Microdialysis Extraction Research Articles

In Situ Inner Lumen Attachment of Heparin to Poly(ether sulfone) Hollow Fiber Membranes Used for Microdialysis Sampling.

An in situ chemical surface modification method to attach heparin to the inner lumen of a single hollow fiber poly(ether sulfone) (PES) membrane incorporated into a commercial microdialysis sampling device is described. The immobilization process uses gentle, room-temperature conditions with the enzyme laccase (E.C. 1.10.3.2) and 4-hydroxybenzoic acid (4HBA). The resulting functionalized inner membrane surface with a carboxylic acid functional groups allowed for (1-ethyl-3-(3-(dimethylamino)propyl) carbodimide)/ N-hydroxysuccinimide) EDC/NHS chemistry to attach heparin to the membrane surface. X-ray photoelectron spectroscopy measurements suggested successful attachment of 4HBA polymers and heparin onto the PES membrane. The microdialysis extraction efficiency after membrane surface modification was measured with model compounds fluorescein isothiocyanate (FITC)-labeled dextrans and lysozyme and the cytokines acidic fibroblast growth factor (aFGF) and CXCL1 (KC/GRO). This work demonstrates an in situ method to modify commercially available PES hollow fiber microdialysis membranes with amine or carboxylic acid functional groups.

Effects of tissue trauma on the characteristics of microdialysis zero-net-flux method sampling neurotransmitters

Microdialysis has been used for studying neurochemistry in brain regions that respond to afferent inputs or administered drugs. As the knowledge derived from and concerning microdialysis grows, so do the concerns over its invasiveness and, hence, the credibility of resulting data. Recent experimental and theoretical studies impugned the validity of the microdialysis zero-net-flux (ZNF) method in measuring brain extracellular neurotransmitters, suggesting that the tissue trauma resulting from probe implantation seriously compromises its worth. This paper developed a theoretical model to study the influences of two categories of tissue trauma on microdialysis ZNF operation: (1) morphological alterations in tissue extracellular structure and (2) physiological impairment of neurotransmitter release and uptake processes. Model results show that alterations of tissue extracellular structure negligibly affect the accuracy of the ZNF method in determining the basal level of extracellular neurotransmitter but do affect the fundamental characteristics of microdialysis: the extraction efficiency and relative recovery. An inhibited or damaged neurotransmitter uptake process always decreases the efficiency of microdialysis extraction, but rise of the relative recovery of neurotransmitters with the same uptake inhibition/damage occurs only when there is far more damage to the neurotransmitter release than to the uptake process in the tissue. A criterion for this rising trend of microdialysis relative recovery is discussed in terms of trauma parameters and neurotransmitter uptake inhibition.

Improving microdialysis extraction efficiency of lipophilic eicosanoids

Microdialysis recovery of the lipophilic analytes prostaglandin B 2, leukotriene B 4 and C 4 was studied in vitro. Relative recovery (RR) through different commercially-available microdialysis probes for prostaglandin B 2 and leukotrienes was examined using different flow rates. The enhancing effect at different concentrations of binding agents such as α, β, γ-cyclodextrins (α, β, γ-CD) on the microdialysis RR for different eicosanoids was evaluated. Small organic molecules such as ethanol, propylene glycol and dimethyl sulfoxide (DMSO) were studied in terms of their effect on enhancing RR. Inclusion of arachidonic acid in either the perfusion fluid or the sample medium caused the microdialysis RR for these hydrophobic analytes to be increased.

Theory relating in vitro and in vivo microdialysis with one or two probes.

In this paper, we further develop the general theory of microdialysis by extending the linear model of Bungay et al. to provide a theoretical basis for in vitro and in vivo microdialysis. Specifically, we considered the effect of active clearance processes on in vivo microdialysis, and thereby elaborated the theory of Benveniste et al. to endogenous compounds. We examined the use of steady state tissue diffusion resistance with negligible clearance processes to interpret microdialysis data. The influence of the tissue properties on the in vitro and in vivo recoveries in dual-probe microdialysis was analyzed and we simulated the effect of the operating parameters on dual probe microdialysis performance. We estimated that the minimum clearance rate constant detectable by microdialysis in a quasi-steady state is about 5.5 x 10(-5) s(-1). This minimum rate constant establishes a criterion, below which inhibition of the active clearance processes does not show detectable influences on the microdialysis extraction efficiency.

In vivo cellular uptake of glutamate is impaired in the rat hippocampus during and after transient cerebral ischemia: A microdialysis extraction study

Using microdialysis in CA1 of the rat hippocampus, we studied the effect of transient cerebral ischemia on in vivo uptake and on extracellular levels of glutamate during, and at different time points after ischemia. 3H-D-aspartate (test substance), and 14C-mannitol (reference substance), were added to the dialysis perfusate, and the cellular extraction of 3H-D-aspartate was calculated from scintillation analysis of fractionated dialysate samples. The extraction of 3H-D-aspartate was studied both in a tracer like condition with a perfusate concentration of 0.2 μM, and in a condition of high saturation level, with 1.0 mM D-aspartate added to the perfusate. In between radioisotope perfusions, dialysate was sampled for analysis of amino acid content by HPLC. During ischemia, extraction of 3H-D-aspartate (0.2 μM) declined to a maximum reduction of 68%. In the hours after ischemia, extraction of 3H-D-aspartate (0.2 μM) was decreased by 32%. In the days after ischemia, there was a progressive decline in extraction of 3H-D-aspartate (1.0 mM), reaching a reduction of 89% on Day 4 after ischemia. Extracellular glutamate remained at control levels at all time points after ischemia. The present study is the first to investigate uptake of glutamate in the intact rat brain in relation to cerebral ischemia. Evidence is provided that uptake of Glu is restrained during ischemia, and that in the hours after ischemia, the extracellular turnover of glutamate is decreased. In the course of the days after ischemia, degeneration of CA1 pyramidal cells occurs concomitantly with a progressive decline in glutamate transport ability, possibly of pathogenetic importance to CA1 pyramidal cell loss. J Neurosci. Res. 66:1118–1126, 2001. © 2001 Wiley-Liss, Inc.

In vivo cellular uptake of glutamate is impaired in the rat hippocampus during and after transient cerebral ischemia: a microdialysis extraction study.

Using microdialysis in CA1 of the rat hippocampus, we studied the effect of transient cerebral ischemia on in vivo uptake and on extracellular levels of glutamate during, and at different time points after ischemia. (3)H-D-aspartate (test substance), and (14)C-mannitol (reference substance), were added to the dialysis perfusate, and the cellular extraction of (3)H-D-aspartate was calculated from scintillation analysis of fractionated dialysate samples. The extraction of (3)H-D-aspartate was studied both in a tracer like condition with a perfusate concentration of 0.2 microM, and in a condition of high saturation level, with 1.0 mM D-aspartate added to the perfusate. In between radioisotope perfusions, dialysate was sampled for analysis of amino acid content by HPLC. During ischemia, extraction of (3)H-D-aspartate (0.2 microM) declined to a maximum reduction of 68%. In the hours after ischemia, extraction of (3)H-D-aspartate (0.2 microM) was decreased by 32%. In the days after ischemia, there was a progressive decline in extraction of (3)H-D-aspartate (1.0 mM), reaching a reduction of 89% on Day 4 after ischemia. Extracellular glutamate remained at control levels at all time points after ischemia. The present study is the first to investigate uptake of glutamate in the intact rat brain in relation to cerebral ischemia. Evidence is provided that uptake of Glu is restrained during ischemia, and that in the hours after ischemia, the extracellular turnover of glutamate is decreased. In the course of the days after ischemia, degeneration of CA1 pyramidal cells occurs concomitantly with a progressive decline in glutamate transport ability, possibly of pathogenetic importance to CA1 pyramidal cell loss.

Enhanced Microdialysis Extraction Efficiency of Ibuprofen in Vitro by Facilitated Transport with β-Cyclodextrin

A novel approach to increase microdialysis recovery (extraction efficiency, E(d)) by facilitated transport through the microdialysis membrane is described. This new approach facilitates mass transport into the microdialysis probe by inclusion of a complexation agent in the microdialysis perfusion fluid. In these studies, β-cyclodextrin (β-CD) (0.25-2.0 w/v%) was included in the microdialysis perfusion fluid consisting of a Ringer's solution (155 mM NaCl, 4.0 mM KCl, 2.4 mM CaCl(2)). β-CD forms known inclusion complexes with 2-(4-isobutylphenyl)propionic acid (ibuprofen). Ibuprofen E(d) was significantly enhanced (1.5-2.0 times) through different microdialysis membrane materials. The effect of microdialysis membrane material (polycarbonate/polyether, AN-69, cuprophan), pH, β-CD concentration, and ibuprofen concentration on the E(d) was examined. Only the polycarbonate/polyether membrane was able to give an E(d) greater than 100%. In general, a maximum increase in E(d) was found when 0.5 w/v% β-CD was included in the perfusion fluid. Variations in the ibuprofen concentration external to the microdialysis probe did not significantly change E(d) when 0.5 w/v% β-CD was included in the perfusion fluid. In contrast to the ibuprofen data, β-CD inclusion in the microdialysis perfusion fluid did not affect antipyrine E(d). Antipyrine does not form known inclusion complexes with β-CD. The ability of β-CD to increase microdialysis E(d) is explained by facilitated transport.

Methods and issues in microdialysis calibration

A review of calibration in microdialysis is presented. This review covers the methodological aspects of microdialysis including how physico-chemical parameters such as analyte diffusion coefficient, kinetics, volumetric flow rates, and membrane length affect calibration. The effects of diffusion versus diffusion coupled with chemical reaction are discussed in relationship to microdialysis extraction fraction and calibration. Resistance theory is used throughout to illustrate how diffusion and chemical reaction affect microdialysis calibration. In vivo calibration methods such as mass transfer coefficient, no-net flux method (Lönnroth method), internal standards (retrodialysis), in vivo delivery, and slow perfusion method are discussed. Applications of these various in vivo calibration methods are also included. Mathematical models are presented as a means to help understand the physical parameters that affect microdialysis calibration. Finally, the challenges in interpreting results from transient methods such as trapping free radicals and in situ metabolism are discussed.

Effect of neostigmine on concentration and extraction fraction of acetylcholine using quantitative microdialysis

A quantitative microdialysis method was used to determine the effect of local perfusion of 0, 100, 200, and 300 nM neostigmine (NEO) on acetylcholine (ACh) extracellular concentration and microdialysis extraction fraction ( E d) in the striatum of the rat. Because of the efficiency of AChE, the inhibition of this enzyme is expected to result in a substantial increase in ACh levels and a decrease in the E d of ACh. The extracellular concentration of ACh increased linearly with increasing concentrations of NEO. The control ACh concentration was determined to be 18.4±11.8 nM ( n=10; mean±S.E.M.) The ACh extracellular concentration for the remaining groups was determined to be 173±14 nM ( n=5), 329±52.5 nM ( n=13), and 581±109 nM ( n=10) for the 100, 200, and 300 nM NEO groups, respectively. Perfusion with 300 nM NEO resulted in a significant reduction in the E d of ACh (64.5±3.5% vs. 43.6±7.5%, P<0.05). In contrast to ACh, perfusion with 0, 1, and 10 μM hemicholinium-3, an inhibitor of high-affinity choline uptake, increased choline levels but did not affect the E d of choline. The effects on E d are consistent with E d being influenced by rapid clearance mechanisms.

Quantitative microdialysis of serotonin and norepinephrine: Pharmacological influences on in vivo extraction fraction

The present study was designed to investigate the potential influence of various neuronal processes including uptake, release and metabolism, on the in vivo microdialysis extraction fraction ( E d) of serotonin (5HT) and norepinephrine (NE). Paroxetine administration decreased the E d of 5HT in the nucleus accumbens from 24 ± 3 to 18 ± 0.2% ( p < 0.05). Similarly, desipramine infusion reduced the NE E d from 35 ± 2 to 26 ± 1% ( p < 0.05). However, perfusion with pargyline or tetrodotoxin had no effect on the E d of either 5HT or NE. Perfusion with agonists for the 5HT, α-adrenergic, D 2 and histamine receptors had no effect on the E d of 5HT. In the same manner, perfusion with the α-adrenergic agonists, methoxamine or clonidine, did not affect the E d of NE. These data are in agreement with experimental results obtained for dopamine in the nucleus accumbens and the theory of quantitative microdialysis which predicts that only changes in the rate of clearance will change E d of monoamines. These results suggest that, like DA, changes in the E d for 5HT or NE are indicative of changes in the reuptake of these neurotransmitters. The results also indicate that pharmacological agents which do not affect uptake have no effect on the extraction fraction.

Anesthetics decreased the microdialysis extraction fraction of norepinephrine but not dopamine in the medial prefkontal cortex

We evaluated the effects of chloral hydrate and pentobarbital sodium on the basal extracellular concentrations of dopamine (DA) and norepinephrine (NA) as well as their in vivo extraction fraction (relative recovery) at the medial prefrontal cortex (mPFC) in rats by using zero-net flux microdialysis method. Adult, male Sprague-Dawley rats anesthetized with either chloral hydrate (400 mg/kg, i.p., with 80 mg/kg i.v. supplements) or pentobarbital sodium (50 mg/kg, i.p., with 10 mg/kg i.v. supplements) were used as treatment groups. Conscious rats were used as a control group. The basal extracellular concentration and in vivo recovery of DA in the conscious group were 2.38 +/- 0.70 nM and 41 +/- 6%. In comparison with the chloral hydrate group (1.51 +/- 0.55 nM and 41 +/- 9%) and the pentobarbital sodium group (2.81 +/- 1.20 nM and 42 +/- 4%), there were no significant effects of anesthesia on the basal extracellular concentration and the in vivo recovery of DA at the mPFC. Additionally, the basal extracellular concentration and the in vivo recovery of NA in the conscious group were 1.59 +/- 0.37 nM and 51 +/- 8%. There also were no significant differences of the basal extracellular concentration of NA among these three groups (chloral hydrate group: 4.38 +/- 1.39 nM; pentobarbital group: 3.67 +/- 0.90 nM). However, the conscious group had a higher in vivo recovery than the two anesthetized groups (chloral hydrate group: 16 +/- 2%; pentobarbital group: 27 +/- 5%).(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of microdialysis extraction fraction of cocaine by the no net flux method under high, low, and zero steady-state cocaine concentrations in rats.

The effect of different steady-state cocaine concentration maintenance on the microdialysis extraction fraction of cocaine (in situ recovery; Ed) in the medial prefrontal cortex (mPFC) was examined. Male, adult Sprague-Dawley rats anesthetized with chloral hydrate were used. All rats were randomly divided into three groups and were given cocaine i.v. infusion of either 0.6 mg/kg/min (group A), 0.3 mg/kg/min (group B), or 0.0 mg/kg/min (group C). A steady-state cocaine concentration in the blood was obtained for both A and B groups. They were 24.3 +/- 2.3 microM, respectively. We then applied the no net flux microdialysis method to determine the Ed of cocaine in vivo for all three groups. Results revealed that there was no significant difference of Ed under these three different infusion regimens (group A: 16 +/- 2%; group B: 17 +/- 1%; group C: 21 +/- 2%). The steady-state cocaine concentration in the mPFC was also estimated to be 17.7 +/- 1.3 microM, 11.3 +/- 2.0 microM, and 0.7 +/- 0.6 microM, respectively. We then concluded that the Ed of cocaine in the mPFC is invariable while the steady-state cocaine concentration in the mPFC was ranging from 0 to 18 microM. This information is useful in the cocaine pharmacokinetic study since scientists are concerned about the variations of Ed while cocaine concentration in the mPFC is changing.

The effect of inhibition of synthesis, release, metabolism and uptake on the microdialysis extraction fraction of dopamine

The present study was designed to determine the effects that synthesis, release, metabolism and uptake have on the in vivo extraction fraction (relative recovery) of dopamine (DA) in the nucleus accumbens of the rat. The extraction fraction and extracellular concentration of DA were established for rats that were perfused with artificial cerebrospinal fluid (aCSF) with or without substances inhibiting synthesis (100 μM α-methylparatyrosine (α-MPT)), release (1 μM tetrodotoxin (TTX)), uptake (1 μM, 20 μM cocaine or 1 μM GBR-12909) or metabolism (100 μM tropolone or 100 μM pargyline) with DA concentrations ranging from 0 to 200 nM. Inhibiting synthesis with α-MPT or release with TTX had no effect on the extraction fraction of DA. Inhibiting intracellular or extracellular metabolism with pargyline or tropolone, respectively, did not cause any changes in the extraction fraction. However, inhibiting uptake with 20 μM cocaine or 1 μM GBR-12909 decreased the extraction fraction by one-third and one-half, respectively. These results provide evidence that uptake is the primary neuronal process affecting the extraction fraction of DA in the nucleus accumbens and indicate that the extraction fraction may be useful as an index of DA uptake in vivo.

Effect of tetrodotoxin and potassium infusion on microdialysis extraction fraction and extracellular dopamine in the nucleus accumbens

The effects of tetrodotoxin (TTX) and K + perfusion on extracellular DA concentration (DA ext) and extraction fraction (also known as relative recovery or E d) were studied using a method for quantitative microdialysis under transient conditions. TTX administered through the microdialysis probe decreased DA ext with no effect on E d. Perfusion with high K + increased DA ext, with no initial effect on E d. However, a decrease in E d was observed under prolonged depolarization. These data provide further evidence that E d may change over the course of drug administration. This method of quantitative microdialysis is a reliable means for measuring changes in DA ext independently of possible changes in microdialysis probe efficiency.