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PubMed2005

LC-MS/MS-analysis of prostaglandin E2 and D2 in microdialysis samples of rats.

For the determination of prostaglandins in microdialysis samples, usually immunoassays are used. However, these assays may show cross-reactivity among various prostaglandins. To overcome this problem a specific method for the determination of prostaglandin E2 and D2 in rat microdialysis samples by using liquid chromatography-electrospay ionization-tandem mass spectrometry (LC-ESI-MS/MS) is described. Prostaglandin E2 and D2 were extracted from microdialysis samples with liquid-liquid extraction using deuterated prostaglandin D2, [2H4]-PGD2, as internal standard. Subsequently, prostaglandins were separated with a phenomenex Synergi Hydro-RP column and determined with a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface operating in negative ionization mode. The method showed a LLOQ of 25 pg/ml for prostaglandin E2 and 50 pg/ml for prostaglandin D2. The applicability of the method is shown in rat spinal cord microdialysis samples following peripheral nociceptive stimulation.

Animals↗
PubMed2006

Serial microPET measures of the metabolic reaction to a microdialysis probe implant.

Despite the widespread use of chronic brain implants in experimental and clinical settings, the effects of these long-term procedures on brain metabolism and receptor expression remain largely unknown. Under the hypothesis that intracerebral microdialysis transiently alters tissue metabolism, we performed a series of 18FDG microPET scans prior to and following surgical implantation of microdialysis cannulae. Parallel microPET measures using the competitive dopamine (DA) D2 receptor antagonist, 11C-raclopride, provided an assay of DA stability in these same animals. 18FDG scans were performed prior to microdialysis cannulation and again at 2, 12, 24, 48, 120, 168, 360 and 500 h (0.2, 0.5, 1, 2, 5, 7, 15 and 25 days). Separate animals received a sham surgery and the control group had no surgical intervention. For the first 24 h (scans at 2, 12 and 24 h post-surgery) uptake was reduced in both hemispheres. However, by 48 h, contralateral uptake had returned to pre-surgical levels. The striking finding was that from 48 to 500 h, the microdialysis cannulation produced widespread ipsilateral reductions in 18FDG uptake that encompassed the entire hemisphere. Despite the extent and persistence of these reductions, 11C-raclopride binding and ECF DA concentrations remained stable.

Animals↗
PubMed2006

Evaluation of an osmotic pump for microdialysis sampling in an awake and untethered rat.

The feasibility of using an osmotic pump in place of a syringe pump for microdialysis sampling in rat brain was investigated. The use of an osmotic pump permits the rat to be free from the constraints of the standard tethered system. The in vitro flow rates of a microdialysis syringe pump (set at 10.80 microl/h) and the osmotic pump (pump specifications were 11.35 microl/h) with no probe attached were compared, yielding results of 10.87 microl/h+/-1.7% and 10.95 microl/h+/-8.0%, respectively. The average of four flow rate experiments in vivo yielded R.S.D.s less than 10% and an average flow rate of 11.1 microl/h. Following the flow rate studies, in vivo sampling of neurotransmitters was accomplished with the osmotic pump coupled to a microdialysis probe implanted in the brain. Finally, after determination of basal levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the rats, the rats were dosed with benserazide followed by l-3,4-dihydroxyphenylalanine (l-DOPA). The results from the dosing study showed at least a 10-fold increase in compounds in the l-DOPA metabolic pathway (DOPAC and HVA) and a slight or no increase in 5-HIAA (serotonin metabolic pathway.) These results indicate that the osmotic pump is a viable alternative to the syringe pump for use in microdialysis sampling.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2004

Binding study of drug with bovine serum album using a combined technique of microdialysis with flow-injection chemiluminescent detection.

Microdialysis coupled with flow-injection chemiluminescence (FI-CL) has been developed to determine the binding parameters of a drug binding to protein by using antibiotic tetracycline hydrochloride binding to bovine serum albumin as a model system. The drug and protein were mixed in different molar ratios in 0.067 M phosphate buffer, pH 7.4, and incubated at 37 degrees C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 microL/min. The concentration of unbound tetracycline hydrochloride in the microdialysate was determined by FIA-CL. In vitro recovery of tetracycline hydrochloride under experimental conditions was 30.0%. The data obtained by the present microdialysis-FI-CL system was analyzed using the Scatchard analysis and Klotz plot. The results show that the Scatchard plot and Klotz plot are linear with good correlation coefficient, indicating a good agreement of the experimental data and to the theoretical equation. The FIA chemiluminescence system combined with microdialysis developed in this work demonstrated its use for determination of interaction between drug and protein by using relatively simple instrument.

Anti-Bacterial Agents↗
PubMed2005

An assessment of calibration and performance of the microdialysis system.

To improve the reliability of microdialysis measurements of tissue concentrations of metabolic substances, this study was designed to test both the performance and the internal validity of the microdialysis methods in the hands of our research group. The stability of the CMA 600 analyser was tested with a known glucose solution in 72 standard microvials and in 48 plastic vials. To evaluate if variation in sampling time makes any difference in sample concentration (recovery), sampling times of 10, 20 and 30 min were compared in vitro with a constant flow rate of 1 microl/min. For testing of sampling times at different flow rates, an in vitro study was performed in which a constant sample volume of 10 microl was obtained. With the no net flux method, the actual concentration of glucose and urea in subcutaneous tissue was measured. The CMA 600 glucose analysis function was accurate and stable with a coefficient of variability (CV) of 0.2-0.55%. There was no difference in recovery for the CMA 60 catheter for glucose when sampling times were varied. Higher flow rates resulted in decreased recovery. Subcutaneous tissue concentrations of glucose and urea were 4.4 mmol/l and 4.1 mmol/l, respectively. To conclude, this work describes an internal validation of our use of the microdialysis system by calibration of vials and catheters. Internal validation is necessary in order to be certain of adequate sampling times, flow rates and sampling volumes. With this in mind, the microdialysis technique is useful and appropriate for in vivo studies on tissue metabolism.

Calibration↗
PubMedDate not supplied

Determination of interstitial rocuronium concentrations in the muscle tissue of anesthetized dogs by microdialysis.

INTRODUCTION: The objective was to establish and validate a microdialysis technique for the quantification of interstitial concentrations of the neuromuscular blocker, rocuronium, in the muscle tissue of dogs under steady-state conditions. METHODS: The standard and combined retrodialysis approaches were used for in vivo microdialysis probe calibration. After induction of anesthesia with pentobarbital (30 mg/kg), the left femoral vein was cannulated and blood drawn for protein binding determination. Microdialysis probes were inserted in the muscle and calibrated in vivo, using vecuronium as the calibrator. Each dog received a short 2-min infusion followed by a 120-min infusion of rocuronium via the right jugular vein and three microdialysis samples were collected at steady-state during a 2-h period. Samples were stored at -70 degrees C until HPLC analysis. RESULTS: Using combined retrodialysis, rocuronium unbound interstitial (C(ISFu)) and venous plasma (C(pssuv)) concentrations are in good agreement; with a ratio C(ISFu)/C(pssuv) of 100+/-11%. Using standard retrodialysis, this ratio was 47+/-7%. CONCLUSIONS: Combined retrodialysis is a more reliable and accurate technique for quantitative assessment of rocuronium interstitial concentrations especially for lengthy anesthetic procedures. These findings have potential implications, as drug concentrations in the site of action would be more relevant for concentration-effect relation of muscle relaxants.

Androstanols↗
PubMed2000

In vivo study of tumor metabolism: an application of new multi-probe microdialysis system in the striatum of freely moving rats grafted with C6 cells.

The purpose of this study is to investigate the in vivo tumoral brain metabolism in free moving rats using microdialysis. Cells from C6 glioma cell line were inoculated in one striatum 15 days before the microdialysis experimentation. Then, using a new system allowing perfusion of several microdialysis probes in free moving rat, normalised dialysate levels of glucose, lactate and pyruvate were monitored in both glioma and control striatum. At the end of the procedure, animals were sacrificed for histological study. Data shows that probe functioning is similar in both tissues. The results for normalised glucose level were in striatum control: 2.14 mM, in tumoral striatum: 1.71 mM (P>0.1); for lactate, respectively, 0.86 and 1.65 mM (P<0.05) and for pyruvate, respectively, 65.56 and 140. 94 microM (P<0.05). This data clearly shows a significant increase of pyruvate and lactate in tumoral striatum compared to normal striatum, correlating previous in vitro studies on glioma metabolism. We conclude that this microdialysis technique is of value in tumoral brain and could constitute an interesting tool for a better understanding of glioma metabolism.

Animals↗
PubMed1997

Extracellular glutamate and dopamine measured by microdialysis in the rat striatum during blockade of synaptic transmission in anesthetized and awake rats.

We investigated the effect of high dose tetrodotoxin (TTX) on microdialysis measurements of extracellular striatal glutamate and dopamine in normal female rats. Both halothane-anesthetized rats with acutely implanted microdialysis probes and awake rats with microdialysis probes implanted for 24 h were tested. Glutamate levels in awake rats were 45% higher than those of anesthetized rats. Extracellular glutamate remained TTX-insensitive regardless of TTX concentration, anesthesia, or time lapsed after probe implantation. In contrast, TTX reduced dialysate dopamine in all TTX concentrations tested. We speculate that the lower glutamate levels in anesthetized rats reflect the effect of anesthesia. Because glutamate is involved, either as a reactant or a product in a variety of reactions critical to intermediary metabolism in the brain, basal dialysate glutamate levels might indirectly reflect brain metabolism. Further, we conclude that extracellular glutamate collected during non-stimulated conditions is TTX-insensitive. The fact that glutamate levels are TTX-independent does not rule out that glutamate is synaptic in origin but rather demonstrates that it is not nerve impulse-dependent. However, the brain interstitial glutamate pool accessible to the microdialysis probe during control conditions is most likely isolated from the synapse, and therefore does not impose a neurotoxic potential.

Anesthesia↗
PubMed1999

Response of amygdalar norepinephrine to footshock and GABAergic drugs using in vivo microdialysis and HPLC.

These studies used in vivo microdialysis and high-performance liquid chromatography (HPLC) to examine levels of norepinephrine (NE) within the amygdala in response to both footshock and GABAergic compounds. In Experiment 1, microdialysis probes were inserted into a previously implanted guide cannula aimed at the amygdala and the level of NE was examined in response to footshock. A 0.55 mA (2 s) footshock induced a significant increase in NE levels when the microdialysis probe was located within the amygdala; levels of NE did not differ from baseline in rats with microdialysis probes located outside the amygdala. Experiment 2 examined the effects of the GABAergic antagonist, picrotoxin, the GABAergic agonist, muscimol, and saline on NE levels in the amygdala. Rats treated systemically with picrotoxin showed a dramatic increase in levels of NE within the amygdala. In contrast, systemic injection of muscimol resulted in decreased levels of NE. These findings are consistent with the hypothesis that drugs that are capable of modulating memory do so by altering levels of NE within the amygdala.

Amygdala↗
PubMed1999

Determination of striatal extracellular gamma-aminobutyric acid in non-hibernating and hibernating arctic ground squirrels using quantitative microdialysis.

This study determined extracellular concentrations of gamma-aminobutyric acid ([GABA](ecf)) in striatum of non-hibernating and hibernating arctic ground squirrels to test the hypothesis that an increase in [GABA](ecf) was associated with profound CNS depression during hibernation. Quantitative microdialysis procedures were employed to circumvent the effects of low temperature on the relative recovery of the analyte across the dialysis membrane and yielded for the first time quantitative in vivo estimates of [GABA](ecf) in any brain region or any species. Laboratory housed, wild caught Arctic ground squirrels (Spermophilus parryii) were implanted intraperitoneally with radio transmitters that enabled the telemetric monitoring of activity and core body temperature (T(b)) and bilaterally implanted with cranial guide tubes that enabled the implantation of microdialysis probes into the striatum. Striatal [GABA](ecf) was determined in unrestrained, non-hibernating ground squirrels (T(b) range 34.7-38.9 degrees C) and hibernating ground squirrels (T(b) range 2.9-3.9 degrees C) using extrapolation to zero flow and very slow flow microdialysis techniques. The results show that [GABA](ecf) in non-hibernating squirrels was 73 nM and this level was decreased by approximately 50% during hibernation thereby suggesting that an increase in [GABA](ecf) does not play a major role in CNS depression during hibernation. The reduction of [GABA](ecf) parallels a decrease in plasma and CSF [glucose] and may be related to a decrease in GABA synthesis or reduced voltage dependent release. This paper demonstrates that measurement of extracellular concentrations of neurotransmitters in animals with vastly different body temperatures is possible using microdialysis techniques of extrapolation to zero flow or very slow flow rates that enable 100% recovery. Such quantitative techniques may prove valuable in the study of the neurochemistry of the cerebral mechanisms of hibernation and tolerance to cerebral ischemia exhibited by hibernating animals.

Adaptation, Physiological↗
PubMed2000

On-line microdialysis-ion chromatographic determination of inorganic anions in olive-oil mill wastewater.

A fully automated method is presented for the determination of inorganic anions in olive-oil mill effluents using on-line dialysis-ion chromatography. The wastewater is first of all sonicated at room temperature to make it homogeneous, then diluted and microdialized. Most of the organic load of the effluents is removed in a few minutes without using reagents, while soluble anion quantitation remains unaffected. The clear solution is analyzed for the inorganic anions content by direct injection on to an ion chromatograph equipped with a conductivity detector. In the absence of standards, the separation efficiency of microdialysis has been investigated by spiking wastewater samples as well as standard oil emulsions with varying amounts of inorganic anions and subjecting them to microdialysis for different periods of time prior to performing instrumental analysis. Excellent spike recoveries and low relative standard deviations are obtained for all the anions if a 10 min microdialysis time is overcame. Chloride, nitrite, nitrate, phosphate and sulphate are not affected by the microdialysis procedure and their recovery is between 96 and 104% in wastewater as well as in standard oil emulsion. Calibration plots are linear over about two orders of magnitude. The dialysis membrane has been replaced after more than 100 analyses. The UV photolysis pre-treatment of the same sample evidences the different information that can be obtained by the two sample pre-treatment procedures.

Anions↗
PubMed2001

Simultaneous monitoring of extracellular glucose, pyruvate, lactate and glutamate in gerbil cortex during focal cerebral ischemia by dual probe microdialysis.

The aim of this study was to monitor dynamic changes in energy-related metabolites in the cortex of gerbils subjected to cerebral ischemia by a dual probe microdialysis technique. Focal cerebral ischemia was produced in anesthetized gerbils by occlusion of the right common carotid artery and the right middle cerebral artery for 60 min. Two microdialysis probes were inserted into both sides of the cortex to simultaneously monitor extracellular glucose, lactate, pyruvate and glutamate. Dynamic and comparative changes in these analytes, on the ipsilateral and contralateral sides of the brain, were simultaneously monitored by liquid chromatography and a microdialysis analyzer. The present study demonstrated decreases in glucose and pyruvate, increases in lactate and glutamate on the ipsilateral side whereas all analytes remain constant on the contralateral side of cortex during cerebral ischemia. In vitro recovery of each microdialysis probe was performed to ensure the quality of experiments. The detection limits of pyruvate, glutamate, lactate and glucose were 0.2, 1.0, 2.0 and 20 microM, respectively. The intra- and inter-assay correlations were less than 5% in standard mixtures and pooled brain dialysates.

Animals↗
PubMed1998

Coupling on-line brain microdialysis, precolumn derivatization and capillary electrophoresis for routine minute sampling of O-phosphoethanolamine and excitatory amino acids.

In previous papers, we described the analysis of excitatory amino acids (EAAs) and catecholamines in microdialysis samples using capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD). In the present paper, we report that an automated analysis of such samples can be easily achieved by on-line coupling of the microdialysis probe with a continuous flow derivatization system and a commercially available CE-LIFD apparatus. Because of the short analysis time (less than 2 min) and high separation efficiency (100-200,000 theoretical plates), high temporal resolution of microdialysis (minute range) is preserved as compared to off-line systems, while both EAAs and O-phosphoethanolamine (PEA) can be simultaneously detected. This new method has been applied to the measurement of these compounds in microdialysis samples from hippocampal slice cultures and striatum of anesthetized rats. Extracellular concentrations of EAAs, but not PEA, increased during perfusion of a solution containing high K+ or a glutamate uptake inhibitor. However, after in vitro ischemia on hippocampal slices, both EAAs and PEA concentrations increased, but with different temporal patterns.

Animals↗
PubMed1999

Simultaneous blood and biliary sampling of esculetin by microdialysis in the rat.

Biliary excretion and intestinal reabsorption in enterohepatic circulation play major dispositional roles for some drugs. To circumvent multiple blood sampling and interruption of enterohepatic circulation in conventional biliary cannulation, the present study utilized the minimally invasive sampling technique of microdialysis in pharmacokinetics and biliary excretion studies. Microdialysis probes were inserted into the jugular vein and bile duct in the anesthetized rat for simultaneous and continuous sampling following intravenous administration of esculetin, a bioactive coumarin derivative. Placements of the microdialysis probes were designed to minimize obstruction to normal flows of the body fluids. Separation and quantitation of esculetin in the dialysates were achieved using high performance liquid chromatography (HPLC) coupled to UV detection. The results indicated higher drug concentrations in the bile than in the blood, suggesting active biliary excretion. The study also provided an example of successful application of in vivo microdialysis as an interesting and feasible alternative for pharmacokinetics and biliary drug excretion studies.

Animals↗
PubMed2000

Microdialysis assessment of shock wave lithotripsy-induced renal injury.

OBJECTIVES: Shock wave lithotripsy (SWL) is the primary treatment modality for managing the majority of symptomatic renal calculi. However, the fundamental mechanisms for stone fragmentation and the resultant morphologic changes that occur are not fully understood. Furthermore, a thorough understanding of the complex biologic pathways involved in SWL-induced renal injury does not exist at present. To elucidate the biologic processes involved in tissue injury after SWL, an animal model was designed to mimic the pathogenesis of high-energy SWL in humans. METHODS: Juvenile female swine were anesthetized, and a midline laparotomy incision was performed to expose the right kidney. Using an introducer apparatus, a microdialysis probe was placed into the renal parenchyma of the right kidney lower pole and a tunnel was generated to exit the distal ends of the inlet and outlet tubing outside the body. After a 72-hour postoperative recovery period, SWL was performed to the lower pole renal region of the kidney, as a microdialysis pump continuously infused dialysate through the inlet tubing. Microdialysis fluids were collected during SWL, and lipid peroxidation, as measured by conjugated diene concentrations, was monitored. RESULTS: All microdialysis probes remained patent for a total of 2000 shock waves. A significant elevation in conjugated diene levels was observed in the SWL versus untreated kidneys after 1000 shock waves were administered (P <0.02). CONCLUSIONS: This animal model is unique in that it represents the first system for the real-time collection of renal interstitial fluids during SWL. Analysis of this fluid may provide insight into the physiologic mechanisms responsible for shock wave-induced renal injury.

Animals↗
PubMed1996

Allergen-induced histamine release in intact human skin in vivo assessed by skin microdialysis technique: characterization of factors influencing histamine releasability.

BACKGROUND: The purposes of the study were to characterize allergen-induced histamine release in intact human skin in vivo by using a novel microdialysis technique and to study covariates influencing histamine releasability. METHODS: Hollow microdialysis fibers were inserted into the upper dermis in 15 timothy-sensitivity subjects. Up to 12 fibers were inserted in each subject. Each fiber was perfused with Krebs-Ringer's solution at a rate of 3.0 microliters/min. Three to four serial dilutions of allergen were applied to the skin by intracutaneous injections or skin prick test above individual fibers. Samples were collected in two 2-minute fractions before skin challenge and in 10 consecutive samples for 20 minutes after skin challenge. Histamine was assayed spectrofluorometrically. RESULTS: A significant dose-response relationship for histamine release was demonstrated with intracutaneous tests and skin prick tests. The time to reach peak histamine release after an intracutaneous test was 4 to 8 minutes, compared with 12 to 14 minutes for a skin prick test. Histamine release correlated significantly with wheal size. Intrasubject coefficient of variation on histamine release was about 20%. A substantial intersubject variation in histamine releasability was observed. Seventy to seventy-five percent of the variation could be accounted for by a combination of gender, total and allergen-specific IgE, and an in vitro basophil histamine release test. CONCLUSIONS: Using a skin microdialysis technique, we have described in detail histamine release in intact human skin by allergen. The microdialysis method proved to be a reproducible technique for monitoring histamine release in allergic skin reactions and for studying histamine releasability of skin mast cells in vivo.

Adult↗
PubMed1997

Methodological considerations of intracerebral microdialysis in pharmacokinetic studies on drug transport across the blood-brain barrier.

For the study of the pharmacokinetics of drugs in the brain a number of in vivo techniques is available, including autoradiography, imaging techniques, cerebrospinal fluid sampling and in vivo voltammetry, which all have their specific advantages and limitations. Intracerebral microdialysis is a relatively new in vivo technique. It permits monitoring of local concentrations of drugs and metabolites at specific sites in the brain which makes it an attractive tool for pharmacokinetic research. In the use of this technique a number of factors should be considered. These include: type of probe, surgical trauma, post-surgery interval, perfusion flow rate, as well as composition and temperature of the perfusion medium. In particular in studies on drug transport across the blood-brain barrier (BBB), effects of insertion of the probe on BBB functionality is important. It appears that BBB functionality is not significantly affected if surgical and experimental conditions are well-controlled. The relationship between dialysate concentrations and those in the extracellular fluid of the periprobe tissue, the recovery of the drug, depends on periprobe processes governing the actual concentration of the drug at that site. These include extracellular-microvascular exchange, metabolism, and diffusion of the drug. Several methods have been proposed to determine recovery values. In particular the no net flux method and the extended no net flux method are useful in practice. Several microdialysis studies on BBB transport of drugs are presented showing that intracerebral microdialysis is capable to assess local BBB transport profiles. Compared with other in vivo techniques, intracerebral microdialysis is the only (affordable) technique that offers the possibility to monitor local BBB transport of drugs in unanaesthetized animals, under physiological and pathological conditions.

Animals↗
PubMed2000

Comparison by microdialysis of striatal L-DOPA after its systemic administration in rats with probes implanted acutely or through a guide cannula.

Different methods of microdialysis probe implantation are utilized according to the purpose and needs of each particular study. However, very few experiments have systematically examined whether these different techniques have an impact on the obtained data. In the present study we examined the influence of two different microdialysis methods - acute probe implantation vs. insertion into a preimplanted guide cannula - on the striatal extracellular availability of systemically administered L-DOPA. Furthermore, we monitored the effects of L-DOPA administration on dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). In rats that received a guide cannula 4 days prior to probe insertion and microdialysis, extracellular L-DOPA concentrations increased to concentrations that were about nine times higher than in rats with acute implantation of a microdialysis probe. Extracellular DOPAC concentrations were also higher in the chronic preparations but dopamine concentrations showed no differences between groups. Our results suggest that the observed differences may be due to inflammatory disruption of the BBB following chronic implantation of a guide cannula.

3,4-Dihydroxyphenylacetic Acid↗