Net Flux Method Research Articles

Small-Signal Modeling of Mutually Coupled Switched Reluctance Motor

A small-signal model based on the net flux method is developed for mutually coupled switched reluctance machines (MCSRMs) to facilitate the design and analysis of the current controller for the drive. The underlying methodology of using net flux allows MCSRM modeling on a per-phase basis which reduces the time involved in characterizing the motor and the number of machine model parameters. A comprehensive theory of modeling the machine and extracting the machine parameters with analytical derivations is provided. Furthermore, a complete design procedure and analysis for a proportional-integrator controller is presented. The derived MCSRM small-signal model is useful to evaluate the controller's current tracking performance, disturbance rejection capability, and response features over a wide speed range. Simulation analysis and experimental results for a 3 phase, 150 W, full-pitched MCSRM drive system are provided both for small-signal model validation and current controller performance evaluation.

Modeling of Mutually Coupled Switched Reluctance Motors Based on Net Flux Method

Modeling and accurate estimation of mutual flux in switched reluctance motors are complex due to interphase flux interactions, magnetic saturation, and harmonics. The mutual flux contributes significantly toward torque production of mutually coupled switched reluctance motors (MCSRMs); therefore, their accurate modeling directly influences the control design. In this article, a net-flux-based machine model is proposed to model MCSRMs. The model utilizes dual-phase excitation and multiphase excitation methods to generate the flux lookup tables from the finite-element model of the MCSRM. The modeling process is simple, as it does not require the segregation of the self- and mutual flux components, and machine performance can be predicted accurately using the net flux. The proposed modeling method is validated by implementing it for a fully pitched and concentrated-wound MCSRM and evaluating accuracy against the finite-element method. Details of the developed model are presented along with its comparison with existing methods. A prototype of a fully pitched MCSRM has been built, and the model is validated experimentally. Results show that the proposed model has excellent accuracy even under saturated operating conditions.

Determination of Whiteners in Cosmetics by Microdialysis and High-Performance Liquid Chromatography

A simple method was developed for the determination of hydrophilic whitening agents (ascorbyl glucoside, magnesium ascorbyl phosphate, arbutin, hydroquinone, and kojic acid) in bleaching cosmetics by high performance liquid chromatography with electrochemical detection. The samples were prepared using microdialysis and were separated within ten minutes using isocratic elution. Linear calibration was achieved for concentrations between 0.10 and 10.00 micromolar. The limits of detection were from 0.6 to 2.0 nanomolar; the recoveries were between 91.43 and 108.04 percent, and the relative standard deviation was less than 6.88 percent. This method was used to determine whitening compounds in several cosmetics with on-line microdialysis and the zero net flux method. The procedure was fast, simple, selective, and suitable for routine analysis.

Relaxation algorithm-based PTV with dual calculation method and its application in addressing particle saltation

This paper proposes a dual calculation method (DCM) that in velocity fields obtained from particle tracking velocimetry detects spurious velocity vectors. The synthetic images were used to test the performance of the DCM. By adding random erasing to the genuine matching data, the influence of noise effect was simulated and analyzed. A comparison with the minimum net-flux method indicates that the DCM is more effective in identifying erroneous vectors as particles having no genuine candidates are more readily identified, thereby, ensuring strong consistent match. The DCM is also found to be more effective in addressing particle saltation, based on the accurately abstracted trajectories, the kinetic parameters of the sand grains are calculated. .

Behavioral and serotonergic consequences of decreasing or increasing hippocampus brain-derived neurotrophic factor protein levels in mice

Antidepressants such as Selective Serotonin Reuptake Inhibitors (SSRI) act as indirect agonists of serotonin (5-HT) receptors. Although these drugs produce a rapid blockade of serotonin transporters (SERTs) in vitro, several weeks of treatment are necessary to observe clinical benefits. This paradox has not been solved yet. Recent studies have identified modifications of intracellular signaling proteins and target genes that could contribute to antidepressant-like activity of SSRI (e.g., increases in neurogenesis and BDNF protein levels), and may explain, at least in part, their long delay of action. Although these data suggest a positive regulation of 5-HT on the expression of the gene coding for BDNF, the reciprocal effects of BDNF on brain 5-HT neurotransmission remains poorly documented. To study the impact of BDNF on serotonergic activity, a dual experimental strategy was used to analyze neurochemical and behavioral consequences of its decrease (strategy 1) or increase (strategy 2) in the brain of adult male mice. (1) In heterozygous BDNF+/− mice in which brain BDNF protein levels were decreased by half, an enhancement of basal extracellular 5-HT levels (5-HText) that induced a down-regulation of SERT, i.e., a decrease in its capacity to reuptake 5-HT, was found in the hippocampus. In addition, the SSRI, paroxetine, failed to increase hippocampal 5-HText in BDNF+/− mice, while it produces robust effects in wild-type littermates. Thus, BDNF+/− mice can be viewed as an animal model of genetic resistance to serotonergic antidepressant drugs. (2) In wild-type BDNF+/+ mice, the effects of intra-hippocampal (vHi) injection of BDNF (100 ng) in combination with a SSRI was examined by using intracerebral microdialysis and behavioral paradigms that predict an antidepressant- and anxiolytic-like activity of a molecule [the forced swim test (FST) and the open field paradigm (OF) respectively]. BDNF induced a rapid and transient increase in paroxetine response on 5-HText in the adult hippocampus, which was correlated with a potentiation of its antidepressant-like activity in the FST. The effects of BDNF were selectively blocked by K252a, an antagonist of its high-affinity TrkB receptor. Such a correlation between neurochemical and behavioral effects of [BDNF + SSRI] co-administration suggests that its antidepressant-like activity is linked to the activation of 5-HT neurotransmission in the adult hippocampus. BDNF also had a facilitatory effect on anxiety-like behavior in the OF test, and paroxetine prevented this anxiogenesis. What was the mechanism by which BDNF exerted these latter effects? Surprisingly, by using zero net flux method of quantitative microdialysis in vivo, we found that an intra-hippocampal BDNF injection in wild-type mice decreased the functional activity of SERT as observed in BDNF+/− mice. However, the decreased capacity of SERT to reuptake 5-HT was not associated to an increase in basal 5-HText in the hippocampus of WT mice. Interestingly, using in situ hybridization experiments indicated that TrkB receptor mRNA was expressed in the hippocampus and dorsal raphe nucleus in adult mice suggesting that the neurochemical and behavioral effects of intra-hippocampal BDNF injection can mobilize both pre- and post-synaptic elements of the brain 5-HT neurotransmission. Taken together, these set of experiments unveiled a relative opposition of neurochemical and behavioral responses following either a decrease (in BDNF+/− mutant mice) or an increase in brain BDNF levels (bilateral intra-hippocampal injection) in adult mice. In view of developing new antidepressant drug strategy, a poly-therapy combining BDNF with a chronic SSRI treatment could thus improve the efficacy of current medications.

Action of thiazide on renal interstitial calcium.

Although thiazides increase urinary sodium excretion, they also decrease urinary calcium excretion. Recent studies in our laboratory have shown that increased dietary salt significantly reduces interstitial fluid calcium in Dahl salt-sensitive (DS) rats, and this was associated with a rise in blood pressure and increased urinary calcium excretion. Owing to the vasorelaxant actions of increased extracellular fluid calcium, we reasoned that the antihypertensive action of hydrochlorothiazide (HCTZ), a commonly used thiazide, may be the result of increased interstitial fluid calcium as a consequence of decreased urinary calcium excretion. To test this hypothesis, DS and Dahl salt-resistant (DR) rats were given high salt alone or in combination with HCTZ for 1 week. Renal cortical interstitial fluid calcium was determined by the zero net flux method. High salt decreased cortical interstitial fluid calcium (1.69 +/- 0.25 vs. 1.13 +/- 0.05 mmol/l; P < 0.05) in DS rats as previously reported; thiazide treatment had no effect on the high salt interstitial fluid calcium response in salt-sensitive animals. However, thiazide decreased interstitial fluid calcium in DS on a normal salt diet. Cortical interstitial fluid calcium was unchanged by dietary salt in DR rats, and thiazide did not alter this interstitial fluid calcium response. We interpret these data to mean that (i) short-term thiazide treatment does not reduce blood pressure by restoring renal cortical interstitial fluid calcium concentration and (ii) a decrease in renal cortical interstitial fluid calcium may not contribute to the increased renal vasoconstriction seen in salt-sensitivity.

Brain-derived neurotrophic factor-deficient mice exhibit a hippocampal hyperserotonergic phenotype

Growing evidence supports the involvement of brain-derived neurotrophic factor (BDNF) in mood disorders and the mechanism of action of antidepressant drugs. However, the relationship between BDNF and serotonergic signalling is poorly understood. Heterozygous mutants BDNF +/- mice were utilized to investigate the influence of BDNF on the serotonin (5-HT) system and the activity of the serotonin transporter (SERT) in the hippocampus. The zero net flux method of quantitative microdialysis revealed that BDNF +/- heterozygous mice have increased basal extracellular 5-HT levels in the hippocampus and decreased 5-HT reuptake capacity. In keeping with these results, the selective serotonin reuptake inhibitor paroxetine failed to increase hippocampal extracellular 5-HT levels in BDNF +/- mice while it produced robust effects in wild-type littermates. Using in-vitro autoradiography and synaptosome techniques, we investigated the causes of attenuated 5-HT reuptake in BDNF +/- mice. A significant decrease in [3H]citalopram-binding-site density in the CA3 subregion of the ventral hippocampus and a significant reduction in [3H]5-HT uptake in hippocampal synaptosomes, revealed mainly a decrease in SERT function. However, 5-HT1A autoreceptors were not desensitized in BDNF +/- mice. These results provide evidence that constitutive reductions in BDNF modulate SERT function reuptake in the hippocampus.

Lactate Concentrations in Incisions Indicate Ischemic-like Conditions May Contribute to Postoperative Pain

The substances in wounds that cause incisional pain and hyperalgesia after surgery are poorly understood. We have developed and characterized rat models for incision-induced pain behaviors and measured increased tissue hydrogen ion concentration. Because lactate may facilitate nociceptor responses to low pH and contribute to ischemic pain mechanisms, we measured tissue lactate after incision of the plantar region of the hindpaw, gastrocnemius muscle, and paraspinal region in halothane anesthetized rats using in vivo microdialysis. Incisions were performed at 1 site (plantar, gastrocnemius, or paraspinal incision) in each rat. The corresponding contralateral side was used as the control. In anesthetized rats, a microdialysis fiber was passed into the incision and the control side. L-Lactate was measured using the lactate oxidase method. Tissue concentration was determined from postoperative day 0 to postoperative day 14 using the no net flux method. Lactate was increased on the day of hindpaw incision to 3.6 ± 1.6 mmol/L compared with control (2.1 ± .6 mmol/L) and remained increased through 7 days. In the gastrocnemius muscle, lactate was increased the day after incision (4.2 ± 1.2 mmol/L vs 1.7 ± .5 mmol/L) until postoperative day 7. On the day of the paraspinal incision, lactate was 3.4 ± 1.1 mmol/L on the operated side and 2.2 ± .6 mmol/L in the control side. Lactate remained increased through postoperative day 8 at the paraspinal incision. These experiments demonstrate that incision of the plantar hindpaw, the gastrocnemius muscle, and the paraspinal region increased tissue lactate concentration. The wound environment contains increased lactate at the same time that pH is decreased; lactate could potentially facilitate nociceptor activation by low pH and contribute to pain after surgery. Perspective This study demonstrates that lactate is increased in wounds when pain behaviors and acid are increased. Lactate and low pH are present in incisions and indicate an ischemic pain mechanism that may contribute to postsurgical pain.

Behavioral and Neurochemical Responses to Cocaine in Periadolescent and Adult Rats

Although recreational drug use by human adolescents is a well-known and long-standing problem, relatively little is known regarding differences in behavioral and physiological responses to abused substances in adolescent vs adult animals. The present study compared effects of the psychomotor stimulant, cocaine, in periadolescent (postnatal days 37-52) and adult (postnatal days 75-90) male Wistar rats. Locomotion and motor stereotypy were recorded after acute and repeated cocaine injections (0, 10, or 20 mg/kg cocaine, intraperitoneal (i.p.), four injections spaced 5 days apart). Spontaneous acquisition of intravenous (i.v.) cocaine self-administration was investigated in two dose groups ( approximately 0.37 or 0.74 mg/kg/infusion) over 14 days. Dopamine levels in the nucleus accumbens were recorded under basal conditions (no net flux method) and after cocaine administration ( approximately 0.37, 0.74, and 2.92 mg/kg/i.v. infusion or 20 mg/kg i.p.) using in vivo microdialysis. The locomotor data are in partial agreement with previous reports of hyposensitivity to acute cocaine in periadolescent vs adult rats; periadolescents were less active overall than adults. Moreover, adult rats exhibited significant locomotor sensitization after repeated injection of 10 mg/kg cocaine, whereas periadolescents required the high dose of 20 mg/kg cocaine to demonstrate sensitization. Neither age group showed sensitization of motor stereotypies. No age-related difference was observed in acquisition of cocaine self-administration, or in basal or cocaine-stimulated nucleus accumbens dopamine. These experiments imply a developmental dissociation between the motor activating and reinforcing effects of cocaine. Similarities in dopamine levels across age groups suggest that age-specific motor responses to cocaine are not mediated by dopamine in the nucleus accumbens.

Intestinal Microdialysis — Applicability, Reproducibility and Local Tissue Response in a Pig Model

Microdialysis has been applied to the intestinal wall for the purpose of monitoring local ischemia. The aim of this study was to investigate the applicability, reproducibility and local response to microdialysis in the intestinal wall. In 12 pigs two microdialysis probes were inserted into the ileal wall, one in the peritoneal cavity and one in the psoas muscle. Relative recovery was measured for all probes by the no net flux method. Metabolic measurements of glucose, lactate and glycerol were performed over six hours. The ileal wall segments containing the probes were processed for histological examination. Large intra- and inter-group differences in the relative recovery were found between all locations. Absolute values of metabolites showed no significant changes during the study period. The lactate in blood was 25-30% of the intra-tissue values. A severe inflammatory reaction was seen in the ileal wall around all probes. Measurement of the relative recovery is essential for valid measurements of metabolites when using microdialysis. The inflammatory reaction around the probe in the intestinal wall is likely to affect metabolism and measurements hereof. Therefore intestinal wall microdialysis seems confined to experimental research, and future studies should consider the intra-peritoneal approach.

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 μl/min. For testing of sampling times at different flow rates, an in vitro study was performed in which a constant sample volume of 10 μl 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.

Quantitative In Vivo Microdialysis in Pharmacokinetic Studies: Some Reminders

This paper reviews the empirical methods of quantitative microdialysis that have been used to interpret the results obtained from pharmacokinetic studies. The concept of extraction efficiency or recovery and the properties of recovery in vivo (variation with flow rate, time dependency and influence of the mode of administration) are considered. The most frequently used methods for determining recovery in vivo are described and evaluated in the light of recent theoretical studies. Specifically, we review the variation of flow rate method, the very slow flow method, the no net flux method and the delivery and retrodialysis methods. Special emphasis is placed on the description of each method, demonstrating its applicability to pharmacokinetic studies conducted under steady-state or transient conditions, and also its limitations. Finally, the more relevant studies that have compared the suitability of these methods are reviewed.

Continuous in vivo Monitoring of Blood Diffusible Calcium Using On-line Microdialysis Sampling Coupled with Flame Atomic Absorption Spectrometry

A direct, rapid and continuous in vivo monitoring of diffusible calcium in the blood of living rabbits has been developed using microdialysis sampling coupled on-line with flame atomic absorption spectrometry. Microdialysates perfused through implanted microdialysis probes were collected with a sample loop on an injection valve and directly introduced into the flame atomizer by a carrier solution. An ultrapure saline solution (0.9% NaCI, pH 7.2) was used as the perfusion solution at a flow rate of 20 microI min(-1) via the microdialysis probe. A 0.1% La solution in 0.5% HNO3 solution was employed as the carrier solution at a nebulizer uptake flow rate of 2.5 ml min(-1). The interval for each determination was 2.5 min (2 min of sampling time, 20 s of read time and 10 s of washing time). The performance characteristics of the on-line microdialysis-FAAS system were validated as follows: linearity range, 0 - 100 mg l(-1); detection limit (3a, n = 7), 3.66 mg l(-1); precision (RSD, n = 50), 6.2%. For the evaluation of analytical accuracy, the proposed on-line method was compared with the in vivo no net flux method. The use of an on-line microdialysis-FAAS system permitted the in situ, dynamic and continuous in vivo monitoring of diffusible calcium in the blood of the living rabbits after CaCl2 administration with a temporal resolution of 2.5 min.

On-line microdialysis sampling coupled with flame atomic absorption spectrometry for continuous in vivo monitoring of diffusible magnesium in the blood of living animals

A novel on-line microdialysis sampling coupled with flame atomic absorption spectrometry (FAAS) with an attractive application is reported. Microdialysates perfused through implanted microdialysis probes were directly introduced into the flame atomizer of a FAAS system using 0.2% HNO 3 as carrier solution at a nebulizer uptake flow rate of 6 ml min −1. The interval for each determination was 90 s (60 s sampling time, 10 s read time and 20 s washing time). The analytical characteristics of the on-line microdialysis–FAAS system were validated as follows: linearity range, 0–300 mg l −1; detection limit (3 σ, n = 7), 0.53 mg l −1; precision (R.S.D., n = 50), 4.1%. By comparing Mg levels in the blood of living rabbits with the results obtained from in vivo no net flux (NNF) method, the accuracy of the proposed on-line method was found to be good. The present method can be successfully applied to the in vivo monitoring of diffusible Mg in the blood of living rabbits after magnesium sulfate (MgSO 4) administration with a temporal resolution of 1.5 min.

Excised porcine skin experimental systems to validate quantitative microdialysis methods for determination of drugs in skin after topical application

Microdialysis is useful as a method to evaluate the disposition of drugs in the skin to design improved transdermal delivery systems (TDDSs). In this study, quantitative microdialysis methods were validated in excised porcine skin experimental systems in vitro. Flurbiprofen (FP), used as a model drug, showed high affinity for the skin tissues in equilibrium states between the medium and skin. The membrane clearances of FP for permeation through the membrane of a dialysis fiber placed in the skin (CL m in S) were lower than that in the medium. The adsorption of components in the skin to the membrane surface of the dialysis fiber and accumulation of FP near the dialysis fiber are the most likely reasons for this. When CL m in S was used to predict the extracellular FP concentration in skin ( C T), the value obtained was lower than that expected from the FP concentration in the medium on the dermis side, which should be equal to C T at equilibrium. In the zero net flux (ZNF) method, in which the concentration difference of perfusate (Δ C) between the inflow and outflow were used to obtain C T, the predicted C T was similar to the expected value. In an in vitro skin permeation experiment, the ZNF method was used for the prediction of C T near the dialysis fiber. The predicted C T was over 10 times higher than the FP concentration in the medium on the dermis side, suggesting a concentration gradient in the dermis. Although the ZNF method is good for predicting the C T in skin, the mass balance has to be considered for the quantitative evaluation of the skin permeation of drugs. In this study, the effect of the mass transfer of FP from the perfusate to the skin on the cumulative amount of FP passing through the skin was relatively low because of the use of suitable solutions as perfusate. The perfusion conditions and schedules should be designed carefully for quantitative evaluations using the ZNF method. These results provide useful information for the in vivo application of quantitative microdialysis to evaluate TDDS.

Selective reduction in ventral hippocampal acetylcholine release in awake galanin-treated rats and galanin-overexpressing transgenic mice

The neuropeptide galanin is an inhibitory modulator of hippocampal acetylcholine (ACh) release and cognitive functions. Anatomical evidence demonstrated some differences between the dorsal and ventral hippocampi notably in the expression of galanin receptor subtypes, and the neuronal population on which galanin-like immunoreactivity is expressed. This is suggestive of a differential role for this peptide in these two areas of the hippocampal formation. Using in vivo microdialysis, we investigated the role of galanin on ACh release in the dorsal and ventral hippocampi. Two models were studied: galanin-administered rats and transgenic mice over-expressing galanin (GAL-tg). In rats, galanin (2.0 and 10.0 μM) infused locally through the dialysis probe induced a significant decrease in ACh release in the ventral hippocampus, confirming previous findings, while no effect was seen in the dorsal hippocampus. Using the no net flux method, a significant reduction in ACh levels was noted only in the ventral hippocampus of GAL-tg compared to wild-type littermates. These results suggest that excess endogenous galanin can suppress basal ACh release, with anatomical specificity, to the ventral hippocampus. These results are of interest in the context of galanin receptor subtypes in the dorsal and ventral hippocampus, and the differential alterations of hippocampal subregions in neurological diseases such as Alzheimer's dementia.

Serotonin transporter in substance P (neurokinin 1) receptor knock-out mice

We recently demonstrated that mice lacking the gene for substance P (neurokinin 1) receptors (NK 1−/−) show improved cortical dialysate serotonin (5-HT) responses to paroxetine [J. Neurosci. 21 (2001) 8188]. To test for changes that may involve the 5-HT transporter (5-HTT) in these mutant mice, in vivo/in vitro studies were performed. Autoradiographic quantification of 5-HTT was performed: [ 3H]citalopram binding did not reveal any modification of 5-HT binding sites in the dorsal raphe nucleus (DRN) of wild-type NK 1+/+ control and mutant NK 1−/− mice. These results were further confirmed by 5-HTT mRNA quantification using competitive reverse transcription and polymerase chain reaction (RT-PCR) assay, which showed similar messenger levels in the DRN of both mice genotypes. The functional status of 5-HTT in vivo was tested by using the zero net flux method of quantitative microdialysis in two serotonergic nerve terminal regions, the frontal cortex and ventral hippocampus, of wild-type NK 1+/+ and NK 1−/− mice. Neither basal extracellular 5-HT levels nor the 5-HT extraction fraction of the probe ( E d an index of 5-HT uptake in vivo) differed between wild-type and mutant mice in the two brain regions studied. These results suggest that no compensatory response to the constitutive deletion of the tachykinin NK 1 receptor involving changes in the activity of the selective 5-HT transporter occurred in the DRN, frontal cortex and ventral hippocampus in mice.

Net uptake of chloride across the posterior gills of the Chinese crab ( Eriocheir sinensis)

Two methods are commonly used for the determination of transbranchial net fluxes of Na + and Cl −: direct analysis of changes in ion concentrations in the external medium using flame spectrophotometry or titration (net flux method), and measurement of unidirectional ion fluxes by means of radioactive tracers (tracer method). When we applied both methods in the same preparation, the isolated perfused posterior gill of freshwater-acclimated Eriocheir sinensis, to determine net fluxes of Cl −, the results differed substantially. In artificial fresh water (AFW) containing NaCl, the net flux method yielded a net uptake, but the tracer method showed a net efflux of Cl −. The net uptake of Cl − was abolished in Na +-free AFW indicating that Cl − uptake is coupled with the uptake of Na +. Applying the tracer method, net efflux of Cl − remained almost unchanged in Na +-free AFW. This suggests the opposite mechanism, i.e. uncoupled uptake of Na + and Cl −. The discrepancy in the results obviously depends on the method employed. Since the data obtained with the net flux method explain the osmoregulatory performance of crabs living in fresh water, we consider this method as appropriate for determining net transbranchial ion fluxes.

Effects of chronic paroxetine treatment on dialysate serotonin in 5-HT1B receptor knockout mice.

The role of serotonin (5-HT)1B receptors in the mechanism of action of selective serotonin re-uptake inhibitors (SSRI) was studied by using intracerebral in vivo microdialysis in conscious, freely moving wild-type and 5-HT1B receptor knockout (KO 5-HT1B) mice in order to compare the effects of chronic administration of paroxetine via osmotic minipumps (1 mg per kg per day for 14 days) on extracellular 5-HT levels ([5-HT]ext) in the medial prefrontal cortex and ventral hippocampus. Basal [5-HT]ext values in the medial prefrontal cortex and ventral hippocampus, approximately 20 h after removing the minipump, were not altered by chronic paroxetine treatment in both genotypes. On day 15, in the ventral hippocampus, an acute paroxetine challenge (1 mg/kg i.p.) induced a larger increase in [5-HT]ext in saline-pretreated mutant than in wild-type mice. This difference between the two genotypes in the effect of the paroxetine challenge persisted following chronic paroxetine treatment. Conversely, in the medial prefrontal cortex, the paroxetine challenge increased [5-HT]ext similarly in saline-pretreated mice of both genotypes. Such a challenge produced a further increase in cortical [5-HT]ext compared with that in saline-pretreated groups of both genotypes, but no differences were found between genotypes following chronic treatment. To avoid the interaction with raphe 5-HT1A autoreceptors, 1 micro m paroxetine was perfused locally through the dialysis probe implanted in the ventral hippocampus; similar increases in hippocampal [5-HT]ext were found in acutely or chronically treated wild-type mice. Systemic administration of the mixed 5-HT1B/1D receptor antagonist GR 127935 (4 mg/kg) in chronically treated wild-type mice potentiated the effect of a paroxetine challenge dose on [5-HT]ext in the ventral hippocampus, whereas systemic administration of the selective 5-HT1A receptor antagonist WAY 100635 did not. By using the zero net flux method of quantitative microdialysis in the medial prefrontal cortex and ventral hippocampus of wild-type and KO 5-HT1B mice, we found that basal [5-HT]ext and the extraction fraction of 5-HT were similar in the medial prefrontal cortex and ventral hippocampus of both genotypes, suggesting that no compensatory response to the constitutive deletion of the 5-HT1B receptor involving changes in 5-HT uptake capacity occurred in vivo. As steady-state brain concentrations of paroxetine at day 14 were similar in both genotypes, it is unlikely that differences in the effects of a paroxetine challenge on hippocampal [5-HT]ext are due to alterations of the drug's pharmacokinetic properties in mutants. These data suggest that there are differences between the ventral hippocampus and medial prefrontal cortex in activation of terminal 5-HT1B autoreceptors and their role in regulating dialysate 5-HT levels. These presynaptic receptors retain their capacity to limit 5-HT release mainly in the ventral hippocampus following chronic paroxetine treatment in mice.

Development of a dog microdialysis model for determining synovial fluid pharmacokinetics of anti-arthritis compounds exemplified by methotrexate.

The purpose of this study was to develop and validate an animal model of drug disposition in synovial fluid (SF) by comparing microdialysis with arthrocentesis using the anti-arthritic drug methotrexate (MTX). Microdialysis probes were calibrated in vitro with the no net flux method using dog synovial fluid. The probes were implanted surgically into the stifle joint space of four dogs and were dialyzed overnight using a portable microinfusion pump. The membrane integrity of the probes was monitored by retrodialysis using an internal standard. After an intravenous bolus of 2.5 mg/kg of MTX, unbound concentrations in synovial fluid, as well as total plasma concentrations, were measured by liquid chromatography tandam mass spectrometer (LC/MS/MS) in samples collected from 0 to 48 h postdose. The probe membrane remained intact at least 48 h after implantation. The mean probe recovery and unbound fraction of MTX in SF were 46.8% and 44.8%, respectively. The unbound fraction of MTX was 44% in synovial fluid. MTX penetrated into the joint space rapidly, with maximal concentrations of 6.6 microM reached at approximately 1 h postdose. The unbound MTX area under the curve in SF was approximately 40% of the total area under the curve in plasma. These data agree well with the previous data obtained for MTX using arthrocentesis. In contrast with arthrocentesis, microdialysis enables the collection of multiple serial SF samples from individual animals with minimal trauma and potential blood contamination. This animal model should prove valuable for studying the disposition of new antiarthritis compounds or biomarkers in SF.