In this interview, discover how Charles River is harnessing the power of microdialysis for drug development and CNS therapies.
Could you please introduce yourself?
My name is Ilona Fist. I currently lead a team of study directors at the Charles River Labs location in Groningen, the Netherlands. I have five years of experience conducting neuroscience and tumor microdialysis studies and my combined knowledge of in vivo and analytical procedures have enabled me to support our customers at a technical and scientific level during the discovery phase of drug development.
What is microdialysis? What are some of the main benefits?
Microdialysis is a catheter-based technique that allows continuous sampling from the interstitial fluid of any target tissue. This technique is mainly based on the principles of osmosis, in which high concentrations from the microenvironment go to the probe. The samples taken can then be analyzed for a wide range of readouts depending on the question being answered. Some common examples of readouts include levels of neurotransmitters and neurometabolites, hormone levels, and detection of therapeutic test articles to interrogate pharmacokinetics and pharmacodynamic information. There is normal microdialysis for neurotransmitters, MetaQuant microdialysis for PK and push-pull microdialysis for larger molecules.
There are three main benefits of microdialysis. The first is that it can be performed in awake, freely behaving animals and samples are collected serially. The second advantage is that it allows you to analyze multiple analytes from one sample. By dividing a sample for different methods, such as HPLC, mass specification or ELISA, you can broaden your biomarker profiling. The final advantage is that you can reduce the number of animals by implanting multiple probes in one animal. This not only reduces the number of animals used in a study, but also allows you to sample from multiple places in one experiment.
What analytical techniques can be used to analyze the samples after they have been collected?
We can use LC-MS methods, but also MSD, ELISA or other existing techniques to analyze these relatively clean samples with little background noise or matrix interference. Using LC-MS we can measure different peptides from the same protein and quantify mutations or specific peptides of interest.
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Can you explain what push-pull microdialysis is? How do you set up a push-pull microdialysis experiment?
Push-pull microdialysis is a technique used to sample large molecules, such as peptides, antibodies and cytokines, which can be either endogenous or therapeutic.
For the push-pull microdialysis we use a specialized Y-shaped probe with a specific membrane with larger pore size. Gravity is used to get optimal flow across the membrane, capturing as many molecules as possible. We determine the recovery percentage across the membrane in vitro experiment before you start in vivo experiments.
The probes will be implanted in the brain region of interest and serial samples can be collected after the recovery period. In addition, a cannula can be placed in the jugular vein to allow blood collection, or in the cisterna magna for CSF collection.
Various routes of compound administration can be used, including traditional routes, as well as intranasal, intrathecal, or other injection methods. Moreover, we can collect different tissues and perform microdissection after the experiment. Therefore, we can extract a wealth of information from a single animal, something I am particularly excited about when using microanalysis in our studies.
The data obtained from these experiments include insights into brain penetration, the relationship between plasma, CSF and the interstitial fluids, as well as the Kpuu.
How does push-pull microdialysis contribute to studying different animal models? Can you share case studies or examples of push-pull microdialysis experiments?
We can perform microdialysis studies in various animal models of Alzheimer’s disease, Huntington’s disease or other models, to assess pharmacodynamics or pharmacokinetics.
Allow me to share with you a PK/PD study in which we measured PK and alpha-synuclein protein levels in rats. We used a push-pull probe in the prefrontal cortex and placed a jugular vein cannula in the same animal for blood sampling. We also placed a cannula in the cisterna magna cerebrospinal fluid sample (CSV).
After administration of a therapeutic antibody, we observed a decrease in alpha-synuclein levels in the microdialysate and cerebrospinal fluid. In the same animals, PK profiles were determined in both plasma and brain microdialysate, reducing the number of animals used in this study.
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What considerations should be made when designing microdialysis experiments?
The main considerations revolve around the technical aspects, especially the choice of membrane and the specific type of molecule to be measured. We must also take into account the available analytical methods. We can also play with the duration of the experiment.
Microdialysis experiments can last from days to even longer periods of time. With additional placement of guide cannulas we can extend this to weeks, allowing us to sample from the same region over time. This capability is invaluable for reducing the number of animals required for experiments.
How many brain areas can you sample in one microdialysis experiment? Is the number consistent between mice and rats?
For rats, we typically use a maximum of two brain regions. We can add a jugular vein or cisterna magna cannulation, a total of three lines. For mice this is limited to two lines. We can still integrate the two brain areas, or one brain area and a jugular vein canularion, but this represents the maximum achievable configuration for mice.
We can also combine different probes per brain region. For example, using a MetaQuant probe in one brain region and a push-pull probe on the other side to capture both the PK and PD profiles of the compound in the same animal. I think this is the true power of microdialysis, as it reduces the number of animals required by consolidating different options within one animal.
How often can you perform microdialysis on the same animal? Is it feasible to perform multiple microdialysis experiments in the same animal over weeks or months?
Yes, it is possible to take samples from the same animal over weeks. However, if the duration can reach months, I recommend splitting it into several groups and performing the operations at later intervals. Nevertheless, we have successfully conducted multi-month experiments with this approach.
How do you ensure that the blood-brain barrier recovers after the experiment?
This is remarkable during the recovery period between the operation and the start of the actual microdialysis. We have historical data that shows that after a certain period of time the blood-brain barrier is restored.
Can you explain in more detail what? in vitro microdialysis is and how is it used to inform in vivo experimental design?
In this setup, the probe is placed in a beaker containing the analyte of interest, and microdialysates are collected. In one in vitro During setup, we typically use different conditions to determine the optimal conditions for the analytes.
This involves using different membranes or adding factors such as BSA to the artificial CSF to improve transfer across the membrane. By comparing these different factors in advance, we aim for the highest possible recovery of the analytes before we proceed to animal testing.
About the speaker
Ilona Vuist currently leads a team of study directors at the Charles River site in Groningen, the Netherlands. She has a background in immunology and cell biology, and experience conducting CNS and tumor microdialysis studies. Her combined knowledge of in vivo and analytical procedures enables her to provide support at both a technical and scientific level during the discovery phase of drug development.
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