What is Microdialysis

What is Microdialysis

Introduction

Microdialysis is a powerful sample collection and administration technique used in scientific research to monitor and analyze the chemical composition of extracellular fluid in various tissues. Microdialysis offers valuable insights into the dynamic changes of neurotransmitters, metabolites, and other molecules within living organisms. In this blog post, we will delve into the intricacies of microdialysis and explore its applications across different fields.

Section 1: Understanding Microdialysis

Principles of Microdialysis

Microdialysis involves the insertion of a semi-permeable probe, known as a microdialysis probe, into the tissue of interest. The probe is perfused with a physiological solution that closely mimics the composition of the extracellular fluid. As the perfusion solution flows through the probe, it creates a concentration gradient that facilitates the diffusion of molecules from the surrounding tissue into the probe. The collected dialysate can then be analyzed to measure the levels of specific analytes.

Probe Design and Materials

Microdialysis probes are available in various designs and materials, each with its own characteristics and applications. The choice of probe design, such as shaft length, diameter, and membrane length, depends on the target tissue. The materials used for the probe membranes can be tailored to suit specific research needs, including biocompatible polymers or glass.

Section 2: Applications of Microdialysis

Neurochemical Research

Microdialysis has revolutionized the field of neuroscience by providing a means to study the dynamic changes in neurotransmitter levels within the brain. Researchers can investigate neurotransmitter release, reuptake, and metabolism in various brain regions. Microdialysis is also invaluable in studying the effects of drugs, diseases, and neuropsychiatric disorders on neurochemical signaling.

Explore Eicom Microdialysis Probes!

Our probes have a 220 µm outer diameter. The probe inlet connects to a syringe pump, which continuously perfuses through the probe.  As the perfusate travels through the probe, analytes cross the membrane and travel out the probe outlet into sample vials on a fraction collector or to a sample loop of an analytical system via a narrow tubing. This valuable tool enables the sampling of rather low molecular weight compounds from within the extracellular space of tissues.

Pharmacokinetics and Drug Development

Microdialysis plays a crucial role in pharmacokinetic studies by allowing researchers to monitor drug concentrations. By continuously sampling from specific tissues, such as brain or muscle, microdialysis provides valuable information on drug distribution, metabolism, and elimination. This data aids in the optimization of drug dosage and formulation during the drug development process.

 

Metabolic Studies

Microdialysis is widely employed in metabolic research to assess tissue-specific metabolism. By measuring the levels of substrate and metabolites, such as glucose, lactate, and amino acids, researchers can gain insights into energy utilization, cellular metabolism, and metabolic disorders. Microdialysis also enables the study of organ-specific metabolism in various tissues, including the liver, adipose tissue, and skeletal muscle.

Environmental Monitoring

Microdialysis finds applications in environmental research for monitoring and analyzing chemical compounds in different environmental matrices. It enables continuous sampling and analysis of pollutants, toxins, or biomarkers in water, soil, air, and biological tissues. Microdialysis contributes to understanding environmental contamination, pollutant fate, and human exposure risks.

Integration with Imaging Techniques

Microdialysis can be combined with imaging techniques, such as fluorescence imaging or mass spectrometry imaging, to provide spatial information about analyte distribution within tissues. This correlation between microdialysis and imaging techniques enables researchers to gain insights into the spatial dynamics and localization of analytes, enhancing their understanding of physiological processes and disease mechanisms. By integrating microdialysis with imaging techniques, researchers can correlate the temporal information obtained through microdialysis with the spatial distribution of analytes. This integration opens up new possibilities for advanced research in fields such as neuroscience, pharmacology, and environmental monitoring.

Scarce Microdialysis Goods May Delay Neuroscience Research

Scarce Microdialysis Goods May Delay Neuroscience Research

Even with thorough planning, global supply chain issues have caused delays in nearly every industry, including lab supplies. Microdialysis probes and other consumables, such as pipette tips, prove difficult for researchers to source due to the globally stressed transit structures. Manufacturing and shipping are complex processes. Combined with labor issues and jammed cargo ports, the pandemic is exposing the shortcomings of a weak supply chain.

Eicom, a world leader in microdialysis and electrochemical detection, is headquartered in Japan. In business since 1986, Eicom would probably be seeing the same delays as other microdialysis providers, except for a well-planned overseas inventory structure.

Eicom’s partner in North America, Amuza, houses microdialysis inventory in San Diego, California.

“Most often Amuza is able to ship microdialysis probes with one or two days,” says CEO, Shinji Azuma. “Serving researchers is our priority.”

A broader reach for materials and a network of partners is thus far succeeding in experiencing minimal supply chain issues for Amuza. Customer Service teams can excel through staying alert for order status changes, managing stock, and keeping customers well informed. Amuza also manages the supply chain by making some items at the San Diego office, such as microdialysis tubing with pre-installed connectors. By customizing microdialysis tubing then preassembling connectors on-site, Amuza has been able to mitigate the pandemic’s effects on sourcing, giving researchers a faster turnaround.

“Throughout the pandemic, we have kept up with microdialysis product orders,” says Office Manager, Ai Cole. “If we do have to inform a customer about a delay, it has been for customs. Fortunately, we have not had the delays we hear others are experiencing.”

Business news outlet, Quartz reports, more than 2 months out, “It’s already too late to ship good to the US in time for Christmas.” Customs has been swamped this past year, and the delays are affecting both holiday gifts and lab essentials needed for research projects.

How Bad Are Delays in Microdialysis Probes?

One researcher mentioned they eventually had to switch vendors, after calling 3 different vendors trying to source microdialysis equipment.

Another said they “had to wait 3 months for a microdialysis syringe pump”.

In other conversations, users have mentioned that some models of microdialysis probes or microdialysis accessories could be back-ordered for months.

When research projects aren’t able to source products necessary for research, science suffers, medicine suffers, humanity suffers. It sounds extreme, but each scientific article published is a result of years of research using lab tools necessary to complete the work.

Without the goods, our scientists lack the tools needed to advance knowledge and discovery that advances human lives.

Most microdialysis probes and accessories are still in stock at Amuza. However, when some inventory does reach low levels, Amuza has still been able to get partial orders out the door quickly. For researchers, Cole suggests, “Ask your supplier. Some suppliers may be able to help you with partial shipments to keep your research going.” At least neuroscience research can stay moving and not halt progress.

What lab products are you finding most difficult to source? Let us know in the comments.

Microdialysis vs Fiber Photometry for Neurotransmitters

Microdialysis vs Fiber Photometry for Neurotransmitters

Microdialysis and Fiber Photometry are both powerful techniques for monitoring the concentrations
of neurotransmitters in vivo, but each technique has very different advantages. Amuza is the only company that provides both types of equipment to the neuroscience community, putting us in a unique position to help you determine when microdialysis or fiber photometry would be the best choice.

MicrodialysisFiber Photometry
Best for
  • Monitoring long-lasting changes
  • Absolute concentrations
  • Many analytes
  • Sub nanomolar analytes
  • Monitoring fast changes
  • Relative concentrations
  • One or two analytes
  • Targeting cell subtypes
SensitivityPicomolarNanomolar
Multiple analytesMany analytes can be measured in each sample with the little added difficultyData processing and experimental protocol are both more difficult
Types of analytesMost types of molecules.Limited by available fluorescent sensors
Type of measurementAbsolute concentration or % change relative to baseline% Change relative to baseline
Data analysisRelatively simpleOften quite complex
Sampling period~30 seconds to hoursMilliseconds
Sampling durationDaysDays, but difficult to monitor slow changes
TargetingBrain region/structureThe brain region, specific projection, cellular subtype, axon vs cell body
Animal movementTether requiredTether or wireless headstage

Multiple analytes

It is routine to measure many different neurotransmitters or amino acids in each microdialysis sample using HPLC chromatography and electrochemical detection. Samples can also be split and stored before analysis so that additional analytes can be added to the protocol at a later date. Commercially available assays also allow panels of multiple peptides and proteins to be measured in each sample.

Measuring multiple analytes simultaneously using fiber photometry is possible, but it requires expressing multiple fluorescent sensors at the target and the data analysis also becomes much more complex

Types of analytes

Microdialysis is extremely flexible and has been used to measure many types of analytes including amino acids, proteins, peptides, neurotransmitters, sugars, and more. If you can assay for it, you can probably sample for it using microdialysis.

Fiber photometry can now measure most of the principal neurotransmitters but is limited by the fluorescent sensors available. The number of sensors is increasing rapidly, but the options are still smaller than those available to microdialysis users.

Type of measurement

Microdialysis allows the monitoring of absolute concentrations or concentrations relative to basal levels in every sample.

Fiber photometry data yields a change in fluorescence data (∆F/F0). This correlates with changes in the concentration of the analyte relative to baseline but typically isn’t used to determine absolute concentrations.

Data Analysis

HPLC software packages already include all of the functions required to automate the processing of microdialysis data, allowing the monitoring of many analytes simultaneously over time. The analysis can be easily taught to new users. In contrast, fiber photometry data processing is still usually a complex operation, with most labs using multiple software packages and writing their own scripts to deconvolve and analyze multiple streams of data. (TeleFipho photometry data only uses fluorescence data from one wavelength, and is easier to process)

Sampling Period and Duration

While sample times can be as short as 30 seconds, microdialysis excels in measuring long-lasting changes in extracellular concentrations, and for determining absolute concentrations for baseline levels. It is quite routine to continuously sample for many hours or even days during a microdialysis experiment. Furthermore, the microdialysis probe can be removed and replaced with a dummy probe for days or weeks between sampling sessions.

Fiber photometry is at the opposite extreme: fluorescence is measured every 1 to 10 ms, allowing it to record events lasting less than a second. But drift, noise, and photobleaching during fiber photometry experiments complicate observation of slow changes over time.

Targeting

Both microdialysis and fiber photometry are used to target a discrete region or structure within the brain. Fiber photometry also allows retrograde or anterograde targeting, so that only specific projections are monitored. Viral vectors that only express the sensor in a specific cellular subtype or target either the axon or cell body can further narrow the scope of the events recorded.

Animal movement

Microdialysis and fiber photometry both typically require a tether during the experiment. TeleFipho is the exception and uses a wireless rechargeable headstage to gather and transmit fiber photometry data.

Do you have a question about microdialysis or fiber photometry?

How to Choose the Best Microdialysis Probe for Your Project

How to Choose the Best Microdialysis Probe for Your Project

Microdialysis Probes How to Choose the Best One

There are many types of microdialysis probes available – some labs even make their own microdialysis probes. This guide can help you choose the right microdialysis probe for your next project. To learn about how microdialysis works and how it can advance your research goals, please start with our overview: What Is Microdialysis?

If you will be doing chronic microdialysis to measure dopamine, serotonin, acetylcholine, amino acids or other small molecules in mouse and rat brains, then, our FZ and CX probes are the best choice. These offer industry-leading recovery rates (the ratio of the concentration of an analyte in a recovered sample to the concentration of the analyte in the area surrounding the microdialysis membrane) for small molecules.

When you are working with mice, we recommend lightweight CX-i probes as your first choice. The CX’s narrow-body allows for bilateral microdialysis, even if your targets are close to the midline. The CX probe snaps into its guide cannula, so it is quick to insert and remove.

For rats and for longer experiments with mice we recommend our newly released FZ probes. The FZ probe is secured in the AG guide cannula by a cap nut (AC-5), locking it into place. This way rats and cannot dislodge the probe by scratching at it. The FZ probe is very similar to our outgoing AZ probe: we kept the best parts of the AZ probe and then made it both easier to use and more robust for use with more active adult rats. The FZ probe uses the same guide cannula and accessories as the AZ probe.

The FZ probe is also offered with a microinjection option: a microinjection cannula (orange) is attached to the MI-AZ probe so that drugs, antibodies, and other molecules can be delivered to the microdialysis site at a specific time(s).

The FZ, MI-AZ, and CX probes are mainly used for chronic microdialysis and use guide cannulae to accurately position the probe during implantation and protect the probe during the experiment. The guide cannula can be surgically implanted well before your actual microdialysis experiment and allows you to quickly implant the probe itself just before beginning an experiment. A dummy probe can be used instead of a microdialysis probe during recovery, conditioning, or while waiting for an injected vector to begin expressing proteins. We recommend using each microdialysis probe only once, as microdialysis membranes may become fouled if used repeatedly. When this happens, the recovery rate of the probe may drop and alter the results of your experiment.

DZ probe

 

For acute microdialysis experiments, where the mouse or rat remains on the stereotaxic frame during the entirety of the experiment, we have the DZ probe. This is our smallest probe, and it is intended to be directly implanted without a guide cannula. While we recommend it for acute experiments, The DZ probe can be used for shorter experiments with freely moving mice.

Researchers use our AtmosLM™ large molecule microdialysis probes to measure levels of peptides, proteins, antibodies, and even ApoE (Apolipoprotein E) particles. These probes have membranes with a molecular weight cut off of one million daltons. The AtmosLM PEP probes are superior to other push-pull probes because we add a vent to equalize pressure between the lumen of the probe and ambient conditions. This stops convection – the bulk transfer of water and solutes being driven across the membrane by pressure – and greatly enhances the accuracy of sampling. We offer these probes with polyethylene – uncharged – and polysulfone – charged membranes, to match your analyte.

If you will need to do MRI or PET imaging studies with the guide cannula and probe in place, we offer C-I probes and guide cannulae. The C-I series are metal-free and won’t interfere with imaging.

OP Probe

TP Probe

All of the probes discussed above were designed for use in the brain, but we also have probes for peripheral tissues. The OP linear probe is designed for dermal use and has a needle at one end to make it easy to implant, and the TP flexible concentric probe can be used in blood vessels and the GI tract.

Many of our probes can be made with different membrane materials. If you would prefer a polyethersulfone (PES) or polyacrylonitrile (PAN) membrane instead of cuprophane (artificial cellulose), please ask us. All of our probes, stereotaxic adapters, and accessories are made to your custom size requirements in just two weeks – even for the smallest orders. We also maintain a large inventory of FZ, CX-i, and PEP probes ready to ship the day you place your order.

If you are planning a new project or having trouble with your current one, please give us a call and we can help you troubleshoot. Eicom has been manufacturing microdialysis probes and equipment for 30 years and we have an integrated support/sales team so you can rely on our expertise.

Questions?

How to Improve Microdialysis Throughput and Success Rates

How to Improve Microdialysis Throughput and Success Rates

There are many ways to speed up your microdialysis experiments and ensure their success.

During surgery:

  • Stereotaxic frames with digital readouts are much quicker to use than ones still equipped with vernier scales: the instant readout and ability to zero each axis speed up each movement of the manipulator. Digital readouts are available as a retrofit for many older stereotaxic frames. Additionally, dual manipulator stereotaxic frames can speed up each surgery by keeping multiple tools accessible at all times.
  • After the initial incision, scratch the surface of the skull with a scalpel: cement will bond more tightly to the roughened surface.
  • Wipe the skull with hydrogen peroxide to help dry it and then wipe with epinephrine to prevent bleeding. For implantation to be successful, the surface of the skull must be dry. If the surface is still damp when cement is applied, the trapped moisture can lead to infection and necrosis of the skull.
  • Switching to UV cure cement lets you avoid mixing a new batch (powder plus solvent) of acrylic cement each time your previous batch hardens. A simple UV light hardens the cement when you need it to – not before.

For sample collection:

  • Use 96 well plates to collect samples instead of individual sample tubes. This eliminates capping, labeling, and handling of individual tubes as samples are collected and moved to storage. Used in conjunction with well plates, self-closing plate seals such as silicone sealing mats and cap mats will protect samples from evaporation and contamination.
  • Use the best fraction collector. The Amuza FC-90 collects from up to four animals/probes simultaneously and never skips a sample. Samples can be collected into 96 well plates or racks of individual tubes, and are kept refrigerated throughout.

And finally – Ask Amuza!
Amuza (formerly Eicom USA) has decades of experience in microdialysis and HPLC-ECD, and we are always ready to help.

 

Eicom AtmosLM Microdialysis Used in Developing Pharmacokinetic Models of Therapeutic Antibody Distribution in the Brain

Eicom AtmosLM Microdialysis Used in Developing Pharmacokinetic Models of Therapeutic Antibody Distribution in the Brain

The brain is a challenging target for therapeutic monoclonal antibodies (mAbs), nanobodies, antibody drug conjugates (ADCs), and other drugs. The blood brain barrier prevents many drugs with otherwise good absorption profiles from crossing into the brain, and also complicates attempts to model how drugs are distributed within the brain.

Prof. Dhaval Shah and PhD student Hsueh-Yuan Chang of the University at Buffalo study the pharmacokinetics/pharmacodynamics (PK/PD) of therapeutic antibodies and ADCs. Their lab recently used the Eicom AtmosLM (large molecule) microdialysis system as a way to quantitate a mAb in multiple brain regions simultaneously, generating data to underpin pharmacokinetic models for the disposition of mAbs in rats (1,2). They found that tissue homogenate and lumbar cerebrospinal fluid samples do not make good proxies for predicting mAb concentrations at their sites of action within the parenchyma of the brain. They also found that the lateral ventricles and the blood-CSF barrier may be an important route for mAb entry.

How Large Molecule microdialysis works

AtmosLM is a push-pull microdialysis system for measuring the levels of large proteins and peptides, as opposed to the catecholamines and other small molecules typically measured by microdialysis. AtmosLM features unique probes that incorporate vents to equalize the pressure inside the membrane of the probe with the outside atmosphere. This prevents ultrafiltration and yields more consistent analyte recovery rates than other push-pull systems. It has been widely used to study levels of Abeta, Tau, synuclein, lipidated ApoE particles, cytokines, and other molecules. Recently, the Derendorf lab
(U of Florida) used AtmosLM to determine tissue interstitial concentrations of mAbs in liver, skin, kidney, and muscle after IV dosing to aid in their development as anticancer drugs.

Microdialysis based PK modeling

Amuza spoke with Hsueh-Yuan (Luke) Chang about how he used AtmosLM in this project, and he also shared several tips for other users of AtmosLM.

Amuza: Could you explain how your PBPK (physiologically-based pharmacokinetic) model can be used by those studying the use of mAb based drugs in the brain?

Hsueh-Yuan: Our current version of the PBKP model was developed to capture nonspecific mAb distribution in the brain and different regions of the brain. Additionally, it can help to quantify the correlation between mAb concentrations in brain CSF and ISF. It may also help to quantify the amount of mAb entering brain parenchyma versus brain CSF compartments.
While the nonspecific mAb PBPK model has not incorporated target binding or receptor-mediated transcytosis yet, both novel delivery mechanisms and target binding kinetics can be mechanistically added into the current basic version of the PBPK model.
The final version of the PBPK model for mAbs may provide an a priori prediction of mAb distribution in the human brain once kon/koff of values of specific mAbs and receptor/target concentrations in the brain have been included. This prediction could be tested in rodents and primates.

Amuza: Central nervous system (CNS) concentrations of mAbs are often determined by taking whole brain homogenate samples or cerebrospinal fluid (CSF) samples from the lumbar region. What are these methods missing?

Hsueh-Yuan: They do not provide direct information of the mAb concentration at the site-of-action as we mentioned in the introduction of the paper. There are many studies suggesting mAb concentrations are different between CSF and ISF.
More importantly, some studies have reported mAb accumulation within brain capillary cells, which may utilize endogenous receptor binding to enhance brain uptake of mAbs.

Shah lab insights for using AtmosLM

Amuza: I’d also like to ask you a few questions about using our AtmosLM system.

You used siliconized sample tubes to prevent adsorption of antibodies in your samples to the plastic. Would blocking the tubes by rinsing with BSA work as well?

Hsueh-Yuan: Yes. However, the storage of low concentration IgG microdialysates requires 0.1-0.15% BSA. BSA is compatible with ELISA. For LC/MS, the BSA method should be replaced.

Amuza: Do you think endogenous IgG could be used similarly to an internal standard to suggest whether or not a microdialysis experiment is working correctly?

Hsueh-Yuan:  Yes, ELISA methods can quantify rat, mouse, or human IgG specifically. They can serve as an endogenous IgG reference, a proxy for calculating in vivo recovery of the mAbs themselves. Hemoglobin can be used, too.

Amuza: During in vitro tests, you were very careful when balancing the flow between the syringe pump (push) and the peristaltic pump (pull), adjusting the peristaltic pump until the ratio of fluid pumped in/out of the probe stayed in the range of 97 – 103%.

What happens if fluid recovery is outside of this range?

Hsueh-Yuan: Then convection [bulk flow of solutes and solvent across a membrane due to a pressure imbalance] will happen.

Amuza: This is indeed a problem. Your paper (1) found that recovery rates were strongly changed when the flow was not properly balanced. This data is available in the supplementary material.

How did you measure the amount of fluid recovered in each sample?

Hsueh-Yuan: By measuring their net weight.

Amuza: With AtmosLM probes, this can also be accomplished by visually monitoring the flow exiting the probe during in vivo experiments. If the peristaltic pump is pulling fluid out of the probe faster than the syringe pump is pushing fluid in, air will enter the system through the vent in the probe and be visible as bubbles in the tubing. If instead the syringe pump is pushing more fluid than the peristaltic pump is removing, the excess fluid will exit the probe through the vent hole. The vent hole is downstream from the membrane, and does not interfere with microdialysis.

What suggestions do you have for others using AtmosLM to study antibody concentrations?

Hsueh-Yuan: 

  • Endogenous IgG or another internal reference should be measured to validate that the BBB is intact.
  • Fresh CSF perfusion buffer should be used. BSA may precipitate if the CSF is left sitting at room temperature.
  • Due to the instability of low concentrations of antibodies in microdialysate, samples should be analyzed ASAP. Alternatively, standards should be diluted to their final concentrations and stored together with the samples until analysis.
  • Always check the inlet and outlet of the probe before connecting the probe to the push-pull system.

Amuza: Do you have future projects in mind for large molecule microdialysis?

Hsueh-Yuan: Yes. We have been working on several projects using AtmosLM microdialysis.

 

  1. Chang, H. Y., Morrow, K., Bonacquisti, E., Zhang, W., & Shah, D. K. (2018, August). Antibody pharmacokinetics in rat brain determined using microdialysis. In MAbs (Vol. 10, No. 6, pp. 843-853). Taylor & Francis.https://doi.org/10.1080/19420862.2018.1473910
  2. Chang, H. Y., Wu, S., Meno-Tetang, G., & Shah, D. K. (2019). A translational platform PBPK model for antibody disposition in the brain. Journal of pharmacokinetics and pharmacodynamics, 1-20.https://doi.org/10.1007/s10928-019-09641-8
  3. Jadhav, S. B., Khaowroongrueng, V., Fueth, M., Otteneder, M. B., Richter, W., & Derendorf, H. (2017). Tissue distribution of a therapeutic monoclonal antibody determined by large pore microdialysis. Journal of pharmaceutical sciences106(9), 2853-2859. https://doi.org/10.1016/j.xphs.2017.03.033

The Amuza FC-90 is a game changer for any microdialysis experiment:

4 channel operation: collect from up to 4 animals simultaneously.

Ideal for large and small molecule microdialysis.

Refrigerated storage in 96 well plates.

Small footprint: 7″ wide.