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So far, magnetic resonance angiography (MRA) of rodents has only been performed by using time-of-flight (TOF) MRI techniques. This is because applications of first-passage contrast agents as in humans are hampered by pronounced physiologic differences (blood volume and heart beat rate). Here we describe the use of low-dose Gd-DOTA to enhance the performance of TOF MRA in rat brain. While no improvement in contrast was achieved, the measuring time could be reduced by almost a factor of three. This decrease in total acquisition time has been used to study the impact of a model of ligatured common carotid on the upper part of the blood system of the rat.

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Glucose is commonly admitted to be the main substrate for brain energy requirement. However, it has been recently proposed that lactate, generated from glucose via glycolysis, would be the oxidative substrate for neurons, particularly during neuronal activation, according to a mechanism called the astrocyte-neuron lactate shuttle hypothesis (ANLSH). In that mechanism, glutamate released in the synaptic cleft during brain activation is taken up by astrocytes. This uptake, via the glutamate/Na(+) transporter, induces the entry of sodium, which is then excluded from the astrocytes via the Na(+)/K(+) ATPase. This exclusion consumes ATP, which stimulates glycolysis and thus lactate formation in astrocytes. This lactate is then transferred to neurons where it is utilized as oxidative substrate. This review tries to gather the recent evidences that support this hypothesis and presents the contribution of NMR to this matter.

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Spatial and temporal control of transgene expression is one of the major prerequisites of efficient gene therapy. Recently, a noninvasive, physical approach has been presented based on local heat in combination with a heat-sensitive promoter. This strategy requires tight temperature control in vivo. Here, we use MRI-guided focused ultrasound (MRI-FUS) with real-time feedback control on a whole-body clinical MRI system for a completely automatic execution of a predefined temperature-time trajectory in the focal point. Feasibility studies on expression control were carried out on subcutaneously implanted rat tumors. A stable modified C6 glioma cell line was used carrying a fused gene coding for thymidine kinase (TK) and green fluorescent protein (GFP) under control of the human heat-shock protein 70 (HSP70) promoter. In vitro studies showed strong induction of the TK-GFP gene expression upon heat shock under various conditions and localization of the protein product in the nucleus. In vivo tumors were subjected to a 3-min temperature elevation using MRI-FUS with a constant temperature, and were analysed 24 hr after the heat shock with respect to GFP fluorescence. Preliminary results showed strong local induction in regions heated above 40 degrees C, and a good correspondence between temperature maps at the end of the heating period and elevated expression of TK-GFP.

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BACKGROUND: Among the techniques used to induce and control gene expression, a non-invasive, physical approach based on local heat in combination with a heat-sensitive promoter represents a promising alternative but requires accurate temperature control in vivo. MRI-guided focused ultrasound (MRI-FUS) with real-time feedback control allows automatic execution of a predefined temperature-time trajectory. The purpose of this study was to demonstrate temporal and spatial control of transgene expression based on a well-defined local hyperthermia generated by MRI-FUS. METHODS: Expression of the green fluorescent protein (GFP) marker gene was used. Two cell lines were derived from C6 glioma cells. The GFP expression of the first one is under the control of the CMV promoter, whereas it is under the control of the HSP70 promoter in the second one and thus inducible by heat. Subcutaneous tumours were generated by injection in immuno-deficient mice and rats. Tumours were subjected to temperatures varying from 42 to 50 degrees C for 3 to 25 min controlled by MRI-FUS and analyzed 24 h after the heat-shock. Endogenous HSP70 expression and C6 cell distribution were also analyzed. RESULTS: The results demonstrate strong expression at 50 degrees C applied during a short time period (3 min) without affecting cell viability. Induced expression was also clearly shown for temperature in the range 44-48 degrees C but not at 42 degrees C. CONCLUSIONS: Heating with MRI-FUS allows a tight and non-invasive control of transgene expression in a tumour.

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Data on motor behavioural disorders induced by systemic 3-nitropropionic acid, an irreversible inhibitor of mitochondrial succinate dehydrogenase and their histopathological correlates in mice, are sparse. We thus further characterised the subacute 3-nitropropionic-acid-induced motor disorder and its time course in C57Bl/6 mice using standard behavioural tests, histopathological correlates and in vivo magnetic resonance imaging. Firstly, we studied two intoxication paradigms (340 and 560 mg 3-nitropropionic acid/kg, 7 days) compared to controls. The low-dose regimen induced only slight motor changes (reduced hindlimb stride length and rearing). The high-dose regimen induced significant (P<0.05) behavioural and sensorimotor integration deficits (pole test, rotarod, stride length, open-field spontaneous activity) but with 37.5% lethality at week one. The clinical motor disorder consisted of hindlimb clasping and dystonia, truncal dystonia, bradykinesia and impaired postural control. Histopathologically, there were discrete lesions of the dorsolateral striatum in 62.5% of mice together with a 32% reduction (P<0.0001) of the striatal volume, reduced caldbindin-D28K immunoreactivity in the lateral striatum, and met-enkephalin and substance P in the striatal output pathways. There was also a significant (P<0.05) 30-40% dopaminergic cell loss within the substantia nigra pars compacta. Secondly, we validated a semi-quantitative behavioural scale to describe the time course of the motor deficits and to predict the occurrence of striatal damage. We sought to determine whether it could also be disclosed in vivo by magnetic resonance imaging. The scale correlated with the striatal volume reduction (r(2)=0.57) and striatal cell loss (r(2)=0.87) but not with the loss of striatal dopaminergic terminals (dopamine transporter binding). Increased T2-signal intensity within the striatal lesion correlated with the cell loss (r(2)=0.66). We conclude that systemic administration of 3-nitropropionic acid in C57Bl/6 mice induces a distinct motor disorder and dose-dependent striatonigral damage, which are potentially useful to model human diseases of the basal ganglia.

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In this study, we examined the effect of creatine ingestion on muscle power output, muscle phosphocreatine resynthesis, inorganic phosphate and pH during repeated brief bouts of maximal exercise. Nine healthy males performed maximal plantar flexion before and after creatine ingestion (20 g x day(-1) for 6 days). The experimental protocol consisted of five 8 s bouts (bouts 1-5) interspersed with 30 s recovery, followed by bouts 6 (8 s) and 7 (16 s) separated by 1 and 2 min, respectively. Muscle phosphocreatine, inorganic phosphate and pH were estimated every 16 s by 31P magnetic resonance spectroscopy. After creatine ingestion, muscle power output increased by approximately 5% (P< 0.05) from bouts 3 to 7 and muscle phosphocreatine resynthesis increased (P< 0.05) during 10 min recovery. The higher phosphocreatine concentration observed after only 30 s of recovery was accompanied by lower inorganic phosphate accumulation and higher pH. Strong correlations were found between exercise power restoration and the corresponding pre-exercise phosphocreatine and inorganic phosphate concentrations and muscle pH after creatine ingestion. The better maintenance of muscle power output observed after creatine ingestion was attributed to a higher rate of phosphocreatine resynthesis, lower accumulation of inorganic phosphate and higher pH.

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Glioma-bearing rats were infused intravenously with a solution containing either [3-(13)C]lactate or both glucose and [3-(13)C]lactate for 20 min or 1 hr. Perchloric acid extracts of healthy and tumoral brain tissues were prepared and analyzed by (13)C- and (1)H-observed (13)C-edited nuclear magnetic resonance (NMR) spectroscopy to determine (13)C-label incorporation into brain tissue and glioma metabolites. Moreover, (13)C enrichments in blood lactate and glucose were determined from (1)H-NMR spectra. In the nontumoral tissue, (13)C labeling of amino acids indicated that [3-(13)C]lactate entered the brain and was metabolized. There was no labeling difference between the contralateral and the ipsilateral hemispheres. Lactate metabolism appeared more specifically neuronal, in agreement with our previous results obtained with normal rat brain (Bouzier et al. [2000] J. Neurochem. 75:480-486). In the glioma tissue, comparison of Ala C3, Glu C4, and Gln C4 labeling indicated that the contributions of blood glutamine and tricarboxylic acid (TCA) cycle to glutamate labeling were about 80% and 20%, respectively, after 1 hr of [3-(13)C]lactate infusion. In contrast, these contributions were about 10% and 90%, respectively, when [1-(13)C]glucose was infused in the absence of lactate. This indicated a major effect of the exogenous lactate on glioma metabolism, which may be due to the following process: The high blood lactate level might hinder the drain of glycolytic lactate produced inside the glioma and thus generate a change in redox potential such that the tumor cells are unable to restore it with oxidative phosphorylation. Thereafter, the high NADH level might inhibit glycolysis and the TCA cycle, and glutamine could become the major carbon source for glutamate labeling.

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This study was designed to test the effects of short-chain fatty acids (SCFA) with an even number of carbon atoms on hepatic energy metabolism. The effect of the SCFA was evaluated by measuring liver ATP content and oxygen consumption. The ATP content was evaluated using (31)P nuclear magnetic resonance in isolated liver from fed rats. In addition, respiratory activity (VO(2)) was assessed using Clark electrodes. The livers were perfused with acetate, butyrate or a medium chain length fatty acid, octanoate, at a concentration of 0.05--5.0 mmol/L. The addition of each substrate enhanced the rate of the net ATP consumption (V(i)), establishing a new ATP steady state that required a perfusion time of > or = 20 min, dependent on the chain length and concentration of the fatty acid (FA). The initial V(i) was unchanged for acetate and the ATP level stabilized at 58% of the initial level. Both butyrate and octanoate induced a dose-dependent increase in V(i). This may reflect an ATP-consuming process for the intracellular pH regulation observed during the acidosis associated with the beta-oxidation pathway. At the new steady state, the ATP concentration was approximately 45% of the initial level for both FA. VO(2) was both rapidly and reversibly increased, and the change was a function of butyrate or octanoate concentration and of the chain length. K(m) values were similar for butyrate and octanoate. Because all of the effects were similar for butyrate and octanoate, in contrast to acetate, we suggest that the impairment of the energy metabolism by butyrate resulted from an increase in the FADH(2)/NADH ratio due to beta-oxidation. In conclusion, the difference in the hepatic oxidation pathways of two products of intestinal fermentation (acetate and butyrate) explains their different actions on energy metabolism.

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BACKGROUND: Metabolic alterations in skeletal muscle associated with malnutrition and the potential reversibility of such alterations during refeeding are not fully understood. OBJECTIVE: We characterized early changes in muscle during refeeding in malnourished, hospitalized elderly subjects. DESIGN: Muscle function, metabolism, and mass were evaluated in 24 clinically stable patients (11 were malnourished) by using isokinetic plantar flexor torque measurements and nuclear magnetic resonance (NMR) imaging for medial gastrocnemius mass assessment and 31P and 13C NMR spectroscopy for inorganic phosphate (Pi), phosphocreatine, and glycogen quantitation. RESULTS: Malnourished subjects had lower muscle mass (P < 0.02) and tended to have lower strength than did control subjects. In malnourished subjects, muscle strength increased after refeeding (P < 0.01) whereas muscle mass was unchanged. The ratio of Pi to ATP was lower in malnourished than in control subjects (P < 0.001) and increased during refeeding (P < 0.01). The mean ratio of phosphocreatine to ATP was lower in malnourished than in control subjects (P < 0.01) and increased to control values after refeeding. Muscle glycogen showed a scattered distribution for malnourished subjects; the mean value did not differ significantly from that of control subjects, either at baseline or after refeeding. CONCLUSIONS: The lower ratio of phosphocreatine to ATP in malnourished subjects could have resulted from either lower total muscle creatine or reduced oxidative capacities. High or normal glycogen associated with a low Pi-to-ATP ratio in malnourished subjects suggested preferential use of lipid over carbohydrate for energy supply, which is known to reduce muscle performance. The data suggest that normalization of muscle metabolite content after refeeding improves muscle strength in malnourished subjects.

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The effects of hypoxia on the metabolism of the central nervous system were investigated in rats submitted to a low oxygen atmosphere (8% O(2); 92% N(2)). [1-(13)C]glucose and [2-(13)C]acetate were used as substrates, this latter being preferentially metabolized by glial cells. After 1-h substrate infusion, the incorporation of 13C in brain metabolites was determined by NMR spectroscopy. Under hypoxia, an important hyperglycemia was noted. As a consequence, when using labeled glucose, the specific enrichment of brain glucose C1 was lower (48.2+/-5.1%) than under normoxia (66.9+/-2.5%). However, relative to this specific enrichment, the (13)C incorporation in amino acids was increased under hypoxia. This suggested primarily a decreased exchange between blood and brain lactate. The glutamate C2/C4 enrichment ratio was higher under hypoxia (0.62+/-0.01) than normoxia (0.51+/-0.06), indicating a lower glutamate turnover relative to the neuronal TCA cycle activity. The glutamine C2/C4 enrichment ratio was also higher under hypoxia (0.87+/-0.07 instead of 0.65+/-0.11), indicating a new balance in the contributions of different carbon sources at the acetyl-CoA level. When using [2-(13)C]acetate as substrate, no difference in glutamine enrichment appeared under hypoxia, whereas a significant decrease in glutamate, aspartate, alanine and lactate enrichments was noted. This indicated a lower trafficking between astrocytes and neurons and a reduced tricarboxylic acid cycle intermediate recycling of pyruvate.

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The novel phosphorylated pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH) was evaluated as a (31)P NMR probe of the pH changes associated with ischemia/reperfusion of rat isolated hearts and livers. In vitro titration curves indicated that DEPMPH exhibited a 4-fold larger amplitude of chemical shift variation than inorganic phosphate yielding an enhanced NMR sensitivity in the pH range of 5.0-7.5 that allowed us to assess pH variations of less than 0.1 pH units. At the non-toxic concentration of 5 mm, DEPMPH distributed into external and cytosolic compartments in both normoxic organs, as assessed by the appearance of two resonance peaks. An additional peak was observed in normoxic and ischemic livers, assigned to DEPMPH in acidic vesicles (pH 5.3-5.6). During severe myocardial ischemia, a third peak corresponding to DEPMPH located in ventricular and atrial cavities appeared (pH 6.9). Mass spectrometry and NMR analyses of perchloric extracts showed that no significant metabolism of DEPMPH occurred in the ischemic liver. Reperfusion with plain buffer resulted in a rapid washout of DEPMPH from both organs. It was concluded that the highly pH-sensitive DEPMPH could be of great interest in noninvasive ex vivo studies of pH gradients that may be involved in many pathological processes.

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The aim of this work was to assess quantitatively and qualitatively the ability of magnetization transfer imaging to follow in vivo remyelination. Demyelination lesions were induced in rats by the injection of L-alpha-lysophosphatidylcholine stearoyl into the corpus callosum and imaging was performed in vivo on a 4.7-Tesla system at different time points. The percentage of magnetization transfer ratio (MTR) decrease was calculated for each animal. To evaluate the MTR findings for remyelination, myelin was quantitated by histological analysis of the lesion size and counting the number of remyelinating axons. An MTR decrease was observed when demyelination was present at 7 days after injection. During the remyelinating phase between day 30 and 40 after injection, contralateral values almost complete returned to normal, thus indicating remyelination. Histologically, at days 30 and 40 after injection, the lesion area was reduced in size and the axons were surrounded by a thin myelin sheath, indicating the remyelination process. Statistical analysis showed that the profile of MTR values was significantly correlated with the course of remyelination. All the MTR changes show a correlation with both myelin damage and repair. In conclusion, the study of the MTR profile in this myelin lesion model demonstrates in vivo the loss of myelin and the presence of spontaneous remyelination. This methodological approach which can also be applied to multiple sclerosis patients to show demyelination, should prove helpful to determine the degree of spontaneous and therapeutically induced remyelination in multiple sclerosis lesions, and thus to validate therapeutic treatments for myelin repair.

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The intracellular mechanisms of regulation of energy fluxes and respiration in contracting heart cells were studied. For this, we investigated the workload dependencies of the rate of oxygen consumption and metabolic parameters in Langendorff-perfused isolated rat hearts.(31)P NMR spectroscopy was used to study the metabolic changes during transition from perfusion with glucose to that with pyruvate with and without active creatine kinase system. The experimental results showed that transition from perfusion with glucose to that with pyruvate increased the phosphocreatine content and stability of its level at increased workloads. Inhibition of creatine kinase reaction by 15-min infusion of iodoacetamide decreased the maximal developed tension and respiration rates by a factor of two.(31)P NMR data were analyzed by a mathematical model of compartmentalized energy transfer, which is independent from the restrictions of the classical concept of creatine kinase equilibrium. The analysis of experimental data by this model shows that metabolic stability-constant levels of phosphocreatine, ATP and inorganic phosphate-at increased energy fluxes is an inherent property of the compartmentalized system. This explains the observed substrate specificity by changes in mitochondrial membrane potential. The decreased maximal respiration rate and maximal work output of the heart with inhibited creatine kinase is well explained by the rise in myoplasmic ADP concentration. This activates the adenylate kinase reaction in the myofibrillar space and in the mitochondria to fulfil the energy transfer and signal transmission functions, usually performed by creatine kinase. The activity of this system, however, is not sufficient to maintain high enough energy fluxes. Therefore, there is a kinetic explanation for the decreased maximal respiration rate of the heart with inhibited creatine kinase: i.e. a kinetically induced switch from an efficient energy transfer pathway (PCr-CK system) to a non-efficient one (myokinase pathway) within the energy transfer network of the cell under conditions of low apparent affinity of mitochondria to ADP in vivo. This may result in a significant decrease in the thermodynamic affinity of compartmentalized ATPase systems and finally in heart failure.

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A method for in-plane imaging of large objects as compared to the RF coil is proposed based on the use of a single specially designed surface coil, without using B(0) gradients. A constant B(1) gradient was generated along the main axis of a ladder-shaped coil, and RF-encoding along the direction of the gradient made it possible to obtain spin-density profiles. Successive acquisitions of profiles obtained by translation of the NMR coil resulted in distorted images-due to the presence of non-zero gradients perpendicular to the constant gradient-that were successfully processed using a mathematical treatment based on linear combinations of calculated altered images from single-pixel objects. Copyright 2000 Academic Press.

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Aminomethylphosphonate (NMePo) and 2-aminoethylphosphonate (NEthPo) were evaluated as alternative pH indicators in the isolated perfused rat liver using (31)P-nuclear magnetic resonance (NMR). NMePo did not distribute within cells and remained in the extracellular space. It exhibited pH titration with a low pK(a) value (5.35). This behaviour makes NMePo useful for extracellular volume or acidic pH determination. In contrast, NEthPo accumulated within cells without altering liver energetic steady state, evaluated from nucleosides triphosphates resonances, even for prolonged (100 min) experiments. Withdrawal of NEthPo from perfusate revealed a residual resonance corresponding to the internalized amount of this phosphonate. This fraction was almost stable vs time and allowed determination of spin-lattice relaxation time constant T(1) within the liver (2.2 +/- 0.3 s; n = 6). Comparison of the titration curves for NEthPo and inorganic phosphate revealed that the accuracy of pH determination within physiologic or acidic range in both cases was comparable. Finally, when extracellular pH was decreased, the NEthPo resonance frequency was found to undergo the same chemical shift variations as observed for cytosolic P(i) signal, which was in good agreement with a cytosolic accumulation of this phosphonate. Therefore, NEthPo could be considered as an interesting cytosolic pH probe suitable for (31)P-NMR measurements, especially when experimental conditions prevent reliable observation of cytosolic Pi resonance.

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Lactate metabolism in the adult rat brain was investigated in relation with the concept of lactate trafficking between astrocytes and neurons. Wistar rats were infused intravenously with a solution containing either [3-(13)C]lactate (534 mM) or both glucose (750 mM) and [3-(13)C]lactate (534 mM). The time courses of both the concentration and (13)C enrichment of blood glucose and lactate were determined. The data indicated the occurrence of [3-(13)C]lactate recycling through liver gluconeogenesis. The yield of glucose labeling was, however, reduced when using the glucose-containing infusate. After a 20-min or 1-h infusion, perchloric acid extracts of the brain tissue were prepared and subsequently analyzed by (13)C- and (1)H-observed/(13)C-edited NMR spectroscopy. The (13)C labeling of amino acids indicated that [3-(13)C]lactate was metabolized in the brain. Based on the alanine C3 enrichment, lactate contribution to brain metabolism amounted to 35% under the most favorable conditions used. By contrast with what happens with [1-(13)C]glucose metabolism, no difference in glutamine C2 and C3 labeling was evidenced, indicating that lactate was metabolized in a compartment deprived of pyruvate carboxylase activity. This result confirms, for the first time from an in vivo study, that lactate is more specifically a neuronal substrate.

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BACKGROUND: Local production of therapeutic proteins, e.g. for cancer treatments, is based on gene therapy approaches and requires tight spatial and temporal control of gene expression. Here we demonstrate the use of local hyperthermia of varying intensity and duration to control the expression of a transgene under control of the thermoinducible hsp70 (heat shock protein) promoter. METHODS: Heat-induced expression of the EGFP (green fluorescent protein) reporter gene was characterized using a stably transfected glioma C6 cell line expressing the EGFP gene under control of the heat inducible minimal hsp70 promoter both in vitro and in vivo for subcutaneous tumors in immunodeficient mice. RESULTS: A heat shock of 20-30 min at 43 degrees C in cell culture led to a maximum EGFP concentration at about 24 h. Heat treatments at higher temperature (up to 48 degrees C) but with shorter durations (down to 30 s) also induced strong EGFP expression. Local heating in situ led to gradients in EGFP expression which decreased with increasing distance from the heat source. CONCLUSION: Local hyperthermia, in combination with a heat sensitive promoter, represents a method for the spatial and temporal control of transgene expression.

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The effects of fatty acids (FA)-carrier, egg-lecithin liposomes (LIPO) as alternative to BSA, on ATP, glycogen and glucose contents in isolated perfused liver of fed rats were non-invasively studied using 31P/13C nuclear magnetic resonance (NMR). Oxidative phosphorylation was studied in isolated mitochondria from the same liver consecutively to the NMR experiments. ATP content decreased slowly and ATP turnover was similar during the perfusion with saline solution (KHB) or LIPO. However, LIPO induced an enhancement of respiratory control ratio in isolated mitochondria. Tissue glycogen and glucose content decreased when FA (linoleate or linolenate) were perfused with defatted BSA (3%) or LIPO (600 mg/l) whereas glucose excretion level was unchanged and lactate excretion tended to increase, reflecting changes in the cytosolic redox state and/or an enhancement of glycolysis. Addition of FA (0.5 or 1.5 mM) to LIPO caused a dramatic fall in liver ATP, a mitochondrial uncoupling and an impairment of the phosphorylation activity. Perfusion with FA (1.5 mM) carried by BSA significantly increased the ATP degradation without change of mitochondrial function. Owing to the higher affinity of BSA than LIPO for FA, these latter could be more easily released from complex LIPO-FA, increasing their uncoupling effect. Hence, the FA concentrations have to be largely decreased from the above currently used concentrations to avoid this effect. It will then be possible to minimize the effector action of FA and to study their more specific metabolic function as fuel. It was concluded that LIPO were appropriate carriers to study the different metabolic effects of FA.

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The application of a heat shock on the human microglial cell line (CHME 5) has been shown to cause cytoskeleton modifications and alterations in phosphorylated metabolite content (Macouillard-Poulletier de Gannes et al., 1998a Metabolic and cellular characterization of immortalized human microglial cells under heat stress. Neurochem. Int. 33, 61-73). In this study, we focused on the possible involvement of mitochondria in this heat stress response. The cell respiratory properties were followed during the recovering period and the possible relationships between mitochondria and the cytoskeleton were studied. We observed that the heat shock induced changes in mitochondrial activity due to protein denaturation, rather than mitochondrial loss. Furthermore, these alterations were correlated with cytoskeleton disorganization since vimentine, tubuline and mitochondria shift, simultaneously, to a perinuclear location. The perturbations of the mitochondrial distribution persisted until cytoskeleton networks had recovered. Nevertheless, the respiratory properties recovered rapidly suggesting a renaturation of mitochondrial proteins in connection with mitochondrial cytoplasmic redistribution.

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