Microdialysis Education

1. What is Microdialysis?

Microdialysis is in vivo bioanalytical sampling technique used to monitor the chemistry of the extracellular space of living tissues. “Micro” refers to the extremely small scale and “dialysis” refers to the movement of chemicals across a permeable membrane.

Using specially designed probes, unbound analytes are continuously sampled. These can include endogenous molecules (e.g. neurotransmitters, hormones, glucose) sampled to assess their biochemical functions, or exogenous compounds (e.g. pharmaceuticals) sampled to determine their distribution within the animal.

Microdialysis provides a preview of what goes on in tissues, before chemical events can be reflected as changes in systemic blood levels.

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2. Principle of Microdialysis Technique

Before a molecule from the blood can enter a cell in an organ, or vice versa, it must first traverse the extracellular space. The extracellular space is filled with a fluid and comprises approximately 20% of the total tissue volume. However, this crucial compartment for chemical communication between cells (including neurotransmission) is experimentally inaccessible by conventional methods of bioanalytical chemistry.

Microdialysis offers the opportunity to assess tissue chemistry with high accuracy, before there are chemical changes occur in the blood. This process is accomplished by implanting a unique catheter—the microdialysis probe—into the tissue of interest.

The microdialysis technique relies on the passive diffusion of substances across a dialysis membrane driven by a concentration gradient. The microdialysis probe is implanted in the target site, generally a blood vessel or tissue. A physiological salt solution (perfusate) is slowly and constantly pumped through the probe’s semipermeable membrane and the solution is equilibrated with the surrounding tissue fluid. When collected at the outlet, the resulting microdialysis solution will contain a representative proportion of the tissue fluid’s molecules. The solution is analyzed to determine the presence of drug or target molecules in the microdialysis sample by suitable analytical techniques

Microdialysis is an exchange of molecules in both directions. As such, it is often used to introduce a substance, such as a drug candidate, into the extracellular space to evaluate regional drug administration. Sampling of endogenous compounds in the extracellular compartments can be performed at the same time.

Because microdialysis is a continuous sampling method, it differs from point sample methods, such as blood sampling. The ability to obtain local measurements in the tissues has led to important discoveries in the detection of tissue changes within the areas of pharmacokinetics, pharmacodynamics, pathology and pathophysiology.

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3. Microdialysis Probes & Membranes

The microdialysis probe is a specially designed catheter with a semipermeable membrane at its tip. It mimics the function of a blood vessel. The probe’s semipermeable membrane allows an exchange of compounds from an area of high concentration to an area of low concentration.

Once the probe is implanted into a target tissue, it is constantly perfused with a physiological salt solution at a low flow-rate (usually less than 2 µl/min). Endogenous substances are filtered by diffusion out of the extracellular fluid into the perfusate within the probe. By reversing the process the probe can be used to locally infuse exogenous compounds, nutrients and drugs for periods of up to several days.

The main design resembles a concentric tube. The perfusion fluid enters through an inner tube, flows to its distal end, exits the tube, and enters the space between the inner tube and the outer dialysis membrane. The direction of flow is now reversed and the fluid moves toward the proximal end of the probe. This is where the “dialysis” takes place, i.e. the diffusion of molecules between the extracellular fluid and the perfusion fluid. Substances from the extracellular fluid of the tissue diffuse across the membrane of the catheter into the perfusion fluid inside the catheter, then to the probe outlet for collection and analysis.

Probes are available with microdialysis membranes of different sizes. Thus it is possible to sample molecules in size ranging from a few hundred Daltons up to about 30,000 Daltons. This makes it feasible to monitor neurotransmission, amino acids, energy metabolites, markers of inflammation, pain, and disease biomarkers, as well as available drug levels in target tissues. 

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4. Microdialysis Research Applications

For more than thirty years, microdialysis has been used to study brain neurophysiology and the release of neurotransmitters, monoamines, and metabolites, amino acids and other small endogenous compounds. With the introduction of microdialysis probes for use in the peripheral organs, microdialysis has seen widespread use in sampling molecules in tissues such as muscle, liver and adipose tissue, as well as in the spinal cord, synovial fluid, vitreous humor and blood, to assess the delivery and distribution of parent drug and metabolites and their effects on endogenous compounds.

a. Drug Screening & Development

Understanding the effect of a drug candidate at its site of action is a critical step in the drug development process. Such information also helps selection of the most promising compounds during screening and determination of doses with optimum therapeutic effect.

The use of microdialysis in drug development can help improve the predictability of clinical outcomes from preclinical studies by providing indicative information at the research stage.

Microdialysis probes can be implanted simultaneously in several organs (including blood) of the same animal. Distribution and time course of free drug (toxin) concentrations are measured in vivo. Pharmacokinetic data can be calculated using theoretical compartment models.

Since the exchange of molecules through the dialysis membrane is in both directions, a microdialysis probe placed in target tissue can be used to continuously:

• Sample unbound drug and/or active metabolites as they arrive to the tissue following systemic administration.
• Deliver drug locally into an organ or tissue through the probe, and simultaneously collect.
• Endogenous target compounds to determine pharmacological effect.
• Assess controlled release of drugs from encapsulation in vivo, within specific tissues.

Microdialysis is a valuable tool for in vivo evaluation studies on drug delivery, drug metabolism, PK/PD, toxicology, bioavailability, bioequivalence and pharmacological efficacy. It is the only technique that gives simultaneous in vivo temporal information on unbound drug and metabolic levels as well as endogenous compounds in target tissues.

b. Psychopharmacology

The mechanisms of drug action on release, uptake and interactions among neurotransmitters and neuromodulators represent the classical application field for microdialysis. Neurochemical correlates to different models of mental disorder, behavioral and cognitive functions can be studied in chronically implanted freely moving animals.

c. Neurophysiology & Neuropathology

Microdialysis has long been used to study brain neurophysiology and the release of neurotransmitters, monoamines and metabolites, amino acids and other small endogenous compounds. It is an excellent tool for monitoring compounds proposed as markers of brain injury. Neurodegenerative diseases, such as ischemia, hypoglycemia and epilepsy, as well as processes related to neuronal plasticity, regeneration, neurotransplantation or tumor growth have been elucidated by microdialysis.

d. Cancer Research

Microdialysis probes are placed in tumor and skin. In these areas, microdialysis is used to collect free available drugs and soluble endogenous factors, including cytokines, to simultaneously assess penetration in vivo in real time. Drugs can be delivered intratumorally or into the dermis through the probe to assess effects on collectable biomarkers.

e. Physiology

Physiological stimuli such as physical exercise, nutrition or stress alter anabolic and catabolic phases of cell biochemistry in peripheral tissues (e.g. muscle, fat). Microdialysis data can serve as a cumulative index of treatment-induced metabolic changes over long time periods.

f. Tissue Engineering

Microdialysis can be adapted as a reliable technique for monitoring cell metabolism and tissue functions. Protein synthesis and energy metabolism within functional tissue can be continuously sampled using microdialysis probes of high molecular weight cut-off. Microdialysis can be then successfully used to monitor metabolism within cultured tissue explants and could be applied to monitor tissue growth in engineered tissues or during tissue repair in vivo.

g. Agriculture & Ecology

Microdialysis is an emerging technique that has been used for in situ and minimally invasive sampling of soil solution solutes. This method has already been used to assess the prevalence and composition of nitrate, ammonium and amino acids in some agricultural soils, showing outstanding results regarding the possible spatial and temporal resolution of the soil solution chemistry. The emergence of the use of microdialysis is this context may become of critical importance in efforts to improve the management of agricultural soils.

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5. Advantages of Microdialysis

Microdialysis is the most flexible in vivo technique and possesses some extraordinary features:

• Spatial resolution, allowing local sampling from very small tissue regions e.g. brain nuclei.
• Time resolution, achieved by sampling minute fractions at high frequency over several days.
• Adaptable for monitoring almost any endogenous or exogenous substance or related metabolites.
• Enables continuous monitoring of intercellular chemical communications.
• Estimation of absolute concentrations of analytes of interest in the extracellular fluid is possible.
• No purification of samples is required prior to analysis.
• There is no risk of enzymatic breakdown of recovered substances.
• Fewer animal experiments are required than other techniques to determine the dose and time profiling of a drug candidate.

Microdialysis is the most physiological way of in vivo sampling, creating minimal adverse tissue reactions, because:

• No macroscopic matter is removed or introduced into the tissue.
• The driving force for sampling is only passive diffusion.
• The semi-permeable membrane of the probe protects the tissue from infection and, on the other hand, does not remove large molecules, such as proteins and enzymes, from the extracellular matrix.
• Samples can be taken continuously from freely moving animals for periods up to several days.

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6. Microdialysis Experimental Design Considerations

There are some concepts and considerations to understand and take into account when designing a microdialysis experiment. Below is a summary list of the more important ones:

1. Properties of the probe membrane

   Consider the molecular weight of the analyte you are collecting. A membrane with a low molecular weight cut off purifies your sample by excluding large molecules, while a high molecular cut off recovers some larger substances, such as peptides or proteins.

2. Type of probe

   Consider the type of tissue or organ you are studying. A stiff probe is suitable for a stereotaxic experiment on the brain while a flexible probe may be better suited for dialysis in a peripheral organ such as adipose tissue, muscle, liver or kidney. A brain probe may require a pre-implanted guide cannula while a subcutaneous probe may be implanted an hour or so before the start of the experiment.

3. Length of the membrane

   Consider your recovery requirements. A longer membrane yields a better recovery of the substances of interest; however, the choice may be limited by the size of the structure you want to study.

4. Perfusion flow

   Use a high flow rate if you want to remove or introduce as many molecules as possible per unit time or a low flow rate if you want to obtain a more concentrated dialysate. It is worth considering that a high flow is liable to disturb the physiology simply because more substances are removed.

5. Composition of the perfusion fluid

   The composition of the perfusate should ideally be as close as possible to the composition of the extracellular fluid. However, you may want to change the concentration of sodium, potassium or calcium in order to influence the cell membrane function in the region you are studying.

6. Time needed to obtain steady state conditions

   The introduction of a probe into the tissue will always cause damage and the recovery of function in the animal will take a certain period of time. An hour or two is often used to reach “baseline conditions”.

7. Use of awake vs. under anesthesia animals

   Using awake animals does not necessarily mean that the conditions are more “normal”. An awake animal is subject to pain and stress that may influence the results as much as the anesthesia.

8. Design of control experiment

   This is certainly one of the most important parts of any experimental design. One may have difficulty in determining the influence of a great number of known or unknown variables in your experiment; however, a well-designed control experiment will take care of many of these problems.

9. Dose-response study

   Microdialysis is an excellent technique for studying drug actions. The ease by which one can follow the time course of local drug concentrations in tissue and drug effects on local physiology is one of the really strong points of the technique. Dose-response experiments are particularly recommended since the qualitative action of a drug often changes as the dose changes.

10. Sample volume required for analysis

    The sample volume and concentration required may depend on the technique used to analyze collected samples. Is a small sample volume and a high concentration (e.g. for HPLC) or a large sample volume and a high amount of the particular compound (e.g. for RIA) required? You may want to choose a low or a high perfusion flow, respectively.

11. Time resolution needed

    Frequent sampling usually means higher perfusion flow in order to get enough sample volume for the analysis. More frequent sampling can provide a more precise measurement (better temporal resolution) of analyte concentration over time. However, there may be a trade-off between time resolution and spatial resolution, which is determined by probe dimensions. Consider how precise a measurement of analyte concentration over time you require.

12. Adequate recovery

    The recovery of a particular substance is defined as the concentration in the dialysate expressed as a percent of the concentration in the interstitial fluid. In other words, the recovery is the expression of the dialyzing properties or efficiency of a microdialysis probe. The recovery of a probe depends on different factors such as the flow rate, membrane properties, analyte properties, and temperature and tissue factors.

    
    

    Probe in vitro recovery tests are conducted to assess the efficiency and dialyzing properties of the microdialysis probe for the particular substance of interest. The purpose of these experiments is to determine if the flow rate and collection period selected yield a sample with a detectable concentration of analyte, as measured by recovery. This is an important step before conducting actual experiments. See Application Note Principles of Recovery in Microdialysis for detailed information

    
    

    The choice of the equipment needed for the in vitro recovery test will depend on the method chosen for carrying out this test. In additional to the chosen probe, the usual equipment used for the in vitro recovery test is a syringe pump for fluid perfusion (CMA 402, CMA 4004), a liquid switch (CMA 110), an in vitro storage stand (CMA 130) and probe clips. The liquid switch allows switching between several perfusion lines (syringes) and a microdialysis probe. An in vitro stand and probe clips are used for storage and during the in vitro test of the probes. The clip makes it easier to handle the probe and reduce the risk of damaging the fragile membrane. The clip is also used when introducing the probe guide in the brain for CNS microdialysis studies.

13. Instrument set-up

    Microdialysis is not only about probes: a whole system is composed of additional components such as the probe accessories, a syringe pump, a fraction collector etc. See next section Microdialysis System Components for more information about the components of a microdialysis set-up.

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7. What Comprises a Microdialysis System?

A microdialysis system collects an analyte of interest from an organ or tissue of an anesthetized or conscious animal. Very simply, a physiological salt solution, or perfusion fluid, is slowly pumped though the microdialylsis probe using a syringe pump and special microsyringes. Samples are continuously collected from the fluid that flows back out of the probe into a microdialysis fraction collector for analysis.

Additional accessories and instruments may be required beyond these basic system components based on (1) the application and (2) the experimental requirements to complete a functional microdialysis system configuration.

This section provides information about the microdialysis system components and configurations using CMA and Harvard Apparatus products as examples.

a. Microdialysis System Components

Microdialysis Probe

The core of the microdialysis system is the microdialysis probe, reviewed in Chapter 2, which is implanted into the tissue. The choice of probe for a microdialysis experiment depends on the application, targeted structure or tissue, the size of molecule under study, as well as other factors. Other important factors related to the probes are the molecular weight cut-off, the membrane, shaft length and membrane material.

CMA Microdialysis Probes
Probe Selection Guide
Probe In Vitro Recovery Test


Syringe Pumps and Accessories

The syringe pump is a critical component of the whole microdialysis fluid circuit. The choice of the syringe pump will depend on the needed flow rates, syringe compatibility, and infusion modes (push and/or pull) for the experiment.

CMA 4004 Microdialysis Touch Screen Syringe Pump
CMA 402 Microdialysis Syringe Pump
CMA Microsyringes


Perfusion Fluid

The composition of the perfusate should ideally be as close as possible to the composition of the extracellular fluid. However, the concentration of the perfusate (sodium, potassium or calcium) can be changed in order to influence the cell membrane function in the region under study.

Perfusion Fluid


Fraction Collector

The choice of fraction collector depends on the sampling frequency and on the stability of the collected sample. Microdialysis sampling and analysis of stable compounds (pharmaceuticals, cell nutrients, metabolites and inorganic ions) usually does not need immediate refrigeration. Unstable compounds require the use of a refrigerated fraction collector enabling the collection of cooled fractions into capped or open vials to prevent chemical degradation and evaporation.

CMA 142 Fraction Collector
CMA 470 Refrigerated Fraction Collector


Probe Accessories

Specific applications may require the microdialysis probe or probe guide to be connected or secured to other system components, such as a stereotaxic frame or an in vitro stand, or cemented to the animal’s skull. A range of accessories is available for these purposes.

Probe Accessories
Stereotaxic Holders


Surgical Instruments, Anesthesia Equipment and Accessories

Most microdialysis experiment require a surgery procedure, especially for the probe implantation (stereotaxic and other surgical instruments). Specific equipment for anesthesia procedure by itself and concomitant animal monitoring (temperature, respiration etc.) are also required for microdialysis studies conducted on an anesthetized animal.

Stereotaxic Instruments
Surgical Instruments
Anesthesia Equipment
Homeothermic Monitoring System


Freely Moving Animal System

Research in behavioral pharmacology, cognitive sciences, toxicology and many other fields requires microdialysis experiments on conscious animals over long periods of time. In this context the basic microdialysis setup can be combined with specific accessories for freely moving animals: containers, food and water accesses, counter-balanced arms, swivel system etc.

CMA 120 System for Freely moving Animals



b. Microdialysis System Configurations

Anesthetized Animals
Freely Moving Animals

A wide variety of configurations exist for carrying out microdialysis experiments depending on application and the experimental requirements. Given the importance of each component of a system on the reliability of the experiment, please contact technical support for assistance in configuring a system that will be suit your experimental needs.

Please find below some examples of standard configurations on anesthetized and freely moving animals using CMA and Harvard Apparatus products.

Microdialysis System Configurations for Anesthetized Animals

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8. Microdialysis Resources

Kho, CM., Enche Ab Rahim, SK., Ahmad, ZA., Abdullah, NS (2017) A Review on Microdialysis Calibration Methods: the Theory and Current Related Efforts. . Mol Neurobiol..54(5): 3506-3527. doi: 10.1007/s12035-016-9929-8.

Erdő, F. (2015) Microdialysis Techniques In Pharmacokinetic and Biomarker Studies. Past, Present and Future Directions A Review . J Clin Exp Pharmacol. 5: 180. doi:10.4172/2161-1459.1000180.

Kehr, J. & Yoshitake, T. (2013) Monitoring molecules in neuroscience: historical overview and current advancements. Frontiers in Bioscience . E5, 947-954, June 1.

Müller, M. (ed.) (2013) Microdialysis in Drug Development . AAPS Advances in the Pharmaceutical Sciences, Series 4. Springer, New York.

de Lange, E.C.M. (2013) Recovery and Calibration Techniques: Toward Quantitative Microdialysis . In: Müller, M. (ed.), Microdialysis in Drug Development. AAPS Advances in the Pharmaceutical Sciences Series, Volume 4. Springer, New York.

Di Giovanni, G. & Di Matteo, V. (eds.) (2013) Microdialysis Techniques in Neuroscience . Neuromethods Series 75, Humana Press, Springer, New York.

Chefer, V.I., Thompson, A.C., Zapata, A. & Shippenberg, T.S. (2009) Overview of Brain Microdialysis . Curr Protoc Neurosci. Apr; Chapter: Unit7.1. doi: 10.1002/0471142301.ns0701s47.

Zapata, A., Chefer, V.I. & Shippenberg, T.S. (2009) Microdialysis in Rodents . Curr Protoc Neurosci. Apr; Chapter 7: Unit7.2. doi: 10.1002/0471142301.ns0702s47.

Kehr, J. (2007) New methodological aspects of microdialysis . In: Westerink, B. & Cremers, T. (eds.) Handbook of microdialysis: Methods, Applications and Perspectives. Elsevier, The Netherlands, pp 111-129.

Kehr, J, & Yoshitake, T. (2006) Monitoring brain chemical signals by microdialysis. In: Grimes CA, Dickey EC & Pishko MV (eds.) Encyclopedia of Sensors, Vol. 6. American Scientific Publishers, USA. 287–312.

Shippenberg, T.S. & Thompson, A.C. (2001) Overview of Microdialysis . Curr Protoc Neurosci. 2001 May; Chapter 7: Unit7.1. doi: 10.1002/0471142301.ns0701s00.

Kehr, J. (1999) Monitoring chemistry of brain microenvironment: biosensors, microdialysis and related techniques. In: Windhorst, U. & Johansson, H. (eds.) Modern techniques in neuroscience research. Springer-Verlag GmbH, Heidelberg, 1149–1198.

Web pages

• Webpage: Wikipedia – Microdialysis
• Webpage: LabAutopedia – Microdialysis: An introduction

Application notes

• Application note: Principles of Microdialysis Recovery
• Application note: In Vitro Recovery Measurement of Peptides.pdf

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