Biomedical subjects

M G Boutelle

Publications and source records attributed to M G Boutelle.

At least 19 recordsLinked to original sources

PubMed2000

Development and comparison of biosensors for in-vivo applications.

Electrochemical biosensors have been of increasing interest, especially those developed to be directly applied in diagnostic areas, such as neuroscience. We have been interested in developing a range of biosensors for monitoring glucose, lactate, pyruvate, and glutamate in order to study on-line both brain function in the laboratory and to monitor brain health in neurointensive care. For a biosensor to function effectively in these situations, it has to combine the following characteristics: quick response and high sensitivity, good reproducibility and adequate stability. In this study we compared the performance of a number of different amperometric biosensors strategies. These included ferrocene mediation of immobilised enzymes (system A), a redox hydrogel based system (system B), and a conducting polymer approach using polyaniline (system C). All assays were operated as flow-injection systems with upstream immobilised enzyme beds if necessary. When calibrated for H2O2 systems A and B reacted quickly enough to give quantitative conversion up to 0.2 mM. Above this concentration the response was limited by horseradish peroxidase enzyme kinetics and eventually enzyme loading. System C showed a restricted H2O2 response. When calibrated for glucose (by use of immobilised glucose oxidase) system B exhibited the highest sensitivity but its analytical range was restricted because the system became limited by H2O2 response. System A had low sensitivity for analyte compared to H2O2 and system B, but a greater useful range. Problems of mediator cycling between the immobilised enzymes are discussed. System C gave an excellent linear range but sensitivity was limited by background noise. Stability and reproducibility of the systems are also described. In conclusion, from this study the ferrocene system proved to be overall most useful and has now been used in the first dual on-line monitoring of glucose and lactate in patients in neurointensive care.

Biosensing Techniques↗

PubMed2000

Dynamic changes in glucose and lactate in the cortex of the freely moving rat monitored using microdialysis.

These experiments for the first time examine simultaneous changes in glucose and lactate in unanaesthetised animals during moderate hypoxia. Unanaesthetised rats were exposed to moderate hypoxia for a period of 15 min by reducing inspired oxygen to 8%. Changes in glucose and lactate were monitored in rat cortex using microdialysis and a novel dual enzyme-based assay. Samples of dialysate collected at 3-min intervals were assayed for both glucose and lactate. There was an early rapid rise of lactate that reached a peak at the end of the period of hypoxia followed by a steep decline. Glucose showed a very much smaller delayed increase that started during the period of hypoxia and continued beyond it. The origin of the rise in glucose is discussed, using the temporal relationship between the lactate and glucose changes.

Adrenergic beta-Antagonists↗

PubMed1999

The role of astrocytes and noradrenaline in neuronal glucose metabolism.

In the classical model the energy requirements during neuronal activation are provided by the delivery of additional glucose directly into the extracellular compartment that results from the increase in local cerebral blood flow (rCBF). The present review proposes that astrocytes play a key role in the response to neuronal activation. Arginine for the synthesis of NO, which has a major role in the increase in rCBF, is released from astrocytes in response to stimulation of astrocytic glutamate receptors. The increased delivery of glucose by the blood stream enters astrocytes, where some of it is converted to glycogen. During neuronal activation there is a decrease in extracellular glucose owing to increased utilization followed by a delayed increase; this results from stimulation of astrocytic beta-adrenergic receptors, which leads to a breakdown of glycogen and the export of glucose.

Animals↗

PubMed1998

An amperometric glucose-oxidase/poly(o-phenylenediamine) biosensor for monitoring brain extracellular glucose: in vivo characterisation in the striatum of freely-moving rats.

Amperometric glucose biosensors based on the immobilization of glucose oxidase (GOx) on Pt electrodes with electropolymerized o-phenylenediamine (PPD) were implanted in the right striatum of freely-moving rats. Carbon paste electrodes for the simultaneous monitoring of ascorbic acid (AA) and/or tissue O2 were implanted in the left striatum. A detailed in vivo characterization of the Pt/PPD/GOx signal was carried out using various pharmacological manipulations. Confirmation that the biosensor responded to changing glucose levels in brain extracellular fluid (ECF) was obtained by intraperitoneal (i.p.) injection of insulin that caused a decrease in the Pt/PPD/GOx current, and local administration of glucose (1 mM) via an adjacent microdialysis probe that resulted in an increase in the biosensor current. An insulin induced increase in tissue O2 in the brain was also observed. Interference studies involved administering AA and subanaesthetic doses of ketamine i.p. Both resulted in increased extracellular AA levels with ketamine also causing an increase in O2. No significant change in the Pt/PPD/GOx current was observed in either case indicating that changes in O2 and AA, the principal endogenous interferents, have minimal effect on the response of these first generation biosensors. Stability tests over a successive 5-day period revealed no significant change in sensitivity. These in vivo results suggest reliable glucose monitoring in brain ECF.

Animals↗

PubMed1998

Continuous monitoring of extracellular glucose concentrations in the striatum of freely moving rats with an implanted glucose biosensor.

We have used a glucose oxidase-based sensor implanted in the striatum of freely moving rats to determine the concentration of extracellular glucose in two distinct ways. With a modification of the zero net flux method, in which different concentrations of glucose are infused through a dialysis probe glued to the biosensor, we calculated the concentration at which there was no change in glucose current by regression analysis; this gave a concentration of 0.351 +/- 0.016 mM. Calculating the concentration from the basal current and the in vitro calibration of the biosensor was not significantly different from this. The basal extracellular glucose concentration determined by either method remained constant over a period of several days. Infusion of 50 microM veratridine through the adjacent dialysis probe caused a steep decrease in glucose current as soon as the drug reached the brain in contrast to the delayed fall (7.5 min) seen with microdialysis in previous experiments from this laboratory. These results demonstrate that this biosensor provides a direct, real-time measure of the extracellular concentration of glucose.

Animals↗

PubMed1997

Measurement of brain tissue oxygen at a carbon past electrode can serve as an index of increases in regional cerebral blood flow.

Simultaneous monitoring of tissue O(2) and regional cerebral blood flow (rCBF) was performed in the striatum of freely-moving rats. Differential pulse amperometry and constant potential amperometry were used to monitor O(2) levels at a carbon paste electrode (CPE), while rCBF values were obtained using the H2 clearance technique. Two forms of behavioural activation were studied and the resultant changes in tissue O(2) and blood flow compared. Both tail pinch and induced grooming produced immediate and parallel increases in O(2) and blood flow which returned to baseline on cessation of activity. These findings indicate that under conditions of physiological stimulation the direct voltammetric measurement of O(2) in brain tissue with a CPE can be used as a reliable index of increases in rCBF, resulting in an improvement in time resolution from 5 min (H2 clearance) to <1 s (amperometry). Because tissue O(2) is a balance between supply by the blood stream and utilisation by the cells, increases in O(2) current are an index of increased blood flow only when supply significantly exceeds utilisation.

Animals↗

PubMed1996

The source of physiologically stimulated glutamate efflux from the striatum of conscious rats.

1. Glutamate in the extracellular compartment of the striatum of freely moving rats was monitored at 5 min intervals using microdialysis and an enzyme-based assay. 2. Basal levels of dialysate glutamate were 3.6 +/- 0.5 microM. Local infusion through the dialysis probe of tetrodotoxin (TTX), cadmium chloride or magnesium chloride produced no reduction in basal levels of glutamate; with the latter two there was, instead, an increase. 3. Neuronal activation stimulated by induced grooming was accompanied by an increase in total glutamate efflux of 47.5 +/- 25.0% above basal level; this increase was not reduced by local infusion of TTX. 4. We propose that the TTX-insensitive release of glutamate in response to physiological stimulation is derived from glial cells and is a Ca(2+)-dependent mechanism triggered by a receptor-mediated release of Ca2+ from internal stores that spreads through the network of astrocytes.

Animals↗

PubMed1996

The mechanisms controlling physiologically stimulated changes in rat brain glucose and lactate: a microdialysis study.

1. This study is concerned with the supply of metabolic substrates for neuronal metabolism. Experiments were carried out to investigate whether mechanisms demonstrated in cultured astrocytes also occurred in vivo; these were cAMP-mediated breakdown of glycogen and glutamate uptake-stimulated release of lactate. 2. In vivo microdialysis was used in freely moving rats. Lactate and glucose in the dialysate were assayed using enzyme-based on-line assays. Drugs were given locally through the dialysis probe. Regional cerebral blood flow was measured using the hydrogen clearance method. 3. There was an increase in dialysate glucose in response to the beta-adrenoceptor agonist isoprenaline and to 8-bromo-cAMP, an analogue of cAMP, the second messenger of beta-adrenoceptor stimulation. The effect of isoprenaline was blocked by the antagonist propranolol. Isoprenaline had no effect on dialysate lactate, which was increased by the glutamate uptake blocker beta-D,L-threohydroxyaspartate (THA). 4. Physiological stimulation of neuronal activity produced an increase in both lactate and glucose. The increase in lactate was depressed in the presence of THA but was unaffected by propranolol. The increase in glucose was blocked by propranolol. Regional cerebral blood flow was increased by physiological stimulation but was unaffected by propranolol. 5. These results demonstrate that physiologically stimulated increases in glucose and lactate in the brain are mediated by different mechanisms.

Adrenergic beta-Agonists↗

PubMed1996

The determination of the extracellular concentration of brain glutamate using quantitative microdialysis.

Quantitative microdialysis with two enzyme-based assays was used to determine the extracellular concentration of glutamate in the striatum of freely moving rats. From the difference between infused and dialysate glutamate a value of 3.0 +/- 0.6 microM for the extracellular glutamate concentration was computed by regression analysis. The in vivo recovery, derived from the slope of the regression line, was 50%.

Animals↗

PubMed1996

Clinical microdialysis: the role of on-line measurement and quantitative microdialysis.

The use of microdialysis in the clinic is examined in the light of lessons learnt from microdialysis in freely moving rats. Changes in concentrations of metabolites are an important index of the state of health of tissues. For effective therapeutic intervention rapid assays are essential Enzyme-based on-line assays for glucose and lactate are described. By combining two of these assays simultaneous measurements of glucose and lactate, sampled at 2 min intervals can be obtained. The relation between dialysate concentrations and the true extracellular concentration of an analyte is dependent on conditions in the tissue sampled and cannot be calculated from in vitro probe recoveries. Furthermore, with acute implantation of the probe and possibly rapidly changing tissue conditions, there will be changes in probe recovery in vivo. Quantitative microdialysis allows the measurement of the true extracellular concentration and the probe recovery in vivo. The clinical applicability of a number of quantitative microdialysis methods is discussed, and three approaches highlighted. By increasing membrane length and reducing flow rate recovery in vivo can be increased to 100%. In this case dialysate concentrations equal extracellular ones. By perfusing an inert exogenous compound an index of changes to extracellular volume and hence tissue oedema can be obtained. In the zero net flux method the infusion of a few concentrations of the analyte under study allows the direct determination of both the ECF concentration and the in vivo recovery. The latter can provide valuable information about changes in the physical as well as chemical state of the tissue. This can guide rapid effective therapeutic intervention.

Animals↗

PubMed1996

Characterization of carbon paste electrodes in vitro for simultaneous amperometric measurement of changes in oxygen and ascorbic acid concentrations in vivo.

Differential-pulse amperometry, an established technique for the in vivo monitoring of dopamine in brain extracellular fluid (ECF), was extended to the simultaneous electrochemical detection of molecular oxygen (O2) and ascorbic acid (AA). Lipid-treated carbon paste electrodes (LCPEs) were characterized in vitro using this technique and found to be ideally suited for the detection of both compounds. For O2 reduction, two equally sized cathodic pulses were applied, the first from a resting potential at -150 to -350 mV, which corresponds to the foot of the reduction wave for O2 at LCPEs, and the second from -350 to -550 mV, which corresponds to the peak of the reduction wave. Following the same criterion, equally sized anodic pulses were then applied from -150 to +50 mV and from +50 to +250 mV for AA oxidation. The complete sequence of pulses for O2 and AA detection lasts 1 s. Interference by O2 with AA currents and vice versa was not a problem. Also, several compounds present in brain ECF were tested and shown not to interfere appreciably with the amperometric signal for either compound. The technique was tested in vivo, and results from behavioural stimulation, achieved by the application of tail pinch, support the conclusion of simultaneous independent detection of changes in O2 and AA at LCPEs.

Animals↗

PubMed1995

Effects of changes in rat brain glucose on serotonergic and noradrenergic neurons.

Microdialysis was used in the freely moving rat to measure the effects of graded changes in brain glucose on the serotonergic and noradrenergic projections to the hippocampus. The concentration of glucose in the dialysate was monitored using an enzyme-based assay. A systemic injection of insulin caused a steep decline in glucose level which was restored to the control level by oral administration of glucose solution. The changes in 5-hydroxytryptamine (5-HT) and noradrenaline were a mirror image of the glucose changes: they rose after insulin injection and returned to control during glucose administration. A delayed increase was shown by 5-hydroxyindoleacetic acid (5-HIAA) which did not return to baseline on glucose administration. The metabolite dihydroxyphenylacetic acid (DOPAC) decreased after insulin administration and increased above control during glucose administration. While the responses of 5-HT, noradrenaline and 5-HIAA to hypoglycaemia resemble those to mild stress, the changes in DOPAC are the reverse of those produced by stress.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1995

The role of N-methyl-D-aspartate receptors in the regulation of physiologically released dopamine.

In vivo voltammetry was used to measure changes in ascorbate, which are an index of changes in the release of glutamate, and microdialysis was used to measure changes in dopamine in the striatum of freely moving rats. A 5 min tail pinch produced a rapid rise in striatal ascorbate paralleled by an increase in motor activity and a slower, more prolonged rise in dopamine. Systemic administration of ketamine or dizocilpine maleate, non-competitive antagonists of the N-methyl-D-aspartate glutamate receptor, produced an increase in the basal level of ascorbate but not dopamine; however, the tail pinch-evoked rise in both ascorbate and dopamine was completely abolished by these drugs. The rise in dopamine was also abolished by local infusion of dizocilpine maleate into the striatum. Local application of N-methyl-D-aspartate produced a dose-dependent increase in dopamine, which was partially reduced in the presence of tetrodotoxin. The results show that the tail pinch-evoked increase in motor activity involves an increase in the release of striatal dopamine which requires the activation of N-methyl-D-aspartate receptors in the striatum. This suggests that phasic increases in striatal dopamine release are triggered by the action of glutamate on dopaminergic nerve terminals.

Animals↗

PubMed1994

On-line measurement of brain glutamate with an enzyme/polymer-coated tubular electrode.

An on-line tubular electrode system is described which provides real time measurements of glutamate in brain microdialysate. It is based on the enzyme glutamate oxidase (EC 1.4.3.11) and the detection of H2O2 on a platinum electrode at 600 mV vs Ag/AgCl. The enzyme is immobilized in a layer of o-phenylenediamine electropolymerized at 750 mV in a phosphate buffer, pH 7.0. The layer is 10-15 nm thick and enables good enzyme loading and fast response time. The ability of the polymer to block out electroactive interferents like ascorbate and uric acid combined with a preoxidation system run at 600 mV resulted in a virtually interference free glutamate assay with a lower detection limit of 0.3 microM in the presence of physiological levels of interferents, a sensitivity of 4 nA/microM, and a linear region up to 30 microM. The system is optimized for use with a microdialysis probe implanted in the brain and perfused at 2 microL/min. It provides a time resolution of < 1 min and has been tested in vivo.

Animals↗

PubMed1994

The physiologically induced release of ascorbate in rat brain is dependent on impulse traffic, calcium influx and glutamate uptake.

Extracellular brain ascorbate fluctuates with neuronal activity. There is previous evidence that the release of ascorbate is triggered by the re-uptake of neuronally released glutamate. This hypothesis predicts that drugs which block the release and re-uptake of glutamate will also block the release of ascorbate. In the present experiments we have used a novel dialysis electrode which allows continuous monitoring of physiologically induced ascorbate release from the striatum in freely moving rats. An infusion of the enzyme ascorbic acid oxidase abolished the increase in oxidation current in response to tail-pinch, which identified it as an ascorbate current. Perfusion with tetrodotoxin reduced the response to 25% and with CdCl2 to 4% of control. Perfusion with the uptake blocker L-trans-pyrrolidine-2,4-di-carboxylate reduced the response to 24% of control. A neuroprotective function for this coupling of ascorbate and glutamate release is discussed.

Animals↗

PubMed1993

Rapid changes in extracellular glucose levels and blood flow in the striatum of the freely moving rat.

The dynamics of regional cerebral blood flow and brain extracellular glucose were studied in the freely moving rat. These two variables were measured in the striatum during and following both mild tail pinch and restraint stress. Blood flow was monitored using a refinement of the hydrogen clearance technique that allowed repeated measurements at 5-min intervals. A slow stream of hydrogen was directed at the rat's snout for 10-20 s through lightweight tubing attached to the animal's head and detected at a chronically implanted platinum electrode. Extracellular glucose was monitored with microdialysis in a separate group of animals using an on-line, enzyme-based assay that provided 2.5-min time resolution. Mean striatal blood flow 24 h following implantation was 89.9 +/- 2.5 ml.(100 g)-1.min-1. A 5-min tail pinch caused flow to increase immediately to 169.5 +/- 20 ml.(100 g)-1.min-1. In contrast, there was no change in blood flow during restraint stress, although there was a small increase following the end of the stress. Significant increases in blood flow were also observed in the striatum during periods of eating and grooming. Extracellular glucose levels increased following both forms of stress, to a maximum of 170 +/- 22% of baseline with restraint compared to 110 +/- 2% with tail pinch. In both cases, the increase occurred after the stress had ended and persisted while blood flow returned to basal levels.

Animals↗

PubMed1993

Physiological stimulation increases nonoxidative glucose metabolism in the brain of the freely moving rat.

The effects of mild stress on nonoxidative glucose metabolism were studied in the brain of the freely moving rat. Extracellular lactate levels in the hippocampus and striatum were monitored at 2.5-min intervals with microdialysis coupled with an enzyme-based flow injection analysis system. Ten minutes of restraint stress led to a 235% increase in extracellular lactate levels in the striatum. A 5-min tail pinch caused an increase of 193% in the striatum and 170% in the hippocampus. Local application of tetrodotoxin in the striatum blocked the rise in lactate following tail pinch and inhibited the subsequent clearance of lactate from the extracellular fluid. Local application of the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801 had no effect on the tail pinch-stimulated increase in lactate in the striatum. These results show that mild physiological stimulation can lead to a rapid increase in nonoxidative glucose metabolism in the brain.

Animals↗

PubMed1993

ATP-sensitive potassium channels and local energy demands in the rat hippocampus: an in vivo study.

Microdialysis coupled with an enzyme-based flow injection analysis was used to monitor brain extracellular lactate and glucose in the freely moving rat. Glucose levels reflect the balance between supply from the blood and local utilisation, and lactate efflux indicates the degree of local nonoxidative glucose metabolism. Local application of tolbutamide, a blocker of the ATP-sensitive potassium channel, decreased extracellular glucose and lactate levels in the hippocampus but not in the striatum. The increase in glucose and lactate levels following mild behavioural stimulation was also reduced by tolbutamide in the hippocampus. Similar effects on both basal and stimulated lactate levels were obtained with local application of 10 mM glucose. These results indicate that ATP-sensitive potassium channels are active under physiological conditions in the hippocampus and that the effects of tolbutamide can be mimicked by physiological glucose levels.

Adenosine Triphosphate↗