Oxygen-sensing microdialysis probe for in vivo use
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Electrochemical conditions were optimized to allow the metal tube used for the shaft of commercial microdialysis (MD) probes to be coated with gold. In in vitro tests with phosphate-buffered Ringer's solution using double differential pulse amperometry (DDPA), the gold-coated shafts were capable of specifically measuring the reduction of oxygen and the oxidation of ascorbic acid in the presence of high concentrations of potentially interfering endogenous substances. By using fixed-potential amperometry (FPA), the gold-plated shaft also measured oxygen with minimal interference from high concentrations of potentially interfering endogenous substances. Concentric design MD probes were constructed that used a metal shaft (O.D = 0.4 mm), fused silica inlet and outlet tubes, and a 1.5 mm dialyzing membrane (O.D = 0.2 mm). A 0.5–0.7 mm gold collar was electroplated onto the metal shaft approximately 0.5 mm above the dialyzing membrane. The nongold outer surface of the MD probe was coated with an insulating polymer. In vivo tests demonstrated that DDPA was not suitable for use with this gold microdialyzing electrode (GMDE). However, brain oxygen levels were satisfactorily measured using FPA. In urethane-anesthetized rats, the reduction current to oxygen in the striatum was increased by brief (1 min) inhalation of O2 or CO2 and decreased by inhalation of N2. Transient application of noxious stimuli (foot pinch) increased cerebral O2, whereas bilateral carotid artery occlusion and death decreased striatal O2. The responses of the GMDE were indistinguishable from the reduction current simultaneously measured from a conventional carbon fiber electrode implanted adjacent to the gold-plated area of the MD shaft. Basal levels of striatal O2 were 20 ± 5 μM (n = 4) for the GMDE and 30 ± 11 μM (n = 3) for the carbon fiber. The GMDE was robust and could be used for at least three animals. This technique can be used to provide information about the oxygen status of the tissue adjacent to the dialyzing membrane without the need for implantation of an additional electrode. J. Neurosci. Res. 63:224–232, 2001. © 2001 Wiley-Liss, Inc.Keywords:
Amperometry
Microdialysis
Microdialysis
Semipermeable membrane
Dialysis tubing
Interstitial fluid
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Microdialysis provides a means of determining nutrient blood flow as well as continuous simultaneous determination of the interstitial concentration of nutrients and metabolites. Microdialysis also allows for local delivery of pharmacological agents to tissue without resultant systemic effects. The potential of microdialysis for exercise studies is clear, yet relatively untapped.
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Interstitial fluid
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In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.
Microdialysis
Interstitial fluid
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Strong evidence supports a central role for beta-amyloid protein (Aß) in the pathogenesis of Alzheimer's disease (AD). Much attention has been focused on toxic forms of soluble and insoluble Aß. Gaining an understanding of the soluble species of Aß that are released from neurons may provide greater insight into the pathophysiology of AD. Previous work has demonstrated the ability to measure extracellular concentrations of soluble Aß in the brains of freely moving animals using in vivo microdialysis (Cirrito et al., 2003). We have compared two distinct microdialysis methods, conventional microdialysis and push-pull microdialysis, to monitor Aß levels in the brain interstitial fluid (ISF) of AD transgenic animals. In addition, we examined the effects of gamma secretase inhibition with ELN44989 on the levels of Aß (1-x) and Aß (1-40). Conventional microdialysis is limited by the membrane cut-off size and generally allows measurement of molecules smaller than 30-60 KDa. Conversely, push-pull microdialysis allows for detection of molecules up to 1-3 MDa offering the advantage of detecting a broader range of soluble oligomeric Aß species. Both methods were employed in this study, followed by ELISA measurement of Aß (1-x) and Aß (1-40). Aß (1-x) and Aß (1-40) in dialysates from conventional microdialysis were 243 pg/ml and 125 pg/ml, respectively. Using push pull microdialysis, levels of Aß (1-x) and Aß (1-40) were notably higher. In both cases, ISF Aß levels were significantly reduced following systemic administration of ELN44989. We conclude that push-pull microdialysis provides distinct advantages over conventional microdialysis for the detection of Aß species in the rodent brain, and can provide greater insight into role of multiple Abeta species (including high molecular weight) to pathological and functional endpoints.
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Amyloid beta
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For some time the subcutaneous (s.c.) tissue has been the target for continuous glucose measurement. The microdialysis technique permits an extracellular region approach, which has been used for about two decades for measuring various metabolites in dialysates obtained from different body regions. By connecting a s.c. implanted microdialysis probe to a flow chamber of an amperometric glucose sensor, the procedure of glucose sensing was transferred to ex vivo. Using this device it was possible to obtain, for up to 24 hours, s.c. tissue glucose profiles of healthy and diabetic people. The microdialysis theory, the calibration process and other microdialysis technique applications are discussed in this paper. Although the combination of the microdialysis technique and amperometric glucose sensing requires certain technical equipment, the combination of microdialysis and glucose sensor seems to be a promising approach to a continuously functioning glucose sensing system.
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Amperometry
Subcutaneous tissue
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We examined the effects of 1-methyl-4-phenylpyridinium ion (MPP+) on the release of DA in rat striatum by the in vivo microdialysis technique. For this study, we made a suitable microdialysis probe from a 22-G needle, microliter pipette tip, silica tube and polyethylene tube. Such a repairable microdialysis probe can be easily made from readily available and inexpensive materials. DA release, as determined by the 3-methoxytyramine level, was dose-dependently increased by MPP+ (1–10 mM). Only the presence of a 1 mM concentration of MPP+ in the dialysate significantly decreased the level of the DA metabolite DOPAC, while administration of higher MPP+ concentrations resulted in no significant change.
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The microdialysis technique can be used to get dialysates of the subcutaneous tissue, which can be continuously measured by an amperometric glucose sensor. In order to get further insight into the microdialysis procedure, we used a steady-state theory for microdialysis to predict the recovery of glucose in the dialysate and compared the results to experimental data obtained by a combination of the microdialysis technique with continuous amperometric glucose sensing. The recovery of glucose obtained in vitro for two different microdialysis probes was close to the theoretical predictions. When quantifying the predictions of the model with regard to the spatial concentration profile in the subcutaneous tissue, it appeared, that the presence of the microdialysis probe depressed the concentration of glucose for 0.2 mm from the probe surface. In a 24 hour in vivo experiment, there were less fluctuations in the sensor signal when the patient was lying in bed compared to the time, when the patient could move freely. In conclusion, the combination of microdialysis and glucose sensor seems to be a promising approach to a continuously functioning glucose sensing system. However, the microdialysis procedure itself disturbs the surrounding of the probe leading to a concentration gradient of glucose. This might explain some differences between the course of blood glucose and the course of subcutaneous glucose, measured by the combination of microdialysis and an amperometric glucose sensor. Further developments of such systems should aim at implanting microdialysis devices which have a minimal influence upon the tissue metabolism.
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Subcutaneous tissue
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