Data Analysis

Microdialysis offers many advantages over the techniques it replaces. For example, in pharmacokinetic studies, precise timed samples can be obtained simultaneously from various sites of the same animal. In pharmacodynamic and physiologic studies

FIGURE 11.7

Analysis of dialysates collected over a 1-min period in the preictal, ictal, and postictal phases of a class 9 seizure in a genetically epilepsy-prone rat. NE (norepinephrine), 5HIAA (5-hydroxyindoleacetic acid), and 5-HT (5-hydroxytryptamine, serotonin) increased in the immediate postictal phase and returned to near normal at 30 min postictal, while DA (dopamine) did not change during or after the seizure.

FIGURE 11.7

Analysis of dialysates collected over a 1-min period in the preictal, ictal, and postictal phases of a class 9 seizure in a genetically epilepsy-prone rat. NE (norepinephrine), 5HIAA (5-hydroxyindoleacetic acid), and 5-HT (5-hydroxytryptamine, serotonin) increased in the immediate postictal phase and returned to near normal at 30 min postictal, while DA (dopamine) did not change during or after the seizure.

in which an endogenous chemical such as a neurotransmitter is measured, the same animal and same implantation site data prior to drug or physiological challenge can serve as its own control.

In the current state of technology, it is relatively easy and reliable to compare the concentration from pretreatment (or baseline) levels and post-treatment levels and interpret them as a ratio of basal levels. However, care should be taken in interpreting the numbers of relative change. While the estimation of extracellular concentration of an endogenous chemical requires independent experiments and thus cannot be combined with other experiments, their importance in interpreting data is well established. There are several methods in practice for estimation of true in vivo recovery (an extracellular concentration). One such method is the "no net flux method" which assumes that, if the concentration inside and outside the fiber is the same, there will be no net exchange of a given chemical. In this method, the substance of interest is added to the perfusion medium in varying concentrations. The values at which there is no difference between analyzed concentrations in perfusion medium and dialysate is considered to be the true extracellular concentration.39 Another method is the "zero flow extrapolation method" which assumes that if the flow rate was nil, the dialysate will equilibrate to the extracellular fluid concentration. In this method the flow rate is reduced sequentially which allows increasing periods of time for the fluid inside the fiber to equilibrate with the extracellular fluid. The flow rate vs. concentration data are plotted to extrapolate the concentration for zero flow rate.40

The relative recovery (or efflux) of probes depends upon many factors.41 While surface area of the fiber and flow rate of the medium can be precisely controlled, the recovery of the dialysate can depend upon the tissue matrix in which the dialysis fiber is placed.42 In vivo recovery of probes may also change in relation to change in concentration, for example following cocaine and amphetamine administration, microdialysis reports a greater increase in dopamine release than was observed by conventional methods, which needs to be carefully interpreted.43 These factors make accurate generalization difficult. In uniform tissue matrices such as blood and brain, it may be possible to use a predetermined factor of the ratio of in vivo and in vitro recovery.44

When it is desirable to measure the concentration of exogenously administered substances such as drugs, an internal standard can be added to the perfusion medium. Since chemical efflux from and influx into the perfusion medium are equivalent passive processes, loss of the internal standard from the perfusion medium and influx of the drug of interest into the dialysate are equal. Thus, loss of the internal standard predicts recovery of the drug of interest. An elegant example of the use of an internal standard for pharmacokinetic studies is provided by the studies of carbamazepine pharmacokinetics carried out by Van Belle et al.4546

When establishing a new application of microdialysis or setting up a new laboratory, repeating published work and performing a number of physiological and pharmacological procedures can validate the system.3435 If the microdialysis application is in the brain, a potassium challenge (replacing the infusion medium with 100 to 50 mM K+ CSF, according to Table 11.3, after baseline has been established) would reveal a rapid neuronal release of all neurotransmitters.47 Using calcium-free medium should reveal a reduction in levels of neurotransmitters that have a calcium-dependent release.35 Pharmacological validation of the identity of an analyte can be done with reuptake blockers in the perfusion medium, which should increase the analyte in the dialysate. For example, addition of fluoxetine to the perfusion medium causes blockade of serotonin reuptake and an increase in the extracellular serotonin.48

Addition of tetrodotoxin in the perfusion medium should successfully reduce the extracellular neurotransmitter levels.3435

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