- Zenon Starčuk, Jr., PhD, is a research team leader at the Institute of Scientific Instruments in the Czech Academy of Sciences in the Czech Republic.
- He is using focused ultrasound to study brain perfusion processes and their therapeutic applications.
- His team developed software to show what is happening in the brain during the delivery of focused ultrasound.
Dr. Starčuk is the head of the Magnetic Resonance and Cryogenics Department, Magnetic Resonance Research Group, and Facility ISI-LMR of infrastructures Czech-BioImaging and Euro-BioImaging. He and a team of researchers at the Institute of Scientific Instruments (ISI) in the Czech Republic are seeking synergetic ways to use MRI and focused ultrasound for the development of new therapeutic applications. If successful, their software, developed for advanced perfusion modeling and validated in animal and human studies, could be valuable for elucidating the perfusion changes induced by the delivery of focused ultrasound, especially when disrupting the blood-brain barrier (BBB). This should help the field better understand whether and how these changes can be used for targeted drug delivery.
To learn more about this preclinical research, which uses an MR-guided FUS Instruments system, we spoke with Dr. Starčuk.
Focused Ultrasound Work
What is your research background?
My primary area of interest has always been in the methodology and technology of magnetic resonance imaging (MRI) and spectroscopy, and application of these tools in biomedical research. Thanks to a major state-funded investment in 2012, we launched a state-of-the-art MRI laboratory equipped with a Bruker Biospec 94/30 scanner. Our small animal research facility allowed us to perform MR measurements in mice and rats for our own methodological research as well as for external clients active in the field of biomedicine, whom we help with testing hypotheses concerning pathophysiology, drug precursors, or therapeutic procedures.
While doing this collaborative work, I could see the importance of transport processes in a living body. I knew that a combination of MR methods could provide information on various aspects of these transport processes, including blood flow in vessels of all sizes, passage of particles across the BBB, and mobility of water molecules inside cells. This chemical MR information can be fused with anatomic information from an MRI. Delivery or flushing of substances in specific volumes by perfusion has been a frequent point of interest for our clients.
When and how did you get interested in focused ultrasound?
I wanted to develop a method that could modify perfusion and BBB function for both basic research into pathophysiology and for the development of therapeutic procedures and drug delivery. Therefore, I became interested in focused ultrasound. It took three years to obtain the funding to purchase a suitable MR-compatible device. After we obtained the FUS Instruments system in 2020, we used it to develop perfusion analysis software to show what is happening in the brain during the delivery of focused ultrasound, especially when disrupting the BBB for drug delivery. Along with our other research activities (in MRI, electron microscopy, coherence optics, electrophysiology, and nano- and quantum technologies), ISI is bringing a new perspective to using focused ultrasound for the study of perfusion processes and their therapeutic applications.
What are your areas of interest along with focused ultrasound?
For us, focused ultrasound is a tool potentially bringing us from observation to a more active position, including intervention. I am interested in the quantitative analysis of perfusion and diffusion, and in spectroscopy. For example, we have completed diffusion studies to analyze the water-molecule mobility changes that occur in Parkinson’s disease using mice models of Parkinson’s disease; we explored changes in perfusion in animal models of schizophrenia and various antipsychotic treatments; and we tested drug transport in cancer models and the efficiency of stroke treatments. We use contrast-based imaging methods, such as dynamic contrast-enhanced (DCE) imaging, which is based on T1 relaxation, dynamic susceptibility contrast (DSC), based on T2 relaxation, and native-state arterial spin labeling (ASL) to monitor transport processes, and we also acquire standard high-resolution anatomical MR images. Focused ultrasound should allow us to controllably interfere with the biological processes we have only observed so far. Now we aim to develop experimental procedures that allow us to properly control the application of focused ultrasound and acquire informative MR images in such a time frame that will allow us to simultaneously apply both techniques in our animal models.
What mechanisms and clinical indications do you study?
Currently, the most important studies concern BBB opening, which we wish to modify by focused ultrasound and quantify the effects (including the dynamics) with MR. Our physiological models parametrize the phenomenon that happens at the BBB between the blood vessels and the interstitial space, which depends on the type of particles that are moving through the bloodstream (e.g., gadolinium, manganese, iron oxides enclosed in or attached to carrier molecular structures, nanoliposomes, polyethylene glycol etc.).
We also contribute experimentally to studies of the function of the BBB and the integrity of the blood vessels, and we are interested in learning how to modify the BBB for therapy. We are developing experimental protocols with concrete applications in mind, such as those of targeted delivery of novel therapeutic drugs across the BBB to fight cancer or poisoning, as suggested by colleagues from the biochemical sector. In preclinical research, we see opening the BBB with focused ultrasound as a method facilitating drug development by decoupling drug delivery and drug action, but there is also translation potential to human medicine.
Furthermore, we would gradually like to get a more comprehensive picture of drug action by integrating spectroscopy with perfusion and diffusion. In perfusion, we can observe the pharmacokinetics of a drug carrier under test, but not the pharmacodynamic impact of the drug distribution. Spectroscopy allows us to observe the local changes in the metabolic profiles the tissues of interest. Unfortunately, small animal spectroscopy lags behind clinical human spectroscopy. Nice human-brain spectroscopic images can be obtained in humans with linear resolution of 2 to 5 mm. However, in small animals, the about 10-fold reduction in size means 1000-fold reduction of signal, assuming similar geometry. Animal systems use magnetic fields about 3 times higher (e.g., 9.4 T instead of 3.0 T) and coils about 10 times smaller, together raising the sensitivity about 30 times, still not compensating for the 1000-fold loss. Therefore, a cryoprobe, increasing the detection sensitivity by another factor 2.5 to 5, is highly attractive. Our cryoprobe was installed in 2021, and it is another piece of essential equipment that we are still learning to use efficiently. With sensitivity boosted up to 150 times, and further accumulation of acquisitions under anesthesia or using thicker slices, we may be approaching, step-by-step, human-scanner sensitivity. Small animal models are still an unavoidable stage of therapy development, but unfortunately, physics is difficult to cheat: the sensitivity is proportional to the magnetic field and inversely proportional to the coil size (roughly), but proportional to the third power of the linear size. It is all about volume, time, and signal-to-noise.
What is the goal of your work?
Besides the general goals mentioned above, we are currently developing three software packages. The first program is for the quantification of perfusion, and we are also working on a web-based version of this program in which we gradually transplant features tested on a locally working prototype. The second software package, jMRUI, serves quantitative spectroscopy and is a joint effort of about a dozen European institutions from France, Belgium, Switzerland, the UK, and others. Our contribution currently consists of coordination of the software development and quantum mechanical simulation for the generation of models of metabolite responses. And finally, we are also developing a general web-based software tool for processing and visualization of the multidimensional complex data produced by MR experiments. This program will allow our clients to access their data in full resolution and dynamic range and to apply our and develop their own customized analysis and browsing perspectives.
How is your research laboratory structured?
We are a group of about 15 people, most of whom are full-time, but we are still struggling with capacities and competences because of the multidisciplinarity of the field and the dedication to both research and client services. Besides two animal caretakers and two technicians, we do not have a firm structure. Instead, the team is “fuzzy-clustered” by the varied competences of its members (perfusion, diffusion, spectroscopy, physiology, pharmacology). This may look disorganized, but it allows us to flexibly mix the competence spectra for specific projects based on specific biological and technical expertise. We are still a small team in which ad hoc task-oriented structuring is feasible. Moreover, I believe it reduces the risk of burnout and loss of critical mass in our key domains. Unfortunately, recruiting new team members is not easy: our universities are inefficient in producing graduates educated in physics and computer programming, which are the key competences in the field of MR. With very limited power to enforce our priorities, we attempt to attract curious good students during their studies, cultivate them to our environment, and show them that academic freedom, friendly atmosphere, and good equipment can outweigh the salaries and vertical carrier paths of the industry. Hopefully one day we’ll also have our double agents who will be able to improve MRI-oriented education. It’s not easy and fast, but not unimaginable.
What is the relationship between your institute and the government?
The Institute of Scientific Instruments, ISI, is a separate legal entity with a statute of a public research institution, as defined by law. It belongs to the family of 54 institutes of the Czech Academy of Sciences (CAS), which acts as a headquarters, coordinator, and a major funding channel. As of 2022, the CAS fits under the competence of the Minister for Science, Research and Innovation, whose role in the system seems to consist in reaching balance between the state budget, state priorities, applicable rules, incentives, and existing resources. The minister is supported by the government’s advisory body called the Research, Development, and Innovation Council, which consists of 17 people recruited from universities, research institutes and industry, prepares the priorities of the country in the areas of research, development, and innovation and also develops evaluation criteria, so it can affect the institutional budget in many ways. Despite this relative closeness to the government, the CAS is not an organizational unit of the state and its operation is not controlled centrally and directly. Nevertheless, an indirect control by the state is executed through public budget allocation to ministries and grant programmes, and finally research institutions and individual grants. The role of private co-funding is much lower. The CAS has a stable and strong position in the country research activities thanks to its results and its integrity, based on its internal democratic mechanism resembling the state (President, Academy Assembly, Academy Council, Science Council), whose participating members have been elected by the electorate from the institutes.
The CAS does not belong to the educational sector, but its institutes have many personal or contractual ties to universities, and a considerable part of its funding stems from grants of the Ministry of Education. Similarly, other grants tie some of our institute’s priorities to the Ministry of Industry and Commerce or the Ministry of Health. Therefore, the formal independence of the state is only relative owing to the fact that about 50% to 75% of our institute’s budget is provided in research grants. For instance, because our group receives support for our service to biomedical research under the Czech-BioImaging infrastructure from the Ministry of Education, it must abide by its rules, which add to the already sufficiently complex legislation. Finding legal ways is often difficult and wastes our capacity. Thanks to the size of the institute (about 250 employees – half of which are researchers), we still enjoy some flexibility within the by-law framework of the institute and we are supported by the institute’s administration. Our group alone cannot affect the rules much because its power may roughly correspond to its about 15% share in ISI’s research staff, but consensual decision-making has been possible so far, partially thanks to the multichannel funding that avoids fierce competition inside the institute (although, on the other hand, devolving funding decisions to grant agencies costs time and precludes strategic planning).
What are your funding sources?
In recent years we have been able to obtain quite reasonable funding for our efforts from various channels of Czechia or the European Union. For our group, about 50% comes from the support of Czech-BioImaging infrastructure by the Ministry of Education, 25% from the institute, and 25% from our own research grants. Unfortunately, the success rate of grant applications is low, which makes long-term planning difficult. The lack of national MR industry is a hindrance to our group’s funding from industrial development grants or direct industrial involvement.
What type of equipment do you have?
For focused ultrasound, we use the FUS Instruments RK-300 preclinical system. Our MRI is a Bruker BioSpec 94 animal scanner, and we normally image mice and rats. We spend roughly 50% of our capacity on client services (i.e., imaging for biomedical researchers) and about 50% for methods development.
Who are your team members?
Our MR group includes:
- Radovan Jiřík, PhD, is a senior specialist in perfusion who is in charge of most animal experiments, coordinator of our perfusion software development and focused ultrasound experiment supervision, teaches MR at the Brno University of Technology.
- Eva Dražanová, MVDr, is a senior veterinarian responsible for animal model preparation, legal work for facility and staff certification, and animal use protocols. She also teaches pharmacology at Masaryk University.
- Ondřej Macíček, PhD, Radim Kořínek, PhD, and Jiří Kratochvíla, PhD, are postdoctoral researchers working on MRI method development for perfusion, diffusion, and fat imaging.
- Jana Starčuková, PhD, is a senior scientist working on spectroscopic quantitation and jMRUI software.
- Iveta Pavlova, Aneta Malá, Amirmohammad Shamaei, and Jiří Vitouš are doctoral students combining study, experiments, and development.
- Tomáš Pšorn develops web software (perfusion and mD browser).
- Zdeněk Dokoupil and Jaroslav Horký are technicians.
- Lucie Krátká supports experimentation; Lucie Pelikánová takes care of animals; Jana Homolová is responsible for sanitation; and Helena Daňková is in charge of administration.
- Zenon Starčuk, PhD, fills all the gaps between, cares of spectroscopic quantitation development, project planning, quality control, training, reporting, and coordination of the group.
- We also have several students on short-term contracts.
Who are your internal and external collaborators?
ISI focuses on measurement instrumentation and methodology, so it has no independent biological research profile. We do, however, direct much of our research to biological applications, which means that we must reach a certain level of understanding of the biological background.
Our client collaborators are typically external. They include research teams from universities or institutes that are active in medical diagnostics, pathophysiology, pharmacology, or biochemistry. We observe their interests and adjust our priorities to their demands. For example, our decision to seek funding for the focused ultrasound equipment was motivated by Dr. Andrew Miller, a guest researcher from the UK working in Czechia, who came with the wish to develop focused ultrasound–supported targeted delivery of liposome-carried drugs. I understood immediately that such equipment would match our development of perfusion measurement and could find application in other research projects as well, so I entered focused ultrasound high on our wish list. It took three years, but finally we can use an MR-compatible focused ultrasound device in our labs.
Prof. Kamil Musílek (Faculty of Science, University of Hradec Králové) has a similar interest, although the drugs and the carrier are different. Assoc. prof. Jana Rudá (Fac. of Medicine, Masaryk Univ., Brno) is a long-term collaborator who is interested in schizophrenia pharmacology. Dr. Jaroslav Turánek (now Masaryk Univ., Brno) has developed an interest in liposome administration and stroke therapy. Others are from St. Anne’s University Hospital in Brno or other Czech university hospitals or research institutes. We have specialized research partners in the fields of perfusion (Dr. T. Taxt, Univ. Bergen, Dr. Standara, Masaryk Memorial Cancer Inst. Brno) and spectroscopy (Dr. R. Kreis, Univ. Bern, Dr. J. Slotboom, INSEL Hospital Bern, Prof. S. Van Huffel, Katholieke Univ. Leuven, Prof. H. Moeller, Max Planck Inst. Leipzig, etc.).
Furthermore, we are part of the Czech-BioImaging infrastructure, which is a Czech version of Euro-BioImaging, or similar to the Australian Imaging Society. Here in Brno, the NMR tradition stemming from our institute has led to a higher concentration of NMR and MR systems, including cutting-edge high-resolution MRI. In the field of in vivo MR research, the most relevant is another institution in Brno called CEITEC, part of Masaryk University; they have two 3T MR scanners fully dedicated for research. Most of their work is currently focused on using functional imaging based on the observation of blood oxygenation changes during brain activity (the BOLD effect). However, they can use the systems very well for other topics and we have always collaborated on various specific projects. As members of the same infrastructure consortia (CzBI and EuBI), we are in close contact.
When considering your focused ultrasound research to date, what are your greatest achievements? Any disappointments?
I must underline that in the field of ultrasound, we are total beginners, and we still are on the learning curve. Due to some technical troubles, COVID-19 restrictions, and a scanner software upgrade in 2021, our initial progress was rather modest and only now do we reach some protocol stability. We feel confident that soon we will be able to achieve reproducible undisputable focused ultrasound effects on perfusion in the mouse brain. The next step will be protocol adjustment and validation in rats.
What do you see as impediments to your success?
There are always a multitude of obstacles of varying importance. For all our work, the key is to maintain a stable core team that is accumulating knowledge and sufficient capacity for scientific work. As main impediments, I would typically name insecurity or insufficiency of funding, shortage of qualified human resources, lack of international salary competitiveness, technical failures, and pervasive bureaucracy (in project management, science evaluation, economic contracts, and foreign education recognition). Today, I must also add COVID-19, the Russian war against Ukraine, and the European Green Deal, which together make long-term conditions more volatile than ever before.
Scientifically, any new topic requires simultaneous approach from our client, the biologist, and our team, the technical provider, which takes time and finds no funding. On the other hand, such mutual education is what makes the work interesting. Our process involves asking what the client wants to observe, identifying suitable observable markers, considering what type of methodological research might be interesting for us, assessing the costs, risks, and benefits, and finally arriving at a common project. For studies involving animals, we prepare the necessary animal use protocols and submit them to the supervising bodies. These processes may be lengthy, but I wouldn’t label them as impediments because they are a normal, unavoidable part of our research.
What is on your research wish list?
We have short-term and long-term wishes, intertwined, with different levels of importance and feasibility. For instance, I would like to see a streamlined protocol for focused ultrasound experiments in mice and rats that include dynamic quantitative MR perfusion imaging. I would love to trust all quantitative data on perfusion, relaxation, and metabolite content. I wish to always know the confidence intervals for all quantitative data. Much better data quality and cleverer data analysis also fit under this umbrella; these may involve use of prior information in quantitation, expressed explicitly mathematically or deeply hidden in artificial intelligence, and better controlled experiments. I wish to be able to save measurement time and avoid bias, which would mean better temporal resolution or better economy.
Among other wishes, I have fast MR spectroscopic imaging, and fast MR signal simulation integrated with quantitation and capable of handling even imperfect acquisition conditions. With better accuracy and precision, I hope we can reliably distinguish drug carriers in blood vessels from those in the interstitial space. And perhaps also from those in the intracellular space. I would like to see all these methods applied for targeted delivery of anti-cancer drugs, which would reduce the side effects of chemotherapy.
When I return back to the ground, I will wish to see MR scanner manufacturers unify and open their data formats, to provide all data and full control of the acquisition, and perhaps move to transferrable MR experiments.
And finally, looking beyond the daily horizon, I would like to understand one day how it comes that our nuclear spins, forming complex coupled systems in the molecules of metabolites, so nicely obey the Schrödinger equation even during the long measurement, obviously without any quantum state demolition, and how the picture continuously changes as we move to optical frequencies. In other words, I’d wish to fill the gap between our quantum description of spin system behavior and the classical detection by Faraday induction. Also, it would be nice if we were able to combine the observation of nuclear spins with the potentially much more sensitive detection of electron spins in radicals.
I have many more wishes than time.
Has the Focused Ultrasound Foundation played a role in your work?
So far, not too much. However, in MR, we have been in the role of a neutral observer. It has been our ambition to provide information that will allow a biologist to understand the processes taking place. Focused ultrasound is getting us beyond the observer paradigm; it gives us the power to change something, which we should be able to immediately test. In fact, this is a revolutionary change of the paradigm.
What comes next?
In the future, we may be able to provide our services to pharmaceutical scientists developing novel drug formulas and testing them in vitro in cell cultures. The next step is testing for in vivo validation, which is a service that we may be able to provide. We would open the BBB, deliver the drug, and then provide evidence for the analysis of the brain changes induced by the drug. I would like to help develop a breakthrough in the way drugs are being delivered, to support precision medicine development. Everyone knows that chemotherapeutics can be devastating to the body. Everyone feels we should provide more selective alternatives. Focused targeting is something that has the promise of medical progress. I hope that we can pave the way in the preclinical space and support its translation to the clinic.
For our group, I do not push for standardized testing services for pharmaceutical companies. There are other imaging laboratories in Czechia (most notably, the CAPI at the 1st Medical Faculty of the Charles University in Prague), whose geographical and institutional position seems more favorable for such work. For our group, the perspective is highly competent support for highly experimental, initial-stage development, and versatile and customized support in small-scale pilot exploratory biomedical experiments. I believe we should keep our focus on MR and focused ultrasound, we should consider intra-institutional synergies (fluorescence endoscopic microscopy, electron microscopy), and cooperate externally with teams experienced in PET or SPECT or other techniques if needed.
Our spectroscopic software is normally presented at www.jmrui.org and www.jmrui.eu, but since the server in Barcelona was encrypted by ransomware last year, these pages are down. Our institute is in the process of producing a modest substitute.
An ISMRM presentation is available at www.ismrm.org for ISMRM members. This is not relevant to focused ultrasound.
A limited support for perfusion analysis (web version = WIP) can be found at http://perflab.cerit-sc.cz/. We have no video yet.
The history of our institute can be found at http://www.isibrno.cz/en/history.