenation with room air (n = 8); 2) the O(2) group: hypoxemia, meconium aspiration, and reoxygenation with 100% O(2) (n = 8); and 3) the control group: meconium aspiration, and reoxygenation with room air (n = 5). Hypoxemia was induced by ventilation with 8% O(2) until the mean blood pressure reached <20 mm Hg or the base excess reached <-20 mM. At this point, reoxygenation was started with either room air or 100% O(2). Three milliliters per kilogram of meconium 110 mg/mL was instilled into the trachea immediately before the start of reoxygenation. The O(2) tension in arterial blood was significantly lower in the room air group; at 5 min of reoxygenation it was 9.1 +/- 0.5 kPa versus 43.5 +/- 6 kPa in the O(2) group (p < 0.05). At 5 min of reoxygenation the tidal volume per kilogram was 12.1 +/- 0.7 mL/kg in the room air group and 13.1 +/- 0.9 mL/kg in the O(2) group (NS). There were no significant differences between the room air and the O(2) groups during 120 min of reoxygenation in mean arterial blood pressure, pulmonary arterial pressure, cardiac index, base excess, or plasma hypoxanthine. In conclusion, hypoxic newborn piglets with meconium aspiration were found to be reoxygenated as efficiently with room air as with 100% O(2).

Pulmonary Circulation and Plasma-Endothelin-1 (P-Et-1) During Hypoxia and Reoxygenation With 21% and 100% O2 in Piglets

Background: We tested the hypothesis that room air normalises pulmonary arterial pressure (PAP) and pulmonary vascular resistance index(PVRI) during reoxygenation as efficient as 100% O2 in hypoxic piglets .Subjects: Eighteen 1-3 d old piglets. Intervention: Hypoxia was induced by ventilation with 8% O2. When mean arterial blood pressure fell below 20 mm Hg or base excess was reduced to <-20 mmol/L a 2-hour reoxygenation-period was started, the piglets were randomly divided into 2 groups; 21% O2 (group 1, n=9) and 100% O2 (group 2, n=9).Results: P-ET-1 decreased during hypoxia from 1.53 ±0.51 ng/L(mean±SD) to 1.17 ±0.55 ng/L (p=0.011) in both groups. At 2 h reoxygenation P-ET-1 increased to 1.92 ±0.90 ng/L (p=0.017) in group 1, and to 1.67 ±0.61 ng/L (p=0.002) in group 2. PAP increased from 24±6 mm Hg at start to 35 ±9 mm Hg at 5 min. reoxygenation in group 1 (p=0.01), and from 27 ±8 mmHg to 31 ±6 (N.S.) in group 2. PVRI decreased from the start of reoxygenation from 0.42 ±0.25 to to 0.15 ±0.11 (p=0.011) and from 0.54 ±0.50 to 0.23 ±0.13(N.S) at 20 min reoxygenation in group 1 and 2, respectively .Conclusion: During reoxygenation PVRI is reduced just as efficient with 21% as 100% O2, an the changes in P-ET-1 are comparable to the changes PVRI, suggesting that changes in PVRI may be mediated by P-ET-1.

Cloning of a Putative Vesicle Transport-related Protein, RA410, from Cultured Rat Astrocytes and Its Expression in Ischemic Rat Brain

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of approximately 70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with these in vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provide insight into a new protein (RA410) that participates in the ischemia-related stress response in astrocytes.

Effect of S-adenosyl-L-methionine on cerebral monoamine turnover after hypoxia in rats

The effects of S-Adenosyl-L-methionine (SAMe) on cerebral monoamine turnover at 60 min of reoxygenation after hypoxia (PaO2, 31-35 mmHg) for 15 min were studied in 44 rats anesthetized with nitrous oxide. The accumulations of monoamine metabolites: 3-methoxy 4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were determined after probenecid. The accumulations of noradrenaline (NA) and dopamine (DA) were also determined after pargyline. In the non-treated group, there was an impairment of degradation of NA to MHPG and of DA to DOPAC or HVA in the control group (no hypoxia). These changes were accompanied by higher levels of NA and DA in the cerebral cortex, hypothalamus and striatum than those of the control group. In the group treated with 100 mg/kg SAMe plus 38 mg/kg mannitol 3 min after the start of reoxygenation, there were no significant changes in these metabolites and amines. Mannitol alone did not cause significant changes. There were no changes in serotonin and 5-HIAA in any of the groups studied. The effects of SAMe were studied in an additional 16 awake rats. MHPG in all regions measured after probenecid in the rats treated with SAMe was higher than that without SAMe. It appears that SAMe ameliorates perturbation of cerebral catecholamine turnover produced by reoxygenation after hypoxia.