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.”
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 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.
Many analytes can be measured in each sample with the little added difficulty
Data processing and experimental protocol are both more difficult
Types of analytes
Most types of molecules.
Limited by available fluorescent sensors
Type of measurement
Absolute concentration or % change relative to baseline
% Change relative to baseline
Data analysis
Relatively simple
Often quite complex
Sampling period
~30 seconds to hours
Milliseconds
Sampling duration
Days
Days, but difficult to monitor slow changes
Targeting
Brain region/structure
The brain region, specific projection, cellular subtype, axon vs cell body
Animal movement
Tether required
Tether 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?
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 releasedFZ 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.
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.
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.
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
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
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 sciences, 106(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.
For approximately 200,000 US veterans, the 1991 Persian Gulf War marked the beginning of their experience with Gulf War Illness (GWI). GWI encompasses a cluster of chronic symptoms including memory and cognitive problems, fatigue, and fibromyalgia.
GWI has long been associated with a combination of several possible contributory factors: the stress of deployment, altered immune function, and exposure to acetylcholinesterase inhibitors (AChEI), but the exact cause or causes have remained elusive. The AChEI pyridostigmine bromide (PB) was administered to soldiers as a prophylactic against the risk of nerve agent weapons, but many veterans were also exposed to AChEI based pesticides, further complicating the etiology of this illness.
To elucidate the relationship between these factors, Dr. Victoria Macht, her advisor Prof. Lawrence Reagan, and colleagues at the University of South Carolina School of Medicine studied rats exposed to pyridostigmine bromide and repeated restraint stress. The rats were then given either an immune challenge or an acute immobilization stress challenge during in vivo microdialysis. It is the first study to use an in vivo method (microdialysis) to show that PB changes the response of the central cholinergic system to both stress and immune challenges, and does so in a brain region specific manner.
By measuring acetylcholine levels via microdialysis and subsequent HPLC-ECD, they found that cholinergic responses were attenuated in the PFC and hippocampus after immobilization stress. Lipopolysaccharide (LPS) was administered as an immune challenge, after which cholinergic responses were attenuated in the hippocampus but not the PFC. These results indicate that PB and stress interact to shift the cholinergic response to future psychological and immunological stressors, providing a potential mechanism for the persistent and exacerbated cognitive symptoms evidenced in soldiers with GWI.
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Mike Churchill: What story do the different responses to the immune challenge and the immobilization challenge tell?
Victoria Macht: By using two different types of challenges, we were able to test both the diversity and consistency of effects of PB and stress on the cholinergic system. LPS is a novel challenge which specifically elicits a response from the innate immune system. The immobilization challenge is more of a psychological stressor, and as it shares some similar qualities with the prior restraint stress, this allowed us to test if rats with PB and restraint stress had impaired neurochemical adaptations to recurrent stressors.
MC: How might these results relate to changes in fear memory and cognitive function?
VM: ACh is an important regulator for a variety of factors in fear memory including coordination of local circuits to help with sensory and cortical processing of stimuli as well as the consolidation process. Interestingly, regional differences in the cholinergic response of the PFC and hippocampus to immobilization stress suggested that PB impairs cortical processing of novel stressful stimuli and impairs the neurochemical adaptation to recurrent stressful stimuli. In our fear conditioning studies, we similarly found impairments in the way PB and stress interacted to impair context and cue related retrieval. This suggested to us that impairments in the function of the cholinergic systems impacts a variety of psychological stressful stimuli, indicating that this is a global deficit in cognitive function rather than a specific deficit to only one type of stressor.
MC: How do the microdialysis results relate to the tests for inflammation you ran?
VM: ACh is really fascinating because while it is not only central in learning and memory, it is also an important negative regulator for the inflammatory response via α7 nicotinic ACh receptors. We found that PB blunted the central cholinergic response to an innate immune challenge, which could suggest an exacerbated chronic inflammatory response in the brain. Interestingly, these microdialysis results for acetylcholine parallel some of our findings with peripheral inflammatory markers. Peripheral levels of c-reactive protein were elevated after the LPS challenge in rats which had received PB, suggesting a dysregulated inflammatory response. While we need to confirm these results with cytokine levels in the brain, our results suggest that impaired cholinergic feedback to inflammatory stimuli could underlie some of the changes in the sensitivity of the immune system which are evident in clinical populations with GWI.
MC: Does PB have to cross the BBB to cause these effects?
VM: It does not. There has been a big debate on this topic. One suggestion was that stress caused a leaky barrier, allowing PB to get through. However, tests on this have been inconsistent on this. What our studies demonstrate is that PB changes the function of the central cholinergic system regardless of whether it is able to get through the BBB.
MC: What will be the next steps for this project?
VM: Prof. Reagan will continue the project: measuring cytokine responses in the brain to see if they match peripheral cytokine responses. There is also an opportunity to see if aging exacerbates the decline of the cholinergic responses and cognitive deficits in our model of GWI. The goal would be to see if animal models of GWI can predict further changes in veterans as they age, and plan treatment accordingly. We have a unique opportunity with this population for the preclinical research on treatments to get ahead of the patient population as they age.
MC: How did you like using the Eicom HTEC HPLC-ECDs in Prof. Jim Fadel’s lab?
VM: It is amazing! I can’t imagine having done these projects without it, and I miss using it. We used the system daily for two years to measure acetylcholine without any real problems. It made my dissertation a much more pleasant experience!
MC: Had you used HPLCs before using the HTEC?
VM: We used a different system before but it was not reliable, so when it was working people felt they had to immediately run all of their samples before it went down again, and watch it all of the time when it was running.
MC: How many samples do you think you ran over the course of this project?
VM: That makes my head spin! We looked at both ACh and glutamate, in two brain regions, each rat underwent microdialysis 2 separate days, there were approximately 8 animals per group, and 4 groups. So at least 3500 samples – plus the pilot study! Plus there were other studies going on during this time which were also using the HTEC.
MC: Where is your career taking you next?
VM: I am now doing a postdoc at UNC Chapel Hill, working with Prof. Fulton Crews, studying the long term effects of binge drinking in adolescents. Interestingly, while this is a different clinical population, changes in the cholinergic system and innate immune system are also common features here.
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The article appears in the April issue of Brain, Behavior, and Immunity:
Pyridostigmine bromide and stress interact to impact immune function, cholinergic neurochemistry and behavior in a rat model of Gulf War Illness
V.A. Macht, J.L. Woodruff, E.S. Maissy, C.A. Grillo, M.A. Wilson, J.R. Fadel, L.P. Reagan
doi: 10.1016/j.bbi.2019.04.015
TaskForcer’s body-supporting cylinder keeps rodents gently restrained so they are more focused on the task at hand. To see how research animals can be trained faster with the TaskForcer, please fill out this form to watch the video.
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The Amuza FC-90 is a game changer for any microdialysis experiment:
