Thiopental attenuates hypoxic changes of electrophysiology, biochemistry, and morphology in rat hippocampal slice CA1 pyramidal cells.

BACKGROUND AND PURPOSE: Thiopental has been shown to protect against cerebral ischemic damage; however, it has undesirable side effects. We have examined how thiopental alters histological, physiological, and biochemical changes during and after hypoxia. These experiments should enable the discovery of agents that share some of the beneficial effects of thiopental. METHODS: We made intracellular recordings and measured ATP, sodium, potassium, and calcium concentrations from CA1 pyramidal cells in rat hippocampal slices subjected to 10 minutes of hypoxia with and without 600 micromol/L thiopental. RESULTS: Thiopental delayed the time until complete depolarization (21+/-3 versus 11+/-2 minutes for treated versus untreated slices, respectively) and attenuated the level of depolarization at 10 minutes of hypoxia (-33+/-6 versus -12+/-5 mV). There was improved recovery of the resting potential after 10 minutes of hypoxia in slices treated with thiopental (89% versus 31% recovery). Thiopental attenuated the changes in sodium (140% versus 193% of prehypoxic concentration), potassium (62% versus 46%), and calcium (111% versus 197%) during 10 minutes of hypoxia. There was only a small effect on ATP (18% versus 8%). The percentage of cells showing clear histological damage was decreased by thiopental (45% versus 71%), and thiopental improved protein synthesis after hypoxia (75% versus 20%). CONCLUSIONS: Thiopental attenuates neuronal depolarization, an increase in cellular sodium and calcium concentrations, and a decrease in cellular potassium and ATP concentrations during hypoxia. These effects may explain the reduced histological, protein synthetic, and electrophysiological damage to CA1 pyramidal cells after hypoxia with thiopental.     

Cancer and thrombosis in women – molecular mechanisms

Both women and men with cancer are at increased risk for developing venous thromboembolism (VTE), a propensity that has been known for many years. Until recently it was assumed, however, that the association between cancer and thrombosis is an epiphenomenon — not causally related to the transforming malignant events. The pathophysiology of thrombosis in patients with cancer is complex involving multiple tumor-related and host-related factors. Several recent studies have provided strong evidence that activation of blood coagulation, perhaps most often mediated by tissue factor (TF)-rich microparticles (MPs), is linked directly to oncogene-induced malignant transformation. In addition, the development of VTE, either before or concurrent with the diagnosis of cancer, appears to predict an aggressive behavior of a tumor, and correlates with increased tumor angiogenesis and early onset of distant metastasis. The regulation of expression of TF in tumor cells is controlled at the molecular level by several oncogenes, as appears to be true for cyclooxygenase-2 (COX-2), an important regulator of platelet function and plasminogen activator inhibitor type 1 (PAI-1), an inhibitor of fibrinolysis. In addition, engagement of protease-activated receptors (PARs) by the TF-factor VIIa complex, factor Xa and/or thrombin, have now been shown to be important for tumor growth, angiogenesis and metastasis. Targeting blood clotting reactions in cancer, therefore, may provide a unique approach to cancer treatment.     

Effect of small changes in temperature on CA1 pyramidal cells from rat hippocampal slices during hypoxia: implications about the mechanism of hypothermic protection against neuronal damage.

Small reductions in temperature have been shown to improve neurologic recovery after ischemia. We have examined the effect of temperature on biochemical and physiological changes during hypoxia using rat hippocampal slices as a model system. The postsynaptic population spike recorded from the CA1 pyramidal cell region of slices subjected to 7 min of hypoxia with hypothermia (34 degrees C) recovered to 73% of its prehypoxic level; slices subjected to the same period of hypoxia at 37 degrees C did not recover. After 7 min of hypoxia ATP fell to 48% of its prehypoxic concentration at 34 degrees C and 30% at 37 degrees C. Potassium fell to 86% during 7 min of hypoxia with hypothermia, this compares to a fall to 58% at 37 degrees C. The increase in sodium after 7 min of hypoxia was also attenuated by hypothermia (133% vs. 163% of its prehypoxic concentration). When the hypoxic period was shortened to 3 min (37 degrees C) the population spike recovered to 94%. If the temperature was increased to 40 degrees C there was only 7% recovery of the population spike after 3 min of hypoxia. With hyperthermia (40 degrees C), ATP fell to 33% after 3 min of hypoxia, this compares to 81% at normothermia. Potassium fell to 76% after 3 min of hypoxia with hyperthermia, this compares to 91% at 37 degrees C. Sodium concentrations increased with hyperthermia before hypoxia, at 3 min of hypoxia there was no significant difference between the hyperthermic and normothermic tissue; there was a large increase in sodium with hyperthermia after 5 min of hypoxia (209% vs. 146%). We conclude that the improved recovery after hypothermic hypoxia is at least in part due to the attenuated changes in ATP, potassium and sodium during hypoxia and that the worsened recovery with hyperthermia is due to an exacerbation of the change in ATP, potassium and sodium concentrations during hypoxia.     

Gamma glutamyl transpeptidase is a dynamic indicator of endothelial response to stroke

Gamma glutamyl transpeptidase (gammaGT) is enriched at the apical surface of the cerebral capillaries that constitute the blood-brain barrier (BBB). This study tested the effects of hypoxia and inflammation on gammaGT activity in mice after stroke induced by transient cerebral artery occlusion (tMCAO) and in cultured cerebral microvessel endothelial cells. In microvessel-enriched preparations from mice after tMCAO, gammaGT activity was higher than in the sham controls in both ipsilateral and contralateral hemispheres from 12 h to 5 days after stroke, but lower at later time points (10-15 days). To identify the roles of different cytotoxic and stimulatory signals in this event, we further studied the dynamic changes of gammaGT activity in rat brain endothelial (RBE4) cells. Tumor necrosis factor alpha and lipopolyssachride significantly increased gammaGT activity in a time-dependent manner, an effect not seen after re-oxygenation. Such endothelial activation correlated with reduced total cellular ATP production. Thus, hypoxia and inflammatory stimulation appeared to have opposite effects on endothelial function. With the co-existence of inflammation and hypoxia in the brain after ischemic stroke, dynamic changes of gammaGT activity reflect evolving changes of endothelial function.     

Cerebral metabolism in hypoxic hypoxia. I. Pattern of activation of glycolysis: a re-evaluation.

In order to evaluate the pattern of activation of glycolysis in cerebral cortex during hypoxic hypoxia, lightly anesthetized rats were subjected to a lowering of arterial Po2 to about 25 mm Hg and brains were frozen in situ for metabolite analyses either 1, 2, 5, 15 or 30 min following the induction of hypoxia. The lactate and pyruvate concentrations increased progressively during the 30 min period of hypoxia. At 1 and 2 min there were decreases in G-6-P and F-6-P, and increases in FDP, DHAP and 3-PG, indicating activation of phosphofructokinase. At 5 min this pattern of changes was less pronounced and at 15 min it was absent in spite of the fact that the lactate and pyruvate concentrations were further increased. At 30 min F-6-P and F-6-P had further increased but the levels of DHAP, FDP and 3-PG were normal. Evidently, phosphofructokinase activation can only be detected in the early stages of hypoxia, i.e. when the maximal increase in glycolytic flux occurs and before there has been a corresponding activation of other rate-limiting enzymatic steps. Signs of activation of phosphofructokinase were observed in the absence of changes in tissue concentrations of ATP or AMP, with minimal elevation of NH4plus, and in spite of increased (or unchanged) levels of citrate. However, since there were small but significant increases in ADP at 1 and 2 min, and pH-independent decreases in phosphocreatine, the results indicate that hypoxia is accompanied by an initial imbalance between production and utilization of ATP. The metabolic consequences of this imbalance (decrease in phosphocreatine, increases in ADP and P1) may be at least partly responsible for activation of phosphofructokinase.     

Levels of adenosine and adenine nucleotides in slices of rat hippocampus

ATP, ADP, AMP, IMP, adenosine, inosine and cyclic AMP were measured in slices of the rat hippocampus maintained in vitro. Immediately following cutting ATP was low (3.5 +/- 0.6 nmol/mg protein) and AMP high (8.6 +/- 0.9 nmol/mg), giving an energy charge of only 0.34 +/- 0.02. Over the next 90 min the energy charge gradually normalized (to 0.92 +/- 0.01), partly due to conversion of AMP to ATP, but mainly to breakdown to adenosine and other purines which were recovered in the incubation medium. Total purine content decreased from approximately 18 to 10 nmol/mg protein in the first hour following cutting. In slices from old rats the energy charge was lower 60 min following preparation than in younger rats, while cyclic AMP and adenosine levels were higher. The adenosine antagonist 8-phenyl-theophylline tended to enhance the recovery of responsiveness after preparation of the slices. Stimulation of excitatory afferent fibers at a frequency of 10 Hz for 5 min did not significantly alter the purine levels in brain slices, while hypoxia decreased the energy charge significantly and tended to increase adenosine levels. These changes occurred somewhat later than the fall in electrophysiological responsiveness. 8-Phenyl-theophylline was able to delay somewhat the decline in the amplitude of synaptic responses under hypoxic conditions. It is concluded that the viable part of the hippocampal slice, which accounts for about half of the tissue, has levels of adenine nucleotides and adenosine which are similar to those found in the intact rat brain. The return of electrophysiological function following slice preparation is paralleled by a normalization of the energy charge, the adenosine level and the concentration of cyclic AMP. The absence of electrophysiological activity following cutting, and the decreases in such responses following either prolonged afferent stimulation or hypoxia may be related to changes in purine concentration in the slice. Although adenosine accumulating in the slice may contribute to the depression of electrophysiological responses it is probably not the major factor responsible for the reduction in synaptic responsiveness.     

Intracellular signalling pathways modulate K(ATP) channels in inspiratory brainstem neurones and their hypoxic activation: involvement of metabotropic receptors, G-proteins and cytoskeleton

K(ATP) channels regulate the neuronal excitability and their activation during hypoxia/ischemia protect neurons. The activation of K(ATP) channels during hypoxia is assumed to occur mainly due to the fall in intracellular ATP levels, but other intracellular signalling pathways can be also involved. We measured single K(ATP) channel currents in inspiratory brainstem neurones of neonatal mice. The activity of K(ATP) channels was enhanced in hypoosmotic bath solutions, or after applying negative pressure to the recording pipette. Cytochalasin B activated K(ATP) channels and prevented the effects of osmo-mechanical stress, indicating that cytoskeleton rearrangements, which occur during hypoxia, contribute to the activation of K(ATP) channels. During hypoxia, extracellular levels of many neurotransmitters increase, leading to activation of corresponding metabotropic receptors that can modulate K(ATP) channels. K(ATP) channels were activated by GABA(B) agonist, baclofen, by mGLUR2/3 agonists and were inhibited by mGLUR1/5 agonists. K(ATP) channels were activated by phorbol esters and were inhibited by staurosporine. These treatments did not occlude the modulating actions of mGLUR agonists, indicating that they are not mediated by protein kinase C. Activator of alpha-subunits of G-proteins Mas 7 increased and their inhibitor GPant-2 decreased the activity of K(ATP) channels. In the presence of either agent, the modulatory actions of baclofen and mGLUR agonists were not observed. We conclude that K(ATP) channels are modulated by G-proteins that are activated by metabotropic receptors for GABA and glutamate and their release during hypoxia complements activation of channels by osmo-mechanical stress and [ATP](i) depletion.     

Cerebral energy metabolism and intracellular pH during severe hypoxia and recovery: a study using 1H, 31P, and 1H [13C] nuclear magnetic resonance spectroscopy in the guinea pig cerebral cortex in vitro

1H and 31P nuclear magnetic resonance spectroscopy was used to study intracellular pH (pHi), high-energy phosphates, lactate, and amino acids in cortical brain slices superfused in Krebs-Henseleit bicarbonate buffer during and after severe hypoxia at 0, 10, and 50 mM glucose. An extensive drop in phosphocreatine (PCr) and a rapid build-up of intracellular lactate and H+ were the first signs of hypoxia. Adenosine triphosphate (ATP) was significantly more resistant to hypoxia provided that glucose was present. In the preparations that had been exposed to hypoxia in the presence of glucose, PCr became detectable within 2 min of reoxygenation, and both PCr and ATP concentrations were restored to 72-80% of normoxic levels within 30 min. Lactate was washed out, and pHi returned to normal within 4-8 min. Using 1-[13C]glucose as a tracer, we demonstrated that the rate of lactate production in the immediate posthypoxic period was at the prehypoxic level, indicating that the elevated lactate during this period was due solely to that produced during hypoxia. During reoxygenation of the preparations that were denied glucose during hypoxia, only 30% of total creatine + PCr and 18% of PCr were restored, and ATP was not detectable. The lactate concentration rose twofold in this period, and pHi became significantly more alkaline than before the hypoxic insult. Thus acute metabolic damage was considerably greater if glucose was absent during the insult, suggesting that either anaerobic ATP production or low pH may exert some protective effect against acute cell damage.     

Gene regulation by hypoxia and the neurodevelopmental origin of schizophrenia

Neurodevelopmental changes may underlie the brain dysfunction seen in schizophrenia. While advances have been made in our understanding of the genetics of schizophrenia, little is known about how non-genetic factors interact with genes for schizophrenia. The present analysis of genes potentially associated with schizophrenia is based on the observation that hypoxia prevails in the embryonic and fetal brain, and that interactions between neuronal genes, molecular regulators of hypoxia, such as hypoxia-inducible factor 1 (HIF-1), and intrinsic hypoxia occur in the developing brain and may create the conditions for complex changes in neurodevelopment. Consequently, we searched the literature for currently hypothesized candidate genes for susceptibility to schizophrenia that may be subject to ischemia-hypoxia regulation and/or associated with vascular expression. Genes were considered when at least two independent reports of a significant association with schizophrenia had appeared in the literature. The analysis showed that more than 50% of these genes, particularly AKT1, BDNF, CAPON, CCKAR, CHRNA7, CNR1, COMT, DNTBP1, GAD1, GRM3, IL10, MLC1, NOTCH4, NRG1, NR4A2/NURR1, PRODH, RELN, RGS4, RTN4/NOGO and TNF, are subject to regulation by hypoxia and/or are expressed in the vasculature. Future studies of genes proposed as candidates for susceptibility to schizophrenia should include their possible regulation by physiological or pathological hypoxia during development as well as their potential role in cerebral vascular function.     

Adenosine released by astrocytes contributes to hypoxia-induced modulation of synaptic transmission

Astrocytes play a critical role in brain homeostasis controlling the local environment in normal as well as in pathological conditions, such as during hypoxic/ischemic insult. Since astrocytes have recently been identified as a source for a wide variety of gliotransmitters that modulate synaptic activity, we investigated whether the hypoxia-induced excitatory synaptic depression might be mediated by adenosine release from astrocytes. We used electrophysiological and Ca2+ imaging techniques in hippocampal slices and transgenic mice, in which ATP released from astrocytes is specifically impaired, as well as chemiluminescent and fluorescence photometric Ca2+ techniques in purified cultured astrocytes. In hippocampal slices, hypoxia induced a transient depression of excitatory synaptic transmission mediated by activation of presynaptic A1 adenosine receptors. The glia-specific metabolic inhibitor fluorocitrate (FC) was as effective as the A1 adenosine receptor antagonist CPT in preventing the hypoxia-induced excitatory synaptic transmission reduction. Furthermore, FC abolished the extracellular adenosine concentration increase during hypoxia in astrocyte cultures. Several lines of evidence suggest that the increase of extracellular adenosine levels during hypoxia does not result from extracellular ATP or cAMP catabolism, and that astrocytes directly release adenosine in response to hypoxia. Adenosine release is negatively modulated by external or internal Ca2+ concentrations. Moreover, adenosine transport inhibitors did not modify the hypoxia-induced effects, suggesting that adenosine was not released by facilitated transport. We conclude that during hypoxia, astrocytes contribute to regulate the excitatory synaptic transmission through the release of adenosine, which acting on A1 adenosine receptors reduces presynaptic transmitter release. Therefore, adenosine release from astrocytes serves as a protective mechanism by down regulating the synaptic activity level during demanding conditions such as transient hypoxia.     

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury

ATP-dependent potassium (K_ATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate K_ATP channels. In addition, the effects of the K_ATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N₂–5% CO₂ instead of 95% O₂–5% CO₂ for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%), probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (300 μM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 μM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 μM) antagonized the effects of diazoxide (300 μM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 μM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO₂. However, only the ATP/ADP ratio was decreased further by diazoxide (300 μM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of K_ATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival via protein and DNA synthesis.     

Brain tumor and seizures: pathophysiology and its implications for treatment revisited

Seizures affect approximately 50% of patients with primary and metastatic brain tumors. Partial seizures have the highest incidence, followed by secondarily generalized, depending on histologic subtype, location, and tumor extent. The underlying pathophysiologic mechanisms of tumor-associated seizures are poorly understood and include theories of altered peritumoral amino acids, regional metabolism, pH, neuronal or glial enzyme and protein expression, as well as immunologic activity. An involvement of changed distribution and function of N-methyl-d-aspartate subclass of glutamate receptors also has been suggested. The often unpredictable responses to seizures after surgical tumor removal add substantial evidence that multiple factors are involved. The therapy of tumor-related seizures is far from perfect. Several factors contribute to these treatment difficulties, such as tumor growth and drug interactions; however, one of the main reasons for poor seizure control may result from the insufficient or even absent influence of the currently available antiepileptic drugs (AEDs) on most of the pathophysiologic mechanisms of tumor-related seizures. Studies are needed to elucidate more clearly the pathophysiologic mechanisms of tumor-related seizures and to identify and develop the optimal AEDs.     

低氧复合运动通过NO-ATF1通路上调大鼠骨骼肌线粒体解偶联蛋白3表达**◆

背景：低氧复合运动可上调解偶联蛋白3的表达,提高骨骼肌线粒体对低氧的抵抗力,但其生物学效应及作用机制尚不清楚。 目的：观察单纯低氧及低氧复合运动对骨骼肌线粒体力能学及解偶联蛋白3表达的影响,并探讨NO-ATF1信号通路在其中的生物学效应。 方法：将60只SD大鼠随机分成常氧对照组、单纯低氧组、低氧复合运动训练组、低氧+ L-NAME组和低氧复合运动训 练+L-NAME组。低氧干预为常压低氧帐篷,模拟11.3%的氧体积分数;运动干预为低氧帐篷内跑台训练;L-NAME干预为饮用水中添加一氧化氮合酶抑制剂左旋硝基精氨酸甲酯。各种干预持续4周,硝酸还原酶法测定骨骼肌一氧化氮含量,荧光素酶发光法检测线粒体ATP合成活力,二氯荧光素法检测线粒体过氧化氢生成速率,实时荧光定量PCR法检测骨骼肌激活转录因子1和解偶联蛋白3 mRNA的表达,Western blot法检测骨骼肌磷酸化激活转录因子1和线粒体解偶联蛋白3蛋白的表达。 结果与结论：低氧复合运动显著上调骨骼肌解偶联蛋白3的表达及线粒体ATP的合成活力,抑制线粒体过氧化氢的产生,同时增加骨骼肌一氧化氮含量及激活转录因子1磷酸化水平,左旋硝基精氨酸甲酯抑制了低氧复合运动对线粒体的保护效应。说明低氧复合运动可通过NO-ATF1途径上调解偶联蛋白3的表达提高骨骼肌线粒体对低氧的抵抗力。 关键词：低氧复合运动;线粒体;一氧化氮;激活转录因子1;解偶联蛋白3;骨骼肌     

Electro-clinical characteristics and postoperative outcome of medically refractory tumoral temporal lobe epilepsy

BACKGROUND: Very few studies have specifically addressed surgical treatment and outcome of patients with tumor-related temporal lobe epilepsy (TLE). AIM: To define the postoperative seizure outcome and the factors that influenced the outcome of patients with tumor-related TLE. MATERIALS AND METHODS: We selected patients whose surgical pathology revealed a temporal lobe neoplasm and who had completed > 1 year of postoperative follow-up. We reviewed the clinical, EEG, radiological and pathological data, and the seizure outcome of these patients and assessed the factors that influenced the outcome. RESULTS: Out of the 409 patients who underwent surgery for refractory TLE during the 8-year study period, there were 34 (8.3%) patients with temporal lobe neoplasms. The median age at surgery was 20 years and the median duration of epilepsy prior to surgery was 9.0 years. MRI revealed tumor in the mesial location in 21 (61.8%) patients. Interictal and ictal epileptiform EEG abnormalities were localized to the side of th lesion in the majority. Mesial temporal lobe structures were included in the resection, if they were involved by the tumor; otherwise, lesionectomy alone was performed. During a median follow-up of 4 years, 27 (79%) patients were completely seizure-free. The only factor that predicted long-term seizure-free outcome was being seizure-free during the first two postoperative years. CONCLUSIONS: Our results emphasize the fact that in patients with tumoral TLE, when the seizures are medically refractory, surgery offers potential for cure of epilepsy in the majority.     

Effects of deprivation of oxygen or glucose on the neural activity in the guinea pig hippocampal slice—intracellular recording study of pyramidal neurons

The block of synaptic transmission and neural activity during deprivation of oxygen or glucose has been simply attributed to the lack of energy due to the disorder of energy production. To clarify the interrelation between neural activity and energy metabolism during hopoxia or glucose deprivation, we studied the changes in ATP levels and electrical events of pyramidal neurons in the CA3 region and [Ca²⁺]i mobilization of the dendritic and cellular region of CA3 area, using guinea pig hippocampal slices. The studies of field potentials and intracellular recording from the pyramidal cell of CA3 area during hypoxia or glucose deprivation revealed that the cessation of synaptic activity and the depolarization of resting potential occurred earlier during hypoxia than during glucose deprivation while the increase of [Ca²⁺]i was slow during hypoxia but rapid during glucose deprivation although the ATP level of CA3 area was maintained at its original level for 20 min during both conditions. When glucose was replaced by lactate, ATP concentration was not reduced but the electrical activity decayed and [Ca²⁺]i increased with the similar time course as observed during lack of glucose, only. These results suggest that different mechanisms underlie the block of synaptic transmission in the CA3 pyramidal neurons during hypoxia and glucose deprivation and that lactate cannot substitute for glucose in the maintenance of neural activity.