Difference method, microdialysis

Graph used to calculate the point of no net flux for dopamine (DA). Using regression analysis, the extracellular concentration of DA is estimated via the difference method [the DA concentration in the perfusate minus the concentration of DA in the dialysate] plotted against the DA concentration in the perfusate. Values above the zero on the y-axis indicate diffusion to the brain, whereas values below the zero indicate diffusion from the brain. The zero point on the y-axis represents a steady state, at which no net flux of DA occurs across the dialysis membrane and represents the extracellular concentration of DA on the x-axis. Figure from Parsons, L.H., Justice, J.B., Jr. (1994). Quantitative approaches to in vivo brain microdialysis. Crit Rev Neurobiol. 8(3) 189-220... 

As the relative recovery will never reach 100%, the dialysate concentrations are only a fraction of the true concentration of the analyte of interest in the surrounding fluid. Before using a microdialysis probe for continuous sampling or monitoring, the trae concentration of the analyte of interest in the surrounding environment and the recoveries at certain perfusate flow rates have to be obtained. There are many different methods for calibration, which are discussed in the following [1]. 

Originating from the neurosciences, the microdialysis technique has been used since several years to monitor drug absorption and disposition or the levels of endogenous substances in the extracellular space of different organs and fluids, such as bone, lung, liver, brain, and blood. The method has evolved from its use in different animal species to the human microdialysis during the late 80s [35],... 

Flow-Based Systems Needle-type sensors with a fluid flowing over the sensor tip seem to resist biofouling and extend sensor lifetime.31 There are numerous methods that have been investigated for flow-based sensors, such as microperfusion systems,75 microdialysis,76 77 and ultrafiltration.78 Reduced fouling was found with an open microflow system where slow flow of protein-free fluid over the sensor surface at the implant site is effected.73 Different from the other flow-based sensors, the open microflow is controlled by the subcutaneous tissue hydrostatic pressure and does not require a pump. 

Sjbberg P. Olofsson IM, Lundqvist T (1992) Validation of different microdialysis methods for the determination of unbound steady-state concentrations of theophylline in blood and brain tissue. Pharm Res 9 1592-1598. 

Somewhat different results were reported from other laboratories n-3 fatty acid deficiency in rats caused a decrease in dopamine level and D2-receptor density in the frontal cortex as well as a decrease in serotonin receptor density (Delion, 1994,1996), whereas fish oil increased the dopamine level (Chalon, 1998). A decrease in the dopamine release from the frontal cortex was also detected by the microdialysis method after thyramine stimulation but not after KCl stimulation (Zimmer, 1998, 2000). In aged rats, however, the monoamine level was not affected, whereas monoamine oxidase activity was decreased during n-3 fatty acid deficiency (Delion, 1997). 

Ischemia may have profound effects on tissue metabolism. On the other hand, different tissues may respond differently (or to a different degree) to the same amount of ischemia. Most methods of studying metabolic changes due to ischemia require termination of the animal, and thus do not allow metabolic and survival studies in the same animal. With microdialysis, both survival and metabolic studies can be conducted. 

The primary focus of this chapter is to highlight current applications and technical advances in LC methods for the analysis of a broad range of neurotransmitters and their metabolites from microdialysis samples. In some cases, LC-based analysis of neurotransmitter tissue content from discrete brain regions is discussed. Neurotransmitter quantifications made with tissue content differ from in-vivo microdialysis measurements in that the levels of the analyte of interest are evaluated at a specific time point corresponding to the time of tissue collection. Thus, tissue content provides a static measure of alterations in the levels of neurotransmitters and their metabolites. Typically, when developing new methods for neurotransmitter analysis, tissue content levels are initially characterized, because their concentration are -10—100-fold greater than measured with in-vivo microdialysis. This is because concentrations of neurotransmitters isolated from tissue are representative of the intracellular levels of a neurotransmitter sequestered and stored within neurons. 

To date, few detection methods are available for analysis of purine molecules in brain microdialysis samples. Most methods infuse purine molecules (retro-dialysis) directly to a brain region of interest then examine alterations in baseline levels of other neurotransmitters, such as DA [150]. When ATP or adenosine is detected in the brain using microdialysis, the dialysate samples are separated and detected by LC connected to either UV absorbance or fluorescence [72—76]. Striatal baseline levels of adenosine have been reported between 40 and 210 nM in the rat, while striatal ATP levels in the mouse are 60—170 nM using chemiluminescence [165]. What is most striking about extracellular adenosine and ATP measurements are the large basal ranges reported, which may be due to differences in LC methods, detector sensitivity, or the derivatiza-tion step required for fluorescence detection. 

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