High temporal resolution delayed analysis of clinical microdialysate streams.

This paper presents the use of tubing to store clinical microdialysis samples for delayed analysis with high temporal resolution, offering an alternative to traditional discrete offline microdialysis sampling. Samples stored in this way were found to be stable for up to 72 days at -80 °C. Examples of how this methodology can be applied to glucose and lactate measurement in a wide range of in vivo monitoring experiments are presented. This paper presents a general model, which allows for an informed choice of tubing parameters for a given storage time and flow rate avoiding high back pressure, which would otherwise cause the microdialysis probe to leak, while maximising temporal resolution.

A method for voltage measurements of cancerous vs non-cancerous omentum.

This paper presents and elaborates upon the practicalities of a method which enables the recording of voltage measurements from omental tissue in patients with advanced ovarian cancer. The key components of the proposed low-cost experimental setup are a tungsten electrode, a Ag/AgCl reference electrode and an instrumentation amplifier. Intriguingly, potential difference recordings between cancerous omentum and tissue culture media and between non-cancerous omentum and media, differ for tissue samples coming from the same patient. Further studies are warranted to assess the potential prognostic value of voltage measurements in cancerous tissue.

Online rapid sampling microdialysis (rsMD) using enzyme-based electroanalysis for dynamic detection of ischaemia during free flap reconstructive surgery.

We describe an enzyme-based electroanalysis system for real-time analysis of a clinical microdialysis sampling stream during surgery. Free flap tissue transfer is used widely in reconstructive surgery after resection of tumours or in other situations such as following major trauma. However, there is a risk of flap failure, due to thrombosis in the flap pedicle, leading to tissue ischaemia. Conventional clinical assessment is particularly difficult in such 'buried' flaps where access to the tissue is limited. Rapid sampling microdialysis (rsMD) is an enzyme-based electrochemical detection method, which is particularly suited to monitoring metabolism. This online flow injection system analyses a dialysate flow stream from an implanted microdialysis probe every 30 s for levels of glucose and lactate. Here, we report its first use in the monitoring of free flap reconstructive surgery, from flap detachment to re-vascularisation and overnight in the intensive care unit. The on-set of ischaemia by both arterial clamping and failure of venous drainage was seen as an increase in lactate and decrease in glucose levels. Glucose levels returned to normal within 10 min of successful arterial anastomosis, whilst lactate took longer to clear. The use of the lactate/glucose ratio provides a clear predictor of ischaemia on-set and subsequent recovery, as it is insensitive to changes in blood flow such as those caused by topical vasodilators, like papaverine. The use of storage tubing to preserve the time course of dialysate, when technical difficulties arise, until offline analysis can occur, is also shown. The potential use of rsMD in free flap surgery and tissue monitoring is highly promising.

Use of rapid sampling microdialysis for intraoperative monitoring of bowel ischemia.

PURPOSE: Intestinal ischemia is a major cause of anastomotic leak and death and remains a clinical challenge as the physician relies on several nonspecific signs, biologic markers, and radiologic studies to make the diagnosis. This study used rapid sampling online microdialysis to evaluate the biochemical changes occurring in a segment of human bowel during and after resection, and assessed for the feasibility and reproducibility of this technique in monitoring intestinal ischemia. METHODS: A custom made, rapid sampling online microdialysis analyzer was used to monitor the changes in the bowel wall of specimens being resected intraoperatively. Two patients were recruited for the pilot study to optimize the analyzer and seven patients undergoing colonic resections were recruited for the data collection and analysis. RESULTS: The concentration of glucose in the extracellular bowel wall fluid decreased transiently after division of individual feeding arteries followed by a rebound increase in the concentration back to baseline concentrations. After completion of resection, glucose concentrations continued to decrease while lactate concentrations increased constantly. CONCLUSION: Rapid sampling microdialysis was feasible in the clinical environment. These results suggest that tissue responds to ischemic insult by mobilizing glucose stores which later decrease again, whereas lactate concentrations constantly increased.

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.

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.

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.

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.

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.

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.

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.

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.

New technologies for amperometric biosensors.

Amperomeric-based detectors have successfully been used as personal monitors for blood glucose levels. However, there is a desire to increase the number of compounds measured in a small blood sample, the speed of detection and enhance the reliability of the measurement. Furthermore, with the increasing use of microdialysis as a clinical sampling method in metabolic medicine, paediatric medicine and neurointensive care, there is a need for rapid on-line detection of analytes such as lactate, glucose and glutamate in low microlitre volume samples. Two approaches to these problems are described. The first uses enzymes immobilized in a packed bed with electrochemical detection of a ferrocene mediator as a flow-injection assay for use with microdialysis. Results from microdialysis of the brain of freely moving rats are described. In the second approach, thin-film techniques are used to fabricate arrays of microdisk and micro line electrodes. The properties of these arrays in free solution and in a flow cell are presented together with an example using multiple arrays to identify an analyte by oxidation potential. Finally, different enzymes are entrapped onto the surface of two arrays by electrochemical polymerization of o-phenylenediamine. The resulting device detects glucose and lactate in real-time.

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.

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%.

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.

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.

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.

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.

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.