Implantation Of Microdialysis Probe Research Articles

Differential susceptibility of astrocytic and neuronal function to 3‐chloropropanediol in the rat inferior colliculus

We have previously shown that systemic administration of S(+)3-chloropropanediol (3-CPD) produces a morphological loss of astrocytes in specific nuclei of the rodent brain that precedes loss of both neurones and endothelial tight junctions. Here, we have evaluated the differential susceptibility of neuronal and astrocytic function to 3-CPD, in order to see if this parallels the morphological selectivity. To do this, we have developed an in vivo method for monitoring astrocyte function over time by giving hourly 20-min bolus challenge exposures to ammonia via an implanted microdialysis probe and measuring the resulting transient increases in the extracellular glutamine : glutamate ratio. These challenge ammonia exposures evoked a stable response for at least 5 h when the probe was implanted in the rat inferior colliculus, but caused no behavioural response or morphological damage. Although 3-CPD produced a rapid and sustained abolition of the ammonia response within 2 h, the field potential response of inferior collicular neurones to sound fell significantly to 75.0 ± 3.9% pre-dose at up to 8 h but then fell markedly, reaching 20.5 ± 3.7% at 2 days. Blood flow in the inferior colliculus also showed only late changes, increasing substantially at 2 days. Astrocyte damage at the EM level was seen from 3 h, followed by loss of astrocytes from 18 h to a minimum of 7 ± 10% control at 3 days. The rapid abolition of the ammonia response suggests that in addition to selective astrocyte death, 3-CPD also produces an earlier impairment of astrocyte function that precedes loss of neuronal function. This initial functional selectivity of 3-CPD provides a potential investigative tool in neurochemical studies of astrocyte-neuronal interactions.

Acute Depression of Energy Metabolism after Microdialysis Probe Implantation is Distinct from Ischemia-Induced Changes in Mouse Brain

The current study used measurements of metabolites and markers of membrane integrity to determine the most suitable time point for microdialysis experiments following probe implantation. Leakage of Evans blue and sodium fluorescein indicated increased BBB permeability only immediately (15 min), but not 1.5 and 24 h following probe implantation. Acute implantation decreased glucose and lactate levels relative to the levels after 24 h (to 13-37% and 25-60%, respectively). No change in extracellular levels of glutamate or glycerol was seen. In comparison to acute probe implantation, the pattern of damage under brain ischemia (middle cerebral artery occlusion) differed: While glucose levels dropped, lactate levels rose after ischemia, and glutamate (tenfold) and glycerol (eightfold) increased sharply. In conclusion, acute implantation of a microdialysis probe causes transient depression of the energy metabolites, glucose and lactate, likely due to injury-induced hypermetabolism. However, no massive tissue damage or severe ischemic conditions around the probe occur.

Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls.

Endocannabinoids play an important role in a diverse range of neurophysiological processes including neural development, neuroimmune function, synaptic plasticity, pain, reward and affective state. This breadth of influence and evidence for altered endocannabinoid signalling in a variety of neuropathologies has fuelled interest in the accurate quantification of these lipids in brain tissue. Established methods for endocannabinoid quantification primarily employ solvent-based lipid extraction with further sample purification by solid phase extraction. In recent years in vivo microdialysis methods have also been developed for endocannabinoid sampling from the brain interstitial space. However, considerable variability in estimates of endocannabinoid content has led to debate regarding the physiological range of concentrations present in various brain regions. This paper provides a critical review of factors that influence the quantification of brain endocannabinoid content as determined by lipid extraction from bulk tissue and by in vivo microdialysis. A variety of methodological issues are discussed including analytical approaches, endocannabinoid extraction and purification, post-mortem changes in brain endocannabinoid content, cellular reactions to microdialysis probe implantation and caveats related to lipid sampling from the extracellular space. The application of these methods for estimating brain endocannabinoid content and the effects of endocannabinoid clearance inhibition are discussed. The benefits, limitations and pitfalls associated with each approach are emphasized, with an eye toward the appropriate interpretation of data gathered by each method.

Long-term calibration considerations during subcutaneous microdialysis sampling in mobile rats

The level at which implanted sensors and sampling devices maintain their calibration is an important research area. In this work, microdialysis probes with identical geometry and different membranes, polycarbonate/polyether (PC) or polyethersulfone (PES), were used with internal standards (Vitamin B 12 (MW 1355), antipyrine (MW 188) and 2-deoxyglucose (2-DG, MW 164)) and endogenous glucose to investigate changes in their long-term calibration after implantation into the subcutaneous space of Sprague-Dawley rats. Histological analysis confirmed an inflammatory response to the microdialysis probes and the presence of a collagen capsule. The membrane extraction efficiency (percentage delivered to the tissue space) for antipyrine and 2-DG was not altered throughout the implant lifetime for either PC- or PES membranes. Yet, Vitamin B 12 extraction efficiency and collected glucose concentrations decreased during the implant lifetime. Antipyrine was administered i.v. and its concentrations obtained in both PC- and PES-membrane probes were significantly reduced between the implant day and seven (PC) or 10 (PES) days post-implantation suggesting that solute supply is critical for in vivo extraction efficiency. For the low molecular weight solutes such as antipyrine and glucose, localized delivery is not affected by the foreign body reaction, but recovery is significantly reduced. For Vitamin B 12, a larger solute, the fibrotic capsule formed around the probe significantly restricts diffusion from the implanted microdialysis probes.

Mechanism-based PKPD modelling of CNS-active drugs

Event Abstract Back to Event Mechanism-based PKPD modelling of CNS-active drugs Elizabeth C. De Lange1* 1 Leiden University, LACDR/Pharmacology, Gorlaeus Laboratories, Netherlands Our ultimate goal is to develop mechanism-based pharmacokinetic (PK)-pharmacodynamic (PD) models to characterize and to predict CNS drug responses in both physiologic and pathologic conditions. To this end, it is essential to have information on the target site (biophase) pharmacokinetics, because it may significantly differ from plasma pharmacokinetics. It is anticipated that the biophase kinetics of CNS drugs in many cases is strongly influenced by transport across the blood-brain barrier (BBB). In vivo microdialysis enables the determination of free-drug concentrations as a function of time in extracellular fluid of the brain, thereby providing important data to determine BBB transport characteristics of drugs. This is important for delineating the mechanisms that govern biophase pharmacokinetics when combined with CNS effect measurements, and therefore provides an indispensable tool in mechanism-based PKPD modeling of CNS drugs. The biophase kinetics of a CNS drug is an important determinant of the time course and intensity of its CNS effects. Biophase pharmacokinetics can be estimated by PKPD modeling by using a hypothetical effect compartment. This hypothetical effect compartment is linked to the plasma compartment by a rate constant for influx and a rate constant for drug efflux such that there is no time delay between the biophase pharmacokinetics in the effect compartment and the time course of the effect. These data on biophase pharmacokinetics can be related to the effect, whereby it is possible to gain information on the true ability of a drug to activate the receptor. This is typically done using the biophase pharmacokinetics of a number of drugs within a same class, in combination with the receptor theory (operational model of agonism).However, the use of the effect compartment model does not provide insight into mechanisms that govern biophase pharmacokinetics which is needed for more generally applicable PKPD models. Apart from plasma pharmacokinetics, mechanisms that govern CNS biophase kinetics include: the rate and extent of blood-brain barrier (BBB) transport; the kinetics of processes of distribution and elimination within the brain; target interaction, and signal transduction (fig 1). All the mechanisms may vary in their contribution to the dose-effect relationship and therefore need to be taken into consideration1. As BBB transport and brain distribution often do not occur instantaneously and to a full extent, this may contribute to a delay of the pharmacologic effect versus time profile relative to the plasma profile. In order to develop more generally applicable models with more power for extrapolation to other conditions, it is of importance to explicitly investigate BBB transport. BBB transport is related to BBB functionality and occurs by passive diffusion, as well as by active transport. Active transport occurs by many membrane transporters such as the Pglycoprotein (Pgp) and the multidrug resistance-associated proteins (MRPs). BBB functionality is dynamically controlled by blood components and the surrounding brain cells by direct contact or indirectly by their extracellular products. Thus, BBB functionality may vary among different physiologic, pathologic, and chronic drug treatment conditions. It is anticipated that such variations in BBB functionality will ultimately affect the biophase kinetics of CNS drugs and therewith the CNS effect-time profile of the drug. In vivo microdialysis is a technique that enables the determination of the free drug in plasma and its concentrations in extracellular fluid (ECF) the brain as a function of time. Herewith, it provides important information for the determination of BBB transport and brain distribution. A prerequisite for this application is that BBB transport characteristics will not be significantly influenced by microdialysis probe implantation and presence in the brain. This was an initial concern. But based on a series of studies performed to validate the usefulness of intracerebral microdialysis in measurements of passive, as well as active, BBB transport, this prerequisite appears to hold, provided that this technique is used under well-controlled surgical and experimental conditions. In this presentation the main principles of mechanism-based PKPD modeling of CNS active drugs will be presented, including arguments for the need to include biophase kinetics, followed by the description of factors that govern biophase kinetics of CNS drugs. Then, two examples on studies in rats that include investigations on the impact of BBB transport will be discussed. The first example is on the EEG effects of opioids2, while the second one deals with BBB transport of L-DOPA and its brain conversion to HVA and DOPAC in Parkinson’s diseased brain3. Acknowledgments We are grateful to GlaxoSmithKline and Eli Lilly for their collaboration and financing of the studies on the opioids and Parkinson’s disease, respectively. Fig 1

Detection of in vivo matrix metalloproteinase activity using microdialysis sampling and liquid chromatography/mass spectrometry.

Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to quantify MMP-1 and MMP-9 substrates and their N-terminal peptide products in samples obtained from implanted microdialysis sampling probes. In vitro studies with purified human MMP-1 and MMP-9 were used to optimize the assay and determine the effectiveness of the local delivery of a broad-spectrum MMP inhibitor, GM 6001. Localized delivery of GM 6001 at 10 microM was sufficient to completely inhibit product formation in vitro. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the subcutaneous tissue. Directly after microdialysis probe implantation, infusions of the MMP-1 and MMP-9 substrates (50 microM each) resulted in recovered product concentrations of approximately 2 microM. During a 50 microM GM 6001 coinfusion with the substrates, a 30% and 25% reduction in product formation for the MMP-1 and MMP-9 substrates was obtained, respectively. Blank dialysates were negative for enzymatic activity that could cleave the MMP substrates. This method allowed for the activity of different MMPs surrounding the microdialysis probe to be observed during in vivo sampling.

Histological studies of the effects of chronic implantation of ceramic-based microelectrode arrays and microdialysis probes in rat prefrontal cortex

Chronic implantation of neurotransmitter measuring devices is essential for awake, behavioral studies occurring over multiple days. Little is known regarding the effects of long term implantation on surrounding brain parenchyma and the resulting alterations in the functional properties of this tissue. We examined the extent of tissue damage produced by chronic implantation of either ceramic microelectrode arrays (MEAs) or microdialysis probes. Histological studies were carried out on fixed tissues using stains for neurons (cresyl violet), astrocytes (GFAP), microglia (Iba1), glutamatergic nerve fibers (VGLUT1), and the blood–brain barrier (SMI-71). Nissl staining showed pronounced tissue body loss with microdialysis implants compared to MEAs. The MEAs produced mild gliosis extending 50–100 μm from the tracks, with a significant change in the affected areas starting at 3 days. By contrast, the microdialysis probes produced gliosis extending 200–300 μm from the track, which was significant at 3 and 7 days. Markers for microglia and glutamatergic fibers supported that the MEAs produce minimal damage with significant changes occurring only at 3 and 7 days that return to control levels by 1 month. SMI-71 staining supported the integrity of the blood–brain barrier out to 1 week for both the microdialysis probes and the MEAs. This data support that the ceramic MEA's small size and biocompatibility are necessary to accurately measure neurotransmitter levels in the intact brain. The minimal invasiveness of the MEAs reduce tissue loss, allowing for long term (> 6 month) electrochemical and electrophysiological monitoring of brain activity.

Microdialysis in Rodents

Microdialysis is an in vivo sampling technique that permits the quantification of various substances (e.g., neurotransmitters, peptides, electrolytes) in blood and tissue. It is also used to infuse substances into the brain and spinal cord. This unit describes methods for the construction and stereotaxic implantation of microdialysis probes into discrete brain regions of the rat and mouse. Procedures for the conduct of conventional and quantitative microdialysis experiments in the awake and anesthetized rodent are also provided.

A modified surgical procedure for microdialysis probe implantation in the lateral ventricle of a FVB mouse

A modified surgical procedure is described to implant a microdialysis probe to sample ventricular cerebrospinal fluid (vCSF) in FVB mice. Microdialysis sampling of drugs in vCSF provides insight into drug penetration into the brain across the blood brain barrier (BBB) and the blood CSF barrier (BCB); however, this method has been reported primarily in larger animal species. Implanting a microdialysis probe in the lateral ventricle of a mouse is technically very challenging. The modification consisted of changes in the stereotaxic coordinates and insertion of the cannula and ultimately the probe at a 20° angle. Exact placement of the probe was confirmed using ultrasound (US), micro‐computed tomography (CT), and histologic review of serial paraffin sections. Additionally, studies of topotecan CSF penetration in the FVB mouse were conducted. With this modified procedure, the ventricular CSF to plasma AUC ratio of unbound topotecan lactone was greater than that previously reported using conventional methods. We speculate this is due to changes incorporated by the modified procedure that places the probe directly into the lateral ventricle allowing sampling of that discrete compartment. Thus, we propose that this modified procedure for placement of the microdialysis probe is superior to the conventional perpendicular method previously reported.

The development of multiple probe microdialysis sampling in the stomach

A multiple probe approach of implanting microdialysis probes into each separate tissue layer would better represent sampling from the stomach. Presently, microdialysis sampling experiments are performed with only a single probe in the submucosa to represent sampling from the stomach tissue. The focus of this research was to develop a four-probe microdialysis sampling design to simultaneously monitor the stomach lumen, mucosa, submucosa and in the blood of the rat. Due to the small outer diameter of the microdialysis probe (350 μm), implantation into each separate layer was achieved with confirmation of probe location from histological examination. To assess the significance of sampling by this approach, multiple probe microdialysis sampling was used to monitor drug absorption in the stomach. Salicylic acid, caffeine and metoprolol were individually dosed to the ligated stomach. Analysis of the dialysate samples was performed by HPLC–UV and concentration–time curves and pharmacokinetics analysis were used to determine differences between the different probe locations.

The Sweet-Blind Mice

Understanding the brain’s reward system, mediated by the neurotransmitter dopamine, has the potential to help address the enormous health risk to humans posed by obesity. Like addictive drugs, good-tasting food increases the accumulation of dopamine in the nucleus accumbens (NAcc) of the ventral striatum. In fact, some mice with defects in the dopamine reward system fail to eat and die of starvation. But besides taste-induced or hedonic responses, animals appear also to sense the nutritional value of ingested food. De Araujo et al. present a series of experiments that implicate the same brain mesolimbic dopamine reward system in both processes. They distinguished the two responses by making knockout mice that lacked a functional TRPM5 (transient receptor potential channel M5) ion channel, which mediates taste sensation in taste receptor cells. Unlike wild-type animals, after being deprived of water, these mutant animals showed no preference for a sucrose solution over plain water. However, over a period of conditioning of 6 days in which mice were presented with bottles containing sucrose solution or water that could be distinguished by their sipper, the starved mutant animals had a distinct preference for the more nutritive solution. The authors implanted microdialysis probes in the NAcc of mutant or wild-type animals and showed that the mutant’s response to the sucrose solution was associated with the release of dopamine similar to that in wild-type mice drinking sucrose solution. Electrophysiological recordings from neurons in the NAcc and orbitofrontal cortex indicated that the increased dopamine release was correlated with activation of the brain reward circuitry. The authors, and Andrews and Horvath in commentary, conclude that both hedonic and nutritional or homeostatic rewards of ingested food are processed similarly, not separately as sometimes thought, in the higher brain centers that process reward responses. I. E. de Araujo, A. J. Oliveira-Maia, T. D. Sotnikova, R. R. Gainetdinov, M. G. Caron, M. A. L. Nicolelis, S. A. Simon, Food reward in the absence of taste receptor signaling. Neuron 57 , 930-941 (2008). [PubMed] Z. B. Andrews, T. L. Horvath, Tasteless food reward. Neuron 57 , 806-808 (2008). [PubMed]

Quantification of Phenol, Phenyl Glucuronide, and Phenyl Sulfate in Blood of Unanesthetized Rainbow Trout by Online Microdialysis Sampling

ABSTRACTFree concentrations of phenol (PH), phenyl glucuronide (PG), and phenyl sulfate (PS) were measured in the bloodstream of unanesthetized rainbow trout by online microdialysis (MD) sampling. Preliminary studies were conducted to optimize the MD system and evaluate three retrodialysis calibration standards: p-nitrophenyl glucuronide (PNPG), p-nitrophenyl sulfate (PNPS), and [14C]-phenol (14C-PH). PG and PNPG exhibited nearly identical dialyzing properties in vitro (saline and plasma) and in vivo (muscle tissue and dorsal aorta). A similar result was obtained for PS and PNPS. In vivo studies were therefore performed using PNPG, PNPS, and 14C-PH as retrodialysis calibrators for PG, PS, and PH, respectively. The utility of MD sampling for kinetic studies with fish was investigated by implanting MD probes into the dorsal aorta of spinally transected rainbow trout. Each fish was then exposed to PH in water in a respirometer-metabolism chamber. The free concentration of PH in blood reached a steady-state level within 12 h of initiating the exposure. A steady state for PS was generally established within 24 h, while free concentrations of PG tended to increase throughout the exposure. Terminal plasma samples were dialyzed using the same probe employed in each experiment. Analyte concentrations determined in this manner were in good agreement with calculated in vivo values. The methods described in this study can be used to collect kinetic data sets of high temporal resolution while eliminating artifacts often associated with conventional blood sampling methods.

Comparative pharmacokinetics and distribution kinetics in brain of phencynonate enantiomers in rats

To investigate the pharmacokinetics in blood and the distribution kinetics in brain of enantiomers of novel anticholinergic agent phencynonate ( N-methyl-9α-(3-azabicyclo[3,3,1]nonanyl)-2′-cyclopentyl-2′-hydroxyl-2′-phenylacetate), we collected blood and implanted microdialysis probes in the cerebral frontal cortex of rats. Phencynonate enantiomers (0.35 mg/kg, i.m.) were then cross administered, and the microdialysates were collected using in situ microdialysis sampling in the brain of freely moving rats, and the concentration of phencynonate enantiomers was determined by the validated method of liquid chromatography–mass spectrometry. Pharmacokinetic parameters were calculated from the blood and the brain dialysate concentrations of phencynonate enantiomers versus time data. The disposition profiles of the phencynonate enantiomers were best fitted to a first order absorption, two-compartment open model in rats. In general, there were some differences when comparing the mean kinetic parameters of S- and R-phencynonate in the blood and brain, but the distinct diversity between individual animals made the statistical difference not obvious. Therefore, stereoselective disposition of phencynonate isomers was not obviously observed in rat.

Application of long-term microdialysis in circadian rhythm research

Our laboratory has pioneered long-term microdialysis to monitor pineal melatonin secretion in living animals across multiple circadian cycles. There are numerous advantages of this approach for rhythm analysis: (1) we can precisely define melatonin onset and offset phases; (2) melatonin is a reliable and stable neuroendocrine output of the circadian clock (versus behavioral output which is sensitive to stress or other factors); (3) melatonin measurements can be performed extremely frequently, permitting high temporal resolution (10 min sampling intervals), which allows detection of slight changes in phase; (4) the measurements can be performed for more than four weeks, allowing perturbations of the circadian clock to be followed long-term in the same animals; (5) this is an automated process (microdialysis coupled with on-line HPLC analysis), which increases accuracy and bypasses the labor-intensive and error-prone manual handling of dialysis samples; and (6) our approach allows real-time investigation of circadian rhythm function and permits appropriate timely adjustments of experimental conditions. The longevity of microdialysis probes, the key to the success of this approach, depends at least in part on the methods of the construction and implantation of dialysis probes. In this article, we have detailed the procedures of construction and surgical implantation of microdialysis probes used currently in our laboratory, which are significantly improved from our previous methods.

Influence of thyrotropin-releasing hormone (TRH) dialyzed into the hippocampus on memory processes in rabbit

There are conflicting reports on the influence of thyrotropinreleasing hormone (TRH) on the process of learning. We decided to study this problem using the paradigm of classical eyeblink conditioning as the animal model of learning processes. During the extinction training TRH in its natural form of pGluTRH was applied into the rabbit hippocampus through a chronically implanted microdialysis probe. A Glu1TRH, analog with less biological potency than TRH as the control of specificity for TRH and a 0.9% NaCl solution as the control for both substances were applied by the same way as pGluTRH. We found that pGluTRH extended the process of extinction and enhanced the further acquisition of the reflex. The analog of TRH, Glu1TRH, was ineffective. Finally, it may be stated that TRH acting in the hippocampus prolonged process of forgetting and improved succeeding learning. The effect was specific and long lasting. Correspondence should be addressed to M. Orlowska-Majdak Email: orlowska@zdn.am.lodz.pl

Bedside monitoring of subcutaneous interstitial glucose in healthy individuals using microdialysis and infrared spectrometry

An IR-spectroscopy-based bedside device, coupled to a subcutaneously implanted microdialysis probe, is developed for quasicontinuous glucose monitoring with intermittent readouts at 10-min intervals, avoiding any sensor recalibration under long-term operation. The simultaneous estimation of the microdialysis recovery rate is possible using an acetate containing perfusate and determining its losses across the dialysis membrane. Measurements are carried out on four subjects, with experiments lasting either 8 or 28 h, respectively. Using the spectral interval data either from 1180 to 950 or 1560 to 1000 cm(-1), standard errors of prediction (SEPs) between 0.13 and 0.28 mM are achieved using multivariate calibration with partial least-squares (PLS) or classical least-squares (CLS) calibration models, respectively. The transfer of a PLS calibration model using the spectral and reference concentration data of the dialysates from the three 8-h-long experiments to a 28-h monitoring episode with another healthy subject is tested. Including microdialysis recovery for the determination of the interstitial glucose concentrations, an SEP of 0.24 mM is obtained versus whole blood glucose values. The option to determine other metabolites such as urea or lactate offers the possibility to develop a calibration- and reagent-free point-of-care analyzer.

Stereotaxic gene delivery in the rodent brain

Stereotaxic surgery has been an invaluable tool in systems neuroscience, applied in many experiments for the creation of site-targeted lesions, injection of anatomical tracers or implantation of electrodes or microdialysis probes. In this protocol, we describe stereotaxic surgery optimized for gene delivery by recombinant adeno-associated viruses and lentiviruses in mice and rats. This method allows the manipulation of gene expression in the rodent brain with excellent spatiotemporal control; essentially any brain region of choice can be targeted and cells (or a subpopulation of cells) in that region can be stably genetically altered at any postnatal developmental stage up to adulthood. Many aspects of the method, its versatility, ease of application and high reproducibility, make it an attractive approach for studying genetic, cellular and circuit functions in the brain. The entire protocol can be completed in 1-2 hours.

Continuous reagent-free bed-side monitoring of glucose in biofluids using infrared spectrometry and micro-dialysis

Quasi-continuous glucose monitoring has been realised for a bed-side device based on reagent-free transmission spectroscopy of microliter dialysate sample volumes. Aqueous glucose solutions and serum ultrafiltrates were used for in vitro testing, whereas in vivo samples were provided by a subcutaneously implanted micro-dialysis probe. Both sample types were transported to a thermostated flow-through micro-cell, housed within the sample compartment of a Fourier-transform mini-spectrometer, by using a programmable fluidic system consisting of a six-port valve with sample loop and a syringe pump. Different options were implemented for spectral evaluation based on multivariate calibration using either partial or classical least squares. The latter approach includes the additional quantification of most relevant dialysate components such as urea, lactate, bicarbonate and others. Automated operator-free in vitro operation with reliable glucose quantification over at least 72 h was tested for analytical performance characterisation. Routines for sensitive air bubble detection by infrared spectrometry including automated removal by the fluidic system have also been implemented.

Pentobarbital inhibits l-DOPA-induced dopamine increases in the rat striatum: An in vivo microdialysis study

Pentobarbital is reported to inhibit ketamine-induced dopamine (DA) release in the rat nucleus accumbens. The accumbens is a part of the limbic dopaminergic system in the brain, and the dopaminergic neural activity of other components may also be sensitive to pentobarbital. We investigated the effect of pentobarbital administration on DA release in the striatum known as DA-rich basal ganglia, and the interaction between pentobarbital and l-DOPA, using in vivo microdialysis techniques. Male SD rats were implanted microdialysis probe into the right striatum. The probe was perfused with modified Ringer's solution and dialysate was directly injected to an HPLC. Every group of rats was consisted of six to seven animals. In the first experiment, rats were given saline, 25 and 50 mg kg −1 pentobarbital. The second, each rat was given a local administration of 2 and 5 μg ml −1 of l-DOPA with perfusate. Finally, other sets of rats were given 5 μg ml −1 of l-DOPA and 25, 50, or 100 mg kg −1 pentobarbital. Pentobarbital anaesthesia decreased the extracellular concentration of DA, and local administration of l-DOPA significantly increased DA concentration. Pretreatment with pentobarbital diminished the l-DOPA-induced DA increase. The results of the present investigation demonstrate that administration of pentobarbital might inhibit dopaminergic neural activity not only in the nucleus accumbens but also in the rat striatum. Pentobarbital anaesthesia antagonizes DA increase induced by l-DOPA and suggests the inhibition of metabolism of l-DOPA. The results of some animal experiments on dopaminergic activity under pentobarbital anaesthesia should be reconsidered.

On-line coupling of microdialysis sampling with high performance liquid chromatography and hydride generation atomic absorption spectrometry for continuous in vivo monitoring of arsenic species in the blood of living rabbits

An automated on-line method has been developed for the continuous in vivo monitoring of arsenic species, including arsenite (AsIII), arsenate (AsV), monomethvlarsonic acid (MMA) and dimethvlarsinic acid (DMA), in the blood of living rabbits. The method is based on microdialysis sampling, high performance liquid chromatography (HPLC) separation and hydride generation atomic absorption spectrometry (HGAAS) detection. Dialysates perfused through an implanted microdialysis probe were collected in the sample loop of an on-line injector for direct and automated injection into an HPLC system hyphenated with HGAAS. Operating under optimal conditions, the detection limit was found to be 2.0, 2.9, 2.4 and 4.2 ng/ml for AsIII, MMA, DMA and AsV, respectively. Typical precision values of 7.0% (AsIII), 7.0% (MMA), 5.1% (DMA) and 8.7% (AsV) were obtained, respectively at a concentration level of 10 ng/ml. Spike recoveries close to 100%, relative to an aqueous standard, were obtained for each species. The average in vivo recoveries of AsIII, MMA, DMA and AsV in the blood of living rabbits were 5.7, 12.4, 8.9, and 11.0%, respectively. The use of an on-line microdialysis–HPLC–HGAAS system permitted the continuous monitoring, with a temporal resolution of 20 min sampling, of four arsenic species in the blood of living rabbits treated with arsenic trioxide. Based on our results, we present here the first finding, to the best of our knowledge of the distribution of arsenite in the blood of a living organism after administration of arsenic trioxide.