Researchers discuss their 2019 study published by Aging, entitled, “Conclusions from a behavioral aging study on male and female F2 hybrid mice on age-related behavior, buoyancy in water-based tests, and an ethical method to assess lifespan.”
Behind the Study is a series of transcribed videos from researchers elaborating on their recent oncology-focused studies published in Aging. A new Behind the Study is released each Monday. Visit the Aging YouTube channel for more insights from outstanding authors.
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Okay. So we met the first time at the Cold Spring Harbor Meeting on Mechanisms of Aging. And me and Julia went there with a lot of data and, if I remember correctly, we just realized that our aging study is so good that we can publish the wild-types only.
I think we realize that it had never been done in hybrid mice in both females and males. So just that we had hybrid mice, but also included both sex, made this study completely new, by just using wild-type mice.
Malin Hernebring
And we were so happy to meet you there, Jamie, and we had a nice discussion over our results and decided to take you on as a collaborator, which we’re very happy about.
Me as well, that was really fantastic, that was fun. I got to learn a lot about hybrid mice, which I had really had never thought about using before, to be perfectly honest. And especially learning about the body composition, which we’re going to talk more about. It was really a fun experience. So thank you. Both of you.
Julia Adelöf
Our pleasure.
Malin Hernebring
So you mentioned Julia, is that another aspect is that we include both sex. We talked about this a little bit before, how the research has changed, so that it has been so very focused on male mice. And now starting to realize the importance of including females in the study, especially since, I mean, women are many of the people that are taking the drugs that are developed.
Julia Adelöf
You see very real test, indeed. I think a lot of things has changed in the past, since we started the study, and nowadays there have been a lot of incentives to include both sex, which is a great thing. Our problem is though that, if you think about it, the tests that we use are predominantly, have been done with male mice for a long time. And that includes behavioral tests. So what was really important with our study, is that we follow these mice, both behaviorally, but also physiologically. And I’m going to come into the mobility test. To address depressive-like behavior, we used a common test called the forced swim test. And in this test, mice are placed in a water tank and then you record how they move in the water.
And both Malin and I had been taking… Were joining swimming teams at the time. And then you really get this feeling for that, people have different floating capacities or they act differently in water. And since we had the physiological data from these mice, we could see that the females had 50%, at the highest, more fat mass than the male mice. And as well, because we were swimming and thinking about this thing…
Malin Hernebring
…If I can just interrupt, because you were saying how, when you were crawling that you had to really have good speed and use your legs a lot to keep floating. While me having, maybe a bit more floating material, did not have that problem at all. So this is really positive of how we came this conclusion. And just thinking about crawling and how you line the water, whereas you’re floating.
Julia Adelöf
And I think one of the greatest things with our studies, that we actually included, the body composition when we were doing the data. And commonly it’s only the body weight, the differs, and we did not see a difference in the body weight. We saw that females and males weighed the same, but they had differences in lean mass and in fat mass. And then when we were looking at this a little bit more in detail, we actually saw that, how much the mice swam in water correlated, to how much percentage fat mass they had.
And this is also has never been shown before. I haven’t found a study where they actually address body composition and floating capacity. And since water tests are used for several different behavioral phenotypes, it’s very important in this study as well, that when you do water tests, that you actually take into account, differences in body weight. If you’re looking at obesity models, and as in our case, we found that there was a sex difference in water, but this sex difference could directly account for, by looking at the difference in the fat mass. So we cannot say, but there’s a strong correlation between immobility or activity in water and body composition.
Malin Hernebring
So we found that 46% in the younger mice, 46% of the difference was caused directly by the [inaudible] it’s…
And I mean, that’s a lot.
Jamie Ross
When you think about it that it really is a lot, because there’s a lot of behavior tests as Julia was mentioning. There’s more to water maze, which people classically use, which of course involves water and the ability to float. And how much muscle mass, how much fat mass you have, really directly can affect your ability to perform that task. Also, the radial arm maze can be filled with water as well. And these are all commonly used cognitive tests, that researchers use. And, again, I’ve never seen it published anywhere, where people are really thinking about how there are sex differences besides, sort of the place cells and how you use your environment. People usually focus on that aspect of the test, not the actual physical aspects.
Julia Adelöf
And I think that’s a challenge that we will see more of, now that both sexes are included in the studies. Because we are so focused on measuring one thing, which is depressive-like behavior in this case. And we don’t think about, “okay, so what are the other sex differences?” Because where you found a difference, and that’s super interesting, but can that actually be explained by something, like in this case, physiological. And that’s why data can be very misinterpreted, if we don’t know what kind of confounding effects, due to other sex effects that is involved in, or is interfering with our results. So I think it will be interesting to see, and I think that our study was early, because we included Themis mice early. But I think this kind of sex-different perspective and the secondary effects, will be even more common in the literature from now on.
Malin Hernebring
I would think so too, and hopefully also… I mean, one of the reasons that we’re doing this now, is to spread this information, so that people will know that there is this correlation. But I’d like to go through a bit, about this reduced exploratory behavior as a conserved hallmark. So one aspect of this study is that, because we are euthanizing animals that are suffering from severe disease, we are looking specifically at aging and not features that come along with decease. So I think this makes our results stronger also, and the fact that what we do see is a reduction in exploratory behavior, while we don’t see any effect on memory, for example, which we were surprised to see.
Interestingly, we neither see an effect in bone density with aging. I mean the old cohort that we’re using, they are not… So they’re at a stage where it’s about 70% survival. So they started to die, but they’re still, relatively healthy. And this was by choice that we chose this, because we didn’t want to have a selection on the cohort, that we looked at. We didn’t want to study the ones that were extremely long-lived only, we wanted to know more about the whole population.
Julia Adelöf
I think it can explain it also, because it’s a difference between aging mice and dying mice.
Jamie Ross
The lifespan and the new way to try to approach it, I think, that’s a really good point. You put an animal in an open field, and an aged animal, and if they have these other comorbidities, you’re not sure if they are moving less, because they actually are moving less. They have sarcopenia and these other issues, or maybe they have some arthritis, or they’re moving less because, wow, they’re actually in pain, because they have a disease.
Julia Adelöf
And that’s a challenge in the aging field, because you want to do research on old mice. But how do you do the discrepancy between dying mice and old mice? And I think one of the reasons why we actually, that we used in our protocol as well. Is that we removed animals with diseases, also helped us forming a cohorts of aging mice and not dying mice.
Malin Hernebring
So basically this method is about giving an estimate of the so-called life span. I mean, traditionally life span experiments are conducted until the animals are… Or they are allowed to live, for as long… That they are considered so sick from disease so that they are not likely to survive for another week and that means that they could go for months or even years with big tumors. Ethically, that’s very problematic, but as we were talking about, it’s also can complicate other analysis of course. But the way that we do this then is that, when we euthanize these animals, we make two curves.
One curve, we count these animals as if they had died from natural causes, and that’s, of course, is an underestimation. And then we make another curve that we calculate these animals as if they were as healthy as their litter mates, which they were not, because they had a terminal illness. So then that’s an overestimation of the life span, so then we have an interval that will be the min and max of this life span. We think this is a really good method, that we hope people will start using, in life span analysis.
Jamie Ross
I think it’s really fantastic, actually. The more and more I think about it, I know that maybe I’m biased. But so many institutions are not allowing researchers, for obvious reasons, to perform life span studies any more because they do not want the animals, understandably so, to be in pain and to be sick. So this is actually a really nice way to say, “Hey, I’m doing a life span analysis, however, I’m going to euthanize the animals who are sick and still get good, solid data that I can use.”
Julia Adelöf
And compared to other aging studies, because that was one of our issues that, we wanted to euthanize our animals upon early signs of disease. But since no other aging studies, that we found had euthanized the same amount or the large percentage of mice, we knew that it will be different because of course they will have a longer life span. But since we created the this, we could actually compare our data with it, even though we use the different protocol, with a lot of other aging studies in the fields, which is also important to know the platform, and to also compare your research against what’s known in the literature.
Malin Hernebring
And to be able to define the age status of your cohorts you’re looking at. I mean, yeah. And this was initially in the paper that we submitted, I don’t remember where now, but it was part of the review… it’s comments, that the animals are probably not feeling so well, because they have such a short life span. And that’s when we started to think about this and to invent this method, basically.
Jamie Ross
Are we going to go back and talk about the exploratory behavior a little bit more? Because one point that I wanted to also mention was, I think that this is a really nice, easy way to monitor what’s going on with the animals because there’s no cognitive testing involved, in the sense that, you don’t have to worry, “Oh, I’m using males or females so I shouldn’t use this for the females, I shouldn’t use this test for the males.” It’s a conserved test, that you can use for both males and females. It’s easy in the sense that it requires, not an absurd amount of equipment. And you put the animals there and then you walk away, and you let them stay in that environment for an hour, hour and a half, and then you look at the first 10, 15 minutes of that exploratory time. And I think that is also something really nice that people … I hope other researchers see and think how they could incorporate that measurement into their aging analysis of the animals.
Julia Adelöf
Yes and that it’s easy to do opens up for a lot of people to do it. But I think also one of the findings of this paper was that we found exploratory behavior to decrease, that we didn’t find learning and memory. And when you look at a lot of behavioral testing, they have an exploratory or exploration component in their tests. And since we found, they could show that, in a hybrid background, in both females and males, this declines. My question, is exploratory behavior and the decline an exploratory behavior, are confounding effects of other results, that has been shown of learning and memory? Because they are based on exploratory components as well. So I think, also when you perform aging studies, you should actually assess the exploratory behavior, to be able to know what your other behavioral tests show, since we have these components integrated into the test.
Jamie Ross
That’s a very good point. We always try to do a light-dark test and some sort of transition test to make sure that they are perceiving their environment correctly and that they move correctly, as well as the open field and other things. So, yeah, I think that’s a really good point to make, that I hope other researchers, who want to study aging more and other age-related diseases, think about.
Julia Adelöf
And also to be cautious about when you do a behavioral test and you have a test battery, is preferable, if you do the open-field tests early on, because you can also see a change. If you do open field test, we learned our lesson. If you do it later, you can actually change the order. So you want to have it early on, in the behavioral test battery. And then you would know also, if you find differences and you need to exclude results from following analyses and tests.
Malin Hernebring
And also that the animals are affected by being handled, so their exploratory behavior will change. We know now it’s hard to compare them.
I had another comment on this…this DXA equipment may not be standard in all laboratories.
Julia Adelöf
For body composition, the DXA. I don’t think everyone knows, the DXA is used for measuring the body composition of mice and rodents in this different type.
Malin Hernebring
Exactly. I don’t know, maybe someone has done this, but I’m just thinking that one way, instead to do this, would be to analyze the density of the mouse. I mean, basically just putting them in water and see how much volume that goes out. And then that would be some kind of measure of what the mouse density is. I’m hoping they’re not taking up.
Julia Adelöf
Mice float, they’re known to float and they don’t do this themselves, but if they’re not naked mice, they do float. And I was thinking, yeah, I think it would be an excellent follow-up to actually look at. And I think it has been done at positioning, so I know that it’s a paper out where they have tried to remove the [inaudible], so they added detergent into the water and then they could see that the position of the mice changed. So it has been shown that the…
Malin Hernebring
…The buoyancy.
Julia Adelöf
…Yeah, but that has not been done in in behavioral tests. So you can see that they do change their position and their angle in the water, but they haven’t correlated this to anything.
Malin Hernebring
Okay. So the take home messages from this work are that we want you all to know that the main conserved hallmark of aging behaviorally, is a decreased exploratory behavior. And we want you to be aware when you’re doing water-based tests to… as a body composition because fat mass can correlate to behavior in water-based tests. And also we want everyone using or doing a lifespan analysis, to think about the ethics surrounding the lifespan analysis and also be aware that there is a method to calculate lifespan, in which animals that suffer from severe disease or from pain, can be euthanized and still give valuable data. I’d like to thank the Swedish Foundation for Strategic Research, SSF for funding Julia’s PhD and also Jamie Ross and contributors to you. I think that’s all we had, right?
Julia Adelöf
Thank you for listening.
Malin Hernebring
Thank you for listening.
Jamie Ross
Thank you.
Click here to read the full study published in Aging.
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