One of the unmet needs in Parkinson’s disease is the lack of reproducible high-throughput cellular models to study α-synuclein biology. In this video, Dr. Folkert Verkaar, Team Leader in Biology at Charles River Leiden, describes the development of a high-content analysis-based assay to measure α-synuclein aggregation in a terminally differentiated cell line. The α-synuclein aggregation assay has been miniaturized to a 384-well plate-based automated assay that was used to test 1,000 compounds as a proof of principle. Learn More

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  • Video Transcript

    My name is Artem Shatillo. I'm from Charles River, Finland, [inaudible] side. I will be presenting a poster of pharmacological MRI as an in vivo imaging platform for the pre-clinical drug discovery in CNS disorders. Over the last few years, we have been implementing the pharmacological MRI method, which is based on the very translational method of functional MRI that is used in clinics.

    There are two different approaches to pharmacological MRI. Depending on the goals of the study, we can use either the relative cerebral blood volume measurement or the blood oxygenation level dependent pharmacological MRI. Both methods are applicable in drug discovery, but they have their own limitations and advantages.

    While RCBV weighted pharmacological MRI has greater spacial resolution and is less sensitive to various FMRI artifacts, both FMRI or both pharmacological MRI is much faster. The time resolution there is around two seconds for the whole brain acquisition, and it has a little bit different profile in terms of the measurement.

    A typical experiment consists of preparation, where the animal is anesthetized and surgically prepared for the measurement. Since our readout is based on very tiny and very minor physiological changes, we need to make sure that experimental conditions are constant, and they are the same basically for all of the animals. And they are constant throughout the whole experiment, which may take up to four hours. For that reason, we have to operate the animal, so we are in certain the femoral artery and vein [facetters] for blood sampling, for control of the blood [gases], and also for artificial ventilation where do tracheostomy.

    This is done for two main reasons. First, in that way we have a handle over the respiratory physiology of the animal, and also we are able to inject our drugs. And we can draw the samples of the blood for, let's say, pharmacokinetic purposes. And also motion is the biggest enemy of any functional imaging. Whenever there is a motion within the acquisition time, it introduces a lot of noise into your data.

    So, this is the typical example of the experimental timeline. We typically do some pretreatment for the animals of the ... Well, using some test compounds or the reference compounds. Then, the animal goes into the surgical procedures, 10 minutes of MRI set up, and then the HMRI starts. Since it's a functional continuous measurement, we start it, and then we follow up around one hour. And the imaging consists usually of baseline, which is typically around five to 10 minutes. Then, there is a contrast agent injection for the RCB pharmacological MRI, or we go straight to the administration of the test compound. This is done during the measurement without interrupting or pausing the procedure, and we can administer the drugs in all possible ways, but not per us. So, we can do intravenous injections. We can do intra peritoneal and inter arterial administrations. After injection is done, the remaining time we're just following up the time course of the measurement. And usually after the data collection, we collect the samples for biomarkers, or like in this case, we have performed HPLC analysis of the prefrontal cortex.

    We used two reference compounds with their antagonists. So, we targeted to different neurotransmitter systems. Dopaminergic system was engaged with amphetamine, one milligram per kilogram IV. And we used a relevant D1 receptor antagonist, the SCH23390. Another system that we engaged here was the noradrenergic system, was Yohimbine, which is an antagonist of Alpha 2 receptors. And the antagonist for that was Medetomidine.

    After 10 minutes of baseline in the animals result, the pretreatment amphetamine induced quite significant signal change of around 50% of RCBB increase. And in animals pretreated with a D1 antagonist, this signal increase was 30% lower. So, that gives us significant window to detect then activation difference in the brain. And in animals that didn't receive any amphetamine injections, or basically it was just a control set of animals with saline entry treatment and saline challenge, we have virtually no response.

    Also, these changes, they can be mapped within the brain. So, showing us like where the target engagement happens, and what is the brain regions that are mainly involved into this, and the ... We can also quantify different barometers from the signal time series. In this case, we'll use the area under the curve quantification for all three groups, and it shows quite significant difference between the controls and the Medetomidine ... Or the amphetamine-treated animals.

    For Yohimbine pharmacological MRI, we had just two groups, and after administration of Yohimbine in not pretreated animals, we can see quite pronounced dropping [bolt] signal, which was around 3.5%, which is considered to be quite strong response for that measurement. And in the animals which were pretreated with Medetomidine, that response was attenuated by the factor of two.

    So as you can see here, there is a quite pronounced difference between the different groups, and because in that particular case, Yohimbine has quite pronounced effect on the basic physiology of the animal in particular, it quite often causes the drop in the mean arterial blood pressure. There is a lot of physiological monitoring involved in this procedure. So what we do routinely, we monitor the temperature of the animal, the respiration, the ECG, the cardiography, and for that particular case, we also can do the continuous mean arterial blood pressure monitoring with the MRI compatible fiber optic sensors.

    In our study, our data shows that there is approximately 20% drop in the systemic blood pressure, but because we have this altered regulation process of ... Altered regulation system in brain that preserves the [homeostageous] sort of, regardless of the changes in the peripheral tissues, so supposedly this drops will not affect our signal change in the brain.

    And after all the data was collected, the samples, the prefrontal Cortex was flash frozen and taken to HPLC analysis because now we have this beautiful possibility in house. So we can do, after functional neuroimaging, we can immediately take the samples of the brain and then take it to all the biomarkers analysis that we please. And the, in this case, we analyze the [neurologic] metabolites and the dopamine metabolites from the prefrontal cortex.

    What it is also possible, because HPLC is giving you the net concentration of the metabolites in your sample, we can also analyze the metabolites in cerebral spinal fluid. So, we can compartmentalize the metabolites to the brain tissue or to the extracellular fluid.

    Overall, this is to show that pharmacological MRI can be a very valuable tool for the drug discovery and for preclinical drug testing. And we believe that functional neuroimaging is the future of the imaging in general and for the drug discovery in particular, because ultimately we are interested how our drugs change the function of the brain. And it's quite rare that we can see any differences in structure.