Anke Tappe-Theodor, PhD, is a habilitated scientist in the Faculty of Pharmacology at Ruprecht Karls University of Heidelberg, Germany. She achieved her PhD at Ruprecht Karls University of Heidelberg and studied synaptic physiology in the spinal cord and forebrain under pain conditions. Her current research interests include preclinical models of neuroscience, comorbidities of pain and other diseases, as well as pharmacological intervention and evaluation at different levels. She critically questions current animal models of pain and the efficacy of the tests to measure spontaneous pain.
Here, Tappe-Theodor chats with Federica Franciosa, a PhD candidate at the University of Bern, Switzerland, and PRF correspondent, to share expert advice that will help researchers identify the most appropriate tools to assess pain in rodents and meet investigators' objectives. Below is an edited transcript of their conversation.
Let’s start from the beginning: how did you become a scientist?
Initially, I did not want to get a PhD. So I obtained a degree to be a biotechnology engineer to work in a lab and conduct valid scientific research. Jobs that were available at the time were in the field of fermentation processes and the food industry. I did not find this work really interesting or fascinating, so I joined a biotech company to work as a technician. I enjoyed working at the bench, and my boss had noticed that I was often doing more, so he suggested I should obtain a PhD.
So I enrolled for a master’s degree, and during that time I got to know Rohini [Kuner] when she was giving a lecture on pain. She was starting her lab, and at the same time, the company I was working for shut down. Rohini’s research ideas were fascinating, so I began to consider pain as my research topic. I started in her lab for my master’s thesis and, in the end, I achieved my PhD that I never really wanted from the beginning. Pain research came into my life accidentally.
I think it’s curious that you weren’t interested in obtaining a PhD, since now it’s so competitive, especially in neuroscience. How did you get through your program as an “unusual” student?
I might say that I probably wasn’t thinking of the consequences. I was certain that I wanted to contribute to a research field where I could help people, and I found pain extremely interesting to investigate. Although it makes you wonder how far we get away from truly helping patients with our preclinical research with optogenetic, chemogenetic, and transgenic modulation of particular neuronal cell types in a specific brain area, for example. But it is necessary on the way to identify new targets and delineate the mechanisms of pain.
Being an “unusual” student was not disadvantageous. I saw and learned a lot from my previous work in that biotech company, and therefore had a good foundation. In general, it is not always the most straightforward way that gets you to your destination or close to what you would like to do.
Can you share a turning point, or a defining moment, in your work so far as a scientist?
It was probably at the point when my main interest shifted to looking closely at the pain models that are currently in use, and the behavioral tasks that are performed to validate them. The model constitutes the basis of all aspects of pain research: to test therapies and to identify novel pathways. Therefore, it is crucial to go back to the mouse or the rat to make it an accurate translational model. We can’t just question the mice about their pain, so we must consider all the relevant factors from a rodent perspective.
Several pain researchers who study chronic pain use inflammatory and neuropathic models indiscriminately, and look for the same behavioral outcomes regarding nociception, motivational, and aversive aspects. Can you comment on how much consistency exists among different labs performing the same type of study?
First, it is a very complex matter due to heterogeneity among labs. It can depend on so many factors like: different housing conditions, potentially the sex of the experimenter, the cage type and housing environment, and even the quality of the food. All of these conditions can influence behavioral outcomes. The use of different mouse strains or substrains can result in different behavioral outcomes. It is important to always keep the rodent perspective in mind. There have been labs addressing these matters in multicenter studies, and other labs have shown that performing experiments according to the mouse’s day-night cycle results in an easier assessment of anxiety- or depression-associated behaviors.
Chronic pain can have different sensory profiles between humans and animals. Do you think that we are looking at the wrong features in the animal? In your opinion, what makes sense to look for in preclinical research?
We need to consider that while interrogating mice on their pain, we do force certain tests onto them, like on a Catwalk. They do the task, or they don’t, whereas a patient can stop walking or doing something whenever they feel like and explain why. With the animals, we must look closely to understand what is going on beyond the cases when pain is manifested by delivering a noxious stimulation. We need to look at parameters that can indicate why the mouse might be lazier, or looks depressed, or less motivated, and it is complicated. Sometimes a mouse is in its corner, which could be pain or depression, but we cannot really tell.
It feels like studying how to generate a proper translational model could be a field of research in itself.
Yes, sure. For example, we often have to isolate mice because they fight or for other reasons, and this acquired isolation severely affects the mouse. We might compare these situations to those of elderly people who are in their hospital rooms or in patient care for months, and it has been shown that they develop depression on top of the initial reason(s) why they are there. Isolation induces stress in mice, is associated with depression, and has been shown to alter an individual’s pain threshold. Mice are quadruped animals that live in cohorts and have a lifestyle that is hard to transpose to humans. If one paw hurts, mice still have three left. A patient with an amputated leg only has one left, and it makes a huge difference. So we need to consider all these factors in our research.
The most utilized quantitative sensory test to assess mechanical pain is the von Frey. Labs generally use manual or automated von Frey hairs (e-vF), and the baseline level for mechanical threshold is substantially different. Accepted values for the manual von Frey are below 1 g of force, whereas for the accepted value for the automated von Frey is 4-5 g. Can you comment on this huge difference?
I use the electronic von Frey, but I have to say I prefer the manual one. The e-vF gives you only one threshold, whereas the manual hairs allow you to decide on the range and measure the progression of allodynia and hyperalgesia. Measuring allodynia accurately is important for treatments where you might have an improvement in a fine range, and this might pass undetected if you’re using the e-vF. The question is what you want to measure. If it’s simply a phenotype, neuropathic pain versus control, for example, then the e-vF is sufficient. However, to understand if there is a subtle difference in the fine range of the filaments, it makes more sense to apply all the hairs from thinnest to thickest. It is time consuming, and you can get a stiff neck during the measurement procedure, but it is necessary to really get the best out of your experiments. Take the time and do things thoroughly.
Females are underrepresented in animal models of disease, and our understanding of female biology is compromised by these deficiencies. Can you comment on the lack of data on females in preclinical research? Do you think it’s time to reevaluate the gender-biased research?
Well, there is a big debate on this. We know from patients that there are differences in pain thresholds between males and females. In fact, the standard chronic pain patient is a 40- to 60-year-old woman, and typically what we use in the lab is a young male mouse to represent whatever happens in the real world. These don’t match. On the other hand, the mouse is the model, and we should be careful when considering the differences already present based upon what mouse strain we use. At some point, people started using more C57/BL6 mice, and some papers fail to report if it’s a J or an N substrain, which can play a significant role in pain outcomes. I think that historically people were not very much aware of this issue. In studies like the characterization of transgenic mice, people were using whatever they could get. Considering the probability of getting a transgenic mouse from a breeding pair and having comparable sets of animals of a certain age, researchers were taking everything they could. Later, they started discriminating between males and females. Again, the question is what you want to look at. To study a specific neuronal subpopulation in any area of the brain, for example, it can be completely fine to start with only males. In general, it is easier to establish a test or to characterize a disease starting with only one gender.
Going back to the person behind the science, what was the most difficult part of your research?
Getting through the FELASA course (Federation of European Laboratory Animal Science Associations)! I could not see blood when I was younger…. I was mainly doing immunohistochemistry and microscopy, so I was wondering whether I had to do the in vivo part. At some point, I was able to start working with living creatures and performing procedures that are way more challenging, such as spinal cord injections in a living mouse. At the FELASA course, I was sent home the first two days because I was feeling heavily sick. Those were two of the hardest days.
Can you tell me if you had some setbacks in your career as a woman in science?
I honestly never had this feeling, maybe because my boss is also a woman. I know that these kinds of problems occur, and I knew that they existed when I went to the mentoring program at the university. The issue is also very present in the clinic environment. However, myself and the women I work with here have never come across such problems.
What is your favorite aspect of your research?
I like working with animals. I find it very challenging to establish new behavioral tests or translate tasks from rat to mouse. For example, it was not straightforward to establish a conditioned place preference test in mice after being published in rats. I like behavioral tests. At some point, I was no longer interested in working on the function of whatever “XY phosphatase,” or some small component that might modulate something here and there…. It is essential but doesn’t drive my interest. I prefer establishing tests, and that might be on a more basic level of research, but it’s also important for other researchers. Without animal models and the appropriate tests, we might not be able to design the right strategy for pain interrogation.
What do you think will be the next “big thing” in pain science?
I think it’s very important to reveal mechanisms of the comorbidities that accompany pain states. It’s relevant right now with the COVID pandemic, as a lot of mental health problems appear to be highlighted. In fact, I am focusing right now on the impact of stress on pain outcomes. I think there are so many comorbidities that accompany pain in daily life that should be investigated more carefully.
What would be your message for new researchers?
Follow your intuition and what your heart is beating for. Obviously, you need to work a lot and find some luck in the topic that you pick. Don’t get pushed to do projects that you don’t like, but understand that sometimes you have to grit your teeth and keep going. I did a lot of calcium imaging during my PhD. We had to write in a calendar the number of hours we were using the microscope, and I realized how long I was working in a room without windows, and how much I liked staying in the sunlight instead. I survived 246 hours of calcium imaging in a dark room within a relatively short time period. Once, as I was leaving late at night, I remember looking at the sky and thinking, “These cells are labeled very nicely!” Stars were shining like they were loaded with Fura-2. It was a sign to start the weekend!
Any parting words?
Look at the big picture, broaden your horizons, and try not to focus too deeply on a small part of the problem. Talk to scientists that approach pain research from other perspectives like psychologists, or scientists who specialize in addiction, for example. Always try to look at the problem from a comprehensive perspective.
Interview by Federica Franciosa, a PhD candidate in the Neuronal Plasticity Group at the University of Bern, Switzerland.