Monitoring dopamine in vivo by microdialysis sampling and on-line CE-laser-induced fluorescence

Microdialysis sampling was coupled on-line to micellar electrokinetic chromatography (MEKC) to monitor extracellular dopamine concentration in the brains of rats. Microdialysis probes were perfused at 0.3 microL/min and the dialysate mixed on-line with 6 mM naphthalene-2,3-dicarboxaldehye and 10 mM potassium cyanide pumped at 0.12 microL/min each into a reaction capillary. The reaction mixture was delivered into a flow-gated interface and separated at 90-s intervals. The MEKC separation buffer consisted of 30 mM phosphate, 6.5 mM SDS, and 2 mM HP-beta-CD at pH 7.4, and the electric field was 850 V/cm applied across a 14-cm separation distance. Analytes were detected by laser-induced fluorescence excited using the 413-nm line of a 14-mW diode-pumped laser. The detection limit for dopamine was 2 nM when sampling by dialysis. The basal dopamine concentration in dialysates collected from the striatum of anesthetized rats was 18 +/- 3 nM (n = 12). The identity of the putative dopamine peak was confirmed by showing that dopamine uptake inhibitors increased the peak and dopamine synthesis inhibitors eliminated the peak. The utility of this method for behavioral studies was demonstrated by correlating dopamine concentrations in vivo and with psychomotor behavior in freely moving rats following the intravenous administration of cocaine. Over 60 additional peaks were detected in the electropherograms, suggesting the potential for monitoring many other substances in vivo by this method.     

In vitro characterization of microdialysis sampling of macromolecules

Experiments were performed to characterize the in vitro collection of macromolecules using microdialysis. Fluorescently labeled proteins and dextrans ranging from 3000 to 150 000 were sampled using a 10-mm, 100 000 molecular weight cutoff, polyethersulfone microdialysis probe. Published models describing microdialysis mass transport of small molecules were examined to determine their appropriateness for sampling of macromolecules. Collection efficiencies, reported as relative recoveries, for macromolecules from 3000 to 70 000 ranged from 5 to 44%. Collection efficiencies determined for microdialysis sampling of macromolecules follow the functionality of published models, although experimental mass transport resistances are to some extent smaller than predicted. Implications of the current study for in vivo microdialysis sampling of cytokines and growth factors are discussed.     

Microfluidic electrophoresis chip coupled to microdialysis for in vivo monitoring of amino acid neurotransmitters

Microfluidic electrophoresis devices were coupled on-line to microdialysis for in vivo monitoring of primary amine neurotransmitters in rat brain. The devices contained a sample introduction channel for dialysate, a precolumn reactor for derivatization with o-phthaldialdehyde, a flow-gated injector, and a separation channel. Detection was performed using confocal laser-induced fluorescence. In vitro testing revealed that the initial device design had detection limits for amino acids of approximately 200 nM, relative standard deviation of peak heights of 2%, and separations within 95 s with up to 30,200 theoretical plates when applying an electric field of 370 V/cm. A second device design that allowed electric fields of 1320 V/cm to be applied while preserving the reaction time allowed separations within 20 s with up to 156,000 theoretical plates. Flow splitting into the electrokinetic network from hydrodynamic flow in the sample introduction channel was made negligible for sampling flow rates from 0.3 to 1.2 microL/min by placing a 360-microm-diameter fluidic access hole that had flow resistance (0.15-7.2) x 10(8)-fold lower than that of the electrokinetic network at the junction of the sample introduction channel and the electrokinetic network. Using serial injections, the device allowed the dialysate stream to be analyzed at 130-s intervals. In vivo monitoring was demonstrated by using the microdialysis/microfluidic device to record glutamate concentrations in the striatum of an anesthetized rat during infusion of the glutamate uptake inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid. These results prove the feasibility of using a microfabricated fluidic system coupled to sampling probes for chemical monitoring of complex media such as mammalian brain.     

In vivo calibration of microdialysis probes for exogenous compounds.

Several approaches for calibrating microdialysis probes for exogenous compounds in vivo are described which avoid the error introduced by in vitro calibration. These methods are based on establishing a steady state of the exogenous compound by a continuous (zero-order) iv infusion. The steady-state concentration is estimated by three methods that directly determine the in vivo concentration. The methods are (a) extrapolation of dialysate concentrations at various flow rates to the concentration at zero flow, (b) dialysis with concentrations of analyte added to the perfusion medium above and below the expected concentration to determine the concentration at no net flux across the membrane, and (c) dialysis at a very slow perfusion rate (57 nL/min) where the recovery is expected to be better than 90%. Using these approaches, the recovery for cocaine in the brain was found to be (8.9 +/- 0.68)%, as compared to an in vitro recovery of (5.1 +/- 0.18)% at 24 degrees C and (7.4 +/- 0.18)% at 37 degrees C, at a perfusion rate of 1.2 microL/min through a 0.3- X 2-mm microdialysis probe. The in vivo concentration of cocaine in the rat brain for an intravenous dose of 0.3 mg/kg per min was found to be 17.1 +/- 1.3 microM.     

Magnetic resonance imaging of a tissue/implanted device biointerface using in vivo microdialysis sampling

Real-time in vivo images of magnetic resonance contrast agent diffusion from implanted microdialysis probes were obtained by magnetic resonance (MR) microscopy. A gadolinium-containing contrast agent (Gd-DTPA) was infused through microdialysis probes implanted into the subcutaneous space of male Sprague-Dawley rats. The infusion of Gd-DTPA alters the T1 relaxation time for water protons near the microdialysis probe, thus causing an increase in brightness around the probe. Steady state concentration profiles of Gd-DTPA around the microdialysis probe were attained within 10 min. The distance for the diffusion of Gd-DTPA away from the probe was calculated to be approximately 1400 microm on the basis of an image intensity analysis. A 5-cm field of view was used with a 256 x 256 matrix, giving a voxel volume of 0.190 mm3 (195 microm x 195 microm x 5,000 microm). These experiments demonstrate the ability of magnetic resonance microscopy to obtain real-time images of Gd-DTPA diffusion around implanted microdialysis probes. This noninvasive technique may be useful for determining how fibrous encapsulation during long-term implantation may affect localized mass transport at a biointerface.     

In vivo measurements of ultrasonic backscattering in blood

Ultrasonic backscattering in blood including its dependence on the hematocrit, plasma proteins, shear rate, and flow disturbance, has been studied extensively theoretically and experimentally in vitro. However, much of the result has never been validated in vivo. To do so, backscattering measurements were made on pigs using a 10-MHz non-focused intravascular transducer in direct contact with blood. The probe was placed in either the abdominal aorta or the inferior vena cava. The backscattering coefficient (BSC) of blood flowing in these vessels as well as downstream from a stenosis was measured using an approach that was originally developed for measurements with focused transducers. With this approach, 6% porcine red cell saline suspensions prepared immediately after each in vivo measurement were used as the reference medium. Result from seven pigs at hematocrits ranging from 29 to 36% (31.9±2.5%) demonstrated that BSC of blood in the vena cava, (4.62±2.06)×10^-5 cm-sr^-1, is consistently higher than that in the aorta, (2.65±1.22)×10 ^-5 cm-sr^-1. The difference has been attributed to the lower shear rate and the formation of red cell aggregation in venous blood. These in vivo results are in agreement with those obtained in vitro. In response to stenoses created by ligating the aorta, backscattering of the blood measured downstream from the stenosis showed that the closer the site of measurement relative to the stenosis, the higher the backscatter, presumably resulting from the higher degree of flow disturbance. In vitro backscattering results on porcine whole blood were also acquired at 20 MHz with a Diasonics intravascular scanner     

Trace-level amino acid analysis by capillary liquid chromatography and application to in vivo microdialysis sampling with 10-s temporal resolution

A sensitive method was developed to determine 16 amino acids, including all the neurotransmitter amino acids and neuromodulators, in physiological samples. Samples were derivatized with o-phthalaldehyde/tert-butyl thiol followed by two scavenging reactions that reduced the chemical background caused by excess derivatization reagent by approximately 90%. A total of 250 nL of the derivatized sample was injected and concentrated onto a 50-micron-inner diameter capillary column packed with 5-micron reversed-phase particles and separated using gradient elution. Analytes were detected amperometrically at a cylindrical 9-micron carbon fiber microelectrode. The combination of on-column concentration, scavenging reactions after derivatization, high sensitivity electrochemical detection, and protocols to minimize amine contamination allowed detection limits of 90-350 pM (20-80 amol) for all the amino acids tested. This method was used to analyze in vivo microdialysate samples from probes implanted in the striatum of anesthetized rats. Probes were perfused at 1.2 microL/min and fractions collected every 10 s. The 200-nL fractions were diluted to 2 microL to facilitate sample handling for off-line analysis. The suitability of this method for simultaneous monitoring of all the major amino acid neurotransmitters with 10-s temporal resolution under basal conditions, during potassium stimulation, and during selective uptake inhibition of gamma-aminobutyric acid is demonstrated.     

In situ and multisubstrate detection of elastase enzymatic activity external to microdialysis sampling probes using LC-ESI-MS

Extracellular proteases play significant roles in mammalian development and disease. Enzymatic activity external to a microdialysis sampling probe can be determined by infusing judicious choices of substrates followed by collecting and measuring the products. Porcine pancreatic elastase was used as a model enzyme with two substrates possessing different cleavage sites, N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (FL-substrate) and N-succinyl-Ala-Ala-Ala-p-nitroanilide (UV-substrate). These substrates were infused through the microdialysis sampling probe to a solution containing elastase. The resulting four products and the remaining two substrates were collected into the dialysate and were subsequently analyzed off-line using liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). All analytes were identified using extracted ion chromatograms of m/z 628 (FL-substrate), m/z 452 (UV-substrate), m/z 471 (N-methoxysuccinyl-Ala-Ala-Pro-Val, FL-NTP), m/z 332 (N-succinyl-Ala-Ala-Ala, UV-NTP), m/z 176 (7-amino-4-methylcoumarin, AMC), and m/z 139 (p-nitroaniline, pNA). FL-NTP and FL-substrate exhibited 10-fold higher ion production as compared to AMC with equimolar standards. Microdialysis sampling combined with LC-ESI-MS detection allowed for in situ determination of the enzymatic activity of a protease external to the microdialysis probe when using different peptide-based substrates.     

On-line coupling of microdialysis sampling with microchip-based capillary electrophoresis

Microdialysis sampling is a technique that has been used for in vivo and in vitro monitoring of compounds of pharmaceutical, biomedical, and environmental interest. The coupling of a commercially available microdialysis probe to a microchip-based capillary electrophoresis (CE) system is described. A continuously flowing dialysate stream from a microdialysis probe was introduced into the microchip, and discrete injections were achieved using a valveless gating approach. The effect of the applied voltage and microdialysis flow rate on device performance was investigated. It was found that the peak area varied linearly with the applied voltage. Higher voltages led to lower peak response but faster separations. Perfusion flow rates of 0.8 and 1.0 microL/min were found to provide optimal performance. The on-line microdialysis/microchip CE system was used to monitor the hydrolysis of fluorescein mono-beta-d-galactopyranoside (FMG) by beta-d-galactosidase. A decrease of the FMG substrate with an increase in the fluorescein product was observed. The temporal resolution of the device, which is dependent on the CE separation time, was 30 s. To the best of our knowledge, this is the first reported coupling of a microdialysis sampling probe to a microchip capillary electrophoresis device.     

In vivo brain microdialysis as a formulation-screening tool for a poorly soluble centrally acting drug

Objective: Efficacy of a formulation of a poorly soluble centrally acting drug was evaluated by measuring dopamine responses using in vivo brain microdialysis.

Methods: Co-crystals (1:1) of carbamazepine and nicotinamide (CBZ–NCT) were complexed with cyclodextrins (γ-CDs) using supercritical fluid processing. Phase solubility and intrinsic dissolution were studied. Pharmacodynamic studies were performed on rats divided into three groups getting either CBZ–NCT in CD (20?mg/kg CBZ), pure CBZ solution or vehicle. A guide cannula was implanted to attach the microdialysis probe. Dialysate samples were analyzed for dopamine levels, which were compared between groups.

Results: The optimized CBZ formulation (5% w/w in γ-CD) with solubility – 10?mg/mL showed stepwise increase in dopamine response (maximum 250% of baseline) compared to neat CBZ or vehicle (p?Conclusion: Hence, brain microdialysis was successfully used to evaluate a dissolution rate enhancing formulation.     

Intravenous microdialysis sampling in awake, freely-moving rats.

Intravenous microdialysis sampling in the awake, freely-moving rat for the determination of free drug concentrations in blood is described. Intravenous microdialysis was performed with a nonmetallic, flexible dialysis probe. The pharmacokinetics of theophylline were determined using both microdialysis sampling and collection of whole blood following an iv dose. There was no difference in the half-life of elimination of theophylline determined by microdialysis, 4.4 +/- 0.4 h, and whole blood sampling, 4.5 +/- 0.7 h. The kinetics of elimination were affected by removing blood samples and by using anesthesia. The half-life of elimination was 4.4 +/- 0.4 h when using simultaneous microdialysis and whole-blood sampling and only 3.0 +/- 0.4 h using microdialysis alone. The half-life of elimination was 17.0 +/- 7.1 h in chloral hydrate anesthesized rats. Microdialysis samples were continuously collected for over 7 h without fluid loss using a single experimental animal. Microdialysis sampling directly assesses the free drug concentration in blood. The extent of theophylline binding to blood proteins was determined in vitro in rat plasma and rat whole blood using both ultrafiltration and microdialysis. Theophylline was (47.3 +/- 1.3)% bound in rat plasma and (52.2 +/- 1.6)% bound in rat whole blood. Microdialysis sampling is a powerful tool for pharmacokinetic studies, providing accurate and precise pharmacokinetic data.     

Improved temporal resolution for in vivo microdialysis by using segmented flow

Microdialysis sampling probes were interfaced to a segmented flow system to improve temporal resolution for monitoring concentration dynamics. Aqueous dialysate was segmented into nanoliter plugs by pumping sample stream into the base of a tee channel structure microfabricated on a PDMS chip that had an immiscible carrier phase (perfluorodecalin) pumped into the cross arm of the tee. Varying the oil flow rate from 0.22 to 6.3 microL/min and sample flow rate from 42 to 328 nL/min allowed control of plug volume, interval between plugs, and frequency of plug generation between 6 and 28 nL, 0.6 and 10 s, and 0.1 and 1.7 Hz, respectively. Temporal resolution of the system, determined by measuring fluorescence in individual sample plugs following step changes of fluorescein concentration at the sampling probe surface, was as good as 15 s. Temporal resolution was independent of both sampling flow rate and distance that samples were pumped from the sampling probe. This effect is due to the prevention of Taylor dispersion of the sample as it was transported by segmented flow. In contrast, without flow segmentation, temporal resolution was worsened from 25 to 160 s as the detection point was moved from the sampling probe to 40 cm downstream. Glucose was detected by modifying the chip to allow enzyme assay reagents to be mixed with dialysate as sample plugs formed. The resulting assay had a detection limit of 50 microM and a linear range of 0.2-2 mM. This system was used to measure glucose in the brain of anesthetized rats. Basal concentration was 1.5 +/- 0.1 mM (n = 3) and was decreased 60% by infusion of high-K(+) solution through the probe. These results demonstrate the potential of microdialysis with segmented flow to be used for in vivo monitoring experiments with high temporal resolution.     

In vivo microdialysis for the evaluation of transfersomes as a novel transdermal delivery vehicle for cinnamic acid

In this study, cinnamic acid-loaded transfersomes were prepared and dermal microdialysis sampling was used in Sprague–Dawley rats to compare the amount of drug released into the skin using transfersomes as transdermal carriers with that released on using conventional liposomes. The formulation of cinnamic acid-loaded transfersomes was optimized by a uniform design through in vitro transdermal permeation studies. Hydration time was confirmed as a significant factor influencing the entrapment efficiency of transfersomes, further affecting their transdermal flux in vitro. The fluxes of cinnamic acid from transfersomes were all higher than those from conventional liposomes, and the flux from the optimal transfersome formulation was 3.01-fold higher than that from the conventional liposomes (p?in vivo microdialysis sampling method revealed that the dermal drug concentrations from transfersomes applied on various skin regions were much lower than those required with conventional liposomes. After the administration of drug-containing transfersomes and liposomes on abdominal skin regions of rats for a period of 10?h, the C_max of cinnamic acid from the compared liposomes was 3.21?±?0.25?μg/mL and that from the transfersomes was merely 0.59?±?0.02?μg/mL. The results suggest that transfersomes can be used as carriers to enhance the transdermal delivery of cinnamic acid, and that these vehicles may penetrate the skin in the complete form, given their significant deformability.     

Effect of sampling on real ocular aberration measurements

The minimum number of samples necessary to fully characterize the aberration pattern of the eye is a question under debate in the clinical as well as the scientific community. We performed repeated measurements of ocular aberrations in 12 healthy nonsurgical human eyes and in 3 artificial eyes, using different sampling patterns (hexagonal, circular, and rectangular with 19 to 177 samples, and 3 radial patterns with 49 sample coordinates corresponding to zeros of the Albrecht, Jacobi, and Legendre functions). For each measurement set we computed two different metrics based on the root-mean-square (RMS) of difference maps (RMS_Diff) and the proportional change in the wavefront (W%). These metrics are used to compare wavefront estimates as well as to summarize results across eyes. We used computer simulations to extend our results to "abnormal eyes" (keratoconic, post-LASIK, and post-radial keratotomy eyes). We found that the spatial distribution of the samples can be more important than the number of samples for both our measured as well as our simulated "abnormal" eyes. Experimentally, we did not find large differences across patterns except, as expected, for undersampled patterns.     

On-line coupling of in vivo microdialysis with tandem mass spectrometry.

The capability of interfacing in vivo microdialysis with mass spectrometry has been demonstrated. The goal of this research was to demonstrate the feasibility of real-time analysis in biological systems using microdialysis in combination with tandem mass spectrometry (MS/MS). Microdialysis sampling was accomplished by surgically implanting a small microdialysis probe into a tissue or area of interest. Molecules diffuse through the membrane of the microdialysis probe due to concentration differences. These molecules are collected in a sample loop and analyzed by tandem mass spectrometry. Sequential injections can be made in as little as 2 min. This capability is advantageous in the study of molecules with very rapid elimination rates. Tris(2-chloroethyl)phosphate (TRCP) was used as a model compound in the development of this analytical technique. As an example of an application of the microdialysis/MS/MS technique, plasma concentration vs time curves were obtained and compared with the plasma concentration profiles obtained using conventional studies. For the microdialysis/MS/MS studies, the average slope from three animals was -0.086 min-1. In comparison, the average slope from four animals from the conventional studies was -0.035 min-1.     

Interleukin-6 collection through long-term implanted microdialysis sampling probes in rat subcutaneous space

Microdialysis sampling is a method that has promise for collection of important signaling proteins such as cytokines that are involved in every aspect of the immune response. The objective of this study was to determine the role of membrane and tissue alterations on the reduction of interleukin-6 (IL-6) relative recovery of microdialysis probes implanted for 3 and 7 days versus probes implanted on day 0 (acute implant or control probe). Lipopolysaccharide (LPS), a bacterial endotoxin, was used to elicit IL-6 production in the animals. Within the same animal, the recovery of IL-6 through control probes implanted the day of sample collection was compared to the 3- or 7-day implanted probes. Two hours post-LPS administration, the IL-6 concentrations obtained from either the 3-day or 7-day implanted probe were reduced by more than 8-fold when compared to the control probe. The IL-6 concentrations obtained for the 3-day versus control probes 2-h post-LPS injection were 730 +/- 310 and 6440 +/- 1550 pg/mL (mean +/- SD, n = 3), respectively. For the 7-day implant, the IL-6 concentration in the dialysis probe obtained at 2-h post-LPS injection was 990 +/- 590 versus 5520 +/- 1430 pg/mL (mean +/- SD, n = 3) for the control. In vitro recovery experiments and scanning electron microscopy images combined with the in vivo data suggest that the decreased IL-6 content in the dialysate was caused principally by tissue alterations or tissue encapsulation rather than membrane blockage with biological components (membrane biofouling).     

In vivo glucose measurement by surface-enhanced Raman spectroscopy

This paper presents the first in vivo application of surface-enhanced Raman scattering (SERS). SERS was used to obtain quantitative in vivo glucose measurements from an animal model. Silver film over nanosphere surfaces were functionalized with a two-component self-assembled monolayer, and subcutaneously implanted in a Sprague-Dawley rat such that the glucose concentration of the interstitial fluid could be measured by spectroscopically addressing the sensor through an optical window. The sensor had relatively low error (RMSEC = 7.46 mg/dL (0.41 mM) and RMSEP = 53.42 mg/dL (2.97 mM).     

In vivo detection of dysplastic tissue by Raman spectroscopy

The detection of dysplasia and early cancer is important because of the improved survival rates associated with early treatment of cancer. Raman spectroscopy is sensitive to the changes in molecular composition and molecular conformation that occur in tissue during carcinogenesis, and recent developments in fiber-optic probe technology enable its application as an in vivo technique. In this study, the potential of Raman spectroscopy for in vivo classification of normal and dysplastic tissue was investigated. A rat model was used for this purpose, in which dysplasia in the epithelium of the palate was induced by topical application of the carcinogen 4-nitroquinoline 1-oxide. High quality in vivo spectra of normal and dysplastic rat palate tissue, obtained using signal integration times of 100 s were used to create tissue classification models based on multivariate statistical analysis methods. These were tested with an independent set of in vivo spectra, obtained using signal collection times of 10 s. The best performing model, in which signal variance due to signal contributions of the palatal bone was eliminated, was able to distinguish between normal tissue, low-grade dysplasia, and high-grade dysplasia/carcinoma in situ with a selectivity of 0.93 and a sensitivity of 0.78 for detecting low-grade dysplasia and a specificity of 1 and a sensitivity of 1 for detecting high-grade dysplasia/ carcinoma in situ.     

Effect of sampling jitter on some sine wave measurements

The effect of sampling jitter on some periodic signal measurements, namely, the amplitude and phase of a pure sinusoid, is investigated. The parameters are determined by the fast Fourier transform (FFT) algorithm, which is used for manipulating samples in a data-acquisition system. Results are expressed in terms of mean values and variances of the measured parameters. Computer simulation results, based on Gaussian jitter, show the changes of amplitude and phase standard deviations versus the changes of standard deviation of jitter and signal amplitude for different numbers of FFT points