Championing all three of the 3Rs in vascular biology

Dr Korff speaks to the NC3Rs about why the 3Rs have always been, and continue to be, integral to his work.

You recently won the Ursula M. Händel Animal Welfare Prize in acknowledgement of your exemplary application of the 3Rs principles. Can you tell us about your experience and how you feel about this recognition?

My laboratory has a focus on vascular biology and tries to identify mechanisms promoting degenerative diseases of arteries and veins both at the cellular and organism level. Over the years our group has developed a number of techniques that allow meaningful experiments, yet reduce the use of laboratory animals or even replace them altogether. This award from the German Research Foundation is a great honour and encouragement, but at the same time it is also a commitment to continue our efforts in this direction.

Why is it important to you that the 3Rs principles are central to your research?

Realisation of the 3Rs principles means respecting laboratory animals. It helps to minimise the number of animals needed and improve their welfare. At the same time it improves the economic and also scientific efficiency of our work, since simple and easy-to-learn experimental techniques are less likely to fail and give more consistent results.

How did your career lead you to working in the area of pathological changes in the vascular system, and what have been your most exciting findings?

My laboratory is mainly interested in the impact of biomechanical forces on the architecture and function of vascular cells. For instance, we analyse mechanisms that are triggered by hypertension in cells composing the vessel wall. During my time as a PhD student I developed a technique mimicking the formation of capillaries in vitro. During that time, I made the exciting observation that suspended endothelial and smooth muscle cells are capable of forming organised three-dimensional aggregates. The architecture of these spheroids resembles that of an artificial inverted globular vessel wall. With my junior professorship I changed gears a little bit and focused on diseases of larger blood vessels.

Have you been successful in your search for alternatives to animal testing?

The capillary sprouting assay, which we developed based on the model just described, and which mimics angiogenesis, (i.e. the formation of new blood vessels,) is now utilised in many laboratories. It allows the researcher to test pro- and anti-angiogenic compounds on human endothelial cells, which can be collected from umbilical cord veins after birth. A protocol for this technique is freely accessibly at www.spherogenex.de, and promoted by some German companies, such as Ibidi in Martinsried or PromoCell GmbH in Heidelberg.

How can cell culture systems be used to investigate vascular change in individual cells?

Basic mechanisms, which do not fully rely on a physiological environment, can be studied through the use of cultured cells. For instance, analysing general effects of distinct cytokines on the proliferation of endothelial cells is a straightforward approach and can be investigated by in vitro experiments. However, the more complex the mechanisms to be investigated are, the more complex the artificial experimental setup has to be. I think that in this respect the cell culture model has not reached its full potential yet. Nevertheless, there are still questions related to multi-tissue interactions or systemic reactions involving different organs that cannot be answered using this relatively simple system.

Can you explain some of the key ways in which you have improved animal welfare in your experiments?

In my lab, we always try to use methods that reduce or refine animal use. For instance, commercially available modern non-invasive analytic methods are applied, such as high resolution ultra-sound analyses. Similar to a prenatal ultrasound test, it is painless and does not require surgery, but you obtain precise information on internal cardiovascular parameters. They can also be performed repeatedly on the same animal at different time points. So you can actually do a time series to look at the effect of a specific treatment, for example, on just a few animals, rather than using one for each time point. This of course reduces the numbers of laboratory animals. Other methods minimise the extent of surgical procedures performed on an animal.

You recent work has studied changes in the wall of a blood vessel in the ear of the mouse. Why is looking at the ear considered a refinement on previous procedures?

A number of different techniques are currently used, depending on the question under investigation. They usually require surgical procedures. We found that some degenerative remodelling processes of the vessel wall, such as varicose vein formation, can be mimicked in the mouse auricle. As all the blood vessels are visible through the skin, this allows for daily imaging and further non-invasive analyses of changes in blood vessel structure and function without sacrificing the animals. Moreover, the possibility to transdermally and locally apply compounds that interfere with detrimental blood vessel remodelling greatly reduce the overall drug dosage to which the animal is exposed. Our mouse auricle model cannot replace all procedures where animals are used in cardiovascular research, but it might encourage other researchers to look into possibilities to adapt the model to their needs.

Your work has been recognised for its potential to minimise the number of animals needed in your research area, how large is this reduction?

This depends on the question we want to address. When submitting an application for a study using animals to the authorities for approval, we have to state the number of animals needed. This number is already based on our refined methods, so it is hard to give an exact figure. A simple example to illustrate the reduction could be as follows; if we want to investigate the growth of blood vessels in five mice each week over a period of 4 weeks by a technique that only allows for invasive analytic methods, 20 animals have to be used in total. If the mouse auricle model is utilised, we only need five mice, since we can apply direct non-invasive imaging methods on the same five animals over the entire period. This is a 75% reduction in the number of animals used.

Can you tell us more about how the 3Rs are supported in Germany and how their importance is promoted?

As animal welfare is a relevant political issue in Germany, the 3Rs principles are promoted on several levels. Researchers who apply and advance the 3Rs may receive recognition through prizes awarded by national organisations such as the German Research Foundation, or by the German federal states. Moreover, the Federal Ministry of Education and Research specifically supports research projects dealing with alternative methods to animal testing. At universities, veterinarians assist researchers who plan a project involving animal testing in designing a protocol that reduces animal use and uses methods that reduce animal suffering.