Microdialysis techniques are used extensively in blood brain barrier analysis, ADME studies, neurotransmitter assays, and other areas of investigation. These applications require fine manipulation of dialysis probes and accurate flow control of perfusate/dialysate delivered to the tissues of interest. The performance of the syringe infusion pump is vitally important, impacting not only analysis of an experimental system but the potential effects of CNS drugs, neurotransmitter modulators, and other compounds in vivo.
Microdialysis is a widely used technique for in vivo testing of endogenous molecules, drug effects, and other vital processes. The technique is minimally invasive, allowing the continuous and free flow of solutes through a probe that is designed to function much like a blood capillary. The various configurations of the probe and shaft, as well as the diminutive size of the probe on the order of µm diameter, lends to versatility both in the range of tissues that can be accessed and the processes that can be measured. This is particularly relevant in brain research, which requires a superior level of accuracy and precision in order to measure complex physiological functions with minimal adverse effects.
Upon insertion of the microdialysis probe in the tissue region of interest, the device is continuously perfused with solution resembling the ionic composition of the extracellular or interstitial space at a low flow rate typically 0.1-5.0 µL/min. Small solutes can freely pass through a dialysis membrane at the probe tip while larger molecules are withheld due to the size cut-off of a particular dialysis membrane. This prevents the entry of cellular debris or proteins into the dialysate, thus simplifying sample analysis. Two main physical processes can be measured by in vivo microdialysis. The low continuous flow rate of fresh solution permits the passive diffusion of solutes down the concentration gradient. Therefore, the technique can be used both for endogenous solute monitoring as well as compound delivery.
The microdialysis probe is composed of two main pieces, a microbore sheathing and a semipermeable membrane or dialysis membrane. The constant flow of ionic solution, meant to mimic the physiological chemical solution of the space measured (e.g. cerebral spinal fluid in brain analysis), creates a concentration gradient allowing the concise spatial and temporal measurement of endogenous molecule such as neurotransmitters, neuropeptides, energy metabolites, and others. It also provides a method to administer accurate doses of drugs and to measure the distribution, metabolic outcome, and breakdown of compounds over time.
Microdialysis technique has several advantages over other sensitive in vivo techniques. Positron Emission Tomography (PET), while non-invasive, is expensive, not amenable to small animal studies, and is limited to just a few neurotransmitters. Microsensors, while high in spatial and temporal sensitivity, suffer again from limitations in the number of neurotransmitter measured as well difficulty in verification of the optimal sensor for given measurements. For these reasons and others, microdialysis applications have been incredibly prolific and have led to tens of thousands of published studies since adoption of the technique.
Another important advantage of the method is the ability to couple with analytical platforms such as HPLC for comprehensive, sensitive, and quantitative analyte measurements. A caveat of this ability is microdialysis devices must function with equivalent levels of sensitivity, accuracy, and precision in order to produce interpretable and actionable results. This performance hinges in large part to the range and integrity of the dialysate flow rate. The flow rate is controlled by the syringe pump, in turn, and the range and fine accuracy of this rate is wholly dependent on this device.
In order to accurately measure analyte concentration changes, the microdialysis probe must be properly calibrated in vivo. When the probe is perfused with solution, the concentration of analyte in the dialysate (Cout) will typically be less than that of the sample (Cin). The relative recovery (R), or the ratio of Cout/Cin, increases with decreased flow rate and is influenced by the geometry of the probe, the temperature, and others factors. For these reasons, the relative recovery alone cannot be used as a baseline calibration
Several more accurate in vivo calibration methods have been developed including: no net flux (NNF), variable flow rate, and low flow rate. The most widespread technique, NNF, involves perfusion of the analyte of interest at various known concentrations through the probe. The concentration of analyte is subsequently measured at the outlet of the dialysate and known concentrations are chosen to encompass the actual analyte concentration in the sample (Cext). The extraction efficiency is a measurement to describe the ability of the tissue to take up material from the probe. Using the following equation, Cext, or the actual concentration of analyte in tissue can be solved: Cin – Cout = E (Cin – Cext).
It is clear that accurate and precise solution delivery is absolutely necessary for calibration and in essence for the measurement and success of in vivo microdialysis applications. The syringe pumps must accommodate a range of flow rates from nL/min to µL/min in scale, with multiple incremental steps. The pumps must handle a range of concentrations, often several orders of magnitude, and thus must be compatible with channel switching and syringe exchange operations. Depending on the application and the interior volume of the probe, the syringe pumps must supply varying amounts of solution and thus accommodate syringes of different sizes and volumes. In certain cases, concentration measurements may involve very minute differences and require a tight range of calibration samples. For these reasons, syringe pumps with minimal step movements, flow rates, and volumetric tolerances are necessary and may mean the difference between experimental success or failure. Finally, programmable syringe pump features permit highly precise and reproducible multistep infusions. This is particularly relevant to animal studies which not only involve complex measurements, but multiple replicates as well.
Microdialysis is widely used in brain studies for many reasons including the analysis of drug penetrance across the blood-brain barrier and other complex applications. The technique is used in many other organ systems as well, for applications ranging from the kinetics of drug distribution to clearance monitoring. Still other applications include glucose monitoring in diabetes patients and artificial or ex vivo organ function.
Microdialysis is the only sampling technique that can continuously monitor drug, biochemical, or metabolite concentrations in vivo in virtually any tissue. The technique can be limited by the probe’s recovery, which is directly dependent on the volume of the probe and the flow rate. The lower the flow rate, the greater the recovery, with the caveat that insufficient flow impacts the temporal resolution of the procedure. Therefore, there is a delicate balance between flow rate and sensitivity regardless of the tissue or application. As such, only syringe pumps with the highest accuracy, precision, and the greatest range can deliver the required performance and the best chances of success.
This article was written by LabX and published in conjunction with Chemyx.