Who funds dog research?

As I move through my training and think ahead to my future career, I wonder: who will pay for all this research I want to do on dogs? I have so many questions to ask!

• What changes happen in the canine brain as it enters, and then leaves, the socialization period?
• How is the brain of a fearful dog different from that of a confident dog?
• What are the genetic differences behind these variations?
• How do environmental differences (prenatal stress, early learning, adult life) change the brain?

In other words, what are the mechanisms in the brain that differ in fearful dogs — receptors, neurotransmitters, synaptic wiring? And how can I learn about them without using invasive (painful and/or terminal) techniques?

Who are the caretakers of Dog, the species, who care about fearfulness? We as dog owners and lovers care, but dog owners and lovers aren’t the ones who are trained to heal unhealthy dogs, to perform research aimed at understanding them, and we (mostly) aren’t the ones who breed them. So who are the groups who are the caretakers of Dog, and what subsets of Dog do they care for?

Veterinarians

We (I am a veterinarian) are trained to heal sick dogs. Relatively few veterinarians perform research compared to those who engage solely in clinical practice. But some do perform research: most commonly as faculty at veterinary schools alongside a clinical practice, or less commonly as researchers without a clinical practice at research instititutions.

Veterinary research, as a result of this strong emphasis on healing the unhealthy, is focused on clinical results. Veterinarians most commonly perform research which asks questions about the effectiveness of particular techniques — medications, surgical approaches, new equipment. Veterinary research very rarely addresses root questions about mechanisms, particularly in the area of behavior. Rather than asking “How are the brains of fearful dogs different?”, veterinary research is more likely to ask how we could fix a fearful dog: “Does this medication make a fearful dog less fearful?”

In fact, as I pursue my mechanism-based questions, I am asked if I miss being a veterinarian. The perception is that because I am engaged in basic, rather than clinical, research, I am no longer working as a veterinarian.

Basic science researchers

If veterinarians do clinical research studies, then who does basic research biomedical studies, studies that look not at how to fix problems but at how the body works? Ph.D. researchers are more likely to do this sort of research, which is why I am currently engaged in obtaining a Ph.D.

Traditionally, Ph.D. researchers have not been interested in dogs. In fact, way back in 2004 when I was originally deciding between a Ph.D. and a D.V.M., I was told by a Ph.D. animal behaviorist, “Ph.D.s don’t study domesticated animals. Veterinarians study those.” (Actually, veterinarians mostly just try to fix unhealthy domesticated animals, not study the healthy ones.)

That perception has changed in a big way in the intervening eleven years. There are now multiple laboratories studying dogs. But where does their funding come from — who cares enough about dogs as dogs, not as models for human problems, to provide the impressive funding needed for a genomics study? (The work I am doing for my Ph.D., sequencing messenger RNA, costs around $45,000.)

The U.S. federal government

The traditional source of funding for basic research is the federal government: the National Institutes of Health for health-based research and the National Science Foundation for more basic research. But these two massive institutions are very much focused on human health — as they should be, as they are funded by the tax dollars of American citizens. The economy can’t support all the research American researchers would like to do, and getting an NIH or NSF grant is becoming more and more difficult as grant funding is cut. Funding to study dogs as models of human disease? Maybe, but isn’t it easier to study laboratory rodents (on which you can perform invasive studies) or work on humans directly? Funding to study dogs as dogs? Go lie down until it passes.

In my experience, the small number of laboratories directly studying dogs are either studying them as models for questions about human health or evolution, operate on a shoestring budget, or have great trouble obtaining funding for what they want to do.

Animal welfare organizations

So who cares about dogs? Animal welfare organizations, some of which are national in scope and do perform research. Some major players in this field are the American Society for the Prevention of Cruelty to Animals (ASPCA), the Center for Shelter Dogs (CSD), and the Humane Society of the United States (HSUS). I am most familiar with the research coming out of the ASPCA and the CSD, and it is exciting stuff. But it is again mostly focused on applied questions: how can we help the shelter dogs in our care?

I reviewed some of the research these two organizations have performed on how to identify and treat food aggression in shelter dogs in my story for the Bark on shelter behavioral assessments. This was ground-breaking research and I am really glad to see it published. But it doesn’t ask the basic (i.e., non-applied) research questions I am interested in: what is it about the brains of these dogs that differs from the brains of dogs without food aggression? That kind of research doesn’t have immediate applied benefit. You can’t take it to a shelter worker with a recommendation about whether or not to put a food aggressive dog on the adoption floor. It is incredibly impressive that these shelter-focused organizations perform any research at all, and it is absolutely appropriate that the research they perform should have a highly applied focus, with clear questions that, when answered, will provide guidance on how to improve the lives of shelter dogs, immediately. They do not have the resources to pursue these sort of mechanism questions that I want to ask, which do not have immediate applicability.

So who cares about understanding how dog brains work, with the hope that that information will provide a base for future applied research? Who cares about the whole species, not just the subset in shelters or the subset in hospitals?

Breed organizations

Breed organizations care very much about the health and welfare of dogs, and in fact have provided funding into the mechanisms behind health issues specific to their breed. A recent paper about associations between spay/neuter status and health issues in Golden Retrievers was partially funded by the American Kennel Club’s Canine Health Foundation (AKC/CHF), and a similar study on Vizslas was funded by the Vizsla Club of America Welfare Foundation. (I blogged about these studies elsewhere.)

These organizations can fund basic research on how and why particular diseases occur in their breeds, and may even be willing to fund expensive genetic studies, such as a recent one on the genetics of cancer in Golden Retrievers, supported in part by both the AKC/CHF and the Golden Retriever Foundation. However, their focus is very much on the problems of a particular breed. My questions are broader: why do dogs of all breeds have different personalities, some more or less fearful? These organizations are really the caretakers of breed subsets of Dog, not of Dog itself.

Who, then?

Who does that leave as a group willing to fund studies on Dog? On problems common to all breeds? On problems which may or may not provide good models for humans? If I hope to one day run a laboratory which studies these problems, who can I hope to help pay for the research?

I would be remiss if I did not mention Morris Animal Foundation here. While their important Golden Retriever Lifetime Study happens to focus on the health issues of a single breed, their mission is to fund research into studies of small animals (dogs and cats), livestock, and wild animals, with no breed limitations. This group is doing important work, and I applaud them.

But one organization is not enough for a laboratory to depend on for survival, especially in these times with research funding so hard to come by. And so I wonder: are we, the dog lovers of the world, the ones to start supporting research into what it is to be a dog? We, who own dogs of all breeds and mixes, with all sorts of problems, who know what problems most plague us as owners — not just medical problems, but behavioral ones?

And so I leave you with my dreams of crowdfunding, in which a researcher proposes a study and asks the public to support it through donations. Such an approach allows the dog community to take the task of answering basic questions about Dogness into their own hands. This direct connection between a researcher and the community affected by their research is a new benefit of this age of social media. Is this approach right for this particular problem? Time will tell.

Can prenatal stress be reversed?

I was scanning the titles of new journal articles a while back, and came across one that made me think, hey, that may be about rats, but it is totally relevant to dogs. And then I thought, why don’t I teach a class on it? Read and interpret this really interesting journal article with a group of dog trainers and dog lovers?

I will be teaching the class Prenatal Stress and Anti-Depressants for APDT the week of November 18 (and you are invited to take it). This post will be used as reading material for it. In the class, we will talk about this article and what conclusions we can draw from it and apply to dogs. So I may not draw as many the conclusions for you in this post as I usually do; the plan is for the students to do that together in class. But it was a fascinating paper and there’s lots of good material in it, so read on if you want a conclusion-free summary of it!

Pereira-Figueiredo I., Juan Carro, Orlando Castellano & Dolores E. Lopez (2014). The effects of sertraline administration from adolescence to adulthood on physiological and emotional development in prenatally stressed rats of both sexes, Frontiers in Behavioral Neuroscience, 8 DOI: http://dx.doi.org/10.3389/fnbeh.2014.00260


Why prenatal stress?
So what’s prenatal stress and why is it important to dogs? The authors of the article don’t provide much background on this phenomenon, but it’s an interesting one: when a mammal undergoes unusually high stress during her pregnancy, the personality of her offspring can be affected. We believe that the stress hormones rising in her bloodstream can pass through her placenta to the fetus or fetuses, and can change how their brains develop at this very early stage of life. That’s prenatal stress: stress experienced before birth.

Part of what this paper investigates is exactly how prenatal stress affects the developing personality, because we don’t yet fully understand how this stuff works. In general, though, we expect prenatally stressed animals to be more anxious and less confident than animals who were not prenatally stressed.

Does prenatal stress affect dogs? We don’t know for sure, but we think it is something that can affect most or all mammals. Would it happen commonly? Hard to say, but I imagine a pregnant dog who is stray or in a shelter and highly stressed, and I wonder what effects this might have on her puppies.

What can you do about it?
If you have a puppy that you believe was prenatally stressed and whose personality you thought was adversely affected, what are your options? Careful socialization and good enrichment are always an excellent choice, but in some cases you might consider medication. This study looks at whether a particular anti-depressant, sertraline, might help change the individual’s personality long-term if given in adolescence. Sertraline is an SSRI, in the same class of medications as Prozac. These are widely used medications believed to be fairly safe, but one of the questions these researchers ask is whether it is safe when given throughout an animal’s entire adolescence.

SSRIs such as sertraline affect the levels of serotonin in your brain. Serotonin is a chemical which affects mood; depressed people tend to have less of it, as do aggressive people. As a result, it is the target of a fair number of anti-depressants, which work to increase its levels. Prenatal stress is known to disrupt the serotonin system in the brain, so a medication which affects serotonin is a reasonable choice for prenatally stressed individuals.

So the idea is: give these prenatally stressed animals a medication which increases their serotonin levels while they are adolescents and their brains are still developing. The hope is that they will develop into more normal adults than they would have without the medication. So, exactly how do you investigate such a question?

Methods: how the study worked
First, the researchers stressed some pregnant rats by putting them in clear tubes to restrain them, and shining bright light on them. This was repeated for forty-five minutes at a time, three times a day. They also kept control rats, who were not stressed during their pregnancy. The pups born to these two sets of rats were then in two categories: prenatally stressed pups and non-stressed pups.

The rat pups began their anti-depressant treatment with sertraline when they were one month old. Now there were four groups of rat pups:

prenatal stress anti-depressants	prenatal stress no anti-depressants
no prenatal stress anti-depressants	no prenatal stress no anti-depressants

Having these four groups allowed the researchers to pick apart the two different effects, the effect of prenatal stress and the effect of anti-depressants during adolescence.

The pups were tested at two months of age, the beginning of rat adolescence, to see if the prenatal stress had affected their personalities. They were assessed for how they dealt with startling noises and being exposed to open space (scary for a rat!). Their blood was also tested to see how their immune systems were developing, because immune systems develop differently in animals who have been subjected to high stress. All these tests were run again one month later, at the end of rat adolescence, to see how the anti-depressant given throughout adolescence had affected treated rats compared to the control groups.

Results: what they found

• Although we may think of prenatal stress as mostly affecting an animal’s behavior, it’s been shown to also affect metabolism, so this study looked at birth weight. Interestingly, prenatal stress only reduced the birth weight of the female rat pups, not the males. The weights of these females had equalized compared to non-stressed pups by weaning age. After weaning, though, the prenatally stressed females continued to gain weight and ended up heavier as adults than the non-stressed females. When prenatally stressed females were given the anti-depressant sertraline, however, this weight difference was reduced.
• The pups were also tested for their startle response when they heard a sudden sound. Prenatally stressed rat pups did seem to have a larger startle amplitude (size) compared to controls, but this wasn’t statistically significant, and was not reversed by sertraline treatment.
• Prenatally stressed females did not habituate to the startling sound after several exposures as well as rats from other groups did; treatment with sertraline reversed this effect.
• The pups’ behavior in an open space was tested. No difference was seen between prenatally stressed and non-stressed pups, except in males on their first time being tested (not on later tests).
• In the open space test, only non-stressed females explored more (became more confident) on repeated testing; males and prenatally stressed females did not become more confident with repeated exposure to the open space.
• The pre-natally stressed rats showed a significant decrease in their number of white blood cells. This change was reversed when they were treated with sertraline.

Discussion: what does it all mean?
The study’s main conclusions are that effects of prenatal stress can be seen in rats, and that giving sertraline during adolescence did not harm them.

The rapid weight gain in the pre-natally stressed females is an effect that’s been seen before, and seen in humans. Children born with low birth weights often grow to have issues with their weight and can suffer from diseases related to a poorly regulated metabolism. This loss of control of energy balance has been associated with dysregulation of serotonin in humans, adding additional support to the choice of sertraline, an anti-depressant which interacts with the serotonin system.

It is interesting that no anxiety-like behavioral changes were seen in the prenatally stressed rats. Prenatal stress is known to cause anxious personalities in many cases. However, these rats were as confident (or as anxious!) in the open space test as rats who had not been prenatally stressed. The researchers comment that this particular test has been done on prenatally stressed rats in other studies, and that those rats didn’t show anxiety in the open space test either, so this does seem to be a real result, rather than a statistical error.

They did see some differences, though. Male rats who had been prenatally stressed did show some additional reluctance to explore (i.e. anxiety) on their first day only in the open space test. After that they explored equal amounts compared to other groups.

The researchers also note that while most of the rats that they tested were equally anxious on all days that they were tested in the open space, females who had not been prenatally stressed appeared to begin to explore more on repeated tests, as if they were learning to be less anxious as their surroundings became more familiar. This was not the case in male rats or in rats who had been pre-natally stressed.

Remember also that female rats who were prenatally stressed did not habituate to startling sounds as well as rats from other groups. Is it possible that with this particular model of prenatal stress, the personality effects of prenatal stress appear not as classical anxiety, but as difficulty habituating to new situations or stressors such as loud noises?

Finally, the researchers looked at effects of pre-natal stress on the immune system, and found significant effects (decreased numbers of white blood cells) which were reversed by treatment with the anti-depressant sertraline. Why did they care about the immune system? Because the immune system and the stress system are closely intertwined. Stressed animals show changes in the numbers of their white blood cells just as the prenatally stressed rats did. The researchers were using the changes in the immune system as markers for changes in the stress system.

There are two possibilities for why these prenatally rats showed stress-associated changes in their immune systems: either because they themselves had high stress levels, or because their immune systems were developing prenatally (in utero) in a high stress environment due to their mother’s stress levels. Either way, it is interesting that treatment with sertraline reversed these effects, suggesting that it may have either changed current stress levels in these adolescent rats (even though the only serious stressors they had undergone were before their birth!), or had counteracted other effects from that prenatal stressor.


Conclusions
It can be hard to know exactly what conclusions to draw from a scientific paper. What do you think? What are the most important findings in this paper (maybe just two or three of them)? Do you think those findings are real phenomena, or maybe just statistical mistakes? If they’re real, do you think they can be extrapolated from rats to humans or dogs?

Shades of grey in the ethics of animal research

This afternoon I read Animal Research: Animal Welfare vs. Animal Rights, by Walter Jessen of Highlight Health. The article addressed some myths about animal research, explaining why it is necessary and what we do to maintain the health of research animals. In many ways, it was a typical volley in the debate between scientists and animal rightists about animal research.

I have issues with the arguments used by both sides of this debate, which miss a lot of shades of gray in the question about whether it is wrong to perform research on animals. The article ably addressed some of the myths and mis-framings presented by the animal rights side of the debate. That is important communication to provide. However, it introduced some mis-framings of its own, and I’d like to address those here, while recognizing that the article is completely factually correct.

Animal rights vs. animal welfare

Jessen starts by defining his terms (“animal rights” and “animal welfare”). He chooses the most extreme position for animal rightists, describing them as people who “reject eating any animal as food, abstain from taking any over-the-counter drug and/or prescription medication, and refuse all vaccinations and/or medical treatment.”

In my experience, that description characterizes a truly extreme group. It leaves out those who take a more graded approach, such as a belief that animals shouldn’t be eaten, but may be used in some forms of research. This may be an attempt to frame the argument in terms of scientists versus fringe crazies. What about the people who fit in neither camp? Isn’t that where most of us lie, accepting medical treatments but feeling somewhat uncomfortable about animal research? Can’t scientists address that discomfort without resorting to the suggestion that anyone who feels that animal research may be wrong must reject all medical treatment?

Immoral and necessary

Jessen argues that animal research is necessary because “animal systems provide invaluable and irreplaceable insights into human systems.” That is completely true (and I would add that animal research provides invaluable and irreplaceable insights into animal systems as well. It isn’t only useful in human medicine). Jessen doesn’t go so far as to say that because animal research is necessary, choosing to perform it is also moral. But that seems to me to be the point of his article: we don’t need to feel bad about this; this is necessary. But just because something is necessary doesn’t mean we should be completely comfortable with it. Can’t animal research be both necessary and immoral? Can’t we choose as a society to do it without absolving ourselves from feeling disturbed by it? When we read about an interesting experiment that involved animal research, can’t we think how cool the results are and how we feel sorry for the animals?

A mountain climber is stranded in a snowstorm. Help won’t come for days. His companion dies. Our hero eats his friend so that he won’t starve to death himself. Immoral, but necessary. He does it, and he knows it is the right choice. But he feels terrible about doing it. Perhaps he will take care to provide for his companion’s children.

A single mother in a country that does not have welfare loses her job through no fault of her own. Her savings are gone. She loses her home. Living on the street, she steals food to give her children. She knows it is wrong, and she feels bad about it. But she also knows it’s the right choice.

Recently I had elective surgery. After the surgery, I chose to use painkillers that surely had been tested on animals. I know that animal research is wrong, and I feel bad about it. But I also know that it is the right choice to continue to learn about how to keep humans and animals healthy.

The debate about animal research is often presented as two choices: it’s wrong, and we should never do it; or it is necessary, so we shouldn’t feel bad about it. We should feel bad about it. And we should continue to do it. (But we should do it somewhat differently than we do now. Read on.)

Are research animals comfortable?

Jessen writes that  “the vast majority of biomedical research does not result in significant discomfort or distress to research animals.” I think he is addressing the misconception that research animals spend the majority of their lives in a great deal of pain, and wants to make the point that they are kept mostly pain-free. However, he writes that “57 percent of all research procedures with animals involved no more than slight or momentary pain or distress.” I note that that leaves 43 percent of procedures that do involve more than momentary pain or distress. That’s a significant fraction.

He also writes that “thirty-eight percent of the research procedures employed anesthesia and post- operative painkillers.” I should hope that painkillers were employed where indicated! But I also would like to point out that painkillers don’t remove all the pain. Would you choose to undergo an unnecessary surgery if you were offered the best possible pain relief afterwards? What if you were offered the surgery but had to recover in a hospital where no one spoke your language, and you had no way of requesting more painkillers if the standard dose was not enough for you?

I’d also like to move the discussion away from pain, which both sides of the debate are (in my opinion) overly focused on. Pain is sometimes necessary in research. What isn’t always necessary is keeping animals in cramped quarters lacking environmental enrichment. I have seen facilities where the mice and rats lack even an exercise wheel, and where there are multiple animals in a cage so small I wouldn’t feel comfortable keeping even a single pet rat in it. That’s not necessary; that’s a choice, due to the fact that more space and more enrichment is expensive.

Again, I don’t think the argument has to be so black and white. We don’t have to argue that research animals are in constant, excruciating pain (because that is untrue). But we can also admit that research animals do often (possibly 43 percent of the time) experience pain as part of the research process. We can feel bad about that, but still do it, because the alternative is so unthinkable. And we can accept that if we’re asking these animals to give up their lives, we don’t also need to ask them to live in such unenriched environments. They require mental stimulation just like we do. If they were so different from us, we wouldn’t be using them to model us.

The indifference of researchers

Jessen argues that “researchers are deeply concerned about the condition of the animals they study.” I agree. Researchers aren’t unfeeling monsters, and I wish that the people arguing on the other side of this divide would stop trying to make them out to be.

However, I think researchers often (not always, but often) subscribe to the black and white version of this particular moral dilemma: it is necessary, so I don’t need to feel bad about it. I have every respect for and sympathy with someone who is living day in, day out with animals that they know will die at the end of the experiment. It would be easier if the cages were big enough, if the animals didn’t lack enrichment, if the painkillers were always sufficient and took away all the pain, if the animals had some place to go after non-terminal experiments. But despite my sympathy for the research community, I would like to see a little more effort on their part to address some welfare issues, and to recognize the moral difficulties inherent in animal research. I do think that it is possible to do animal research, and still sympathize with the viewpoint of people out there who find the idea of animal research disturbing. It should be disturbing. Even though it is necessary.


Moving forward

Am I saying that everyone who benefits from animal research should be guilt-stricken all the time? Of course not. That wouldn’t be very productive. I am saying we should recognize the shades of grey. And that, as a society, we should feel bad enough about animal research to make improving lab animal welfare more of a priority. More of the money that goes to research could go to making the lives of research animals more comfortable. (If a pet rat lives in a bigger cage, so should a research rat!) If they are giving their lives for us, and we recognize that that is wrong, we should also recognize that it is our responsibility to make their lives not just as pain free as possible, but as good as possible. We’re doing a decent job on the former. I don’t think we are working anywhere near hard enough on the latter.

Why cortisol sucks as a measurement of stress

Standing in the ward of a veterinary hospital, you see a dog jumping up and down in his run, barking. Is he distressed at being here? Or is he just barking to get attention? Obviously, you decide to perform a research study on dogs in the hospital, to measure their stress levels. How are you going to go about getting some sort of numeric measurement of stress, so that you can perform statistics on your data and publish it in a journal?

People have approached similar problems in a lot of ways. One of the most common answers is to measure the dog’s cortisol levels. (Or corticosterone, if it’s actually a rat, mouse, rabbit, or bird.) This is the approach I’m using; cortisol is in saliva, which is why I spent so much time over the last year trying to get dogs to drool more.

What is cortisol, actually? There was an excellent post on mindhacks.com recently about cortisol and how science journalists sometimes misrepresent it. I’m going to use dogs as my examples, but what I have to say is just as relevant to studies on humans, if that is your cup of tea. It might help you to understand some of the news stories floating around about various things which “raise cortisol levels.”

Cortisol is a hormone made by your adrenal glands. Your adrenals sit next to your kidneys, but they produce cortisol in response to hormones released from your brain in stressful situations. So we like to measure cortisol levels because they tend to increase when the brain is sending out “I’m stressed” messages.

OK, but what is stress? What I care about, and what many people who measure cortisol care about, is psychological distress — being yelled at, being scared you’re going to be eaten by a predator, being left in a loud veterinary hospital with no familiar faces around you. Stress is a lot of other things as well, however. It is hunger, illness, feeling too cold, having exercised recently. In fact, cortisol has a normal rise and fall over the course of the day to help your body know that it is time to be awake or to go to sleep. Your adrenals also produce it to help you deal with anything which requires some extra energy. You may need that extra energy for a good reason, such as competing in an athletic event. “Good” stressors like that are known as eustressors. So if you’re going to use cortisol to measure stress, you are going to be measuring both eustress and normal daily stress like hunger, in addition to whatever source of distress you may be interested in.

Knowing this, you’ll try to design your experiment to work around the problem. You’re interested in whether dogs find their time in a veterinary hospital to be distressing. So you will try to remove eustressors from the equation — you will make sure that none of our dogs have exciting things like getting fed or taken for walks happen while we’re studying them. You will also make sure that all of your study dogs are healthy, since illness can raise cortisol levels. And you will measure the dogs’ cortisol levels at exactly the same time of day, because of cortisol’s diurnal cycle. (There is some very interesting debate about whether dogs, unlike all other mammals which have been studied, actually don’t have a daily cycle of cortisol. One theory is that they don’t because they sleep most of the day.) Now you believe you're just measuring distress.

Cortisol is still an awfully bad way to measure distress! Males and females react to stressors in different ways. (This has mainly been reported in humans, but it’s been said that studies of stress in rats which are limited to males miss an important segment of the population.) Age has something to do with cortisol levels as well, though mainly just in the very young and very old. In dogs, it is an open question of whether breed matters, but I’m guessing it does, since personality affects cortisol responses to stress in humans.

So you control for that, too. You get a bunch of dogs of the exact same age, gender, and breed. They are all laboratory animals, so you can be reasonably sure their histories are the same, and you aren’t going to find out at the end that half of them have spent more time in a veterinary hospital than others. (This wasn’t the direction I chose, but some studies do give it a go, using laboratory beagles of similar ages and only one gender. There are obvious ethical implications here, but that’s a post for another time.) You put these dogs in a veterinary hospital and measure their cortisol levels. Now are you measuring their reaction to the hospital setting?

Maybe. The next problem is that all these animals have their baseline “unstressed” cortisol level set at a different point. We don't understand all the genetics having to do with how this system works, but we are learning. We do know that a cortisol level that indicates stress (good or bad) in one individual might indicate total relaxation in another. Many studies deal with this problem by looking only at changes in cortisol levels. They measure cortisol before and after the stressor, and look at the difference, rather than at absolute levels. So let’s assume you can do this in your hospitalized dogs. You keep them in one environment for a few weeks or months, until they have time to settle in and relax, and you keep track of their average cortisol levels there. Then you put them in the hospital and look for a difference. Now are you measuring their reaction to the hospital?

You probably are, but what exactly are they reacting to? Something which is a stressor for one individual isn’t necessarily a big deal for another. For example, the Trier Social Stress Test (TSST) is a test specifically designed to raise cortisol levels in humans. It’s used to study things like how gender affects responses to stress. You stress the person out by making them do some public speaking and public arithmetic. But only 70% of people who take the TSST actually have increased cortisol levels compared to just before they took the test. Doing arithmetic in front of a hostile audience just isn't alarming for some people.

In the case of your hospitalized dogs, some don’t like the noise, and some don’t like being in a cage, and some don’t like having other dogs around, and some don't like all of the above. But some think it’s awesome to be in such an exciting environment with so much going on. The hospital isn’t just one big stressor, it is a lot of different little ones.

If cortisol is such a bad way to measure distress, why do we use it? Unfortunately, it is still the best understood method we have. There are lots of other methods, but they all have their own problems. It’s a good idea to use at least two methods together, actually.

So what do you do? Give up? My approach has been to cross my fingers (maybe close my eyes) and just proceed. I think a lot of research involves just circling around a problem, picking away at it until it starts to give in. Studies of stress may not be able to give precise answers to questions about stressors. But if enough of them are done, our picture of how the stress response works will continue to get clearer and clearer. It’s really hard to know what is going on in the mind of a member of a different species; it can even be hard to know what’s going on inside the mind of a member of your own species. We just have to keep trying.

[ETA: See the follow-up post, “Why do other measurements of stress suck worse than cortisol?”]

Nest making, oxytocin, and social bonding

I encountered Nest making and oxytocin comparably promote wound healing in isolation reared rats [1] while reading about how stress affects wound healing, and it drew me in with its lure of drawing connections between nest making and oxytocin. Oxytocin does a lot of things in the body, but what this paper was interested in was its participation in social bonding. You all must already know the coolest story about oxytocin, the story about the two species of voles. The species are almost identical. One species mates for life, one doesn’t. The one that does has more receptors in its brain for oxytocin than the one that doesn’t.[2]

Because of the promise of learning more about oxytocin, I chose this paper for my second and final journal club presentation. The second time is a lot less scary than the first, I learned, and preparation goes faster.

This paper took a while to get to oxytocin, so let me start at the beginning. The authors had run three separate experiments which the paper covered. In the first study, Assessment of wound healing due to Nestlet treatment, they gave some rats a burn on their backs. (It was a pretty serious burn; two of the rat pups died of shock after receiving it.) Then they put them into three groups. One group of rats lived in sets of three (“group housed”). One group of rats lived alone (“isolation housed”). And one group of rats lived alone, but were given Nestlets, which are little cotton squares which can be used to make nests. They compared the healing rates of the three groups. The group housed rats healed the fastest; the isolation housed rats which had Nestlets to use to make nests healed almost as fast (statistically there was no significant difference); and the isolation housed rats without Nestlets healed most slowly.

They asked themselves: why does nest making help rats heal better? It seems like the mechanism has to be central (in the brain, rather than in the rest of the body), since a nest is really environmental enrichment. In their second experiment, Time series analysis of wound healing and comparison of wound healing with Nestlets to wound healing with oxytocin, they repeated their first experiment, but this time with a fourth group of rats: isolation housed rats who were injected with oxytocin once a day for several weeks. Why oxytocin? The idea seemed to be that rats build nests together, and if a rat lives alone, building a nest is still somehow a social act. Since oxytocin is released during social bonding, a dose of oxytocin might theoretically substitute for having other rats around or for nest building. And indeed, the rats given oxytocin healed faster, with rates similar to the rates of rats given Nestlets.

Finally, in Assessment of behavior and brain changes due to Nestlet administration, they had four groups of rats: group housed rats without Nestlets; group housed rats with Nestlets; isolation housed rats with Nestlets; isolation housed rats without Nestlets. No rats were burned in this experiment. They demonstrated gene expression changes (mRNA changes) in the brains of rats given Nestlets, and demonstrated behavior changes (decreased hyperactivity in an “open field test”).

They conclude by suggesting that Nestlets do affect wound healing rates; these changes seem to happen because of changes in the brain (though that isn’t proven); and this particular model of wound healing and Nestlet administration might be a good model for studying stress impairment of physical health in humans.

I had gone into this paper enthusiastically, because oxytocin is probably my favorite hormone. I came out the other end bothered on several levels, but decided to present the paper at journal club anyways, because it seemed like a good exercise. The attendees seemed as bothered by the paper as I was, and contributed more issues with it than those I’d come up with; actually, the presentation felt really enjoyable to me as a result.

What bothered me? To start, why were the rats not given any analgesics at all? Perhaps the researchers felt that pain killers would have confounded their study design in some way, but I would have been interested to know what way. As it stood, I wondered if they simply hadn’t thought of it.

Can you really say that the effects of nest making are centrally mediated (take place in the brain) just because they are similar to the effects of oxytocin? Can you even say that the effects of nest making are related to the effects of social interactions? No, you cannot. Perhaps the tool used here for measuring rate changes in wound healing was so insensitive that any number of changes would look similar. Perhaps the changes were in fact very similar but caused by very different things. (One possibility proposed by someone at journal club: the rats which were able to nest improved because the nests helped them keep warm better, something which group housed rats use each other to do.) To their credit, the authors of this paper did state that they hadn’t proven that the effects of Nestlets were centrally mediated or even related to the effects of oxytocin administration. But if they knew that their study wasn’t going to answer that question, why include the oxytocin group at all? Or, if they really were interested in comparing the two mechanisms, why not include an oxytocin group in the experiment in which they looked at behavior changes and mRNA changes in the brain?

The studies certainly did demonstrate that oxytocin administration improves wound healing in isolation reared rats. But does this have to do with the fact that oxytocin is associated with bonding? It might have been interesting to see if oxytocin administration improved wound healing in group housed rats as well.

The studies also demonstrated that providing Nestlets does improve healing rates in isolation housed rats. However, one rat researcher at journal club pointed out that, in fact, they couldn’t even say that “nest building” caused the improvement. Perhaps it was just the exercise of moving the Nestlets around the cage. She suggested a control group which was given exercise wheels.

The suggestion that this model could be useful to study the physical effects of stress in humans gave me pause, as well. Different kinds of stress have different effects. This kind of social isolation stress isn’t necessarily going to affect a human the same way some other kind of stress might, like job-related stress. In fact, one journal club attendee pointed out that the slower wound healing rates in the isolation housed rats might have less to do with social isolation stress, and more to do with the fact that group housed rats are able to lick each other’s wounds. (None of us knew if rats actually did that, though.)

As a veterinary student, I was also a little saddened by the fact that this paper was never placed by its authors into the context of laboratory animal medicine. Though the purpose of the paper was to illustrate a model that is useful for human medicine, I felt at least a sentence could have been devoted to explaining the context of enrichment for laboratory animals and why it is important.

At the end of the paper, the authors explained some future directions for their research, including giving oxytocin antagonists to rats with Nestlets. This would prevent oxytocin from acting in those rats. If the effects of the Nestlets were blocked in those rats (i.e., if their wound healing slowed), then we might conclude that oxytocin had something to do with the mechanism of Nestlets’ effects. That would be an interesting next step and would definitely clarify the Nestlet/oxytocin relationship, if there is one. I wish that they had waited to publish this paper until they could include those findings; as it is, the paper felt somewhat incomplete.

We talked a little bit about the journal that this paper was published in: PLoS ONE, an open access journal. One attendee felt that open access journals are likely to publish lower quality papers. I hope that’s not actually true, since I believe open access to be important. If this paper weren’t open access, I wouldn’t be able to provide a link to its full text in this blog post!

Of course, the paper did have some interesting things to say. Nest making improves wound healing in socially isolated rats. That’s interesting! (But we don’t really have any idea why it does this, at least not based on this paper.) Oxytocin also improves wound healing in socially isolated rats, which I find even more interesting. I’m curious whether oxytocin has been used in wound healing experiments in the past. I know that research has suggested in the past that happily bonded people are healthier (though I don’t know anything about this field and therefore hesitate to try to find a reference for this assertion — if you have a good one, let us know in the comments). I’ve always wondered why. Do people living alone find taking care of themselves harder? They might be more likely to have trouble getting to the hospital, or lack caretakers when they’re ill, or just lack someone to tell them “You’re sick, go to bed.” This study suggests that loneliness might actually affect health more directly. Of course, the complete social isolation these rats experienced might have different effects from the milder isolation of someone who lives alone but works in an office. And healing from burn wounds is not the same thing as general health. It’s still food for thought.

I was definitely dissatisfied with this paper, but the presentation was fun. The attendees felt engaged and interested. I hadn’t realized before how useful it can be to present a paper that you have some issues with. It’s not something I’d do every time, but it’s a useful trick to know.

[1] Vitalo A, Fricchione J, Casali M, Berdichevsky Y, Hoge EA, Rauch SL, Berthiaume F, Yarmush ML, Benson H, Fricchione GL, & Levine JB (2009). Nest making and oxytocin comparably promote wound healing in isolation reared rats. PloS one, 4 (5) PMID: 19436750 [Free full text]

[2] Young, L. (1998). Neuroendocrine bases of monogamy Trends in Neurosciences, 21 (2), 71-75 DOI: 10.1016/S0166-2236(97)01167-3 [Abstract at oxytocin.org] [Pop-sci summary]

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