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Using low sample volumes to better understand brain diseases

Marlien van Mever delved into the analysis of tiny samples, cerebrospinal fluid from transgenic mouse models for example. She validated methods that can now be used to study brain diseases such as migraine and epilepsy. Van Mever will receive her PhD on 14 June.

A powerful way to understand how the human body works is metabolomics: the study of metabolites, or substances that the body makes. A metabolome is the complete collection of metabolic products that a particular organism makes. 'By analysing the metabolome, you gain insight into the physiological status of an organism,' says Marlien van Mever.

Metabolomics is a promising technique for neuroscience research into brain diseases. By finding outwhich amino acids are present in a sample, or substances from a cell's respiratory cycle for example, you know more about its functioning and any defects in it. 'For brain research, it is difficult that you cannot take research samples from living brains so easily,' said Van Mever, who conducted her research at the Metabolomics and Analytics Centre of LACDR, the Leiden Academic Centre for Drug Research.

Less than a drop

But it can be done, she explains. 'For example, we can take a little brain fluid from laboratory animals. This is often done via microdialysis, a widely used technique in neuroscience to monitor brain chemistry.' Researchers then use a small tube with a semi-permeable membrane to collect small molecules in living, freely moving animals. 'From a rat you usually obtain about 30 microlitres per sample, in mice it will be a maximum of 5 microlitres per sample.' By comparison, a drop of water contains 10 to 50 microlitres.

Alternative to laboratory animals

Working with laboratory animals is often a necessary evil in brain disease research, but alternatives are emerging. 'There are upcoming systems in which cells from patients are not grown flat on a dish, but in 3D cell culture on a microfluidic chip. These systems are becoming increasingly popular in biomedical research and may ultimately reduce the need for laboratory animals.'

Valuable technique

Van Mever worked on developing several methods to analyse micro samples. 'I focused on both sample preparation and sample analysis.' She investigated the analysis method CE-MS. 'That has been getting more attention in recent years as a suitable technique for metabolomics.'

CE-MS stands for capillary electrophoresis and mass spectrometry. In capillary electrophoresis, researchers guide a mixture to be separated under the influence of an electric field through a capillary, a tube as narrow as a hair. Differences in charge and size of molecules cause them to move through the capillary at different speeds, thus separating them from each other. Mass spectrometry then shows which and how many molecules were in the sample. 'The combining of those two techniques turns out to be sensitive enough to measure the substances we want to measure.'

For neuroscience research, CE-MS is a valuable technique because you have enough sample quantities starting from a few microlitres. Based on Van Mever's results, CE-MS is now actually usable to analyse relevant biological samples within neuroscience research. Van Mevers' supervisor is already applying the technique. Rawi Ramautar, a researcher at the Metabolomics and Analytics Centre where Van Mever did her research, writes: ‘Currently, we are using CE-MS to analyse metabolites in mouse brain fluid. In doing so, we are investigating epilepsy, together with the Free University of Brussels. Once we have published this research and presented it at conferences, I expect more interest in this method.'

Challenge and opportunity: the corona crisis

With all that work on tiny samples, you do expect ups and downs. For Van Mever, the biggest challenge during her PhD project was the covid-19 crisis. 'Due to lockdowns, it was often not possible to work in the lab. That caused delays for my experimental plans.' Teaching largely went on as usual, online or hybrid, and Van Mever played a big part in that. 'I enjoyed my time in the lab, but I also loved teaching .'

Supervisor Ramautar praises his PhD student for her creative and efficient approach. 'Marlien is not only a creative bioanalytical chemist, but she also took initiatives to conduct experiments in a smart way. She used an efficient approach called  Design-of-Experiments. This left her with spare time that she happily devoted to teaching. Besides her fine dissertation, she also obtained her University Teaching Qualification.'

Text: Rianne Lindhout

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