Metabolism in pedicled and free TRAM flaps: a comparison using the microdialysis technique

The most common complication in flap surgery is of a circulatory nature. Impeded blood flow leads to altered metabolism in the tissue. Possible metabolic differences between different zones of the transverse rectus abdominis muscle (TRAM) flap were studied and the metabolism of pedicled and free TRAM flaps was compared intraoperatively and postoperatively. The method used was microdialysis, which is a useful technique for following local metabolic changes continuously in various tissues.Twenty-two patients with a pedicled or free TRAM flap were monitored using the microdialysis technique. Two microdialysis catheters were placed subcutaneously in the flap (zone I and zone II), and a third one was placed subcutaneously in the flank to serve as a control. The flaps were monitored intraoperatively and postoperatively for 3 days with repeated analyses of extracellular glucose, lactate, and glycerol concentrations. An additional analysis of pyruvate was performed in some patients to calculate the lactate-to-pyruvate ratio.This study showed that glucose, lactate, and glycerol change in a characteristic way when complete ischemia (i.e., complete inhibition of the blood circulation) is present. A slower stabilization with prolonged metabolic signs of ischemia, such as lower glucose and higher lactate and glycerol concentrations, was seen in zone II compared with zone I, and more pronounced metabolic signs of ischemia, but with a faster recovery, were detected in the free TRAM flap group than in the pedicled TRAM flap group. The fact that the metabolites returned to normal earlier in free flaps than in pedicled flaps may indicate that free TRAM flaps sustain less ischemic damage because of better and more vigorous perfusion.     

Evaluation of monoaminergic neurotransmitters in the acute focal ischemic human brain model by intracerebral in vivo microdialysis

The release of neurotransmitters principally glutamate during cerebral ischemia has been extensively studied. It is well recognized that ischemia induced release of glutamate plays a key role in “excitotoxic” neuronal death. The role of monoaminergic neurotransmitters is however unclear. The purpose of this study was to evaluate the extracellular norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA) and serotonin (5-HT) under varied degrees of ischemia in the acute focal ischemic model of the human brain by in-vivo microdialysis. The ischemic response of these amines was correlated with the glutamate levels. Our study concludes that these amines and metabolites can be detected in the human “stroke” model. No marked fluctuations were noted in the levels of norepinephrine and DOPAC. However, significant changes to partial and total ischemia were noted in the extracellular levels of 5-HIAA and 5-HT. These compounds showed a dramatic increase with the onset of ischemia with higher detectable levels in the partial ischemic state in comparison to the total ischemic dialysate levels. The exact role played by the differential increase in the levels of 5-HT to the other catecholamines in the pathogenesis of ischemic neuronal damage remains unclear and warrants further study.     

脑内谷氨酸、乳酸以及葡萄糖对中重型脑外伤患者病情的评价意义

目的:应用微透析技术对于中重型脑外伤患者进行持续脑内谷氨酸、乳酸以及葡萄糖,分析结果以评价以上因素与患者病情的关系。方法:选择我院2006年3月-2009年11月颅脑外科和ICU收治的急性颅脑损伤患者32例,根据GCS分为重度昏迷组和中度昏迷组,均行急诊手术治疗,并在手术直视下置入微透析探针,置入后第4天拔除,定时收集透析液约10μl,于术前以及术后第1、2、3、4天收取标本并立即送检,分别检测患者标本中的谷氨酸、乳酸和葡萄糖含量,并结合患者预后进行分析。结果:中度昏迷组乳酸与谷氨酸值在手术后呈进行性下降,与术前比较,术后第2、3、4天差异有统计学意义(P<0.05),乳酸值的变化与谷氨酸变化趋势相近,与术前比较,在术后第3、4天差异有统计学意义(P<0.05),葡萄糖值与术前比较,术后第2、3、4天差异有统计学意义(P<0.05);重度昏迷组谷氨酸、乳酸和葡萄糖与术前比较,三者均在第4天出现有统计学意义的变化。重度昏迷组谷氨酸测量值在各个观察点均高于中度昏迷组测量值(P<0.05),乳酸值亦明显高于中度昏迷组测量值(P<0.05),葡萄糖测量值两组术前测量值差异无统计学意义(P>0.05),自术后第1天始,中度昏迷组各个时间点测量值明显高于重度昏迷组。结论:结合患者的GCS评分,应用微透析技术实时监测患者脑内谷氨酸、乳酸以及葡萄糖的含量变化,能很好的把握患者的病情,有效指导临床治疗。     

Effects of magnesium sulfate on energy metabolites and glutamate in the cortex during focal cerebral ischemia and reperfusion in the gerbil monitored by a dual-probe microdialysis technique

Previous studies have demonstrated that magnesium sulfate has cytoprotective properties for treating experimental rat brain injuries. The aim of this study is to evaluate changes in energy-related metabolites and glutamate in the cortex of gerbils subjected to focal cerebral ischemia with the pretreatment of magnesium sulfate. The focal cerebral ischemia was produced by the occlusion of the right common carotid artery and the right middle cerebral artery for 60 mins. A significant decrease in infarct size was found in the magnesium sulfate treated group when compared to the controls. Two microdialysis probes were inserted bilaterally into the cortex to monitor extracellular glucose, lactate, pyruvate and glutamate during cerebral ischemia and reperfusion periods. The present study showed a dynamic decrease of glucose (10% of the baseline), pyruvate (15% of the baseline), and an increase of lactate (200% of the baseline) and glutamate (1400% of the baseline) on the ipsilateral side during ischemia in the control group. Magnesium sulfate significantly preserved glucose (up to 50% of the baseline) and pyruvate (70% of the baseline) levels in the ipsilateral side during ischemia. There was significant attenuation in the elevation of glutamate and lactate (500% and 150% of the baseline, respectively) when treatments of magnesium sulfate were administered. No significant influence on these neurochemicals in the contralateral side was observed in either group. These results suggest that both the preservation of cellular energy metabolism, and the attenuation of glutamate release during cerebral ischemia and after restoration of reperfusion may contribute to the neuroprotective effects of magnesium sulfate.     

Metabolic responses to exhaustive exercise change markedly during the protracted non-trophic spawning migration of the lamprey <Emphasis Type="Italic">Geotria australis</Emphasis>

Adults of the Southern hemisphere lamprey Geotria australis were subjected to an exercise/recovery regime at the commencement and end of their 12–15 month non-trophic, upstream spawning migration. In early (immature) migrants and pre-spawning females, muscle glycogen was markedly depleted during exercise, but became rapidly replenished. As muscle lactate rose during exercise and peaked 1–1.5 h into the recovery period, and therefore after muscle glycogen had become replenished, it cannot be the direct source for that replenishment. However, both plasma lactate and glycerol (but not muscle glycerol and glucose) rose sharply during exercise and then declined markedly during the first 0.5 h of recovery and thus exhibited the opposite trend to that of muscle glycogen, implying that these limited pools of glycogenic precursors contribute to glycogen replenishment. Although plasma glucose rose following exercise, and consequently could also be a precursor for muscle glycogen replenishment, it remained elevated even after muscle glycogen had become replenished. While resting pre-spawning females and mature males retained high muscle glycogen concentrations, this energy store became permanently depleted in females during spawning. In mature males, muscle glycogen remained high and lactate low during the exercise/recovery regime, whereas muscle glycerol declined precipitously during exercise and then rose rapidly. In summary, vigorous activity by G. australis is fuelled extensively by anaerobic metabolism of glycogen early in the spawning run and by pre-spawning females, but by aerobic metabolism of its energy reserves in mature males.     

脑内谷氨酸、乳酸以及葡萄糖对中重型脑外伤患者病情的评价意义

目的：应用微透析技术对于中重型脑外伤患者进行持续脑内谷氨酸、乳酸以及葡萄糖,分析结果以评价以上因素与患者病情的关系。方法：选择我院2006年3月-2009年11月颅脑外科和ICU收治的急性颅脑损伤患者32例,根据GCS分为重度昏迷组和中度昏迷组,均行急诊手术治疗,并在手术直视下置入微透析探针,置入后第4天拔除,定时收集透析液约10μl,于术前以及术后第1、2、3、4天收取标本并立即送检,分别检测患者标本中的谷氨酸、乳酸和葡萄糖含量,并结合患者预后进行分析。结果：中度昏迷组乳酸与谷氨酸值在手术后呈进行性下降,与术前比较,术后第2、3、4天差异有统计学意义（P〈0．05）,乳酸值的变化与谷氨酸变化趋势相近,与术前比较,在术后第3、4天差异有统计学意义（P〈0．05）,葡萄糖值与术前比较,术后第2、3、4天差异有统计学意义（P〈0．05）;重度昏迷组谷氨酸、乳酸和葡萄糖与术前比较,三者均在第4天出现有统计学意义的变化。重度昏迷组谷氨酸测量值在各个观察点均高于中度昏迷组测量值（P〈0．05）,乳酸值亦明显高于中度昏迷组测量值（P〈O．05）,葡萄糖测量值两组术前测量值差异无统计学意义（P〉0．05）,自术后第1天始,中度昏迷组各个时间点测量值明显高于重度昏迷组。结论：结合患者的GCS评分,应用微透析技术实时监测患者脑内谷氨酸、乳酸以及葡萄糖的含量变化,能很好的把握患者的病情,有效指导临床治疗。     

Influence of Perfusate Glucose Concentration on Dialysate Lactate, Pyruvate, Aspartate, and Glutamate Levels Under Basal and Hypoxic Conditions: A Microdialysis Study in Rat Brain

Abstract: The aim of this study was to evaluate the influence of perfusion media with different glucose concentrations on dialysate levels of lactate, pyruvate, aspartate (Asp), and glutamate (Glu) under basal and hypoxic conditions in rat brain neocortex. Intracerebral microdialysis was performed with the rat under general anesthesia using bilateral probes (o.d. 0.3 mm; membrane length, 2 mm) perfused with artificial CSF containing 0.0 and 3.0 m M glucose, respectively. Basal dialysate levels were obtained 2 h after probe implantation in artificially ventilated animals. Dialysate levels of glucose were also measured for the two different perfusion fluids. The mean absolute extracellular concentration of glucose was estimated by a modification of the no-net-flux method to be 3.3 mmol/L, corresponding to an average in vivo recovery of 6% for glucose. Hypoxia was induced by lowering the inspired oxygen concentration to 3%. Hypoxia caused a disturbance of cortical electrical activity, evidenced by slower frequency and lower amplitudes on the electroencephalogram compared with prehypoxic conditions. This was associated with significant elevations of lactate, Asp, and Glu levels. There were no statistically significant differences in dialysate metabolite levels between the two perfusion fluids, during either normal or hypoxic conditions. We conclude that microdialysis with glucose-free perfusion fluid does not drain brain extracellular glucose in anesthetized rats to the extent that the dialysate lactate, pyruvate, Asp, and Glu levels during basal or hypoxic conditions are altered.     

Brain distribution of carboxy terminus of Hsc70-interacting protein (CHIP) and its nuclear translocation in cultured cortical neurons following heat stress or oxygen–glucose deprivation

Carboxy terminus of Hsc70-interacting protein (CHIP) is thought to be a cytoprotective protein with protein quality control roles in neurodegenerative diseases and myocardial ischemia. This study describes the localization of CHIP expression in normal rodent brain and the early CHIP response in primary cultures of cortical neurons following ischemic stress models: heat stress (HS) and oxygen–glucose deprivation (OGD). CHIP was highly expressed throughout the brain, predominantly in neurons. The staining pattern was primarily cytoplasmic, although small amounts were seen in the nucleus. More intense nuclear staining was observed in primary cultured neurons which increased with stress. Nuclear accumulation of CHIP occurred within 5–10 min of HS and decreased to baseline levels or lower by 30–60 min. Decrease in nuclear CHIP at 30–60 min of HS was associated with a sharp increase in delayed cell death. While no changes in cytoplasmic CHIP were observed immediately following OGD, nuclear levels of CHIP increased slightly in response to OGD durations of 30 to 240 min. OGD-induced increases in nuclear CHIP decreased slowly during post-ischemic recovery. Nuclear CHIP decreased earlier in recovery following 120 min of OGD (4 h) than 30 min of OGD (12 h). Significant cell death first appeared between 12 and 24 h after OGD, again suggesting that delayed cell death follows closely behind the disappearance of nuclear CHIP. The ability of CHIP to translocate to and accumulate in the nucleus may be a limiting variable that determines how effectively cells respond to external stressors to facilitate cell survival. Using primary neuronal cell cultures, we were able to demonstrate rapid translocation of CHIP to the nucleus within minutes of heat stress and oxygen–glucose deprivation. An inverse relationship between nuclear CHIP and delayed cell death at 24 h suggests that the decrease in nuclear CHIP following extreme stress is linked to delayed cell death. Our findings of acute changes in subcellular localization of CHIP in response to cellular stress suggest that cellular changes that occur shortly after exposure to stress ultimately impact on the capacity and capability of a cell to recover and survive.     

<Emphasis Type="Italic">Ginkgo biloba</Emphasis> extract (EGb761) and FK506 preserve energy metabolites in the striatum during focal cerebral ischemia and reperfusion in gerbils monitored by microdialysis

Cell death after cerebral ischemia is mediated by the accumulation of excitatory amino acids, calcium influx into cells and the generation of free radicals. The aim of this study was to evaluate changes in energy-related metabolites in the striatum of gerbils subjected to focal cerebral ischemia after pretreatment withGinkgo biloba extract (EGb761), a well-known antioxidant, and FK506, a calcium-dependent phosphatase calcineurin inhibitor. Ischemia was induced by occlusion of the right common carotid artery and the right middle cerebral artery for 60 min. A microdialysis probe was inserted into the right striatum to monitor extracellular glucose, lactate and pyruvate levels. This study showed decreases in glucose (10% of the baseline), pyruvate (20% of the baseline) and lactate (60% of the baseline), and a 5-fold increase in the lactate to pyruvate ratio during ischemia in the control group. Both EGb761 treatment and the combination (EGb761 and FK506) therapy significantly preserved glucose (50% of the baseline) and pyruvate (60% of the baseline) levels during ischemia. The marked increase in the lactate to pyruvate ratio was not observed in the combination group. These results suggest that preservation of cellular energy metabolism during cerebral ischemia and after restoration with reperfusion may contribute to the neuroprotective effects of EGb761 and FK506.     

On‐line glucose and lactate monitoring in rat striatum: effect of malonate and correlation with histological damage

Microdialysis of glucose, lactate and glycerol was performed to monitor brain insults and to predict brain injury in a rat model using the mitochondrial toxin malonate (5–100 mm ). Striatal dialysates were analyzed off‐line using a CMA 600 microdialysis analyzer or on‐line using flow‐injection analysis and biosensors for glucose and lactate. Histological damage was evaluated using stereological principles. Lactate (baseline ca. 1 mm ) was dose‐dependently increased, reaching a maximum of five‐ to six‐fold increase, whereas glucose (baseline 1–2 mm ) was decreased (>50%) by malonate >20 mm . These changes were reversible upon perfusion with normal Ringer's. Transient increases in glycerol (four‐ to eight‐fold) were only observed in some rats, and were not dose‐dependent. Histological damage was related to the perfused malonate concentration, but was not significantly correlated with lactate or glycerol changes.     

Administration of Thimerosal to Infant Rats Increases Overflow of Glutamate and Aspartate in the Prefrontal Cortex: Protective Role of Dehydroepiandrosterone Sulfate

Thimerosal, a mercury-containing vaccine preservative, is a suspected factor in the etiology of neurodevelopmental disorders. We previously showed that its administration to infant rats causes behavioral, neurochemical and neuropathological abnormalities similar to those present in autism. Here we examined, using microdialysis, the effect of thimerosal on extracellular levels of neuroactive amino acids in the rat prefrontal cortex (PFC). Thimerosal administration (4 injections, i.m., 240 μg Hg/kg on postnatal days 7, 9, 11, 15) induced lasting changes in amino acid overflow: an increase of glutamate and aspartate accompanied by a decrease of glycine and alanine; measured 10–14 weeks after the injections. Four injections of thimerosal at a dose of 12.5 μg Hg/kg did not alter glutamate and aspartate concentrations at microdialysis time (but based on thimerosal pharmacokinetics, could have been effective soon after its injection). Application of thimerosal to the PFC in perfusion fluid evoked a rapid increase of glutamate overflow. Coadministration of the neurosteroid, dehydroepiandrosterone sulfate (DHEAS; 80 mg/kg; i.p.) prevented the thimerosal effect on glutamate and aspartate; the steroid alone had no influence on these amino acids. Coapplication of DHEAS with thimerosal in perfusion fluid also blocked the acute action of thimerosal on glutamate. In contrast, DHEAS alone reduced overflow of glycine and alanine, somewhat potentiating the thimerosal effect on these amino acids. Since excessive accumulation of extracellular glutamate is linked with excitotoxicity, our data imply that neonatal exposure to thimerosal-containing vaccines might induce excitotoxic brain injuries, leading to neurodevelopmental disorders. DHEAS may partially protect against mercurials-induced neurotoxicity.     

Ischemic injury of the liver in a porcine model of cardiac death assessed by in vivo microdialysis

This study aims to evaluate the ischemic injury of the liver in a porcine model of cardiac death assessed by in vivo microdialysis. A porcine model of cardiac death was established by the suffocation method. Metabolic indicators were monitored using the microdialysis technique during warm ischemia time (WIT) and cold ischemia time (CIT). Pathological changes in ischemic-injured livers were observed by haematoxylin–eosin staining. The predictive values of biochemical parameters regarding the liver donor were evaluated by receiver operating characteristic curve analysis. All statistical analyses were conducted using the SPSS 18.0 software (SPSS Inc, Chicago, Illinois, USA). The degree of warm ischemic injury of the livers increased with prolonged WIT. Serum glucose, glycerol, pyruvate, lactic acid levels and lactate-to-pyruvate (L/P) ratio increased gradually during WIT. Results from Pearson correlation analyses indicated that serum lactate level and L/P ratio were positively associated with the degree of warm ischemic injury of the livers. The degree of cold ischemic injury of the livers gradually increased after 12 h CIT. Serum glucose, lactic acid and L/P ratio achieved a peak after 6–8 h of CIT, but gradually decreased with prolonged CIT. The peak of glycerol occurred after 8 h of CIT, while no changes were found with prolonged CIT. Serum pyruvate level exhibited an increasing trend after 12 h CIT. Our results confirmed that serum glucose and lactate levels were negatively correlated with cold ischemic injury of the liver. However, serum glycerol and pyruvate levels showed positive correlations with cold ischemic injury of the liver. The liver donor was unavailable after 30 min WIT and 24 h CIT. The cut-off value of serum lactate level for warm ischemic injury of the livers was 2.374 with a sensitivity (Sen) of 90 % and specificity (Spe) of 95 %; while the L/P radio was 0.026 (Sen = 80 %, Spe = 83 %). In addition, the cut-off values of serum glucose, lactate, glycerol and pyruvate levels for cold ischemic injury of the livers were 0.339 (Sen = 100 %, Spe = 77 %), 1.172 (Sen = 100 %, Spe = 61 %), 56.359 (Sen = 100 %, Spe = 65 %) and 0.020 (Sen = 100 %, Spe = 67 %), respectively. Our findings provide empirical evidences that serum glucose, lactate levels and L/P ratio may be good indicators for the degree of warm ischemic injury of the livers after cardiac death; while serum glucose, lactate, glycerol and pyruvate levels may be important in predicting cold ischemic injury.     

Effects of Retigabine on the Neurodegeneration and Extracellular Glutamate Changes Induced by 4-Aminopyridine in Rat Hippocampus <Emphasis Type="Italic">In Vivo</Emphasis>

We have previously shown that microdialysis perfusion of the K⁺ channel blocker 4-aminopyridine (4-AP) in rat hippocampus induces convulsions and neurodegeneration, due to the stimulation of glutamate release from synaptic terminals. Retigabine is an opener of the KCNQ2/Q3-type K⁺ channel that possesses antiepileptic action and may be neuroprotective, and we have therefore studied its effect on the hyperexcitation, the neuronal damage and the changes in extracellular glutamate induced by 4-AP. Retigabine and 4-AP were co-administered by microdialysis in the hippocampus of anesthetized rats, with simultaneous recording of the EEG, and the extracellular concentration of glutamate was measured in the microdialysis fractions. In 70–80% of the rats tested retigabine reduced the 4-AP-induced stimulation of glutamate release and prevented the neuronal damage observed at 24 h in the CA1 hippocampal region. However, retigabine did not block the EEG epileptic discharges and their duration was reduced in only 20–25% of the tested animals. We conclude that the neuroprotective action of retigabine is probably due to the blockade of the 4-AP-induced stimulation of glutamate release. This inhibition, however, was not sufficient to block the epileptic activity. Special issue dedicated to Dr. Simo S. Oja     

Food Deprivation Protects the Rat Striatum Against Hypoxia-Ischemia Despite High Extracellular Glutamate

Abstract: In a model that combines hypoxia with ischemia, the relationship between histological outcome, evoked rise in blood glucose, and striatal glutamate release was investigated in the 24-h food-deprived and normally fed rat. Food deprivation protected the dorsolateral striatum very effectively, as was shown with a silver stain. An on- line monitoring technique based on microdialysis showed that, in the protected condition, more glutamate was re- leased into the striatal extracellular space than in the com- promised condition. The possibility that the microdialysis results were influenced by a difference in shrinking of the extracellular space following food deprivation was ex- cluded by the measurements of whole-tissue impedance. During the hypoxic-ischemic challenge, blood glucose rose in normally fed rats, but was suppressed almost com- pletely after food deprivation. These results led us to con- clude that, in our model of hypoxia-ischemia, the amount of glutamate released is not related directly to the extent of brain damage, but the increase in blood glucose may determine at least part of the brain damage.     

Allopregnanolone Potentiates the Glutamate-Mediated Seizures Induced by 4-Aminopyridine in Rat Hippocampus in vivo

Excitatory and inhibitory neurotransmission in the central nervous system can be modulated by neurosteroids. We previously found that in rat hippocampal slices allopregnanolone (3α-hydroxy-5α-pregnan-20-one), a positive GABA_A receptor modulator, suppresses the epileptic discharges induced by 4-aminopyridine (4-AP), a convulsant K⁺ channel blocker that stimulates glutamate release. Here, we tested the action of allopregnanolone on the epileptogenic and excitotoxic effects of the intrahippocampal administration of 4-AP in vivo. Drugs were perfused by a microdialysis cannula-electrode in the dorsal hippocampus and the EEG was recorded. Extracellular levels of aspartate, glutamate and GABA were analyzed by HPLC in the microdialysis fractions, and 24 h after the experiment the hippocampus was studied histologically. 4-AP induced intense epileptic discharges, increased the extracellular levels of aspartate, glutamate, and GABA by 383, 420, and 245%, respectively, and produced a notable neurodegeneration in CA1 and CA3 areas. Allopregnanolone administration alone did not affect the electrical activity, amino acids levels or cellular morphology, but when co-infused with 4-AP incremented 55–77% the duration of the epileptic discharges, and potentiated 32–49% the release of glutamate in comparison with 4-AP alone. The 4-AP-induced neurodegeneration was not modified by allopregnanolone. The NMDA receptor antagonist MK-801 protected against the epilepsy and neurodegeneration produced by 4-AP, and allopregnanolone did not affect this protection. We conclude that, differently from the observations in vitro, allopregnanolone potentiated the stimulatory effect of 4-AP on glutamate release and that this may explain the potentiation of the epileptogenic effect of 4-AP in vivo.     

Topical glucose and accumulation of excitotoxic and other amino acids in ischemic cerebral cortex.

Pre-ischemic hyperglycemia aggravates brain damage due to transient global ischemia as demonstrated by exacerbation of brain lesions. Lactacidosis and elevated glutamate levels have been implicated as mechanisms of the increased damage. Our objective was to determine the effects of different levels of glucose (0, 66.5, 450 mg/dL) in cortical superfusates on the ischemia/reperfusion-evoked release of amino acids from the rat cerebral cortex. Physiologic levels of glucose significantly reduced the amount of aspartate, glutamate and gamma-aminobutyric acid and the supra-physiologic levels of glucose reduced the amount of aspartate and phosphoethanolamine released from the cortex during ischemia/reperfusion in comparison with no glucose. The decrease in glutamate release may be due to increased availability of glucose for glycolysis with the subsequent formation of ATP and lactate, which has been shown to act as an energy source for neurons. The decreased levels may also reflect the continued energy-dependent uptake of glutamate by glial cells.     

Effect of Ageing and Ischemia on Enzymatic Activities Linked to Krebs’ Cycle,Electron Transfer Chain,Glutamate and Aminoacids Metabolism of Free and Intrasynaptic Mitochondria of Cerebral Cortex

The effect of ageing and the relationships between the catalytic properties of enzymes linked to Krebs’ cycle, electron transfer chain, glutamate and aminoacid metabolism of cerebral cortex, a functional area very sensitive to both age and ischemia, were studied on mitochondria of adult and aged rats, after complete ischemia of 15 minutes duration. The maximum rate (V _max) of the following enzyme activities: citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs’ cycle; NADH-cytochrome c reductase as total (integrated activity of Complex I–III), rotenone sensitive (Complex I) and cytochrome oxidase (Complex IV) for electron transfer chain; glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases for glutamate metabolism were assayed in non-synaptic, perikaryal mitochondria and in two populations of intra-synaptic mitochondria, i.e., the light and heavy mitochondrial fraction. The results indicate that in normal, steady-state cerebral cortex, the value of the same enzyme activity markedly differs according (a) to the different populations of mitochondria, i.e., non-synaptic or intra-synaptic light and heavy, (b) and respect to ageing. After 15 min of complete ischemia, the enzyme activities of mitochondria located near the nucleus (perikaryal mitochondria) and in synaptic structures (intra-synaptic mitochondria) of the cerebral tissue were substantially modified by ischemia. Non-synaptic mitochondria seem to be more affected by ischemia in adult and particularly in aged animals than the intra-synaptic light and heavy mitochondria. The observed modifications in enzyme activities reflect the metabolic state of the tissue at each specific experimental condition, as shown by comparative evaluation with respect to the content of energy-linked metabolites and substrates. The derangements in enzyme activities due to ischemia is greater in aged than in adult animals and especially the non-synaptic and the intra-synaptic light mitochondria seems to be more affected in aged animals. These data allow the hypothesis that the observed modifications of catalytic activities in non-synaptic and intra-synaptic mitochondrial enzyme systems linked to energy metabolism, amino acids and glutamate metabolism are primary responsible for the physiopathological responses of cerebral tissue to complete cerebral ischemia for 15 min duration during ageing.     

HO-1 expression in primary cultured astroglial cells

Microdialysis of glucose, lactate and glycerol was performed to monitor brain insults and to predict brain injury in a rat model using the mitochondrial toxin malonate (5–100 m m ). Striatal dialysates were analyzed off-line using a CMA 600 microdialysis analyzer or on-line using flow-injection analysis and biosensors for glucose and lactate. Histological damage was evaluated using stereological principles. Lactate (baseline ca . 1 m m ) was dose-dependently increased, reaching a maximum of five- to six-fold increase, whereas glucose (baseline 1–2 m m ) was decreased (>50%) by malonate >20 m m . These changes were reversible upon perfusion with normal Ringer's. Transient increases in glycerol (four- to eight-fold) were only observed in some rats, and were not dose-dependent. Histological damage was related to the perfused malonate concentration, but was not significantly correlated with lactate or glycerol changes.     

Intra- and extra-cellular flux distributions of TCA and glyoxalate cycle and vancomycin production of <Emphasis Type="Italic">Amycolatopsis orientalis</Emphasis> grown in different glycerol concentration

The relationship between tricarboxylic acid (TCA) and glyoxalate cycle and the effect of their metabolites levels on the vancomycin production of Amycolatopsis orientalis were investigated in different concentration of glycerol (2.5–20 g/l). Intracellular glycerol levels increased with respect to increases in glycerol concentrations of the growth medium. Extracellular glycerol levels decreased slowly up to 24 h while uptake rates were increased during 36–48^th h for 10 and 15 g/l and during 36–60^th h at 20 g/l of glycerol. Intracellular citrate, α-ketoglutarate, fumarate levels increased up to 10 g/l glycerol concentration. However, intracellular succinate and malate levels were increased up to 15 g/l glycerol. Extracellular citrate, α-ketoglutarate, succinate and malate levels increased with respect to increases in glycerol concentration. The highest α-ketoglutarate dehydrogenase activity was determined at 15 g/l glycerol. Isocitrate lyase activity showed a positive correlation with the increases in glycerol concentration of the growth medium. Vancomycin production increased with the increases in glycerol concentration from 5 to 10 g/l. These results showed that A. orientalis grown in glycerol containing medium used glyoxalate shunt actively instead of TCA cycle which supports precursors of many amino acid which are effective on the antibiotic production.     

An in vivo microdialysis characterization of the transient changes in the interstitial dialysate concentration of metabolites and cytokines in human skeletal muscle in response to insertion of a microdialysis probe

Skeletal muscle has recently been described as an endocrine organ, capable of releasing cytokines and regulators of metabolism. Microdialysis of the interstitial space of skeletal muscle enables analysis of the release of such cytokines. The purpose of this study was to determine the transient changes in concentration of metabolites and cytokines in human skeletal muscle in a 7 h period following the insertion of a microdialysis probe. In total, sixteen microdialysis catheters were inserted into the vastus lateralis of male participants (age 26.2 ± 1.35 y, height 180.8 ± 3.89 cm, mass 83.9 ± 3.86 kg, BMI 25.7 ± 0.87 kg m⁻², body fat 26.1 ± 3.0%). Serial samples were analyzed by micro-enzymatic and multiplexed immunoassay. Muscle interstitial glucose and lactate levels remained stable throughout, amino acid concentrations stabilized after 2.5 h, however, insertion of a microdialysis catheter induced a 29-fold increase in peak IL-6 (p < 0.001) and 35-fold increase in peak IL-8 concentrations (p < 0.001) above basal levels 6 h post insertion. In contrast to stable amino acid, glucose and lactate concentrations after 2 h, commonly reported markers of tissue homeostasis in in vivo microdialysis, the multi-fold increase in IL-6 and IL-8 following insertion of a microdialysis catheter is indicative of a sustained disturbance of tissue homeostasis.