The rough guide to the stress response

[Note: This post is intended as reading material for my upcoming online course, "Canine Hormones: From molecules to behavior." This is an entirely online course offered through APDT, begins Februrary 11, and is worth 12 CEUs. I posted with more information. I encourage you to sign up!] 

The series of organs working together to form to stress response are called the hypothalamic-pituitary-adrenal (HPA) axis. This post is a reference to them. The major players are:

The hypothalamus

The hypothalamus
Licensed under CC BY-SA 2.1 jp
via Wikimedia Commons

What it is: part of the brain, an important link between the nervous system and the endocrine (hormonal) system

What it does in the HPA: in response to input from other parts of the brain, releases cortocotropin-releasing hormone (CRH) into blood vessels which take it directly to the pituitary and not into the rest of the body

The pituitary
Source:
Emplacement de l'Hypophyse
Patrick J. Lynch, medical illustrator
via Wikimedia

The pituitary

What it is: a little gland hanging off the bottom of the brain. Some people consider it part of the brain and some don't.

What it does in the HPA: in response to hormones coming through the blood directly from the hypothalamus (not going out through the rest of the body first), sends adrenocorticotropic hormone (ACTH) out to the rest of the body

The adrenals

What they are: small organs next to the kidneys responsible for sending all kinds of important hormones out into the body

What they do in the HPA: in response to ACTH in the bloodstream, release cortisol into the bloodstream so that it can alert different organs and tissues around the body to the need to respond to a stressor

The minor players

Those are the three organs which are part of the name of the stress response: the hypothalamic-pituitary-adrenal (HPA) axis. But to some extent, humans just chose those three as the central parts of the axis because we understood their functions first. Other organs are important in the functioning of the stress system too.

The hippocampus

What it is: a part of the brain associated with learning and memory

What it does in the HPA: assesses the amount of cortisol in the bloodstream and sends a negative feedback message to the hypothalamus to tell it to slow down the HPA axis (resulting, eventually, in the release of less cortisol from the adrenals). This is probably part of how socialization works: the hippocampus undergoes epigenetic changes early in life which make it more or less able to send the “slow down” message to the hypothalamus and put the brakes on the stress response.

The amygdala

What it is: a part of the brain associated with fear

What it does in the HPA: the amygdala is part of the system that sends that initial message of fear when an animal encounters something scary, triggering the initial HPA axis stress response.

The liver

What it is: an organ that makes a lot of useful substances used for various things in the body

What it does in the HPA: makes corticosteroid-binding globulin (CBG), the little protein that carries cortisol around in the blood stream. CBG does more than just ferry cortisol about; it actively spits it out in locations where it's needed, and when an animal has very low levels of CBG, the entire HPA axis becomes less reactive. Very young animals have low levels of CBG, which may contribute to their early lack of fear.

The stress of life

[Note: This post is intended as reading material for my upcoming online course, "Canine Hormones: From molecules to behavior." This is an entirely online course offered through APDT, begins Februrary 11, and is worth 12 CEUs. I posted with more information. I encourage you to sign up!]

Stress isn't good or bad. Stress is life. Stress is some change in your environment that means your body has to work a little harder. Stress is a blast of cold, missing a meal, not getting enough sleep. But stress is also going for a run, seeing a loved one after a long absence, thinking through a hard problem and getting it right. All of these things might mean your body has to rev up: your heart might beat faster, you might spend less energy on digestion, your immune system might adapt to meet expected coming challenges. An extra challenge can be good or bad. If life were one long nap, it wouldn't be much of anything.

Your body uses the hormone often referred to as the stress hormone, cortisol, to manage its response to stress. Going to an agility trial today? Need a little more cortisol to deal with all the extra energy you're going to spend. Cortisol affects our bodies profoundly, regulating our immune systems, our metabolism, and our behavior. Without enough of it, we would die, and indeed there is a disease (called Addison's in humans and hypoadrenocorticism in dogs, but it is the same thing) which is simply a lack of sufficient cortisol. Without treatment, it is often fatal.

But we think of stress as a bad thing, and indeed when it goes on too long, it is. Our bodies have developed to expect brief, passing stressors. A predator's attack. A few days of icy weather. Then safety and warm sun. When stress goes on and on, our bodies try to adapt, but high cortisol levels over weeks or months have side effects. Our immune systems become suppressed -- look at any college campus during final exams and you'll see rampant sneezing and coughing as students' high cortisol levels leave them unable to fight off infections. Our metabolism changes — we store fat for famines that never come, and eventually succumb to diabetes. And our brains suffer: ongoing high levels of cortisol can actually cause certain parts of our brains (associated with learning and memory) to become smaller, and other parts (associated with fear) to become larger. Ongoing stress leads to depression.

The stress system is among the more complex of the various hormonal systems. It is called the hypothalamic-pituitary-adrenal (HPA) axis because of the three main organs which secrete its hormones: the hypothalamus (part of the brain), the pituitary (maybe part of the brain and maybe a little stalk hanging off of it, depending on who you ask), and the adrenals (tiny glands down by the kidneys). But there are all kinds of other parts to this system: other parts of the brain which feed in to it from the top level (the hippocampus, site of learning and memory and many other things; the amygdala, site of fear and many other things); corticosteroid binding globulin, the little carrier protein which carries cortisol around in the blood and is made by the liver. The reproductive hormones, estrogen and testosterone, also affect the HPA axis. So does serotonin, the chemical targetted by so many anti-depressants.

All of these different parts of the system work together to regulate the amount of cortisol in the bloodstream: up when there is stress (good or bad), down when there is not. Our bodies are a chemical soup of hormones and these hormones are both cause and effect: cortisol rises when we experience stress. But high levels of cortisol also seem to cause us to feel distress. The stress system is enormously complicated, as mysterious in some ways as the brain itself, and yet a huge part of what makes each of us who we are. Different cortisol profiles (usually high, usually low, very reactive, very unreactive) are associated with different personality types in animals: bold, shy, proactive, reactive. The human research has some more complex findings but the basic truth remains that our personalities are, in part, chemical. We are our hormones.

[The title of this post, The Stress of Life, is also the title of a book by Hans Selye, who first isolated cortisol.]

Shelter dogs, movement, and stress

The always-awesome group of researchers at the Center for Shelter Dogs (associated with the Animal Rescue League of Boston, MA) has just published a new paper. They took a bucketful of different kinds of stress measurements of dogs in their shelter and looked to see if there were correlations between the different kinds. They are working on the same problem that I tackled in my Master’s work: it is awfully hard to tell which dogs in a population are stressed; can we use some kind of easy marker (like observing behavior) to do it? They got similar results to mine: yes, it’s super hard! There is no silver bullet answer. But they provide some interesting insight into how to move forward in the quest to improve stress detection methods.

Jones S., Dowling-Guyer S., Patronek G.J., Marder A.R., Segurson D’Arpino S. & McCobb E. (2014). Use of Accelerometers to Measure Stress Levels in Shelter Dogs, Journal of Applied Animal Welfare Science, 17 (1) 18-28. DOI: 10.1080/10888705.2014.856241

The biggest contribution this paper makes, I think, is the use of an accelerometer to test activity levels in shelter dogs. They attached this device to the collars of dogs when they first came in to the shelter, and got a report of how much the dogs moved around over the course of 24 hours. Because cortisol is such a difficult measurement of stress to interpret, it’s important to supplement cortisol measurements with other measurements, to sort of triangulate your answer. Movement in the kennel is one that I haven’t seen measured before in shelter dogs, and is something that might provide some interesting answers: do dogs move around a lot when they are stressed (pacing) or very little (depression)? I’m really happy to see this new measurement entering the shelter dog literature.

The researchers also attempted to provide a stress score of the dogs by watching them and subjectively assessing stress levels based on a minute’s worth of behavior. This is the holy grail of stress studies: can we look at a dog and tell how stressed it is? If so, we wouldn't need to do all this correlation with cortisol levels. I think we all want to say that someone who really understands dog body language can tell if a dog is stressed by looking at it. I know that I believe that I can estimate the average dog's stress level by looking at it. If you see a dog with low body posture, refusing to meet your eyes, maybe shaking, it’s stressed, right? How hard is that?

Well, if you try to compare your observations (particularly over a very short period of time — in my work, I had more luck with 20 minutes than with 2), the answer is, it’s extremely hard. In this study, they tried to assess an average level of stress in the dogs by taking multiple cortisol measurements, both salivary and urinary. Salivary cortisol changes so very fast that I wouldn’t expect it to correlate well to a day’s worth of exercise, or even to behavioral observations unless they were taken within just a few minutes of the saliva sample, but as this study involved multiple saliva samples over time, I was interested to see if a better average measure was obtained. The researchers also compared salivary cortisol samples with urinary cortisol samples, and since urine builds up in the bladder over time, I’d expect urinary samples to produce a better average as well.

So what did they find? To the question of movement (measured by the accelerometer) vs cortisol (salivary and urinary):

• Maximum activity level correlated with salivary cortisol (p = 0.025)
• Maximum activity level did not correlate with urinary cortisol
• Mean (average) activity level correlated with mean urinary cortisol (p = 0.028)
• Mean activity level did not correlate with salivary cortisol

To the question of the behavioral scoring: no correlation with cortisol of either kind.

So how we interpret all this?

First off, this study ended up enrolling only 13 dogs, taking quite a few measurements of each dog (I haven’t actually covered all of their findings here), and trying to draw conclusions. On the one hand, I want to emphasize that this is how stress studies in dogs are done. I did exactly the same thing in my stress study of hospitalized dogs. It is just extremely hard to enroll enough dogs. If you read the paper itself you get a feel for what the primary researcher went through, as she lists the reasons she had to exclude dogs from the study. It reminded me of my intense frustration during my Master’s work as I had to give up on dog after dog for a variety of reasons. This is par for the course in all these studies: it is almost impossible to get the time and funding to enroll enough subjects to have solid statistical findings. So I am not in any way criticizing these researchers, who did a great job introducing some interesting findings.

But on the other hand, it’s important to recognize that with so many questions and such a small sample set, it is almost impossible to trust the statistical significance of the results. If you do a study in which you ask 100 questions, and you set your p value at 0.05 (which is usual), then you are saying that you expect 5 of your answers to look significant even though they are not. That's what a p value is: setting the bar at which you accept a few false positives. One way around this is to have more subjects, which will lower your p values. Then you can say you'll only accept p < 0.01 or something even more stringent. This makes your findings a bit more trustworthy.

For the number of questions this study asked and the size of their sample set, I would take their findings with a grain of salt. Does activity level correlate with cortisol level? I think it's likely that it does. But I also think that what this study tells us is “this is an interesting area which is worth more study,” not “you should trust that these findings are absolutely true.”

Moreover, what are high cortisol levels telling us about dogs who move around a lot? Exercise itself can increase cortisol levels. This can be a good stressor. So are these dogs distressed or not? This is a problem which is going to be very hard to pick apart. I think a lot of the dogs in a shelter who pace incessantly are indeed very distressed, but some, as this paper points out, are coping with their distress by means of that exercise and are doing better than the dogs who don’t move. The paper also asks the question about how to interpret movement in small versus large dogs. Small dogs simply have relatively more room to move in a little shelter kennel. So how does that change the equation?

As for the lack of correlation between the behavioral stress score and cortisol levels: I feel pretty confident interpreting that one despite my comments about statistics above, because this is a question that has been asked before, and is always answered the same way. We can’t tell what a dog’s cortisol level will be by looking at its behavior. Why not? Is it because we don’t know what the dog's inner experience is (we don’t know if the dog is feeling stress)? Or is it the cortisol level that is lying to us, doing a terrible job of telling us about stress levels, and we’re interpreting the behavior just fine? We don't know, but we do keep trying to find out. Hopefully one day we will.

In the end, I enjoyed this paper. Kudos to the researchers for exploring stress levels in a variety of ways, instead of just one or two. This study was well designed, in my opinion; it just needed a lot more dogs. Hopefully this group or another one will be able to pull together a bigger study going forward.

Designing stress studies, part 3: how do you get the pee?

Having discussed how to choose what substance to test for cortisol (blood, saliva, urine, feces, hair), and how to get the blood or saliva, I now move on to how to collect the —

Urine

I don’t have any personal experience with collecting urine for stress studies. How hard can it be, though, right? I certainly was sent to collect urine from patients fairly frequently as a vet student, and have fond memories of chasing male dogs around a yard with a cup while they would spray just two or three drops at a time. The best vet clinics have long-handled soup ladles which you can use to collect the pee. I have certainly never used my own soup ladle to collect pee from my own dogs to take in for analysis when they were doing poorly.

One of my professors this past semester analyzed estrogen in baboon urine. Apparently one waits on the ground while the baboon is in a tree and watches. Eventually the baboon pees out of the tree. It falls on the ground and voila. Confused, I asked, “But doesn’t it soak into the ground? How do you collect it?” She explained that usually it fell onto a leaf and you could use a syringe to get it from there. I thought to myself: your world is not my world.

Getting pee from cats is a whole separate story. You provide them with a litter box with nonabsorbable pellets, and collect the pee from that. It sounds simple in practice, but in my experience many cats will refuse to pee on a non-absorbable surface.

Of course, if all else fails, you can extract urine directly from the bladder of a dog or cat using a needle. This procedure, called a cystocentesis, obviously requires trained personnel, who may not be available to all studies.

No post on pee would be complete without input from the queen of pee, Julie Hecht. When asked, Julie had quite a bit of advice about urine collection in dogs. She pointed out that when the study in question is being performed using laboratory animals rather than pets, you can teach the dogs to pee on command. This is super convenient, but you’re less likely to have that option with pet dogs. She listed some pitfalls that she found with colleting pee from pet dogs:

• Timing! If you need to collect pee before and after the particular event that you’re studying, it is problematic if the animal doesn’t feel the need to go at the right time.
• If you are out walking with the owner and the dog, try not to act weird. Dogs notice when you act weird. Then they don’t feel like peeing. So make casual conversation, even though all you are thinking about is collecting that lovely, lovely pee.
• Wind sucks. Wear plastic gloves.
• If a dog has a lot of fur, finding the urine stream can be hard. She says succinctly: “That stinks.”

So that is the lowdown on pee collection, and the conclusion of my series on designing stress studies!

Designing stress studies, part 2: how do you get your sample?

I recently posted about how to choose what bodily substance to use to test for cortisol in a stress study: blood, saliva, urine, feces, or hair. Once you have your substance of choice, though, you have to actually extract it from the dog. This can present more or fewer challenges, you know, depending.

Blood

When people first started measuring cortisol, they used blood to do it. Blood is where cortisol shows up first. All the other substances that we measure cortisol in have had their cortisol levels compared to blood cortisol levels, to make sure that they correlate strongly. Researchers had to do studies to prove that these other substances worked for this measurement, which cost a lot of effort and money. They did this because blood is pretty hard to get hold of, in most cases. Sticking a needle in a dog will usually stress it out, and it's hard to get the blood extracted before the stress of the restraint starts changing the blood cortisol levels.

But even aside from that, sometimes a blood draw is simply out of the question. For my Master’s work, I had to cold-call hospital clients and convince them to let me enroll their dog (already in the hospital for some procedure or other, in other words, already having a bad day) in my study. If I had told them that the dog would need a blood draw too, I guarantee that most of them would have said no.

In a comment on the previous post in this series, Tegan pointed out that animals can be trained to submit calmly to blood draws. For some studies, this approach would be invaluable. For my study, again, it wouldn’t have worked. Training an animal to accept a needle is an arduous process, and I had access to those dogs once, on one night. For most shelter dog studies, this would also be an impossible hurdle. But it’s a pretty cool thing to do, if you can do it.

I wish I had a video of another approach to stress-free blood draws. I have seen other vets slide a needle into the lateral saphenous vein, the vein that bulges out of the side of a dog’s hind leg just above the hock. If the dog is distracted (say by someone feeding it), a competent venipuncturist can get it done using this vein with little to no stress. I have seen this technique used in shelter dogs who would not allow restraint for a more traditional draw. But it takes a dog with short, smooth fur and a particularly lovely bulgey vein. It does not work in little dogs. And it definitely requires a competent person to do the draw. After a few years of practice in blood draws, I was just getting to the point during my internship where I could do this one. There can’t be too much poking around to find the vein, or the game is up.

(I did find a video of a technician drawing from the lateral saphenous of a dog who is lying on his side, with an assistant holding off. This is the same vein as the one I am talking about, but in the procedure I’ve seen, the dog can be standing and you actually don’t need someone else to hold off the vein. You come at the vein from above, not below, in a standing dog. Just in case any of you blood-drawers out there want to try this yourself.)

Since blood was such a pain to get, people started trying other substances, figuring anything had to be easier than a blood draw.

Saliva

Saliva is now used much more often than blood in human cortisol studies. You hand a person a cup and they drool into it. No needles, no added stress. Dogs are not so easy. You can’t ask a dog to drool into a cup; you have to get the drool out yourself.

For my study, I used Sorbettes, also known as eye sponges. The instructions say to put one Sorbette into the dog’s mouth for 30-60 seconds, and voila, it has enough saliva on it for an assay. You then put the Sorbette into a tube and spin the tube in a centrifuge to get the saliva out. You only need 25µg, which is hardly anything! What could go wrong.

Sorbettes

First of all, when you are analyzing the saliva later on, you use 25µg per well in the plate of saliva samples, and you get one cortisol value per well. But it turns out that the assay is fairly imprecise, and gets it wrong a decent percent of the time, sometimes close to 10% of the time. So it makes sense to use two wells per sample (now we are at 50 µg per dog). This way, if you get two very different answers for your two wells, you know that the assay went wrong and not to use one of the samples. Wait, which sample is good and which sample is bad? To avoid that problem, just use three wells per sample (now 75µg per dog). Then you can throw out the bad one and keep the two good ones. I had to do this maybe 4-5 times total out of my 90-odd samples. Every time, I was really glad that I had three wells. With two wells I would have had to discard that sample (and that dog) from the study. With one well I would have included bad data in my results.

So 75µg is still not all that much saliva, but it turns out that it is enough to be pretty difficult to get, especially from dogs who are stressed out in a hospital. I used three Sorbettes and rolled them around in the dogs’ mouths for up to four minutes, at which point I had to stop in case the stress of restraint was affecting the cortisol levels. Even then, I had a lot of dry sponges. It was incredibly disheartening. In the end, we saved most of my samples by a) diluting them and changing our calculations, and b) showing the dogs cans of cat food to make them salivate.

I am currently engaged in an email exchange with other researchers who are having similar problems, particularly in small breed dogs and puppies. These days, the new tech to use to get saliva out of dogs is a small rope which the dog can chew on. I like that better than the little sponge-on-a-stick, which dogs could possibly break off and swallow (I had one come perilously close to doing just that). But even so, the problem of getting enough spit remains.

Could you give the dogs food? There is a study suggesting that cheese will not interfere with the cortisol assay, and would be safe to give. [1] It makes me nervous, though.

Could you condition the dogs to salivate when you present the little rope? This is currently under discussion, but some of us are concerned that messing around with the dog’s experience of sampling would invalidate the sample. It’s worth a small study to test it out, though, for sure. I hope someone does it.

By the way: I heard a story, which may be apocryphal, but I will repeat it anyways (and maybe someone out there can corroborate): supposedly a rhino salivary cortisol study used the procedure of collecting saliva with a very long-handled spoon. If true, it is awesome.

To come: urine, feces, and hair, oh my.

References

[1] Ligout S., Wright H., van Driel K., Gladwell F., Mills D.S. & Cooper J.J. (2010). Reliability of salivary cortisol measures in dogs in training context, Journal of Veterinary Behavior: Clinical Applications and Research, 5 (1) 49. DOI: 10.1016/j.jveb.2009.09.027

Designing stress studies, part 1: what do you sample?

Apparently I am an expert in designing stress studies in dogs using cortisol, because I have published one paper about it. Here are some of the words of wisdom I have to share from my extensive experience. You may also be interested in my previous post from several years ago, Why cortisol sucks as a measurement of stress. As I have so many words of wisdom to share, I am going to start with a post just on what you should sample in order to get some cortisol levels. (I intend more posts to follow. But you know how these things go.)

You can measure cortisol in blood, saliva, urine, feces, or hair. We consider the blood (plasma) measurement to be the gold standard: when the adrenals release cortisol, they release it into the blood. This is the hardest to get (you have to stick a needle into the dog) and the fastest to change. Blood cortisol starts increasing only 3 minutes after the onset of a stressor. Practically, this means that since sticking a needle into a dog is likely to stress the dog, you have to complete the blood draw (probably including catching and restraining the dog, unless it is a very mellow dog) in under 3 minutes! [1] This can be possible to do with some dogs and impossible with others. Either way, it requires someone who is very competent at blood draws.

After cortisol is released into the blood, it diffuses into the saliva. This process takes about a minute, so you should collect the saliva less than 4 minutes after you stress the dog by restraining it. [1] If the dog really doesn’t mind the restraint, you can take longer, but I found that sticking things in a dog’s mouth to collect saliva tended to get them excited. In a hospital, just walking into the dog’s run got most dogs excited!

Blood and saliva are the best ways to measure the immediate response to a stressor: take a baseline measurement (in under 3-4 minutes), stress the dog, wait some period of time, then take the post-stress measurement (in under 3-4 minutes, in order to be sure you’re measuring the correct stressor). Taking a single measurement of blood or saliva is not going to tell you as much: there is no known baseline of cortisol for any species, including dogs. It varies too much hour to hour, not to mention that some individuals just start at a different level when they are unstressed. [2]

So take one sample before the stressor starts. After the stressor starts, how long do you wait to sample again? Definitely the same amount of time for each dog. Studies have mapped the time course of cortisol’s rise and fall after a stressor: it seems to go up for an hour or so and then come back down [3]. This is almost certainly dependent on the stressor, of course. My personal rule of thumb is that 20 minutes is a good amount of time to wait to make sure that the cortisol levels have come up enough to be a good reflection of the dog’s reaction to the stressor you’re measuring. (So, just to be super clear: the 3-4 minute rule is just about the beginning of the rise in cortisol levels. The rise will continue for a while.)

If you are interested in how an animal is responding to a chronic stressor, like a few days or weeks in a shelter environment, you’ll be more interested in some measurement of cortisol which covers a longer time period than 20 minutes. Saliva and blood are awful for this kind of study, because their cortisol levels change so fast that you aren’t getting a good overall picture of daily cortisol level; you’re getting more of a snapshot. You could take hourly samples, but that would be difficult in terms of collection and expensive in terms of analysis.

For this kind of study, most people use urinary cortisol. Technically this is the cortisol to creatinine ratio: what is the ratio of cortisol to a standard urine molecule, creatinine? Measuring cortisol this way standardizes your measurement so that it isn’t affected by how dilute the urine is. Urinary cortisol levels will provide something like an average cortisol measurement over however long the dog has been filling up its bladder, probably about 4-6 hours. Urinary cortisol has  been used as a measurement for chronic stress in shelter dogs [4], where you are interested in average stress levels, not an immediate stress response. (For more on measuring stress in shelter dogs using cortisol, see the excellent recent review by Hennessy. [5])

One interesting study looked at elevations in urinary cortisol after dogs had had a trip to a veterinary clinic [6]. In this case, I worry that measuring a specific stressor that has a beginning and an end prior to urine collection is difficult with this method. When did the dogs start making that urine? Before they got stressed, while they were stressed, after they stopped being stressed? When you are comparing different dogs’ urinary cortisol, are you comparing the same thing?

I rarely see studies using fecal cortisol to assess stress in dogs, beyond the proof of concept study [2]; these studies are mostly done in wild animals, because poop is the only thing you can easily collect from them. I have always thought that fecal cortisol might actually be a really good approach to stress measurement in shelter dogs, though: easier to collect than urine, and measuring a longer period of time than urine (since dogs urinate more often than they defecate), so therefore presumably getting a better average. Today as I was looking on Mendeley for some references for this post, I encountered a new study using fecal cortisol to assess stress in cats. [7] Cool.

You can actually measure cortisol in hair as well! I have not seen this done in dogs. It would be a good measurement of even longer term stress levels, over months. One fascinating study measured cortisol levels in archaeological hair, to determine cortisol levels in prehistoric humans. [8]

So, in summary: saliva or blood are good samples to take for a response to an acute stressor, usually one you have control over. Take a sample before the stressor begins and then about 20 minutes after the stressor has begun. Be careful to take your samples very promptly to make sure you are not measuring the stress of the sampling. Urine and feces are better measurements for chronic stressors, and provide a several hour summary of what the cortisol has been doing in the dog’s blood. You can take just one sample of these to compare to your control group.

References

[1] Kobelt A.J., Hemsworth P.H., Barnett J.L. & Butler K.L. (2003). Sources of sampling variation in saliva cortisol in dogs, Research in Veterinary Science, 75 (2) 157-161. DOI: 10.1016/S0034-5288(03)00080-8

[2] Schatz S. & Palme R. Measurement of faecal cortisol metabolites in cats and dogs: a non-invasive method for evaluating adrenocortical function., Veterinary research communications, PMID: 11432429

[3] Vincent I.C. & Michell A.R. (1992). Comparison of cortisol concentrations in saliva and plasma of dogs, Research in Veterinary Science, 53 (3) 342-345. DOI: 10.1016/0034-5288(92)90137-Q

[4] Stephen J.M. & Ledger R.A. (2006). A longitudinal evaluation of urinary cortisol in kennelled dogs, Canis familiaris, Physiology & Behavior, 87 (5) 911-916. DOI: 10.1016/j.physbeh.2006.02.015

[5] Hennessy M.B. (2013). Using hypothalamic–pituitary–adrenal measures for assessing and reducing the stress of dogs in shelters: A review, Applied Animal Behaviour Science, 149 (1-4) 1-12. DOI: 10.1016/j.applanim.2013.09.004

[6] Vonderen I.K., Kooistra H.S. & Rijnberk A. (1998). Influence of Veterinary Care on the Urinary Corticoid: Creatinine Ratio in Dogs, Journal of Veterinary Internal Medicine, 12 (6) 431-435. DOI: 10.1111/j.1939-1676.1998.tb02146.x

[7] Gourkow N., LaVoy A., Dean G.A. & Phillips C.J.C. (2014). Associations of behaviour with secretory immunoglobulin A and cortisol in domestic cats during their first week in an animal shelter, Applied Animal Behaviour Science, 150 55-64. DOI: 10.1016/j.applanim.2013.11.006

[8] Webb E., Thomson S., Nelson A., White C., Koren G., Rieder M. & Van Uum S. (2010). Assessing individual systemic stress through cortisol analysis of archaeological hair, Journal of Archaeological Science, 37 (4) 807-812. DOI: 10.1016/j.jas.2009.11.010

Open access dog salivary cortisol data

I finally got around to sharing the data from my study of dog salivary cortisol levels on figshare. I have meant to do this for months. Particularly, I wanted to do it so that I could wear the cool “I’m a figsharer!” t-shirt that Mark Hahnel gave me at scio13. How embarrassing would it be to wear that shirt and have someone ask what you shared and have to admit that you still haven't actually shared anything? But I am a figsharer now. So if you want numbers, go check it out.

Oh, and in case you’re interested in the associated paper, that’s here (but, sadly, not open access):

Hekman, Jessica P., Alicia Z. Karas, and Nancy A. Dreschel. “Salivary cortisol concentrations and behavior in a population of healthy dogs hospitalized for elective procedures.” Applied Animal Behaviour Science (2012). http://dx.doi.org/10.1016/j.applanim.2012.08.007

Will we ever be able to measure cortisol in real time?

In my Copious Free Time (CFT), I sometimes like to try to figure out how close we are to implementing some of the crazy technology I’d love to use in research. I want to learn more about the canid stress response, as a way of learning about canid domestication (domesticated animals have blunted stress responses, and this may be part of why they are so accepting of novelty and so easy to socialize). The hormone that most people use to study the stress response is cortisol.

I have written in the past about some of the many problems with studying cortisol. Two of those problems are

• Getting hold of cortisol (from blood or even saliva) without increasing the animal’s stress and therefore invalidating your study, and
• Measuring cortisol frequently enough to actually be able to track its very rapid changes in the bloodstream (changes on the order of minutes, continuing to occur and be important over the course of hours).

What we really need, obviously, is a Star Trek-style tricorder that we can point at an animal and ask “what is this animal’s blood cortisol level just now? And how about now?” So recently I was wondering how close we were to this technology.

I asked a friend who works in research imaging. She obligingly sent me a review paper to read, about studying dopamine levels in humans using PET. The problem this paper addresses is getting at the dopamine levels in the brain without having to slice open the skull (something we definitely don’t like to do in humans — and although we might be willing to do it in rats or mice, it is going to be hard to retest the same animal later to see how its dopamine levels have changed, seeing as how a common side effect of skull sliceage is death). This is a pretty cool technology. It goes something like this:

• Inject the individual with a radiotracer which is attached to dopamine agonist or antagonist. The agonist or antagonist will attach to dopamine receptors, and the radiotracer will allow us to use PET to monitor how much of it is attached in the part of the brain that we care about.
• Monitor the changes in the radiotracer in the region of interest. As dopamine levels in that region increase, the unlabelled dopamine will bump more and more labelled agonist or antagonist off of the receptors, which will mean there will be less radiotracer in the region. Less tracer implies more actual dopamine. Do math.

Egerton A., Mehta M.A., Montgomery A.J., Lappin J.M., Howes O.D., Reeves S.J., Cunningham V.J. & Grasby P.M. (2009). The dopaminergic basis of human behaviors: A review of molecular imaging studies, Neuroscience & Biobehavioral Reviews, 33 (7) 1109-1132. DOI: 10.1016/j.neubiorev.2009.05.005

You could use something similar to monitor cortisol binding in the brains of dogs. That would be very interesting, actually, but the studies I tend to envision are more concerned with cortisol amounts that are released from the adrenals. We are actually in a better position here with cortisol, compared to the suckers studying dopamine in the brain: dopamine is released in the brain and stays in the brain, so you never get a chance to see it in the bloodstream. The bloodstream is actually easier to get at than the brain, obviously.

Conversely, cortisol comes from the adrenal glands (way down near the kidneys, far from the brain). The brain sends a signal to the adrenals via very long nerves, and then the adrenals release more or less cortisol, for a longer or shorter period of time. It’s the “more” or “less”, “longer” or “shorter” that are interesting. I actually don’t know enough about where cortisol binds to say if using a radiotracer-labelled cortisol agonist or antagonist, to sit on binding sites, would be interesting, but I suspect this is not the right direction for this technology. Cortisol binds in organs all over the body and affects a lot of processes. Unlike with dopamine, where researchers are interested in very specific (hence small) brain areas, we would want to scan the whole body for cortisol binding.

The radiotracer idea is interesting, though. Maybe we could attach a radiotracer to one of the precursors of cortisol, like cholesterol? We would inject labelled cholesterol. The adrenals would take it up and convert it to cortisol. Then when they released cortisol, we could see the label spreading across the body. No need to measure binding. We could in fact just scan one part of the body where there is a lot of blood — a vein coming out of the adrenals? — to watch cortisol levels rise and fall. The downside: the use of PET to monitor the changes in the radiotracer label. PET is expensive and it requires the subject to hold... perfectly... still. Something dogs are not very good at doing.

What I really wanted, I decided, was something that works sort of the way a pulse oximeter works. Pulse oxes are little devices that you hook up to an animal while it is under anesthesia to monitor their blood oxygenation (you know, to tell if they are dying or not, something which ironically is often easier to tell just by looking at the animal, but we use the things anyways). These devices work by shining a light through an area of non-pigmented skin (such as the tongue, an unpigmented paw pad, or if all else fails, a vulva) and measuring how much hemoglobin (hence oxygen) is in the blood based on color. Could some such device measure amounts of tracer label?

I was letting these ideas percolate and considering how I might write them up for you, dear readers, when I completely by chance came across the following announcement: Sano Intelligence is working on a wearable patch which will continuously monitor blood chemistry.

A wearable patch! That’s actually a much better solution to this problem. It operates wirelessly, so you slap it on (at a cost of $1-2 per patch for materials, though much more in the end to the company to pay for development costs, I imagine) and then remotely monitor changes in blood sugar, electrolytes, and — cortisol? Of course the company does not mention cortisol as one of the substances the patch would monitor. I wonder if there is any reason it couldn’t be included, though. It would help if I had any idea how this patch worked. The company asserts that it’s non-invasive and does not hurt to apply. So how does it get at the substances in the bloodstream? Apparently the company isn’t saying until the patch is released.

So now I wait. If any of you out there in internet land know more, or have thoughts on how this might work, let me know!

Links post

I’m catching up on science blogs reading after a few days off. I was awfully busy recovering from defending my thesis! I am now almost done with the MS part of my dual-degree program, though there will be some thesis edits to do. Then I settle in for the final two years of the DVM program.

Anyways, links!

• The PepsiGate linkfest (A Blog Around the Clock): so comprehensive, he even linked to me.
• Mesozoic Blogosphere (Chasmosaurus): David Orr considers the usefulness of topic-based networks. The comments suggest aggregators to achieve this goal.
• “Dominance” mythologies, Suzanne Hetts (The Other End of the Leash): More on dominance theory in dog training from Patricia McConnell
• Tick news? It ain’t good, Dr. Flea tells AVMA audience (Pet Connection)
• Environmental enrichment is key to happy, healthy animals (Pet Connection): This seemed like a relevant link after The Thoughtful Animal’s recent post about behavioral differences in pigs in enriched environments.
• On detecting stress endocrines in hamster poop (C6-H12-O6): need I say more?
• Learning to speak dog (Dog Star Daily): the usefulness of understanding canine body language, and some good pointers
• You are what you eat – how your diet defines you in trillions of ways (Not Exactly Rocket Science): Nice post about how populations of gut bacteria are influenced by diet in different life stages and in different cultures. “As we learn more about our bacterial partners, we might eventually find ways of influencing them to improve our health, just as breast milk appears to selectively nourish helpful species.” He suggests inoculating people with appropriate gut bacteria, which makes me a little sad. I’d rather see people change their eating habits. Anyone for some research on the effects of fresh whole foods on populations of gut bacteria?
• Under Pressure: The Search for a Stress Vaccine (Wired): What is it today with links to articles about fixing problems with injections? Actually, this is a really good article about Robert Sapolsky, who did ground-breaking work on the effects of chronic stress on health. Apparently Sapolsky is now working on a vaccine to counter the neural effects of chronic stress. I have to admit that I find that a little scary. It sounds like a great answer to the problem of a society full of highly-stressed people, but the stress response is so complex and affects so many parts of our metabolism that it just can’t work without horrible side effects, can it? (The article addresses some of the issues.)
• Virginia Heffernan Is Our Target Audience (Uncertain Principles): For those who don’t know the background, Heffernan wrote a piece in the New York Times in which she criticized Scienceblogs.com for having some snarky people on it, and said as a result of its tone, she didn’t find it to be a good place to go to learn about science. Various science bloggers have opined that she’s dumb and no one should change what they are doing. Here, Uncertain Principles suggests perhaps science bloggers should be trying harder to speak to this particular audience. I’m not going to write a whole blog post about it, but I vote with UP and the others who’ve voiced this particular opinion. Who cares who’s right? The important thing is getting your message across, and it’s pretty clear that some members of the audience find a less snarky message to be easier to absorb.

What could poison ivy possibly have to do with the stress response?

Hyperactive immune system + big back yard + hard-to-find poison ivy + dog who likes to roll in plants in back yard + dog zombie who likes to let her dog sleep on her bed... It’s all bad, people. I’ve thought of various ways I could turn my bad fortune into blog material, but I don’t have the energy due to side effects from various medications (prednisone can upset your stomach; it can also make it hard to sleep, and overdosing on sleeping pills will apparently also upset your stomach), so I’m just going to distill out some facts, inspired by the joyful weekend I’ve had.

• It isn’t the urushiol oil itself on the poison ivy which makes you itch; it is your immune system’s extreme response to it. This may seem like splitting hairs, but it is an important distinction when the rash continues to spread for days (in my case, more than a week). Is the problem that you are being continually exposed? (Dog + yard — this was possible in my case.) Or is the problem that your immune system has become so overstimulated that it is simply continuing to make rashes here and there, whether or not there is any good reason to?
• If the rash just continues to spread, it is worth trying to convince a doctor to give you prednisone. Prednisone is an artificial imitation of our very favorite hormone, cortisol. Because chronic stress suppresses the immune system, when we need to suppress the immune system we can do so by telling the body that it is under extreme amounts of stress. The doses of prednisone that are given in this case are really large compared to the amount of cortisol you might normally expect to see circulating around your blood system. This is why it is important to taper off your dose of prednisone. Your body notices that it is pumped brim-full of glucocorticoid substitute, and stops making cortisol itself. So if you stop taking prednisone suddenly, you could suffer from the effects of a sudden deficiency of glucocorticoids in your system. They are stress hormones, but we have a little bit of stress every day, and so they are actually vital for proper bodily functioning.
• What are the side effects of so much stress hormone in your body likely to be? A lot of the background reading for my thesis involved the effects of endogenous (natural) glucocorticoids on health outcomes, and I did some side reading about the effects of artificial glucocorticoids. So, as you can imagine, I asked my doctor what side effects I might see. He allowed as how my immune system would be suppressed (that was the whole point, after all), and so if I had a fever or some such, I should let him know. For the next few days, I was hyperactive, almost manic, and had extreme difficulty sleeping. I wondered if this was just psychosomatic, due to my knowledge that I was full of stress hormones, and my personal obsession with them. I finally did some research online and discovered that no, these were side effects common to this medication, about which my doctor had failed to warn me. When you are very stressed, you need lots of energy (to run away from the predator which your body assumes is pursuing you). Therefore, your body elevates your blood sugar, mobilizing storage reserves if necessary. This may account for my jumpiness.
• A side note about poison ivy and life with dogs... I know how reactive I am to the stuff, and I am very careful not to touch it. If I had seen any of it in my yard, I would have noticed it. When you live with dogs, it’s important to remember that they may get it on their fur. They may not show signs themselves, but will carry it into the house and give it to you as a present. I don’t actually know that this is what happened, but I suspect. Today I made a hand-made haz-mat suit and toured my yard, killing anything that had three leaflets, then washed everything I could think of, including my dog. Apparently Palmolive is a great way to cut urushiol oil. I also use Tecnu, which is marketed for the purpose, though I hate the smell of it. I was tempted to put this task off until my boyfriend was in town, as he is less reactive to poison ivy, but decided it was best to deal with it while I was still on high doses of prednisone, in case of a reaction.

If this post sounded slightly manic, thank the pred. I have about another week to taper down.

Why do other measurements of stress suck worse than cortisol?

After an overwhelming number of requests (2) for a sequel to my post Why cortisol sucks as a measurement of stress, I am obliging. The fact that I am in the middle of writing this particular section of my thesis and need some high-level perspective on it might also have something to do with it. So: why do other measurements of stress suck worse than cortisol?

When I left you, you were trying to design a study of stress in hospitalized dogs using cortisol as your marker of psychological distress. You were confounded by the fact that cortisol measures both psychological and physiological distress, and that it varies a lot between individuals. I haven’t been around to keep an eye on you lately, so you have started investigating other approaches to measuring stress other than cortisol.

Cortisol is a messenger used by the HPA (hypothalamic-pituitary-adrenal) axis, for the brain to send a message about stress levels out to the body, for the body to pass that message along to the organs that need to change their operations as a result, and for the body to then report back to the brain that the message has been received, so the brain can stop yelling about it. There are multiple levels in this axis; cortisol comes from the bottom-most level, the adrenals. Why not go up one level, to the pituitary? It is actually in the brain, so it is closer to the source of the message and might be less distorted by the game of telephone.

The hormone that the pituitary gland releases as part of the HPA axis is ACTH (adrenocorticotropic hormone, or “the hormone that makes the adrenal cortex change”). ACTH causes cortisol release. Why don’t you measure ACTH release directly? Unfortunately, ACTH can only be measured in the blood; it doesn’t get into the saliva. (Or urine, hair, or feces, three other places you can go to get an estimate of cortisol levels.) The owners of your hospitalized dogs aren’t going to be happy if you tell them you need to draw blood from their dogs for your study. And remember, you’d have to draw the blood pretty quickly in order to get it before the brain mounted a stress response as a result of having a needle stuck into the body. Cortisol levels change in under three minutes. I don’t actually know how long it takes ACTH levels to change, but I will hazard a guess that since they are farther up the telephone chain, they change faster.

What about farther down the chain? CBG (corticosteroid binding globulin, a.k.a. transcortin) is a protein that carries cortisol around in the blood. The body uses CBG as a way of regulating the stress response. When there is less CBG, cortisol is more able to jump inside cells and do its work. OK, no one actually uses CBG to measure stress levels, because we have no real idea how it works. But it is a very cool system that I’m really curious about. And stress researchers would do well to remember that it is there. If the dogs you are studying are very sick, they might not be able to make as much CBG as a healthy dog would, and that would affect their cortisol levels.

That pretty much exhausts using the HPA. Luckily there is an entire second axis for you to mine: the SAM (sympatho-adrenomedullary) axis. This is the series of chemicals that regulate the well-known “fight or flight” response. This particular game of telephone includes adrenaline (epinephrine), the effects of which which many people enjoy abusing when they go on roller coasters. This axis works much more quickly than the HPA. If you hear a sudden loud noise, you will get an adrenaline rush within a second. So you can try to measure adrenaline levels in the blood, but there is just no way you will be able to get the blood out fast enough to not have the stress of the needle (damn needle) affecting them. If you had a very controlled population of animals, with catheters already placed that they were used to, so that you could draw out blood without stressing them, that might work, assuming you could catch the animals without stress. (Catch a mouse without stressing it: difficult. Catch a dog without stressing it: actually, when I went into the runs with the hospitalized dogs I was studying, they definitely experienced eustress, or happy stress.)

You can also measure adrenaline levels in pee! This turns out not to be useful, though. Adrenaline levels go up and down, as we’ve said, very quickly, in response to individual stressors. Pee collects all those changes and averages them out over however many hours (say six). So this approach is definitely not good for measuring responses to specific stressors, like a sudden loud noise. It might be better at measuring something longer term (hey, like the response to being in a hospital!) but initial studies haven’t shown it to work very well at that, either. Adrenaline is just the most interesting when you can map it as it goes up and down, not when you have to look at an average and guess about what was smoothed out.

What about the other end of the SAM? When you get an adrenaline rush, you have some physical changes. Among many other things, your heart rate gets faster. Can you measure that? Well, again, good luck measuring that in a dog without having the excitement of interacting with a human confound your measurement! And heart rate is very sensitive to physical changes; you might be measuring whether the dog is standing up versus lying down, rather than its level of distress.

It turns out that what is a better way to measure physiologic changes from SAM activation is heart rate variability. Your heart rate normally speeds up a little when you breathe in, and slows down a little when you breathe out. (I actually did notice this in a dog once, in a lab where I was supposed to be learning how to find abnormal heart rhythms, and I had to call a vet over to ask if it was actually normal, because it sounded so weird once I noticed it.) When you are stressed (physically or psychologically), this variability goes away. This is not a bad way to measure stress, but you can’t measure it with a stethoscope; you have to hook up equipment to the dog in the form of a little vest with a monitor attached. This is expensive (too expensive for you to use, because your project is on a shoestring budget!). You would also have to get the dog used to the vest, so that you were sure you wouldn’t be measuring stress from having clothing on when the dog is used to being naked. It is therefore not a good measurement for hospitalized dogs on their first day in the hospital, but it is a good measurement for some studies. It’s best when used in conjunction with cortisol, so that the two measurements can catch each other’s mistakes.

That uses up the SAM, but there is a system that is the opposite of the SAM. When your body is not in “fight or flight” mode, it is in “rest and digest” mode. This mode is regulated by the parasympathetic branch of the ANS (autonomic nervous system). (The SAM is the sympathetic branch of the ANS.) Can you measure parasympathetic activity? It should increase when stress decreases, and vice versa. It turns out that when your body is thinking it’s time to rest and digest, it releases a digestive protein into your saliva, known as α-amylase. This protein is useful for pre-digesting carbohydrates. More α-amylase suggests less stress. And it’s even in the saliva, so it can be measured non-invasively! You are very excited until you find a paper from the 1950s (I am not kidding) which is the last time anyone bothered to look for α-amylase in dog saliva. Dogs don’t make it. Because they are not meant to eat lots of carbs? Oh wait, this isn’t a post about nutrition.

(For those of you who say “OK, but what about measuring stress via α-amylase in humans?” — I didn’t delve any deeper into this one after I learned it wasn’t useful in dogs. My guess is that it suffers from similar problems to measuring cortisol: it measures more than just [lack of] distress. It also has been less widely used than cortisol, so we understand its pitfalls less. This would be another good measurement to use as a complement to measuring cortisol. If you want to use it in humans, read lots studies that have used it before you commit.)

So much for the ANS. But you know that increases in stress cause decreases in parts of the immune system. In fact, that’s partly why we care about stress in hospitalized dogs — stressed dogs may not heal as quickly or as well. Can we measure the immune system?

We can. Your saliva normally contains a kind of antibody called IgA. This presumably provides a first line of defense against the bugs on your food. When you are stressed, you make less of it. (At a guess, this is because when you’re running from a lion, you’re not likely to be eating. You’re more likely to be getting bitten, so your immune system needs to focus on defenses against open wounds instead of microbes in food.) Salivary IgA is known as “sIgA.” Can you measure that in dogs? You can, and it is being fairly widely used in humans, in fact. Only some initial work has been done on it in dogs, though. It seems to be prey to some of the same issues cortisol is — varying regularly throughout the day, varying irregularly between individuals — so it’s not yet clear if it’s really a better option. It might be a good way to go for a long term project. For something short, though, it might be better to stick with what is well-understood.

Are there any other ways to measure immune system function as it relates to stress? As I said, your immune system reorients when it thinks you’re running from a lion, to protect against open wounds. It does this in part by packing the blood full of a kind of white blood cell called a neutrophil. Neuts are the first line of defense against microbes coming in through open wounds. You can measure their ratio to another kind of white blood cell, a lymphocyte, to measure stress. A greater N : L (neutrophil : lymphocyte) ratio implies greater stress levels. In some ways, this is a really great measure of stress, because it takes a little while — an hour or so — for the N : L ratio to change after a stressor. So when dogs first come in to the hospital, if you can get blood right away, you could actually measure their unstressed baseline. A later blood sample could provide a comparison. Then you could ignore all that annoying individual variability, because you would be measuring the difference pre- and post-stressor in the same individual. I would have loved to have use this measurement.

But, as always, good luck getting an owner to consent to not one but two unnecessary blood draws. I am not sure I would have felt good about adding that much stress to an already stressed dog’s hospital visit, either. For a different kind of study, this might be a really good option, though as always, it measures the effects of multiple systems, so there is going to be some extra variability to account for.

And that is why, though cortisol is a really appalling way to try to measure stress (looking at my salivary cortisol data right now, I keep saying “why does anyone use this hormone?!”), it is still the most widely used approach. As we learn more about how all these systems interact, it is possible that some day we will develop a method of taking multiple kinds of measurements and basically triangulating distress. Or maybe we’ll develop hand-held fMRI scanners and be able to directly measure activation of specific parts of the brain. For now, we are stuck with spit.

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?”]

But what do you do with the spit after you get it?

Last night, before bed, I collected some saliva from my dog Jack and my roommate’s dog Casey. Jack has, by this point, become inured to the idea that occasionally I am going to grab his muzzle and stick a sponge on a stick inside his lip and swab around for two minutes. He was ready for bed when I did it last night and decided he might as well just doze through it.

Casey is another matter. When I cornered him and grabbed his muzzle, he freaked out.

Casey: OH MY GOD I THINK I AM GOING TO DIE

Me: Casey, I have done this to you five times. Why do you always act like it is going to hurt?

Casey: IT DOES HURT IT HURTS HORRIBLY THE PAIN IS BLINDING

Me: I have done it to myself. I know it doesn’t hurt.

Casey: IT HURTS DOGS! I CAN’T HOLD MYSELF UP ANY MORE

Me: I have done it to about 30 dogs and it didn’t seem to hurt any of them much.

Casey: Oh.

Most dogs are annoyed when I try to insert the swab, but then discover it doesn’t hurt and just deal with it. Casey inevitably squeezes his eyes shut, hyperventilates, and sometimes even collapses to the ground. (You might at this point ask “how do you know there isn’t something special about Casey?”, to which I would reply that, while there are certainly many special things about Casey, he also reacts this way to having his nails clipped, so I am pretty sure he is not experiencing any unusual physical sensations when I collect his saliva.)

I imagined Casey asking me “Why are you doing this to me?” and what my answer might be: “To find out how stressed you are.” As bizarre as that might be from a dog’s perspective, in fact my plan was to analyze his saliva to find out how much cortisol was in it. Cortisol has been called the “stress hormone,” and is an indicator of how much stress an animal is under.

Because I’ve never worked in a lab before, I wanted to do a few sample assays with unimportant data (i.e., saliva from my own dog that is easy to collect) before using the irreplaceable data collected from hospital dogs. It has taken me four months to successfully enroll 24 dogs; if I waste those samples, I am SOL. (Six dogs a month? Well, I’m a lot better at it than I used to be, and I had to take some time off in the middle. I am actually averaging 3-4 dogs a week at this point.) I performed my test assay this morning.

The cortisol assay is a kit costing a couple of hundred dollars which I purchased from the ever-helpful Salimetrics. It is an ELISA, or enzyme-linked immunoabsorbent, assay. The main equipment in the kit is a little plate (I was surprised by how tiny it was) made up of 96 wells, into each of which one might pour a very small amount of liquid. Each of the wells has some cortisol antibodies in the bottom (or so I’m told, since obviously antibodies are too small to see). These will grab on to any cortisol they see and hold on to it.

When I got into the lab this morning, I took the kit out of the fridge, and some older samples out of the freezer, so that they could warm up to room temperature. Then, while last night’s samples from Casey and Jack centrifuged, I sat down with the lab tech’s computer and figured out where everything was going to go on the plate. Aside from all my samples, I had to have room for the standards, which are samples given to you with the kit containing a known amount of cortisol. If your assay doesn’t tell you that the well with the 3.000 standard has about 3.000 µg/dL of cortisol in it, you know you’ve done something wrong. There are also the controls, which contain an arbitrary “high” and “low” amount of cortisol, and are also useful for telling you when you’ve messed up.

Then there are the “zero” and “blank” wells. The zero wells contain nothing but the diluent, or the liquid used to dilute some of the chemical agents in the kit. When the calculations are done at the end, you need to have this well to be able to tell the difference between a real effect and an effect caused by the diluent. And, finally, there are the blank wells, which don’t have any cortisol binding antibodies in them, and therefore will behave differently than the zero wells when the plate is read.

For me, the hardest part of the process is making sure that everything goes where it’s supposed to. This is more complicated than it sounds, involving lots of finding the right well in a small plate that is packed with small wells; remembering to leave space for a sample that I’m going to add later, after it has been diluted; getting the standards and controls in the right places; etc. Doing something that doesn’t require a lot of brainpower but does require a lot of precision, over and over and over, is hard for me.

When all the standards, controls, and samples were in place, and diluent was in the zero wells and the blank wells, I added conjugate to everything. The “conjugate” is cortisol which is attached to an enzyme. Of course, the whole point of the exercise is that you are putting in your saliva samples which contain cortisol, and now you’re adding this new source of cortisol as well. The two sources of cortisol are going to compete for the chance to bind to the cortisol-binding antibodies in the bottom of each well. When there is more cortisol in the sample, less of the conjugated cortisol (with its attached enzyme) can bind, and vice versa.

I put the plate with its samples and conjugate on to a rotator. This is a small device with a flat top which basically waves the plate around so that its contents mix. Every time I use it I am terrified that the plate is going to go flying off and spread my samples across the lab floor. It has not happened yet.

After 55 minutes for the whole competition thing to happen, I washed the plate off (four times), which includes blotting: picking the plate up, turning it upside down, and slamming it on to an absorbent pad. This is also a scary procedure, especially as sometimes strips of wells break off and you have to figure out in which direction to reattach them. Attach them upside-down, and you won’t know which sample is which.

At this point, in theory, the wells were mostly full of bound cortisol. Some of the cortisol, which came from the conjugate I added, had enzyme bound to it. Some of the cortisol, which came from my samples, did not. The wells which contained samples high in cortisol had less of the bound enzyme, and the wells which contained samples low in cortisol had more of the bound enzyme. Next I added TMB (tetramethylbenzidine), which reacts with the bound enzyme to change its color. Obviously, the wells with more bound enzyme will change to a different color than the wells with less.

More plate rotating is next, and then a brief time (sequestered in the dark) for the plate to let the reaction run its course. When I took the plate out of its light-proof bag at the end of this waiting period, the wells were all full of a lovely blue liquid, and different wells were visibly different shades. I added stop solution to stop the reaction, which immediately changed the color to yellow. And then I put the plate in the plate reader, which is a small machine attached to a Windows computer on the lab tech’s desk. It made some thumping noises as it determined the optical density of each well — its color, expressed as a number. It relayed these numbers to the waiting computer, which did the calculations to convert the numbers into concentrations, and I exported the results into Excel.

So how did I do? This was actually my second attempt at doing this assay. Both times, the results were fairly close to what I expected, based on the standards. However, part of what I wanted to figure out today was whether I could dilute small samples and still calculate values which were close to undiluted values. (In other words, if I dilute one sample by 50%, and multiple the calculated concentration by 2, will the result be similar to what I get when I put an undiluted version of that sample in a different well?) My dilutions were way off, so that’s something I need to figure out. I’m hoping to find a lab on campus which does these assays frequently, and ask if I can work with them for a little while, to get some experience.

By the way, the above implies that I competently performed this entire operation on my own, which is completely untrue: I worked under the close guidance of the extremely talented hospital lab technician. I am very lucky that she enjoys teaching; she told me today that she once considered being a school teacher. Her patience and good nature made the day much more enjoyable than it might have been.