thumbnail|Microdialysis probes manufactured by CMA Microdialysis AB, Kista, Sweden

Microdialysis is a minimally-invasive sampling technique that is used for continuous measurement of free, unbound analyte concentrations in the extracellular fluid of virtually any tissue. Analytes may include endogenous molecules (e.g. neurotransmitter, hormones, glucose, etc.) to assess their biochemical functions in the body, or exogenous compounds (e.g. pharmaceuticals) to determine their distribution within the body. The microdialysis technique requires the insertion of a small microdialysis catheter (also referred to as microdialysis probe) into the tissue of interest. The microdialysis probe is designed to mimic a blood capillary and consists of a shaft with a semipermeable hollow fiber membrane at its tip, which is connected to inlet and outlet tubing. The probe is continuously perfused with an aqueous solution (perfusate) that closely resembles the (ionic) composition of the surrounding tissue fluid at a low flow rate of approximately 0.1-5μL/min. Once inserted into the tissue or (body)fluid of interest, small solutes can cross the semipermeable membrane by passive diffusion. The direction of the analyte flow is determined by the respective concentration gradient and allows the usage of microdialysis probes as sampling as well as delivery tools. and dialysis sacs were implanted into animal tissues, especially into rodent brains, to directly study the tissues' biochemistry. Further improvement of the dialytrode concept resulted in the invention of the "hollow fiber", a tubular semipermeable membrane with a diameter of ~200-300μm, in 1974. Today's most prevalent shape, the needle probe, consists of a shaft with a hollow fiber at its tip and can be inserted by means of a guide cannula into the brain and other tissues. An alternative method, open flow micro-perfusion (OFM), replaces the membrane with macroscopic openings which facilitates sampling of lipophilic and hydrophilic compounds, protein bound and unbound drugs, neurotransmitters, peptides and proteins, antibodies, nanoparticles and nanocarriers, enzymes and vesicles.

Microdialysis probes

thumb|right|upright=1.8|Schematic illustration of a microdialysis probe

There are a variety of probes with different membrane and shaft length combinations available. The molecular weight cutoff of commercially available microdialysis probes covers a wide range of approximately 6–100kD, but also 1MD is available. While water-soluble compounds generally diffuse freely across the microdialysis membrane, the situation is not as clear for highly lipophilic analytes, where both successful (e.g. corticosteroids) and unsuccessful microdialysis experiments (e.g. estradiol, fusidic acid) have been reported. However, the recovery of water-soluble compounds usually decreases rapidly if the molecular weight of the analyte exceeds 25% of the membrane’s molecular weight cutoff.

Recovery and calibration methods

Due to the constant perfusion of the microdialysis probe with fresh perfusate, a total equilibrium cannot be established. The recovery can be determined at steady-state using the constant rate of analyte exchange across the microdialysis membrane. The rate at which an analyte is exchanged across the semipermeable membrane is generally expressed as the analyte’s extraction efficiency. The extraction efficiency is defined as the ratio between the loss/gain of analyte during its passage through the probe (C<sub>in</sub>−C<sub>out</sub>) and the difference in concentration between perfusate and distant sampling site (C<sub>in</sub>−C<sub>sample</sub>).

In theory, the extraction efficiency of a microdialysis probe can be determined by: 1) changing the drug concentrations while keeping the flow rate constant or 2) changing the flow rate while keeping the respective drug concentrations constant. At steady-state, the same extraction efficiency value is obtained, no matter if the analyte is enriched or depleted in the perfusate. the dynamic (extended) no-net-flux method, and the retrodialysis method. The proper selection of an appropriate calibration method is critically important for the success of a microdialysis experiment. Supportive in vitro experiments prior to the use in animals or humans are therefore recommended. Today's area of application has expanded to monitoring free concentrations of endogenous as well as exogenous compounds in virtually any tissue. Although microdialysis is still primarily used in preclinical animal studies (e.g. laboratory rodents, dogs, sheep, pigs), it is now increasingly employed in humans to monitor free, unbound drug tissue concentrations as well as interstitial concentrations of regulatory cytokines and metabolites in response to homeostatic perturbations such as feeding and/or exercise.

When employed in brain research, microdialysis is commonly used to measure neurotransmitters (e.g. dopamine, serotonin, norepinephrine, acetylcholine, glutamate, GABA) and their metabolites, as well as small neuromodulators (e.g. cAMP, cGMP, NO), amino acids (e.g. glycine, cysteine, tyrosine), and energy substrates (e.g. glucose, lactate, pyruvate). Exogenous drugs to be analyzed by microdialysis include new antidepressants, antipsychotics, as well as antibiotics and many other drugs that have their pharmacological effect site in the brain. The first non-metabolite to be analyzed by microdialysis in vivo in the human brain was rifampicin.

Applications in other organs include the skin (assessment of bioavailability and bioequivalence of topically applied dermatological drug products), and monitoring of glucose concentrations in patients with diabetes (intravascular or subcutaneous probe placement). The latter may even be incorporated into an artificial pancreas system for automated insulin administration.

Microdialysis has also found increasing application in environmental research, sampling a diversity of compounds from waste-water and soil solution, including saccharides, metal ions, micronutrients, organic acids, and low molecular weight nitrogen. Given the destructive nature of conventional soil sampling methods, microdialysis has potential to estimate fluxes of soil ions that better reflect an undisturbed soil environment.

Critical analysis

Advantages

  1. To date, microdialysis is the only in vivo sampling technique that can continuously monitor drug or metabolite concentrations in the extracellular fluid of virtually any tissue. Depending on the exact application, analyte concentrations can be monitored over several hours, days, or even weeks. Free, unbound extracellular tissue concentrations are in many cases of particular interest as they resemble pharmacologically active concentrations at or close to the site of action. Combination of microdialysis with modern imaging techniques, such positron emission tomography, further allow for determination of intracellular concentrations.
  2. Insertion of the probe in a precise location of the selected tissue further allows for evaluation of extracellular concentration gradients due to transporter activity or other factors, such as perfusion differences. It has, therefore, been suggested as the most appropriate technique to be used for tissue distribution studies.
  3. Exchange of analyte across the semipermeable membrane and constant replacement of the sampling fluid with fresh perfusate prevents drainage of fluid from the sampling site, which allows sampling without fluid loss. Microdialysis can consequently be used without disturbing the tissue conditions by local fluid loss or pressure artifacts, which can occur when using other techniques, such as microinjection or push-pull perfusion.
  4. The semipermeable membrane prevents cells, cellular debris, and proteins from entering into the dialysate. Due to the lack of protein in the dialysate, a sample clean-up prior to analysis is not needed and enzymatic degradation is not a concern.

Limitations

  1. Despite scientific advances in making microdialysis probes smaller and more efficient, the invasive nature of this technique still poses some practical and ethical limitations. For example, it has been shown that implantation of a microdialysis probe can alter tissue morphology resulting in disturbed microcirculation, rate of metabolism or integrity of physiological barriers, such as the blood–brain barrier. While acute reactions to probe insertion, such as implantation traumas, require sufficient recovery time, additional factors, such as necrosis, inflammatory responses,
  2. Microdialysis has a relatively low temporal and spatial resolution compared to, for example, electrochemical biosensors. While the temporal resolution is determined by the length of the sampling intervals (usually a few minutes), the spatial resolution is determined by the dimensions of the probe. The probe size can vary between different areas of application and covers a range of a few millimeters (intracerebral application) up to a few centimeters (subcutaneous application) in length and a few hundred micrometers in diameter.
  3. Application of the microdialysis technique is often limited by the determination of the probe’s recovery, especially for in vivo experiments. Determination of the recovery may be time-consuming and may require additional subjects or pilot experiments. The recovery is largely dependent on the flow rate: the lower the flow rate, the higher the recovery. However, in practice the flow rate cannot be decreased too much since either the sample volume obtained for analysis will be insufficient or the temporal resolution of the experiment will be lost. It is therefore important to optimize the relationship between flow rate and the sensitivity of the analytical assay. The situation may be more complex for lipophilic compounds as they can stick to the tubing or other probe components, resulting in a low or no analyte recovery.

References