Interaction between Mesocortical and Mesothalamic Catecholaminergic Transmissions Associated with NMDA Receptor in the Locus Coeruleus.

Noncompetitive N-methyl-D-aspartate/glutamate receptor (NMDAR) antagonists contribute to the pathophysiology of schizophrenia and mood disorders but improve monoaminergic antidepressant-resistant mood disorder and suicidal ideation. The mechanisms of the double-edged sword clinical action of NMDAR antagonists remained to be clarified. The present study determined the interaction between the NMDAR antagonist (MK801), α1 adrenoceptor antagonist (prazosin), and α2A adrenoceptor agonist (guanfacine) on mesocortical and mesothalamic catecholaminergic transmission, and thalamocortical glutamatergic transmission using multiprobe microdialysis. The inhibition of NMDAR in the locus coeruleus (LC) by local MK801 administration enhanced both the mesocortical noradrenergic and catecholaminergic coreleasing (norepinephrine and dopamine) transmissions. The mesothalamic noradrenergic transmission was also enhanced by local MK801 administration in the LC. These mesocortical and mesothalamic transmissions were activated by intra-LC disinhibition of transmission of γ-aminobutyric acid (GABA) via NMDAR inhibition. Contrastingly, activated mesothalamic noradrenergic transmission by MK801 enhanced intrathalamic GABAergic inhibition via the α1 adrenoceptor, resulting in the suppression of thalamocortical glutamatergic transmission. The thalamocortical glutamatergic terminal stimulated the presynaptically mesocortical catecholaminergic coreleasing terminal in the superficial cortical layers, but did not have contact with the mesocortical selective noradrenergic terminal (which projected terminals to deeper cortical layers). Furthermore, the α2A adrenoceptor suppressed the mesocortical and mesothalamic noradrenergic transmissions somatodendritically in the LC and presynaptically/somatodendritically in the reticular thalamic nucleus (RTN). These discrepancies between the noradrenergic and catecholaminergic transmissions in the mesocortical and mesothalamic pathways probably constitute the double-edged sword clinical action of noncompetitive NMDAR antagonists.

A simple blood microdialysis in freely-moving rats for pharmacokinetic-pharmacodynamic modeling study of Shengmai injection with simultaneous determination of drug concentrations and efficacy levels in dialysate.

Microdialysis is a powerful in vivo sampling technique for pharmacokinetic-pharmacodynamic (PK-PD) modeling of drugs in pre-clinical and clinical studies. However, the noticeable limitations of previous studies using microdialysis were that animals anesthesia in the whole experiment and the combination of microdialysis and blood sampling for drug and (or) effect detection, which can obviously influence PK and PD behavior of drugs. In this study, a simple blood microdialysis sampling system in freely-moving rats was established for simultaneous study of PK and PD of Shengmai injection (SMI) effect on inducing real-time nitric oxide (NO) release on isoproterenol (ISO) induced myocardial ischemia rats. The LC-MS/MS and HPLC with fluorescence detection (HPLC-FLD) methods were developed to determine ginsenside Rg1, Rg2, Re, Rf, Rb1, Rd and Rc, the main effective components of SMI, and NOx-, the main oxidation products of NO, in dialysates respectively. Through simultaneous determination of drug concentrations and NO efficacy levels in dialysate, the developed methods were successfully applied to set up concentration-time and effect-time profiles followed by PK-PD modeling of SMI effect on inducing NO release after intravenous administration of 10.8 mL kg-1 SMI in myocardial ischemia rats. The PK-PD modeling characterized the dose-effect relationships of SMI and behaved good prediction ability. The established blood microdialysis in freely-moving rats is an appealing technology for rational PK-PD studies when selecting suitable blood endogenous micromolecule as effect marker.

Microdialysis as a New Technique for Extracting Phenolic Compounds from Extra Virgin Olive Oil.

The amount and composition of the phenolic components play a major role in determining the quality of olive oil. The traditional liquid-liquid extraction (LLE) method requires a time-consuming sample preparation to obtain the "phenolic profile" of extra virgin olive oil (EVOO). This study aimed to develop a microdialysis extraction (MDE) as an alternative to the LLE method to evaluate the phenolic components of EVOO. To this purpose, a microdialysis device and dialysis procedure were developed. "Dynamic-oil" microdialysis was performed using an extracting solution (80:20 methanol/water) flow rate of 2 µL min-1 and a constant EVOO stream of 4 µL min-1. The results indicated a strong positive correlation between MDE and the LLE method, providing a very similar phenolic profile obtained with traditional LLE. In conclusion, the MDE approach, easier and quicker in comparison to LLE, provided a reliable procedure to determine the phenolic components used as a marker of the quality and traceability of EVOO.

Investigating local and long-range neuronal network dynamics by simultaneous optogenetics, reverse microdialysis and silicon probe recordings in vivo.

BACKGROUND: The advent of optogenetics has given neuroscientists the opportunity to excite or inhibit neuronal population activity with high temporal resolution and cellular selectivity. Thus, when combined with recordings of neuronal ensemble activity in freely moving animals optogenetics can provide an unprecedented snapshot of the contribution of neuronal assemblies to (patho)physiological conditions in vivo. Still, the combination of optogenetic and silicone probe (or tetrode) recordings does not allow investigation of the role played by voltage- and transmitter-gated channels of the opsin-transfected neurons and/or other adjacent neurons in controlling neuronal activity. NEW METHOD AND RESULTS: We demonstrate that optogenetics and silicone probe recordings can be combined with intracerebral reverse microdialysis for the long-term delivery of neuroactive drugs around the optic fiber and silicone probe. In particular, we show the effect of antagonists of T-type Ca(2+) channels, hyperpolarization-activated cyclic nucleotide-gated channels and metabotropic glutamate receptors on silicone probe-recorded activity of the local opsin-transfected neurons in the ventrobasal thalamus, and demonstrate the changes that the block of these thalamic channels/receptors brings about in the network dynamics of distant somatotopic cortical neuronal ensembles. COMPARISON WITH EXISTING METHODS: This is the first demonstration of successfully combining optogenetics and neuronal ensemble recordings with reverse microdialysis. This combination of techniques overcomes some of the disadvantages that are associated with the use of intracerebral injection of a drug-containing solution at the site of laser activation. CONCLUSIONS: The combination of reverse microdialysis, silicone probe recordings and optogenetics can unravel the short and long-term effects of specific transmitter- and voltage-gated channels on laser-modulated firing at the site of optogenetic stimulation and the actions that these manipulations exert on distant neuronal populations.

Simultaneous percutaneous implantation of a microdialysis probe for monitoring perineural concentrations of local anaesthetics during peripheral nerve block in rabbits.

OBJECTIVE: To develop a technique that allows simultaneous percutaneous implantation of both a microdialysis probe and injection catheter in order to monitor the perineural pharmacokinetics of local anaesthetics (LA) after a femoral block. STUDY DESIGN: Prospective experimental study. ANIMALS: Five anaesthetized male New Zealand rabbits with a mean ± SD weight of 3.2 ± 0.2 kg. METHODS: After femoral nerve localization by electrostimulation, an injection catheter and a microdialysis probe were slowly and simultaneously inserted into a cannula left into place in the perineural region. Both were then secured into place, after removal of the cannula. At the end of the experiment, methylene blue was injected to confirm the distance from the femoral nerve during subsequent postmortem anatomical dissection of the injection site. RESULTS: Staining was adequate and the catheter found to be located within 4 mm of the femoral nerve in three out of five rabbits. CONCLUSIONS AND CLINICAL RELEVANCE: This procedure allows direct implantation of a microdialysis probe near the injection site of LA during a femoral nerve block without loosing nerve localization accuracy. This procedure has been used successfully to monitor the regional pharmacokinetics of LA after a peripheral nerve block.

Reliability of the microdialysis pump CMA 107 under hyperbaric conditions.

OBJECTIVE: Microdialysis measurements of extracellular substances under hyperbaric conditions were manifold used in several investigations. However, to our knowledge there is no analysis, which verified the applicability of microdialysis pumps under hyperbaric conditions. Thus, a goal of this study was to investigate the reliability of the microdialysis pump (MDP) CMA 107 in a hyperbaric environment up to 2.4bar absolute pressure. METHODS: The CMA 107 with a perfusion rate of 2microL/min was stored in a decompression chamber. The ambient pressure was increased from 1 to 2.4bar absolute within 15min, maintained for 90min and then decreased to 1bar within 15min. The vials were changed every 15min, weighed before as well as after collecting the sample volume and the absolute recovery of glutamate, pyruvate, lactate, glucose and glycerol was determined. The same setup was performed under normobaric conditions. RESULTS: The pumping capacity was 1.7% greater than expected under normobaric conditions, 36.5% less than expected in the compression phase, 10.5% less than expected in the isopression phase and 26.3% greater than expected in the decompression phase under hyperbaric conditions. The absolute recoveries under hyperbaric conditions were affected during the isopression phase with a deviation from -6 to +20% compared to normobaric environments. CONCLUSION: The study demonstrated that an absolute ambient pressure up to 2.4bar did influence the pumping capacity and the reliability of the absolute recovery. These results need to be taken into consideration when interpreting microdialysis studies performed under hyperbaric conditions.

AAPS-FDA workshop white paper: microdialysis principles, application and regulatory perspectives.

Many decisions in drug development and medical practice are based on measuring blood concentrations of endogenous and exogenous molecules. Yet most biochemical and pharmacological events take place in the tissues. Also, most drugs with few notable exceptions exert their effects not within the bloodstream, but in defined target tissues into which drugs have to distribute from the central compartment. Assessing tissue drug chemistry has, thus, for long been viewed as a more rational way to provide clinically meaningful data rather than gaining information from blood samples. More specifically, it is often the extracellular (interstitial) tissue space that is most closely related to the site of action (biophase) of the drug. Currently microdialysis (microD) is the only tool available that explicitly provides data on the extracellular space. Although microD as a preclinical and clinical tool has been available for two decades, there is still uncertainty about the use of microD in drug research and development, both from a methodological and a regulatory point of view. In an attempt to reduce this uncertainty and to provide an overview of the principles and applications of microD in preclinical and clinical settings, an AAPS-FDA workshop took place in November 2005 in Nashville, TN, USA. Stakeholders from academia, industry and regulatory agencies presented their views on microD as a tool in drug research and development.

Measurement of GABA and glutamate in vivo levels with high sensitivity and frequency.

In the present protocol, we demonstrate a high-performance liquid chromatography (HPLC) system that enables detection of very low amounts of gamma-aminobutyric acid (GABA) (0.03 pmol) and glutamate (0.8 pmol). The HPLC system consists of two pumps, an electrochemical detector, a high-pressure six-way switching valve, a guard column, a microbore column, and a column oven. A microdialysis probe was implanted in the right parietal cortex in rats. Dialysates were collected every 5 min and were split into two equal aliquots for separate analysis of GABA and glutamate. After derivatization with o-phthalaldehyde (OPA), samples were isocratically separated and purified by the guard column. To make the peak of GABA or glutamate appear in an opportune place in a chromatogram, a six-way switching valve was used to control the eluate containing GABA or glutamate to be led to the microbore column and electrochemical detector. By the use of this system, decrease in extracellular concentration of GABA, which precedes the appearance of electrical discharge initiated by hyperbaric oxygen (HBO2) exposure, was detected by microdialysis at the time resolution of 5 min.

Effects of triiodothyronine and fluoxetine on 5-HT1A and 5-HT1B autoreceptor activity in rat brain: regional differences.

The thyroid hormone triiodothyronine (T3) augments and accelerates the effects of antidepressant drugs. Although the majority of studies showing this have used tricyclics, a few studies have shown similar effects with the selective serotonin re-uptake inhibitor (SSRI) fluoxetine. In this study we investigated the effects of fluoxetine (5 mg/kg), T3 (20 microg/kg) and the combination of these drugs, each administered daily for 7 days, on serotonergic function in the rat brain, using in vivo microdialysis. Fluoxetine alone induced a trend towards desensitization of 5-HT1A autoreceptors as shown by a reduction in the effect of 8-OH-DPAT to lower 5-HT levels in frontal cortex, and desensitized 5-HT1B autoreceptors in frontal cortex. The combination of fluoxetine and T3 induced desensitization of 5-HT1B autoreceptors in hypothalamus. Since there is evidence linking hypothalamic function and depression, we suggest that this effect may partly account for the therapeutic efficacy of the combination of an SSRI and T3.

Review of microdialysis in brain tumors, from concept to application: first annual Carolyn Frye-Halloran symposium.

In individuals with brain tumors, pharmacodynamic and pharmacokinetic studies of therapeutic agents have historically used analyses of drug concentrations in serum or cerebrospinal fluid, which unfortunately do not necessarily reflect concentrations within the tumor and adjacent brain. This review article introduces to neurological and medical oncologists, as well as pharmacologists, the application of microdialysis in monitoring drug metabolism and delivery within the fluid of the interstitial space of brain tumor and its surroundings. Microdialysis samples soluble molecules from the extracellular fluid via a semipermeable membrane at the tip of a probe. In the past decade, it has been used predominantly in neurointensive care in the setting of brain trauma, vasospasm, epilepsy,and intracerebral hemorrhage. At the first Carolyn Frye-Halloran Symposium held at Massachusetts General Hospital in March 2002, the concept of microdialysis was extended to specifically address its possible use in treating brain tumor patients. In doing so we provide a rationale for the use of this technology by a National Cancer Institute consortium, New Approaches to Brain Tumor Therapy, to measure levels of drugs in brain tissue as part of phase 1 trials.

Plant tissue-based chemiluminescence flow biosensor for determination of unbound dopamine in rabbit blood with on-line microdialysis sampling.

A novel plant tissue-based chemiluminescence (CL) biosensor for dopamine combined with flow injection analysis is presented in this paper. The potato roots act as molecular recognition elements. Dopamine is oxidized by oxygen under the catalysis of polyphenol oxidase in the tissue column to produce hydrogen peroxide, which can react with luminol in the presence of peroxidase of potato tissue to generate CL signal. The CL emission intensity was linear with dopamine concentration in the range of 1x10(-5)-1x10(-7) g/ml and the detection limit was 5.3x10(-8) g/ml (3sigma) with a relative standard deviation of 1.7%. Combined with microdialysis sampling, the biosensor was applied to monitor the variation of dopamine level in the blood of rabbit after the administration of dopamine to demonstrate the favorable resolution and reliability of the system for in vivo on-line monitoring.

Clinical cerebral microdialysis: a methodological study.

OBJECT: Clinical microdialysis enables monitoring of the cerebral extracellular chemistry of neurosurgical patients. Introduction of the technique into different hospitals' neurosurgical units has resulted in variations in the method of application. There are several variables to be considered, including length of the catheter membrane, type of perfusion fluid, flow rate of perfusion fluid, and on-line compared with delayed analysis of samples. The objects of this study were as follows: 1) to determine the effects of varying catheter characteristics on substance concentration; 2) to determine the relative recovery and true extracellular concentration by varying the flow rate and extrapolating to zero flow; and 3) to compare substance concentration obtained using a bedside enzyme analyzer with that of off-line high-performance liquid chromatography (HPLC). METHODS: A specially designed bolt was used to conduct two adjacent microdialysis catheters into the frontal cortex of patients with head injury or poor-grade subarachnoid hemorrhage who were receiving ventilation. One reference catheter (10-mm membrane, perfused with Ringer's solution at 0.3 microl/minute) was constant for all studies. The other catheter was varied in terms of membrane length (10 mm or 30 mm), perfusion fluid (Ringer's solution or normal saline), and flow rate (0.1-1.5 microl/minute). The effect of freezing the samples on substance concentration was established by on-line analysis and then repeated analysis after storage at -70 degrees C for 3 months. Samples assayed with the bedside enzyme analyzer were reassessed using HPLC for the determination of glutamate concentrations. CONCLUSIONS: Two adjacent microdialysis catheters that were identical in membrane length, perfusion fluid, and flow rate showed equivalent results. Variations in perfusion fluid and freezing and thawing of samples did not result in differences in substance concentration. Catheter length had a significant impact on substance recovery. Variations in flow rate enabled the relative recovery to be calculated using a modification of the extrapolation-to-zero-flow method. The recovery was approximately 70% at 0.3 microl/minute and 30% at 1 microl/minute (10-mm membrane) for all analytes. Glutamate results obtained with the enzyme analyzer showed good correlation with those from HPLC.

On-line monitoring of substrate delivery and brain metabolism in head injury.

Head injury is associated with complex pathophysiological changes in metabolism. The objective of the study was to investigate these changes by applying on-line bedside monitoring of cerebral metabolism using microdialysis. Following approval by the Local Ethics Committee and consent from the next of kin, a microdialysis catheter was inserted into the frontal cortex of patients with severe head injury. Twenty-one patients were studied for 102.3 +/- 26.9 hours (mean +/- 95% confidence interval; total 89.4 patient monitoring days). The overall cerebral glucose (mean of means) was 1.63 +/- 0.31 mM with periods of undetectable glucose recorded. The cerebral lactate and lactate/pyruvate ratio were 4.69 +/- 0.61 mM and 29.9 +/- 3.73 respectively. Patients who died (n = 4) or who were severely disabled (not proceeding to rehabilitation, n = 5) had a tendency towards lower glucose (1.39 +/- 0.35 mM), higher lactate (5.10 +/- 1.02 mM) and higher lactate/pyruvate ratios (35.5 +/- 7.67) compared to patients with good outcome (home or proceeding to rehabilitation, n = 12, glucose 1.80 +/- 0.49 mM, lactate 4.38 +/- 0.85 mM, lactate/pyruvate ratio 27.9 +/- 4.33). Trends in these metabolic parameters relating to outcome were identifiable. In the majority of patients, cerebral glutamate levels (overall mean of means 9.47 +/- 4.59 microM) were initially high and then declined to stable levels. Patients in whom the glutamate level remained elevated or in whom secondary rises in glutamate were seen had a poor outcome. The application of bedside analysis of microdialysis enables the progress of the patient to be monitored on-line. In addition to establishing trends of improving and deteriorating metabolism, the technique has the potential to monitor the effects of therapeutic manoeuvres on the biochemistry.

Intracerebral microdialysis of glutamate and aspartate in two vascular territories after aneurysmal subarachnoid hemorrhage.

Cerebral ischemia associated with subarachnoid hemorrhage may have severe consequences for neuronal functioning. The excitatory amino acid neurotransmitters glutamate and aspartate have been shown to be of particular importance for ischemia and ischemic neuronal damage. For seven patients who underwent early surgery for ruptured intracranial aneurysms, intracerebral microdialysis of glutamate and aspartate was performed to monitor local metabolic changes in the medial temporal (all seven patients) and subfrontal cortex (Patients 4 through 7). Samples were collected every 30 or 60 minutes, using an autosampler. The results show that extracellular glutamate and aspartate concentrations can rise to very high levels after surgery for subarachnoid hemorrhage and aneurysm. These increased levels of excitatory amino acids correlated well with the clinical course and neurological symptoms of the patients. Simultaneous sampling from two vascular territories (middle cerebral artery and anterior cerebral artery) also showed that a rise in extracellular glutamate and aspartate in one territory is not necessarily parallel with a rise in the other. The application of the microdialysis technique with an on-line assay system might be of value in the future for continuous monitoring of ischemic events to optimize treatment with, for example, blockers of glutamatergic neurotransmission.

Recovery characteristics of a rigid, nonmetallic microdialysis probe for use in an electromagnetic field.

It is well known that metal objects perturb electromagnetic fields. Therefore, a conventional metal microdialysis probe cannot be used to determine the bioeffects of electromagnetic radiation. Using fused-silica tubing, we developed an inexpensive nonmetallic, rigid microdialysis probe for use in electromagnetic radiation research or during magnetic resonance imaging. This probe has a concentric tube design, with the membrane length adjustable to the size of the area to be dialyzed. The probes tested had regenerated-cellulose membranes that were 3 mm in length. This report describes how to make this probe. Average relative recovery rates at flow rates of 2.0, 1.0, and 0.5 microliters/min were 21%, 27%, and 42%, respectively. These rates were slightly lower than the 30%, 42%, and 68% obtained with the commercially available metallic CMA10 microdialysis probe with a 3 mm membrane. This may be due to the fused-silica probe and CMA10 probe being made with different types of dialysis membranes.