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.

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.

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.

The effects of anxiolytic and anxiogenic benzodiazepine receptor ligands on motor activity and levels of ascorbic acid in the nucleus accumbens and striatum of the rat.

The effects of the anxiolytic benzodiazepine flurazepam and the anxiogenic beta-carboline N-methyl-beta-carboline-3-carboxylate (FG 7142) were measured in unanaesthetised rats. Changes in motor activity, using a Doppler-shift microwave device, and in the extracellular concentration of ascorbate in the striatum and nucleus accumbens, using linear sweep voltammetry with carbon paste electrodes, were monitored continuously over a period of 7 days. Both motor activity and release of ascorbate were greater during the dark than the light period; regression analysis showed a high correlation coefficient for motor activity vs release of ascorbate. The two drugs caused similar changes in this diurnal pattern. A single intraperitoneal injection of either flurazepam or FG 7142 at the end of the light period was followed by a reduction in the nocturnal rise of motor activity and of levels of ascorbate in both the nucleus accumbens and striatum. However, whereas the correlation coefficient for motor activity vs the level of ascorbate in both the nucleus accumbens and striatum remained high after the injection of flurazepam, there was a breakdown of the correlation on the day after the injection of FG 7142, followed by recovery.

Dopamine in the basal ganglia and benzodiazepine-induced sedation.

The effects of the anxiolytic benzodiazepine flurazepam on motor activity and the turnover of dopamine were measured in rats. Changes in motor activity were measured using a doppler-shift device; changes in extracellular homovanillic acid (HVA), monitored by linear sweep voltammetry with carbon paste electrodes implanted in the striatum and nucleus accumbens and ex vivo measurements of changes in 3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratios in the striatum and nucleus accumbens were used as indices of changes in the turnover of dopamine. Injection of vehicle increased the nocturnal rise in the concentration of HVA and the ex vivo DOPAC/DA ratio in the nucleus accumbens. Injection of flurazepam decreased the nocturnal rise in HVA and DOPAC/DA ratio in the nucleus accumbens below control levels. There was also a decrease in the nocturnal rise in motor activity. Neither injection of vehicle nor injection of flurazepam caused changes in either the concentration of HVA or the DOPAC/DA ratio in the striatum. The correlation coefficient for motor activity compared to concentration of HVA remained high for the nucleus accumbens but was reduced for the striatum after administration of flurazepam. The results suggest that the sedative effect of flurazepam may be due to an action on the mesolimbic but not the nigrostriatal dopaminergic pathway.

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.

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.

Rapid changes in striatal ascorbate in response to tail-pinch monitored by constant potential voltammetry.

The first peak in the voltammogram recorded with linear sweep and a carbon paste electrode implanted in the rat striatum is due to the oxidation of ascorbic acid. When the potential is held at a level slightly positive to this peak a current is recorded which is abolished by the microinjection of ascorbic acid oxidase in the vicinity of the electrode; this suggests that it is due to the oxidation of ascorbate. This current shows the same diurnal variation as the size of the ascorbate peak and its rise and fall coincides with the onset and offset of motor activity. A tail-pinch applied through a paper clip causes an immediate rise in the ascorbate current which begins to fall as soon as the paper clip is removed. Measurement of the ascorbate current at constant potential provides a technique for monitoring rapid changes in extracellular brain ascorbate in response to physiological stimuli.

Effect of diazepam on behaviour and associated changes in ascorbate concentration in rat brain areas: striatum, n. accumbens and hippocampus.

The effect of diazepam on spontaneous and tail-pinch-induced behaviour was monitored together with the measurement of extracellular ascorbate using constant potential voltammetry with carbon paste electrodes. Diazepam (3 mg/kg) was followed by eating during the 1st hour after administration in non-food-deprived rats and a reduction in the behaviour triggered by a mild tail-pinch 90 min after drug administration. There was no change in ascorbate concentration in parallel with the spontaneous eating; however, the brisk increase in ascorbate concentration in striatum, nucleus accumbens and hippocampus, which accompanies the tail-pinch, was decreased in size and duration after diazepam. This effect was blocked by the central benzodiazepine receptor antagonist Ro15 1788 (5 mg/kg).

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.

In vivo neurochemical effects of tail pinch.

Tail pinch in the rat gives rise to a well characterised pattern of behaviour which includes gnawing, licking and eating. We have used both in vivo voltammetry and microdialysis to monitor neurochemical changes which accompany the behavioural response to a 5-min tail pinch. Tail pinch resulted in a increase of extracellular 5-hydroxytryptamine and a smaller and more delayed increase of 5-hydroxyindole acetic acid in the hippocampus. In the striatum there was a rise of both extracellular dopamine and ascorbate. With a recently developed constant potential voltammetric technique we can continuously monitor changes in extracellular ascorbate. Using this technique we found a very rapid rise in ascorbate current during a 5-min tail pinch; the current began to decline as soon as the clip was removed. The high time resolution of the technique also allowed us to record similar ascorbate changes during a 0.5-s tail pinch.

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.

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.

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%.

An amperometric enzyme electrode for monitoring brain glucose in the freely moving rat.

Brain glucose concentration was measured with an amperometric enzyme electrode using glucose oxidase (EC 1.1.3.4) irreversibly adsorbed onto an organic conducting salt. The responses of the electrode and its stability both in vitro and in vivo are described. Parellel changes in brain glucose and blood glucose (measured in samples from an implanted intra-atrial cannula) following injections of insulin are reported.

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.

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.

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.

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.